Liposomes useful for drug delivery
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15 claims: 10 independent, 5 dependent
- 1ZASTRZEŻENIA PATENTOWE 1. Kompozycja ^^w/i^r^ającLa liposomy w podłożu, przy czym liposom zawiera 1,2distearoilo-SN-fosfatydylocholinę, cholesterol I N-(omega-metoksy-poll(gllkol etylenowy)oksykarbonylo)-1,2-dIstearoIlofosfatydyloetanoloamInę w stosunku molowym 3:2:0,015, gdzie wewnątrz liposomu zamknięte są irinotekan i oktosiarczan sacharozy.
- 2Kompozycja według zastrz. 1, pry czym trielydoamon również jest zamknięty wewnątrz liposomu.
- 3Kompozycja farmaceutyczna zawierająca kompozycję iiposomu według zastrz.1 oraz farmaceutycznie dopuszczalny nośnik i substancję buforową.
- 4Kompozycja farmaceutyczna według zastrz. 3, przy czym współczynnik pH wynosi pomiędzy 6,0 a 7,5.
- 5Kompozycja farmaceutyczna według zastrz. 3 albo 4, przy czym jest ona sporządzona w formie zastrzyku.
- 6Kompozycja według zastrz. 1 lub kompozycja farmaceutyczna według któregokolwiek z zastrz. 3-5, przy czym N-(omega-metoksy-poli(glikol etylenowy)oksykarbonylo)-1,2-distearoilofosfatydyloetanoloamina jest N-(omega-metoksypoli(glikolem etylenowym) (masa cząsteczkowa 2000) oksykarbonylo)-1,2distearoilofosfatydyloetanoloaminą. Merrimack Pharmaceuticals, Inc. Stany Zjednoczone Ameryki Pełnomocnik:120 EP 1 746 976 B1 Z-15531 121 ΕΡ 1 746 976 Bl Ζ-15531 2/29 Figura 3 Figura 4 122 EP 1 746 976 Bl Z-15531 3/29 Czas po wstrzyknięciu (godziny) Figura 5 Objętość guza, mm : 40 50 Czas po inokulacji guza, dni Liposomy TEA-SOS, 50 mg/kg Liposomy TEA-SOS, 25 mg/kg Próbka kontrolna roztworu soli w wodzie Pozbawiony irynotekanu, 50 mg/kg Liposomy TEA-Pn, 50 mg/kg Liposomy TEA-Pn, 25 mg/kg Figura 6 123 Ό V) ο ΐϊ £ .ο" Ό3 Ό ω Ε C 'E N J-ί—«ł.| -ii .t—i.-/ Próbka kontrolna roztworu soli w wodzie *—Wolny lek, 50 mg/kg A—TEA-Pn;50 mg/kg —A—TEA-Pn;25 mg/kg —·—TEA-SOS;50 mg/kg —Θ—TEA-Pn;25 mg/kg 20 30 40 50 60 70 dzień po inokulacji guza Figura 7 124 ΕΡ 1 746 976 Bl Z-15531 5/29 Czas po wstrzyknięciach, godziny Figura 8A Figura 8B 125 EP 1 746 976 Bl Z-15531 ń/29 Żywotność komórek, % w nieleczonei próbie kontrolnej Figura 9 Figura 10 126 EP 1 746 976 Bl Z-15531
- 77/29 Figura 11 Figura 12 127 ΕΡ 1 746 976 Bl Ζ-15531
- 88/29 Czas (dni) Figura 13A 25 35 45 55 Czas (dni) Figura 13B 128
- 99/29 129 ΕΡ 1 746 976 Bl Ζ-15531
- 1010/29 130
- 1111/29 Lipid lub ΑΡΕ (%ID) żywotność komórki (%) Figura 16 Figura 17 131 ΕΡ 1 746 976 Bl Ζ-15531
- 1212/29 Figura 18 132 EP 1 746 976 Bl Stosunek leku/lipidu, % wartość początkowego %I.D. we krwi zastrzyku Z-15531
- 1313/29 czas po wstrzyknięciu, godziny Figura 20 133 ΕΡ 1 746 976 Bl Ζ-15531 Figura 21 Figura 22 134 ΕΡ 1 746 976 Bl Ζ-15531 15/29 Figura 23 Figura 24 135 ΕΡ 1 746 976 Bl Ζ-15531 16/29 Figura 25 Figura 26 17/29 Figura 27 Figura 28 137 EP 1 746 976 Bl Z-15531 18/29 22,0 21,0 20,0 19,0 18,0 17,0 16,0 15,0
- 1414,0 masa ciała (g) _-*-Ls VRB I t |I I I I | I I I I | t « I I | I I | I I « | I I I I » I I I | I-1 ί I | 20 25 30 35 40 45 50 55 dzień po inokulacji guza Figura 29 138 EP 1 746 976 Bl Z-15531 19/29 Objętość guza (mm A 3) Roztwór soli w wodzie Ls VRB 4 mg/kg -±— Ls VRB 6 mg/kg -X—wolne VRB 6 mg/kg -ete-wolne VRB 8 mg/kg -♦—wolne VRB 12 mg/kg 10 15 20 25 30 35 dzień po inokulacji guza Figura 30 139 σ) ro ro Ό ro ω ro Ε 20/29 —♦— Roztwór soli w wodzie Ls VRB 4 mg/kg —a— Ls VRB 6 mg/kg —°—wolne VRB 6 mg/kg —ώ—wolne VRB 8 mg/kg —•—wolne VRB 12 mg/kg J I I I I-1-1-1-1-1-1-1-1-1-1 20 30 dzień po inokulacji guza Figura 31 EP 1 746 976 Bl Z-15531 2500 τ 2000 -co ε Ε, ro Ν =3 σι :g 1000 α Ο 1500 -500 -20 140 21/29 —O” Próbka kontrolna roztworu soli w wodzie Wolny lek -H- F5-ILs-VRB TEA-SOS -A-F5-ILs-VRB TEA - Pn *+ 430 50 czas po inokulacji guza, dni Figura 32 Figura 33 Objętość guza (mm 3 ) 141 22/29 Figura 35 142 23/29 143 EP 1 746 976 Bl Z-15531 24/29 %ID we krwi Objętość guza (mm3) 20 30 40 50 60 Czas po inokulacji guza, dni Figura 37 czas po wstrzyknięciu, godziny Figura 38 144 25/29 czas po wstrzyknięciu, godziny Figura 39 Figura 40 145 ΕΡ 1 746 976 Bl czas po wstrzyknięciu, godziny Z-15531 Figura 41 czas po wstrzyknięciu, godziny Figura 42 146 27/29 4-— Próbka kontrolna roztworu soli w wodzie Zmiana masy ciała (%) objętość guza, mm Figura 43 Ll .t.1 | I t I I | I I t I | I I I I J t I I I | I I I I j I I I I | I I ! i ! 1 I I 1 | I t t 1 | 1 1 I I j 5 10 15 20 25 30 35 40 45 50 55 Czas po rozpoczęciu okresu leczenia (dni) Figura 44 147 2000 28/2 U objętość guza (mm 3 ) 1500 1000 500 - -I—|I I-i—I|I I I I J 1-1-1 I J I-1 i I j I i I t|1 I I l J t i I i j·!!:!
- 1515 20 25 30 35 40 45 50 Czas po implantacji guza (dni) Figura 45 148 ΕΡ 1 746 976 Bl Ζ-15531 29/29 Figura 46 Figura 47
Independent claims15
834 paragraphs in 2 sections, as filed
The present invention relates generally to the field of liposomes, and more particularly to liposome compositions useful in providing therapeutic or diagnostic individuals.
Background of the Invention
Liposomes or bilayer lipid vesicles have been used or proposed for use in a variety of applications in scientific, industrial and medical research, in particular for use as carriers of diagnostic or therapeutic substances in vivo. See, for example: Lasic, D. Liposomes: from physics to app / ications. Elsevier, Amsterdam, 1993. Lasic, D, and Papahadjopoulos, D., ed. Medisai Applications of Liposomes Elsevier, Amsterdam, 1998. Liposomes are usually characterized by having an internal space isolated from the external environment via a membrane of one or more bilayers forming a microscopic pouch or bubble. Bilayer liposome membranes are typically formed by lipids, i. amphiphilic molecules of synthetic or natural origin, which contain a spatially separated hydrophilic and hydrophobic region. See Lasic D., 1993, supra. The bilayer liposome membranes can also be made by amphiphilic polymers and surfactants (polymersomes, niosomes). A liposome typically serves as a carrier for an individual such as, without limitation, a chemical compound, a combination of compounds, a supramolecular complex of synthetic or natural origin, genetic material, a living organism, a part or derivative thereof that is capable of possessing a useful property or exerting a useful activity. To this end, the liposomes are prepared to contain the desired species in a suitable form. The process of introducing the desired species into the liposome is often referred to as "loading." The inserted liposome species can be completely or partially inserted into the internal space of the liposome, in the bilayer membrane of the liposome, or bound to the outer surface of the liposome membrane. The incorporation of species into liposomes is also referred to as encapsulation or closure, the three terms being used interchangeably in the present invention while retaining the same meaning. The intention of encapsulating an individual in the liposome is often to protect the individual against a destructive environment, while providing the possibility of exerting activity mainly in the area or in an environment in which such activity is beneficial, but to a lesser extent in other areas where the activity may be unhelpful or unwanted. This phenomenon is referred to as delivery. E.g,
Ideally, such liposomes can be formulated to contain compounds (i) with high loading efficiency, i.e. a high percentage of encapsulated species relative to the amount taken in the encapsulation process; (ii) a high number of encapsulated species per unit of liposomal bilayer material; (iii) in a high concentration of the encapsulated species; and (iv) in a stable form, i.e. a small release (leak) of the contained species upon storage or substantially prior to the liposome in the region or environment in which the encapsulated liposome species is capable of exerting the intended activity.
Documents WO2005 / 002546, WO98 / 17256, US4321259, US5316771, US5785987 and US6110491 disclose liposomes to be encapsulated and methods for their production.
Accordingly, there is a need in the art for providing various liposomal compositions that are useful in a delivery system of various compounds, especially therapeutic, diagnostic or imaging units.
Summary of the Invention
The present invention is based on a finding which treats substituted ammonium salts and polyanions useful for loading and retaining individuals inside liposomes. Accordingly, the present disclosure provides methods and liposome compositions useful for delivery of a plurality of entities, particularly therapeutic entities, i.e. units useful in diagnosing, prognosing, examining, imaging, treating or preventing an undesired condition, i.e., a disease in a living body, such as man, plant or animal.
The invention provides a composition comprising liposomes in an environment in which the liposome comprises 1,2-distearoyl-SN-phosphatidylcholine, cholesterol and N- (omega-methoxy-poly (ethylene glycol) oxycarbonyl) -1,2-distearoylphosphatidylethanolamine in a molar ratio of 3: 2: 0.015, where irinotecan and sucrose octasulphate are enclosed within the liposome.
The invention also provides a pharmaceutical composition comprising a liposome composition of the invention and a pharmaceutically acceptable carrier and a buffer substance.
In one embodiment, the present disclosure provides a composition comprising a liposome in an environment in which the interior of the liposome comprises substituted ammonium.
<sup>R</sup>and
wherein at least one of the organic groups has a secondary or tertiary carbon directly bonded to the ammonium nitrogen. Preferably, the substituted ammonium compound encapsulated in the liposomes has a negative acid logarithm (deprotonation) of the dissociation constant (pKa) of at least about 8.0, at least about 8.5, at least about 9.0, at least 9.5, or at least 10.0, like approxare reformed in an aqueous solution at room temperature.
In another embodiment, the present disclosure provides a composition comprising a liposome in a substrate, wherein the internal space of the liposome comprises a polyanion and wherein the polyanion is a polyol or polyated sugar. The liposome preferably contains a transmembrane gradient capable of loading the individual into the liposome. In one embodiment, the transmembrane gradient is a gradient of an ammonium, a quaternary ammonium or a primary, secondary or tertiary substituted ammonium compound having a negative acid logarithm (deprotonation) of the dissociation constant (pKa) of at least about 8.0 in dilute aqueous solution at ambient temperature. , at least about 8.5, at least about 9.0, at least
9.5, or at least 10.0. The liposome optionally contains a closed species, e.g. therapeutic agents, a detectable label or whole cationic organic molecules.
In still another embodiment, the composition provided in the present disclosure further comprises an individual encapsulated in the liposomes of the disclosure. Preferably the individual is encapsulated in the inner space of the liposome. For example, the inner space of the liposome further comprises anti-cancer therapeutic agents and the level of toxicity of the composition for the subject is at least equal to or less than the level of toxicity of the anti-cancer therapeutic agent administered to the subject without the composition.
In yet another embodiment, the composition provided by the present disclosure is a liposome composition comprising a camptothecin compound. The composition has an antitumor effect at least two times, four times or ten times higher than that of camptothecin given at similar doses in the absence of the composition, while the toxicity level of the composition is not exceeded, is at least two times or at least four times less than the toxicity camptothecin compound administered at similar doses in the absence of the composition. In one embodiment, the camptothecin compound is a prodrug and is contained in liposomes in an amount of at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.5 mg, or at least 1 mg per day. 1 mg membrane of liposomal material, e.g. lipids. The camptothecin compound is preferably encapsulated in the liposome, substantially within the interior space of the liposome. In one case, the camptothecin compound is irinotecan (CPT-11).
In yet another embodiment, the composition provided by the present disclosure is a liposome composition of alkaloids of a rose dye or its derivatives. The composition has the property of 24-hour drug retention within the liposome and after 24 hours of in vivo mammalian blood exposure of at least 50%, at least 60% or at least 70% of the initial drug loading. The alkaloids of the rose dye or its derivatives are preferably encapsulated in the liposome, substantially inside the interior space of the liposome. One of these exemplary mammals is the rat. Examples of alkaloids of the rose dye and its derivatives are vincristine, vinblastine and vinorelbine.
In yet another embodiment, the present disclosure provides a method for containing an individual in a liposome. The method comprises contacting the liposomes of the present disclosure with an individual, e.g. a curative or detectable species.
Preferably, the reaction is carried out under conditions where the concentration of the substituted ammonium or polyanion of the present disclosure in the substrate is less than in the interior space of the liposomes. In one embodiment, the liposome composition remains in contact with the individual in an aqueous environment.
In yet another embodiment, the present disclosure provides a method for containing an individual in a liposome. The method comprises contacting a liposome-containing composition according to the present disclosure with an initial species in which the unit is capable of transforming into an individual under defined conditions and providing conditions within the liposome where transformation of the initial individual into an individual within the liposome. In one case, the individual is an organic compound, as well as the initial individual in its basic derivative.
In yet another embodiment, the present disclosure provides a kit for encapsulating individuals into liposomes. The kit includes a container with liposomes according to the present disclosure and, optionally, a container containing an individual and / or instructions for the user, e.g. for encapsulation of the individual.
Short Description of Drawings
Figure 1 shows the pharmacokinetics of lipid lipid (circle) and drug (triangles) blood after intravenous bolus delivery of CPT-11 loaded liposomes to a rat. Liposomes are loaded using the TEA-Pn method (see example 9).
Figure 2 shows the dynamics of the rat's liposomal lipid drug ratios in vivo after intravenous bolus delivery of CPT-11 loaded liposomes using the TEA-Pn method (see Example 9).
Figure 3 shows the efficacy of anti-tumor free CPT-11 and liposomal CPT-11 against human tumor xenografts BT-474 in nude mice. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 10).
Figure 4 shows the dynamics of animal body weight during the treatment of tumors
BT-474 in nude mice using free CPT-11 or liposomal CPT-11. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 10).
Figure 5 shows the dynamics of the rat's liposomal lipid drug ratios in vivo after intravenous bolus delivery of CPT-11 loaded liposomes using TEA-SOS methods. (See example 14).
Figure 6 shows the efficacy of anti-tumor free and liposome CPT-11 against human HT-29 colon tumor xenografts in nude mice. The inscription on the board indicates how the medicine was loaded and the dose administered during the injection. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 15).
Figure 7 depicts animal body weight dynamics when treating HT-29 tumors in nude mice using a slow or liposomal formulation of CPT-11. Error bars represent the standard deviation of the data. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 15).
Figure 8A shows the pharmacokinetics of blood lipid lipid after intravenous bolus injection of Topotecan loaded liposomes. The inscription on the board indicates the way of loading and the content of the drug in liposomes. (See example 24).
Figure 8B shows the dynamics of the rat's liposomal lipid drug ratios in vivo after intravenous bolus administration of Topotecan-loaded liposomes. The inscription on the board indicates the loading method and content of the drug in liposomes. (See example 24).
Figure 9 depicts in vitro cytotoxicity of free, liposomal or HER2-targeted immunoliposomal Topotecan (TEA-Pn method) against SKBr-3 breast cancer cells. (See example 27).
Figure 10 depicts in vitro cytotoxicity of free, liposomal or HER2-targeted immunoliposomal Topotecan (TEA-SOS method) against SKBr-3 breast cancer cells. (See example 32).
Figure 11 shows the efficacy of the anti-cancer formulation of Topotecan (TPT) against human breast tumor xenografts BT-474 in nude mice. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 29).
Figure 12 shows the dynamics of animal body weight during the treatment of tumors
BT-474 in nude mice using free Topotecan (TPT) or anti-HER2 immunoliposomal Topotecan (F5 ILs-TPT). A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 29).
Figure 13A shows the efficacy of the anti-cancer formulation that is Topotecan against human tumor xenografts BT-474 in nude mice. Free Topotecan (free TPT) or liposomal Topotecan (Ls-TPT) were administered at one eighth their maximum tolerated dose. Error bars represent the standard deviation of the data. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 31).
Figure 13B shows the efficacy of the anti-cancer preparation that is Topotecan against human tumor xenografts BT-474 in nude mice. Free Topotecan (free TPT) or liposomal Topotecan (Ls-TPT) were administered at one-fourth of their maximum tolerated dose. Error bars represent the standard deviation of the data. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 31).
Figure 13C shows the efficacy of the anti-cancer preparation that is Topotecan against human tumor xenografts BT-474 in nude mice. Free Topotecan (free TPT) or liposomal Topotecan (Ls-TPT) were administered at one-half of their maximum tolerated dose. Error bars represent the standard deviation of the data. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 31).
Figure 13D shows the efficacy of the anti-cancer preparation that is Topotecan against human tumor xenografts BT-474 in nude mice. Free Topotecan (free TPT) or liposomal Topotecan (Ls-TPT) were administered at their maximum tolerated dose. Error bars represent the standard deviation of the data. A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 31).
Figure 14 depicts the dynamics of the average body weight of an animal when treating BT-474 tumors in nude mice with free Topotecan (free TPT) or liposomal Topotecan (Ls-TPT) administered at the maximum tolerated dose.
A "control sample" means mice treated only with a drug-free substance and liposomes. (See example 31).
Figure 15 depicts the cytotoxicity of free 6- (3-aminopropyl) -lixycin (free AE), liposomal 6- (3-aminopropyl) -lixycin (Ls-AE) or the immunotrophic 6-aminopropyl) ellipticine-directed HER2 (F5 ILs -AE) against in vitro BT-474 breast cancer cells. (See example 35).
Figure 16 depicts in vitro cytotoxicity of free 6- (3-aminopropyl) ellipticine (free APE), liposomal 6- (3-aminopropyl) -lixycin (Ls-APE) or EGFR-targeted immunoliposomal 6- (3-aminopropyl) -lixycin (C225) -ILs-APE) against low (MCF-7) or high (MDA-MB468) breast cancer cells expressing the EGF receptor. (See example 36).
Figure 17 shows the pharmacokinetic characteristics of liposomal 6 [beta] -aminopropyl) ellipticine (APE) liposomal blood: pharmacokinetics of the lipid liposome (table A, open circles), drug (table A, filled circles) and dynamics of the lipid lipid ratio (Table B) after intravenous administration of the APE liposome bolus to the rat. (See example 37).
Figure 18 shows blood pharmacokinetic characteristics of vinorelbine formulated in liposomes (Ls-VRB) and anti-HER2 immunoliposomes (F5-ILs-VRB): lipid lipid pharmacology of the lipid (array A), drug (table B) and dynamics of the drug-liposome ratio lipid (Table C) after intravenous administration of vinorelbine liposomes to the rat. (See example 43).
Figure 19 shows the pharmacokinetics of blood lipid lipid after intravenous bolus injection of liposomes loaded with vinorelbine. Liposomes are loaded using initially closed triethylammonium dextran sulfate (DS-TEA), ammonium dextran sulfate (DS-A) or ammonium sulphate (SA). (See example 44).
Figure 20 shows the dynamics of the rat's liposomal lipid drug ratios in vivo after intravenous bolus delivery of vinorelbine-loaded liposomes using initially closed compounds such as triethylammonium dextran sulfate (DS-TEA), ammonium dextran sulfate (DS-A) and ammonium sulphate ( ARE). (See example 44).
Figure 21 shows the pharmacokinetics of lipid liposome blood after intravenous bolus injection of liposomes loaded with vinorelbine. Liposomes are loaded using initial triethylammonium sucrose (TEA-SOS) sucrose octasulfate and have an average size as indicated in the table. (See example
45).
Figure 22 shows the dynamics of the rat's liposomal lipid drug ratios in vivo after intravenous bolus injection of vinorelbine-loaded liposomes. Liposomes are loaded using initial triethylammonium sucrose (TEA-SOS) sucrose octane and have an average size as indicated in the table. (See example 45).
Figure 23 shows the pharmacokinetics of lipid lipid in rat after intravenous bolus injection with vinorelbine formulated in liposomes (Ls-VRB) or anti-HER2 immunoliposomes (F5-ILs-VRB) using the TEA-SOS method. (See example 46).
Figure 24 shows the dynamics of drug ratios on rat liposomal lipid in vivo after intravenous bolus administration of liposomal liposomes-loaded liposomes (Ls-VRB) or anti-HER2 immunoliposomes (F5-ILs-VRB) using the TEASOS method. (See example 46).
Figure 25 depicts in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls-VRB) or HER2-directed immunoliposomal vinorelbine (F5-ILs-VRB) against overexpressing human breast cancer cells MDA-MB-453. (See example 48).
Figure 26 depicts in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls-VRB) or HER2-directed immunoliposomal vinorelbine (F5-Ils-VRB) against overexpressed HER2 of non-small cell lung CaLu-3. (See example 49).
Figure 27 depicts in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls-VRB / SOS-TEA) or HER2-targeted immunoliposomal vinorelbine (F5-Ils-VRB / SOS-TEA) against overexpressed HER2 to human SKBr-breast cancer cells 3. (See example 50).
Figure 28 shows the anti-tumor efficacy of free vinorelbine (free VRB) or liposomal vinorelbine (Ls VRB) against human HT-29 colon carcinoma xenografts in nude mice. "Saline solution in water" means mice treated only with drug-free substance and liposomes. Error bars represent the standard deviation of the data. (See example 51).
Figure 29 shows the dynamics of average body weight during the treatment of HT29 tumors in nude mice with free vinorelbine (free VRB), liposomal vinorelbine (Ls VRB), or only substance (saline solution in water). Error bars represent the standard deviation of the data. (See example 51).
Figure 30 shows the antitumor efficacy of free vinorelbine (free VRB) or liposomal vinorelbine (Ls VRB) in the syngeneic C-26 model of mouse colorectal cancer. The dose of the drug for injection was indicated in the text on the blackboard. Error bars represent the standard deviation of the data. "Saline solution in water" means mice treated only with drug-free substance and liposomes. (See example 52).
Figure 31 shows the dynamics of average body weight during the treatment of murine syngeneic C-26 colon tumors with a variety of free vinorelbine (free VRB), liposomal vinorelbine (Ls VRB) or only substance (saline solution in water). The dose of the drug for injection was indicated in the text on the blackboard. (See example 52).
Figure 32 shows the antitumor efficacy of free vinorelbine (free drug) or scFv F5-HRP conjugated anti-HER2 immunoliposomal vinorelbine prepared by the TEA-SOS (F5-ILs-VRB TEA-SOS), anti-HER2 immunoliposomal vinorelbine method developed by the TEA- Pn (F5-ILs-VRB TEA-Pn) against HER overexpression of human breast cancer xenografts (BT-474) in nude mice. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 53).
Figure 33 shows the dynamics of average body weight in the treatment of mice overexpressing HER2 human breast cancer xenografts (BT-474) with free vinorelbine, scFv F5 conjugated, anti-HER2 immunoliposomal vinorelbine prepared using the TEA-SOS method, anti-HER2 immunoliposomal vinorelbine prepared using the TEA-Pn method or only the substance itself. For explanation of symbols, see subtitles for Figure 32. (See also example 53).
Figure 34 shows the antitumor efficacy of free vinorelbine (free drug) or scFv F5 conjugated anti-HER2 immunoliposomal vinorelbine prepared using a different amount of PEG lipid against HER overexpression of human breast tumor xenografts (BT-474) in nude mice. Error bars are standard deviations of data. "Substance control" means mice treated only with a drug-free substance and liposomes. (See example 54).
Figure 35 depicts the anti-tumor efficacy of free vinorelbine (free NAV), liposomal vinorelbine (NAV Lip) or conjugated FC225Fab 'with anti-EGFR immunoliposomal vinorelbine (C225-NAV Lip) against overexpressing EGFR human glioblastoma xenografts (U87) in nude mice. "Saline solution in water" means mice treated only with drug-free substance and liposomes. (See example 55).
Figure 36 shows the lipid lipid pharmacology of the lipid and drug drug dynamics / lipid lipid in the rat's blood following bolus administration of doxorubicin formulated in iiposomes using the triethylammonium sulphate method. (See example 56).
Figure 37 shows anti-tumor efficacy of liposomal doxorubicin (Ls-Dox) or conjugated scFv F5, anti-HER2 immunoliposomal doxorubicin (F5 ILs-Dox) prepared using different amount of PEG lipid against HER overexpression of human breast tumor xenografts (BT-474) in nude mice . The inscription on the board shows the amount of PEG lipids expressed in mole. % in phospholipids of liposomes. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 57).
Figure 38 shows the pharmacokinetics of porphyrinic blood of vinblastine in a rat.
(See example 58).
Figure 39 shows the dynamics of the drug / liposomal lipid rat ratio in the rat's blood after intravenous bolus administration of vinblastine liposomes. (See example 58).
Figure 40 depicts in vitro cytotoxicity of free vincristine (free VCR), liposomal immunoglobulin (Ls-VCR) or HER2-directed immunoliposomal vincristine (F5-ILs-VCR) against overexpressed HER2 to human SKBr-3 breast cancer cells. (See example 61).
Figure 41 shows the pharmacokinetics of blood in the lipid lipid of the rat after intravenous bolus delivery of vincristine formulated in liposomes of various mean sizes (as indicated in the panel text). (See example 62).
Figure 42 shows the dynamics of the drug / lipid lipid ratio in the blood of the rat after intravenous bolus delivery of vincristine formulated in liposomes of various mean sizes (as indicated in the panel text). (See example 62).
Figure 43 shows the anti-tumor efficacy of free vincristine (free VCR), liposomal vincristine prepared by the triethylammonium citrate method (Ls-VCR citrate), liposomal vincristine prepared by sucrose triethylammonium octosulfate (Ls-VCR SOS) or scFv F5 conjugated anti-HER2 immunoliposomal vincristine prepared by the triethylammonium process. sucrose octostearate (F5 IL1-VCR SOS) against overexpression HER2 human breast cancer xenografts (BT-474) in nude mice. "Saline control in water" means mice treated only with drug-free substance and liposomes. (See example 64).
Figure 44 shows the dynamics of mean body weight in the treatment of mice overexpressing HER2 human breast cancer xenograft (BT-474) with free vincristine (slow VCR), liposomal vincristine prepared with triethylammonium citrate (Ls-VCR citrate), liposomal vincristine prepared with the triethylammonium octamulphate process sucrose (Ls-VCR SOS) or conjugated scFv F5, anti HER2 immunoliposomal vincristine prepared by the triethylammonium sucrose octosulfate method (F5 IL1-VCR SOS), or using only the substance (control sample of saline solution in water). (See example 64).
Figure 45 depicts the anti-tumor efficacy of free vincristine (vincristine), liposomal vincristine (nt-vcr) or C225 Fab 'conjugated anti-EGFR immunoliposomal vincristine (C225-vcr) against overexpressing EGFRvIII human brain tumor xenograft (U87) in nude mice. "Saline solution in water" means mice treated only with drug-free substance and liposomes. (See example 65).
Figure 46 shows the pharmacokinetics of blood in CPT-11 and the dynamics expressed as a percentage of CPT-11 present in the active form (lactone) in rat's blood after intravenous bolus administration of liposomal CPT-11. (See example 69).
Figure 47 shows the pharmacokinetics of kiwifruit in CFT-II and the dynamics expressed as a percentage in the CPT-11 present in the active form (lactone) in the blood of srcrurt after intravenous administration of the CPT-11 solution (free CPT-11). (See example 69).
Description of Preferred Exemplary Executions
The present disclosure generally relates to methods and liposome compositions useful for delivery of a plurality of species, particularly therapeutic agents and imaging agents. The discovery of the present disclosure is that substituted ammonium and polyanion salts are useful for loading and maintaining species, e.g. an intra liposome compound. Accordingly, the present disclosure provides a liposome composition and kits comprising substituted ammonium salts and / or polyanions and methods for making such liposomal compositions.
According to one aspect of the present disclosure, it provides a liposome composition comprising in its interior space one or more substituted ammonium compounds of the formula
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wherein each of R 1, R 2, R 3 and R 4 is an independent hydrogen atom or an organic group, and in which at least one of R 1, R 2, R 3 and R 4 is an organic group such as alkyl, alkylidene, heterocyclic alkyl, cycloalkyl , aryl, alkenyl, or a cycloalkenyl group derived from a hydroxy-substituted group, optionally including a S, O or N atom in its hydrocarbon chain, e.g. to form an ether (including acetal or ketal), ester, sulfide (thioether) amine or amide linkage . If fewer than three of R1, R2, R3 and R4 are organic groups, at least one, preferably two of the organic groups of the disclosure have secondary or tertiary carbon atoms (e.g., a carbon atom having 2 or 3 carbon-carbon bonds), directly related to the ammonium nitrogen atom, e.g. substituted ammonium is a sterically blocked ammonium. In general, the presence of a titratable ammonium such as unsubstituted ammonium ion (NH4 +) as well as primary or secondary straight-chain alkyl ammonium ions in the inner surface of the liposome according to the present disclosure is known to provide increased hermeticity to weak amphiphilic bases, e.g. via the "active" mechanism, " remote "or" targeted transmembrane gradient "loading (Haran, et al., Biochim Biophys. Acta, 1993, v. 1152, pp. 253-258, Maurer-Spurej, et al., Biochim. Biophys. Acta, 1999, pp. 1416, pp. 1-10). However, these ammonia compounds have hydrogen atoms that are easily introduced into the nucleophilic displacement reaction and then chemically react differently with liposome-enclosed units, and are therefore capable of impairing the chemical integrity of the individuals during or after the loading (closure) process. Thus, it is desirable for a closed substituted ammonium compound to be more chemically inert, to be poor in chemical function, resulting in instability and ease of reacting with the liposomal components that can be contained in the hermetic species. Unexpectedly, it has been found that liposome compositions containing in their internal space substituted tertiary and quaternary ammonium salts which have no substitutable hydrogen atom or sterically blocked primary or secondary ammonium in which access to the ammonium bicarbonate atom is hindered by being sterically blocked by adjacent large organic group such as, for example,
In one of the embodiments disclosed herein, the liposome-enclosed substituted ammonium compound is pharmaceutically inert, i.e., it does not elicit an undesirable physiological response when administered to a live subject e.g. in humans or animals, within the amount of liposome membrane material that is sufficient. to provide an effective dose of the individual encapsulated in the liposome. In another embodiment, the substituted ammonium has an acceptable level of toxicity to the subject. Usually an acceptable level of toxicity means that the toxic dose, e.g. the maximum tolerated dose (MTD) or doses causing 50% mortality (LD50) in substituted ammonium is at least twice, at least four times, at least eight times or at least ten times greater than a toxic dose in an individual encapsulated in a liposome, e.g. drug loaded inside the liposome. For example, triethylammonium sulfate has an acceptable level of toxicity because its LD50 is about 40 times higher than LD50 in doxorubicin, an anti-cancer drug. The level of toxicity or physiological responses at the substituted ammonium as well as units contained in the liposomes, if they are no longer known, can be readily determined using standard techniques well known to those skilled in the biomedical art. See, for example, SC Gad. Drug Safety Evaluation, Wite · /, New York, 2002. One method for quantifying the toxicity of a free and / or a liposomally-formed drug is described in Example 16 herein. because its LD50 is about 40 times higher than LD50 in doxorubicin, an anti-cancer drug. The level of toxicity or physiological responses at the substituted ammonium as well as units contained in the liposomes, if they are no longer known, can be readily determined using standard techniques well known to those skilled in the biomedical art. See, for example, SC Gad. Drug Safety Evaluation, Wite · /, New York, 2002. One method for quantifying the toxicity of a free and / or a liposomally-formed drug is described in Example 16 herein. because its LD50 is about 40 times higher than LD50 in doxorubicin, an anti-cancer drug. The level of toxicity or physiological responses at the substituted ammonium as well as units contained in the liposomes, if they are no longer known, can be readily determined using standard techniques well known to those skilled in the biomedical art. See, for example, SC Gad. Drug Safety Evaluation, Wite · /, New York, 2002. One method for quantifying the toxicity of a free and / or a liposomally-formed drug is described in Example 16 herein. they can be easily established using standard techniques well known to those skilled in the biomedical field. See, for example, SC Gad. Drug Safety Evaluation, Wite · /, New York, 2002. One method for quantifying the toxicity of a free and / or a liposomally-formed drug is described in Example 16 herein. they can be easily established using standard techniques well known to those skilled in the biomedical field. See, for example, SC Gad. Drug Safety Evaluation, Wite · /, New York, 2002. One method for quantifying the toxicity of a free and / or a liposomally-formed drug is described in Example 16 herein.
In one preferred embodiment, the substituted organic groups of R1, R2, R3, or R4 have size and physicochemical properties sufficient to provide such conditions that the substituted ammonium forms in the aqueous medium a substantially true (molecular) solution, but not two-layered micelles or the like. self-organizing structures. Accordingly, the substituted ammonium is preferably somewhat dispersed or substantially dispersed in the bilayer portion of the liposomes, thereby minimizing the risk of destabilization, dissolution or permeability of the encapsulated ammonium liposomes.
The organic group in substituted ammonium is typically a hydrocarbon containing up to 8 carbon atoms, up to 6 carbon atoms or up to 4 carbon atoms, and in its entirety, the substituting groups contain up to 18, up to 16, up to 12, or up to 9 carbon atoms. These displaceable hydrocarbon groups include a combination of primary, secondary or tertiary carbon atoms joined together, as well as cycloalkyl groups that are connected at their ends directly to the ammonium nitrogen, forming heterocycles or with the carbon atom of the substituent group of ammonium bicarbonate. Such substituted alkyl groups may also contain heteroatoms, e.g., oxygen, nitrogen or sulfur in carbon chains forming a functional group, e.g., ether, acetal, amine or sulfides, as well as one functional group, e.g., a hydroxyl group bonded to the alkyl chain. coal.
In another embodiment, the substituted ammonium is: a heterocyclic ammonium, e.g. ammonium, in which at least two of the substituents R 1, R 2, R 3, or R 4 form a ring; sterically blocked primary ammonium; or sterically blocked secondary ammonium. In general, sterically blocked primary or secondary ammonium includes any substituted ammonium with one or two substituents R 1, R 2, R 3, and R 4 together with alkyl groups that spatially densify the molecule, e.g. any substituted ammonium with one or two substituents R 1, R 2 , R3, and R4 together with one or two cycloalkyl groups or an alkyl group containing at least one secondary or tertiary alkyl carbon atom linked to an ammonium substituted nitrogen atom. Examples of such heterocyclic sterically blocked primary and secondary amines include, without limitation, isopropylethylammonium, isopropylmethylammonium, diisopropylammonium, te / p-butylethylammonium, dicyclohexylammonium, protonated morpholine forms, pyridine, piperidine, pyrrolidine, piperazine, citid-butylamine, 2-amino-2-methylpropanol-1, 2-amino-2- methyl-1,3-propanediol and tris16 (hydroxyethyl) aminomethane. These substituted ammonium compounds are generally commercially available in the form of various salts or they can easily be made from the corresponding amines by neutralization with acids.
In yet another embodiment, the substituted ammonium is a tertiary or quaternary ammonium including, without limitation, trimethylammonium, triethylammonium, tributylammonium, diethylmethylammonium, diisopropylethylammonium, triisopropylammonium, N-methylmorpholine, N-hydroxyethyl piperidine, N-methylpyrrolidine and N, N'-dimethylpiperazine, tetramethylammonium, tetraethylammonium and tetrabutylammonium. These substituted ammonium compounds are generally commercially available in the form of various salts or they can easily be made from the corresponding amines by neutralization with acids.
In yet another embodiment, the substituted ammonium compound is a cationic compound in its entirety, i.e. under hermetic conditions, typically in an aqueous solution with a pH between pH 2 and pH of about 8, it possesses a positive charge, e.g., as a result of ionization (protonation). nitrogen atom.
In yet another embodiment, the substituted first-, second- or tertiary ammonium compound encapsulated in liposomes has a negative acid logarithm (deprotonation) of the dissociation constant (pKa) of at least about 8.0, at least about 8.5, at least about 9 , At least 9.5, or at least about 10.0, as determined in dilute aqueous solution at room temperature (typically 25 ° C). The pKa factor is a well-known characteristic of ammonium compounds that are generally characterized by strong basic properties, and methods for determining pKa are conventional and routine in the art. The pKa values for many amines and their protonated (ammonium) forms are summarized in the textbooks of chemistry and pharmacology. See, for example, IUPAC Handbook of Pharmaceutical Salts, edited by PH Stahl and CG Wermuth, Wiley-VCH, , 2002; CRC Handbook of Chemistry and Physics, 82nd edition, edited by DR Lide, CRC Press, Florida, 2001, pp. 8-44 to 8-56. Generally, higher pKa is characterized by stronger rules. Exemplary substituted ammonium compounds as well as unsubstituted ammonium (denoted as a conjugated amine base) have the following pKa values: pyrrolidine, 11.31; piperidine, 11.12; diisopropylamine, 11.05; diethylamine, 10.93; triethylamine, 10.75; dimethylamine, 10.73; tert-butylamine, 10.68; cyclohexylamine, 10.66; methylamine, 10.66; ethylamine, 10.65; propylamine, 10.54; isopropylamine, 10.53; Netylpiperidine, 10.45; dicyclohexylamine, 10.4; N-methyl piperidine, 10.38; diethylmethylamine, 10.35; dimethylpropylamine, 10.15; trimethylamine, 9.8; piperazine, 9.73 (1), 5.33 (II); 2-amino-2-methylpropanol, 9.69; N, N'-dimethylpiperazine, 9.66 (I), 5.2 (II); Diethyl- (2-hydroxyethyl) amine, 9.58; ethanolamine, 9.5; Nyridoxyethylpyrrolidine, 9.44; diethanolamine, 9.28; ammonia, 9.27; dimethyl- (2-hydroxyethyl) amine, 8.83; 2-amino-2-methylpropanediol-1.3, 8.8; morpholine, 8.5; tris (hydroxymethyl) aminomethane, 8.3; N-methylglucamine, 8.03; triethanolamine, 7.76; Netylmorpholine, 7.67; N-hydroxyethylmorpholine, 7.39; imidazole, 7.03; pyridine, 5.23. As a rule, substitution of an alkyl or cycloalkyl group with hydrogen in an ammonium compound increases the pKa value. In particular, many functional groups, e.g. hydroxyl or ether in a substituted alkyl group or in the presence of an aromaticity in the nitrogen-containing heterocyclic group, reduce the pKa value relative to similarly substituted ammonia, without a hydroxyl or ether function. Compounds with more than one ammonium group usually have a pKa value of the second and subsequent ammonium group much lower than the first one. Surprisingly, it has been found that substituted ammonia with a higher pKa value, i.e. formulated by more strong basic amines, are more effective than those formulated with weaker amines stabilizing the drug inside liposomes. For example, both the tributylammonium and SOM trDH salts (pKa = 10.75) were significantly more effective than the corresponding triethanolammonium salts (pKa = 7.76), in stabilizing the irinotecan within the liposomes in vivo (example 73). they are more effective than those formulated with weaker amines stabilizing the drug inside liposomes. For example, both the tributylammonium and SOM trDH salts (pKa = 10.75) were significantly more effective than the corresponding triethanolammonium salts (pKa = 7.76), in stabilizing the irinotecan within the liposomes in vivo (example 73). they are more effective than those formulated with weaker amines stabilizing the drug inside liposomes. For example, both the tributylammonium and SOM trDH salts (pKa = 10.75) were significantly more effective than the corresponding triethanolammonium salts (pKa = 7.76), in stabilizing the irinotecan within the liposomes in vivo (example 73).
The substituted ammon contained in the liposome composition may be in any preferred form, e.g. a salt. Preferred salts include pharmaceutically acceptable salts. See, for example, PH Stahl, CG Wermuth (ed.), Handbook of Pharmaceutical Salts, Wii ^^^ - ZC ^ IH, Weinheim, 2002. In one embodiment, substituted ammonium is a salt containing one or more polyanions. Optimally, the counterion (anion), in the substituted ammonium salt, renders the water-soluble salt, is pharmaceutically inert, capable of forming deposits or gels when they come into contact with the therapeutic species or the detectable species and / or is less permeable through the liposome membrane than the substituted ammonium or its undisputed amine forms. In general, the substituted ammonium salt forms a true inner liposome solution, e.g. spatial and does not form a significant amount of a condensed phase, such as micelles, bilayers, gels, or crystalline phases. The relative amount of substituted ammonium and the salt-forming anion, e.g., polyanion, is at or near the stoichiometric equilibrium point and typically has a pH in the range of 3-9, more usually in the pH range 4-8, depending, for example, on the dissociation constant of the conjugated substituted ion base. ammonium.
In general, the substituted ammon is inside, i.e. in the inner space of the liposomes. In one embodiment, the substituted ammonium is partially or substantially completely removed from the external environment surrounding the liposomes. Such removal of the substituted ammonium can be carried out by any of the preferred means known to those skilled in the art, e.g. by dissolution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, precipitation, etc.
According to another aspect of the present invention, a liposome composition comprising a polyanion is provided. The polyanion of the present disclosure may be any preferred chemical species with more than one negatively charged group resulting in a negative net ionic charge of more than two units in the interior of the liposomes, e.g., aqueous, spatial. The polyanion of the present disclosure may be a divalent, trivalent, multivalent, polymeric multivalent polyanionated polyol or polyanionized sugar. Sulfate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, citrate are, without limitation, exemplified by di and trivalent anions. In one preferred embodiment, the polyanion of the present invention is a polyanionic polymer having an organic (carbon) or inorganic main chain and a plurality of anionic functional groups, e.g. groups capable of negative ionization in an inert aqueous solution and ready to be integrated into or attached to the main chain. A polymer is a natural or synthetic compound, usually of high molecular weight, consisting of repeating connected units, each of which is a relatively light and simple molecule. Examples of polyanionic polymers are polyphosphate, (poly) vinyl sulfate, (poly) vinyl sulfonate, anionic polyacrylic polymers, anionic, e.g. polysulfonated polyamines, such as polysulfonated poly (ethylenimines); polysulfurized, polycarboxylated or polyphosphorylated polysaccharides; acid polyamino acids; polynucleotides; other polyphosphorylated, polysulfurized, polysulphonated, polycarbonated or polycarboxylated polymers. Such multivalent anions and polymers are well known in the art and many of them are commercially available. The polymeric anion of the present disclosure is preferably biodegradable, i.e. capable of degrading to non-toxic units in a living body. An exemplary biodegradable polymeric anion is polyphosphate. that is, capable of decomposing into non-toxic units in the living body. An exemplary biodegradable polymeric anion is polyphosphate. that is, capable of decomposing into non-toxic units in the living body. An exemplary biodegradable polymeric anion is polyphosphate.
In another preferred embodiment, the polyanion is a polyanionated polyol or polyanionic sugar. A polyol is an organic molecule having a plurality of hydroxyl groups bonded to e.g. a linear, branched or cyclic carbon backbone. Thus, the polyol can be characterized as a polyhydroxylated compound. Preferably, most of the carbon atoms in the polyol are hydroxylated. Polyols (polyatomic alcohols) are molecules well known in the art. Both simple (linear or branched) and cyclic polyols may be used. Exemplary polyols of the present disclosure include, without limitation: ethylene glycol; glycerol, threitol, erythritol, pentaerythritol, mannitol, sorbitol, sorbitol, sorbitan, xylitol, lactitol, maltitol, fructitol and inositol. Sugar usually contains cyclic acetal, a cyclic ketal, ketone or aldehyde group or its adduct, in the group of mainly hydroxyl carbon atoms which are interconnected. Sugars are often found compounds. Hydrolysis of sugars in the aquatic environment leads to the formation of units called monosaccharides. Typically in an aqueous solution, monosaccharide molecules with five or six carbon atoms form a cyclic hemiacetal with an annular structure. Preferably, the sugars of the present disclosure are monosaccharides or disaccharides, i.e. they consist of one or two monosaccharide units, each containing from three to seven, and preferably from three to six, carbon atoms. Exemplary sugars of the present disclosure are, without limitation, hexose monosaccharides such as glucose (dextrose), galactose, mannose, fructose; pentose monosaccharides such as xylose, ribose, arabinose and disaccharides such as lactose, trehalose, sucrose, maltose and cellobiose. Compounds containing several connected sugar units forming a ring (cyclodextrins) and their derivatives may also be used. One of the ways of obtaining polyols is the reduction of sugars. More advantageous, however, are more stable "non-reducing" and non-metabolic disaccharides, such as sucrose or trehalose. Various polyols, monosaccharides, disaccharides are commercially available.
The polyanionic polyol or sugar contains a hydroxyl group wholly or partially modified or replaced with an anionic (anionic) group. Thus, the polyanionized polyol or polyanionized sugar comprises a polyol moiety or a sugar moiety together with the anionic groups bound thereto. Exemplary anionic groups include, without any limitation, carboxylate, carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfate, sulfonate, nitrate and borate. It is preferred that the at least one anionic group in the polyanionic sugar or polyol is a stronger anionic group, i.e. more than 50% of the group is ionized over a wide pH range e.g. 3 to 12, preferably from 2 to 12, while in the environment aqueous, or optionally at a constant dissociation constant (pKa) of 3 or less, preferably of 2 or less. The polyanionation of the polyol or sugar can be carried out by various chemical processes known in the art. For example, as a result of the reaction of polyols and / or sugars with sulfur trioxide or chlorosulfonic acid in pyridine or 2-picoline, it causes some or all of the hydroxyl groups to be esterified together with sulfuric acid residues (sulphates), thereby providing polysulphate of sugar or polyol . Exemplary sulfated sugars of the present invention are sulfated sucrose, including, without limitation, sucrose hexasulfate, sucrose heptadiate sulphate and sucrose octane sulfate (see Ochi K., et al., 1980, Chem. Pharm. as a result of the reaction of polyols and / or sugars with sulfur trioxide or chlorosulfonic acid in pyridine or 2-picoline, some or all of the hydroxyl groups are esterified together with sulfuric acid residues (sulfates), thereby providing a polysulfate of sugar or polyol. Exemplary sulfated sugars of the present invention are sulfated sucrose, including, without limitation, sucrose hexasulfate, sucrose heptadiate sulphate and sucrose octane sulfate (see Ochi K., et al., 1980, Chem. Pharm. as a result of the reaction of polyols and / or sugars with sulfur trioxide or chlorosulfonic acid in pyridine or 2-picoline, some or all of the hydroxyl groups are esterified together with sulfuric acid residues (sulfates), thereby providing a polysulfate of sugar or polyol. Exemplary sulfated sugars of the present invention are sulfated sucrose, including, without limitation, sucrose hexasulfate, sucrose heptadiate sulphate and sucrose octane sulfate (see Ochi K., et al., 1980, Chem. Pharm.
Bull., 28, pp. 638-641). The sulphated sugar of the present invention is sucrose octane sulfate. Similarly, the reaction with phosphorous oxychloride or diethylchlorophosphate in the presence of a basic catalyst results in the formation of polyphosphorylated polyols or sugars. Polyphosporated polyols are isolated from natural sources. For example, inositol polyphosphates such as inositol hexaphosphate (phytic acid) are isolated from corn. Various suitable sulfated, sulfonated and phosphorylated sugars and polyols preferred for carrying out the present disclosure are disclosed e.g. in U.S. Patent No. 5,783,568 and 5,281,237. Surprisingly, it has been found that polyanionated polyhydroxylated compounds together with strong acids during particular dissociation steps e.g. groups with a pKa of less than about 3.0, more preferably less than about 2.0 such as, for example, monoester sulphates (pKa 1.0 or less), provide encapsulation of liposomes with better drug maintenance than polyanionated polyhydroxylated compounds having weakly acidic dissociation steps , e.g., phosphate monoesters (step 1, pKa about 1.5, step 2, pKa about 6.7, see Stahl and Wermuth, Op. cit, 2002). Example 73 illustrates this discovery. The complexation of polyols and / or sugars with more than one boric acid molecule also results in the polyanionation (polyborane) of the product. The reaction of polyols and / or sugars with carbon disulfide in the presence of alkali metals results in the formation of polyanionated (polydithocarbonates, poloxanthomas) derivatives. The polyanionated derivatives of polyols or sugars can be isolated as the free acid and neutralized with a suitable base, e.g. an alkali metal hydroxide, ammonium hydroxide, or preferably an amine substituted, e.g. an amine corresponding to the substituted ammonium of the present disclosure, in pure form or as a substituted ammonium hydroxide providing a polyanionic salt of substituted ammonium according to the present disclosure. Optionally, the sodium, potassium, calcium, barium or magnesium salts of polyanionic polyol / sugar may be isolated, and then they may be converted to a preferred form, e.g. a substituted ammonium salt by any known method, for example by ion exchange. an amine corresponding to the substituted ammonium of the present disclosure, in pure form or as a substituted ammonium hydroxide providing the polyanionic salt of the substituted ammonium of the present disclosure. Optionally, the sodium, potassium, calcium, barium or magnesium salts of polyanionic polyol / sugar may be isolated, and then they may be converted to a preferred form, e.g. a substituted ammonium salt by any known method, for example by ion exchange. an amine corresponding to the substituted ammonium of the present disclosure, in pure form or as a substituted ammonium hydroxide providing the polyanionic salt of the substituted ammonium of the present disclosure. Optionally, the sodium, potassium, calcium, barium or magnesium salts of polyanionic polyol / sugar may be isolated, and then they may be converted to a preferred form, e.g. a substituted ammonium salt by any known method, for example by ion exchange.
The polyanion according to the present disclosure usually has a charge density of at least two, three or four negatively charged groups per unit, e.g., for one carbon atom in a ring or carbon chain or per monosaccharide unit in sugar. The polyanionized sugar or cyclic polyol according to the present disclosure preferably comprises at least 75% of the polyalkonated hydroxyl groups available, and even more preferably 100% available polyalkonated hydroxyl groups. In addition, polyanionation within liposomes usually occurs at a level that is compatible with this phenomenon or facilitates the delivery and release of the encapsulated individual within the liposomes, at the site of its intended effect, but simultaneously reduces the amount of prematurely released individual, i.e. before the liposome reaches the target site where it will start. work.
The degree of polyanionation within the liposomes can be used to regulate the release characteristics, e.g., release frequency and kinetics of the enclosed unit within liposomes. In general, the degree of polyanion can be assessed on the basis of the amount of polyanionized sugar or polyol with respect to the total amount of ion (s) or, in the case of polyanion, the only type of anion, the polyanion percentage relative to the total polyanionic polyanionic capacity, e.g. polyanionic sugar or polyol, or mixtures inside liposomes. In one embodiment, the polyanionized sugar or polyol is mixed with one or more other anions and a smaller amount of polyanionated sugar or polyol and the more other anions the faster the individual is released from the liposomes.
Typically, if the entrapped individual is released from the liposomes at its intended site of action too slowly, the desired rate of the individual can be achieved by using a mixture of polyanionated sugar or polyol with one or more other single or polyvalent anions e.g. chloride, sulfate, phosphate, etc. Optional , mixtures of polyanionized sugar or polyols with different degrees of polyanion can be used. In one embodiment, the degree of polyanionation inside the liposomes is from 0.1% to 99%, 10% to 90% or 20% to 80% of the total amount of anions within the liposomes, e.g. with a closed species.
In general, the liposome composition may contain one or more polyanions in any preferred form, e.g. in the form of an acid or salt containing a polyanion and a cation. The amount of polyanion, e.g. a polyanionized sugar or polyol, may be stoichiometrically balanced or different from the amount of cation. In one embodiment, the liposome composition comprises one or more polyanion salt of a given cation in which there is a concentration gradient of cation or a pH gradient present in the liposome membrane. In another embodiment, the liposome composition comprises at least one or more substituted ammonium polyanion salt. In yet another embodiment, the liposome composition comprises a polyanion inside liposomes, whereas the polyanion in the liposome containing medium is partially or substantially removed by any suitable means known to those skilled in the art, e.g. dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, absorption, precipitation, etc. In yet another embodiment liposomes with a closed polyanion, e.g., a polyanionated polyol or sugar, also have a transmembrane gradient effective to maintain the substance in the liposomes. Examples of such transmembrane gradients are the pH gradient, the electrochemical potential gradient, the ammonium ion gradient, the substituted ammonium ion gradient, or the solubility gradient. The gradient of substituted ammonium typically includes a substituted form of an ammonium ion containing at least one CN bond, such as primary, quaternary, tertiary or quaternary ammonium groups. Methods of creating transmembrane gradients are routine in the field of liposomes.
According to yet another element of the present disclosure, the liposome composition of the present disclosure comprises one or more substituted amines and / or polyanions according to the present disclosure and chemical or biological species, e.g., therapeutic entities or detectable entities. For example, an individual contained in the liposome composition of the present disclosure may be a therapeutic agent, ink, dye, magnetic compound, fertilizer, bait, biocatalyst, taste sensation or substance-modifying fragrance, bleach, or any unit that is detectable by any beneficial agents known in the art. in the field of technology, e.g. by magnetic resonance imaging (MRI), imaging, fluorescence / luminescence imaging or nuclear imaging techniques. preferably,
In one embodiment, the individual contained in the liposome composition of the present disclosure is a therapeutic agent.
In another embodiment, the individual contained in the liposome composition is an anti-cancer unit. A partial list of some of the commonly approved (or in active development) anticancer drugs on sale is classified as follows.
The following classes are distinguished based on the structure: Fluoropyrimidines - 5-FU, fluorodeoxyuridine, Ftorafur, 5'-deoxyfluorouridine, UFT, S-1 capecitabine; pyrimidine nucleosides - deoxycytidine, cytosine arabinoside, 5-azacytosine, gemcitabine, azacytosine-arabinoside; Purines - 6-mercaptopurine, thioguanine, azathioprine, allopurinole, cladribine, fludarabine, pentostatin, 2-chloro-adenosine; Platinum analogs - cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin, CI-973, JM-216; Anthracyclines / anthraquinones - doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone; Epipodophyllotoxins - etoposides, teniposides; Camptothecin - irinotecan,
Topotecanes, Lurtotecans, Silatanes, 9-amino Camptothecin, 10,11-Methylenedioxy Camptothecin, 9-Nitro Camptothecin, TAS 103, 7- (4-methyl-piperazino-methylene) -10, 11-ethylenedioxy-20 (S) -camptothecin, 7 - (2-N-isopropylamino) ethyl) -20 (S) -camptothecin; Hormones and hormone analogs - diethylstilbestroles, tamoxifenes, toremifenes, tolmudeks, thymitaq, flutamides, bicalutamides, finasterides, estradiols, trioxyphenes, droloxifenes, medroxyprogesterone acetates, megesterol acetates, aminoglutethimides, testolactones and others; Enzymes, proteins and antibodies - asparaginases, interleukins, interferons, leuprolides, pegaspargases, and others; Alkaloids of rose vinca, vincristine, vinblastine, vinorelbine, vindesine; Taxanes-paclitaxel, docetaxel.
The following classes are distinguished on the basis of the mechanism: Anti-hormones - See the classification of hormones and analogues of anastrozole hormones; Antipolates - methotrexates, aminopterin, trimetrexata, trimethoprim, pyritrexim, pyrimethamine, edatrexate, MDAM; Anti-microtubule agents - taxanes and alkaloids of rose dye; Alkylating agents (classical and non-classical) - nitrogen mustards (mechlorethamine, chlorambucils, melphalan, uracil mustards), oxazaphosphates (ifosfamide, cyclophosphamides, perfosphamides, trofosfamels), alkylsulfonates (busulfans), nitrosoureas (carmustines, lomustines, streptozycins) thiotepa, dacarbazine and other; Antimetabolites - purines, pyrimidines and nucleosides mentioned above; Antibiotics - anthracyclines / anthracquinones, bleomycins, dactinomycins, mitomycins, filamycins, pentostatin, streptozocins; Topoisomerase inhibitors - camptothecin (Topo I), epipodophyllotoxins, m-AMSA, ellipticine (Topo II); Antiviral agents - AZT, zalcitabine, gemcitabine, didanosine and others; Various cytotoxic agents - hydroxyureas, mitotans, fusion toxins, PZAs, briostatines, retinoids, butyric acids and its derivatives, pentosans, fumagilates and others.
In addition to the above, the anti-cancer units include, without limitation, any topoisomerase inhibitors, vinca alkaloids, e.g., vincristine, vinblastine, vinorelbine, vinflunine and vinpocetine, depolymerization microtubules or destabilizing agents, microtubule-stabilizing agents, e.g. taxanes, aminoalkyls or aminoacyl analogs of paclitaxel or docetaxel, e.g., 2 '- [3- (N, N-diethylamino) propionyl] paclitaxel, 7- (N, N-dimethylglycyl) paclitaxel and 7-Lalanylaclitaxel, alkylating agents, agents with binding receptors, tyrosine kinase inhibitors , phosphatase inhibitors, cyclin-dependent kinase inhibitors, enzyme inhibitors, aurora kinase inhibitors, nucleotides, polynucleotides and farnesyltransferase inhibitors.
In another embodiment, the individual contained in the liposome composition of the present disclosure is a therapeutic agent with anthracycline compounds or derivatives thereof, camptothecin compounds or derivatives thereof, elliptic compounds or derivatives thereof, alkaloids of a rose dye or their derivatives, wortmannin, its analogues and derivatives or compounds pyrazolopyrimidine with aurora kinase inhibitory properties.
In yet another embodiment, the individual contained in the liposome composition of the present disclosure is an anthracycline drug, doxorubicin, daunorubicin, mitomycin C, epirubicin, pirarubicin, rubidomycin, carcinomycin, Nacetyladriamycin, rubidazone, 5-imidodaunomycin, N-acetyl-dunomicin, daunoril, mitoxantrone; camptothecin, camptothecin, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11 methylene-dioxycamptothecin, 9-amino-10,11 methylene-dioxycamptothecin, 9-chloro-10,11-methylene-dioxycamptothecin irinotecan, topotecan, lurtotecan, silatate (7- (4-methylpiperazinomethylene) -10, 11-ethylenedioxy-20 (S) -camptothecin, 7- (4-methylpiperazinomethylene) -10,11-methylenedioxy-20 (S) -camptothecin, 7- (2-N-isopropylamino) ethyl) - (20S) - camptothecin; ellipticine compound, ellipticine,
In yet another embodiment, the individual contained in the liposome of the present disclosure is a pharmaceutical unit including, without limitation, any of the following: antihistamine derivatives of ethylenediamines (bromenamines, diphenhydramines); antiprotozers: quinolones (iodoquinol); amidines (pentamidine); anthelmintics (pyrantel); anti-schistosomal drugs (oximinin); antifungal triazole derivatives (flonicazoles, itraconazoles, ketoconazoles, miconazoles); antimicrobial cephalosporins (cefazolin, c-e-lixidone, cefotaxime, cetazazidime, cefuroxime);
antibacterial beta-lactam derivatives (aztrepam, cefmetazole, cefoxitin);
antibacterial agents from the erythromycin group (erythromycin, azithromycin, clarithromycin, oleandomycin); penicillins (benzylpenicillin, phenoxymethylpenicillin, cloxacillin, methicillin, naphthline, oxacillin, carbenicillin); tetracylina; other antibacterial antibiotics, novobiocin, spectinomycin, vancomycin; antimycobacterial drugs: aminosalicylic acid, capreomycin, ethambutol, isoniazid, pyrazinamide, rifabutin, rifampicin, clofazimine; antiviral adamantans: amantadine, rimantadine; quinidine derivatives: chloroquine, hydroxychloroquine, primaquinone, quinone; antibacterial quinones: ciprofloxacin, enoxacin, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin; sulfonamides; antibacterial drugs for the urinary tract: urotropin, nitrofurantoin, trimethoprim; nitroimidazole: metronidazole; cholinergic quaternary ammonium compounds (ambetinium, neostigmine, physostigmine); aminoacridine against Alzheimer's disease (tacrine); anti-Parkinson's drugs (benztropine, biperiden, procyclidine, trihexyphenidyl); anti-muscarinic agents (atropine, hyoscyamine, scopolamine, propantelin); adrenergic dopamine (albuterol, dobutamine, ephedrine, adrenaline, noradrenaline, isoproterenol, metaproterenol, salmeterol, terbutaline); ergotamine derivatives; muscle relaxants or curacine series; muscle relaxants with central effect; baclofen, cyclobenzaprin, dantrolene; nicotine; beta-blockers (acebutil, amiodarone); benzodiazepines (diltiazem); anti-arrhythmic drugs (disopyramide, enkainide, local anesthetic series - procaine, procainamide, lidocaine, flecainide), quinidine; ACE inhibitors: captopril, enelaprilat, fosinoprol, quinapril, ramipril; anti-lipid drugs: fluvastatin, gemfibrozil, HMG-coA reductase inhibitors (pravastatin); antihypertensive drugs: clonidine, guanabenz, prazosin, guanethidine, granadril, hydralazine; and drugs that do not increase coronary vessels: dipyridamole.
According to the present disclosure, an individual included in the liposome composition of the present disclosure may also be a pre-species, e.g. a prodrug or agent that is capable of transforming into the desired species at one or more conversion steps under conditions where there is a change in pH or enzymatic activity. cleavage of the labile bond. Such conversion may occur after release of the prodrug within the liposome at the intended drug / liposome site of action. However, the initial species can be converted to the desired active species within the liposome of the present disclosure before using liposomes as a delivery agent, e.g. by administering this drug to a patient. For example, an individual can be modified in a pre-case like that, that it may be easier to be loaded into liposomes, and then it can be converted back into the desired unit when it is already within the liposomes of the present disclosure. Thus, according to the present disclosure, entities that are not substantially susceptible to "active", "remote" and other gradient-based loading methods can be effectively loaded into liposomes, e.g., into the inner space of liposomes in their unmodified form.
Global cationic compounds, i.e., compounds capable of achieving a positive net ion charge in accordance with the conditions prevailing during the loading of the liposome, in particular compounds containing a titratable amine are known for their effectiveness during loading into the liposomes exhibiting an ion-transmembrane gradient. If the individual is an organic compound and is not a global cationic compound with a titratable amine and its derivatives have suitable ionic properties, it may be prepared by a preferred modification, e.g. according to the methods described in Woodle et al. in WO 96/25147. For example, an amino group can be introduced by esterification of a hydroxyl group in a unit that contains an amino acid. Optionally, a hydrophobic group can be incorporated into the water-soluble compound, in order to facilitate its division into the membrane of the liposome, and then translocate the membrane into the intraliposomal chamber, i.e. inside the liposomes. Another useful modification to form the initial species for loading into the liposome is the formation of the adduct of the carbonyl group, e.g. hydrazone, oxime, acetal or ketal. The modified group containing an amino group may be subjected to hydrolysis or another chemical method that will divide the modified compound after loading the modified compound in the liposomes of the present invention. Typical intraliposomal processes that reproduce the individual from the initial unit are hydrolysis, photolysis, radiolysis, thiolysis, ammonolysis, reduction, substitution, oxidation or elimination. These processes can be achieved without limitation, by changing the pH or by enzymatic activity.
For example, paclitaxel and docetaxel, non-ionic units, are converted to their 2 '(diethylaminopropionyl) - or 7' - (diethylaminopropionyl) esters, which are weak bases (initial species). After loading into the liposomes by any known method including without limitation an "active", "remote", "transmembrane gradient" or "resolution based on solubility" by the method and / or methods of the present disclosure, intraliposomal 2 '- (diethylaminopropionyl) -paclitaxel it is converted to the original paclitaxel, thereby stimulating its hydrolysis by raising the pH above 7.0. In this way, liposomes that encapsulate the neutral taxane molecules in their internal space are able to get the drug / lipid by more than 0.05 moles per mole lipid liposome,
The liposomes contained in the liposome composition of the present disclosure can be any known or later discovered liposome in the art. In general, the liposomes of the present invention may have any liposome structure, e.g., structures having an internal space separated from the external environment by one or more lipid bilayers, or any microcapsules that have a semipermeable membrane with a lipophilic central portion in which the membrane separates the interior. The bilayer lipid may have any system of amphiphilic molecules characterized by a hydrophilic portion (hydrophilic moiety) and a hydrophobic portion (hydrophobic moiety). Typically, amphiphilic molecules in the double layer are arranged in two-dimensional sheets in which the hydrophobic moiety is oriented inwardly of the sheet, while the hydrophilic residues are directed outwards. The amphiphilic liposome-forming molecules may be any known or later discovered amphiphilic molecules, e.g., fats of synthetic or natural origin or biocompatible lipids.
Liposomes can also be formulated by amphiphilic polymers and surfactants, e.g., polymerosomes and niosomes. For the purposes of this disclosure, without any limitation, such liposome-forming materials are also referred to as "lipids".
According to the present invention, the liposomes contained in the liposome composition of the present invention may also be targeted liposomes, e.g. liposomes containing one or more target groups or biodistribution modifiers on the surface of liposomes. The targeted moiety can be any agent that is capable of specifically binding or interacting with the desired target. In one embodiment, the targeted moiety is a ligand. The ligand of the present invention is preferably combined with and / or internalized into a cell in which the encapsulated species in the liposome has the desired effect (target cells). A ligand is typically a member of a binding pair in which the second member is present on or in target cells or tissue containing target cells. Examples of ligands preferred for the present invention are: folic acid, proteins, e.g., transferrin, growth factor, enzyme, peptide, receptor, antibody or antibody fragment such as Fab ', Fv, single chain Fv, single domain antibodies or any other polypeptides containing sequences binding antigens (CDRs) to the antibody molecule. A targeted ligand liposome in which the targeted moiety is an antibody or a fragment of its directed binding antigen is called the immunoliposome. In a preferred embodiment, the liposome transporting a targeted moiety, e.g. a ligand, is internalized by the target cell. In yet another embodiment, the targeted moiety is a ligand that specifically interacts with a tyrosine kinase receptor, such as EGFR receptor HER2, for example. HER3, HER4, PD-GFR, VEGFR, bFGFR or IGFR. In yet another embodiment, the targeted species specifically interacts with a growth factor receptor, an angiogenic factor receptor, a transferrin receptor, a cell adhesion molecule or a vitamin receptor.
According to another embodiment, the liposomes contained in the liposome composition show a gradient of transmembrane concentration of the substituted ammonium and / or polyanion. Preferably, the higher concentration is in the inner space of the liposomes. In addition, the liposome composition may contain one or more transmembrane gradients in addition to the gradient formed by the substituted ammonium and / or polyanion. For example, the liposomes contained in the liposome composition may additionally include a pH transmembrane gradient, an ionic gradient, an electrochemical potential gradient, and / or a solubility gradient.
According to yet another embodiment of the present invention, the liposome composition of the present invention may be provided in a kit comprising a liposome container and optionally a container with unit and instructions, e.g. procedures or information regarding the use of the liposome composition in one or more applications. Such instructions may be provided by any medium, e.g. paper copy, electronic media or access to a database, or a web page containing such instructions.
The liposome membrane composition of the present invention may be made by any convenient known method or subsequently discovered by one skilled in the art. In general, various lipid components may be used to obtain liposomes. Lipid components typically include, but are not limited to: (1) uncharged lipid components, e.g. cholesterol, ceramide, diacylglycerol, acyl (poly) ether or alkyl poly (ether); (2) neutral phospholipids, e.g.
diacylphosphatidylcholine, sphingomyelin and diacylphosphatidylethanolamine, (3) anionic lipids, e.g. diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatid, cardiolipin, diacylphosphatidylinositol, diacylglycerol, hemisuccinate, diacylglycerol, hemiglutarate, cholesterol hemisuccinate, cholesterol, ketoglutarate, and the like; (4) conjugated polymer lipids, e.g., N [methoxy (polyethyleneglycol) diacylphosphatidylethanolamine, poly (ethylene glycol) diacylglycerol, poly (ethylene glycol) -ceramide, and (5) cationic lipids, e.g., 1,2, diacyl; -3-trimethylammonium propane (DOTAP), dimethyl dioctadecylammonium bromide (DDAB), 1,2-diacyl-sn-glycero-3-ethylphosphocholine Derivatives of these lipids with substituted monoacyl, as well as analogs with mono-di-alkyl can also be used.
The various lipid components may be selected to fill, modify or impart one or more desired functions. For example, a phospholipid may be used as the main vesicle-forming lipid. Cholesterol incorporation is useful for maintaining membrane stiffness and reducing drug leakage. Lipid conjugated polymers can be used in liposome formulations to increase circulating stability by reducing the clearance of liposomes in the liver and spleen, or to improve the stability of liposomes from aggregation during storage in the absence of circulatory effects. While the incorporation of lipid-PEG in the amount of 1 mole% or above of the lipid liposome is ensured and a prolonged circulation time of the liposomes in the blood is ensured (see, e.g., U.S. Patent 5,013,556), Surprisingly, it has been found that the liposomes of the present invention are quite long in circulation, and the addition of lipid-PEG to a liposome composition only prolongs durability and viability in circulation less than twice, if at all. In addition, lipids (titrimetric) that can modulate the charge can be used to deliver units encapsulated in liposomes for purposes such as cytosolic or nuclear regions by facilitating the escape of certain routes to the endosomal pathway.
In one embodiment, the liposomes of the present disclosure include lecithin, cholesterol and an amphipathic polymer. Lecithin contained in liposomes may be natural lecithin, hydrogenated natural lecithin, synthetic lecithin, 1,2-distearoyl-lecithin, dipalmitoyl lecithin, dimyristoyl lecithin, dioleoyl lecithin, 1-stearoyl-2-oleoyl lecithin or 1 palmitoyl-2-oleoyl lecithin, wherein the amphipathic polymer can be a derivative of a polyethylene glycol lipid, e.g. polyethylene glycol phosphatidyl ethanolamine, polyacylate diacylglycerol glycol or a polyethylene glycol derivative of ceramide, wherein the poly (ethylene glycol) part has a molecular weight of about 250 to about 20,000, most typically about 500 to about 5000. In another embodiment, the ratio of lecithin and cholesterol in the liposomes of the present disclosure is about 3: 2 per mole. In yet another embodiment, the amphipathic polymer is at least 0.1 mol% of liposome-forming lipids in the liposomes of the present disclosure. In yet another embodiment, the amount of amphipathic polymer is between 0.1% and 1% by molar lipid-forming liposomes in the liposomes of the present disclosure. Preferably, the amphipathic polymer is an inert polymer, i.e. it has an ionic net charge of zero at loading conditions, for example PEG-diacylglycerol, PEG-dialkylglycerol or ceramide-PEG. Surprisingly, it has been found that the inclusion of ionically neutral amphipathic lipids into PEG lipids of about 5.7 mol% of the total lipid amount with high loading efficiency of liposomes, e.g.
In yet another embodiment, the liposomes of the present disclosure comprise a prodrug with camptothecin, such as irinotecan, and contain lecithin and cholesterol, e.g. in a ratio of about 3: 2 per mole, and an amphipathic polymer, e.g. in an amount of at least 0.1 mole percent. and less than 1% lipids forming liposomes.
The liposomes of the present invention may be prepared by any method that is known or will be known in the art. See, for example, G.
Gregoriadis (publisher), Liposome Technology, vol. 1-3, 1st edition, 1983; 2nd edition, 1993, CRC Press, Boca Raton, FL. Examples of suitable methods for preparing a liposome composition of the present invention include extrusion, reverse phase evaporation, sonication, solvents (e.g., ethanol), injection, microfluidization, detergent dialysis, ether injection, and dehydration / hydration. The size of the liposomes can be controlled by controlling the pore size of films used at low pressure or pressure profiles and the amount of permeated material used in microfluidization or other suitable methods. In one embodiment, the desired lipids are first hydrated by thin-film hydration or by injecting ethanol, and then they are sorted by extrusion through membranes of a specific pore size; most often by size 0.05 pm, 0.08 pm, or 0.1 pm.
A liposome composition comprising substituted ammonium and / or polyanion salts according to the present disclosure within liposomes can be made by any suitable means, e.g. by forming liposomes in the presence of a substituted ammonium and / or polyanion according to the present disclosure, e.g., in salt form. The substituted ammonium and / or polyanion outside the liposomes can be removed or diluted either after the liposomes have been made, or before loading or closing the desired unit. Optionally, a liposome composition comprising a substituted ammonium and / or polyanion according to the present disclosure may be made by the ion exchange method directly or indirectly by an intermediate free-acid step of the substituted ammonium gradient of the present disclosure, e.g. a substituted ammonium salt of a polyanionized sugar or polyol. Such liposomes can be neutralized with an amine or a salt thereof with a volatile acid, e.g. a carbonate. The resulting liposome solution can be used directly or optionally, and the salt contained therein can be removed, if desired, e.g. by evaporation, followed by crystallization and dissolution in an aqueous medium.
Preferably, the liposome composition of the present disclosure has a transmembrane gradient of substituted ammonium and / or polyanion, e.g., the concentration of substituted ammonium and / or polyanionic salt within the liposome is higher, usually at least 100 times higher than the concentration of substituted ammonium and / or polyanion in the external environment liposome.
In one embodiment, the concentration of substituted ammonium and / or polyanionic salt within the liposome is at least 100 times greater than the concentration of substituted ammonium and / or polyanionic salt in the external liposome environment and is at least at a concentration of about 10 mM, 50 mM, 0.1 M , 0.2 M, 0.5 M, 0.6 M, 0.7 M, or 1.0 M, wherein the molar concentration is calculated based on the substituted ammonium. In another embodiment, the concentration of substituted ammonium and / or polyanionic salt within the liposome is at least 100 times greater than the concentration of substituted ammonium and / or polyanionic salt in the external liposome environment and is at least at a concentration of about 0.65 M or about 1.0 M .
In addition, the liposome composition of the present invention generally has an external pH value that is compatible with or useful to maintain the stability of the desired species during the loading process, along with high loading efficiency, e.g. above 90% closure. Preferred pH values are in the range, for example, from 4 to 7, or from 4.5 to 6.5. In particular, according to the present invention, the loading of irinotecan is best achieved at the pH of the external environment in the range of from about 4.0 to about 7.0, more preferably between about 5.0 and 6.5. Loading of a derivative of a rose dye, e.g. vincristine, vinorelbine or vinblastine, is best achieved at a pH of about 5.0-7.0, preferably at a pH of about 6.5.
According to the present disclosure, the desired species can be loaded or encapsulated in liposomes by incubating the desired species with the liposomes of the present disclosure in an aqueous environment at a suitable temperature, e.g. at a temperature above the lipid transition phase during loading, which is then reduced below the transition phase, when the individual is already loaded. The incubation time usually depends on the nature of the lipid components of the unit to be loaded into the liposomes and the incubation temperature. Typically, the incubation time is from a few minutes to several hours. Because a high closing efficiency is achieved, which is more than 85%, typically more than 90%, there is usually no need to remove unfinished units. However, if desired, the unfinished units can be removed from the composition by various means such as, for example, size exclusion chromatography, dialysis, ultrafiltration, adsorption or precipitation. It has surprisingly been found that maintaining a low ionic strength during the incubation of the entity, such as, in particular, a camptothecin derivative or alkaloid derivative of the rose dye with the liposomes of the present disclosure, occurs by increasing the ionic strength at the end of the incubation, resulting in higher loading efficiency, better removal of the unopened drug and better stability of liposomes during the aggregation process. Typically, the incubation is carried out, for example, in an aqueous solution, with an ionic strength of less than 50 mM NaCl, or more preferably, less than 30 mM NaCl. After incubation, concentrated salt, e.g. NaCl, the solution can be added to increase the ionic strength to higher than 50 mM NaCl values or more preferably to values greater than 100 mM NaCl. Without being bound by theory, it can be hypothesised that the increase in ionic strength helps in dissociating the unit from the liposome membrane, leaving essentially all units hermetically sealed in the internal liposomal space.
In general, the unit to lipid ratio, e.g., the loading ratio of the drug obtained after loading the unit depends on the amount of unit enclosed within the liposomes; the closed concentration of substituted ammonium and / or polyanion, e.g. salt, physicochemical properties of the closed unit and the type of counterion (anion), e.g. polianiou. Due to the high loading efficiency of the composition and / or the method of the present invention, the unit to lipid ratio for the liposome encapsulation unit is over 80%, over 90%, and typically more than 95%, where this ratio is calculated on the basis of the amount the individual and lipid liposome taken during loading ("input" ratio). In fact, almost 100% (quantitative) encapsulation is common. The ratio of the individual to the lipid in liposomes can be characterized in terms of body mass index (amount by weight of the individual per mass or molar unit of the lipid liposome), or molar ratio (number of moles of the individual to mass or molar unit of the lipid liposome). One unit of the ratio of the individual to the lipid can be converted into other units by routine calculations as given in the examples below. The weight ratio of the individual in the liposomes of the present invention is typically at least 0.05, 0.1, 0.2, 0.35, 0.5, or at least 0.65 mg of unit per mg of lipid. Depending on the molar ratio in which the ratio of the individual to the lipid of the present invention is at least from about 0.02 to about 5, preferably at least 0.1 to about 2, more preferably from about 0.15 to about 1.5 moles of drug per mole of lipid liposome. In one embodiment, the ratio of the individual to the lipid, e.g. the drug load ratio of camptothecin derivatives is at least 0.1, e.g. 0.1 mole camptothecin derivative per one mole lipid liposome, preferably at least 0.2. In another embodiment, the ratio of unit to lipid, e.g., the amount of drug loaded is at least about 300 mg unit (e.g., alkaloids of a rosette dye or derivatives thereof) per mg lipid forming liposome. In yet another embodiment, the ratio of unit to lipid, e.g., the amount of drug loaded is at least about 500 mg of unit (e.g., camptothecin or prodtothecin prodrug) per mg lipid-forming lipid. It has surprisingly been found that the invention can maintain a stable and close to quantitative liposomal hermetization of irinotecan,
If the liposome contains a phospholipid, it is convenient to express the content of the individual in units of weight of the drug amount per molar unit of the phospholipid liposome, e.g., mg of drug / mmol of phospholipid. However, one skilled in the art will know that the drug content can be equivalently expressed independently of the presence of phospholipids in the liposomes, and can also be equivalently expressed in the molar amount of drug per unit (mass or molar) lipid liposome content. For example, liposomes containing 3 parts by mole distearoylphosphatidylcholine (DSPC, molecular weight 790), 2 parts mole cholesterol (molecular weight 387) and 0.015 parts mole poly (ethylene glycol) - distearoylphosphatidylethanolamine (PEG-DSPE, molecular weight 2750) and a drug containing doxorubicin (molecular weight 543,
(a) Obliteration of the normalized and molar molarities of components I of the lposome Ipid to a molar unit of a phospholipid liposome (DSPC and PEG-DSPE in this example) by dividing the amount of moles of the component by the total molar amount of phospholipid liposomes:
DSPC 3 / (3 + 0.015) = 0.99502
Cholesterol 2 / (3 + 0.015) = 0.66335
PG-DSPE 0.015 / (3 + 0.015) = 0.00498 (b) ΟόΝ ^ εηιε I iś m ^^ s ^ I compactes I lpids corresponding to the first and in a molar unit of phospholipid liposomes and molecular weight components:
Total amount of lipid, mg / mmol phospholipid = 0.99502x790 + 0.66335x387 + 0.00498x2750 = 1056,48 (c) Observation and iodine of the soil Ieku nn exe of the mass I Ζο0ο I lpids divide the drug content in units of mass into the phospholipid molar unit by the number obtained in step (b):
Doxorubicin, mg / mg total lipid = 150 / 1056,48 = 0,14198.
(d) Observation of the molecular weight of the yeast I mass by the molecular weight of the drug (in this case 543.5) by dividing the number obtained in step (c):
Doctorubicin, mmol / g total lipid = 0.14198 / 543.5x1000 = 0.261.
(e) Calculation of the molar part of phospholipids in the lipid lipid matrix:
The molar part of phospholipids = (total moles of phospholipids) / (total moles of lipids) = (3 + 0.015) / (3 + 2 + 0.015) = 0.6012.
(f) Calculation of the molar ratio of doxorubicin to total lipid.
Doxorubicin, mol / mol total amount of lipids = (phospholipid molar part) x (doxorubicin, g / mol phospholipids) / (doxorubicin molecular mass) = 0.6012x150 / 543,5 = 0.162
Thus, the relationship between drug to lipid ratio and drug to phospholipid expressed in different units is easy to determine. As used herein, the term "lipid" includes, without limitation, any film-forming components of liposome membranes such as, for example, polymers and / or detergents.
A liposome that contains a closed substituted ammonium and / or a polyanion salt solution of the present disclosure typically has an osmotic strength (osmolality) that maintains liposome stability from osmotic damage (edema and / or rupture) without loss of capacity in the liposomes. In one embodiment, the osmolality of the liposome composition of the present disclosure is in the range of 0.1 to 1.5 mol / kg, or preferably 0.2 to 1.0 mol / kg. It has surprisingly been found that the liposomes of the present disclosure are stable against the adverse effect of the high intraliposomal osmotic force on drug loading. A high intraliposomal osmolality of 0.727 mol / kg was well tolerated,
In general, the liposome composition of the present invention is very stable during storage, e.g., measured by the percentage of encapsulated individual released outside of liposomes or internal liposomes after a period of initial loading of the individual within the liposomes of the present invention. For example, the liposome composition of the present invention is stable at 4 ° C for at least 6 months, e.g., less than 10% of the encapsulated species is released 6 months after the initial loading of an individual. In one embodiment, the liposome composition of the present invention is stable at 4 ° C for at least 2 years, e.g., less than 20% of the encapsulated species is released 2 years after the initial loading of the individual.
It is advantageous for the individual encapsulated in the liposome to retain its hermeticity in the liposome until it reaches the site of its intended action, e.g. in the case of a liposomal anticancer drug administered to a cancer patient. The liposomes of the present invention exhibit surprising stability against the release (leakage) of the encapsulated species in vivo, e.g. in the blood of a mammal. The exposure time needed for 50% release of the encapsulated individual, e.g. the drug, from the liposomes (half-release time) in the rat's blood in vivo was more than 24 hours. In particular, liposomes loaded with a drug with a vinca alkaloid, e.g. vinblastine, vincristine and vinorelbine, showed significant stabilization prior to drug leakage in vivo during half-time release for at least 24 hours, and the amount of unit remained hermetically sealed after 24 hours in blood in vivo and was at least about 50% of the pre-drug value. Typically it has been observed that the release time is more than 33 hours or the amount of the encapsulated species remaining after 24 hours in the blood in vivo of at least about 60%; and it is even common that half-release time is more than 46 hours or the amount of encapsulated species remaining after 24 hours in the blood in vivo of at least about 70% of the value prior to administration. Sometimes, the half-time of release for the encapsulated drug in the blood in in vivo was over 93 hours and even over 120 hours. Liposomes loaded with camptothecin derivatives such as topotecan and irinotecan also showed exceptional in vivo stability in the blood, with 79-85% of the initial drug content, which remained hermetic after 24 hours. It is unusual that the liposomes of the present invention, while having such a low release rate of the drug in vivo in the blood circulation, have demonstrated a significant antitumor activity exceeding that of the free drug (i.e., administered in the form of a solution).
The liposomes of the present invention provide an unexpected combination of high efficacy of the closed therapeutic agent and low toxicity. In general, the activity of the therapeutic entity encapsulated in the liposomes of the present invention, e.g., the antitumor activity of irinotecan in mammals, is at least equal, at least twice as high or at least four times as high as that of the therapeutic species when administered in such a mammal. the same amount using a non-liposomal routine, e.g. without applying the liposome composition of the present invention, while the toxicity of the liposome encapsulated individual is not exceeded, is at least two times, at least three times or at least four times lower than the same individual treated at the same dose and schedule, but in a free, non-humified form. For example, it is well known from published other methods that liposomal encapsulation of anti-cancer camptothecin derivatives leads to increased toxicity (lower maximum tolerated dose, lethal dose by 50%) compared to non-aerosolized drug. See U.S. Patent No. 6,355,268; U.S. Patent No. 6,465,008; Colbern, et al. Clinical Cancer Res. 1998, vol. 4, pp. 3077-3082; Tardi, et al. Cancer Res, 2000, v. 60, pp. 389-3393; Emerson, et al. Clinical Cancer Res. 2000, vol. 6, pp. 2903-2912. Liposomally-encapsulated prodrugs of camptothecins such as iriotetra (CPT-11), which is water-soluble, cationic camptothecin prodrug derivatives have a substantially higher activity, e.g. at least 4 times, and even 10-fold greater antitumor activity, as assessed in vivo in the tumor model, than the drug in the absence of a liposomal formulation, e.g. a free form (solution). This is all the more significant because irinotecan requires enzymatic activation, e.g. by endogenous action of a non-specific carboxyl esterase, but according to the present invention encapsulated essentially in the inner space of the liposome. On the other hand, it has surprisingly been observed that the toxicity of a camptothecin prodrug such as CPT-11 in liposome form (mass ratio drug / lipid above 0.1, e.g., 0.2-0.6 or more) according to the present invention is more than 2 times, over 3 times, and even more than 4 times lower than the free (non-aerosolized) prodrug CPT-11. In addition, long-term release of the drug from the CPT-11 liposome in vivo has been achieved, of which more than 50%, and even more than 70% (79-86%) of the original drug content still remaining in liposomes 24 hours after administration into the bloodstream and with a half-time release greater than 24 hours, typically over 48 hours. The prolonged remanence of the drug in liposomes in vivo was associated with increased antitumor activity. Surprisingly, it was observed that the slowest release of CPT-11 in vivo and the highest anti-tumor activity is in liposomes containing a low molecular polyanionated sugar derivative (sucrose octasulfate) and not in a polymeric anion (polyphosphate) (example 15). The prolonged remanence of the drug in liposomes in vivo was associated with increased antitumor activity. Surprisingly, it was observed that the slowest release of CPT-11 in vivo and the highest anti-tumor activity is in liposomes containing a low molecular polyanionated sugar derivative (sucrose octasulfate) and not in a polymeric anion (polyphosphate) (example 15). The prolonged remanence of the drug in liposomes in vivo was associated with increased antitumor activity. Surprisingly, it was observed that the slowest release of CPT-11 in vivo and the highest anti-tumor activity is in liposomes containing a low molecular polyanionated sugar derivative (sucrose octasulfate) and not in a polymeric anion (polyphosphate) (example 15).
According to another embodiment of the present invention, the liposome composition of the present invention can be provided in the form of pharmaceutical compositions comprising a liposome composition of the present invention and a carrier, e.g., a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are isotonic saline, isotonic dextrose, isotonic sucrose, Ringer's solution and Hanks' solution. A buffer substance can be added to provide the optimum pH for storage stability. For example, a pH of between about 6.0 to about 7.5, more preferably a pH of about 6.5, is optimal for the lipid membrane stability of the liposome and provides excellent retention of the enclosed species. Histidine, hydroxyethylpiperazine-ethyl sulphonate (HEPES), ethyl morpholipoate (MES), succinate, tartrate, citrate, typically at a concentration of 2-20 mM are exemplary buffer substances. Other suitable carriers include, for example, water, a buffered aqueous solution of 0.4% NaCl, 0.3% glycine and the like. Proteins, carbohydrates or polymeric stabilizers and tonicity agents may be added, e.g. gelatin, albumin, dextran or polyvinyl pyrrolidone. The tonicity of the composition may be adjusted to a physiological level of 0.25-0.35 mole / kg with glucose or more inert substances, such as lactose, sucrose, mannitol or dextrin. These compositions can be sterilized by conventional well-known sterilization techniques, e.g. by filtration. The resulting aqueous solutions can be packaged for use or filtered under sterile conditions and freeze-dried,
The liposome pharmaceutical compositions can also contain other pharmaceutically acceptable excipients required to approximate physiological conditions, such as pH adjusting agents and buffering agents, tonicity agents and the like, e.g. sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. In addition, the liposome suspension may contain protective lipid agents that protect lipids from free radicals and damage of peroxy lipids during storage. Suitable lipophilic free radical quenchers are alpha-tocopherol and water-soluble iron-containing chelating agents such as ferrioxamine.
The concentration of liposomes of the present invention in liquid pharmaceutical formulations can vary over a wide range, i.e. from less than about 0.05%, usually or at least about 2-10%, to as much as 30 to 50% by weight, and will be selected primarily all thanks to the volume of fluids, viscosity etc. according to the particular mode of administration chosen. For example, the concentration may be increased to reduce the loaded fluid associated with the treatment. This may be particularly beneficial in patients with atherosclerosis associated with congestive heart failure or severe hypertension. Optionally, the pharmaceutical compositions comprising the irritating lipids can be diluted to low concentrations to reduce inflammation at the site of administration.
The amount of liposome pharmaceutical composition will depend on the particular therapeutic entity encapsulated inside the liposomes, the condition of the disease being treated, the type of liposomes used and the diagnosis of the clinician. In general, the amount of liposomal pharmaceutical composition administered will be sufficient to provide an effective therapeutic dose to a given therapeutic species.
The amount of liposome pharmaceutical composition necessary to deliver an effective therapeutic dose can be determined by in vitro and in vivo routine testing, methods known in the study of drugs. See, for example, DBBudman, AHCalvert, EKRowinsky (publishers). Handbook of Anticancer Drug Development, LWW, 2003. Effective therapeutic dosages for various therapeutic species are well known to those skilled in the art; and in accordance with the present invention, the therapeutic entity provided by the liposome pharmaceutical composition of the present invention provides at least the same or twice as, 4-fold or 10-fold higher activity than that obtained by administering the same amount of therapeutic species in routine non-liposome preparations .
Typically, the liposome pharmaceutical composition of the present invention is formulated for topical use or for injection, or as a liquid solution or suspension. However, solid forms suitable for dissolving or suspending the substance in the liquid prior to injection may also be prepared. The composition can be formulated in the form of a tablet in an acid-proof coating or a gel capsule according to known methods in the art.
The liposome composition of the present invention may be administered in any manner that is medically acceptable and which may depend on the injury and the condition being treated. Possible drug delivery routes include parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intrathecal, intrathecal, or other routes, as well as orally, nasally, ocular, rectally, vaginally, topically, or to the lungs, e.g. by inhalation. In the case of delivery of liposomal drugs formulated in accordance with the invention for tumors of the central nervous system, a slow, prolonged intracranial infusion of liposomes directly into the tumor (enhanced delivery by convection or CED) is preferred. See Saito, et al., Cancer Research, vol. 64, pp. 2572-2579, 2004; Mamot, et al., J. Neuro-Oncology, vol. 68, p. 1-9, 2004. The compositions may also be administered directly to the surface of the tissues. Prolonged release, pH-dependent release or other administration e.g. specific chemical or release under mediating environmental conditions are also specifically described in the present invention e.g. by means of such agents as subcutaneous injections or decomposable implants.
Examples
The following examples are intended to illustrate, but not to limit the invention in any way, shape or form, directly or indirectly. While these examples are typical examples that may be used, other procedures, methods, or techniques known to those skilled in the art may be used.
Example 1. Preparation of substituted ammonium salts. useful trialkylammonium and dialkylammonium sulfate solutions for loading drugs (e.g., doxorubicin) into liposomes were prepared by diluting sulfuric acid with water to a concentration of 0.25 M, followed by titration of a sulfuric acid solution with one of the different amines. The substituted amines used in this example were triethylamine, trimethylamine, dimethylamine, diethylamine or diethanolamine. After the addition of the amines is complete, the resulting solution is diluted to a final concentration of 0.2 M of the substituted ammonium salt. Osmolality was determined using an osmometer and dew point determination.
The properties of the prepared solutions of substituted alkylammonium sulfate are shown in Table 1 below.
Table 1. Properties of various solutions of dialkylammonium sulphate and trialkyl ammonium
<td>salt</td><td>Osmolality, mmol / kg</td><td>pH</td>
<td>Dimethylammonium sulphate</td><td>472</td><td>5.65</td>
<td>Dimethyl ethanol ammonium sulphate</td><td>509</td><td>5.72</td>
<td>Diethylammonium sulphate</td><td>519</td><td>5.85</td>
<td>Trimethylammonium sulphate</td><td>497</td><td>5.81</td>
<td>Triethylammonium sulphate</td><td>559</td><td>5.33</td>
Example 2. Preparation of liposomes with closed salts of dialkyl ammonium and trialkyl ammonium and loading substances into these liposomes.
Distearoylphosphatidylcholine (DSPC), cholesterol (Chol) and N- (methoxy-poly (ethylene glycol) oxycarbonyl) -distearoylphosphatidylethanolamine (PEG-DSPE) (made from polyethylene glycol with a molecular weight of 2,000) were co-dissolved in chloroform at a molar ratio 3: 2: 0.015, then chloroform was removed at 55-60 ° C on a rotary evaporator. The dried lipid layer was hydrated in a solution of one of each dialkyl or trialkylammonium sulfate mentioned in example 1 at 60 ° C for 30 minutes. The lipid suspension was pressed under pressure through two stacked filters with an etched polycarbonate membrane with a pore size of 0.1 μm (Corning Nuclepore). The size of the liposomes was approximately 110-120 nm and was dependent on a method called quasi-elastic light scattering. Non-aerosolized trialkylammonium or dialkylammonium salts were removed from the external environment of liposomes by gel filtration using a crosslinked dextran gel (Sephadex G-75, Amersham Pharmacia Biotechnology), the column washed with HEPES buffered saline pH 7.2-7.4, and liposomes they were collected in an empty space in a column. USP doxorubicin hydrochloride (lyophilized with a powder containing 5 parts by weight of lactose per part of doxorubicin) was added to liposomes at a concentration of 150 μg drug / pmol phospholipid liposome. The mixture was incubated at 55 ° C for 45 min, cooled on ice for 10 minutes, and the non-moisturized drug was removed by gel permeation chromatography using a Sephadex G-75 column eluting with physiological buffer buffered with HEPES at pH 7.4. The presence of free doxorubicin (characterized by the fact that a slower moving red band appears) was visually undetectable. Purified liposomes loaded with doxorubicin were analyzed for phospholipid and doxorubicin according to examples 70 and 71 (spectrophotometric method). The obtained loading efficiency of the drug is shown in Table 2.
Table 2. Loading of doxorubicin into liposomes with a closed solution of dialkyl- and trialkylammonium salts. Drug / phospholipid input ratio of 150 μg / umol.
<td>The salt enclosed in the liposome:</td><td>Drug / phospholipid ratio in liposomes (pg / pmol)</td><td>Closing efficiency (%)</td>
<td>Trimethylammonium sulphate</td><td>140.74 ± 10.35</td><td>93.8 ± 5.7</td>
<td>Triethylammonium sulphate</td><td>163.81 ± 16.41</td><td>109.2 ± 11.6</td>
<td>Diethylammonium sulphate</td><td>158.16 ± 18.34</td><td>105.4 ± 7.8</td>
<td>Dimethyl ethanol ammonium sulphate</td><td>155.08 ± 8.51</td><td>103.4 ± 11.6</td>
Example 3. Preparation of liposomes containing various dialkyl, trialkyl and heterocyclic salts with substituted ammonium sulfate and loading of doxorubicin in these liposomes.
The substituted ammonium sulfate solutions were prepared as in Example 1, using commercially available amines with substituted alkyl, hydroxyalkyl and heterocyclic. The liposomes were formulated as in Example 1, except that instead of the hydration step of lipid coatings, pure lipids were dissolved in ethanol (approximately 100 μl ethanol for every 50 pmol phospholipids) and mixed with a solution of substituted ammonium salt at 60-65 ° C. such that the resulting lipid dispersion contains about 10% by volume of ethanol.
The loading of doxorubicin was performed by adding a solution of doxorubicin (2 mg / ml in HEPES buffered saline at pH 6.5) to the liposome in a ratio of 155 pg drug / pmol phospholipid liposome (PL) and heated at 58 ° C for 45 minutes in a hot bath water. The resulting liposomes were separated from any non-humified doxorubicin residues and analyzed for drug and lipid content as in Example 1. The results are shown in Table 3.
Table 3. Loading of doxorubicin into liposomes with closed sterically blocked alkyls, dialkyls, trialkyls and heterocyclic substituted ammonium sulphate salts.
<td>Amina used for preparation of substituted salt ammonium</td><td>osmolality mmol / kg</td><td>loading the medicine, mg / mole phospholipid</td><td>Effectiveness loading,%</td>
<td>trimethylamine</td><td>497</td><td>149.4 ± 7.9</td><td>96.4 ± 4.9</td>
<td>triethylamine</td><td>559</td><td>149.6 ± 6.9</td><td>96.5 ± 4.3</td>
<td>dimethylethanolamine</td><td>509</td><td>163.1 ± 6.6</td><td>105.3 ± 4.5</td>
<td>dimethylamine</td><td>472</td><td>158.6 ± 7.4</td><td>102.3 ± 4.9</td>
<td>diethylamine</td><td>519</td><td>156.7 ± 13.0</td><td>101.1 ± 8.5</td>
<td>diisopropylamine</td><td>533</td><td>159.9 ± 6.2</td><td>103.2 ± 4.1</td>
<td>Tris (hydroxymethyl) minometan</td><td>423</td><td>179.9 ± 15.3</td><td>116.1 ± 11.5</td>
<td>1-Piperidine</td><td>506</td><td>153.5 ± 7.1</td><td>99.0 ± 4.5</td>
<td>4-methylmorpholine</td><td>465</td><td>152.4 ± 9.8</td><td>98.3 ± 6.2</td>
<td>piperidine</td><td>479</td><td>158.5 ± 12.5</td><td>102.3 ± 8.2</td>
<td>1 Metylopyrolidyna</td><td>492</td><td>153.6 ± 12.3</td><td>99.1 ± 7.8</td>
<td>dimethylpiperazine</td><td>378</td><td>158.0 ± 6.5</td><td>101.9 ± 4.3</td>
Example 4. Preparation of a solution of trieMoammonium polyphosphate (TEA-Pn).
Linear poly (sodium phosphate) having 13-18 phosphate units per molecule (glass phosphate; CALGON®, obtained from Sigma Chemical Company) was dissolved in water at a concentration of about 1.3 M phosphate. The solution was passed through a column filled with 120 ml sulfonated polystyrene-divinylbenzene copolymer, an ion exchange resin in the form of a granulate (Dowex 50Wx8-200, Dow Chemical Co.) in the hydrogen form. The column was previously equilibrated with an aqueous solution of 3 - 3.6 M HCl to bring the resin to hydrogen form and washed with deionized water to obtain a neutral pH. Fifteen ml of sodium polyphosphate solution was loaded onto the column and washed with deionized H2O. The eluent from the column was observed using a conductivity detector. The outflow from the column corresponding to the peak conductivity was titrated with pure triethylamine to pH 5.5-6.0. The solution was analyzed for sodium residuals by potentiometric method using a sensitive sodium glass electrode and for phosphate content using inorganic phosphate analysis as in Example 1. A solution containing residual sodium content below 1% was diluted to a final concentration of 0.55 M. The solution typically has TEA concentration of 0.520.55 M, pH 5.5-6.0, and osmolality is 430-480 mmol / kg.
Example 5. Removal of unopened polyphosphate salts from liposomal preparations.
Liposomes (size 120 nm) with a closed fluorescent marker 8-hydroxypyrin trisulfonate were prepared according to Kirpotin et al., Biochemistry 36: 6675, 1997 and mixed with a sodium polyphosphate solution. The mixture was loaded onto a size column excluding cross-linked dextran granules (Sephadex G-75), 6% agarose granules (Sepharose 6B-CL), or 4% agarose granules (Sepharose 4B-CL), all from Amersham Pharmacia and then eluted with MES-dextrose buffer (pH 5.5). The effluents were analyzed for phosphate content using the Bartlett phosphate test (1959) and the liposome content using spectrofluorimetry. The tested gel permeation chromatography vehicles, Sepharose CL-6B, ensure complete separation of the polyphosphate from the liposomes in a sample / column volume ratio of 13.
Example 6. Preparation of a solution of sucrose triethylammonium sulfate (TEASOS).
Sodium sucrose octane (equivalent to 144.8) is the sodium salt of the sucrose derivative in which all hydroxyl groups have formulated esters of sulfuric acid. Sucrose octostearate (SOS), the sodium salt has been purchased from Toronto Research Chemicals, Toronto, Canada, p / n S699020. Six grams of sodium sucrose sodium sulfate was dissolved in 16.57 ml of deionized water to obtain a final concentration of about 2.5 N sulfate groups. The solution was treated by ion exchange as in Example 4. The octosulfatesaccharose acid solution was obtained as the effluent of the ion exchange column and then titrated with pure triethylamine until pH 5.7 (neutralization point) and the pH and osmolality of the solution were determined. The resulting solution had a calculated triethylammon concentration of 0.643 M, pH 5, 7 and osmolality 530 mmol / kg. The presence of residual sodium was undetectable by potentiometry (less than 0.1%).
Example Ί. Liposomes loaded with irinotecan (CPT-11) using substituted ammonium salts: preparation and release of the drug in vitro in the presence of blood serum.
In this example, both sulfates, citrates, pyrophosphates, triphosphates and linear polyphosphates (13-18-mer) as well as anions in liposomes in which the substituted solutions of ammonium salts are closed are tested. Phosphate polymers have been chosen for their biodegradability because polyphosphates occur naturally in cells unlike other synthetic polymeric anions (polyacrylate, dextran sulfate, and the like). In addition, the viscosity of low molecular weight polyphosphate solutions was lower than with other polymers, making polyphosphates easier to handle.
The following materials were used for the preparation of salt solutions.
1. Sodium phosphate, NaO- [PO3Na]<sub>n</sub>-Na, n = 13-18, purchased from Sigma (product No. P8510, "Phosphate Glass, Practical Grade", also known as sodium hexametaphosphate or under the trademark CALGON);
2. Pentasodium tripolyphosphate, Na5P3O10, purchased from Sigma (Product No. T5883); 3. Tetrasodium pyrophosphate decahydrate, Na4P2O7<sup>-</sup>10H2O purchased from Sigma (Product No. P-9146).
4. Dowex 50Wx4 ion exchange resins (4% cross-linked sulfonated polystyrene resin, mesh size 100-200) purchased from Sigma (product number 50X4-200) or Dowex HCR-W2 (8% crosslinked sulfonated polystyrene resin with mesh size 50-100 ) purchased from Sigma (product No. I-8880) were used interchangeably. The resins were washed by decanting in the following order: three times deionized water, twice with 1N HCl (3x more by volume relative to the resin), three times with water, twice with 1N NaOH, three times with water, three times with 1N HCl and three times with water. After decanting, the resin was in the H + form.
5. Trimethylamine (TMA), 40% aqueous solution from Aldrich Chemical Co. (product No. 43, 326-8). The concentration was determined by acid titration at about 5.9 N.
6. Triethylamine (TEA), 99% HPLC purity from Fisher (Product No. 04884). The concentration was determined by acid titration at about 6.9- 7.1 N.
The water was purified by reverse osmosis, ion exchange and removal of organic compounds to obtain the "16-18 MOhm" quality.
Aqueous solutions of pyrophosphate, triphosphate and polyphosphate salts were prepared by the ion exchange method. Sodium polyphosphate solutions (3 g in 25 ml water), sodium pyrophosphate (4 g in 27 ml water) or sodium polyphosphate (6.7 g in 30 ml water) were loaded onto a column containing 100 ml (bed volume) ion exchange resin prepared as above. The column was washed with water and the fractions were collected. Fractions showing acidic pH (pH <3) were combined. A triplicate portion of the 0.5-mL pooled fraction containing phosphate acid was diluted in 20 mL of water and titrated with 0.100 N NaOH to a final pH point of 4.5-5.0 (Fisher analytical solution) to determine normality. The combined ion exchange fractions were titrated with trimethylamine (to obtain a trimethylammonium salt) to pH 5.4-5.5.
Trimethylammonium and triethylammonium sulphates were prepared by diluting 1.39 ml of concentrated (17.9 M) sulfuric acid in 80 ml of water and titrated with a dilute solution of pure triethylamine or a solution of aqueous trimethylamine under pH-meter control to reach the equivalence point (pH 5.1 -5.5). When using water, the volume was brought to 100 ml.
A solution of trimethylammonium citrate was prepared by dissolving 1.572 g of citric acid monohydrate, ACS from Sigma (product No. C-1909) in 20 ml of water, and then the aqueous solution of trimethylamine was titrated to the equivalence point. When using water, the volume was brought to 25 ml.
The solutions are filtered through a cellulose acetate filter 0.2 μm using positive pressure. The osmolality and pH of the solutions were measured using a vapor pressure osmometer and a calomel glass electrode for pH adjustment. Normality of the anion in phosphate solutions was determined by spectrophotometric assay with phosphomolybdate blue (see example 70), after acid hydrolysis (5 min, 100 ° C, 3N H2SO4). Only acidic functional groups that are substantially ionized at a pH of 5.5 are considered when anion normality is achieved. Normality of the cation was determined based on the trialkylammonium base added. The solutions obtained have the following properties (Table 4):
Table 4. Properties of solutions of substituted ammonium salts for loading CPT-11 into liposomes.
Salt correctness of cation regularity of anion pH
Osmolality (mmol / kg)
<td>TMA citrate</td><td>0.58</td><td>0.60</td><td>5.1</td><td>791</td>
<td>TMA sulfate</td><td>0.50</td><td>0.50</td><td>5.4</td><td>625</td>
<td>TMA pyrophosphate</td><td>0.44</td><td>0.54</td><td>5.4</td><td>651</td>
<td>TMA triphosphate</td><td>0.57</td><td>0.68</td><td>5.4</td><td>695</td>
<td>TMA polyphosphate</td><td>0.49</td><td>0.58</td><td>5.5</td><td>336</td>
<td>TEA sulfate</td><td>0.54</td><td>0.50</td><td>5.35</td><td>719</td>
Cholesterol and DSPC were purchased from Avanti Polar Lipids, Alabaster, Alabama, USA. PEGDSPE (PEG MW 2,000) was purchased from Shearwater Polymers, Huntsville, AL, USA. DSPC, cholesterol and PEG-DSPE in a weight ratio of 3: 1: 0.1 (at a molar ratio of approximately 3: 2: 0.03) were dissolved in chloroform (Fisher; Optima grade, stabilized with amylene) in 60 mg / ml DSPC . The solution was placed in Pyrex 30 mg DSPC tubes (0.5 ml per tube) and slowly evaporated under reduced pressure using a rotary evaporator at 60 ° C. The lipid membranes were dried under vacuum (100 microns mercury, oil pump) for 30-60 minutes at room temperature.
Dry lipid membranes were hydrated by gentle shaking in the aforementioned aqueous salt solutions at 60 ° C for 15-20 minutes. The lipids form a milky suspension (multilamellar vesicles). This milk suspension was subjected to five freezing cycles in a mixture of dry ice and isopropanol (-80 ° C, 3 minutes) and thawing in a water bath at 60 ° C for 3 minutes. The lipid suspension was then extruded 10 times (double impacts) through two stacked polycarbonate membrane filters (Nucleopore, Whatman, 0.1 μm pore size) and heated by hand-operated reciprocating extruders (Avanti Polar Lipids) at 60 ° C .
The extruded liposomes were stored at 60 ° C for five minutes, and the reaction was quenched in ice water (0-4 ° C) for five minutes. The liposomes were then separated from the saline solution forming a gradient to the MES-dextrose loading buffer (50 g / L dextrose ACS, 0.975 g / L 2- (N-morpholino) ethanesulfonic acid (MES) and sufficient 5M NaOH to bring the pH to 6, 4) by means of gel chromatography on a column
Sephadex G-75. Liposomes appear in the void fraction (at 30% deposition of the column bed).
The formulation CPT-11 (irinotecan hydrochloride) containing 0.860 mg of base CPT11 per mg of solid was dissolved in 0.001N HCl to give a stock solution
16.5 mg / ml CPT-11 bases. This solution is mixed with the liposomes in MESdecrose buffer to achieve a ratio of 150 pg of CPT-11 to pmol of phospholipid liposomes.
The mixture was incubated at 55 ° C in a water bath with occasional gentle shaking (approximately once every five minutes) for 30 minutes and then rapidly quenched in ice water (0-4 ° C). The liposomes were separated from the encapsulated drug by gel chromatography on a Sephadex G-75 column using MES-dextrose as eluent. The encapsulated drug was determined by a spectrophotometric assay (example 71) and the phospholipids were determined using an extraction assay (example 70).
In vitro release of the loaded CPT-11 liposomes in the presence of 50% human plasma was tested as follows. Frozen human donor plasma was thawed at 37 ° C, centrifuged at 14.500 g for 10 minutes and filtered through a 0.45 μm syringe filter with cellulose acetate. CPT-11 loaded liposome preparations were sterilized by passing them for 0.2 pm sterile syringe filter with surface-active cellulose acetate (SFCA). 0.5 ml liposomes were mixed with 0.5 ml plasma in a 1.5 ml sterile Eppendorf copolymer tube, sealed and incubated on a shaker at 37 ° C for 24 hours. Instead of liposomes, the blank sample contained 0.5 ml of sterile MES-dextrose. The liposomes were isolated by gel chromatography on a pearly cross-linked 2% agarose gel (Sepharose CL-2B, Pharmacia; 10 ml bed volume) with 144 mM NaCl, 5 mM HEPESNa, buffer (HBS-5) pH 7.4. Liposomes appeared in the void fraction, while proteins from plasma and the released drug (if any) were delayed by the gel. The liposome fractions were analyzed for CPT-11 and phospholipids and the drug / phospholipid ratio (base ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing samples. The percentage of residual drug in liposomes after incubation was determined by dividing the drug / lipid baseline ratio by the drug / lipid entry ratio (drug / lipid ratio before incubation with plasma). The loading and release data are summarized in Table 5. 5 mM HEPESNa, buffer (HBS-5) pH 7.4. Liposomes appeared in the void fraction, while proteins from plasma and the released drug (if any) were delayed by the gel. The liposome fractions were analyzed for CPT-11 and phospholipids and the drug / phospholipid ratio (base ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing samples. The percentage of residual drug in liposomes after incubation was determined by dividing the drug / lipid baseline ratio by the drug / lipid entry ratio (drug / lipid ratio before incubation with plasma). The loading and release data are summarized in Table 5. 5 mM HEPESNa, buffer (HBS-5) pH 7.4. Liposomes appeared in the void fraction, while proteins from plasma and the released drug (if any) were delayed by the gel. The liposome fractions were analyzed for CPT-11 and phospholipids and the drug / phospholipid ratio (base ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing samples. The percentage of residual drug in liposomes after incubation was determined by dividing the drug / lipid baseline ratio by the drug / lipid entry ratio (drug / lipid ratio before incubation with plasma). The loading and release data are summarized in Table 5. The liposome fractions were analyzed for CPT-11 and phospholipids and the drug / phospholipid ratio (base ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing samples. The percentage of residual drug in liposomes after incubation was determined by dividing the drug / lipid baseline ratio by the drug / lipid entry ratio (drug / lipid ratio before incubation with plasma). The loading and release data are summarized in Table 5. The liposome fractions were analyzed for CPT-11 and phospholipids and the drug / phospholipid ratio (base ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing samples. The percentage of residual drug in liposomes after incubation was determined by dividing the drug / lipid baseline ratio by the drug / lipid entry ratio (drug / lipid ratio before incubation with plasma). The loading and release data are summarized in Table 5.
Table 5. Loading CPT-11 into liposomes with tertiary alkylammonium salts and in vitro drug release in the presence of human plasma.
<td>Closed solution</td><td colspan="2">Before incubation with plasma</td><td colspan="2">After incubation with plasma</td>
<td>salt</td><td>ratio drug / lipid</td><td>effectiveness encapsulation (%)</td><td>ratio drug / lipid</td><td>residue hermetic drug (%)</td>
<td>TMA sulfate</td><td>127.2 ± 5.6</td><td>84.8 ± 3.8</td><td>132.1 ± 6.9</td><td>103.8 ± 10.0</td>
<td>TMA pyrophosphate</td><td>136.2 ± 9.0</td><td>90.8 ± 6.0</td><td>132.3 ± 5.0</td><td>97.1 ± 10.1</td>
<td>TMA triphosphate</td><td>132.9</td><td>88.6</td><td>129.2</td><td>97.3</td>
<td>TMA-Pn</td><td>134.4 ± 9.3</td><td>89.6 ± 6.2</td><td>135.0 ± 7.4</td><td>100.4 ± 12.4</td>
<td>TEA sulfate</td><td>131.1 ± 6.5</td><td>87.4 ± 4.4</td><td>125.2 ± 5.0</td><td>95.5 ± 8.6</td>
Example 8. Stability of liposomes in vividated CPT-11 uses salts of pyrophosphate, triphosphate, polyphosphate, citrate and trialkyl ammonium sulfate.
On the other hand, liposomes with camptothecin may show an acceptable drug leak in the blood plasma in vitro, the drug may leak faster in the bloodstream in vivo.
Therefore, a panel of liposomal preparations with CPT-11 was visualized for drug stability in the bloodstream in vivo by means of a single point during the mouse test.
The liposomes were prepared and loaded with CPT-11 as described in Example 6 with the following modifications. Instead of using PEG-TDWP from Shearwater Polymers, similar PEG-TDWP from Avanti Polar Lipids was used. In order to obtain a quantitative lipid liposome in the matrix in blood / tissue samples, a radioactive label was not included [<sup>3</sup>H] -heter of hexadecyl cholesterol ([<sup>3</sup>H] -CHE; (Amersham, USA) to a solution of chloroform with lipids at 0.25 mCi / mmol phospholipids. The lipid solutions were spilled into Pyrex tubes of 12 mg DSPC / tube, and the lipid membranes were formulated by a rotary evaporator / vacuum drying. The lipid membranes were hydrated in 0.7 ml forming a gradient of substituted solutions of ammonium salts. The concentration of lipids in liposomes with closed phosphate-containing salts was determined by radioactivity by scintillation counting. Formulations without closed phosphate-containing salts were also tested for phospholipids without extraction as described in Example 70 and were used as standards for measuring lipid radioactivity. A portion of the medicinal liposome mixture prepared for loading has been preserved and tested to confirm the pre-loading ratio of CPT11 added to the lipid liposome before loading ("input ratio"). The averaged volume of agents and the standard deviation of the particle size distribution of liposomes are determined by quasulicic light scattering (QELS) using the Gaussian model. The properties of these liposomes are summarized in Table 6.
Table 6. Characterization of loading CPT-11 to [<sup>3</sup>H] -CHE-labeled liposomes for stability testing in vivo
<td>Closed solution salt</td><td>ratio drug / lipid before loading</td><td>ratio drug / lipid after loading</td><td>effectiveness loading (%)</td><td>Size liposomes, (mean ± SD) nm</td>
<td>TMA citrate</td><td>159.2 ± 3.5</td><td>156.7 ± 3.6</td><td>98.5 ± 4.4</td><td>122.1 ± 25.3</td>
<td>TMA sulfate</td><td>156.1 ± 2.5</td><td>156.1 ± 3.1</td><td>100.0 ± 3.6</td><td>122.2 ± 28.4</td>
<td>TMA pyrophosphate</td><td>164.6 ± 5.8</td><td>156.6 ± 4.3</td><td>95.2 ± 6.0</td><td>121.1 ± 19.9</td>
<td>TMA triphosphate</td><td>163.6 ± 5.7</td><td>156.0 ± 3.2</td><td>95.3 ± 5.3</td><td>122.4 ± 12.9</td>
<td>TMA polyphosphate</td><td>170.5 ± 8.0</td><td>162.4 ± 4.0</td><td>95.3 ± 6.8</td><td>123.0 ± 12.7</td>
<td>TEA sulfate</td><td>153. ± 3.3</td><td>154.9 ± 4.9</td><td>101.0 ± 5.3</td><td>121.1 ± 18.0</td>
Six-week-old CD-1 female mice (Charles River) were injected via the tail vein with 10 mg / kg CPT-11 (0.2 mg CPT-11 / mouse) lipids in duplicate. Eight hours later, the mice were anesthetized and exsanguinated by open-heart puncture. Blood was collected into heparinized syringes (10-20 μΙ per 1000 U / mL USP heparin) and transferred to weighed tubes containing 0.4 mL of phosphate buffered saline (PBS) containing 0.04% EDTA (Gibco BRL) and kept on ice. . The tubes were weighed to determine the weight of the blood samples, the blood cells were separated by centrifugation at 9,000 g for 5 minutes and the supernatants containing the diluted PBS plasma were stored for further drug and lipid liposome studies. CPT-11 was quantitated using a fluorometric assay (example 71). The lipid liposome was quantified by (quenching) the scintillation counting of radioactivity. Norms of radioactivity of phospholipid liposomes were counted in parallel with plasma samples. The percentage of the drug that remains encapsulated has been calculated by dividing the drug / radioactivity ratio in plasma samples by the drug / radioactivity ratio of the injected liposomes. Due to the rapid removal of free CPT-11 from blood (see example 69) and known stability [ The percentage of the drug that remains encapsulated has been calculated by dividing the drug / radioactivity ratio in plasma samples by the drug / radioactivity ratio of the injected liposomes. Due to the rapid removal of free CPT-11 from blood (see example 69) and known stability [ The percentage of the drug that remains encapsulated has been calculated by dividing the drug / radioactivity ratio in plasma samples by the drug / radioactivity ratio of the injected liposomes. Due to the rapid removal of free CPT-11 from blood (see example 69) and known stability [<sup>3</sup>H] -CHE against lipid exchange, test readings are considered to indicate blood content in liposomal CPT-11 and lipids.
The percentage of injected lipid dose (% ID) remaining in circulation was calculated assuming that 100% of the bolus injected entered the circulation; a blood volume of 6.3% of the body weight of the mouse and the hematocrit of 45%. The results are summarized in Table 7.
Table 7. In vivo stability of CPT-11 encapsulation and lifetime of circulating lipids loaded with CPT-11 in mice at a given time point (8 hours) after injection. % ID,% of the injected dose.
<td>The salt is enclosed in the liposome</td><td>Drug / lipid ratio,% value initial injection</td><td>Lipid liposome,% ID in the blood</td>
<td>TMA citrate</td><td>80.2 ± 7.8</td><td>18.8 ± 3.4</td>
<td>TMA sulfate</td><td>70.1 ± 4.8</td><td>23.6 ± 1.8</td>
<td>TMA pyrophosphate</td><td>67.3 ± 9.2</td><td>23.2 ± 3.1</td>
<td>TMA triphosphate</td><td>70.6 ± 6.0</td><td>24.9 ± 8.2</td>
<td>TMA polyphosphate</td><td>107.5 ± 8.9</td><td>24.3 ± 3.4</td>
<td>TEA sulfate</td><td>76.6 ± 13.1</td><td>23.6 ± 0.1</td>
All preparations showed the level of the encapsulated drug after 8 hours in the blood in vivo, at 70-80% of the level before injection, while the polyphosphate containing liposomes were the most stabile (the encapsulated drug remained at around 100%). Example 9. Pharmacokinetics of CPT-11 liposomes blood that were made using triethylammonium polyphosphate.
The CPT-11 liposomal formulation using the triethylammonium polyphosphate salt was prepared as described in Example 3. Lipids - DSPC, cholesterol and N- (methoxy-poly (ethylene glycol) (molecular weight 2000) oxycarbonyl) -DSPE (PEG-DSPE) ( all from Avanti Polar Lipids, Inc.) - they were combined in the form of dry powders in a molar ratio of 3: 2: 0.015 and dissolved in 100% ethanol (USP grade, about 0.15 ml / 100 mg of lipids) at 62- 65 ° C. For pharmacokinetic studies, ether<sup>3</sup> H-cholesterol hexadecyl (<sup>3</sup>H15 CHE, obtained from Amersham Pharmacia) was added to the lipids at 0.5 mCi / mmol phospholipids as a radioactive non-replaceable lipid tag. The aqueous TEA-Pn solution (0.5 M triethylammonium pH 5.7-6.2) was prepared as in Example 4. A solution of TEA-Pn (10 volumes of ethanol added) was mixed with the lipid solution at 60-65 ° C. and stirred at this temperature until a homogeneous milk suspension is obtained in the form of multilamellar vesicles. This slurry was 15-folded with 2 stacked filters with an etched polyvinyl chloride membrane.carbonate (Corning Nuclepore) with a pore size of 100 nm, using a pressure extruder with argon (Lipex Biomembranes) at 60-65 ° C, and the resultant monolayer liposomes were quickly cooled on ice and allowed to reach ambient temperature. Ethanol and the excluded polyphosphate salt were removed by gel chromatography on an Etepharo C CL-4B column that was eluted with MES-delstrose buffer (5 mM MES, g / l dextrose, pH adjusted to 6.5 with NaOH).
CPT-11 stock solution (irinotecan hydrochloride) containing 20 mg of irinotecan base in water was added to the liposomes in a drug / lipid ratio of 150-200 mg / mmol phospholipids, and then this mixture was incubated with occasional mixing for 45 -60 minutes at 60 ° C. 62 ° C. The incubation mixture was quickly cooled and incubated for 10 minutes at 0 ° C and then allowed to reach ambient temperature. 1/20 of a volume of 2.88 M NaCl was added to adjust to physiological ionic strength and improve the membrane-induced capacity of CPT-11 (as opposed to an encapsulated drug inside the liposome). The non-hyperbarized drug was removed by gel chromatography on a Sephadex G-25 or G-75 column (Amersham Pharmacia), which was eluted with HBS-6 buffer, 5 (5 mM 2- (4- (2-hydroxyethyl) piperazine) ethylsulfonic acid (HEPES), 144 mM NaCl, pH 6.5). The liposome fractions eluted in the empty space were combined, sterilized by filtration through 0.2 micron pores and stored at 4-6 ° C before further use. Liposomes are characterized by lipid concentration, drug concentration and particle size as in example 7. Liposomes have an average size of 108 nm and a CPT-11 content of 139 ± 18 mg of base CPT-11 per mmol phospholipids.
The persistence of the lipid liposome and liposome in the blood drug and the release characteristics of the drug with the liposomes in vivo were tested in female Sprague-Dawley rats (190-210 g), with catheters permanently attached to the central veins. Rats were injected with 0.2-0.3 ml 3H-CHE bolus of labeled liposomes with irinotecan (0.05 mmol phospholipids or 7-8 mg CPT-11 per kg body weight). Blood samples (0.2-0.3 ml) were made at various times after injection using heparinized syringes. The collected blood volume was supplemented with phosphate buffered saline. Blood samples were diluted with 0.3 ml of ice-cold PBS containing 0.04% EDTA, weighed, and the blood cells were separated by centrifugation. The liquid supernatants were collected and assayed for CPT-11 using the fluorometric procedure of example 71 and for the lipid lipid marker by scintillation counting of radioactivity using conventional methods. Preparations of liposomes with known drug and<sup>3</sup>The H-CHE-lipid concentration was used as the reference standard. The radioactivity standards contain the same amount of dilute plasma of rats to calculate them for hardening. The amount of CPT-11 and lipid liposome in the blood was calculated assuming that the volume of blood was expressed in ml, as 6.5% of body weight in grams and hematocrit of 40%. The total amount of lipids and drug in the blood was expressed as% of the injected dose (% ID,% ID) and plotted on the graph considering the time after injection. The percentage of drug remaining in the liposomes was calculated by dividing the drug / lipid ratio in the blood samples by the drug / lipid ratio in the injected liposomes (taken as 100%). Because the graphs generally showed good compatibility with the mono-dispersive kinetics (half-log linearity), the half-life of the drug in the blood, lipids and drug release from liposomes were calculated with the best fit of data to the monoexponential equation of natural disintegration using the TREND options of the Microsoft EXCEL computer program (Microsoft Corp., USA). The results are shown in Figure 1. From the best fit parameters, half-life of blood lipids and drug was 16.4 hours and 6.61 hours, respectively. Under these conditions, free CPT-11 was cleared from circulation very quickly (see example 69). 4 hours and 6.61 hours. Under these conditions, free CPT-11 was cleared from circulation very quickly (see example 69). 4 hours and 6.61 hours. Under these conditions, free CPT-11 was cleared from circulation very quickly (see example 69).
The drug / lipid ratio in the blood showed a two-phase nature of CPT-11 released from liposomes (figure 2). During the first 24 hours, drug release occurred linearly over time (R = 0.992), giving evidence that the release kinetics were zero order. Only about 75% of the released drug at the 24-hour point had a further release that was non-linear. For 24 hours, liposomes released the drug at a constant rate of about 3.6% of the initial loading / hour. Thus, 50% of the drug was released within approximately 14 hours. The release of a (zero-order) drug (drug) is effective and gives an extraordinary quality in the prolonged release of preparations, because the rate of drug release remains constant at all times.
Example 10. Antitumor efficacy of CPT-11 liposomes prepared using triethylamine polyphosphate against breast cancer xenografts in nude mice
The anti-tumor efficacy of CPT-11 liposomes was examined in a human breast cancer model BT-474, an estrogen-dependent adenocarcinoma that is an overexpressor C ErbB2 (HER2) receptor. BT-474 cells were obtained from the American Type Culture Collection (Rockville, MD). A subordinate BT-474 with a higher tumor growth rate was established due to the rapidly growing xenograft tumor grown as described below. The cells were grown in vitro in RPMI-1460 medium with 10% fetal calf serum, 0.1 mg / ml streptomycin sulphate and 100 U / ml penicillin G in T-150 flasks and separated in a 1: 3 ratio weekly. NCR nu / nu female mice (4-6 weeks old, Taconic Farms) were implanted subcutaneously (at the base of the tail) with 0.72 mg granules of 173-estradiol (Innovative Research of America, Inc.).<sup>7</sup> BT474 in cell culture medium. Tumor progression was monitored by palpation and measurements by tumor vernier along the largest (length) and smallest (width) axis twice a week. Tumor sizes were determined twice weekly by measuring the caliper using a pattern (Geran, RI, et al., 1912 Cancer Chemother Rep. 3: 1-88):
Tumor volume = [(length) x (width)<sup>2</sup>] /
On day 13 after implantation, the tumor reached an average size of 200 mm<sup>3</sup>and the animals were randomly assigned to three groups of 13-15 each.
The liposomal CPT-11 was prepared as in Example 8 (drug / phospholipid ratio 192 mg / mmol, mean liposome size was 86.8 nm). Free and liposomal CPT-11 were diluted with MES-dextrose to 5 mg / ml CPT-11 base. The animals were injected via the tail vein with free CPT-11, liposomal CPT11, or only substance on days 14, 18, 21 and 25 after tumor implantation. Drug-containing formulations were administered at a dose of 50 mg CPT-11 / kg per injection, which is the average of the doses reported in the literature and studies of CPT-11 on rodent tumor models.
To assess the toxicity associated with treatment, the animals were also weighed twice a week. Observations were carried out up to 60 days after implantation, at which time the granules of estrogen supplementation were exhausted. The average tumor volumes in the whole group, depending on the time, were plotted on the graph. As shown in Figure 3, while free CPT-11 reduced tumor growth rate, tumors significantly decreased in the group receiving liposomal treatment. While on day 36 in the control group, the tumors reached the maximum acceptable size, which was on average 3,500 mimf, and on day 46 in the free drug group, tumors had an average volume of about 1,000 mm 3, at the same time point none of the animals in the liposomal group it did not have a palpable tumor.
Treatment-related toxicity was assessed on the basis of animal body weight dynamics (figure 4). None of the groups showed any significant toxicity. The weight of animals in the control group increased steadily. There was a slight decrease in the mean body weight of animals receiving CPT-11 liposomal treatment, by approximately 3.3%, on the last day of treatment. This weight loss has been restored to normal and the animals have reached their previous weight. This decrease in mean body mass carried out with the Student's t-test was not statistically significant in relation to the weight before treatment (p = 0.274). Thus, all treatments were tolerated and without significant toxicity.
Thus, the CPT-11 liposomal formulation obtained by loading the drug through a pre-enclosed sterically blocked substituted ammonium salt (a triethylammonium) of a polyanionic, biodegradable polymer (polyphosphate) showed prolonged blood viability, stable release properties and increased anti-tumor activity in the tumor model under study without apparent increased toxicity .
Example 11. Comparative evaluation of CPT-11 loaded liposomes prepared using pre-enclosed triethylammonium salts: effect of liposomal size, drug / lipid ratio and nature of the pre-enclosed anion.
Two prototypical preparations of CPT11 loaded liposomes were prepared, one using liposomes with pre-closed TEA-Pn and the other with pre-closed TEA-SOS. The preparation of these liposomes included the following production steps.
1) The combination of lipids by dissolution in ethanol. The com position of the lipid matrix consists of 1,2-distearoyl-SN-phosphatidylcholine (DSPC) (molecular weight 790), 3 molar portions (59.8 mol%); Cholesterol (Chol) (MW 387) 2 parts by mole (39.9 mol%); and N- (omega-methoxy-poly (ethylene glycol) oxycarbonyl) -1,2-distearoylphosphatidylethanolamine (MW 2787) (PEGDSPE) 0.015 part by mol (about 0.3 mol%). DSPC and PEG-DSPE were purchased from Avanti Polar Lipids, Birmingham, Alabama. Cholesterol (the highest degree of purity) was purchased from Calbiochem. The dry lipids were weighed with an accuracy of ± 0.1 mg in a borosilicate glass container and combined in absolute ethanol in a ratio appropriate for the lipids in the dispersion step described below. Due to high DSPC transition temperatures (55 ° C),
2) Prepared TEA-Po and TEA-SOS solutions. Sodium polyphosphate (n = 13-18) was from Sigma Chemical Co., p / n P 8510. Sodium sucrose octane was purchased from Toronto Research Chemicals, Toronto, Canada, p / n S699020. The salts were weighed and dissolved in water to provide 1.2-2.5 N solutions. Dowex 50Wx8-200 anionites or Dowex HCR-W2 form H + (available from Sigma) were used to convert the sodium salts into free acids. Before the first use, the resins were rinsed by mixing with 3 volumes of the following solutions, followed by decanting: (1) 1.0-1.2 M aqueous HCl solution 2 times; (2) Water 2 times; (3) 1.0-1.2 M aqueous NaOH solution 2 times; (4) Water 2 times; (5) 1.01.2 M aqueous HCl solution 2 times. The resin suspension washed in water was filled under the influence of gravity in a suitable column size to obtain at least 8 ml of filled resin for each ml of sodium salt solutions. The resin was further equilibrated by passing 2 column volumes of 3.0-3.6 M with a HCI aqueous solution, followed by 5 column volumes with water, until the leakage conductance fell below 1 micro-S. After use, the columns were regenerated by successive passes: 1-1.2 M HCl - 3 column volumes; 3.0-3.6 M HCl - 2 column volumes; water - at least 5 column volumes, until the leakage conductivity drops below 1 pS and stored in 0.2 m filtered water at room temperature. Pn and SOS sodium solutions were applied to the dehydrated column surface (approximately 1 ml for every 4 ml volume filled with resin) and gravity flow was allowed to run at about 1-2 ml / min for a resin granulate of 75-150 ml. The column was washed with water. The eluate was tested for conductivity. Fractions with 10 mS or higher conductivity were collected. If more concentrated polyacids are required, the collection can start at 20-50 mS, but at a cost of a slightly higher loss of salt forming the gradient. In the case of phosphoric acid, the collected solution is kept in a freezer (0-4 ° C) up to the amine titration stage due to hydrolytic instability of the phosphodiester bond at low pH. The collected eluates will have a pH less than 0.7 (typically about 0.4) and a conductivity of about 120-200 mS. Possibly, the amine titration step is carried out without delay due to the persistence of polyphosphates at low pH. HPLC-grade triethylamine (purity 99.5 +%) from Fisher, p / n 04884 was used for titrating the acid solutions obtained from ion exchange. The level of TEA purity normality was determined by potentiometric titration. A 0.1 ml portion of TEA (0.100 ml) was added to 20 ml of water in triplicate. The portions were titrated in 0.1 N HCl standard solution to the pH endpoint (glass electrode) of 5.5-6.0. The calculated normality (7.07 N) was close to the theoretical value of 7.17N. The measured amount of polyphosphoric acid (Pn) or sucrose octasulfate (SOS) solution was titrated with pure TEA under pH control (glass electrode). Thorough mixing was required to disperse the amine. The titration end point was at pH 5.6-6.2. The volume of added TEA was registered correctly. The volume of the titrated solution was measured and the TEA concentration was calculated based on the volume of added TEA and normality. Water was added as needed to adjust the TEA concentration required to obtain 0.55 ± 0.05 N or N 0.65 ± 0.03 as indicated below. The amount of residual sodium in the obtained TEA-Pn or TEA-SOS solutions was determined potentiometrically using a glass selective electrode (Corning). One ml of the solution is diluted with 19 ml of water and the sodium concentration is determined by the growth method according to the electrode manufacturer's instructions. The amount of residual sodium was less than 1 mM, typically less than 0.5 mM. The obtained TEAPn or TEA-SOS solutions were passed through a 0.2 μm sterile cellulose acetate filter using the overpressure channel. The final pH and osmolality of the solutions were measured and recorded. To measure the pH we use a calomel micro-combined glass electrode, and for osmolality, vapor pressure / dewpoint osmometer. The solutions were stored in a refrigerator until they were used.
3). Forming a lipid dispersion in a gradient-forming buffer by mixing the ethanolic lipid solution with a gradient forming buffer. The lipids were dispersed in a gradient salt forming solution using the ethanol mixing method. All steps were carried out at 60-65 ° C. The fats were dissolved in 100% USP ethanol at a concentration of about 0.5-0.6 M DSPC in a chemically resistant glass pear and tube flask. Gradient salt solution (TEA-Pn or TEA-SOS) was preheated to 60-65 ° C and added to the ethanolic lipid solution at once, and the ingredients were mixed thoroughly by centrifugation and / or vortexing. The final amount of ethanol was about 10% by volume. For preparations on a scale exceeding 0.1 mmol of phospholipid, the resulting suspension was placed on a rotary evaporator at 60-65 ° C and placed in a vacuum with rotation until the evolution of ethanol was stopped, which manifests itself to form a foam. For a 0.1 mmol scale of phospholipid or less, ethanol was not removed from the lipid dispersion at this stage. The resulting lipid suspensions were stored at 60-65 ° C and used immediately in the extrusion step.
4). Sequential extrusion of the lipid dispersion with pink pores. For the suspension of lipid volumes up to 1 ml, we used a manual reciprocating extruder supplied by Avanti Polar Lipids. The extruder is equipped with 19 mm membrane etched filters and thermostatted by a strong heating block of metal. Up to a volume of 1 to 10 ml, we used a thermostated, with the possibility of gas pressure control, an extruder with one-way flow from Lipex Biomembranes. The extruder is equipped with filtering membranes with a size of 25 mm. Lipid suspensions were repeatedly extruded at 60-65 ° C using manual feed or argon gas pressure, by using 2 stacked polycarbonate filtration membranes (filters from Corning-Nuclepore and Osmonics Corp.). were equally favorable) with a nominal pore size of 100 nm, 80 nm or 50 nm. In the case where the liposomes reached the desired size, the extrusion was stopped at the step at 100 nm, 80 nm or 50 nm. The exact type of filters and the number of extruders for each experiment. are indicated below. The extruded liposomes are kept at 60-65 ° C for about 15 minutes and rapidly cooled to 2-4 ° C in an ice bath. After about 15 minutes. in an ice bath, the liposomes were allowed to reach room temperature. The extruded liposomes are kept at 60-65 ° C for about 15 minutes and rapidly cooled to 2-4 ° C in an ice bath. After about 15 minutes. in an ice bath, the liposomes were allowed to reach room temperature. The extruded liposomes are kept at 60-65 ° C for about 15 minutes and rapidly cooled to 2-4 ° C in an ice bath. After about 15 minutes. in an ice bath, the liposomes were allowed to reach room temperature.
5). Removal of the extralorpion buffer against gradient and migration of the drug buffer to be neutralized. The non-aerosol gradient forming salts were removed and the liposomes transferred to the drug with loading buffer by size exclusion chromatography (SEC). Tangential flow filtration, hollow fiber dialysis from the second scalable stage can be used in a higher scale production. It is preferred to ensure complete removal of the extraliposomal polyanion by treatment of the liposomes with an anion exchange resin (e.g., Dowex-1 or Dowex-2 quaternary ammonium crosslinked polystyrene granules). The drug with loading buffer contained 50 g / L of anhydrous dextrose USP and certified 5 mM HEPES for tissue culture in water, adjusted to a pH of 6.5 with NaOH. The buffer was vacuum filtered through a 0.2 micron nylon filter (Whatman). The extruded liposomes were chromatographed on a Sepharose CL-4B column (Pharmacia) and eluted with the drug with loading buffer. Liposomes appeared in the void fraction and were then collected, depending on the turbidity of the eluate, in a volume of about 2x of this use. The eluted liposomes were tested for phospholipid concentrations according to example 70, the particle size was measured with QELS and stored at 46 ° C. depending on the turbidity of the eluate in a volume of about 2x this application. The eluted liposomes were tested for phospholipid concentrations according to example 70, the particle size was measured with QELS and stored at 46 ° C. depending on the turbidity of the eluate in a volume of about 2x this application. The eluted liposomes were tested for phospholipid concentrations according to example 70, the particle size was measured with QELS and stored at 46 ° C.
6) Incubation of liposomes with I and I. The base of CPT-11 (irinotecan hydrochloride) was prepared just before mixing with the liposomes by dissolving irinotecan hydrochloride in water until a concentration of 20 mg / ml basic drug was obtained. The pH of the solution was in the range of 4.0 and 5.0. The drug solution was filtered through a sterile 0.2 micron polyethersulfone (PES) filter with overpressure. Portions of liposomes in the drug with the loading buffer prepared in the above step, were mixed at room temperature with a substituted solution of irinotecan to achieve drug / lipid ratio in the range of 0.15-0.55 g of drug per mmol of phospholipid liposome. If this is the case, the detailed entry ratios in the drug / lipid ratio are given below. The pH values of the mixtures were adjusted to 6.5 with 1 M NaOH, the mixtures in glass vials were incubated in a thermostatic water bath at 58-62 ° C with slow stirring for 30-45 minutes, then rapidly cooled in an ice-water bath (0-2 ° C) and left at this temperature for 15 minutes. The liposomes were then allowed to warm to room temperature to the next step (removal of the non-aerosolized drug and transfer to buffer for further storage). This step resulted in the effectiveness of encapsulation, which now was more than 95%, typically 98-100% in the whole range of the drug / lipid drug test. The liposomes were then allowed to warm to room temperature to the next step (removal of the non-aerosolized drug and transfer to buffer for further storage). This step resulted in the effectiveness of encapsulation, which now was more than 95%, typically 98-100% in the whole range of the drug / lipid drug test. The liposomes were then allowed to warm to room temperature to the next step (removal of the non-aerosolized drug and transfer to buffer for further storage). This step resulted in the effectiveness of encapsulation, which now was more than 95%, typically 98-100% in the whole range of the drug / lipid drug test.
7). Removal of non-pressurized CPT-11, transfer of iiposomes in storage buffer, final filtration and storage. The non-aerosolized drug was removed and the liposomes were transferred to the storage buffer by size exclusion chromatography. The storage buffer contains 20 mM HEPES, 135 mM NaCl, pH 6.5 (adjusted with NaOH) in water and prior to use has been vacuum filtered through a 0.2 micron filter. Gel chromatography on a Sephadex G-75 column (Amersham Pharmacia Biotech) was performed essentially as described in step 2 above. The CPT-11 liposomes were eluted from the column (void fraction) and examined for phospholipid liposomes. and CPT-11 (spectrophotometrically, see examples 70 and 71), and volume weighed median particle sizes using QELS. The concentration of the drug was adjusted, if necessary, to be in the range of 2.0-4.0 mg / ml. The liposomes were filtered through a 0.2 micron sterile polyethersulfone filter and aseptically separated into sterile polypropylene tubes (Corning Cryo-Vials) or into 4-ml glass PTFE lined vials with borosilicate caps at approximately 70-80% of the vial volume. The vials were closed under aseptic conditions (air), determined and stored at 4-6 ° C.
Example 12. Effect of drug / lipid ratio on drug loading and in vivo drug retention with TEA-Pn-containing liposomes
Liposomes with a closed aqueous 0.65 N TEA-Pn solution, pH 6.1, osmolality 531 mmol / kg, were prepared according to the procedure in Example 11. The lipid dispersion was extruded ten times with two stacked polycarbonate pore size filters. 100 nm. The lipid liposome matrix also includes [<sup>3</sup>H] -CHE 0.5 mCi / mmol phospholipids. The size of liposomes prior to drug loading was 98.5 ± 34.3 nm. Liposomes were loaded in the initial drug to phospholipid ratio of 200, 300, 400 and 500 mg CPT-11 / mmol phospholipid, respectively. Amounts of drug and phospholipid in liposomes were determined by the spectrophotometry of example 71 and by extraction and digestion with the blue phosphomolybdate of example 72.
In order to evaluate the release rate of the drug in vivo, the method of Example 8 was used. The liposomes were injected via the tail vein of 6-week-old Swiss Webster mice (body weight 18-22 g) at a dose of 5 mg CPT-11 / kg. At 8 and 24 hours after injection, mice in groups of 3 were anesthetized and bled by open-heart puncture. The blood was mixed with 0.4 ml of ice-cold 0.04% EDTA in PBS, the blood cells were separated by centrifugation, and the plasma concentration of CPT-11 was measured with a spectrofluorometer as described in example 71. Lipids were determined by measuring the amount [<sup>3</sup>H] -CHE using (corrected) scintillation fluid counting, and the amount of retained drug in liposomes was calculated by dividing the drug / lipid ratio by the drug / lipid ratio in the injected liposomes. Because there was a rapid removal of free CPT-11 from the blood, this resulted in low blood levels and this allows to assume that all tested drugs were in liposomal form.
The results are shown in Table 8. Differences between drug retention between groups were not statistically significant. As a result of these studies, it was found that by increasing the loading of the drug to 500 mg / mmol no negative impact on the loading of the drug or stability in vivo. This ratio was adopted for use in further studies.
Table 8. Effect of drug / lipid ratio on drug loading and in vivo drug retention in TEA-Pn liposomes containing irinotecan.
<td colspan="3">Drug / lipid ratio, mg / mmol phospholipid</td><td colspan="2">Drug remaining in liposomes,% value initial injection</td>
<td>Entrance</td><td>exit</td><td>% of loading</td><td>After 8 hours</td><td>After 24 hours</td>
<td>200</td><td>208.4</td><td>104.2</td><td>54.6 ± 9.9</td><td>9.72 ± 2.23</td>
<td>300</td><td>286.3</td><td>95.4</td><td>85.2 ± 14.3</td><td>14.52 ± 2.51</td>
<td>400</td><td>348.8</td><td>87.2</td><td>81.5 ± 18.3</td><td>17.31 ± 6.14</td>
<td>500</td><td>518.9</td><td>103.8</td><td>66.8 ± 19.6</td><td>13.47 ± 1.44</td>
Example 13. Efficacy of loading CPT-11 into TEA15 SOS-containing liposomes: effect of the size of liposomes and drug retention in vivo in mice.
Liposomes containing closed solutions prepared as in Example 11, using a gradient forming solution with 0.643 N TEA-SOS pH 5.7 and osmolality 530 mmol / kg. The lipid dispersion was extruded ten times through two stacked polycarbonate filters with pores of 50 nm, 80 nm and 100 nm. The lipid-lipid matrix also includes [<sup>3</sup>H] -CHE 1., 5 mCi / mmol of foffoolipiprophate. Dimensions of:
liposomes were determined by dynamic light scattering. The liposomes were loaded with CPT-11 at the initial drug to phospholipid ratios of approximately 550 mg irinotecan / mmol phospholipid. Drug loaded liposomes were measured by QELS and tested as described in Examples 70 and 71.
Swiss Webster mice (8-10 weeks old, average 27-30 g) were injected via the tail vein with these liposomal preparations with CPT-11 at a dose of mg / kg. Mice were sacrificed at 24 hours and blood was collected and tested for CPT11 and lipid liposome as in Example 11. The results are summarized in Table 9.
Table 9. Loading of irinotecan and retention of the drug in vivo in TEA-SOS liposomes.
<td>Pore size membranes (through which it is embossed substance), nm</td><td>Size liposomes, nm average SD</td><td>Loading the drug, mg irinotecan / mmol phospholipid</td><td>The drug that remained in mouse liposomes after 24 hours. % value initial injection</td>
<td>50</td><td>87.6 ± 28.1</td><td>579.31 ± 24.2</td><td>79.2 ± 3.8</td>
<td>80</td><td>98.5 ± 15.1</td><td>571.1 ± 69.7</td><td>82.6 ± 2.1</td>
<td>100</td><td>110.8 ± 25.2</td><td>567.7 ± 37.7</td><td>86.2 ± 2.7</td>
Unexpectedly, liposomes with a triethylammonium salt of sucrose octostearate, a non-polymeric polyanionated organic hydroxylated organic compound (sugar) provides much better (4-5 times) in vivo retention of the drug in liposomes compared to similar liposomes with a polyanionic polymer (polyphosphate).
Example 14. Pharmacokinetics of SOS-TEA liposomes in blood loaded CPT-11 in rats.
Liposomes (pore size of the membrane during 100 nm extrusion) were prepared as described in Example 12. Liposomes were administered intravenously at a dose of 10 mg.
CPT11 / kg for two nine-week-old female Sprague Dawley rats (Harlan) (body weight about 200 g) using a 10 mg CPT-11 / kg fixed dose catheter (17.6 pmol phospholipids / kg). Blood samples were collected at fixed time points and analyzed for drug content and lipid liposome as in Example 9. Data were expressed as% of injected lipid / ml of plasma and% drug retention within the liposome at each time point, function versus time plotted on the graph after the injection, the half-lives for the lipid liposome, as well as the half-lives for drug release from liposomes, all calculated for the best fit to the monoexpotent kinetic model (Figure 5). The half-life of drug release from CPT-11 loaded TEA-SOS iosomes was 56.8 hours,
Example 15. Antitumor activity of free CPT-11 and hermetized CPT-11 in TEA-Pn and liposomes containing TEA-SOS in athymic nude mice with subcutaneous human colon carcinoma (HT-29) xenografts.
The liposomes were prepared as in Example 11, using a TEA-Pn solution with 0.65 M TEA pH 6.1 and Osmolality 531 mmol / kg, or a TEA-SOS solution with 0.643 M TEA pH 5.7 and osmolality 530 mmol / kg. The extrusion includes 10 passes through two stacked polycarbonate films with a pore size of 100 nm. The obtained TEA-Pn and TEASOS liposomes were respectively 112.3 ± 15.5 nm and 120.5 ± 42.5 nM (mean ± SD of the size distribution), respectively. The liposomes were loaded with CPT-11 at a drug / phospholipid entry ratio of 500 mg / mmol. The obtained liposomes had a drug content of 465.6 ± 26.5 (93% loading efficiency) 499.9 ± 22.5 mg (100% loading efficiency) CPT-11 / mmol phospholipid for TEA-Pn and TEA SOS.
HT-29 cells were obtained from American Type Culture Collection, Rockville, MD and expanded in DMEM medium supplemented with 10% fetal bovine serum, 50 U / ml penicillin G and 50 mg / ml streptomycin sulphate at 37 ° C, 5% CO 2, according to the supplier's recommendations. NCR nu / nu homozygous athymic nude male mice (6 weeks old, weight at least 16 g) were supplied from Charles River. Mice were implanted subcutaneously in the right flank with 0.1 ml of a suspension containing 5 x 10<sup>6</sup> cells suspended in culture medium without antibiotics. Eleven days later, animals having tumors from 150 mm in size<sup>3</sup> and 350 mm<sup>3</sup> were assigned to treatment groups according to the following method. The animals were classified according to tumor size and divided into 6 groups according to declining trend (tumor size). The six treatment groups comprised of 11 animals / groups were formulated by random selection of one animal from each tumor size class, so that in each treated group all tumor sizes were equally represented. Beginning on day 13, the animals received four tail vein injections at 4-day intervals with the following preparations: 1) Control (HEPES-buffered saline at pH 6.5); 2) free CPT-11 50 mg / kg, administered as a freshly prepared solution of 5 mg / ml non-buffered physiological saline; 3) TEA-Pn liposomal CPT-11 at a dose of 25 mg / kg per shot; 4) TEA-Pn liposomal CPT-11 at 50 mg / kg for injection; 5) TEA-SOS liposomal CPT-11 at a dose of 25 mg / kg per shot; 6) TEA-SOS liposomal CPT-11 at a dose of 50 mg / kg for injection. The weight of the animals and the size of the tumor were monitored twice a week as described in Example 10. The weight of the tumor was subtracted from the weight of the animal in order to obtain the body weight of the animal. Animals were observed for 60 days after tumor implantation. When the tumors in the group reached 20% of the body weight of mice, animals in the group were killed. In some groups, total tumor regression occurred without evidence of tumor re-growth at the end of the study. The tissues of the animals at the tumor inoculation site were collected and stored for pathological analysis to examine remaining tumor cells. 6) TEA-SOS liposomal CPT-11 at a dose of 50 mg / kg for injection. The weight of the animals and the size of the tumor were monitored twice a week as described in Example 10. The weight of the tumor was subtracted from the weight of the animal in order to obtain the body weight of the animal. Animals were observed for 60 days after tumor implantation. When the tumors in the group reached 20% of the body weight of mice, animals in the group were killed. In some groups, total tumor regression occurred without evidence of tumor re-growth at the end of the study. The tissues of the animals at the tumor inoculation site were collected and stored for pathological analysis to examine remaining tumor cells. 6) TEA-SOS liposomal CPT-11 at a dose of 50 mg / kg for injection. The weight of the animals and the size of the tumor were monitored twice a week as described in Example 10. The weight of the tumor was subtracted from the weight of the animal in order to obtain the body weight of the animal. Animals were observed for 60 days after tumor implantation. When the tumors in the group reached 20% of the body weight of mice, animals in the group were killed. In some groups, total tumor regression occurred without evidence of tumor re-growth at the end of the study. The tissues of the animals at the tumor inoculation site were collected and stored for pathological analysis to examine remaining tumor cells. The weight of the tumor was subtracted from the weight of the animal in order to obtain the weight of the animal. Animals were observed for 60 days after tumor implantation. When the tumors in the group reached 20% of the body weight of mice, animals in the group were killed. In some groups, total tumor regression occurred without evidence of tumor re-growth at the end of the study. The tissues of the animals at the tumor inoculation site were collected and stored for pathological analysis to examine remaining tumor cells. The weight of the tumor was subtracted from the weight of the animal in order to obtain the weight of the animal. Animals were observed for 60 days after tumor implantation. When the tumors in the group reached 20% of the body weight of mice, animals in the group were killed. In some groups, total tumor regression occurred without evidence of tumor re-growth at the end of the study. The tissues of the animals at the tumor inoculation site were collected and stored for pathological analysis to examine remaining tumor cells.
The results of this study are shown in Figures 6 and 7. Free CPT-11 had only a minor effect on tumor growth. All liposomes had a pronounced effect causing regression of the tumor, however, later in the period the tumor regrowth occurred in most animals. A dose of 50 mg / kg was more effective than 25 mg / kg for both TEA-Pn and CPT-11 liposomes with TEA-SOS. The average times of doubling tumors were calculated based on the tumor size data (Figure 7) and were: control group - 4.2 days; Free drug group, 50 mg / kg 4.8 days; Group with liposomal TEA-Pn, 25 mg / kg - 43.6 days; Group with liposomal TEA-Pn, 50 mg / kg - 47.5 days; Liposomal TEA-SOS at a dose of 25 mg / kg - 48.2 days, and the liposomal drug TEA-SOS at a dose of 50 mg / kg - over 56 days (the doubling time has not been reached). Thus, CPT-11 liposomes prepared according to the present invention were at least 10-fold more active than the free drug, due to the dose itself and the schedule of administration. Surprisingly, the CPT-11 liposomes from TEA-SOS were clearly more effective at reducing tumor growth than the liposomes of CPT-11 with TEA-Pn administered at the same dose. Whereas in the groups treated with the free drug and the TES-Pn liposomal drug at a dose of 50 mg / kg per injection, there were no animals without re-growth of the tumor, in the groups receiving a dose of 25 mg / kg of each liposome preparation, one animal (9.1%) was tumor-free at the end of the study, and in the 50 mg / kg treated group of CPT-11 with TEA-SOS at the end of the study 4 animals (36.4%) were tumor free with no signs of tumor regrowth. due to the dose itself and the schedule of administration. Surprisingly, the CPT-11 liposomes from TEA-SOS were clearly more effective at reducing tumor growth than the liposomes of CPT-11 with TEA-Pn administered at the same dose. Whereas in the groups treated with the free drug and the TES-Pn liposomal drug at a dose of 50 mg / kg per injection, there were no animals without re-growth of the tumor, in the groups receiving a dose of 25 mg / kg of each liposome preparation, one animal (9.1%) was tumor-free at the end of the study, and in the 50 mg / kg treated group of CPT-11 with TEA-SOS at the end of the study 4 animals (36.4%) were tumor free with no signs of tumor regrowth. due to the dose itself and the schedule of administration. Surprisingly, the CPT-11 liposomes from TEA-SOS were clearly more effective at reducing tumor growth than the liposomes of CPT-11 with TEA-Pn administered at the same dose. Whereas in the groups treated with the free drug and the TES-Pn liposomal drug at a dose of 50 mg / kg per injection, there were no animals without re-growth of the tumor, in the groups receiving a dose of 25 mg / kg of each liposome preparation, one animal (9.1%) was tumor-free at the end of the study, and in the 50 mg / kg treated group of CPT-11 with TEA-SOS at the end of the study 4 animals (36.4%) were tumor free with no signs of tumor regrowth.
The drug has some toxicity. Animals receiving free CPT-11, but not liposomal CPT-11, experienced a temporary disease state (loss of alertness, humped posture, matted fur, decreased mobility) for about one hour after drug administration. Animals receiving free CPT-11 suffered a permanent loss of about 6% of body weight during treatment and were no longer recovered, animals receiving both CPT-11 liposomal preparations experienced a transient decrease in body weight between the second and third injection, on average about 5% (at dose 25 mg / kg) and about 9% (at 50 mg / kg) of pre-treatment values and finally reached normal weight. Accordingly, the toxicity of the liposomal drug was no more than a free (non-liposomal) drug, and the efficacy of the liposomal drug was substantially higher. The loss of weight was restored to the initial state when drug treatment was terminated and all animals regained weight without fatal disease or poisoning. Then, the animals gained weight simultaneously with tumor regression. In the control group treated with saline in water, animals that developed large tumors evidently experienced weight loss due to cancer-related complications. In general, a liposomal drug formulation that was loaded into liposomes containing pre-closed polyanionic sugar (sucrose octasulfate) was found to be the most effective while having less toxicity than the non-liposomal drug. the animals gained weight simultaneously with tumor regression. In the control group treated with saline in water, animals that developed large tumors evidently experienced weight loss due to cancer-related complications. In general, a liposomal drug formulation that was loaded into liposomes containing pre-closed polyanionic sugar (sucrose octasulfate) was found to be the most effective while having less toxicity than the non-liposomal drug. the animals gained weight simultaneously with tumor regression. In the control group treated with saline in water, animals that developed large tumors evidently experienced weight loss due to cancer-related complications. In general, a liposomal drug formulation that was loaded into liposomes containing pre-closed polyanionic sugar (sucrose octasulfate) was found to be the most effective while having less toxicity than the non-liposomal drug.
Example 16. Toxicity of free and liposomal CPT-11 in mice.
The high toxicity of free CPT-11 and liposomes to encapsulated CPT-11 prepared according to the present invention were compared by determining the maximum tolerated dose (MTD) after a single administration of a normal intravenous (immunocompetent) injection mice.
The following materials were used:
1) Preparation of CPT-11 (hydrochloride and prothotan) containing irinotecan hydrochloride 98.9% by HPLC and at a humidity of 7.6%. In this study, the drug preparations were made on the "as is" basis, without the correction of humidity or irinotecan base content.
2) Example 11 (cs-CPT-11) was prepared (ak (ak in 11 using a DSPC lipid matrix of 200 parts by mol, cholesterol 133 parts by mole, PEGDSPE 1 part mole; sealed TEA-SOS solutions having 0.65 M TEA, pH 6.4, drug loaded into liposomes in 5 mM HEPES buffer, 5% dextrose, pH 6.5 at 60 ° C for 30 min in the entry drug / lipid ratio 500 mg drug / mmol phospholipid The loading efficiency was> 99% Liposome size (mean volume ± standard deviation using QELS): 101 ± 37 nm Liposomes were formulated in the substance, 20 mM HEPES-Na, 135 mM NaCl, pH 6 The concentration of the drug in the injectables was as indicated in the tables below.
3) rootwith with free CF'T-1 ^ 1 ^. Free sublingual solution and desalination (desirably made by dissolving irinotecan hydrochloride in a 5% dextrose aqueous solution at 22 mg / ml and sterilized by 0.2 pm filtration. This solution was diluted with 5% sterile dextrose prior to injection.
4) Animals. Female mice, Swiis Websser, 6-8 (ygodni życia, podhodziły from Harlan, USA.
The determination of the MTD is essentially the result of the protocol adopted by
United States National Cancer Institute Developmental Therapeutics Program. The protocol included the following three stages:
Stage 1): Scope of searching for a stage with a dose escalation factor of 1.8. Groups of two animals injected in the tail vein with an increased dose of free or liposomal irinotecan starting at a dose of 60 mg / kg and continued with a dose escalation rate of up to 1.8 to early mortality or lethal mortality (up to> 1 day after injection) were observed in each animal. In one of the steps below, the lethal / lethal dose was recorded.
Step 2): Scope of searching for a stage with a dose escalation factor of 1.15. Groups of two animals injected in the tail vein with an increased dose of free or liposomal irinotecan starting from the dose in Example 1 and continued with a dose escalation rate of up to 1.15 until early mortality or lethal mortality (up to> 1 day after injection) were observed in each animal. In one of the steps below, the lethal / lethal dose was recorded in the initial MTD.
Step 3): Verification stage. A group of 5 animals was injected intravenously. (tail vein) irinotecan in free or liposomal form in the initial MTD determined in Step 2.
The animals were observed for 7 days, the body weight of the animal is recorded twice a week and compared to the weight before injection. The general health of animals was observed (vigilance, care, feeding, droppings, skin, fur and mucous membrane condition, walking, breathing, posture). If there was no mortality, progressive morbidity or weight loss greater than 15% in relation to pre-injection body weight during the follow-up period, the dose was considered normal as an acute single MTD injection. If any of these symptoms occur, the experiment is repeated at the next lower dose with a factor of 1.15.
To obtain additional statistics for the verification phase, the dynamics of body weight of surviving animals occurred for up to 11 days after the injection. A dose greater than 324 mg / kg of liposomal irinotecan could be administered due to limitations in concentration and injection volumes. The results are shown in Table 10.
Table 10. A prospective study of MTD of CPT-11 formulations in mice.
RESULTS
<td colspan="2">Stage 1 increase of the dose by a factor of 1.8</td>
<td colspan="2">Body weight of the animal on the day after the injection</td>
<td colspan="2">drug injection concentration Volume of the mouse 0 1 2 (g) 4 (g) 5 6 (g) 7 11 (g)</td>
<td>Dose</td><td>injection medicine no. (g) (g) (g) (g)</td>
<td colspan="2">(mg / kg) (mg / ml) (μΙ)</td>
<td>Ls- 60</td><td>8 150 1 19.2 18.0 None 20.3 20.6 20.6 20.0 19.7</td>
<td>CPT11</td><td>2 19.7 19.3 data 20.6 20.4 19.6 19.7 20.7</td>
<td></td><td>lack</td>
<td></td><td>data</td>
<td>100</td><td>12 165 1 19.5 18.6 None 19.6 20.0 20.1 19.4 19.9</td>
<td></td><td>2 20.1 18.9 data 20.2 21.5 22.2 21.8 22.5</td>
<td></td><td>lack</td>
<td></td><td>data</td>
<td>180</td><td>22 165 1 19.4 18.4 None 18.9 19.7 20.5 19.5 20.5</td>
<td></td><td>2 20.0 19.3 data 19.6 20.6 21.4 21.6 21.7</td>
<td></td><td>lack</td>
<td></td><td>data</td>
<td>324</td><td>30.6 210 1 21.8 21.2 21.2 none 20.2 None 20.2 None</td>
<td></td><td>2 21.6 20.4 21.3 data 20.8 data 21.4 data</td>
<td></td><td>lack none missing</td>
<td></td><td>data data</td>
<td>free 60</td><td>8 150 1 20.6 20.4 none 22.1 22.1 22.2 22.0 22.5</td>
<td>CPT11</td><td>2 19.5 19.1 data 20.1 20.3 20.4 20.5 21.1</td>
<td></td><td>lack</td>
<td></td><td>data</td>
<td>100</td><td>12 165 1 19.3 death after 1-2 minutes after injection</td>
<td></td><td>2 20.1 death after 1-2 minutes of injection</td>
<td></td><td>3 19.9 death after 1-2 minutes of injection</td>
After injection, all mice treated with free CPT11 were sick, had shallow breath for about 1 hour, and then returned to their previous state. After injection, all mice treated with Ls-CPT11 showed no change.
Stage 2 increase the dose by a factor of 1.15
Body weight of the animal on the day after the injection
<td>bow</td><td>injection Dose (Mg / kg)</td><td>concentration drug (Mg / ml)</td><td>Volume injection (ΜΙ)</td>
<td>free CPT11</td><td>60</td><td>8</td><td>150</td>
<td></td><td>70</td><td>8</td><td>175</td>
<td></td><td>80</td><td>8</td><td>200</td>
<td></td><td>90</td><td>12</td><td>150</td>
<td></td><td></td><td>8</td><td>225</td>
<td>mouse No.</td><td>0 (g)</td><td>1 (g)</td><td>2 (g)</td><td>5 (g)</td><td>7 (g)</td>
<td>3</td><td>19.9</td><td>20.0</td><td>20.9</td><td>19.9</td><td>21.3</td>
<td>4</td><td>19.5</td><td>18.7</td><td>19.4</td><td>18.8</td><td>18.9</td>
<td>5</td><td>20.9</td><td>20.0</td><td>20.6</td><td>19.3</td><td>20.4</td>
<td>6</td><td>22.3</td><td>21.8</td><td>22.4</td><td>22.4</td><td>22.8</td>
<td>7</td><td>20.6</td><td>19.9</td><td>20.1</td><td>19.9</td><td>20.9</td>
<td>8</td><td>20.6</td><td>20.8</td><td>21.1</td><td>20.7</td><td>21.4</td>
death after 1-2 minutes after injection death after 1-2 minutes after injection death after 1-2 minutes after injection
Stage 3 Correctness
Body weight of the animal on the day after the injection
<td>bow</td><td>injection Dose (Mg / kg)</td><td>concentration drug (Mg / ml)</td><td>Volume injection (ΜΙ)</td><td>mouse no</td><td>0 (g)</td><td>3 (g)</td><td>5 (g)</td><td>7 (g)</td>
<td>free CPT11</td><td>80</td><td>8</td><td>200</td><td>1</td><td>20.2</td><td>19.3</td><td>20.0</td><td>21.7</td>
<td></td><td></td><td></td><td></td><td>2</td><td>20.5</td><td>20.6</td><td>20.5</td><td>21.2</td>
<td></td><td></td><td></td><td></td><td>3</td><td>20.7</td><td>20.6</td><td>20.8</td><td>21.9</td>
<td></td><td></td><td></td><td></td><td>4</td><td>20.8</td><td>21.4</td><td>22.1</td><td>23.0</td>
<td colspan="4"></td><td>5</td><td>21.9</td><td>21.9</td><td>21.6</td><td>21.5</td>
<td>Ls-CPT11</td><td>324</td><td>365</td><td>180</td><td>6</td><td>21.0</td><td>20.0</td><td>20.1</td><td>20.2</td>
<td></td><td></td><td></td><td></td><td>7</td><td>20.4</td><td>20.4</td><td>20.2</td><td>19.2</td>
<td></td><td></td><td></td><td></td><td>8</td><td>20.4</td><td>19.8</td><td>20.3</td><td>20.7</td>
<td></td><td></td><td></td><td></td><td>9</td><td>20.9</td><td>19.9</td><td>20.5</td><td>21.5</td>
<td></td><td></td><td></td><td></td><td>10</td><td>20.7</td><td>19.5</td><td>19.8</td><td>20.2</td>
Thus, when the MTD of free CPT-11 was 80 mg / kg, the MTD of the liposomal CPT11, unexpectedly, was not reached, even at the highest administered doses of 324 mg / kg. Accordingly, encapsulation of the CPT-11 liposome according to the present invention has reduced drug toxicity of at least 4-fold.
Example 17. Stability of storage of TEASOS loaded CPT-11 liposomes against drug leakage.
Five batches of liposomal CPT-11 were made using the TEA-SOS method (Example 11), at the input drug / lipid ratio 500-550 mg / mmol phospholipid. Liposomes were prepared by membrane extrusion through a polycarbonate membrane with a pore size of 80 nm or 100 nm as indicated in the table below. The liposomes were passed through a sterile 0.2 filter and stored at 3.4-14.5 mg / ml CPT-11 in 135 mM NaCl, 20 mM HEPES-Na pH 6.5 (storage buffer) at 4- 8 ° C. After the indicated storage time, the leaked drug was removed by gel chromatography on a Sephadex G-75 column using buffer for storage as an eluent.
Concentrations of drug and phospholipids in liposomes before and after gel permeation chromatography were determined by spectrophotometry and using the acid blue phosphomolybdate digestion method, respectively, as described in examples 70 and 71. The CPT-11 liposomes prepared according to the present invention were very solid. The CPT-11 leak of iiposomes during storage was less than 5% in 6 months (Table 10).
Table 11. Stability of encapsulation of CPT-11 liposomes during storage (data are mean ± SE).
<td>Party</td><td>Pore size</td><td>The concentration of CPT-11,</td><td>Time</td><td>% residue</td>
<td>liposome nr</td><td>(through which he is extruded substance), nm</td><td>mg / ml</td><td>storage, months</td><td>hermetic drug</td>
<td>1</td><td>80</td><td>3.44 ± 0.06</td><td>6</td><td>99.02 ± 3.77</td>
<td>2</td><td>80</td><td>7.88 ± 0.19</td><td>6</td><td>102.38 ± 4.78</td>
<td>3</td><td>100</td><td>4.57 ± 0.06</td><td>6</td><td>96.38 ± 4.69</td>
<td>4</td><td>100</td><td>4.62 ± 0.11</td><td>6</td><td>95.72 ± 4.36</td>
<td>5</td><td>80</td><td>14.52 ± 0.42</td><td>3</td><td>103.4 ± 5.92</td>
Example 18. Liposomes loaded with Topotecan.
The TEA-Pn solution and the TEA-SOS solution encapsulated in liposomes were prepared as in Example 11. The stock solution of Topotecan hydrochloride (GlaxoSmithKline, PA, USA) was prepared immediately before mixing with the liposomes by dissolving the topotecan hydrochloride in water at 15-20 mg / ml. counting from the actual Topotecan HCl content. The pH was adjusted to 3.0 with 1 N HCl. The drug solution was filtered through a sterile 0.2 micron polyethersulfone (PES) filter with overpressure. Portions of liposomes containing TEA-Pn or TEA-SOS in drug with loading buffer were mixed at room temperature with a substituted HCL Topotecan solution to achieve the drug / lipid entry ratio in the range of 0.15-0.45 g / mmol phospholipid liposome. A preferred ratio was 0.35 g of HCL Topotecan per mmol of phospholipid liposome. The mixtures in glass containers were incubated in a thermostatic water bath at 5562 ° C with slow stirring for 30-60 minutes, then rapidly cooled in a water bath with ice (0-2 ° C) and left at temperature for 5-15 min. This step led to an encapsulation efficiency of 89-90% (TEA-Pn gradient) or 97-100% (TEA-SOS gradient). The undersized Topotecan was removed and the liposomes were transferred to the storage buffer by size exclusion chromatography. Prior to use on the column, the ionic strength of the liposome formulation was increased by mixing with 1/20 volume then quickly cooled in a water bath with ice (0-2 ° C) and left at temperature for 5-15 min. This step led to an encapsulation efficiency of 89-90% (TEA-Pn gradient) or 97-100% (TEA-SOS gradient). The undersized Topotecan was removed and the liposomes were transferred to the storage buffer by size exclusion chromatography. Prior to use on the column, the ionic strength of the liposome formulation was increased by mixing with 1/20 volume then quickly cooled in a water bath with ice (0-2 ° C) and left at temperature for 5-15 min. This step led to an encapsulation efficiency of 89-90% (TEA-Pn gradient) or 97-100% (TEA-SOS gradient). The undersized Topotecan was removed and the liposomes were transferred to the storage buffer by size exclusion chromatography. Prior to use on the column, the ionic strength of the liposome formulation was increased by mixing with 1/20 volume
2.88 M sodium chloride, then the mixture was incubated for about 15 minutes. Surprisingly, it was found that adjusting the ionic strength of the liposome substrate from a low value during loading (typically equivalent to less than 20 mM NaCl) to a higher value above 20 mM NaCl, preferably up to 50 mM NaCl and above, improved the deprivation of nonhermeredic drug and increased the stability of Topotecan. encapsulated in liposomes prior to aggregation, it is possible that by facilitating the removal of membrane-bound Topotecan, in contrast to drugs in encapsulated in the interior of liposomes. The rest of the procedure is described in example 11, step 7. For the results, see table 12 below.
Example 19. Preparation of anti-HER2-immunoliposomal preparations
Topotecan.
Immunoliposomes Topotecan specially assimilated by HER2-overexpressing HER2-surface receptor tyrosine kinase (C-ErbB-2) onkoprotein were prepared by coupling the Topotecan liposomes to anti-HER2 with a single-chain fragment of human Fv, F5 antibody selected from the phage library due to high assimilation in HER2 overexpressing cells (Poul, et al., 2000, J. Molecular Biology, pp. 301, pp. 1491-1161). F5 is a 27 kDa protein that binds to the extracellular region of the HER2 receptor with a colummation of about 150 nm, resulting in rapid bioavailability (Neve, et al., 2001, Biophys. Biochim Res. Commun., 280, p.274- 279). For liposome coupling, methods of US Patent No. 6,210,707 and Nielsen, et al. (2002), Biochim. Biophys. Acta, 1591, p.109118, they were basically adhered to. A hydrophilic lipopolymer coupled to F5 was first made. The end of the F5 amino acid carbon chain had an additional terminal cysteine group (F5Cys). The F5Cys structure was expressed in E. coli and isolated from the bacterial lysate by Protein A using column chromatography. Eluted Protein A fractions were adsorbed on an anion exchange resin to remove pyrogens and host DNA and thiol-treated reducing agent to release the thiol group of the final cysteine. The decreased F5Cys was further purified by ion-exchange chromatography using SP Sepharose Fast Flow (Amersham Pharmacia). The purified protein has been coupled with a thiol reacting with a lipid-poly (ethylene glycol) linkage, N- (3- (Nmaleimido) propionylamido) -poly (oxyethylene) oxycarbonyl) -1, 2-distearoylphosphatidylethanolamine (MAL-PEG-DSPE), a PEG derivative with molecular weight 2000 (Figure 4.1), commercially produced by Avanti Polar Lipids Inc., Alabama, USA. Proteins and linker were incubated in buffered aqueous solution at a molar ratio of 1: 4, and unreacted linker was quenched with 1 mM cysteine. During the reaction, the final cysteine F5Cys is attached by a covalent bond to the maleimide group of the linker. The obtained F5-PEG-DSPE conjugate was soluble in water in the form of micelles having high molecular weight (500-850 kDa) and was separated from the unreacted protein (about 25%) by size exclusion chromatography. The amount of protein in the purified conjugate was determined by UV spectrophotometry at 280 nm and the amount of connector was determined using a spectrophotometric method, identical to the phospholipid used for the quantitative approach (see example 70). The purified F5-PEG-DSPE conjugate was stable in water, fully immunoreactive and stable to denaturation and loss of reactivity, for at least 1 hour at 65 ° C and at least 3 months at 37 ° C.
To prepare anti-HER2 immunoliposomal Topotecan, liposomes loaded
The topotecan of Example 18 was mixed with F5-PEG-DSPE in buffered saline at a ratio of 15 micrograms protein to 1 micromole phospholipid (approximately 45 duplicates
F5 on iiposom). The mixture was incubated for 40 min. at 60 ° C, cooled on ice and subjected to column chromatography with Sepharose CL-4B (cross-linked 4% agarose granulate, Amersham Pharmacia) to remove micellar remains of the conjugate, unconjugated protein and traces of the extraliposomal drug that could be released during incubation. F5-PEG-DSPE loaded liposomes were eluted with 5 mM HEPES buffered,
144 mM NaCl, pH 7.4, then collected in an empty column volume, sterile filtered and separated for storage (4-6 ° C). The amount of Fos loaded liposomes was typically> 80% of the added conjugate. It was determined by SDS-PAGE with liposomes with a quantitative Coomassie-stained dye F5 by densitometry. The concentration of drug and lipids in immunoliposome preparations has been determined in a similar way to other non-targeted liposomes. The properties of the Topotecan liposome and F5 immunoliposomes (examples 18-19) are summarized in Table 12.
Table 12. Characterization of Topotecan liposome and immunoliposomes.
<td>Salt closed in the liposome</td><td>addition F5 scFv:</td><td colspan="2">Drug / lipid ratio, g / mol phospholipid</td><td>% hermetization</td><td>Size liposomes, mean ± SD, nm</td>
<td></td><td></td><td>Entrance</td><td>exit</td><td></td><td></td>
<td>TEA-Pn</td><td>No</td><td>173.6</td><td>155.2 ± 5.9</td><td>89.4 ± 3.4%</td><td>96.4 ± 38.7</td>
<td>TEA-Pn</td><td>Yes</td><td>173.6</td><td>156.2 ± 5.2</td><td>90.0 ± 3.0%</td><td>96.2 ± 33.8</td>
<td>TEA-SOS</td><td>No</td><td>347.2</td><td>340.8 ± 14.7</td><td>98.2 ± 4.2%</td><td>99.1 ± 32.6</td>
Example 20. Effect of pH loading buffer and drug / lipid ratio in Topotecan loading to liposomes.
Liposomes (DSPC / Chol / PEG-DSPE, molar ratio 3: 2: 0.015) with closed 0.5N TEA-Pn, pH 6.2, osmolality 413 mmol / kg, were prepared using the ethanol injection method 18 d), then were extruded 5 times from two stacked polycarbonate filters with a pore size of 100 nm and 10 times with a pore size of 50 nm. The loading buffer was 5 mM MES, 50 g / L dextrose and was adjusted to various pH values in the range 5.0-6.5. The size of the liposomes was 73.1 ±
21.3 nm and measured using QELS. Liposomes were loaded by mixing the Topotecan stock solution (20 mg / ml) with liposomes in the loading buffer at the input drug to phospholipid ratio of 100 mg / mmol, incubating the mixture at 60 ° C for 45 minutes, quenching on ice for 15 minutes and removing non-humidified drug using a Sephadex G-75 column eluting with 20 mM HEPES, 135 mM
NaCl at pH 6.5. Topotecan and phospholipids were quantified by spectrophotometry (Examples 70 and 71). The results (Table 13) showed that the Topotecan loading was almost quantitative in the pH range 5.5-6.5.
Table 13. Effect of pH loading buffer on% encapsulation of Topotecan in iiposomes with closed TEA-Pn.
<td>Loading buffer pH</td><td>% hermetization</td>
<td>5.0</td><td>50.1 ± 2.1</td>
<td>5.5</td><td>97.2 ± 8.1</td>
<td>6.0</td><td>115.5 ± 15.0</td>
<td>6.5</td><td>102.1 ± 8.1</td>
The effect of drug to lipid ratio (0.15-0.45 mg / mmol phospholipid) on the loading efficiency was also investigated. Closed TEA-Pn liposomes (0.5 M TEA, pH 5.8, 480 mmol / kg osmolality) were prepared as described above except for the last ten-fold extrusion step through two stacked polycarbonate filters with a pore size of 0 , 08 pm. The loading took place at a pH of 6.5. The size of the liposomes was 93.1 ± 15.1 nm and was measured using QELS. The results (Table 14) showed that the loading efficiency of the drug is above 85% in the whole range of studies on drug / lipid ratios.
Table 14. Effect of the drug / lipid ratio on the efficacy of encapsulation of Topotecan in TEA-Pn-containing liposomes.
<td colspan="2">Topotecan / phospholipid ratio, mg / mmol</td><td rowspan="2">% hermetization (Mean ± SE)</td>
<td>Acceptance ratio</td><td>The ratio of efficiency (after loading)</td>
<td>168.2</td><td>166.9 ± 11.1</td><td>99.2 ± 6.6</td>
<td>224.4</td><td>232.5 ± 47.6</td><td>103.7 ± 21.2</td>
<td>280.3</td><td>253.5 ± 19.8</td><td>90.4 7.0</td>
336.4 298.3 ± 18.0 88.7 ± 5.3
392.5 361.2 ± 36.8 92.0 ± 9.4
448.5 394.9 ± 29.5 88.0 ± 6.6
Example 21. Stability of Topotecan liposomes in vitro in the presence of plasma.
Liposomes (DSPC / Chol / PEG-DSPE, molar ratio 3: 2: 0.015), with a closed 0.5 NPn TEA, pH 6.2, osmolality 413 mmol / kg, were prepared as described in Example 18. Liposomes 96.4 ± 29.3 nm, were prepared by extruding ten times through two stacked polycarbonate filters with a pore size of 100 nm. For the quantification of lipid liposomes in plasma, it has been included [<sup>3</sup>H] -CHE in a lipid solution with a concentration of 0.5 pCi / pmol DSPC. The topotecan was loaded at pH 6.0 at 58 ° C for 45 minutes in a drug / phospholipid ratio of 150 mg / mmol. The loading efficiency was 148.48 ± 10.26 pg Topotecan / pmol phospholipid (99.0 ± 6.8%).
Liposomes were incubated with 50% human plasma in a multiwell microdialysis device (Spectra-Por MicroDialyzer 10-well, Spectrum, USA). The human donor plasma was diluted in an equal volume of HEPES buffered saline (20 mM HEPES, 135 mM NaCl), pH 6.5 containing 0.02% sodium azide and added to the bottom dialyzer reservoir (32 ml). Wells (0.4 ml) were separated from the reservoir by means of a poly-carbonate membrane with pores of 30 nm, resulting in a free flow of plasma proteins and small molecules, but not liposomes. The liposomes were mixed with the calculated amounts of plasma and HEPES-buffered saline until a concentration of 2.5 mM phospholipid and 50% plasma volume was obtained. The device was incubated at 37 ° C and the contents of the tank were mixed slowly. After 8 hours of incubation, the content of the lower reservoir was changed to fresh 50% of the plasma. At the indicated time points (see below), 50 μΙ aliquots were taken from wells and subjected to column chromatography containing 2.2-2.4 ml Sepharose CL-2B, eluent of HEPES buffered saline solution in water to separate liposomes from plasma proteins and free the drug. Liposomes were collected in fractions of empty space. Topotecan was quantified by fluorometry using 384 nm excitation and 524 nm emission after dissolving the plasma samples in a 90% aqueous solution of isopropanol with 0.1 N HCl, and the lipids were quantified by scintillation counting [ 50 μΙ aliquots were taken from wells and subjected to column chromatography containing 2.2-2.4 ml Sepharose CL-2B, eluent of HEPES buffered saline solution in water to separate liposomes from plasma proteins and free drug. Liposomes were collected in fractions of empty space. Topotecan was quantified by fluorometry using 384 nm excitation and 524 nm emission after dissolving the plasma samples in a 90% aqueous solution of isopropanol with 0.1 N HCl, and the lipids were quantified by scintillation counting [ 50 μΙ aliquots were taken from wells and subjected to column chromatography containing 2.2-2.4 ml Sepharose CL-2B, eluent of HEPES buffered saline solution in water to separate liposomes from plasma proteins and free drug. Liposomes were collected in fractions of empty space. Topotecan was quantified by fluorometry using 384 nm excitation and 524 nm emission after dissolving the plasma samples in a 90% aqueous solution of isopropanol with 0.1 N HCl, and the lipids were quantified by scintillation counting [<sup>3</sup>H] -CHE (toughened hardening). The specific ratio of drug to lipid over time was compared to the initial ratio before incubation to obtain% Topotecan, which remained closed at each time point. After 8 hours of incubation, the amount of drug remaining in liposomes was about 55% of the initial value (Table 15).
Table 15. In vitro release of Topotecan from liposomes loaded with a TEA-Pn gradient in 50% human plasma at 37 ° C.
<td>Incubation time, hours</td><td>% residual hermetic drug</td>
<td>1</td><td>95.5 ± 5.4</td>
<td>4</td><td>76.8 ± 7.3</td>
<td>8</td><td>55.9 ± 4.1</td>
<td>24</td><td>55.4 ± 16.8</td>
Example 22. Topotecan liposomes with a closed TEA-Pn gradient in different drug / lipid ratios: Retention and viability of the in vivo drug circulation in mice.
Liposomes (DSPC / Chol / PEG-DSPE in a molar ratio of 3: 2: 0.015 containing [<sup>3</sup>H] -CHE 0.5 mCi / mmol DSPC) with encapsulated gradient salt solutions (0.5
N TEA-Pn at pH 6.2, osmolality 413 mmol / kg) were prepared as in Example 18 by 12-fold extrusion through two stacked polycarbonate filters with a pore size of 100 nm. The size of the liposomes was 107.7 ± 19.1 nm and was measured using QELS. Liposomes in 5 mM HEPES, 50 g / L dextrose at pH 6.5, were mixed with a basic aqueous solution of Topotecan (20 mg / mL) at drug / phospholipid ratios in the range of 130-360 pg / pmol, followed by incubation of the mixture at 58 ° C for 45 minutes, then placed on ice for 15 minutes and the non-aerosolized drug was removed by chromatography on a Sephadex G-75 column. Twelve week old female FvB mice were injected with liposomes via the tail vein at a dose of 5 mg Topotecan per kg body weight (about 0.2 mg Topotecan / animal) in triplicate. In the times indicated, Typically for 8 hours or 24 hours after injection, the mice were anesthetized, exsanguinated and blood samples were tested for drug and lipid liposome as in Example 8. The results are shown in Table 16. After 24 hours, about 6-32% of the initial drug loading it remains encapsulated. Higher drug loads (> 200 mg / mmol phospholipids) resulted in longer retention of drugs.
Table 16. In vivo, retention and viability in the circulation of a prototype drug loaded with Topotecan liposomes by the TEA-Pn gradient method for various drug / lipid ratios.
Ratio of encapsulated. Remaining lipid in circulation,%. Remaining encapsulated
<td rowspan="2">drug / phospholipid, mg / mmol</td><td colspan="2">injected dose</td><td colspan="2">Topotecan, initial% loading</td>
<td>After 8 hours</td><td>After 24 hours</td><td>After 8 hours</td><td>After 24 hours</td>
<td>127.2 ± 10.9</td><td>36.1 ± 2.0</td><td>18.7 ± 8.1</td><td>51.7 ± 7.1</td><td>6.72 ± 2.5</td>
<td>207.2 ± 21.6</td><td>32.1 ± 5.2</td><td>9.84 ± 1.88</td><td>75.6 ± 13.0</td><td>13.8 ± 3.5</td>
<td>301.3 ± 24.5</td><td>34.4 ± 3.2</td><td>8.04 ± 4.25</td><td>79.2 ± 4.2</td><td>25.6 ± 4.4</td>
<td>360.3 ± 35.6</td><td>33.6 ± 2.4</td><td>8.68 ± 4.96</td><td>73.5 ± 7.0</td><td>32.3 ± 9.8</td>
Example 23. In vivo, drug retention and viability in the circulation of Topotecan loaded liposomes using various closed ammonium and triethylammonium salts.
Liposomes consisting of DSPE, cholesterol, PEG-DSPE (3: 1: 0.1 by weight), and containing [<sup>3</sup>H] -CHE at 0.22 mCi / mmol DSPE, were prepared as in Example 18, except that the extrusion step contained 10 passes through 2 stacked filters with 200 nm pore size, 10 passes through 2 stacked filter pore size 100-nm and 20 passes through 2 stacked filters with a pore size of 50 nm. Liposomes contained the following salt solutions:
A 0.5 N dextran sulphate ammonium solution (A-DS) was prepared from dextran sulfate sodium (MW 5,000) from Sigma and converted to an ammonium salt according to an ion-exchange procedure similar to the method of Example 4. A solution of sulfuric acid with dextran immediately titrated in a 12.4 M aqueous solution of ammonia. The A-DS solution had a pH value of 55 and (56 and an osmolality of 208 mmol / kg.
0.48 N of sucrose ammonium octasulphate (A-SOS) was prepared as in Example 6, but ammonium hydroxide was used for the titration. The solution had a pH of 6.27 and an osmolality of 258 mmol / kg.
0.47 M triethylammonium sucrose octachulfate (TEA-SOS) was prepared as in Example 6. The solution had a pH of 6.6 and an osmolality of 297 mmol / kg.
Topotecan was loaded into liposomes in an aqueous solution of 10 mM MES25 na, 50 g / liter dextrose, pH 6.5, by incubating the liposomes with the drug at 61-62 ° C, and the drug / phospholipid entry ratio was 346 ± 1 mg / mmol. for 40 minutes and then incubating on ice for 10 minutes. The liposomes were purified from the non-aerosolized drug by chromatography on a Sephadex G-25 column, eluent-aqueous solution of 2 mM histidine, 144 mM NaCl, pH 6.6 (HCl).
Female Swiss Webster mice from seven to nine weeks of age 5 were injected via the tail vein with 5mg Topotecan liposomal preparations
Topotecan per kg body weight (about 0.2 mg topotecan / animal) in triplicate. After 8 or 24 hours after the injection, the blood was collected and tested for Topotecan and lipid liposome as in Example 22.
The results are shown in Table 17 below. While all three liposomal preparations showed circulating stability very close to liposomes, having approximately 2328% of the injected dose remaining in the blood 24 hours after injection, unexpectedly the drug retention in TEA-SOS and A-SOS liposomes was better than in A-DS liposomes. both in size (approximately 2-fold increase in drug retention) and statistical significance (significance at the confidence level and 95% by the 2-sided paired Student's t test for p = 0.0257 and p = 0.00995, respectively; and test U Mann, where the difference was significant in the variable α = 0.01). The retention of the drug in TEA-SOS containing Topotecan liposomes was better than in A-SOS containing Topotecan liposomes.
Table 17. In vivo drug retention and maintenance of iiposomes
Topotecan prepared using TEA-SOS, ammonium SOS (A-SOS), ammonium dextran sulfate (A-DS).
Gradient Ratio Efficiency Amount remaining in Remaining drug / phospholipid loading,% iiposome, circulation,% hermetic, mg / mmol nm of initial dose of Topotecan,% initial loading
After 8 After 24 After 8 After 24 hours hours Hours
<td>A-DS</td><td>288.1 ± 20.6</td><td>83.3 ± 6.0</td><td>76.9 ± 22.7 43.7 ± 1.2</td><td>27.7 ± 1.5</td><td>43.6 ± 6.8</td><td>18.7 ± 1.5</td>
<td>A-SOS</td><td>346.2 ± 14.3</td><td>100.0 ± 4.1</td><td>99.7 ± 28.9 42.3 ± 2.2</td><td>23.4 ± 2.0</td><td>53.3 ± 0.8</td><td>31.3 ± 3.2</td>
<td>TEA-SOS</td><td>340.8 ± 14.7</td><td>98.5 ± 4.2</td><td>99.1 ± 32.6 42.1 ± 2.3</td><td>23.0 ± 2.9</td><td>57.0 ± 5.6</td><td>38.1 ± 6.1</td>
Example 24. Pharmacokinetics of drug and lipid in blood in liposomal Topotecan in rats
The parameters of viability in the circulation and release of Topotecan were evaluated in rats. Liposomes (DSPC / cholesterol / PEG-DSPE molar ratio 3: 2: 0.015) were prepared by the ethanol / extrusion mixing method and loaded with Topotecan using a TEA-Pn gradient or a sucrose octasulfate TEA gradient (TEA-SOS) as described in the example 18 and loaded in different drug / lipid ratios (15-450 mg / mmol phospholipid). For the lipid matrix in quantitative terms, the lipid liposome contained [3H] -CHE in 0.5-1.5 mCi / mmol DSPC. female Sprague Dawley rats (6-8 weeks old, body weight about 200g) with permanent central venipuncture and intravenous injection. (via a catheter) with Topotecan liposome at a dose of 4 - 5 mg / kg body weight. The catheter was rinsed with saline in water. In selected periods (up to 48 hours after injection) blood samples (0.2-0, 3 ml) were withdrawn through a catheter in heparinized syringes, mixed with 0.4 ml of cold phosphate buffered saline with 0.04% EDTA, blood cells separated by centrifugation, and supernatants (diluted PBS plasma) were examined for lipids with 3H- CHE counting of radioactivity (hardening corrected) and for Topotecan by fluorometry (example 71). The test results were corrected for plasma dilution, then calculated based on the weight of the blood sample obtained and the assumption that the hematocrit was 40%. The total dose of drug and blood lipids was assessed based on blood volume calculated as 6.5% body weight. The percentage of Topotecan retained in liposomes was calculated by comparing the drug / lipid ratio over a given period to the drug / lipid ratio in the injected liposomes. Table 18 below summarizes the half-life of lipid and drug in blood and the half-life for drug release, as well as other properties of liposomes. Pharmacokinetic curves (PK) are shown in Figures 8A (lipid) and 8B (drug / lipid ratio). In conclusion, the blood PK curves for both drug and lipid fit well with a single exponent model (R.<sup>2</sup> 0,9840,999). Despite their size of 90-100 nm, and a very small amount of PEGylated lipids (0.3 mol%), liposomes unexpectedly exhibited good circulating lifetime (the serum half-life of the lipid component ranged from 11 to 16 hours). The slowest release of Topotecan (at a partial time of 22.9 hours) was observed with loaded liposomes using the TEA-SOS method.
Table 18. Circulation half-life (t1 / 2), lipid, drug and partial time dimension for drug release from prototype Topotecan liposomes in rats.
Closed Loading. Size of salt and concentration of Topotecan, liposomes, mg / mmol nm
Injected tt / 2 ti / 2 lek, ti / 2 Number of dose, lipid, hours of animal release mg / kg eeku hour, in given
<td></td><td>phospholipid</td><td colspan="2">(Mean ± SD )</td><td colspan="3">hours</td><td>group</td>
<td>TEA-Pn 0.5 N</td><td>124.3 ± 9.7</td><td>92.3 ± 23.3</td><td>4</td><td>15.8</td><td>4.13</td><td>5.34</td><td>3</td>
<td>TEA-Pn 0.5 N</td><td>360.3 ± 35.6</td><td>107.7 ± 19.1</td><td>5</td><td>12.8</td><td>6.06</td><td>9.97</td><td>2</td>
<td>TEA-SOS 0.643N</td><td>439.2 ± 15.9</td><td>108.8 ± 13.4</td><td>5</td><td>10.8</td><td>7.36</td><td>22.87</td><td>2</td>
Example 25. Stability of the drug against leakage during storage of liposomes
topotecan
Samples of several prototype preparations prepared for the above-described studies were stored at 4-6 ° C for various times to assess the storage stability of the encapsulated Topotecan against drug leakage from the liposomes. Liposomal samples were run on Sephadex G-75 columns, eluted with 20 mM HEPES, 135 mM NaCl, pH 6.5 to remove extraliposomal drug and analyzed for drug content by spectrophotometry and lipid counters using [3 H] -CHE counting of radioactivity. The results (Table 19) indicate good retention of Topotecan in liposomes during storage.
Table 19. Drug retention in the prototype of Topotecan liposomes during storage.
<td>Gradient salt in liposome</td><td>Size liposomes, mean ± SD nm</td><td>Initial loading drug, mg drug / mmol phospholipid</td><td>Time storage, months</td><td>Loading the drug after storage as% initial</td>
<td>TEA-Pn 0.500 N pH 6.2</td><td>96.4 ± 29.3</td><td>148.5 ± 10.3</td><td>8</td><td>101.6 ± 5.5</td>
<td>TEA-Pn 0.500 N pH 6.2</td><td>107.7 ± 19.1</td><td>127.2 ± 10.9</td><td>6</td><td>94.6 ± 6.2</td>
<td>TEA-Pn 0.500 N pH 6.2</td><td>107.7 ± 19.1</td><td>207.2 ± 21.6</td><td>6</td><td>113.9 ± 9.4</td>
<td>TEA-Pn 0.500 N pH 6.2</td><td>107.7 ± 19.1</td><td>301.3 ± 24.5</td><td>6</td><td>112.9 ± 9.3</td>
<td>TEA-SOS 0.643 N pH 5.6</td><td>108.8 ± 13.4</td><td>439.2 ± 15.9</td><td>2</td><td>97.8 ± 9.4</td>
Example 26. In vitro absorption of liposomal and immunoliposomal Topotecan by tumor cells overexpressing HER2.
This study refers to the capacity of Topotecan loaded with anti-HER2immunoliposomes prepared according to the disclosure to provide Topotecan, in particular to cells overexpressing HER2 in cell culture. (immuno) liposomes were prepared and loaded with Topotecan using the TEA-Pn method of Example 19. Human HER-2 overexpressing breast cancer cells (SKBr-3, ATCC) were cultured in modified McCoy 5A (without tricine) supplemented with 10% serum. fetal calf, 50 pg / ml streptomycin sulphate and 50 U / ml penicillin G (total growth medium in culture medium) in T-75 flasks at 37 ° C, 5% CO 2 until confluent. Cells were harvested by trypsinization, implanted in 24 well cell culture plates at 150,000 cells / well at 0, 5 ml of total culture medium and allowed to acclimate them overnight. The medium was replaced with 0.5 ml of total growth medium containing Topotecan formulations at the selected concentration in the range of 0.01-0.1 mM phospholipid. Three wells were used for each condition. Control wells were incubated in the absence of drug and / or liposomes (in order to obtain background readings for drug designation). The plates were incubated with slow stirring at 37 ° C, 5% CO 2 for 4-8 hours. The media was aspirated and the cells were rinsed 4 times with 1 ml portions of a cold Hank's solution containing Ca and Mg salts. The cells were then dissolved by adding 0.1 ml of 1% Triton X-100 in water and the amount of drug in cell lysates was determined fluorometrically (example 71). A standard curve in the range of 102500 ng Topotecan / well was obtained and fitted to the second order polynomial (to include quenching itself at higher drug concentration) after subtracting the background of cell autofluorescence. When a microplate fluorimeter was used, a 400/30 nm pore size filter was used for excitation and 530/25 nm for emission. Both the cuvette and microplate fluorimeter gave the same results.
The results of the two experiments are summarized in Table 20 below. A prominent cellular absorption of the liposomal drug with targeted HER2 was seen (50-300 times higher than the undirected liposomal Topotecan). Interestingly, the absorption of free Topotecan was significantly lower than that of the targeted HER2 immunoliposomal topotecan. This can be explained by the rapid hydrolysis of the camptothecin lactone ring of the Topotecan molecule in the cell growth medium in the presence of serum, to form a carboxylate form of the drug that may have lower cell permeability and lower cytotoxicity. In summary, the ability to target and assimilate ligand-conjugated immunoliposomes, used to deliver Topotecan intracellularly, has been confirmed.
Table 20. In vitro cell uptake of Topotecan liposomes and TEA-Pn anti-HER2 immunoliposomes (ND, not determined). Characteristics of liposomes see table 12.
<td rowspan="2">Concentration liposomes, nM phospholipids</td><td rowspan="2">Concentration topotecan pg / ml</td><td rowspan="2">Time exposure hours</td><td colspan="3">Absorption of Topotecan by SK-Br-3 cells, ng / 100 000 cells</td>
<td>undirected liposomes</td><td>F5Immunoliposomy</td><td>Free drug</td>
<td>0.1</td><td>15.5</td><td>4</td><td>1.45 ± 0.09</td><td>163 ± 5.7</td><td>no data</td>
<td>0.01</td><td>1.55</td><td>4</td><td>0.185 ± 0.03</td><td>60.2 ± 2.0</td><td>no data</td>
<td>0.033</td><td>5.0</td><td>8</td><td>3.62 ± 2.03</td><td>169.6 ± 13.7</td><td>5.56 ± 0.91</td>
Example 27. Liposomal and immunoliposomal cytotoxicity of Topotecan against HER2 overexpressing tumor cells in vitro.
When the anti-HER2 capacity of Topotecan immunoiipooms for intracellular drug delivery to HER2 overexpressing tumor cells was determined (Example 26), it was important to ensure that internalized liposomes could release the drug in an active form. To this end, the cytotoxicity of free Topotecan in vitro (i.e., Topotecan prepared as a solution), liposomal Topotecan and anti-HER2-immunoliposomal Topotecan was examined. Topotecan liposomal formulations were prepared and cultured and SKBrr-3 cells were harvested as described in Example 26. Cells were transplanted into 96 well cell culture plates at 5,000 cells in 0.1 ml total culture medium, triplicate and allowed to acclimatize through the night. Most of the edges of the rows and columns of tiles remained empty. Sterile prepared Topotecan liposomes, immunoliposomes and free drug (freshly prepared by diluting a 20 mg / ml Topotecan baseline solution at pH 3 in a non-buffered saline solution in water to 2 mg / ml) were diluted with the total drug agent to obtain concentrations of 90, 30, or 10 pg / ml and serially diluted in the medium using factor 3. The well medium was replaced with 0.2 ml drug / liposome solutions and incubated at 37 ° C, 5% CO 2 for a specified time (4-6 hours) . One well in each row was incubated with a drug-free agent to serve as an untreated control. The medication containing the medium was aspirated from the wells, the cells were rinsed with 0.2 ml of drug-free a 0, 2 ml of fresh drug-free medium was added to all wells. The plates were incubated for 4 days at 37 ° C,
5% CO2. Without changing the agent, 0.03 ml of a 2 mg / ml solution of tetrazolium dye (thiazolyl blue MTT) (Sigma Chemical Co.) in serum free medium was added to each well. The plates were incubated for an additional 2-3 hours at 37 ° C, 5% CO 2. The agent was aspirated and the wells filled with 0.2 ml of 70 vol%. Aqueous solution of isopropanol, 0.075 N HCl and gently mixed until dissolution of the formazan dye (15-30 min). The optical density in formazan solutions was determined using a microplate photometer at 540 nm. Cell viability as% of the untreated control was calculated as the ratio of the optical density in the experimental wells to the optical density in the wells containing the untreated cells corrected for background.
The results are shown in Figure 9. A dose of a drug that causes a 50% growth inhibition (IC50) for free Topotecan or a non-targeted liposomal Topotecan above 30 pg / ml; for F5 immunoliposomal Topotecan, 0.15 pg / ml. These results are consistent with data containing the absorption of a targeted drug.
Example 28. Comparative stability and pharmacokinetics of liposomal and F5-immununoliposomal Topotecan in blood in mice.
Topotecan liposomes containing the radioactive lipid [3H] -CHE tag at 1.5 mCi / mmol phospholipid were prepared according to examples 11 and 19 using the procedure of mixing and extruding the lipid ethanol solution according to the following conditions: 0.423 N formaldehyde salt precipitate: 0.643 N sucrose octasulphate sucrose ; extruded polycarbonate film: 15 passes through 2 stacked PCTE filters with a pore size of 80 nm; Topotecan loading: drug / phospholipid entry ratio of 350 mg / mmol (calculated for the basic-free Topotecan); F5 scFv coupling was performed as described in Example 19. Liposomes had the following properties:
The size was measured using QELS: the average mass was 101.2 nm; standard deviation 20.1 nm.
Encapsulation of the drug: Topotecan liposomes (Topo-Ls) 359.3 ± 27.4 mg / mmol phospholipid; F5scFv Topotecan Immunoliposomes (Topo-F5-ILs) 326.3 ± 15.9 mg / mmol phospholipid.
The study was performed essentially as in Example 22. Groups of nine Swiss Webster mice (8-10 weeks of age, 24-27 g) were injected via the tail vein Topo-Ls, Topo-F5ILs or freshly prepared Topotecan 1 mg / ml in unbuffered saline in water at a basic dose of Topotecan of 5 mg per kg body weight (corresponding to a lipid dose of 14-16 pmoles phospholipid / kg body weight). Within 1 hour, 8 hours and 24 hours at the time points after injection, 3 animals per time point were exsanguinated by open-heart puncture under ketamine / xylazine anesthesia, blood was collected in tubes containing PBS-EDTA and tested for Topotecan (fluorometry) and lipid liposome (scintillation counting of radioactivity). The amounts of drug and lipid remaining in the blood at specific time points were calculated from the administration of the dose as 100%, assuming that the amount of blood per animal is 6.3% of body weight and the fraction of blood cells concentrate is 45%. The amount of residual drug encapsulated in liposomes at each time point was calculated for each animal individually by comparing the ratio of radioactivity in the drug / lipid plasma samples with the injected liposomes. The amount of free Topotecan in the plasma samples collected at 1 hour after injection was less than 1% of the injected dose (in fact, below our detection limit) by our test method; therefore, further time points of the free Topotecan group were not investigated. Because of the quick removal of blood and low blood levels with free Topotecan, we assumed
The results are summarized in Table 21 below. Interestingly, liposomes prepared according to the disclosure retain 79-85% of the initial drug loading even at 24 hours after injection into the bloodstream of the animals. Differences between the mean values of lipid plasma or drug between liposomal groups and immunoliposomal groups were in the range of 1.8-13.6% and were close to or within the error range of the test. The probabilities of a null hypothesis between a group of liposomes and immunoliposomes with respect to drug or lipid values at each time point were calculated using the Student's t test, ranging from 0.543-0.938. We can conclude that differences in the levels of drug residues in the blood or lipids between the two preparations were insignificant and statistically indistinguishable.
Table 21. Amounts of lipid liposomes, Topotecan and Topotecan remaining hermetically sealed in the plasma liposome at various time points after intravenous injection.
<td>Time after</td><td>Lipid,%</td><td>Drug,% injected</td><td>Drug / lipid,% value</td>
<td>injection</td><td>injected dose</td><td>dose</td><td>the initial injection</td>
Conjugated F5 with liposomal Topotecan (Topo-F5ILs):
<td>1 hour</td><td>57.58 ± 4.95</td><td>55.45 ± 7.23</td><td>96.14 ± 7.32</td>
<td>8 hours</td><td>35.37 ± 3.84</td><td>34.18 ± 5.87</td><td>96.31 ± 11.92</td>
<td>24 hours</td><td>15.51 ± 11.84</td><td>12.30 ± 9.02</td><td>79.36 ± 8.03</td>
<td colspan="4">liposomal Topotecan (unconjugated) (Topo-Ls):</td>
<td>1 hour</td><td>58.88 ± 9.51</td><td>57.63 ± 9.45</td><td>97.90 ± 5.29</td>
<td>8 hours</td><td>39.61 ± 1.99</td><td>38.82 ± 1.49</td><td>98.06 ± 4.44</td>
<td>24 hours</td><td>15.84 ± 3.85</td><td>13.45 ± 2.64</td><td>85.25 ± 7.03</td>
Example 29. Antitumor efficacy of liposomal and anti-HER2-immunoliposomal Topotecan in a BT-474 xenograft model.
In this study, we used the first prototype of the Topotecan immunoliposomes, which uses a triethylammonium polyphosphate gradient to encapsulate the drug. The liposomes were prepared essentially according to the methods of Examples 11 and 19. Lipid components of the DSPC matrix (Avanti Polar Lipids, 3 parts by mole), Cholesterol (Calbiochem, 98.3%, 2 parts by mole) and methoxy-PEG (2000) -DSPE (Avanti Polar Lipids, 0.015 mol part) - were combined with 100% ethanol USP to obtain a solution containing 0.5 mM phospholipid at 60 ° C. The ethanolic lipid solution was diluted at 60 ° C with an aqueous solution of triethylammonium polyphosphate (0.608 M triethylamine, 0.65 N phosphate, pH 6.1, osmolality 531 mmol / kg), thoroughly mixed and extruded 10 times with 2 stacked polycarbonate membranes with a pore size of 100 nm (Nuclepore Corning) using a thermostated gas-pressure extruder (Lipex Biomembranes) at 60 ° C. The extruded liposomes were cooled on ice and the non-thermrhized triethylammonium polyphosphate was removed by gel chromatography in Sepharose CL-4B using 5% dextrose-5 mM HEPES-Na buffered pH 6.5 as eluent. The size of the liposomes was 103.8 ± 35.1 nm and was measured using QELS. The liposomes in this buffer were incubated with Topotecan hydrochloride at 60 ° C for 30 minutes in a ratio of 0.35 mg of basic Topotecan to pmol phospholipid. At the end of the incubation, the liposomes were cooled on ice and chromatographed on a Sephadex G-75 column, the eluent being 20 mM HEPES-Na, 135 mM NaCl, pH 6.5, to remove the non-aerosolized drug. The content of the drug was determined by fluorometry and the lipid content by means of a phosphate test as previously reported. The liposomal Topotecan thus obtained has 365.4 ± 23.1 mg of basic
Topotecan per mmol of phospholipid. To make HER2-targeted topolecan immunoliposomes, a portion of the Topotecan liposomal formulation was incubated with the purified scFv F5 anti-HER2 conjugate and maleimido-PEG-DSPE connector essentially as described in Example 19. Briefly, the F5-PEG-DSPE conjugate in 10% aqueous sucrose solution -10 mM sodium citrate solution, pH 6.5, combined with Topotecan liposomes in a ratio of 15 mg protein per mmol phospholipid liposome and incubated at 60 ° C for 30 minutes. The incubation mixture was cooled in ice and chromatographed on a Sepharose CL-4B column, eluent being 20 mM HEPES-Na, 135 mM NaCl, pH 6.5, to remove the un-introduced scFv conjugate. After this additional incubation, the drug-lipid ratio decreased by 14%.
Preparations of the Topotecan liposome and immunoliposome containing 1-2 mg / ml of Topotecan were passed through a 0.2 micron sterile syringe filter, aseptically separated into polypropylene vials and stored at 4-6 ° C for up to one month prior to use.
The free Topotecan was freshly prepared by dissolving the Topotecan hydrochloride in powder form at a dose of 2 mg / ml in 5% dextrose and sterilized by passing through a 0.2 micron syringe filter.
Human breast cancer xenograft (BT-474) with overexpression HER2 was determined as described in example 10. On day 13 after tumor implantation, animals having tumors in the 120-350 mm cubic range were selected and randomly assigned to 3 groups with the treatment applied and 1 control group of 12 animals in each. On days 14, 18 and 21 after tumor implantation, the mice were treated with intravenous injections (tail vein) with Topotecan formulations at an injection dose of 5 mg / kg body weight, or with an equal volume of saline solution. The general health of the animals was monitored daily. Tumor sizes and body weights were monitored twice a week up to 53 days after tumor implantation. Animals whose tumors reached 20% of body weight, or those with progressive weight loss of 20% or more, have been killed.
Figures 11 and 12 show data on tumor growth and body weight, respectively. Topotecan liposomal formulations were more active in inhibiting tumor growth than the free drug, and the F5 oriented liposomal formulation was more active than the non-targeted one. The average tumor sizes at the end of the observation period were significantly different between the groups (p values for unpaired 2-tailed Student's t-test were 1.2x10-6 for free drug versus immunoliposomal drug, 0.000114 for free drug versus liposomal drug and 0.00718 for the liposomal drug in the ratio of immunoliposomal drug). Thus, the liposomally-encapsulated Topotecan was more active than the free drug, and the anti-HER2 immunoliposomal Topotecan was more active than the undirected liposomal drug. In the liposomal and immunoliposomal groups after initial regression, tumor regrowth occurred within 10 days after the last drug administration. Tumor regression did not occur in the free drug group. It was noted that liposomal topotecan formulations at a given dose were more toxic than with the free drug. Gastrointestinal toxicity occurred. The animals receiving liposomal Topotecan showed diarrhea and weight loss, an average of about 14% at the highest point. While in the non-targeted liposomal group, the animals returned to health, with the exception of one (12.5%), which had 15% permanent weight loss at the end of the study, in the F5 targeted group, five animals (41.6%) developed a deadly condition and died; and two more (16.7%) had a permanent weight loss of about 15%.
Example 30. Maximum tolerated dose (MTD) of free and liposomal Topotecan in mice injected intravenously in a 3-week series.
In this study, a Topotecan liposome formulation prepared as in Example 29 was used except that the triethylammonium polyphosphate solution was replaced with a sucrose octane solution of triethylammonium containing 0.65 M triethylammonium at pH 6.2; and for extrusion, 80 nm was used instead of filters with a polycarbonate membrane with a pore size of 100 nm. The volume-weighted liposome size was determined by the method of acid-elastic light scattering in approximately the Gauss model (QELS) application and was 95.1 ± 19.6 nm (mean ± SD); the drug / lipid ratio was 369.1 ± 18.3 mg / mmol phospholipid. Five six-week-old SwissWebster female mice (18-20 g) in groups of two received three intravenous (in the tail vein) injections with free or liposomal Topotecan once a week, from a dose of 2 mg / kg of basic Topotecan per injection and increasing in each subsequent group by a factor of 1.8 in the dose of 37.8 mg / kg. Immunoliposomal Topotecan was not included in this study. Animal body weight and general health were monitored daily. Progressive weight loss by more than 20% or natural death at any time in any two animals in the group during the ten days from the start of treatment was considered an indicator toxic dose. According to animal mortality and data, MTD weights were determined to be in the range 11.7-21 mg / kg relative to free Topotecan and 2.0-3.6 mg / kg in relation to the liposomal (Prototype 2) of Topotecan. In the second study, mice received free injections, liposomal or F5-immunoliposomal Topotecan (prepared from the liposomal Topotecan in this example as described in example 29), at doses of 2.0 mg / kg (liposomal / immunoliposomal Topotecan) or 12 mg / kg (free Topotecan), and increased in each successive group by a factor of 1.15 until a dose similar to the upper range of the predetermined interval of MTD is reached. The maximum dose that did not cause death or mortality in any of the animals was considered MTD and determined to be 18.4 mg / kg for free Topotecan, 3.0 mg / kg for Liposomal Topotecan and 3.0 mg / kg for immunoliposomal topotecan. Thus, the liposomal Topotecan showed greater toxicity than the free drug. and increased in each subsequent group by a factor of 1.15 until a dose similar to the upper range of the predetermined interval of MTD is obtained. The maximum dose that did not cause death or mortality in any of the animals was considered MTD and determined to be 18.4 mg / kg for free Topotecan, 3.0 mg / kg for Liposomal Topotecan and 3.0 mg / kg for immunoliposomal topotecan. Thus, the liposomal Topotecan showed greater toxicity than the free drug. and increased in each subsequent group by a factor of 1.15 until a dose similar to the upper range of the predetermined interval of MTD is obtained. The maximum dose that did not cause death or mortality in any of the animals was considered MTD and determined to be 18.4 mg / kg for free Topotecan, 3.0 mg / kg for Liposomal Topotecan and 3.0 mg / kg for immunoliposomal topotecan. Thus, the liposomal Topotecan showed greater toxicity than the free drug. 0 mg / kg for immunoliposomal Topotecan. Thus, the liposomal Topotecan showed greater toxicity than the free drug. 0 mg / kg for immunoliposomal Topotecan. Thus, the liposomal Topotecan showed greater toxicity than the free drug.
Example 31. Efficacy of anti-tumor liposomal Topotecan in a BT-474 xenograft model in the range of 0.125-1.0xMTD
In the present study, Topotecan liposomes and F5-immunoliposomes from example 30 were used. Subcutaneous BT474 xenografts were cultured in nude mice as in Example 29. On day 18 after tumor cell inoculation, animals with tumors (105-345 mm cubic, medium about 200 cubic mm) were randomly divided into treatment groups of 6 animals / group, and in the control group - 8 animals / group. The animals received free or liposomal Topotecan at 1xMTD, 0.5xMTD, 0.25xMTD or 0.125xMTD in three intravenous injections. (to the tail vein) on days 19, 23 and 27 after tumor implantation. The control group received saline injections. Tumor size and body weight of animals was monitored as in Example 29. In order to obtain weight measurements of the animal, tumor mass (calculated from the size of tumors, assuming a tumor density of 1.0), it was subtracted from the total weight of the animal. All drug preparations in MTD showed antitumor activity (Figures 13A-13D). There was no significant difference in efficacy between free and liposomal drug administered in relevant or identical MTD fractions (1/2, 1/4 and 1/8). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). 0) has been subtracted from the total weight of the animal. All drug preparations in MTD showed antitumor activity (Figures 13A-13D). There was no significant difference in efficacy between free and liposomal drug administered in relevant or identical MTD fractions (1/2, 1/4 and 1/8). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). 0) has been subtracted from the total weight of the animal. All drug preparations in MTD showed antitumor activity (Figures 13A-13D). There was no significant difference in efficacy between free and liposomal drug administered in relevant or identical MTD fractions (1/2, 1/4 and 1/8). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). All drug preparations in MTD showed antitumor activity (Figures 13A-13D). There was no significant difference in efficacy between free and liposomal drug administered in relevant or identical MTD fractions (1/2, 1/4 and 1/8). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). All drug preparations in MTD showed antitumor activity (Figures 13A-13D). There was no significant difference in efficacy between free and liposomal drug administered in relevant or identical MTD fractions (1/2, 1/4 and 1/8). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14). Thus, liposomal encapsulation of the drug using a TEA-SOS gradient gradient resulted in about a 6-fold increase in anti-tumor activity, but also a similar increase in drug toxicity. Animal body weight dynamics showed that all treatments were non-toxic except for treatment with free Topotecan in MTD, which showed transient weight loss (about 15% of the pre-treatment value) that was later resolved (Figure 14).
Example 32. In vitro cytotoxicity and preparation of Topotecan liposomes prepared by the method of sucrose triethylammonium sucrose closing.
The liposomal Topotecan was prepared essentially according to the procedure of Example 18, using a closed TEA-SOS solution of 643 mM TEA, pH 5.7, osmolality 530 mmol / kg, and drug / phospholipid ratio of 170 mg / mmol. Liposomes contained 155 mg drug / mmol of phospholipid; 90% loading efficiency, particle size 105 nm. These liposomes were incubated with a micellar solution of the F5-PEG-DSPE conjugate at about 30 scFv on liposomes (15 mg antibodies / mmol phospholipid) at 60 ° C for 1 hour, essentially as described in Example 19. Liposomes with conjugated antibody were separated by help
SEC using Sepharose CL-4B and forming a buffered HBS-6.5 HEPES saline solution in water. There were no detectable changes in the drug / lipid ratio during the anti-HER2 scFv (F5) addition.
The absorption of Topotecan preparations by tumor cells is determined as follows. human HER2 overexpressing adenocarcinoma cells (SK-Br-3,
ATCC HTB-30) were seeded into 24-well cell culture plates at 150,000 cells / well and acclimated overnight. The cells were incubated (in triplicate) with F5-directed and non-targeted liposomal Topotecan in whole culture medium at a liposome concentration of 0.1 mM and 0.01 mM for 4 hours at 37 ° C. The cells were washed 4 times with Hanks' balanced salt solution, dissolved in 0.1% Triton X-100 - 70% acidified 1: 10 mixture of isopropanol, and the number of cells bound to Topotecan per well was determined fluorometrically. The results (mean ± standard error) are summarized in Table 22. Targeted liposomes delivered 100-300 times more drug to target cells than non-targeted liposomes.
Table 22. Absorption of liposomal Topotecan with Sk-Br-3 breast cancer cells.
<td>Preparation</td><td>Absorption of topotecan at 0.1 mM phospholipid, ng / well</td><td>Absorption of topotecan at 0.01 mM phospholipid, ng / well</td>
<td>Undefined liposome</td><td>4.76 ± 0.24</td><td>0.607 ± 0.088</td>
<td>Targeted HER2 liposome</td><td>53.8 ± 13.7</td><td>197.0 ± 4.6</td>
<td>Ratio:</td><td>112.1 ± 8.6</td><td>324 ± 55</td>
<td>Directed / undirected</td><td></td><td></td>
The cytotoxicity of these Topotecan preparations against SKBr-3 breast cancer cells was determined as described in Example 27. SKBr-3 cells were implanted in 96 well plates at 5,000 cells / well, acclimated overnight and incubated with increasing concentrations (0.004-30 pg). ml) of free, liposomal, or F5-immunoliposomal Topotecan in cell growth medium for 4 hours at 37 ° C. The drug containing medium was removed and the cells were allowed to grow in drug-free medium for 72 hours. The amount of viable cells in each well was determined using the tiazolyl blue (MTT) study of tetrazol and expressed as a% control (untreated) cell values. The results are shown in Figure 10. Topotecan immununolyposomes were more cytotoxic (IC 50 0.15-0,
Example 33. Stability in v / vo of Topotecan Hposomes of various sizes.
TEA-P containing liposomes were prepared as in Example 22 using 12-fold extrusion through a polycarbonate membrane with a pore size of 100 nm or an additional 12-fold through a polycarbonate membrane with a pore size of 50 nm. Topotecan (TPT) was added in a ratio of 150 pg / pmol phospholipid. The loading was completed at 58 ° C after 45 minutes in a water bath and then by cooling in ice. The loading efficiency for 50-nm- and 100 nm-extruded liposomes was 126.80 ± 19.24 pg TPT / pmol PL (84.5 ± 12.8%) and 148.48 ± 10.26 pg TPT / pmol PL ( 99.0 ± 6.8%), respectively. Female Swiss Webster mice received three intravenous injections of one of the two Ls-TPT preparations at a dose of 5 mg TPT / kg in groups of three. Mice were euthanized after 6 h, and then blood was collected. Plasma was analyzed for TPT and lipid liposomes,
Table 23. Stability of different sizes of loaded Ls-TPT in v / vo by means of the TEA-Pn closure method.
<td>Size liposome, nm</td><td>Plasma drug,% injected dose</td><td>Lipid liposome w plasma,% injected dose</td><td>Drug / lipid ratio,% initial value injection</td>
<td>74.2 ± 21.6</td><td>32.93 ± 1.97</td><td>45.7 ± 2.2</td><td>72.06 ± 5.51</td>
<td>96.4 ± 29.3</td><td>33.26 ± 3.56</td><td>37.6 ± 5.3</td><td>88.41 ± 15.68</td>
Example 34. Synthesis and encapsulation of 6- (3-aminopropyl) ellipticine liposome (6APE).
6- (3-aminopropyl) ellipticine was prepared from ellipticine using a two-step method according to the procedure described by Snabel et al. J. Med. Chem. 1986, v. 29, pp.1321-1322. 501.4 mg of basic ellipticine (NSC 71795) (Aldrich Chemical Co.) were mixed with approximately 100 mg of sodium hydride (Sigma; washed with anhydrous petroleum ether) in 5 ml of dry dimethyl formamide (DMF) at room temperature for 30 minutes. To this mixture was added dropwise a solution of 678 mg of N-bromopropylphthalimide (Aldrich) with 2 ml of dry DMF. The purple colored reaction mixture was stirred under argon overnight, treated with 1 ml of water and poured into 60 ml of water. The mixture was extracted twice with 25 ml of methylene chloride, the extract was dried over anhydrous sodium sulphate and passed through a layer of neutral aluminum oxide. The aluminum oxide layer was washed twice with 10 ml of methylene chloride and the combined filtrates and washes were concentrated to dryness under vacuum. The product was stirred overnight with 20 ml of absolute ethanol and 2 ml of anhydrous hydrazine at room temperature. The resulting suspension was filtered under vacuum conditions, the yellow filtrate was diluted with 50 ml of 0.2 N NaOH and extracted with two portions (75 ml and 50 ml) of chloroform. The chloroform extract was dried over Na 2 SO 4 and concentrated under vacuum. The crude product (yield 408 mg) was subjected to column chromatography of silica 60, eluted isocratic with a mixture of chloroform and methanol (7: 3 by volume) and saturated with dry trimethylamine. It was shown that the eluted fractions in the second yellow tape, subsequent to unreacted ellipticine, they contain the desired compound in approximately 30% yield. The structure was confirmed by H-NMR. TLC: Rf 0.29-0.31 (silica 60; CHCl3-MeOH 7: 3 by volume, saturated with trimethylamine). Ellipticine, Rf 0.81-0.83. The obtained compound was converted into the dihydrochloride salt by dissolving the salt in anhydrous ethanol and titrating with 6 N HCl in anhydrous isopropanol. Orange crystals of 6-APE dihydrochloride (NSC 176328), filtered, washed with ether and dried under vacuum. The dihydrochloride yield was 86%. The obtained compound was converted into the dihydrochloride salt by dissolving the salt in anhydrous ethanol and titrating with 6 N HCl in anhydrous isopropanol. Orange crystals of 6-APE dihydrochloride (NSC 176328), filtered, washed with ether and dried under vacuum. The dihydrochloride yield was 86%. The obtained compound was converted into the dihydrochloride salt by dissolving the salt in anhydrous ethanol and titrating with 6 N HCl in anhydrous isopropanol. Orange crystals of 6-APE dihydrochloride (NSC 176328), filtered, washed with ether and dried under vacuum. The dihydrochloride yield was 86%.
Liposomes were prepared by hydrating pure lipid DSPC, cholesterol and PEG (molecular weight 2,000) -DSPE (molar ratio 3: 2: 0.015) in a solution of trimethylammonium polyphosphate (TMA-Pn) in 0.5 M TMA, pH 5.6, at 60 ° C, followed by six rapid freezing (-78 ° C) and thawing cycles (60 ° C) and ten extrusion through two stacked filters with a pore size of 50 nm. The undersized TMA-Pn was removed using a Sepharose CL-4B column eluting with HEPES dextrose (5 mM HEPES, 5% dextrose, pH 5.5). The size of the liposomes was 85.7 ± 32.1 nm.
A concentrated 6-APE solution (10 mg / ml) was added to liposomes containing TMA-Pn liposomes at a drug to phospholipid ratio of 100 pg APE / pmol phospholipid, the mixture was incubated at 58 ° C for 45 minutes and then rapidly quenched on ice by 15 minutes. The non-humussed drug was removed by gel permeation chromatography on a Sephadex G-75 column eluted with HEPES-dextrose buffer (5 mM HEPES-Na, 5% dextrose, pH 6.5). APE encapsulated in liposomes was quantified by spectrophotometry as described in example 71, phospholipid liposomes were determined using the extraction assay of example 70. The encapsulation of the drug was virtually quantitative.
Example 35. Preparation of HER2-directed immunoliposomal 6APEs and cytotoxicity of 6-APE preparations against BT-474 cells of HER2 overexpressing breast cancer.
Liposomes with encapsulated 6-APE (Ls-APE) were prepared as in Example 34 above. Anti-HER2 Immunoliposomes with encapsulated 6-APE (F5 ILs-APE) were prepared from Ls-APE by the method of Example 19. The MTT-based cell viability test of Example 27 was used to determine the cytotoxicity of 6-APE provided as a solution, Ls-APE APE or HER2-targeted F5-ILS-APE anti-human (BT474) human HER2 overexpressing breast cancer. The cells were exposed on medium containing the drug for 6 hours after incubation in drug-free medium for 3 days. The results are shown in Figure 15. IC50 for free APE is 0.26 pg APE / ml, F5-ILs-APE was 0.756 pg APE / ml, and for non-targeted Ls-APE it was 51.0 pg APE / ml. It was 67,
Example 36. EGFR-directed preparations immunoliposomal 6-APE and cytotoxicity against tumor cells in vitro.
6-APE loaded liposomes were prepared as described in Example 34. EGFR-directed immunoliposomes were prepared by attaching EGFR-specific Fab 'antibody fragments as follows. C225 specific EGFR IgG (cetuximab, ERBITUX ™, Imclone Systems) was digested with pepsin to produce fragments (Fab ') 2. Purified (Fab ') 2 fragments were reduced by treating them with 10-20 mM 2-mercaptoethylamine for 15 minutes at 37 ° C, and Fab' fragments were purified by gel filtration using Sephadex G-25. The presence of reactive thiol groups was typically about 0.9 thiol groups per protein molecule (quantitatively using an Ellmann reagent). C225Fab 'were covalently coupled to the amphiphilic Mal-PEG-DSPE link (Avanti Polar Lipids, AL) in an aqueous solution at pH 6.2-6.5 and a 1: 4 protein-molar ratio, for 2-4 hours at room temperature or overnight at 4-6 ° C to form C225Fab'-PEG-DSPE coupled with 30-50% protein yield. The micelle-forming conjugate was separated from unreacted protein by column chromatography excluding 3% agarose - 4% polyacrylamide gel granulate (Ultrogel AcA34, obtained from Sigma Chemical Co.) eluted with HBS-6.5 buffer. The conjugate was recovered in the empty space fractions. Immunoliposomal 6-APE was prepared by incubating these liposomes with C225 Fab'-PEG-DSPE with a drug loaded with liposomes in a ratio of 30 mg C225 protein / mmol phospholipid liposome for 30 minutes at 60 ° C,
MDA-MB-468 cells of human breast cancer overexpressing EGFR and MCF-7 cells of low EGFR EGFR (ATCC, Rockville, MD) were cultured in the culture medium recommended by the supplier, cytotoxicity of free, liposomal and anti-EGFR immunoliposomes was examined 6 -APE against these cells according to the method described in Example 27. Cells were incubated in drug-containing medium for 6 hours and then for 3 days after incubation in drug-free medium. The results are shown in Figure 16. In MDA-MB-468, IC50 cells for free 6-APE were about 0.1 pg / ml, and for C225-ILs-APE about 0.9 pg / ml. In MCF-7, IC 50 cells were 0.1 pg / ml for free 6-APE were approximately 0.5 pg / ml, and for C225-ILs-APE approximately 14 pg / ml.
IC50 Ls-APE in both cell lines was> 30 pg / ml. Thus, EGFR-targeted immunoliposomes loaded with 6-APE exhibited cytotoxic antigen specific activity in MDA-MB-468 breast cancer cells overexpressing EGFR, but not in MCF-7 breast cancer cells overexpressing EGFR. In MCF7 cells, targeted and non-targeted 6-APE liposomes were equally active.
Example 37. Pharmacokinetics of 6-APE liposomes in rats.
Liposomes with a closed TEA-Pn solution (557 mM phosphate groups, 500 mM TEA, pH 5.8 and osmolality 480 mmol / kg) and a composition of lipids DSPC, cholesterol and PEG-DSPE (molar ratio 3: 2: 0.015) were prepared as in example 11 above. The ethanolic lipid solution was combined at 60 ° C with 10 volumes of TEA-Pn aqueous solution and extruded ten times with two stacked polycarbonate membranes with a pore size of 80 nm. The non-pressurized TEA-Pn was removed using a Sepharose CL-4B column eluting with MES dextrose (5 mM MES, 5% dextrose, pH 5.5). The size of the liposomes was 92.3 ± 23.3 nm and was measured using QELS. Non-replaceable radioactive lipid tracer [<sup>3</sup>H] -CHE was included in the lipid matrix at a concentration of 0.5 mCi / mmol phospholipid. The liposomes were loaded with 6-APE as described in example 34.
A pharmacokinetic study followed the protocol of Example 9. Female Sim Albino rats (9 week old, 200 g) were injected intravenously with a dose of 10 mg 6-APE / kg. Blood was collected at fixed time points and plasma analyzed for 6-APE using fluorometry. Plasma portions (0.05-0.2 mL) were mixed in 1-2 mL with 90% aqueous isopropanol, 0.1 N HCl, and 6-APE was quantified by fluorescence as in Example 71. The lipid was quantified through [<sup>3</sup>H] -CHE scintillation counting of radioactivity.
The results are shown in Figure 17. Blood half-life (t1 / 2) was 13.7 hours and lipid liposomes 16.6 hours (Panel A). The half-life of drug released from liposomes was 77.9 hours, showing significant stability of encapsulation (panel B).
Example 38. Liposomal synthesis and hermetization 2- (2- (N, N-diethylamino) ethyl) ellipticine (2-DAE).
2- (2- (N, N-diethylamino) ethyl chloride ellipticine (NSC 359449) is an antitumor derivative of ellipticine which is prepared by alkylation of ellipticine with 2- (N, N-diethylamino) ethyl chloride in methanol in the presence of triethylamine (see Snare drum, LM, Angelo, M., Fry, DM, and Worth, DFJ Med. Chem. 1986, 29: 1321-1322.) Liposomes containing closed TEA-Pn were prepared as described in example 37. 2DAE.2HCl was incubated with liposomes. TEA-Pn in 5 mM HEPES-Na, 5% dextrose, pH 7.4, in a ratio of 2-DAE to phospholipid 100 pg / pmol The amount of drug loaded was 88.2 pg APE / pmol PL (efficiency 88.2% ).
Example 39. Pharmacokinetics of liposomal 2-DAE in rats.
The pharmacokinetics of liposomal 2-DAE in blood (example 38) were tested in rats as in Example 37. tt / 2 2-DAE lasted 17.8 h and the lipid matrix of the liposome,
18.2 h (A). The half-life of drug release from liposomes in the blood was tt / 2 = 677 h (B).
Thus, these liposomes were extremely stable against drug leakage in the bloodstream.
Example 40. Loading of vinorelbine into liposomes by the TEA-Pn method. The effectiveness of pHL
The liposomes were prepared by the ethanol injection method as in Example 11 using a TEA-Pn solution of 0.608 M TEA, 0.65 M phosphate groups at pH 6.1 and osmolality of 531 mmol / kg, and a lipid suspension 15 extruded through two stacked pellets. stack polycarbonate membranes with a pore size of 100 nm. The liposomal size obtained was 108.3 ± 17.1 nm and was measured using QELS. Vinorelbine (VRB) in the form of vinorelbine 10 mg / ml USP base mixture was added to liposomes in an aqueous solution of 5 mM HEPES-Na, 5% dextrose, pH 6.5, at a drug to phospholipid ratio of 350 pg / pmol, pH was brought to the desired value with 1-5 N NaOH, and the mixture was incubated at 58 ±
2 ° C for 30 minutes. The mixture was then ice-cooled for 15 minutes and the non-moisturized drug was removed through a Sephadex G-75 column by gel permeation chromatography, eluting with HBS-6.5 buffer (20 mM HEPES-Na, 135 mM NaCl, pH 6.5). Portions of purified liposomes were then dissolved in acidic isopropanol and examined for vinorelbine using 270 nm spectrophotometry. Phospholipid liposomes were quantified using the Bartlett phosphate test (1959) after extraction with methanol and chloroform.
The calculated drug to lipid ratios after loading are shown in Table 24. The loading of vinorelbine was quantitative (i.e., practically 100%) and regardless of the pH in the range studied.
Table 24. and ^ n ^ k of vinorelbine to iiposomes with closed TIA - Pn at various pH values of external buffer
<td>pH</td><td>Drug to phospholipid ratio (pg / pmol)</td><td>Loading efficiency (%)</td>
<td>4.5</td><td>351.2 ± 52.88</td><td>100.4 ± 15.2</td>
<td>5.0</td><td>347.6 ± 6.35</td><td>99.3 ± 1.8</td>
<td>5.75</td><td>35.2 ± 11.2</td><td>101.5 ± 3.2</td>
<td>6.25</td><td>377.0 ± 21.5</td><td>107.7 ± 6.6</td>
<td>7.0</td><td>374.3 ± 29.58</td><td>106.9 ± 9.0</td>
Example 41. Liposomal vinorelbine prepared by the TEA-Pn method in different drug / lipid ratios: encapsulation efficacy and in vivo stability in mice.
Liposomes with a closed TEA-Pn solution were made according to an example
40, except that [<sup>3</sup>H] -CHE was included in the lipid matrix at 1.5 mCi / mmol phospholipid. The size of the liposomes was 98.5 ± 34.3 nm and was measured using QELS. The liposomes were mixed with vinorelbine bitartrate of USP in an aqueous 5 mM buffer solution
HEPES-Na, 5% dextrose, pH 6.5, drug to phospholipid 150-450 mg VRB / mmol and incubated at 58 ± 2 ° C for 30 minutes. There was no pH correction following the addition of the drug. Liposomes loaded with vinorelbine (Ls-VRB) were isolated and the drug and phospholipids were screened as in Example 40.
Six-week old female Swiss Webster mice (Harlan Bioresearch) were injected intravenously in groups of three Ls-VRB-Pn at a dose of 5 mg / kg of VRB. The lipid dose varied depending on the loading degree and could be determined from the calculated drug to lipid ratios. At 8 hours or 24 hours after injection, the animals were anesthetized, exsanguinated, and blood was collected on ice in weighed tubes containing known amounts of PBS with 0.04% EDTA. Blood cells were separated by centrifugation, supernatants were analyzed for lipid liposomes by [<sup>3</sup>H] -CHE scintillation counting of radioactivity and for vinorelbine by HPLC as follows. The samples were seeded with vinblastine (internal standard) and extracted with diethyl ether, evaporated and the residues dissolved in a mobile phase consisting of 50 mM aqueous triethylammonium acetate (pH 5.5) and acetonitrile (58:42 by volume). The samples were loaded onto a C 18 reverse phase silica column (Supelco C-18 column, 250 mm x 4 mm id, 5 μm particle size) preceded by C-18 column cover. The column was isocratically eluted in the above mobile phase at a flow rate of 1.0 ml / min. VRB was detected using an absorbance detector at 280 nm.
Typical retention times for VRB and vinblastine (internal standard) were 9.1 min and 7.8 min, respectively.
The results are shown in Table 25. The loading efficiency decreased with increasing drug / lipid ratio, practically at 100% at a dose of 150 mg / mmol to about 66% at a dose of 450 mg / mmol. It has been found that the addition of vinorelbine bitartrate in a ratio of more than 250 mg vinorelbine to mmol of phospholipid resulted in a significant acidification of the liposomal suspension (pH <4,0), which led to reduced loading efficiency. Thus, the need to control the pH during the loading phase of the drug has been established. Amounts of the liposome matrix were detected in the blood after 8 hours and were 30.4 ±
6.6% of the injected dose (% id) 38.6 ± 5.2% id with no apparent reference to the absolute amount of lipid injected. After 24 hours, it was found that from 6.4% ID to 14.8% ID of the lipid matrix detectable in the blood is present. The amount of drug that remained encapsulated after 8 hours differed and ranged from 37% to 63%. However, 24 hours after the injection, the drug levels fell below the limit of detection by analytical method.
Table 25. Efficacy of encapsulation and drug retention of an in-drug Ilpurotic vinorelbine prepared at different drug / lipid ratios using the TEA-Pn method (without pH regulation in the loading buffer). Drug retention data are mean ± SD (N = 3).
The ratio of vinorelbine / phospholipid% drug remaining hermetically sealed at 8 hours after injection
<td>entrance,</td><td>output</td><td>Effectiveness</td>
<td>mg / mmol,</td><td>mg / mmol,</td><td>encapsulation,%</td>
<td>calculated</td><td>measured</td><td></td>
<td>150</td><td>156</td><td>104.0</td><td>36.6 ± 4.2</td>
<td>250</td><td>238</td><td>95.2</td><td>56.3 ± 1.3</td>
<td>350</td><td>260</td><td>74.3</td><td>65.9 ± 2.3</td>
<td>450</td><td>299</td><td>66.4</td><td>63.0 ± 4.1</td>
Example 42. Loading of vinorelbine into liposomes by the TEA-SOS method in different drug / lipid ratios.
TEA-SOS liposomes for loading into the drug were prepared as in Example 5 with the exception that a TEA-SOS solution with 0.65 M TEA, pH 5.4, osmolality and 521 mmol / kg was used instead of a TEA-TEA solution. Pn and the liposomes were extruded through a polycarbonate membrane with a pore size of 80 nm. The size of the liposomes was 86.6 ± 12.9 nm and was measured using QELS. VRB was added to the liposomes in an aqueous solution of 5 mM HEPES-Na, 5% dextrose, pH 6.5, in different drug to phospholipid ratios, and then the mixture was incubated at 60 ° C for 30 minutes. The loaded VRB liposomes were isolated and examined as in example 40.
The calculated drug to lipid ratios in VRB liposomes are shown in Table 26. Interestingly, unlike the polymer loading anion anion, loading of vinorelbine in liposomes with polyanionated sugar (sucrose octasulfate) was practically quantitative, irrespective of the drug / iipide ratio db 450 mg VRB / mmoles phospholipid and only slightly smaller for (88%) 550 mg VRB / mmol phospholipid.
Table 26. Dependence of vinorelbine loading on liposomes in relation to drug to lipids
<td colspan="2">The ratio of vinorelbine / phospholipid, mg / mmol</td><td rowspan="2">Loading efficiency (%)</td>
<td>total</td><td>Encapsulated in liposomes</td>
<td>150</td><td>159.9 ± 11.5</td><td>106.6 ± 8.1</td>
<td>250</td><td>255. ± 12.4</td><td>102.2 ± 5.1</td>
<td>350</td><td>381.8 ± 16.3</td><td>109.1 ± 5.1</td>
<td>450</td><td>456.1 ± 29.5</td><td>101.4 ± 6.6</td>
550
486.2 ± 26.0
88.4 ± 4.2
Example 43. Preparation of HER2-targeted immunoliposomes trapped in vinorelbine using the TEA-On method and comparative pharmacokinetics of vinorelbine-targeted liposomes of vinorelbine in rats.
The anti-HER2 scFv F5-PEG-DSPE conjugate was prepared as in Example 19. HER2-directed vinorelbine immunolipsomes were prepared by incubating non-targeted vinorelbine liposomes (Example 41, loading drug / phospholipid ratio was 350 mg / mmol) with F5-PEG conjugate. DSPE (Example 19) in a 20 mM aqueous solution buffered with HEPES-Na, 135 mM NaCl, pH 6.5 in a protein / phospholipid ratio of 15 mg / mmol at 60 ° C for 30 minutes. The unconverted F5 conjugate was removed by gel chromatography on a Sepharose 4B column eluting with the same buffer. Non-targeted liposomes (Ls-Pn-VRB) and HER2-targeted (F5-ILs-Pn-VRB) were intravenously administered to female Albino rats (8-9 weekly, 200 g) per 5 mg VRB / kg dose. Blood samples were taken at various time points as described in Example 9 and analyzed for VRB and lipid liposome as in Example 41. The half-life of lipid lipids in the blood and the release time of 50% drug were calculated from the concentration versus time graphs or drug ratio plots. / lipid against time, respectively, by finding the best match to the monoexpotent kinetics by means of Microsoft Excel (Microsoft Corp.) the TREND spreadsheet function. The results (Figure 18) indicated that both targeted and non-targeted vinorelbine liposomes had identical drug and lipid pharmacokinetics with a lipid half-life of about 12.1 hours and 50% drug release over about 4.3 hours. The half-life of lipid lipids in the blood and the release time of 50% of the drug were calculated from plots against time or drug / lipid ratio vs. time, respectively, by finding the best match to the monoexpotent kinetics using the Microsoft Excel (Microsoft Corp.) TREND spreadsheet function. The results (Figure 18) indicated that both targeted and non-targeted vinorelbine liposomes had identical drug and lipid pharmacokinetics with a lipid half-life of about 12.1 hours and 50% drug release over about 4.3 hours. The half-life of lipid lipids in the blood and the release time of 50% of the drug were calculated from plots against time or drug / lipid ratio vs. time, respectively, by finding the best match to the monoexpotent kinetics using the Microsoft Excel (Microsoft Corp.) TREND spreadsheet function. The results (Figure 18) indicated that both targeted and non-targeted vinorelbine liposomes had identical drug and lipid pharmacokinetics with a lipid half-life of about 12.1 hours and 50% drug release over about 4.3 hours.
Example 44. Preparation and in-vivo comparison of the stability of vinorelbine liposomes prepared with ammonium salts and substituted ammonium salts.
Ammonium dextran sulphate solution (DS-A) pH 5.8, 0.65 M NHU, osmolality from 390 mmol / kg and triethylammonium dextran sulphate solution (DS-TEA) at pH 6.0, 0.65 M NH4 + o osmolality 465 mmol / kg was prepared from dextran sulfate with molecular weight 10,000 (Sigma Chemical Co.), according to the method described in example 4, using titration with 12.4 M aqueous ammonia solution or pure triethylamine, respectively. Ammonium sulphate (SA), aqueous solution 325 mM, pH 5.1, osmolality 703 mmol / kg, prepared with an analytical grade of ammonium sulphate. All three solutions contained less than 1% Na + total cation. Liposomes that enclose these solutions were made using the method of mixing and extrusion with ethanol from Example 41 (DSPC / Cholesterol / PEG-DSPE, molar ratio 3: 2: 0.015). Radioactive lipid tracer [<sup>3</sup>H] -CHE was included in the lipid matrix at a concentration of 1.5 mCi / mmol phospholipid. The extrusion step consisted of passages through two stacked polycarbonate membranes with a pore size of 0.1 μm. VRB was added to liposomes in an aqueous solution of 5 mM HEPES-Na, 5% dextrose, pH 6.5, at a drug to phospholipid ratio of 350 pg / pmol, the pH was adjusted to 6.5 with 1 N
NaOH and the mixture was incubated at 58-60 ° C for 30 minutes. The reaction mixture was then cooled on ice for 15 minutes, and the non-thermographed drug was removed through a Sephadex G-75 column by gel permeation chromatography, eluting with an aqueous solution of 20 mM HEPES-Na, 135 mM NaCl, pH 6.5. The purified liposomes loaded with vinorelbine were examined spectrophotometrically for VRB and phospholipid using the Bartlett phosphate test (1959) (see examples 70, 71). The pharmacokinetics of the liposomal lipid and drug in blood were tested in rats as in Example 43.
The results are shown in Figure 19-20 and in Table 27. The liposomes loaded with triethylammonium dextran sulfate were compared with the liposomes loaded with the ammonium dextran sulfate salt. Surprisingly, liposomes loaded with the triethylammonium salt were much more stable than those loaded with the ammonium salt. The pharmacokinetics of the liposomal carrier alone was similar in three different formulations and was therefore mainly dependent on the lipid composition. The leakage of vinorelbine loaded with Ls-VRB using triethylammonium dextran sulfate was about three times slower than with those liposomes loaded with ammonium dextran sulfate. Liposomes loaded with ammonium sulphate had the fastest rate of drug leakage.
Table 27. Comparative in vivo stability of drug encapsulation in liposomes using closed ammonium salt and substituted salts.
<td>Preparation, salt</td><td>Size</td><td>half-life</td><td>Time to 50% release of the drug</td>
<td>closed in</td><td>liposomes, nm,</td><td>lipid matrix</td><td>in blood, hours</td>
<td>liposome</td><td>mean ± SD (incl using QELS)</td><td>in blood, hours</td><td></td>
<td>DS-TEA</td><td>120.8 ± 28.5</td><td>9.5 ± 3.3</td><td>66.3 ± 13.4</td>
<td>DS-A</td><td>107.8 ± 15.4</td><td>11.2 ± 0.6</td><td>22.9 ± 1.7</td>
<td>ARE</td><td>114.5. ± 15,6</td><td>10.7 ± 0.2</td><td>1.77 ± 0.16</td>
Example 45. In vivo stability and preparation of lixosomes loaded with vinorelbine of various sizes.
[<sup>3</sup>H] -CHE-labeled liposomes (1.5 mCi / mmol phospholipid) with closed triethylammonium sucrose octosulfate (0.65 M TEA, pH 6.4, osmolality
502 mmol / kg) were prepared by the method of mixing and extrusion with ethanol from example 11. The extrusion step contains passages through two stacked polycarbonate films with a pore size of 0.05, 0.08 or 0.1 μm. The loading of vinorelbine, vinorelbine liposomes isolation and liposome characteristics were described in example 40. female albino rats (8-9 week old, 200 g) were used to study the stability of liposomes in vivo. Lipid liposomes and drug pharmacokinetics in rats were tested as in Example 43.
The results are shown in Figure 21, 22 and Table 28 below. The liposomes extruded through polycarbonate filters with a pore size of 0.05, 0.08 and 0.1 μm filters were compared and presented to have similar pharmacokinetics of the drug and liposomal carrier as well as a similar degree of leakage. Drug release from blood liposomes is characterized by 50% release over a period of approximately 40-80 hours, well over 24 hours.
Table 28. Characteristics of vinorelbine liposomes.
<td>Size liposomes, nm, mean ± SD (incl using QELS)</td><td>Loading drug, mg / mole phospholipid</td><td>Effectiveness loading,%</td><td>Half-life matrix lipid in the blood, hours</td><td>Time up to 50% release of the drug in blood, hours</td>
<td>87.6 ± 28.1</td><td>352.4 ± 13.9</td><td>100.7 ± 4.0</td><td>14.6 ± 0.7</td><td>39.7 ± 3.1</td>
<td>98.5 ± 15.1</td><td>322.6 ± 22.7</td><td>92.2 ± 6.5</td><td>13.0 ± 0.2</td><td>47.9 ± 3.8</td>
<td>109.6 ± 24.6</td><td>357.0 ± 10.5</td><td>102.0 ± 3.0</td><td>14.3 ± 0.3</td><td>78.0 ± 1.4</td>
Example 46. Preparation of HER2-targeted vinorelbine liposomes to the TEA-SOS closure and HER2 pharmacokinetics of scFv-directed immunoliposomal vibrorelbine and non-targeted immunoliposomal vinorelbine in rats.
The liposomes were prepared, loaded with vinorelbin at a drug / phospholipid ratio of 350 mg / mmol, and analyzed as described in Example 43, except that the TEA-SOS solution of Example 45 was replaced by a TEA-Pn solution. The extrusion step included passages through polycarbonate filters with a pore size of 0.08 microns.
The size of the liposomes was 95.0 ± 26.0 nm and was measured using QELS. F5scFv anti-HER2 conjugated vororelbine immunoliposomes were prepared from these vinorelbine liposomes, and the pharmacokinetics of lipid lipids in the blood and drug with the HER2 and non-targeted vinorelbine liposome was examined in rats as described in Example 43. The circulating half-life of the lipid liposome was 11, 4 hours and 10.3 hours, and the release time of 50% of the drug was 30.9 hours and 30.3 hours for F5-ILs-VRB and Ls-VRB, respectively. Thus, the pharmacokinetics of the lipid and the drug Ls-VRB and F5-ILs-VRB were very similar, indicating that the introduction of the scFv-PEG-DSPE conjugate did not affect the clearance of the carrier alone or cause increased leakage of drug from the carrier in the circulation (figs 23, 24).
Example 47. Preparation and pharmacokinetics of vinorelbine liposomes containing non-ionic lipid derivatives derived from poly (ethylene glycol).
Methoxy-PEG (molecular weight 2,000) of a degraded synthetic C20 -ceramide (ceramide-PEG) was obtained from Northern Lipids, Inc., Canada. Methoxy-PEG (molecular weight 2,000) -distearoylglycerol (PEG-DSG) (SUNBRIGHT GS20) was from the company NOF Corp., Japan.
Liposomes containing a lipid composition of DSPC, cholesterol and PEG-lipids (ceramide-PEG or PEG-DSG) in a molar ratio of 3: 2: 0.3 and a closed solution of TEA-SOS (0.65 M TEA, pH 6.4, osmolality 502 mmol / kg) were prepared by the ethanol mixing / extrusion method of Example 11. The extrusion step involves 2 passes through two stacked polycarbonate filters with a pore size of 0.2 μm and 10 passes through the same filters with a pore size of 0.08. pm. The liposomes were loaded with vinorelin at a drug / phospholipid ratio of 350 mg / mmol, characterized by size, drug and lipid concentration and their pharmacokinetic properties were tested in rats as in Example 46. Both formulations showed increased circulation time of the lipid matrix and slow drug release in vivo
Table 29. Characterization of vinorelbine liposomes with various PEG-lipids.
<td>lipids-PEG Size</td><td>Loading</td><td>Effectiveness</td><td>half-life</td><td>Time up to 50%</td>
<td>liposomes,</td><td>drug, mg / mole</td><td>loading%</td><td>matrix</td><td>release of the drug</td>
<td>nm, mean ±</td><td>phospholipid</td><td></td><td>lipid w</td><td>in blood,</td>
<td>SD (for</td><td></td><td></td><td>blood, hours</td><td>hours</td>
<td>using QELS)</td><td></td><td></td><td></td><td></td>
<td>PEG-ceramide</td><td>103.3 ± 30.9</td><td>291.4 ± 18.0</td><td>83.26 ± 5.14</td><td>14.0</td><td>102.7</td>
<td>PEG-DSG</td><td>101.3 ± 20.1</td><td>359.3 ± 7.2</td><td>102.7 ± 2.1</td><td>15.1</td><td>24.6</td>
Interestingly, the increased PEGylation of these liposomes (the PEG lipid content was about 5.7 mol% of the total lipid) practically had no effect on the viability of the liposomes in the bloodstream compared with similar size matched liposomes containing low PEGylation of 0.3 mol. % of total lipids (example 45, 109.6 nm, tt / 2 = 14.3 hours, 98.5 nm, t 1/2 = 13.0 hours).
Example 48. Preparation of HER2-targeted liposomal vinorelbine and cytotoxicity of free, HER2-directed and non-targeted liposomal vinorelbine against MDA-MB-453 cells in vitro.
Liposomes loaded with vinorelbine (Ls-VRB) were prepared as in Example 42 (without<sup>3</sup>H] -CHE) using a drug loading at pH 6.0 and 350 pg vinorelbine / pmol phospholipid. Anti-HER2 immunoliposomal vinorelbine (F5-ILs-VRB) was prepared by incubating these liposomes with the F5-PEG-DSPE conjugate as described in Examples 19 and 42 above, except that they were not added [<sup>3</sup>H] -CHE. The "free" vinorelbine was prepared by diluting vinorelbine vinegar with 10 mg / ml USP solution with cell culture medium.
MDA-MB-453 are human breast adenocarcinoma cells (American Type Culture Collection, Rockville, MD) in which the HER2 receptor overexpression is slightly affected (approximately 3x10<sup>4</sup> do1x10<sup>5</sup> copies / cell). Cytotoxicity of VRB delivered as a free drug as an un-targeted liposomal vinorelbine or HER2-focused (F5) immunoliposomal vinorelbine against MDA-MB-453 cells was designated as described in Example 27, except cells were plated in 96 well microtiter plates in Growth conditions recommended by the supplier (Leibowitz L-15 with 10% fetal calf serum without supplementation of CO2) at a density of 10,000 cells / well, and drug formulations were added in a gradation series of 1: 3, starting at 0.03-0.1 mg / ml. Cell survival data were plotted against drug concentration (Figure 25) and the drug concentration required to reduce cell viability by 50% (IC50) were estimated from the plots. IC50 vinorelbine liposome directed to F5 at concentration 0, 06 pg / ml) was similar to free drug (0.07 pg / ml) and substantially lower than non-targeted liposomes (2.2 pg / ml). It is a 37-fold increase in activity resulting from specific targeted drug delivery to cancer cells.
Example 49. Cytotoxicity of free, HER2-directed and non-targeted liposomal vinorelbine against CaLu-3 cells in vitro.
The liposomes and methods of the previous example (example 48) were used to study the free vinorelbine cytotoxicity, Ls-VRB and F5-ILs-VRB in CaLu-3 cells of human HER2 overexpressing non-small cell lung cancer (American Type Culture Collection, Rockville, MD). Cells were grown in RPMI-1460 medium with 10% fetal calf serum in the presence of 5% CO2. The results are shown in Figure 26. The IC50 for free VRB was 1.2 pg / ml, 10 pg / ml for F5-ILs-VRB and 50 pg / ml of non-targeted Ls-VRB. This is a 5-fold increase in drug activity in liposomes understood as a function of targeted delivery to cells.
Example 50. Cytotoxicity of free, HER2-directed, non-targeted liposomal vinorelbine against SKbr-3 cells in vitro.
The liposomes and methods of Example 48 were used to study the free vinorelbine cytotoxicity, Ls-VRB and F5-ILs-VRB in SKBr-3 cells of human HER2 overexpressing breast cancer (American Type Culture Collection, Rockville, MD), except that cells grown in McCoy 5A medium supplemented with 10% fetal calf serum in the presence of 5% CO2, then seeded at 5,000 cells / well, and the drug was incubated with the cells for 6 hours.
The results are shown in Figure 27. The IC50 for free VRB was 0.28 pg / ml, 0.17 pg / ml for F5-ILs-VRB and 0.8 pg / ml of non-targeted Ls-VRB. This is a 4.7-fold increase in drug activity understood as a function of targeted delivery to cells.
Example 51. Efficacy of anti-cancer liposomal vinorelbine in vivo in human HT29 colon carcinoma xenografts in mice.
Small unilamellar vesicles of liposomes (93.2 ± 26.4 nm with QELS) were prepared from distearoylphosphatidylcholine, cholesterol, PEG-DSPE (molar ratio 3: 2: 0.045) by hydration from a concentrated ethanolic solution in an aqueous solution of triethylammonium succinate Octane (0) , 6 M triethylamine pH 5.7-6.2) followed by repeated extrusion through polycarbonate films (pore size 100 nm), removal of extraliposomal polyanion salt and loading with vinorelbine by incubation with liposomes in isoosmotic buffer pH 6.5 drug / lipid ratio of 325 mg VRB / mmol phospholipid at 60 ° C as described in Example 42.
100
Homozygous females of nude BALB / c mice (6-8 weeks old, weighing 17-20 g) were injected subcutaneously in the lumbar region with 1x10<sup>6</sup> human HT-29 colon carcinoma cells (American Type Culture Collection, Rockville, MD). Starting from the 16th day after tumor inoculation, when the tumor reached a mean diameter of 5-8 mm, the mice were randomly divided into three groups of six animals each and treated with free or liposome vinorelbine at a dose of 5 mg / kg via the tail vein every three days for a total of four injections. In the control group, mice were treated with an equal volume of saline solution. The tumor size of each mouse was measured using a vernier caliper and the tumor volume was calculated using the following formula: (tumor length) x (tumor width)<sup>2</sup>/ 2. To assess the toxicity associated with treatment, the animals were also weighed twice a week. Liposomal vinorelbine was found to be much more effective in inhibiting the growth of HT-29 cancer cells than free vinorelbine, causing tumor regression, while in the free drug group, tumors always continued to grow (Figure 28). There were slight changes in body weight in animals during treatment, indicating that treatment is well tolerated and that liposomalisation does not increase drug toxicity (Figure 29).
Example 52. Efficacy of anti-cancer liposomal vinorelbine in vivo against syngeneic mouse C-26 colon cancer.
Liposomal vinorelbine and free vinorelbine were prepared as in Example 48. Male BALB / c mice (6-8 weeks old, weighing 17-20 g) were vaccinated subcutaneously with 2x10<sup>5</sup> mouse C-26 colon carcinoma cells. On day 17 after inoculation, when the tumor diameter reached 5-8 mm, the mice were randomly divided into six treatment groups of five animals / group. The tumor was injected to the mice via the tail vein with free vinorelbine at a dose of 6 mg / kg, 8 mg / kg or 12 mg / kg, and liposomal vinorelbine at a dose of 4 mg / kg or 6 mg / kg every three days, for a total of four injections. . In the control group, the mice were treated with an equal volume of normal saline solution in water. Tumor size and animal body weight were monitored as in Example 51. Liposomal vinorelbine, even at a dose of 4 mg / kg, was significantly more effective in reducing tumor growth than free drug at a dose of 12 mg / kg (Figure 30). The body weight of the animal during the treatment showed slight changes (<10% decrease), which indicated that
Example 53. Efficacy of HER2-directed HER2-directed anti-tumorigenic vinorelbine in a xenograft of human BT-474 human breast tumors:
the loading efficiency of the counterion.
The VRB loading size of 99.5 ± 10.2 nm was prepared using the TEA-Pn method from Example 41 and the TEA-SOS from Example 42, respectively
101 except that [<sup>3</sup>H] -CHE has not been added. VRB was loaded at a drug / phospholipid ratio of 350 mg / mmol. HER2-directed liposomal vinorelbine was formulated by incubating these liposomes with the F5-PEG-DSPE conjugate (see example 19) as described in Example 43. Human HER2 overexpressing HER2 precancerant breast cancer cells were cultured in homozygous nude mice as described in Example 10. On day 25 after tumor cell insertion, when the tumors reached a volume of about 200 mm 3 (size range 144-309 mirP), the mice were randomly divided into four groups of five animals / group and treated intravenously with 5 mg / kg free VRB, F5 -ILs-VRB with Pn as the counterion, or F5-ILs-VRB with SOS as the counterion, at a dose of 5 mg / kg for one week for a total of three injections. The control group received injections of equal volume from normal saline. Tumor weight and animal body weight were monitored as in Example 10. Liposomal HER2 targeted liposomalate loaded with sucrose octasulfate was significantly more effective in reducing tumor growth than the same targeted liposomal unit loaded with poly (phosphate), and both immunoliposomal preparations were significantly more effective than free vinorelbine, given at a dose of 5 mg VRB / kg (figure 32). Mice treated with the drug showed slight changes in body weight, indicating that treatment is well tolerated (Figure 33). The HER2-targeted liposomal loading loaded with sucrose octasulfate was significantly more effective in reducing tumor growth than the same targeted liposomal unit loaded with poly (phosphate), and both immunoliposomal preparations were significantly more effective than free vinorelbine, given at 5 mg VRB / kg (figure 32). Mice treated with the drug showed slight changes in body weight, indicating that treatment is well tolerated (Figure 33). The HER2-targeted liposomal loading loaded with sucrose octasulfate was significantly more effective in reducing tumor growth than the same targeted liposomal unit loaded with poly (phosphate), and both immunoliposomal preparations were significantly more effective than free vinorelbine, given at 5 mg VRB / kg (figure 32). Mice treated with the drug showed slight changes in body weight, indicating that treatment is well tolerated (Figure 33).
Example 54. Efficacy of HER2-directed HER2-targeted anti-cancerous vinorelbine in vivo in a xenograft of human BT-474 breast tumors in mice:
PEGylation efficiency.
The DSPC liposomes and cholesterol in a molar ratio of 3: 2 were prepared according to example 48 by hydration of the DSPC lipid matrix, cholesterol and PEGdistearoylglycerol with a PEG molar mass of 2,000 (GS-20, NOF Corp., Japan) in a molar ratio of 3: 2: 0.015 ( "0.5% PEG") or 3: 2: 0.3 ("10% PEG") using an ethanolic solution in an aqueous triethylammonium sucrose octane solution followed by extrusion through a film according to example 48. VRB was loaded into liposomes at a ratio of drug / phospholipid 350 mg / mmol. Immunoliposomal F5-directed vinorelbine was formulated by incubating these liposomes with the F5-PEG-DSPE conjugate (example 19) as described in Example 43. Nude mice bearing BT-474 xenografts were cultured and treated intravenously with free VRB, F5-ILs- VRB- "0,
102
Example 55. Efficacy of anti-tumor lipid immunity directed against EGFR in vivo in a human xenograft xenograft of U87 in mice.
Liposomes (size 86.6 ± 12.9 nm measured with QELS) with an encapsulated 0.65 M TEA-SOS solution were prepared and loaded with VRB according to example 42. Anti-EGFR immunoliposomal VRB (C225Fab'-ILs-VRB) was prepared by incubating VRB liposomes with a PEG-DSPE conjugate against EGFR anti-EGFR antibody fragments as described in Example 36.
NCR male mice nu / nu (5-6 weeks of age, weighing 17-20 g) were injected subcutaneously in the lumbar region of 1x10<sup>7</sup> cells (ATCC) of human U87 glioblastoma suspended in culture medium, in a total volume of 150 μΙ. When the tumor reached an average size of 250 mm<sup>3</sup>, mice were randomly divided into four groups of 10-12 animals. Mice treated with three week intravenous injections of "free" VRB, non-targeted Ls-VRB or C225Fab'-ILs-VRB at a dose of 5 mg VRB / kg. The control group received injections of equal volume from saline. Tumor size and body weight of the animals were monitored as in Example 10. C225-Fab'-ILs-VRB was noticeably more effective in inhibiting the growth of human brain tumor EGFR overexpressing than either non-targeted liposomal vinorelbine or free vinorelbine in an equal dose (FIG. ).
Example 56. Preparation and pharmacokinetic properties of encapsulated doxorubicin in liposomes using the triethylammonium sulfate method.
Liposomes with various lipid matrix compositions (as indicated in the table below) were formulated as described in Example 2. N-Glutaryl-DSPE (Glu-DSPE) available from Avanti Polar Lipids, AL, USA. Pure lipid film formulated from the lipid solution in chloroform in a rotary evaporator, the volatiles were removed under vacuum conditions (90 μιτι Hg, 2 hours), the lipid membrane was hydrated with a solution of triethylammonium sulphate (TEA-SO4) (0.65 N TEA), subjected to six cycles of rapid freezing and thawing and 10 extrusion through two stacked polycarbonate filters with a pore size of 0.1 μιτι and ten times through pores with a size of 0.05 μιτι. To quantify the lipid matrix in blood samples, [3H] -CHE was included in the lipid matrix at a concentration of 0.51,5 mCi / mmol phospholipid. Liposomes with closed TEA-SO4 solution were loaded with doxorubicin according to example 2. Liposomes in saline solution in HEPES buffered water (20 mM HEPES-Na, 135 mM NaCl, pH 6.5) were incubated with doxorubicin hydrochloride (drug / phospholipid ratios 140-170) mg / mmol) at 60 ° C for 45 minutes, then quenched on ice and non-sealed doxorubicin was removed by gel permeation chromatography. doxorubicin
103 was determined spectrophotometrically (example 71) and the phospholipid was determined by the method
Bartlett (example 70). The properties of the resulting liposomes are summarized in Table 30 below.
Table 30. Liposomal properties of doxorubicin in various liquid compositions.
Lipid composition (molar ratio) Liposome size, nm Drug / phospholipid (mean ± SD after (mg / mmol) using QELS)
<td>DSPC / Chol / PEG-DSPE (3: 2: 0.015)</td><td>81.8 ± 27.3</td><td>163.6 ± 4.4</td>
<td>DSPC / Chol (3: 2)</td><td>79.1 ± 27.9</td><td>137.0 ± 17.5</td>
<td>DSPC / Chol / Glu-DSPE (2.85: 2: 0.15)</td><td>83.6 ± 27.2</td><td>141.7 ± 10.4</td>
<td>DSPC / Chol / PEG-DSPE (2.7: 2: 0.3)</td><td>83.7 ± 23.1</td><td>175.0 ± 6.8</td>
The pharmacokinetics of these liposomes in the blood containing doxorubicin and the 2.7: 2: 0.3 lipid composition were tested in rats following a single intravenous dose of 5 mg doxorubicin / kg, as described in Example 9. Liposomes remained in circulation for a long time (the half-life was around 28 hours) (figure 36). The stable ratio of doxorubicin to phospholipid indicates that the formulation was unusually stable to the drug spillage in circulation, losing less than 25% of the drug over the 48-hour period.
Example 57. Lipsomes loaded doxorubicin and anti-HER2 immunoliposomes prepared by the TEA-sulfate method: preparation and anti-tumor efficacy in vivo against human HER-overexpressing HER2 xenografts.
Liposomes loaded with doxorubicin having various lipid properties and compositions (listed in the table below) were prepared as described in Example 56. Anti-HER2 immunoliposomes loaded with doxorubicin were prepared from doxorubicin loaded liposomes by co-incubation with anti-HER2 scFv F5-PEG25 DSPE conjugate (about 30 scFv / liposome) as described in Example 19. NCR nu / nu mice were grown with subcutaneous human breast cancer xenograft (BT-474) that were treated (in groups of 10-12 animals) liposomal or anti-human -HER2 immunoliposomal doxorubicin at a dose of 5 mg / kg, once a week for three weeks in total, when the tumors reached an average volume of 200 mm<sup>3</sup>, tumor growth and animal body weight were monitored as
104 are described in example 29. For doxicated formulations of doxorubicin, lipid compositions not containing PEG-DSPE, 0.5 mol% PEG-DSPE, or 10 mol% PEG-DSPE, have been tested; for F5-immunolipsomal doxorubicin, preparations with 0.5 mole% PEG-DSPE and 10 mole% PEG-DSPE were tested (here the quantitatively expressed PEG-DSPE is expressed as mole%% phospholipid liposome). The results (Figure 37, Table 31) showed that all doxorubicin treatments were effective in delaying tumor growth. Based on tumor size at day 53 after implantation, differences in inhibition of tumor growth among all three groups treated with a non-targeted liposome were not statistically significant (ANOVA p = 0.081), but doxorubicin immunoliposom was significantly more effective than non-targeted liposomal doxorubicin (ANOVA p = 5.5 x 10 '<sup>10</sup>), the "10% PEG-DSPE" preparation was more effective than "0.5% PEG-DSPE" (Student's t test p = 0.027). In the F5-ILs group "10% PEG-DSPE", tumors regress to 1 mm<sup>3</sup> or less in 67% of animals, while in the F5-ILs group "0.5% PEG-DSPE" only in 9%. In the control group (treated with saline solution in water), tumors exceeded the limit of 15% of body weight in 38-43 days.
Table 31. Investigating the anti-tumor efficacy of liposomal doxorubicin in vivo. liposome characteristics and test results.
<td>Lipid composition</td><td>Size liposomes, nm (mean ± SD)</td><td>Ratio drug / phospholipid, mg / mmol (mean ± SD)</td><td>Medium size tumor at 58 days mm3 (average ± SEM)</td>
<td>DSPC / Chol / PEG-DSPE (3: 2: 0.015)</td><td>83.4 ± 23.3</td><td>136.7 ± 6.7</td><td>490 ± 74</td>
<td>DSPC / Chol (3: 2)</td><td>80.5 ± 26.6</td><td>151.2 ± 1.9</td><td>587 ± 61</td>
<td>DSPC / Chol / PEG-DSPE (2.7: 2: 0.3)</td><td>81.0 ± 24.7</td><td>140.1 ± 4.2</td><td>365 ± 60</td>
<td colspan="2">DSPC / Chol / PEG-DSPE (3: 2: 0.015) + F5 Not measured</td><td>140.7 ± 2.8</td><td>119 ± 39</td>
<td>scFv-PEG-DSPE</td><td></td><td></td><td></td>
<td colspan="2">DSPC / Chol / PEG-DSPE (2.7: 2: 0.3) + F5 Not measured</td><td>132.9 ± 2.2</td><td>15.5 ± 7.6</td>
<td>scFv-PEG-DSPE</td><td></td><td></td><td></td>
Example 58. Preparation of liposomal vinblastine and vinclasnine liposomal pharmacokinetics in rats.
Liposomes with closed aqueous TEA-SOS solution (0.65 M TEA, pH 6.4, osmolality 502 mmol / kg) and size 99.5 ± 10.2 nm (mean ± SD calculated using QELS) were prepared according to the method from example 11, using twice
105 extrusion with two stacked polycarbonate membranes with a pore size of 0.2 [mu] m and ten times with polycarbonate membranes with a pore size of 0.08 [mu] m. Vinblastine (VBL) in the form of USP vinblastine sulfate was added at a drug to phospholipid ratio of 150 mg / mmol. The pH of the drug / liposome mixture was adjusted to 6.5 with 1 N NaOH, and the mixture was then incubated at 60 ° C for 30 minutes. The reaction mixture was then cooled on ice for 15 minutes and the non-moisturized drug was removed through a Sephadex G-75 column by gel permeation chromatography, eluting with a 5 mM HEPES-Na solution, 135 mM NaCl, pH 6.5. The purified liposomes were analyzed for VBL spectrophotometrically and for phospholipids by Bartlett's method, as in Examples 70 and 71. [<sup>3</sup>H] -CHE was included in the formulation in a ratio of 1.5 mCi / mmol phospholipid. Liposomal vinblastine contained 152.4 ± 12.0 mg VBL / mmol phospholipid (encapsulation quantitatively).
The pharmacokinetics of liposomal vinblastine in blood in female Albino rats (8-9 weeks of age, body weight 200 g) at 5 mg VBL / kg tested as described in Example 9. Quantified vinblastine in blood plasma samples as described in Example 41 ( using vorgebra as an internal standard). Vinblastine liposomes showed a good viability in circulation (plasma half-life of the lipid component of 12.8 ± 0.04 hours) (figure 38) and very good stability against drug efflux from liposomes, more than 70% of initial vinblastine loading remained hermetically sealed after 24 h (figure 39). The time after injection needed to achieve the release of 50% of the encapsulated drug was 40.6 ± 1.2 hours.
Example 59. Preparation of liposomes loaded with vincristine using the TEA-SOS method and effect of pH on loading efficiency.
Liposomes size 86.6 ± 12.9 nm (calculated with QELS), lipid composition DSPC / Chol / PEG-DSPE in a molar ratio of 3: 2: 0.015 and closed aqueous solution of TEA-SOS (0.65 M TEA, pH 5.4, osmolality 521 mmol / kg) were prepared according to the method of Example 11, using an extrusion step comprising passing through two stacked polycarbonate membranes with a pore size of 0.08 μm. Vincristine (VCR) was added to liposomes in an aqueous solution of 5 mM HEPES-Na, 5% aqueous dextrose buffer, pH 6.5, at a drug to phospholipid ratio of 350 vincristine pg / pmol phospholipid, pH was adjusted to the ratio indicated with 1 N NaOH and the mixture was incubated at 60 ° C for 30 minutes, cooled on ice for 15 minutes, and liposomes were separated from the non-aerosolized drug by Sephadex G-75 column by gel permeation chromatography, eluting with HBS-6.5 (20 mM HEPES-Na, 135 mM NaCl, pH 6.5). The purified liposomes were analyzed for vincristine using spectrophotometry
106 using absorbance at 265 nm after dissolving in acidic isopropanol and for phospholipids using the Bartlett phosphate test (1959).
The results are shown in Table 32 below. The loading of the drug was above 5 90% in the pH range 4.5-7.5 and practically quantified at pH 5.0-7.5. At pH 3.5, which is observed in the liposome mixture after drug addition, but without pH regulation, the loading was significantly less efficient.
Table 32. pH dependence of vincristine loaded into liposomes with closed
TEA-SOS.
<td>pH</td><td>Drug / phospholipid ratio, pg / pmol</td><td>Loading efficiency (%)</td>
<td>3.5</td><td>39.7 ± 4.9</td><td>11.3 ± 0.2</td>
<td>4.5</td><td>327.2 ± 20.6</td><td>93.5 ± 5.4</td>
<td>5.0</td><td>360.6 ± 5.8</td><td>103.0 ± 1.7</td>
<td>5.5</td><td>371.2 ± 30.2</td><td>106.1 ± 9.1</td>
<td>6.0</td><td>347.7 ± 20.4</td><td>99.3 ± 5.8</td>
<td>6.5</td><td>347.7 ± 20.9</td><td>99.4 ± 5.9</td>
<td>7.0</td><td>377.3 ± 22.2</td><td>107.8 ± 6.8</td>
<td>7.5</td><td>371.5 ± 24.9</td><td>106.1 ± 7.6</td>
Example 60. Preparation of liposomes loaded with vincristine using the TEA-SOS method: effect of drug / lipid ratio on loading efficiency.
Liposomes containing SOS-TEA were prepared as in Example 59 and loaded with vincristine sulfate at drug to phospholipid ratio of 150-550 pg vincristine / pmol phospholipid at pH 6.5 according to the procedure of Example 59. Liposomes purified from non-aerosolized drug were analyzed for VCR by spectrophotometry and for phospholipid liposomes using Bartlett's test (1959). The loading efficiency of the drug was over 90% in the entire range of the drug / lipid drug test and practically quantified was between 150-450 pg vincristine / pmol phospholipid (table 33).
Table 33. Loading of vincristine into liposomes by the TEA-SOS method in different drug / lipid ratios.
107
<td>The drug input ratio to phospholipid (pg / pmol)</td><td>Input ratio encapsulated drug to phospholipid (pg / pmol)</td><td>Loading efficiency (%)</td>
<td>150</td><td>163.6 ± 6.6</td><td>109.0 ± 4.8</td>
<td>250</td><td>251.1 ± 17.0</td><td>100.5 ± 6.8</td>
<td>350</td><td>347.7 ± 20.9</td><td>99.4 ± 5.9</td>
<td>450</td><td>452.0 ± 18.8</td><td>100.4 ± 4.2</td>
<td>550</td><td>521.6 ± 24.9</td><td>94.8 ± 4.3</td>
Example 61. Preparation of immunoliposomal vincristine I liposomal cytotoxicity and immunoliposomal vincristine against tumor cells in vitro.
Liposomal vincristine (Ls-VCR) was prepared as described in Example 59, stating the amount of leUu / phospholipid 350 mg / mmol. F5-immunoiiposomal vincristine with specific HER2 (F5 ILs-VCR) was prepared from liposomal vincristine by co-incubation with anti-HER2 scFv F5-PEG-DSPE conjugate as described in example 19. "Free" vincristine solution (VCR) was prepared by dilution of USP vincristine sulfate in water followed by sterile filtration. cytotoxicity
VCR, Ls-VCR and F5-ILs-VCR against SKBr-3 (ATCC) cells of human HER overexpressing HER2 breast were determined by MTT based on a cell viability assay using the procedure of Example 27, in which cells were inoculated into 96-well plates. for microtitering at 5,000 cells / well, they were acclimated overnight and incubated with drug-containing medium for 4 hours, followed by incubation in drug-free medium for 3 days. The results are shown in Figure 40. The IC50 for the free VCR was 75 pg / ml, 11 pg / ml for F5-ILs-VCR and 3 pg / ml for Ls-VCR. The targeted liposomal vincristine prepared according to the disclosure was 6.8 times more active than the free drug and 273 times more active than the undirected liposomal drug, showing that
Example 62. Pharmacokinetics of Ls-VCR in blood in rats.
Liposomes with closed solution of SOS-TEA (0.65 M TEA, pH 5.8, osmolality
530 mmol / kg) and the lipid composition DSPC / Chol / PEG-DSPE (molar ratio 3: 2: 0.015), also containing [<sup>3</sup>H] -CHE at 1.5 mCi / mmol phospholipid were prepared as described in Example 11, using an extrusion step involving passage through two stacked polycarbonate films with a pore size of 80 nm and 100 nm.
108
Liposomes were loaded with VCR at pH 6.5, drug / phospholipid ratio
350 mg / mmol as described in Example 59. The VCR loaded liposomes were administered intravenously to albino female rats (180-220 g) at a dose of 5 mg VCR / kg, and the pharmacokinetics of the drug in the blood and lipid liposome were tested as described in the example
9. The amount of VCR in the blood samples was quantified by HPLC as described in Example 41, except that the volume ratio of aqueous triethylammonium acetate (pH 5.5) and acetonitrile in the mobile phase was 65:35. The typical retention time for the VCR was 8.8 minutes. The results are shown in Figure 41 and Table 34. Both formulations have an extended lifetime in circulation (blood half-life 12-17 hours).
Liposomal vincristine was extremely stable against drug leakage in both preparations (the half-life was over 120 hours) (Figure 42).
Table 34. Characterization of liposomes loaded vincristine at 350 mg / mmol phospholipid using the TEA-SOS method.
<td>Pore size</td><td>Size</td><td>Loading</td><td>ti / 2p lipid,</td><td>ti / Σβ VCR,</td><td>tt / 2 release</td>
<td>(through which he is</td><td>liposomes, nm</td><td>drug, mg / mole</td><td>hours</td><td>hours</td><td>VCR, hours</td>
<td>extruded</td><td>(mean ± SD)</td><td>phospholipid</td><td></td><td></td><td></td>
<td>substance), nm</td><td></td><td></td><td></td><td></td><td></td>
<td>80</td><td>101.2 ± 20.2</td><td>347.7 ± 20.93</td><td>17.5 ± 1.5</td><td>16.0 ± 2.0</td><td>> 120</td>
<td>100</td><td>125.6 ± 32.0</td><td>366,8 ± 18.11</td><td>12.1 ± 0.7</td><td>12.0 ± 0.8</td><td>undetectable</td>
Example 63. Pharmacokinetics of Ls-VCR in blood in rats at various drug / lipid ratios.
Liposomes with closed solution of SOS-TEA (0.65 M TEA, pH 6.4, osmolality 485 mmol / kg) and lipid composition DSPC / Chol / PEG-DSPE (molar ratio 3: 2: 0.015), also containing [<sup>3</sup>H] -CHE at 1.5 mCi / mmol phospholipid were prepared as described in Example 11, using an extrusion step involving passage through two stacked polycarbonate films with a pore size of 50 nm and 80 nm. Liposomes loaded with VCR at pH 6.5 as described in Example 59 by adding a stock aqueous solution of 20 mg / mL VCR sulfate in the calculated drug / lipid ratio of 100, 200 or 350 mg / mmol phospholipid. The loading efficiency of the drug was over 96% for all preparations. VCR loaded liposomes were administered intravenously to female albino rats (8-9 weeks old, weight 190-220 g) at a dose of 5 mg VCR / kg, and the pharmacokinetics of the drug in the blood and lipid liposome were tested as described in Example 62. The results are presented in table 35.
It is stable in all examined sizes and drug to lipid ratios (half-life of drug release in 93 hours).
Table 35. Characterization of liposomes loaded with vincristine using the TEA-SOS method at different drug / lipid ratios.
<td>Size</td><td>Size</td><td colspan="2">VCR, mg / mmol phospholipid</td><td>t1 / 2 lipid, t1 / 2 VCR, t1 / 2</td>
<td>pores (by Which is</td><td>liposomes, nm (average</td><td>added</td><td>encapsulated</td><td>hour of release drug, hours</td>
<td>extruded substance),</td><td>± SD)</td><td></td><td></td><td></td>
<td>nm</td><td></td><td></td><td></td><td></td>
<td>50</td><td>76.8 ± 27.2</td><td>100</td><td>96.1 ± 3.0</td><td>35.6 ± 2.7 30.3 ± 4.0 227 ± 96</td>
<td></td><td></td><td>200</td><td>193.3 ± 3.9</td><td>20.8 ± 2.2 18.4 ± 0.7 244 ± 130</td>
<td></td><td></td><td>350</td><td>375.2 ± 10.0</td><td>24.8 ± 0.9 19.6 ± 0.9 93.2 ± 6.7</td>
<td>80</td><td colspan="2">101.6 ± 25.3 100</td><td>104.5 ± 2.1</td><td>33.0 ± 7.6 26.8 ± 4.8 153 ± 10</td>
Example 64. Preparation of HER2-targeted liposomal vincristine and anti-tumor efficacy of non-targeted and HER2-targeted anti-tumor liposomal vincristine against HER2 overexpressing human breast cancer.
Liposomes loaded with vincristine (Ls-VCR-SOS) by the TEA-SOS method were prepared according to example 63 (omitting the component [<sup>3</sup>H] -CHE) using membrane extrusion with a pore size of 50 nm and drug loading in the drug / phospholipid 100 mg / mmol ratio. F5 immunoliposomal vincristine (F5-ILs-VCR) was produced by incubating Ls-VCR-SOS with anti-HER2 scFv F5-PEG-DSPE conjugate (example 19) as described in example 43. Liposomes loaded with vincristine using TEAcytrate (Ls -VCR-Cit) were prepared in a similar way to Ls-VCR-SOS liposomes, with the exception that a solution of triethylammonium citrate (prepared by titration with an aqueous citric acid solution with pure triethylamine, pH 5.1 and adjusted 0.65 M concentration triethylamine) was replaced with a TEA-SOS solution. The design of the study and treatment results from the description in Example 10. Subcutaneous xenografted human breast cancer BT-474 tumors were cultured in nude mice and when tumors reached 250 mm 3 (range 144-309 mm 3), mice in the eight to nine group were treated with free VCR, Ls-VCR or F5-ILs-VCR in a week with a VCR dose of 2 mg / kg, for a total of three weeks starting from day 19 after vaccine inoculation. Tumor size and body weight of the animals were monitored as described
110 in Example 10. In the control group, mice were treated with an equal volume of physiological saline. Differences in tumor size between treatment groups were statistically evaluated on day 63 after tumor implantation using the Mann-Whitney test. The dynamics of mean tumor size in groups is shown in Figure 43. F5-ILs-VCR showed maximum efficacy compared to both Ls-VCR or free VCR, resulting in complete tumor regression on six days in six of eight animals (75%). Ls-VCR-Cit was also effective, resulting in a complete tumor regression still observed on day 63 in two out of nine animals (22%), but was less effective than F5-ILs-VCR (p <0.005). Ls-VCR-SOS and free VCR were equally effective (p> 0.2) and less effective than the use of one of F5-ILs-VCR or Ls-VCR-Cit. So, unexpectedly, with targeted delivery to the cell, the liposomal encapsulated drug with the multivalent anion of the present disclosure has been shown to be more effective than the liposomal encapsulated drug delivered via the non-binding anion. Animal body weight dynamics showed that all VCR liposomal preparations were less toxic than free VCR, resulting in less weight loss during treatment (Figure 44).
Example 65. Preparation of EGFR-targeted liposomal vincristine and anti-tumor efficacy of anti-tumor EGFR-targeted liposomal vincristristin targeted at EGFR.
The vincristine-loaded liposomes (Ls-VCR) were prepared by the TEA-SOS method as in Example 64. Immunoliposome vincristine targeted at EFGR was prepared by co-incubating liposomes with the anti-HER2 Fab 'C225Fab-PEG-DSPE conjugate as described in example 36.
Female NCR nu / nu mice (5-6 weeks old, weighing 17-20 g) were injected subcutaneously in the lumbar area with 0.15 ml of cell growth medium containing 1x10<sup>7</sup> U87 human glioma cells stably expressing the epidermal growth factor (HER1) EGFRvIII mutation receptor. On day 11, when the tumor reached an average size of 300-400 mm<sup>3</sup>, mice were randomly divided into four groups of 10-12 animals / group. Treatment with free VCR (vincristine sulfate 1 mg / ml saline solution in water), Ls-VCR or C225Fab-ILs-VCR in an intravenous dose of 1.5 mg / kg administered on days 11, 18 and 25 after tumor inoculation. Mice in the control group were treated with the same injections with an equal volume of ordinary saline in water. The size of the tumor and the weight of the mice were monitored as in Example 10. The results are shown in Figure 45. All animals treated with VCR showed inhibition of tumor growth as compared to control animals. There was no significant one
111 difference between the free VCR and Ls-VCR treated groups. C225Fab-ILs-VCR focused on
EGFR was more effective than free or non-targeted liposomal VCR.
Example 66. Preparation of liposomes with closed triethylammonium solution of inositol hexaphosphate (TEA - HP).
The polyanionic polyol, dodecasodium salt of inositol hexaphosphate (-HP) was obtained from Sigma (St. Louis, MO). An aqueous solution containing 0.65 M triethylammonium and 0.681 M phosphate groups at pH 6.5, with an osmolality of 718 mmol / kg, was prepared by a Dowex 50Wx8-200 ionisable crosslinked with a sulfonated polystyrene resin and then titrated with pure TEA and diluted with water according to the procedure from Example 4. The residual sodium content was less than 1% of all cations. Dry lipids (150 pmol DSPC, 100 pmol Chol, 0.75 pmol PEG-DSPE) were dissolved in 0.5 mL of 100% ethanol USP at 60 ° C and mixed with a 4.5 ml solution of triethylammonium inositol hexaphosphate preheated to the same temperature. The ethanol was partially removed on a rotary evaporator at 30-40 mm Hg and a temperature of 40-45 ° C until the mixture started boiling. The lipid suspension was then extruded 15 times at 60-65 ° C with two stacked polycarbonate membranes with a pore size of 0.1 μm. The resulting liposomes were 104.3 ± 39.0 nm and were measured using QELS. The insoluble triethylammonium -HP was removed by gel chromatography on a Sepharose 4B column eluted with 5 mM HEPES-Na, 5% dextrose, buffer pH 6.5, and liposomes were quantified by phospholipid concentration using the Bartlett method along with the extraction of example 70.
Example 67. Loading of drugs into liposomes with a closed TEA-HP solution.
The liposomes of example 67 were loaded with CPT11 or vinorelbine. Vinorelbine was loaded at a drug to phospholipid ratio of 175 or 350 g / mol, and CPT11 at a ratio of 250 or 500 g / mol. Drugs were added to the liposomes in HEPES-dextrose buffer (Example 67) in the drug / phospholipid input ratios indicated below (see Table 36). If necessary, the pH value is adjusted to 6.5-6.8 using 1 N NaOH. The mixtures were incubated at 60 ° C for 30 minutes, chilled on ice for 15 minutes, and then chromatographed on a Sephadex G-25 column with filter gel, eluted with 5 mM HEPESNa, 145 mM NaCl, pH 6.5. Portions of purified liposomes were dissolved in acidified methanol and analyzed by spectrophotometry (example 71). The phospholipid was quantified by Bartlett's (1959) method with extraction (example 70). Both drugs were loaded quantitatively (i.e., practically 100%) into liposomes as shown in Table 36 below.
112
Table 36. Properties of drugs loaded into liposomes with closed inositol hexaphosphate.
<td>Bow</td><td>Input ratio drug / lipid, g / mol phospholipid</td><td>The ratio of encapsulation drug / lipid, g / mol phospholipid</td><td>Effectiveness loading,%</td>
<td>vinorelbine</td><td>175</td><td>175.3 ± 8.0</td><td>100.2 ± 4.5</td>
<td>vinorelbine</td><td>350</td><td>352.3 ± 11.8</td><td>100.6 ± 3.3</td>
<td>CPT-11</td><td>250</td><td>265.1 ± 11.2</td><td>106.1 ± 4.7</td>
<td>CPT-11</td><td>500</td><td>518.7 ± 27.8</td><td>103.7 ± 5.8</td>
Example 68. Chemical stability of free or liposomal CPT-11 in the presence of mouse plasma in vitro.
In the body, CPT-11, which is a prodrug, undergoes a chemical treatment to form the active metabolite of a drug known as SN-38. Both SN-38 and CPT-11 are also converted from their active lactone species to inactive products known as SN-38 or CPT-11 carboxylates. This example examines the effect of CPT-11 liposomalisation in accordance with the present invention on the chemical conversion of CPT11 in these products in the presence of blood plasma. Liposomes containing trimethylammonium succinate closed acetate (0.65 M TEA, pH 6.4, osmolality 485 mmol / kg) and lipid composition DSPC, cholesterol and PEG-DSPE in a molar ratio of 3: 2: 0.015 were prepared according to example 11 using tenfolds extrusion through two stacked polycarbonate filters with a pore size of 0.08 pm. Liposomes were 87.4 ± 19 in size, 2 nm and measured using QELS. CPT-11 was loaded in an amount of approximately 500 mg of basic CPT-11 / mmol phospholipid liposome by incubation in aqueous 5 mM HEPES20 Na, 5% dextrose, pH 6.5, 60 ° C for 30 minutes and then by cooling in ice for 15 minutes. The CPT-11 loaded liposomes were then purified on a Sephadex G-75 column which was eluted with saline in HEPES buffered water (5 mM HEPES, 145 mM NaCl, pH 6.5). The obtained CPT-11 liposomes had 565.5 ± 20.1 mg CPT-11 / mmol phospholipid. The free CPT-11 solution was prepared with freshly dissolved Irinotecan hydrochloride USP at a dose of 1 mg / ml in 144 mM aqueous NaCl, acidified to pH 3 with HCl solvent. Ten-pl portions of free or liposomal CPT-11 or free CPT-11 were mixed with 90 μl of heparin-stabilized plasma (Harlan Bioproducts, USA) and incubated at 37 ° C in a shaking water bath. At a given time point, liposome samples, in triplicate,
113 were chromatographed on a Sepharose CL-4B size exclusion column (2 ml bed volume) eluted with HBS-6.5 and drug-containing fractions detected by fluorescence. The first (empty volume) and the second (final) drug-containing peak were collected and considered as liposomal and released drug-drug fractions. The samples were extracted at
400 μΙ ice-cold methanol by vortexing for 10 seconds, then centrifuged at 14,100 x g for 5 minutes. The supernatants were analyzed for CPT-11 and its HPLC conversion products, using a modification of the Warner and Burke method, J Chromatogr, Ser. B Biomed. Sci. Appl. 1997, vol. 691, pp. 161-71. The mobile phase consisted of 3% triethylammonium acetate pH 5.5 (solution A) and acetonitrile (solution B) delivered at a dose of 1.0 ml / minute in a linear gradient of 20 vol%. B to 50% vol B in 14 minutes. The eluted products were detected by fluorescence at excitation at 375 nm and 500 nm emission. The retention time was 5.3 min (CPT-11 carboxylate) 6.8 min (SN-38 carboxylate), 9.3 min (CPT-11) and 11.0 min (SN-38). The results (Table 37) showed that while releases from the free CPT-11 and CPT-11 liposomes were converted,
Table 37. Conversion of free and liposomal CPT-11 into the SN-38 form and a carboxylate form in mouse plasma in vitro.
<td rowspan="2">A sample</td><td rowspan="2">Time, hours</td><td>CPT-11,%</td><td colspan="3">SN-38,%</td>
<td>lactone</td><td>carboxylate</td><td>lactone</td><td>carboxylate</td>
<td>Free CPT-11</td><td>2</td><td>1.9 ± 0.4</td><td>35.2 ± 1.9</td><td>4.4 ± 0.1</td><td>58.4 ± 2.1</td>
<td></td><td>12</td><td><0.1</td><td>11.5 ± 0.9</td><td>9.9 ± 0.8</td><td>78.6 ± 1.3</td>
<td></td><td>24</td><td><0.1</td><td><0.1</td><td>22.5 ± 9.8</td><td>77.5 ± 9.8</td>
<td>Ls-CPT-11</td><td>12</td><td>97.7 ± 0.1</td><td><0.1</td><td>2.3 ± 0.1</td><td><0.1</td>
<td>(Encapsulated)</td><td>24</td><td>97.7 ± 0.1</td><td><0.1</td><td>2.3 ± 0.1</td><td><0.1</td>
<td>Ls-CPT-11</td><td>12</td><td>60.5 ± 10.4</td><td>25.0 ± 7.1</td><td>5.0 ± 0.3</td><td>9.5 ± 3.0</td>
<td>(freed)</td><td>24</td><td>78.3 ± 6.7</td><td>14.0 ± 5.2</td><td>6.5 ± 0.5</td><td>1.2 ± 1.7</td>
Example 69. Chemical Stability of free or lipomic CPT-11 in rats in vivo.
The liposomal CPT-11 was prepared as in Example 68 with triethylammonium sucrose acetate sulfate 0.65 M TEA, pH 6.4
114 and an osmolality of 502 mmol / kg. The liposome was 98.5 ± 18.4 nm and the CPT-11 encapsulation was 510.1 ± 16.5 mg CPT-11 / mmol phospholipid. Liposomal and free CPT-11 were administered to female Albino rats (180-220 g) intravenously at a dose of 25 mg / kg, permanently attached to central venous catheters, blood samples were collected at intervals of 48 hours. Blood samples were mixed with ice-cold PBS containing 0.04% EDTA and rapidly centrifuged to remove blood cells. Aliquots of liquid supernatants were analyzed for CPT-11, SN-38, and their carboxylate forms by HPLC, as in Example 68 above. The results are shown in FIGS. 46 and 47. In contrast, the free CPT-11 was purified very quickly, being undetectable after 30 minutes, the liposomal CPT-11 was maintained in circulation (t / 2 15.2 hours) from 37, 8% of the drug in the blood after 24 hours and approximately 10% of the drug was still in circulation after 48 hours. There was no detectable transformation of the liposomal form of CPT-11 either to SN-38 or to the carboxylate form of CPT-11. Free CPT-11, i.e. administered as a solution, removed from the circulation fairly quickly (half-life of about 16 minutes) and was significantly converted to the drug carboxylate form.
Example 70. Quantification of phospholipid liposomes.
Modified acid digestion -1 way of blue. The phosphomolybdate method. This method is modified after Bartlett (1959). 10-20 ml portions of liposomes were placed in heat-resistant glass test tubes, etched by heating with 0.5 ml of 10 N sulfuric acid for 2 hours at 110-130 ° C, milled by addition of 50 ml of 9% hydrogen peroxide solution and heated for an additional 30 minutes. minutes until the disappearance of hydrogen peroxide so that it is not detectable by the indicator which was the litmus paper. The etched samples at room temperature were diluted in 1 ml of 0.2% aqueous ammonium molybdate, mixed with 0.1 ml of a 5% aqueous ascorbic acid solution and incubated in a water bath for 10 minutes.
Modified acid digestion - II way of blue phosphomolybdate. This method is a modification of the method developed by Morrison (1964). 5 μΙ portions of liposomes containing 1-10 mM phospholipid were mixed with 60 μl concentrated sulfuric acid and 10 μl with a 30% solution of hydrogen peroxide in heat-resistant glass test tubes. The mixtures were heated at 200-220 ° C for 10 minutes, diluted with 0.7 μΙ of deionized water, mixed with 10 μΙ of a 10% aqueous solution of sodium sulfite, incubated in a water bath for 5 minutes and cooled to ambient temperature. 200 μΙ of a 2% aqueous solution of ammonium molybdate was added
115 and 10 μΙ of a 10% aqueous solution of ascorbic acid, the samples were incubated in a water bath for 10 minutes. The samples were quickly cooled to ambient temperature, and the reduced absorbance of the phosphomolybdate complex was set at 825 nm relative to the blank. The amount of phospholipids was determined from a standard curve obtained over the same period using standard solutions containing 2, 4, 6, 8 and 10 mM dihydrogen phosphate.
Extraction method. 25-100 μΙ portions of liposomes were extracted 3 times with 200 μΙ parts of a mixture of methanol and chloroform (1: 2 by volume). The organic phases are connected in a test tube made of heat-resistant glass, and the solvents are removed under vacuum. The residue was treated with 10 N sulfuric acid and then tested for phosphorus based on method I above.
Unless otherwise indicated, the analytical data are shown as the mean ± standard error in a series of triplicates.
Example 71. Quantification of drugs in liposomes.
Spectrophotometric quantitative approach. Portions of liposomes (10-50 μΙ) were mixed with 1 ml of 70 vol%. an aqueous solution of isopropanol containing 0.075-0.1 N HCl, the absorbance for the blank was measured at the following wavelengths: doxorubicin, 485 nm; CPT-11 and topotecan, 372 nm; ellipticine, 306 nm, vinorelbine, 270 nm; vincristine and vinblastine, 265 nm. The amount of drug is determined by simultaneous comparison with the standard curve.
Fluorometric quantitative approach. Portions of liposomes containing samples (e.g., blood plasma) were diluted with acidified isopropanol (0.02-0.1 ml aliquots: 1 ml 70% isopropanol-0.075 N HCl;> 0.1 ml portions: 90% isopropanol-0.1 N HCl up to 1 ml). If protein precipitation occurs, samples are incubated on ice for 1-2 hours and clarified by centrifugation for 10 minutes with 12.100xg. Fluorescence of the supernatants was measured at the following wavelengths: CPT-11, excitation 370 nm, emission 423-425 nm;
Topotecan, boost 380-385 nm, emission 520-525 nm; ellipticine, excitation 306 nm, emission 520 nm. The amount of drug was calculated from the standard curves simultaneously after subtracting the value for the fluorescence assay.
Example 72. Effect of lipopolymers on the loading of vinorelbine into liposomes.
Liposomes consisting of 200 parts by weight of DSPC, 133 parts by mole of cholesterol and poly (ethylene glycol) (molecular weight 2,000) PEG-DSPE lipid derivatives (1116 parts by mole) or PEG-DSG (20 parts by mole) and containing an encapsulated solution 0.65 M TEA-SOS were prepared according to the method of Example 11, using a membrane extrusion step with a pore size of 80 nm. The liposomes were loaded with vinorelbin at a drug / phospholipid ratio of 350 mg / mmol and purified from the non-humified drug according to the method of Example 40. The liposomes were tested for drug and lipid content as described in Examples 70, 71 and for liposomal size using QELS using a Gaussian volume-weight approximation. The results (Table 38) showed that while the anionic derivative of PEG, PEG-DSPE,
Table 38. Properties of vinorelbine liposomes prepared by the SOS TEA method in various amounts of PEG-lipid derivatives.
<td>PEG-lipids</td><td>amount of lipids, PEG, mole % total lipids</td><td>Size liposomes, nm (average SD)</td><td>Loading the drug, mg / mole phospholipid</td><td>Effectiveness loading,% encapsulation</td>
<td>PEG-DSPE</td><td>0.3</td><td>108 ± 32</td><td>359.5 ± 17.8</td><td>102.7 ± 5.2</td>
<td>PEG-DSPE</td><td>0.6</td><td>110 ± 18</td><td>346.6 ± 14.5</td><td>99.0 ± 4.1</td>
<td>PEG-DSPE</td><td>1.8</td><td>104 ± 35</td><td>332.0 ± 14.0</td><td>94.9 ± 3.8</td>
<td>PEG-DSPE</td><td>2.9</td><td>94 ± 33</td><td>259.8 ± 9.5</td><td>74.2 ± 2.0</td>
<td>PEG-DSPE</td><td>4.0</td><td>100 ± 36</td><td>155.4 ± 7.0</td><td>44.4 ± 0.9</td>
<td>PEG-DSPE</td><td>5.7</td><td>103 ± 31</td><td>61.2 ± 5.2</td><td>17.5 ± 0.3</td>
<td>PEG-DSG</td><td>5.7</td><td>97 ± 36</td><td>362.7 ± 14.2</td><td>103.6 ± 4.2</td>
Example 73. Effect of an intraliposomal drug encapsulating agent on viability of CPT-11 in blood in mice.
Liposomes with closed 0.65 N triethylammonium (TEA) solutions or triethanolammonium salts (TEOA) inositol hexaphosphate (IHP, phytic acid) or sucrose octarnate were prepared and loaded with CPT-11 at 500 g / mol phospholipid using the general procedure of Example 66 Liposomes were administered intravenously to Swiss-Webster mice at a dose of 5 mg CPT-11 / kg body weight. Twenty-four hours later, mice were anesthetized and bled out by open-heart puncture. Blood was collected and analyzed
In terms of the content of CPT-11 in the blood plasma by HPLC as described in example 68, the amount of drug was expressed as% of the injected remaining dose in the blood (% ID).
TEOA-IHP was less effective in improving the durability of the drug in the bloodstream than TEA-IHP, TEOASOAS and TEA-SOS (Table 39).
Table 39. CPT-11 retention in blood 24 hours after intravenous administration of CPT-11 liposomes.
<td>The drug is closed by an intraliposomal drug</td><td>% ID remaining in the blood</td>
<td>TEOAE-IHP</td><td>2.74 ± 0.54</td>
<td>TEA-IHP</td><td>5.86 ± 0.20</td>
<td>TEOAE-SOS</td><td>7.03 ± 0.17</td>
<td>TEA-SOS</td><td>11.32 ± 0.46</td>
Example 74. Loading of the drug into liposomes containing 1.05 N sucroseyl sucrose octasulphate
Aqueous solution of 1.05 N diethylammonium sucrose octosulfate (DEA-SOS), pH 6.0, osmolality 727 mmol / kg, was prepared using the ion exchange / titration method of example 6 with pure diethylamine (99.5% purity). The lipid matrix with 3 molar parts of DSPC, 2 parts by mole of Cholesterol and 0.015 parts by mole of PEG2000-DSPE, were formulated in liposomes (volume mean by 92.4 nm) in the presence of DEA-SOS and CPT-11 solution, which were loaded into liposomes at various drug / lipid entry ratios using the method of Example 11. The nonhermered drug was removed by gel permeation chromatography and the amount of encapsulated drug per lipid unit determined (drug / lipid drug ratio). The effectiveness of encapsulation was calculated as a% of the drug / lipid starting ratio relative to the input ratio. The results are shown in Table 40. The maximum loading level was about 1.76 moles of drug per mole of phospholipid (1.67-1.70 mole drug / g total lipid), which is in good agreement with the amount (1.78 moles of diethylamine / mole of phospholipid) depending on the content of diethylamone in liposomes assuming a stoichiometric exchange of intraliposomal diethylammonium ions for drug molecules and estimated intraliposomal volume of encapsulated substance of approximately 1.7 l / mole phospholipid.
Table 40. Loading of CPT-11 in liposomes DSPC / Chol / PEG-DSPE containing 1.05
N DEA-SOS.
118
<td>Acceptance ratio drug / lipid, mol / g</td><td>The ratio of performance drug / lipid, mol / g</td><td>Efficiency of encapsulation,%</td>
<td>1.25</td><td>1.247 ± 0.038</td><td>99.8 ± 3.0</td>
<td>1.50</td><td>1.534 ± 0.052</td><td>102.3 ± 3.5</td>
<td>1.80</td><td>1.669 ± 0.043</td><td>92.7 ± 2.4</td>
<td>2.06</td><td>1.690 ± 0.054</td><td>82.0 ± 2.6</td>
<td>2.20</td><td>1.704 ± 0.062</td><td>77.5 ± 2.8</td>
<td>2.42</td><td>1.685 ± 0.103</td><td>69.6 ± 4.3</td>
Unless otherwise indicated, the analytical data are shown as the mean ± standard error in a series of triplicates. The rat plasma pharmacokinetic data is the mean ± standard error in a series of two replicates.
Merrimack Pharmaceuticals, Inc. United States of America Plenipotentiary:
119
EP 1 746 976 Z-15531
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Numbers
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Titles2
- English
- LIPOSOMES USEFUL FOR DRUG DELIVERY
- Polish
- LIPOSOMY PRZYDATNE W SYSTEMIE PODAWANIA LEKÓW
Classification
- CPC, 68
- A61K9/0019
- A61K9/1271
- A61K31/4745
- A61K9/127
- A61K47/6913
- Y10S977/773
- Y10S977/906
- Y10S977/907
- A61K31/4375
- A61K31/475
- A61K31/704
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- C07H13/12
- A61P31/04
- A61P31/12
- A61P35/00
- A61P43/00
- A61K38/00
- A61P1/00
- A61P1/04
- A61P1/08
- A61P1/10
- A61P1/12
- A61P1/14
- A61P1/16
- A61P1/18
- A61P3/00
- A61P3/04
- A61P3/10
- A61P3/12
- A61P5/50
- A61P7/10
- A61P9/00
- A61P9/04
- A61P9/10
- A61P11/00
- A61P11/06
- A61P13/00
- A61P13/08
- A61P13/12
- A61P15/10
- A61P25/00
- A61P25/04
- A61P25/18
- A61P25/20
- A61P25/22
- A61P25/24
- A61P25/28
- A61P27/16
- A61P29/00
- A61P35/02
- A61P35/04
- C07K7/08
- C07K14/245
- A61K47/50
- C07K7/06
- A61K9/50
- A61K45/06
- A61K47/10
- A61K47/30
- A61K47/36
- A61K38/10
- A61K47/60
- A61K9/1277
- A61K47/26
- A61K9/1278
- A61K47/24
- A61K47/28
- IPC, 2
- A61K9 127
- A61K31 4745