Liposomes useful for drug delivery

Field of the Invention

This invention relates generally to the field of liposomes, and more specifically to liposome compositions useful for the administration of therapeutic or diagnostic entities.

Lasic physics, D, to and

of

Background of the Invention

Liposomes, or lipid bilayer vesicles, have been used or proposed for use in a variety of research, industry and medical applications, particularly for use as carriers of therapeutic or diagnostic compounds in vivo. See, for example: Lasic, D. Liposomes: from applications. Elsevier, Amsterdam, 1993.

Papahadjopoulos, D., eds. Medical Applications

Liposomes. Elsevier, Amsterdam, 1998. Liposomes are usually characterized by having an inner space isolated from an outer environment by a membrane of one or more bilayers forming a microscopic sac, or vesicle. The liposome bilayer membranes are typically formed of lipids, i.e. amphiphilic molar molecules of synthetic or natural origin, which comprise spatially separated hydrophilic and hydrophobic domains. See Lasic D., 1993, supra. The liposome bilayer membranes may also be formed by amphiphilic and surfactant polymers (polymerosomes, niosomes). A liposome typically serves as a vehicle of a

Como1746976 entity such as, without limitation, a chemical compound, a combination of compounds, a supramolarecular complex of synthetic or natural origin, a genetic material, a living organism, a portion thereof or a derivative thereof, which is capable of having a property useful or engaging in a useful activity. For this purpose, liposomes are prepared to contain the desired entity in a form incorporated into liposomes. 0 The process of incorporating a desired entity into a liposome is often referred to as loading. The liposome incorporated entity may be located wholly or partially within the interior space of the liposome, within the liposome bilayer membrane or associated with the outer surface of the liposome membrane. Incorporation of liposome entities is also referred to as encapsulation or entrapment, and these three terms are used interchangeably with the same meaning in the present invention. Often, the intention of encapsulating an entity in the liposome is to protect it from the destructive environment while providing the possibility for the encapsulated entity to perform its activity, particularly in the location or environment where such activity is advantageous, but less elsewhere. where it may be useless or undesirable. This phenomenon is referred to as administration. For example, a pharmaceutical substance within the liposome may be protected from destruction by enzymes in the body, but may be released from the liposome and provide treatment at the disease site.

Ideally, these liposomes may be prepared to include the desired compound (i) with high loading efficiency, i.e. high percentage of the encapsulated entity.

ΡΕ1746976 in relation to the amount used in the encapsulation process; (ii) high amount of encapsulated entity per unit of liposome bilayer material; (iii) at a high concentration of the encapsulated entity and (iv) in a stable form, i.e. with little release (leakage) of an encapsulated entity during storage or generally before the liposome appears at the place or environment where the liposome entrapped entity is expected to exert its intended activity. WO2005 / 002546, WO98 / 17256, US4321259, US5316771, US5785987 and US6110491 disclose encapsulation liposomes and production methods.

Therefore, there is a need in the art to provide various liposome compositions which are useful for the administration of a variety of compounds, especially therapeutic, diagnostic or imaging entities.

Summary of the Invention

The present invention is based on the discovery that substituted ammonium and polyanion are useful for carrying and retaining entities within the present liposome disclosures. Accordingly, it provides methods and liposome compositions useful for the administration of a variety of entities, especially therapeutic entities, i.e. entities useful in the diagnosis, prognosis, testing, screening, treatment or prevention of an undesirable condition, for example a disease, in a living organism, such as a human being, a plant or an animal.

The invention provides a composition comprising a liposome in a medium, wherein the liposome comprises 1,2distearoyl-SN-phosphatidylcholine, cholesterol and N- (omegamethoxy-poly (ethylene glycol) oxycarbonyl) -1,2ΡΕ1746976 distearoylphosphatidyl ethanolamine in a molar ratio of 3: 2: 0.015, and are trapped within the irinotecan liposome and sucrose octasulfate.

The invention also provides a pharmaceutical composition comprising the liposome composition of the invention, a pharmaceutically acceptable carrier and a buffer substance.

In one embodiment, the present disclosure provides a composition comprising a liposome in a medium, wherein the interior of the liposome contains a substituted ammonium (I) wherein each of R 1, R 2, R 3 and R 4 is independently a hydrogen or a group. including up to 18 carbon atoms in which at least one of R1, R2, R3 and R4 is an organic group, wherein the organic group is independently a hydrocarbon group having up to 8 carbon atoms, e is an alkyl, alkylidene, heterocyclic alkyl, cycloalkyl, aryl, alkenyl or cycloalkenyl group or a hydroxy-substituted derivative thereof, optionally including within their hydrocarbon chain an S, O or N atom forming an ether bond, ester, thioether, amine or amide, wherein at least three of R 1, R 2, R 3 and R 4 are organic groups, or the substituted ammonium is a sterically hindered ammonium, such as when at least one of the organic groups has a secondary or tertiary carbon atom directly attached to the ammonium nitrogen atom. Preferably the encapsulated substituted ammonium compound

ΡΕ1746976 in liposomes have a negative logarithmic acid dissociation constant (pKa) (deprotonation) 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 as determined in an aqueous solution at room temperature.

In another embodiment, the present disclosure provides a composition comprising a liposome in a medium, wherein the interior space of the liposome contains a polyanion, and wherein the polyanion is a polyanionized polyol or polyanionized sugar. The liposome preferably contains a transmembrane gradient capable of loading an entity into the liposome. In one embodiment, the transmembrane gradient is a gradient of an ammonium, a quarternary ammonium or a substituted primary, secondary or tertiary ammonium compound having in a dilute aqueous solution at room temperature a negative logarithm of the acid dissociation constant (pKa) (deprotonation) 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. The liposome optionally contains an entrapped entity, for example a therapeutic, a detectable marker or a globally cationic organic molarecule.

In yet another embodiment, the composition provided by the present disclosure further comprises a liposome-encapsulated entity of the present disclosure. Preferably, the entity is encapsulated within the interior space of the liposome. For example, the interior space of the liposome further comprises an antineoplastic therapeutic, and wherein the level of toxicity of the composition to an individual is at least equal to or less.

ΡΕ1746976 that the level of toxicity of the antineoplastic therapeutic administered to the individual without the composition.

In yet another embodiment, the composition provided by the present disclosure is a liposomal composition comprising a camptothecin compound. The composition has an anticancer activity at least two, four, or ten times greater than that of the similarly administered camptothecin compound in the absence of the composition, while the toxicity of the composition does not exceed, is at least twice or at least four times lower than the toxicity of the drug. camptothecin compound administered similarly in the absence of the composition. In one embodiment, the camptothecin compound is a prodrug and is contained in the liposome in 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 1 mg of liposome membrane materials, for example lipids. The camptothecin compound is preferably encapsulated in the liposome within substantially 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 liposomal composition of a vinca alkaloid or a derivative thereof. The composition has 24-hour drug retention within the liposome after 24 hours of in vivo mammalian blood exposure of at least 50%, at least 60% or at least 70% of the parent drug loading. The vinca alkaloid or a derivative thereof is preferably encapsulated in the liposome within substantially the interior space of the liposome. An example of the mammal is a rat. Exemplary vinca alkaloids and derivatives are vincristine, vinblastine and vinorelbine.

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In yet another embodiment, the present disclosure provides a method for encapsulating an entity in a liposome. The method comprises contacting the liposomes of the present disclosure with an entity, for example, a therapeutic or detectable entity. Preferably, contact is made under conditions where the concentration in the medium of the substituted ammonium or polyanion of the present disclosure is less than that of the interior space of the liposomes. In one embodiment, the liposome composition is contacted with an entity in an aqueous medium.

In yet another embodiment, the present disclosure provides a method for encapsulating an entity in a liposome. The method comprises contacting the liposome-containing composition of the present disclosure with a pre-entity, wherein the pre-entity is capable of being converted to an entity under a condition, and providing the condition within the liposome that converts the pre-entity. entity to entity within the liposome. In one case, the entity is an organic compound and the pre-entity is a basic derivative thereof.

In yet another embodiment, the present disclosure provides a kit for making liposome encapsulated entities. The kit comprises a liposome container of the present disclosure, and optionally a container containing an entity and / or instructions for the user, for example to encapsulate an entity.

Brief Description of the Drawings

Figure 1 shows the blood pharmacokinetics of liposome lipid (circles) and drug (triangles) after

ΡΕ1746976 intravenous bolus administration to a rat of CPT-11 loaded liposomes. Liposomes are loaded using the TEA-Pn method (see Example 9).

Figure 2 shows the dynamics of the liposome drug-to-lipid ratio in a rat's blood in vivo following intravenous bolus administration of the CPT-11 loaded liposome using the TEA-Pn method (see Example 9).

Figure 3 shows the antitumor efficacy of free CPT-11 and liposomal CPT-11 against BT-474 human breast cancer xenografts in nude mice. Control means mice treated with drug-free vehicle and liposome only. (see Example 10).

Figure 4 shows the dynamics of animal body weights during treatment of nude BT-474 tumor bearing mice with free CPT-11 or liposomal CPT-11. Control means mice treated with drug-free vehicle and liposome only. (see Example 10).

Figure 5 shows the dynamics of the liposome drug-to-lipid ratio in a rat's blood in vivo following intravenous bolus administration of the CPT-11 loaded liposome using the TEA-SOS method. (see Example 14).

Figure 6 shows the antitumor efficacy of free and liposomal CPT-11 against HT-29 human colon cancer xenografts in nude mice. The legend on the panel indicates the drug loading method and the dose administered by injection. Saline Control means mice treated with drug-free vehicle and liposome only. (see Example 15).

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Figure 7 shows the dynamics of animal body weights during treatment of HT-29 tumor-bearing nude mice with CPT11 free or liposomal formulations. Error bars represent the standard deviation of the data. Saline Control means mice treated with drug-free carrier and liposome only. (see Example 15).

Figure 8A shows the pharmacokinetics of blood liposome lipid following intravenous bolus administration to a rat of topotecan-loaded liposomes. The legend on the panel indicates the drug loading method and the liposome drug content. (see Example 24).

Figure 8B shows the dynamics of the drug-lipid liposome ratio in a rat's blood in vivo following intravenous bolus administration of topotecan-loaded liposomes. The legend on the panel indicates the drug loading method and the liposome drug content. (see Example 24).

Figure 9 shows the in vitro cytotoxicity of HER2-directed free liposomal or immunoliposomal topotecan (TEA-Pn method) against SKBr3 breast carcinoma cells. (see Example 27).

Figure 10 shows the in vitro cytotoxicity of HER2-directed free liposomal or immunoliposomal topotecan (TEA-SOS method) against SKBr3 breast carcinoma cells. (see Example 32).

Figure 11 shows the antitumor efficacy of various topotecan (TPT) formulations against xenografts of

ΡΕ1746976 BT-474 human breast cancer in nude mice. Saline Control means mice treated with drug-free carrier and liposome only. (see Example 29).

Figure 12 shows the dynamics of animal body weights during treatment of nude BT-474 tumor bearing mice with free topotecan (TPT), liposomal topotecan (Ls-TPT) or antiHER2 immunoliposomal topotecan (F5 ILs-TPT). Control means mice treated with drug and liposome free vehicle only. (see Example 29).

Figure 13A shows the antitumor efficacy of topotecan formulations against BT-474 human breast cancer xenografts in nude mice. Free topotecan (free TPT) or liposomal topotecan (Ls-TPT) were administered at one-eighth of their maximum tolerated doses. Error bars represent the standard deviation of the data. Control means mice treated with drug and liposome free vehicle only. (see Example 31).

Figure 13B shows the antitumor efficacy of topotecan formulations against BT-474 human breast cancer xenografts in nude mice. Free topotecan (free TPT) or liposomal topotecan (Ls-TPT) were administered to one of their maximum tolerated doses. Error bars represent the standard deviation of the data. Control means mice treated with drug and liposome free vehicle only. (see Example 31).

Figure 13C shows the antitumor efficacy of topotecan formulations against BT-474 human breast cancer xenografts in nude mice. Free Topotecan (TPT)

Livre1746976) or liposomal topotecan (Ls-TPT) were administered at half of their maximum tolerated doses. Error bars represent the standard deviation of the data. Control means mice treated with drug-free vehicle and liposome only. (see Example 31).

Figure 13D shows the antitumor efficacy of topotecan formulations against BT-474 human breast cancer xenografts in nude mice. Free topotecan (free TPT) or liposomal topotecan (Ls-TPT) were administered at their maximum tolerated doses. Error bars represent the standard deviation of the data. Control means mice treated with drug-free vehicle and liposome only. (see Example 31).

Figure 14 shows the dynamics of mean body weights during treatment of BT-474 nude tumor-bearing mice with free topotecan (free TPT) or liposomal topotecan (Ls-TPT) administered at their maximum tolerated doses. Control means mice treated with drug-free vehicle and liposome only. (see Example 31).

Figure 15 shows the cytotoxicity of free (free EA) 6- (3-aminopropyl) ellipticine, liposomal 6- (3-aminopropyl) -elipticine (Ls-AE) or targeted immunoliposomal 6- (3-aminopropyl) -elipticine -HER2 (F5 ILs-AE) against BT-474 breast carcinoma cells in vitro. (see Example 35).

Figure 16 shows the aminopropyl) -elipticine aminopropyl) -elipticine aminopropyl) -elipticine in vitro 6- (3-free (free APE), 6- (3-liposomal (Ls-APE) or targeted 6- (3-immuno-liposomal) cytotoxicity). EGFR

ΡΕ1746976 (C225-ILs-APE) against EGF receptor low (MCF-7) or high expression (MDA-MB468) breast carcinoma cells. (see Example 36).

Figure 17 shows the blood pharmacokinetic attributes of liposomal formulated 6- (3-aminopropyl) elipticin (APE): blood pharmacokinetics of liposome lipid (Panel A, empty circles), drug (Panel A, filled circles), and the dynamics of the liposome drug-lipid ratio (Panel B) following intravenous bolus administration to a rat liposome of APE. (see Example 37).

Figure 18 shows the blood pharmacokinetic attributes of liposome formulated vinorelbine (Ls-VRB) and anti-HER2 immunoliposomes (F5-ILs-VRB): blood pharmacokinetics of liposome lipid (Panel A), drug (Panel B) , and the dynamics of the liposome drug-to-lipid ratio (Panel C) following intravenous bolus administration to a rat of vinorelbine liposomes. (see Example 43).

Figure 19 shows the pharmacokinetics of blood liposome lipid following intravenous bolus administration to a rat of vinorelbine-loaded liposomes. Liposomes are loaded using pre-trapped triethylammonium dextran sulfate (DS-TEA), ammonium dextran sulfate (DS-A) or ammonium sulfate (SA). (see Example 44).

Figure 20 shows the dynamics of the liposome drug-lipid ratio in a rat's blood in vivo following intravenous bolus administration of vinorelbine-loaded liposomes using dextran sulfate.

ΡΕ1746976 Pre-trapped triethylammonium (DS-TEA), dextran ammonium sulfate (DS-A) or ammonium sulfate (SA). (see Example 44).

Figure 21 shows the pharmacokinetics of blood liposome lipid following intravenous bolus administration to a rat of vinorelbine-loaded liposomes. Liposomes are loaded using pre-trapped triethylammonium sucrose octasulfate (TEA-SOS) and are of average size as indicated in the panel legend, (see Example 45).

Figure 22 shows the dynamics of the liposome drug-lipid ratio in a rat's blood in vivo following intravenous bolus administration of vinorelbine-loaded liposomes. Liposomes are loaded using pre-trapped triethylammonium sucrose octasulfate (TEA-SOS) and are of average size as indicated in the panel legend, (see Example 45).

Figure 23 shows the pharmacokinetics of blood liposome lipid in rats following intravenous bolus administration of liposome-formulated vinorelbine (Ls-VRB) or anti-HER2 immunoliposomes (F5-ILs-VRB) using the TEA-SOS method. . (see Example 46).

Figure 24 shows the dynamics of the drug-lipid liposome ratio in a rat's blood in vivo following intravenous bolus administration of liposome-formulated vinorelbine (Ls-VRB) or anti-HER2 immunoliposomes (F5-ILsVRB) using the TEA-SOS method. (see Example 46).

Figure 25 shows in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls-VRB) or targeted immunoliposomal vinorelbineΡΕ1746976

HER2 (F5-Ils-VRB) against HER2 overexpressed MDA-MB-453 human breast cancer cells. (see Example 48).

Figure 26 shows the in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls-VRB) or HER2-directed immunoliposome vinorelbine (Ca5u-3 human non-small cell lung cancer cells) with HER2 overexpression. (see Example 49).

Figure 27 shows the in vitro cytotoxicity of free vinorelbine (free VRB), liposomal vinorelbine (Ls VRB / SOSTEA) or HER2-directed immunoliposomal vinorelbine (F5-ILs VRB / SOS-TEA) against human breast cancer cells SKBr -3 with HER2 overexpression. (see Example 50).

Figure 28 shows the antitumor efficacy of free vinorelbine (free VRB) or liposomal vinorelbine (Ls VRB) against HT-29 human colon cancer xenografts in nude mice. Saline means mice treated with drug-free carrier and liposome only. Error bars represent the standard deviation of the data. (see Example 51).

Figure 29 shows the dynamics of mean body weights during treatment of HT-29 tumor-bearing nude mice with free vinorelbine (free VRB), liposomal vinorelbine (Ls VRB) or vehicle only (saline). 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 a

ΡΕ1746976 model of syngeneic C-26 murine colon carcinoma. The caption on the panel indicates the dose of drug by injection. Error bars represent the standard deviation of the data. Saline means mice treated with drug-free vehicle and liposome only. (see Example 52).

Figure 31 shows the dynamics of mean body weights during treatment of mice bearing syngeneic C-26 murine colon carcinoma tumors with various doses of free vinorelbine (free VRB), liposomal vinorelbine (Ls VRB) or vehicle only (saline). ). The caption on the panel indicates the dose of drug by injection (see Example 52).

Figure 32 shows the antitumor efficacy of free vinorelbine (drug free) or scFv F5-conjugated anti-HER2 immunoliposomal vinorelbine prepared by a TEA-SOS method (F5-ILs-VRB TEA-SOS) or immunoliposomal anti-HER2 vinorelbine scFv F5 conjugate prepared by a TEA-Pn (F5-ILs-VRB TEA-Pn) method against human breast carcinoma xenografts (BT-474) with HER2 overexpression in nude mice. Saline Control means mice treated with drug-free vehicle and liposome only. (see Example 53).

Figure 33 shows the dynamics of mean body weights during treatment of mice bearing human breast carcinoma xenografts (BT-474) with free vinorelbine HER-overexpressed, anti-HER2 immunoliposomal vinorelbine prepared using scFv F5 a TEA-SOS method, scFv F5-conjugated anti-HER2 immunoliposomal vinorelbine prepared by a TEA-Pn method or with vehicle only.

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For explanation of the symbols, refer to the caption in figure 32. (See also example 53).

Figure 34 shows the antitumor efficacy of free (drug free) vinorelbine or scFv F5-conjugated anti-HER2 immunoliposome vinorelbine prepared using various amounts of overexpressed human breast carcinoma (BT-474) xenografts HER2 in nude mice. Error bars are the standard deviation of the data. Vehicle Control means mice treated with drug-free vehicle and liposome only. (see Example 54).

Figure 35 shows the antitumor efficacy of free (free NAV) vinorelbine, liposomal vinorelbine (NAV Lip) or FC225Fab ​​'(C225-NAV Lip) anti-EGFR conjugated anti-EGFR vinorelbine against human glioblastoma (U87) xenografts EGFR expression in nude mice. Saline means mice treated with drug-free vehicle and liposome only. (see Example 55).

Figure 36 shows the pharmacokinetics of blood liposome lipid and the dynamics of the drug / lipid ratio in a rat's blood following intravenous bolus administration of liposome-formulated doxorubicin using the triethylammonium sulfate method. (see Example 56).

Figure 37 shows the antitumor efficacy of liposomal doxorubicin (Ls-Dox) or scFv F5-conjugated anti-HER2 immunoliposomal doxorubicin (F5 ILs-Dox) prepared using various amounts of PEG-lipid against

ΡΕ1746976 Human breast carcinoma xenografts (BT-474) with HER2 overexpression in nude mice. The panel legend shows the amount of PEG-lipid expressed in molar% of liposome phospholipids. Saline Control means mice treated with drug-free carrier and liposome only. (see Example 57).

Figure 38 shows the pharmacokinetics of liposomal vinblastine in rat blood (see Example 58).

Figure 39 shows the dynamics of the liposome drug / lipid ratio in the blood of a rat following intravenous bolus administration of liposomal vinblastine. (see Example 58).

Figure 40 shows in vitro cytotoxicity of free vincristine (free VCR), liposomal vincristine (Ls-VCR) or HER2-directed immunoliposomal vincristine (F5-ILs-VCR) against overexpressed human breast cancer cells SKBr-3 from HER2. (see Example 61).

Figure 41 shows the pharmacokinetics of liposome lipid in rat blood following intravenous bolus administration of vincristine formulated in different medium sized liposomes (indicated in the panel legend). (see Example 62).

Figure 42 shows the dynamics of the liposome drug / lipid ratio in the blood of a rat following intravenous bolus administration of vincristine formulated in different medium sized liposomes (indicated in the panel legend). (see Example 62).

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Figure 43 shows the antitumor efficacy of free vincristine (free VCR), liposomal vincristine prepared by the triethylammonium citrate method (Ls-VCR citrate), liposomal vincristine prepared by the triethylammonium sucrooctasulfate method (Ls-VCR SOS) scFv F5-conjugated anti-HER2 immunoliposome vincristine, prepared by the triethylammonium sucrooctasulfate (F5 ILs-VCR SOS) method against human breast carcinoma xenografts (BT-474) with HER2 overexpression in nude mice. Saline Control means mice treated with drug-free carrier and liposome only. (see Example 64).

Figure 44 shows the dynamics of mean body weights during treatment of mice bearing human breast carcinoma xenograft (BT-474) with HER2 overexpression with free vincristine (free VCR), liposomal vincristine prepared by the citrate citrate method. triethylammonium (Ls-VCR citrate), liposomal vincristine prepared by the triethylammonium sucrooctasulfate (Ls-VCR SOS) method, scFv F5-conjugated anti-HER2 immunoliposome vincristine, prepared by the triethylammonium sucrooctasulfate method (F5 ILs-VCR SOS), or with vehicle alone (saline control). (see Example 64).

Figure 45 shows the antitumor efficacy of free vincristine (vincristine), liposomal vincristine (nt-vcr) or C225 Fab '(C225-vcr) anti-EGFR conjugated anti-EGFR vincristine against human brain cancer xenografts (U87) with EGFRvIII overexpression in nude mice. Saline means mice

ΡΕ1746976 treated with drug and liposome free vehicle only. (see Example 65).

Figure 46 shows the pharmacokinetics of blood CPT-11 and the percentage dynamics of CPT-11 present in the active form (lactone) in the blood of a rat following intravenous bolus administration of liposome CPT-11. (see Example 69).

Figure 47 shows the pharmacokinetics of blood CPT-11 and the percentage dynamics of CPT-11 present in the active form (lactone) in the blood of a rat following intravenous bolus administration of the CPT-11 solution (CPT-11). free). (see Example 69).

Description of Preferred Embodiments

The present disclosure generally relates to methods and liposome compositions useful for the administration of a variety of entities, especially therapeutic and imaging agents. It is the discovery of the present disclosure that substituted ammonium and polyanion are useful for loading and retaining entities, for example the compound, within the liposomes. Accordingly, the present disclosure provides liposome compositions and kits containing substituted ammonium and / or polyanion and methods of preparing such liposome compositions.

According to a feature of the present disclosure, it provides a liposome composition containing in its interior space one or more substituted ammonium compounds of a formula

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R₄—N — R₂

Go₃ (I) wherein each of R 1, R 2, R 3 and R 4 is independently a hydrogen or an organic group, and wherein at least one of R 1, R 2, R 3 and R 4 is an organic group such as an alkyl, alkylidene group , heterocyclic alkyl, cycloalkyl, aryl, alkenyl or cycloalkenyl, a hydroxysubstituted derivative thereof, optionally including within their hydrocarbon chain an atom of S, O or N, for example forming an ether bond thereon (including an acetal or ketal) , ester, sulphide (thioether), amine or amide. If less than three of R1, R2, R3 and R4 are organic groups, then, according to the disclosure, at least one, and preferably two, of the organic groups have a secondary or tertiary carbon atom (i.e. carbon atoms). respectively having 2 or 3 carbon-carbon bonds) attached directly to the ammonium nitrogen, ie the substituted ammonium is a sterically hindered ammonium. In general, the presence of titratable ammonium, such as unsubstituted ammonium ion (NH®) as well as primary and secondary straight chain alkylammonium ions within the liposome interior space of the present disclosure is known to provide better encapsulation of amphiphilic bases. by, for example, an active, remote or transmembrane gradient driven loading mechanism (Haran, et al., Biochim. Biophys. Acta, 1993, v. 1152, p. 253-258; Maurer-Spurej, et al. ., Biochim. Biophys. Acta, 1999, v. 1416, p. 1-10). However, these ammonia compounds have hydrogen atoms that readily enter nucleophilic substitution reactions and, on the other hand, react chemically with entrapped entities in the

ΡΕ1746976 liposomes and therefore are capable of impairing the chemical integrity of entities during or after the liposome loading (entrapment) process. Accordingly, it is desirable that an entrapped substituted ammonium compound be more chemically inert, devoid of chemical functions that are unstable or readily reactive with liposome components, which may include an encapsulated entity. Unexpectedly, we have discovered that liposome compositions which comprise within their interior space a substituted tertiary and guaternary ammonium which has no substitutable hydrogen, or a sterically hindered primary or secondary ammonium, where access to an ammonium hydrogen atom is sterically hindered. a bulky neighboring organic group having one or two secondary or tertiary carbon atoms attached to ammonium nitrogen, show not only excellent carrying capacity of entities, but also increased stability of the entrapped entity in the liposome, for example a drug, against premature release from the liposome into the living organism.

In an embodiment disclosed in the present invention, the substituted ammonium compound entrapped in the liposome is pharmaceutically inert, i.e. does not elicit an adverse physiological response when administered to a living individual, for example, a human or animal, within a amount of liposome membrane material that is sufficient to deliver an effective dose of the entrapped entity in the liposome. In another embodiment, the substituted ammonium has an acceptable level of toxicity to an individual. Normally, an acceptable level of toxicity means that the toxic dose, for example a maximum tolerated dose (DMT) or a dose that causes 50% mortality.

ΡΕ1746976 (DL50), of substituted ammonium is at least twice, at least four times, at least eight times, or at least ten times higher than the toxic dose of a liposome entrapped entity, for example, a drug, charged within. of liposomes. For example, triethylammonium sulfate has an acceptable level of toxicity as its LD50 is about 40 times higher than the LD50 of doxorubicin, an anticancer drug. Toxicity levels or physiological responses of substituted ammoniums as well as entities of interest, if not yet known, can be readily established by routine techniques well known to those skilled in the biomedical art. See, for example, SC Gad. Drug Safety Evaluation, Wiley, New York, 2002. A method for quantifying the toxicity of free and / or liposome formulated drug is described in Example 16 of the present invention.

In a preferred embodiment, the R 1, R 2, R 3 or R 4 substitutable organic groups have sufficient size and physicochemical properties to ensure that the substituted ammonium forms substantially a true (molar) solution but not micelles in aqueous environment. , bilayers or similar self-assembling structures. Therefore, the substituted ammonium preferably has little or substantially no distribution in the bilayer portion of the liposomes, thus minimizing the risk of destabilization, solubilization or permeabilization of the substituted ammonium-trapping liposomes.

The substituted ammonium organic group is usually a hydrocarbon containing up to and including 8 carbon atoms, up to 6 carbon atoms or up to 4 carbon atoms, and all substituent groups contain up to and including

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18, up to 16, up to 12 or up to 9 carbon atoms. Such substitutable hydrocarbon groups include any combination of interconnected primary, secondary or tertiary carbon atoms, as well as cycloalkyl groups attached at their termini directly to ammonium nitrogen to form a heterocycle, or to a carbon atom of an ammonium hydrogen substitutable group . Such substituted alkyl groups may also include heteroatoms, for example oxygen, nitrogen or sulfur in their carbon chains forming a functional group, for example an ether, acetal, amine or sulfide group, as well as forming a functional group, for example, a hydroxyl group attached to the carbon chain of the alkyl. Examples of the organic group of the present disclosure include, without limitation, hydroxy-substituted alkyls, alkylidenes, heterocyclyl, cycloalkyl, aryl, alkenyl, cycloalkenyl or derivatives thereof, for example, a hydroxy-substituted alkylidene forming an inclusive ring of N in substituted ammonium.

In another embodiment, the substituted ammonium is: a heterocyclic ammonium, that is, an ammonium in which at least two of R 1, R 2, R 3 or R 4 form a ring; a sterically hindered primary ammonium; or a sterically hindered secondary ammonium. In general, a sterically hindered primary or secondary ammonium includes any ammonium substituted with one or two of the R 1, R 2, R 3 and R 4 groups substituted with some groups which sterically agglomerate the molecule, e.g. R 3 and R 4 substituted with one or two cycloalkyl groups or alkyl groups having at least one carbon atom of the secondary or tertiary alkyl attached to the substituted ammonium nitrogen. Examples of such

Heterocyclic, sterically hindered primary ammoniums and sterically hindered secondary ammonium include, without limitation, isopropylethyl ammonium, isopropylmethylammonium, diisopropylammonium, tert-butylethylammonium, protonated forms of morpholine, pyridine, piperidine, pyrrolidine, amino-pyrazine, pyrrolidine -2-methylpropanol-1,2-amino-2-methylpropandiol-1,3 and tris- (hydroxyethyl) aminomethane. These substituted ammonium compounds are generally commercially available in the form of various salts, or are readily prepared from their corresponding amines by acid neutralization.

In yet another embodiment, the substituted ammonium is a tertiary or quaternary ammonium, including, without limitation, trimethylammonium, triethylammonium, tributylammonium, diethylmethylammonium, diisopropylethylammonium, triisopropylammonium, N-methylmorpholinium, Nhydroxyethylpiperidinium, N-methylpyrrolidine , N'dimethylpiperazinium, tetramethylammonium, tetraethylammonium and tetrabutylammonium. These substituted ammonium compounds are generally commercially available in the form of various salts, or are readily prepared from their corresponding amines by acid neutralization.

In yet another embodiment, the substituted ammonium compound is a globally cationic compound, that is, under the conditions of encapsulation of the entity, usually in aqueous solution at a pH between about pH 2 and about pH 8, is charged. positive liquid, for example, as a result of ionization (protonation) of the nitrogen atom.

ΡΕ1746976

In yet another embodiment, the substituted primary, secondary or tertiary ammonium compound encapsulated in the liposomes has a negative logarithm of the acid dissociation constant (pKa) (deprotonation) of at least about 8.0, at least about 8.5. at least about 9.0, at least 9.5 or at least about 10.0 as determined in an aqueous solution diluted at room temperature (usually 25 ° C). The pKa parameter is a well known feature of ammonium compounds which generally characterizes the strength of their basic properties, and methods for determining pKa are conventional and routine in the art. PKa values ​​for many amines and their protonated forms (ammoniums) are tabulated in reference books on chemistry and pharmacology. See, for example, IUPAC Handbook of Pharmaceutical Salts, ed. by PH Stahl and CG Wermuth, Wiley-VCH, 2002; CRC Handbook of Chemistry and Physics, 82.^The Edition, ed. by DRLide, CRC Press, Florida, 2001, p. 8-44 to 8-56. In general, a larger pKa characterizes stronger bases. Exemplary substituted ammonium as well as unsubstituted ammonium compounds (listed as their conjugated amine bases) 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; Nmethylpiperidine, 10.38; diethylmethylamine, 10.35;

dimethylpropylamine, 10.15; trimethylamine, 9.8; piperazine, 9.73 (I), 5.33 (II); 2-amino-2-methylpropanol, 9.69; N, N'dimethylpiperazine, 9.66 (I), 5.2 (II); diethyl- (2hydroxyethyl) amine, 9.58; ethanolamine, 9.5; Nhydroxyethylpyrrolidine, 9.44; diethanolamine, 9.28; ammonia,

ΡΕ1746976

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; N-ethylmorpholine, 7.67; Nhydroxyethylmorpholine, 7.39; imidazole, 7.03; pyridine, 5.23. As a general rule, substitution of the alkyl or cycloalkyl group with a hydrogen in an ammonium compound increases the value of pKa. Namely, multiple hydroxyl or ether functions in the substituent alkyl groups, or the presence of aromaticity in a nitrogen-containing heterocyclic group reduces the value of pKa relative to similar ammonia substituted without hydroxyl or ether functions. Compounds with more than one ammonium group usually have the pKa of the second and subsequent ammonium group much lower than that of the first. We found unexpectedly that, in stabilizing the drug within the liposomes, ammonia substituted with higher pKa values, i.e. formed by more strongly basic amines, was more effective than that formed by weaker amines. For example, in stabilizing irinotecan within liposomes in vivo, the triethylammonium IHP and SOS salts (pKa = 10.75) were particularly more effective than the corresponding triethanolammonium salts (pKa = 7.76) (Example 73).

The substituted ammonium contained in the liposomal composition may be in any suitable form, for example salt. Suitable salts include pharmaceutically acceptable salts. See, for example, PHStahl, CG Wermuth (eds), Handbook of Pharmaceutical Salts, Wiley-VCH, 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 water with soluble salt, is pharmaceutically inert, capable of forming precipitates or

ΡΕ1746976 gels when in contact with a therapeutic or detectable entity, and / or is less permeable through the liposome membrane than the substituted ammonium or its undissociated amine form. In general, the substituted ammonium salt forms a true solution in the intraliposomal, e.g. aqueous, space and does not form a significant amount of a condensed phase, such as a micelle, bilayer, gel or crystalline phase. The relative amount of a substituted ammonium and salt-forming anion, for example a polyanion, is or is close to the stoichiometric equivalence point, and usually has a pH in the range of 3-9, most often pH 4-8. depending, for example, on the dissociation constant of the conjugated base of the substituted ammonium ion.

In general, substituted ammonium is contained within, that is, within the inner (inner) space of the liposomes. In one embodiment, the substituted ammonium is partially or substantially completely removed from the outer environment surrounding the liposomes. Such removal may be accomplished by any suitable means known to one of skill in the art, for example, dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, precipitation, etc.

According to another feature of the present disclosure, it provides a liposome composition containing a polyanion. The polyanion of the present disclosure may be any suitable chemical entity with more than one negatively charged group, resulting in a negative net ionic charge of more than two units in the interior space of the liposome, for example, aqueous. The polyanion of the present disclosure may be an anion.

ΡΕ1746976 divalent, a trivalent anion, a polyvalent anion, a polyvalent polymeric anion, a polyanionized polyol or a polyanionized sugar. Examples of such di and trivalent anions are, but are not limited to, sulfate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate and citrate. In a preferred embodiment, the polyanion of the present disclosure is a polyanionic polymer having an organic (carbon) or inorganic backbone and a plurality of anionic functional groups, that is, ionizable functional groups for a negative charge in a neutral aqueous solution, and integrated or attached to the main chain. A polymer is a naturally occurring or synthetic compound, usually of high molecular weight, consisting of repeating units each linked with a relatively light and simple molarecule. Exemplary polyanionic polymers are polyphosphate, polyvinyl sulfate, polyvinyl sulfonate, anionized polyacrylic polymers, for example anionized polysulfonated polyamines, such as polysulfonated poly (ethylene imine); polysulfated, polycarboxylated or polyphosphorylated polysaccharides; the acid polyamino acids; polynucleotides; other polyphosphorylated, polysulfated, polysulfonated, polyborated or polycarboxylated polymers. Such anions and polyvalent polymers are well known in the art and many are commercially available. A polymeric anion of the present disclosure is preferably biodegradable, i.e. capable of disintegrating into non-toxic units in the living organism. The exemplary biodegradable polymer anion is polyphosphate.

In another preferred embodiment, the polyanion is a polyanionized polyol or a polyanionized sugar. A polyol is an organic molarecule that has a plurality

ΡΕ1746976 of hydroxyl groups attached to, for example, a straight, branched or cyclic carbon backbone. Thus, a polyol may be characterized in other terms as a polyhydroxylated compound. Preferably, most carbon atoms in a polyol are hydroxylated. Polyols (polyatomic alcohols) are molar molecules well known in the art. Linear (straight or branched) and cyclic chain polyols may be used. Exemplary polyols of the present disclosure are, but are not limited to: ethylene glycol, glycerol, treitol, erythritol, pentaerythritol, mannitol, glucitol, sorbitol, sorbitan, xylitol, lactitol, maltitol, fructitol and inositol. A sugar usually comprises a cyclic acetal, a cyclic ketal, a ketone or an aldehyde group, or an adduct thereof, within a group of predominantly hydroxylated interconnected carbon atoms. Sugars are often naturally occurring compounds. The hydrolysis of sugars in aqueous medium leads to units called monosaccharides. Typically, in an aqueous solution, a monosaccharide sugar molecule of five or six carbon atoms forms a cyclic hemiacetal, a ring structure. Preferably, the sugars of the present disclosure are monosaccharides or disaccharides, i.e. consist of one or two monosaccharide units, each having from three to seven, preferably from three to six carbon atoms. Exemplary sugars of the present disclosure are, but are not limited to, monosaccharide hexoses such as glucose (dextrose), galactose, mannose and fructose; monosaccharide pentoses such as xylose, ribose, disaccharide arabinose such as lactose, trehalose, sucrose, maltose and cellobiose consisting of

The

They may also be used for the various ring-linked sugar units (cyclodextrins) and their

ΡΕ1746976 derivatives. Sugar reduction is a method for obtaining polyols. More stable non-reducing and non-metabolizable disaccharides such as sucrose or trehalose are preferred. Various polyols, monosaccharides and disaccharides are commercially available.

with groups a polyol carboxylate exemplifying anionic groups the

Anionic groups any limitation, thiocarbonate,

A polyol or polyanionized sugar is a polyol or sugar having its anionic (anionized) or fully modified or substituted hydroxyl groups. Thus, a polyanionized or polyanionized sugar comprising a polyol moiety or a sugar moiety together with therein include, without carbonate, dithiocarbonate, phosphate, phosphonate, sulfate, sulfonate, nitrate and borate. It is preferred that at least one anionic group of a polyanionized sugar or polyol is a strongly anionic group, that is, it is more than 50% ionized over a wide pH range, e.g. pH 3-12, preferably pH 2- 12, when in aqueous medium, or alternatively a log of the dissociation constant (pK_The) of 3 or less, preferably 2 or less. Polyanionization of a polyol or sugar can be accomplished by a variety of chemical processes well known in the art. For example, the reaction of polyols and / or sugars with sulfur trioxide or chlorosulfonic acid in pyridine or 2-picoline results in some or all of the hydroxyl groups esterified with sulfuric acid (sulfated) residues to provide a polysulfated sugar or polyol. Sulfated sugar exemplifying the present disclosure is sulphated sucrose, including, but not limited to, sucrose hexasulfate,

ΡΕ1746976 sucrose heptasulfate and sucrose octasulfate (see Ochi. K., et al., 1980, Chem. Pharm. Buli., V.

28, p. 638-641). The sulfated sugar of the present invention is sucrose octasulfate. Similarly, reaction with phosphorus oxychloride or diethylchlorophosphate in the presence of base catalyst leads to polyphosphorylated polyols or sugars. Polyphosphorylated polyols are also isolated from natural sources. For example, inositol polyphosphates such as inositol hexaphosphate (phytic acid) are isolated from maize. A variety of sulfated, sulfonated and phosphorylated sugars and polyols suitable for the practice of the present disclosure are disclosed, for example, in US Patent 5,783,568 and US Patent 5,281,237. It has been unexpectedly found that polyanionized polyhydroxylated compounds with only strongly acidic dissociation steps, for example, groups having pKa less than about 3.0, preferably less than about 2.0, such as, for example, sulfate monoesters (pKa of 1.0 or less) provide liposomal encapsulation with better drug retention than polyanionized polyhydroxylated compounds also having weakly acidic dissociation steps, as phosphate monoesters (step 1, pKa of about 1.5; step 2, pKa of about 6.7; see Stahl and Wermuth, Op. cit., 2002). Example 73 below illustrates this finding. Complexation of polyols and / or sugars with more than one boric acid molarecule also results in a polyanionized (polyborate) product. The reaction of polyols and / or sugars with carbon disulphide in the presence of alkali results polyanionized (polydithiocarbonates,

A derivative in polyxanthogenate derivatives of polyol or polyanionised sugar may be isolated as an acid

It is free and neutralized with a suitable base, for example with an alkali metal hydroxide, ammonium hydroxide or, preferably, with a substituted amine, for example, an amine corresponding to a substituted ammonium of the present disclosure, in pure or as a substituted ammonium hydroxide providing a substituted ammonium polyanionic salt of the present disclosure. Alternatively, a sodium, potassium, calcium, barium or magnesium salt of a polyanionized polyol / sugar may be isolated and converted to a suitable form, for example an ammonium salt form substituted by any known method, for example by ion exchange.

The polyanion of the present disclosure typically has a charge density of at least two, three or four negatively charged groups per unit, for example, per carbon atom or ring in a carbon chain or per monosaccharide unit in a sugar. The polyanionized cyclic sugar or polyol of the present disclosure preferably has at least 75% of the available polyanionized hydroxyl groups and more preferably 100% of the available polyanionized hydroxyl groups. In addition, polyanionization within the liposomes is usually at a level that is compatible or facilitates administration and release of the entrapped entity within the liposomes at the site of its intended action, but decreases the premature release of the entrapped entity, i.e. for the liposome to reach its intended site of action.

The degree of polyanionization within the liposomes can be used to regulate the release characteristics, for example the release rate and kinetics of an entity.

ΡΕ1746976 trapped within the liposomes. In general, the degree of polyanionization may be assessed on the basis of the amount of sugar or polyanionized polyol relative to the total amount of anion (anions) or, in the case of polyanion being the only anion type, the percentage of polyanionization which refers to the amount of anion. total polyanionization ability of the polyanion, for example sugar or polyanionized polyol or a mixture thereof within the liposomes. In one embodiment, the polyanionized sugar or polyol is mixed with one or more different anions, and the lower the amount of sugar or polyanionized polyol relative to the amount of other anion (s), the faster the release of the anion (s). liposome entity.

Normally, if an entrapped entity is released very slowly from the liposomes at the site of its intended activity, the desired release rate of the entity can be achieved using a polyanionized sugar or polyol mixture with one or more different monovalent or polyvalent anions, for example, chloride, sulfate, phosphate, etc. Alternatively, mixtures of sugar or polyanionized polyols with varying degrees of polyanionization may be used. In one embodiment, the degree of polyanionization within the liposomes is between 0.1% and 99%, 10% and 90%, or 20% and 80% of the total (anion) anion (s) within the liposomes. , for example, with an entrapped entity.

In general, the liposome composition may contain one or more polyanions in any suitable form, for example, as an acid or salt comprising a polyanion and a cation. The amount of polyanion, for example sugar or polyanionized polyol may be stoichiometric

ΡΕ1746976 equivalent or different from cation quantity. In one embodiment, the liposome composition contains one or more polyanion salts of a cation, wherein a cation concentration gradient or a pH gradient is present throughout the liposome membrane. In another embodiment, the liposome composition contains one or more substituted ammonium polyanion salts. In yet another embodiment, the liposome composition contains the polyanion within the liposomes, while the polyanion in the liposome-containing medium is partially or substantially removed by any suitable means known to one of skill in the art, for example, dilution, chromatography. ion exchange, size exclusion chromatography, dialysis, ultrafiltration, absorption, precipitation, etc. In yet another embodiment, the liposome with the entrapped polyanion, for example a polyanionized polyol or a polyanionized sugar, also has a transmembrane gradient effective in retaining substances within the liposome. 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. A substituted ammonium gradient typically includes a substituted form of the ammonium ion comprising at least one CN bond, such as primary, quaternary, tertiary or quaternary ammonium. Methods of creating transmembrane gradients are routine in the liposome technique.

According to yet another feature of the present disclosure, the liposome composition of the present disclosure contains one or more substituted ammoniums and / or polyanions of the present disclosure and a chemical or biological entity, for example, a therapeutic entity or

ΡΕ1746976 therapeutic, fertilizer, detectable. For example, the entity contained in the liposome composition of the present disclosure may be an ink, dye, magnetic compound, bait, biocatalyst, taste or odor modifying substance, bleach or any entity known to resonance imaging is detectable by any suitable means. in the art, for example magnetic imaging (MRI), optical imaging, fluorescent / luminescent imaging or nuclear techniques. Conveniently, a contained or chargeable entity for the liposome composition of the present disclosure is a weakly basic (membrane-permeable) lipid entity, for example, an amine or nitrogen-based entity.

In one embodiment, the entity contained in the liposome composition of the present disclosure is a therapeutic agent.

In another embodiment, the entity contained in the liposomal composition is an anticancer entity. A partial list by classification of some of the commonly known commercially approved (or actively developing) antineoplastic agents is as follows.

Structure-based classes: fluoropyrimidines - 5-FU, fluorodeoxyuridine, ftorafur, 5'-deoxyfluorouridine, UFT, Sl capecitabine; pyrimidine deoxycytidine nucleosides, cytosine arabinoside, 5-azacytosine, gemcitabine, 5-azacytosine arabinoside; purines - mercaptopurine, thioguanine, azathioprine, allopurinol, cladribine, fludarabine, pentostatin, 2-chloro adenosine;

ΡΕ1746976 analogues of platinum-cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin, CI973, JM-216; anthracyclines / anthracenediones - doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone; epipodophyllotoxins - etoposide, teniposide; camptothecins-irinotecan, topotecan, lurtotecan, silatecan, 9-amino camptothecin, 10,11-methylenedioxy camptothecin, 9-nitro camptothecin, TAS 103, 7- (4-methylpiperazine methylene) 10,11-ethylenedioxy-20 (S ) -camptothecin, 7- (2-Nisopropylamino) ethyl) -20 (S) -camptothecin; hormones and hormonal analogues - diethylstilbestrol, tamoxifen, toremifene, tolmudex, timitaq, flutamide, bicalutamide, finasteride, estradiol, trioxifene, droloxifene, medroxyprogesterone acetate, megesterol acetate, aminoglutethimide, testolactone and others; enzymes, proteins and antibodies - asparaginase, interleukins, interferons, leuprolide, pegaspargase and others; vinca alkaloids - vincristine, vinblastine, vinorelbine, vindesine; taxanes - paclitaxel, docetaxel.

Mechanism-based classes: anti-hormones - see classification for hormones and hormonal analogues, anastrozole; antifolates - methotrexate, aminopterin, trimethrexate, trimethoprim, piritrexim, pyrimethamine, edatrexate, MDAM; antimicrotubule agents - taxanes and vinca alkaloids; alkylating agents (classical and non-classical) - nitrogen nitrogen compounds (mechlorethamine, chlorambucil, melphalan, uracil mustard), oxazaphosphorins (ifosfamide, cyclophosphamide, phosphosamide), alkylsulfonates (busulfan), nitrosureas (carmustine, tanthospine) , dacarbazine and others; antimetabolites - purines, pyrimidines and nucleosides, listed above;

antibioticsΡΕ1746976 anthracyclines / anthracenediones, bleomycin, dactinomycin, mitomycin, plicamycin, pentostatin, streptozocin; topoisomerase inhibitors - camptothecins (Top I), epipodophyllotoxins, m-AMSA, ellipticines (Top II); antivirals - AZT, zalcitabine, gemcitabine, didanosine and others; Miscellaneous cytotoxic agents - hydroxyurea, mitotane, fusion toxins, PZA, bryostatin, retinoids, butyric acid and derivatives, pentosan, fumagillin and others.

In addition to the above, an anticancer entity includes, without limitation, any vinca alkaloid inhibitor, for example topoisomerase, vincristine, vinpocetin, microtubule or vinblastine depolymerising agent, vinorelbine, destabilizing vinflunine, microtubule stabilizing agent, for example, taxane, aminoalkyl or aminoacyl analog of paclitaxel or docetaxel, for example 2 '- [3- (N, N-diethylamino) propionyl] paclitaxel, 7- (N, N-dimethylglycyl) paclitaxel and 7-L-alanylpaclitaxel, alkylating agent, receptor binding agent, tyrosine kinase inhibitor, phosphatase inhibitor, cyclin dependent kinase inhibitor, enzyme inhibitor, aurora kinase inhibitor, nucleotides , polynucleotides and farnesyltransferase inhibitor.

In another embodiment, the entity contained in the liposome composition of the present disclosure is a therapeutic agent of anthracycline compounds or derivatives, camptothecin compounds or derivatives, ellipticine compounds, vinca alkaloids or derivatives, vortmanine, analogs thereof and pyrazolopyrimidine derivatives or compounds having aurora kinase inhibiting properties.

ΡΕ1746976

In yet another embodiment, the entity contained in the liposome composition of the present disclosure is an anthracycline, doxorubicin, daunorubicin, mitomycin C, epirubicin, pirarrubicin, rubidomycin, N-acetyladriamycin, rubidazone, 5imidodaunomycin, Nimidodaunomycin drug daunoriline, mitoxantrone; a compound of camptothecin, camptothecin, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11methylenedececane-1-chlotececane 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; a compound of ellipticine, ellipticine, 6-3aminopropyl-ellipticine, 2-diethylaminoethyl-ellipticinium and salts thereof, datelipid, reteliptin.

In yet another embodiment, the liposome entity of the present disclosure is a pharmaceutical entity, including, but not limited to, any of the following: ethylenediamine antihistamine derivatives (bromphenamine, diphenhydramine);

antiprotozoans: quinolones (iodoquinol); amidines (pentamidine); anthelmintics (pirantel); antischistosomal drugs (oxaminiquine); antifungal derivatives of triazole (fliconazole, itraconazole, ketoconazole, miconazole); antimicrobial cephalosporins (cefazolin, cefonicide, cefotaxime, ceftazimide, cefuoxime); antimicrobial beta-lactam derivatives (aztreopam, cefmetazole, cefoxitin); antimicrobial drugs

ΡΕ1746976 erythromycin group (erythromycin, azithromycin, clarithromycin, oleandomycin); penicillins (benzylpenicillin, phenoxymethylpenicillin, cloxacillin, methicillin, naphcillin, oxacillin, carbenicillin); tetracyclines; other antimicrobial antibiotics, novobiocin, spectinomycin, vancomycin; antimicobacterial drugs: aminosalicylic acid, capreomycin, ethambutol, isoniazid, pyrazinamide, rifabutin, rifampin, clofazime; antiviral adamantanes: amantadine, rimantadine; guinidine derivatives: chloroguine, hydroxychloroguine, promaguine, gionone; antimicrobial gionolones: ciprofloxacin, enoxacin, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin; sulfonamides; urinary tract antimicrobials: methenamine, nitrofurantoin, trimethoprim; nitroimidazoles: metronidazole; cholinergic guaternary ammonium compounds (ambetinium, neostigmine, physostigmine); anti-Alzheimer's aminoacridines (tacrine); anti-Parkinson's drugs (benztropine, biperiden, procyclidine, trihexylenidyl); antimuscarinic agents (atropine, hyoscyamine, scopolamine, propantelin); adrenergic dopamines (albuterol, dobutamine, ephedrine, epinephrine, norepinephrine, isoproterenol, metaproperenol, salmeterol, terbutaline); ergotamine derivatives; myorelaxants or curare series; centrally acting myorelaxants; baclofen, cyclobenzepine, dentrolene; nicotine; betaadrenobloggers (acebutyl, amiodarone); benzodiazepines (dithiazem); antiarrhythmic drugs (diisopyramide, encidine, local anesthetic series - procaine, procainamide, lidocaine, flecaimide), guinidine; ACE inhibitors: captopril, enelaprilat, fosinoprol, guinapril, ramipril; antilipidemics: fluvastatin, gemfibrozil, HMG-CoA (pravastatin) inhibitors;

ΡΕ1746976 hypotensive drugs: clonidine, guanethidine granadril, hydralazine; coronary arteries: dipyridamolar.

guanabenz, prazocin, and non-vasodilators According to the present disclosure, the entity contained in the liposome composition of the present disclosure may also be a pre-entity, for example, a prodrug or an agent that is capable of being converted to a desired entity. after one or more conversion steps under a condition, such as a change in pH or an enzymatic cleavage of a labile bond. Such conversion may occur upon release of the prodrug from within the liposome at the desired site of drug / liposome action. However, the pre-entity may be converted to the desired active entity within the liposomes of the present disclosure prior to use of the liposomes as an administration vehicle, for example administration to a patient. For example, an entity may be modified to a pre-entity so that it is easier to load on liposomes and then converted back to the desired entity when within the liposomes of the present disclosure. Thus, according to the present disclosure, entities which are generally not subject to active, remote or other gradient-based loading methods may be effectively loaded into the liposomes, for example, into the interior space of the liposome, in its unchanged, native form.

Globally cationic compounds, that is, compounds capable of achieving a positive net ionic charge under liposome loading conditions, especially compounds containing a titratable amine, are known as

Carregar1746976 load effectively on liposomes that exhibit transmembrane ion gradients. If an entity of interest is an organic compound and is not a globally cationic compound containing a titratable amine, a derivative thereof having the necessary ionic properties may be prepared by suitable modification, for example according to the methods described. in Woodle et al. in WO 96/25147. For example, an amino group may be introduced by esterification of a hydroxyl group of the entity with an amino acid. Alternatively, a hydrophobic group may be introduced into a water-soluble compound to aid in its partitioning in the liposome membrane and subsequent displacement across the membrane to the intraliposomal compartment, that is, within the liposomes. Another useful modification for creating a liposome-loading preent is the formation of a carbonyl group adduct, for example a hydrazone, an oxime, an acetal or a ketal. A group containing a modified amino may be hydrolyzed or chemically divided from the modified compound after loading of the modified compound on the liposomes according to the present invention. Typical processes for regenerating the entity from a pre-entity within the liposomes are hydrolysis, photolysis, radiolysis, thiolysis, amonolysis, reduction, substitution, oxidation or elimination. These processes can be performed without limitation by changing the pH or by an enzymatic action. For example, paclitaxel or docetaxel, nonionic entities, are converted to their 2 '- (diethylaminopropionyl) - or 7' (diethylaminopropionyl) esters, which are weak bases (presets). After being loaded into the liposomes by any known method, including but not limited to gradient-based remote active method

In transmembrane or solubility gradient based methods and / or the methods of the present disclosure, intraliposomal 2 '(diethylaminopropionyl) -paclitaxel is converted to the original paclitaxel encouraging its hydrolysis by increasing the pH above pH 7.0. Thus, a liposome with a taxane neutral molarecule encapsulated in its inner space with a drug / lipid ratio of more than 0.05 moles per molar of the liposome lipid is obtained without the aid of hydrophilic covalent modifications of the taxane molarecule. (e.g., by PEG binding), taxane cyclodextrin complexes or micelle forming surfactants, taxane solubilizers.

Liposomes contained in the liposome composition of the present disclosure may be any known or later discovered liposome in the art. In general, the liposomes of the present invention may have any liposome structure, for example, structures having an interior space isolated from the outer environment by one or more lipid bilayers, or any microcapsule having a semipermeable membrane with a central lipophilic part where the membrane encloses an interior. A lipid bilayer may be any arrangement of amphiphilic molar molecules, characterized by a hydrophilic part (hydrophilic portion) and a hydrophobic part (hydrophobic portion). Normally, the amphiphilic molar molecules in a bilayer are arranged in two-dimensional sheets, in which the hydrophobic portions are oriented inward while the hydrophilic portions are oriented outward. Amphiphilic molarecules forming liposomes may be known or later discovered amphiphilic molarecules, for example, lipids of synthetic origin or

ΡΕ1746976 natural or biocompatible lipids. Liposomes may also be formed of amphiphilic and surfactant polymers, for example, polymerosomes and niosomes. For purposes of the present disclosure, without limitation, these liposome-forming materials are also referred to as lipids.

In accordance with the present invention, the liposomes contained in the liposome composition of the present invention may also be targeted liposomes, for example, liposomes containing one or more targeting moieties or liposome surface-modifying modifiers. A targeting moiety may be any agent that is capable of specifically binding or interacting with a desired target. In one embodiment, a targeting moiety is a ligand. 0 The ligand according to the present invention preferably binds and / or internalizes in a cell in which the liposome entrapped entity exerts its desired effect (a target cell). A ligand is usually a member of a binding pair, wherein the second member is present on or in target cells or tissue comprising the target cell. Examples of suitable ligands for the present invention are: folic acid, protein, for example transferrin, growth factor, enzyme, peptide, receptor, antibody or antibody fragment, such as Fab ', Fv, single chain Fv, single domain or any other polypeptide comprising antigen binding (CDR) sequences of an antibody molarecule. A ligand-directed liposome in which a targeting moiety is an antibody or target antigen-binding fragment thereof is called an immunoliposome. In a preferred embodiment, the liposome which

ΡΕ1746976 carries a targeting moiety, for example a ligand, is internalized by a target cell. In yet another embodiment, a targeting moiety is a ligand that specifically interacts with a tyrosine kinase receptor such as, for example, EGFR, HER2, HER3, HER4, PD-GFR, VEGFR, bFGFR or IGFR receptors. In yet another embodiment, the targeting moiety specifically interacts with a growth factor receptor, an angiogenic factor receptor, a transferrin receptor, a cell adhesion molarecule, or a vitamin receptor.

According to another embodiment, the liposomes contained in the liposome composition have a transmembrane concentration gradient of a substituted ammonium and / or polyanion. Preferably, the highest concentration is in the inner (inner) space of the liposomes. In addition, the liposome composition may include one or more transmembrane gradients in addition to the gradient created by the substituted ammonium and / or polyanion. For example, the liposomes contained in the liposome composition may additionally include a transmembrane pH gradient, an ion gradient, an electrochemical potential gradient and / or a solubility gradient.

In accordance with 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 the entity and instructions, for example, procedures or information. relating to the use of the liposome composition in one or more applications. These instructions may be provided by any means, eg copying

ΡΕ1746976 on paper, electronic media or access to a database or web site containing the instructions.

The liposome membrane composition of the present invention may be made by any suitable method known or later discovered by one of skill in the art. In general, a variety of lipid components can be used to make liposomes. Lipid components usually include, but are not limited to, (1) uncharged lipid components, for example, cholesterol, ceramide, diacylglycerol, acyl (polyethers) or alkylpoly (ethers); (2) neutral phospholipids, for example diacylphosphatidylcholine, sphingomyelin and diacylphosphatidylethanolamine; (3) anionic lipids, for example, diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidate, cardiolipin, diacylphosphatidylinositol, diacylglycerolhemisuccinate, diacliglycerolhemiglurate, cholesterylhemisuccinate, cholesterylhemylate and similar; (4) polymer conjugated lipids, for example, N- [methoxy- (poly (ethylene glycol) diacylphosphatidylethanolamine), poly (ethylene glycol) diacylglycerol, poly (ethylene glycol) ceramide; and (5) cationic lipids, for example 1,2-diacyl-3-trimethylammonium propane (DOTAP), dimethyldiketadecylammonium bromide (DDAB) and 1,2-diacyl-sn-glycero-3-ethylphosphocholine. Monacyl-substituted derivatives of these lipids as well as di and monoalkyl analogs will also be employed.

Various lipid components may be selected to satisfy, modify or confer one or more desired functions. For example, the phospholipid may be used as the major vesicle-forming lipid. The inclusion of cholesterol is useful for maintaining membrane stiffness and

ΡΕ1746976 decrease drug leakage. Polymer-conjugated lipids may be used in the liposomal formulation to increase circulation time by reducing liver and spleen clearance of liposomes, or to improve the stability of liposomes against aggregation during storage in the absence of the extension effect. of circulation. While it is stated that the inclusion of PEG-lipids in the amount of 1 or more mole% of liposome lipid prolongs the blood circulation time of liposomes several times (see, for example, US Patent 5,013,556), we have surprisingly found that The liposomes of the present invention are of quite long circulation, and the addition of PEG-lipid to the liposome composition only prolonged circulation longevity by less than twice, if at all. In addition, charge-modulating lipids (titratable) may be used to help deliver liposome-encapsulated entities to cytosolic or nuclear targets, facilitating some classes of entities that escape the boundaries of the endosomal pathway.

In one embodiment, the liposomes of the present disclosure include lecithin, cholesterol and an amphipathic polymer. Lecithin included in the liposomes may be a natural lecithin, a hydrogenated natural lecithin, a synthetic lecithin, 1,2-distearoyl lecithin, dipalmitoyl lecithin, dimyristoyl lecithin, diololyl lecithin, l-stearoyl-2-oleolylecithin or lecithin. 2oleoyl lecithin, while the amphipathic polymer may be a polyethylene glycol lipid derivative, for example polyethylene glycol phosphatidylethanolamine, polyethylene glycol diacylglycerol or polyethylene glycol ceramide derivative, wherein the poly (ethylene glycol) moiety has a molar weight of about 250

ΡΕ1746976 to about 20,000, more commonly from about 500 to about 5,000. In another embodiment, the ratio of lecithin and cholesterol in the liposomes of the present disclosure is about 3: 2 per molar. In yet another embodiment, the antipathetic polymer is at least 0.1 mol% of the liposome-forming lipid in the liposomes of the present disclosure. In yet another embodiment, the amount of an antipathetic polymer is between 0.1 mol% and 1 mol% of liposome-forming lipid in the liposomes of the present disclosure. Preferably, the antipathetic polymer is a neutral polymer, that is, under drug loading conditions, the net ionic charge of zero, for example PEG-diacylglycerol, PEGdialkylglycerol or PEG-ceramide. It has been unexpectedly found that inclusion of ionically neutral antipathic lipids to a PEG-lipid content of about 5.7 mol% of total lipid produces a high efficiency liposome loading of, for example, vinca alkaloids such as vinorelbine, whereas in the case of anionically charged PEGDSPE the loading efficiency visibly decreases to a PEG-lipid content of 1.6 mol% or more (Example 72).

In yet another embodiment, the liposomes of the present disclosure contain a camptothecin prodrug, such as irinotecan, and are composed of lecithin and cholesterol, for example, in a ratio of about 3: 2 per molar, and an antipathetic polymer. for example in an amount of at least 0.1 mol% or less than 1% of the liposome-forming lipid.

Liposomes of the present invention may be made by any method known or to be known in the art.

Técnica1746976 technique. See, for example, G. Gregoriadis (editor), Liposome Technology, vol. 1-3, I^The edition, 1983; 2^The 1993 edition, CRC Press, Boca Raton, FL. Examples of suitable methods for making the liposome composition of the present invention include extrusion, reverse phase evaporation, sonication, solvent injection (e.g., ethanol), microfluidization, detergent dialysis, ether injection and dehydration / rehydration. 0 Liposome size can be controlled by controlling the pore size of the membranes used for low pressure extrusions, or the pressure and number of passages used in microfluidization or any other suitable methods. In one embodiment, the desired lipids are first hydrated by thin film hydration or by injection of ethanol and subsequently sized by extrusion through membranes of a defined pore size; most commonly 0.05 pm, 0.08 pm or 0.1 pm.

Liposome compositions containing the substituted ammonium and / or polyanion of the present disclosure within the liposomes may be made by any suitable method, for example, liposome formation in the presence of substituted ammonium and / or polyanion of the present disclosure, for example. for example in the form of salt. Substituted ammonium and / or polyanion outside the liposomes may be removed or diluted either after liposome formation or prior to loading or entrapment of a desired entity. Alternatively, the substituted ammonium-containing and / or polyanion-containing liposome composition of the present disclosure may be made directly by the ion exchange method or by an intermediate free acid step having a substituted ammonium gradient of the present disclosure, e.g. ammonium salt

ΡΕ1746976 substituted for polyanionized sugar or polyol. Such liposomes may be neutralized using the amine or its salt with a volatile acid, for example carbonate. The resulting liposome solution may be used directly or, alternatively, the salt contained therein may, if desired, be removed, for example, by evaporation and crystallization followed by dissolution in aqueous medium.

Preferably, the liposome composition of the present disclosure has a transmembrane concentration gradient of substituted ammonium and / or polyanion, for example, the concentration of the substituted ammonium salt and / or polyanion within the liposome is usually at least 100 times higher. higher than the concentration of substituted ammonium and / or polyanion in the medium outside the liposome.

In one embodiment, the concentration of the substituted ammonium salt and / or polyanion within the liposome is at least 100 times greater than the concentration of the substituted ammonium salt and / or polyanion in the medium outside the liposome and is at least one concentration. about 10 mM, 50 mM, 0.1 M, 0.2 M, 0.5 M, 0.6 M, 0.7 M or 1.0 M, where molarity is calculated on the basis of substituted ammonium . In another embodiment, the concentration of the substituted ammonium salt and / or polyanion within the liposome is at least 100 times greater than the concentration of the substituted ammonium salt and / or polyanion in the medium outside the liposome and is at a concentration of about 0.65 M or about 1.0 M.

In addition, the liposome composition of the present invention usually has an external pH that is compatible or useful for maintaining the stability of a desired entity during the loading process.

ΡΕ1746976 with high loading efficiency, eg above 90% trapping. For example, pH in the range 4-7 or 4.5-6.5 is preferred. In particular, according to the present invention, irinotecan loading is best performed at external medium pH in the range from about 4.0 to about 7.0, more preferably from about pH 5.0 to about 6, 5 Loading of a vinca derivative, for example vincristine, vinorelbine or vinblastine, is best performed at a pH of about 5.07.0, more preferably at a pH of about 6.5.

According to the present disclosure, a desired entity may be charged or entrapped in the liposomes by incubating the desired entity with the liposomes of the present disclosure in an aqueous medium at a suitable temperature, for example, a temperature above the phase transition temperature of the liposomes. lipid components during loading, being reduced below the phase transition temperature after loading of the entity. Incubation time is usually based on the nature of the lipid components, the entity to be loaded on the liposomes and the incubation temperature. Normally, incubation times of a few minutes to a few hours are sufficient. Since high entrapment efficiencies of more than 85%, typically over 90% are achieved, there is generally no need to remove the non-entrapped entity. However, if there is such a need, the non-entrapped entity may be removed from the composition by various means, such as size exclusion chromatography, dialysis, ultrafiltration, adsorption or precipitation. It has been unexpectedly found that keeping the ionic strength low during the incubation of an entity, such as a

Camptothecin derivative ΡΕ1746976 or a vinca alkaloid derivative, with the liposomes of the present disclosure, followed by increased ionic strength at the end of incubation, results in higher loading efficiency, better removal of non-entrapped drug, and better stability of the liposomes against aggregation. Typically, incubation is carried out, for example, in an aqueous solution at a lower ionic strength than equivalent to 50 mM NaCl, or more preferably less than equivalent to 30 mM NaCl. After incubation, a concentrated salt solution, for example NaCl, may be added to increase ionic strength to greater than 50 mM NaCl, or more preferably greater than 100 mM NaCl. . Without being bound by theory, we consider that increasing ionic strength aids in the dissociation of the entity from the liposome membrane, leaving substantially the entire entity encapsulated within the liposome interior space.

In general, the entity-to-lipid ratio, for example, drug loading ratio obtained by loading an entity depends on the amount of the entrapped entity within the liposomes, the concentration of substituted ammonium and / or entrapped polyanion, e.g. salt, the physicochemical properties of the entrapped entity and the type of shrinkage (anion) used, for example polyanion. Due to the high loading efficiencies achieved in the compositions and / or methods of the present invention, the entity-to-lipid to liposome entrapped ratio is greater than 80%, greater than 90%, and typically more than 95%. % of entity-to-lipid ratio calculated based on the amount of entity and liposome lipid taken for the loading process (the ratio of

Entrada1746976 entry). In fact, nearly 100% (quantitative) encapsulation is common. The entity-paralipid ratio in liposomes can be characterized in terms of the weight ratio (amount of entity weight by weight or liposome molar unit) or the molar ratio (entity molars by weight or liposome molar unit of liposome) ). One unit of the entity-to-lipid ratio may be converted to other units by routine calculation as exemplified below. The weight ratio of an entity in the liposomes of the present invention is usually at least 0.05, 0.1, 0.2, 0.35, 0.5 or at least 0.65 mg of the entity per mg of lipid. . In terms of the molar ratio, the entity-to-lipid ratio of the present invention is at least about 0.02 to about 5, preferably at least 0.1 to about 2, and with more preferably from about 0.15 to about 1.5 moles of the drug per molar of the liposome lipid. In one embodiment, the entity-to-lipid ratio, for example, the drug loading ratio of camptothecin derivatives is at least 0.1, for example 0.1 moles of camptothecin derivative per one molar lipid. liposome, and preferably at least 0.2. In another embodiment, the entity-to-lipid ratio, for example, the drug loading is at least about 300 mg of entity (e.g., vinca alkaloid or a derivative thereof) per mg of the forming lipid. of liposome. In yet another embodiment, the entity-to-lipid ratio, for example, drug loading is at least about 500 mg of entity (e.g., camptothecin derivative or camptothecin prodrug) per mg of the drug. liposome-forming lipid. Surprisingly, the invention provided stable and near quantitative liposomal encapsulation of irinotecan at a drug-to-lipid ratio of more than 0.8

ΡΕ1746976 mmolar of entity per 1 g of liposome lipid, more than 1.3 mmolar of entity per 1 g of liposome lipid, and even as high as 1.7 mmolar of entity per 1 g of liposome lipid (see Example 74 ).

If the liposome comprises a phospholipid, it is convenient to express the entity content in units of weight (mass) of the drug per molar unit of the liposome phospholipid, for example, mg of drug / mmolar phospholipid. However, one skilled in the art will appreciate that the drug content can be eivalently expressed independently of the presence of phospholipids in a liposome and, moreover, can be eivalently expressed in terms of a molar amount of the drug per unit. (mass or molar) of the liposome lipid content. For example, a liposome containing 3 molar parts of distearoylphosphatidylcholine (DSPC, molar weight 790), 2 molar parts of cholesterol (molar weight 387) and 0.015 molar parts of poly (ethylene glycol) derivatized distearoylphosphatidylethanolamine (PEG-DSPE 27, molar weight) , and containing a doxorubicin drug (molar molecular weight 543.5) at a phospholipid 150 mg / mmole drug / lipid ratio, the same drug content can be eivalently expressed in terms of mg drug / mg total lipid as follows:

(a) Calculate the molar quantities of the liposome lipid components normalized to the liposome phospholipid molar unit (in this example, DSPC and PEGDSPE) by dividing the molar amounts of one component by the total molar amounts of the liposome phospholipids:

ΡΕ1746976

DSPC 3 / (3 + 0.015) = 0.99502 Cholesterol 2 / (3 + 0.015) = 0.666335 PG-DSPE 0.015 / (3 + 0.015) = 0.00498 (b) Calculate the mass amount of the total lipid of the liposome corresponding to a unit molar amount of the liposome phospholipid and the molar weights of the components:

Total lipid, mg / mmolar phospholipid = 0.99502x790 + 0.66335x387 + 0.00498x2750 = 1056.48 (c) Calculate the amount of drug mass per unit mass of the total lipid by dividing the drug content expressed in units of mass per molar unit of phospholipid by the number obtained in step (b):

Doxorubicin, mg / mg total lipid = 150 / 1056.48 = 0.14198.

(d) Calculate the molar amount of the drug per unit mass of the total lipid by dividing the number obtained in step (c) by the molar weight of the drug (in this case, 543.5):

Doxorubicin, mmolar / g total lipid = 0.14198 / 543.5x1000 = 0.261.

(e) Calculate the molar part of the phospholipids in the liposome lipid matrix:

Molar part of phospholipids = (total molars of phospholipids) / (amount of total molar lipids) = (3 + 0,015) / (3 + 2 + 0,015) = 0,6012.

F1746976 (f) Calculate the molar ratio of doxorubicin to total lipid.

Doxorubicin, molar / total lipid molar = (molar part of phospholipids) x (doxorubicin, g / molar phospholipid) / (molar weight of doxorubicin) =

0.6012x150 / 543.5 = 0.166.

Thus, the relationship between drug-to-lipid and drug-to-phospholipid ratio expressed in various units is readily established. As used herein, a lipid includes, but is not limited to, any membrane forming components of the liposome membrane, such as polymers and / or detergents.

The substituted ammonium salt and / or polyanion solution trapped in the liposome of the present disclosure usually has an osmotic force (osmolarity) that helps maintain liposomes stable against osmotic damage (swelling and / or rupture) without sacrificing the carrying capacity of the liposomes. . In one embodiment, the osmolarity of the liposome composition of the present disclosure is in the range 0.1 to 1.5 molar / kg or preferably 0.2 to 1.0 molar / kg. Surprisingly, we have found that the liposomes of the present disclosure are stable against adverse effects of high intraliposomal osmotic force on drug loading. Intraliposomal osmolararities as high as 0.727 molar / kg were well tolerated, resulting in practically quantitative loading of a drug up to the theoretical maximum stoichiometric exchange of intraliposomal substituted ammonium ions with drug molars (in the case of

ΡΕ1746976 irinotecan, a drug molarecule by a substituted ammonium ion), although the osmolarity of the extraliposomal aqueous medium during drug and liposome coincubation is close to the physiological value of about 0.3 molar / kg (Example 74).

In general, the liposome composition of the present invention is quite stable during storage, for example as measured by the percentage of entrapped entity released outside the liposomes or remaining within the liposomes after a certain period of time from initial entity loading. 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, for example, less than 10% of the entrapped entity is released 6 months after the initial loading of the entity. In one embodiment, the liposome composition of the present invention is stable at 4 ° C for at least 2 years, for example, less than 20% of the entrapped entity is released 2 years after the initial loading of the entity.

It is advantageous for a liposome entrapped entity to remain encapsulated in the liposome until it reaches the site of its intended action, for example a tumor, in the case of a liposomal antitumor drug administered to a patient. The liposomes of the present invention have shown surprising stability against release (leakage) of the entrapped entity under in vivo conditions, for example, in the blood of a mammal. The exposure time required for the release of 50% of the entrapped entity, for example the drug, from liposomes (half-release time) in a rat's blood in vivo was more than 24 hours. In particular, liposomes loaded with

ΡΕ1746976 vinca alkaloids, for example vinblastine, vincristine and vinorelbine, showed remarkable stability against drug leakage in vivo, with half-release time of at least 24 hours, or the amount of entity remaining encapsulated after 24 hours. in vivo blood of at least about 50% of the pre-administration value. Normally, the half-release time over 33 hours was observed, or the amount of encapsulated entity that remains encapsulated after 24 hours in blood in vivo of at least about 60%; and half-release time over 46 hours, or the amount of the encapsulated entity after 24 hours in blood in vivo of at least about 70% of the pre-administration value was even common. Sometimes the half-release time for an in vivo blood-encapsulated drug was more than 93 hours, and even more than 120 hours. Liposome loaded with camptothecin derivatives such as topotecan and irinotecan also showed exceptional in vivo blood stability, with 79-85% of the initial drug loading remaining encapsulated after 24 hours. Notably, the liposomes of the present invention, while having a low drug release rate in vivo in the bloodstream, showed significant antitumor activity in vivo, exceeding that of free drug (i.e. administered as a solution).

Liposomes of the present invention have provided an unexpected combination of high trapped therapeutic agent efficiency and low toxicity. In general, the activity of a liposome-encapsulated therapeutic entity according to the present invention, for example, the antineoplastic activity of irinotecan in a mammal, is at least twice as high, or at least four times greater than entity activity

It is therapeutic if administered in the same amount through its routine non-liposome formulation, for example without using the liposome composition of the present invention, while the toxicity of the liposome-encapsulated entity does not exceed at least twice, at least twice. three times, or at least four times smaller than the same therapeutic entity administered at the same dose and time but in a free, unencapsulated form. For example, it is well known that liposomal encapsulation of camptothecin anticancer derivatives by methods published by other authors results in increased toxicity (lower tolerated maximum dose, 50% lower mortality dose) compared to unencapsulated drug. See US Patent 6,355,268; US Patent 6,465,008; Colbern, et al., Clinical Cancer Res. 1998, v. 4, p. 3077-3082; Tardi, et al., Cancer Res., 2000, v. 60, p. 3389-3393; Emerson, et al., Clinical Cancer Res. 2000, v. 6, p. 2903-2912. Liposome-encapsulated camptothecin prodrugs, such as irinotecan (CPT-11), which is a cationic, water-soluble camptothecin prodrug derivative, have substantially greater antitumor activity, for example at least 4-fold, and even 10 times larger, evaluated in an in vivo tumor model than the drug in the absence of a liposomal formulation, for example in a free form (solution). This is all the more remarkable because irinotecan requires enzymatic activation, for example by the action of endogenous, unspecified carboxylesterase, but according to the present invention is substantially encapsulated within the interior space of the liposome. On the other hand, surprisingly, the toxicity of camptothecin prodrug such as CPT-11 in liposomal form (drug / lipid mass ratio greater than

ΡΕ1746976

0.1, for example 0.2-0.6 or more) according to the present invention was more than 2 times, more than 3 times, and even more than 4 times smaller than that of the prodrug Free CPT-11 (unencapsulated). In addition, a prolonged release of drug from CPT-11 liposomes was achieved in vivo, with more than 50%, and even more than 70% (79-86%) of the original drug content remaining in the liposomes 24 hours after administration to the bloodstream, and at half-release times above 24 hours, usually above 48 hours. Prolonged liposome permanence of the drug in vivo was associated with a greater antitumor effect. Surprisingly, slower release of CPT-11 in vivo and higher antitumor activity were observed in liposomes containing a low molecular weight polyanionized sugar derivative (sucrose octasulfate) instead of a polymeric anion (polyphosphate) (Example 15).

According to another embodiment of the present invention, the liposome composition of the present invention may be provided as a pharmaceutical composition containing the liposome composition of the present invention and a carrier, for example, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are normal saline, isotonic dextrose, isotonic sucrose, Ringer's solution and Hanks's solution. A buffer substance may be added to provide an optimal pH for storage stability. For example, a pH between about 6.0 and about 7.5, more preferably a pH of about 6.5, is optimal for liposome membrane lipid stability, and provides excellent retention of entrapped entities. . Exemplary buffer substances are the

41746976 histidine, hydroxyethylpiperazine ethyl sulfonate (HEPES), morpholipoethyl sulfonate (MES), succinate, tartrate and citrate, usually at a concentration of 2-20 mM. Other suitable carriers include, for example, water, buffered aqueous solution, 0.4% NaCl, 0.3% glycine and the like. Proteins, carbohydrates or polymeric stabilizers and tonicity adjusters may be added, for example gelatin, albumin, dextran or polyvinylpyrrolidone. The tonicity of the composition may be adjusted to the physiological level of 0.25-0.35 molar / kg with glucose or a more inert compound such as lactose, sucrose, mannitol or dextrin. These compositions may be sterilized by conventional sterilization techniques, well known, for example by filtration. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous medium prior to administration.

Liposome pharmaceutical compositions may also contain other pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions such as pH adjusting agents and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate. , sodium chloride, potassium chloride, calcium chloride, etc. In addition, the liposome suspension may include lipid protecting agents that protect lipids against free radical damage and lipid peroxidation during storage. Lipophilic free radical suppressors such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

ΡΕ1746976

The concentration of the liposomes of the present invention in fluid pharmaceutical formulations may vary widely, that is usually from less than about 0.05%, or at least about 2-10%, to as much as 30 to 50% by weight, and will be selected primarily by fluid volumes, viscosities, etc., according to the specific mode of administration selected. For example, the concentration may be increased to decrease the fluid load associated with treatment. This may be particularly desirable in patients who have congestive heart failure associated with atherosclerosis or severe hypertension. Alternatively, liposome pharmaceutical compositions composed of irritating lipids may be diluted to low concentrations to reduce inflammation at the site of administration.

The amount of the liposome pharmaceutical composition administered will depend upon the specific therapeutic entity entrapped within the liposomes, the condition of the disease being treated, the type of liposomes used and the judgment of the physician. In general, the amount of the liposome pharmaceutical composition administered will be sufficient to deliver a therapeutically effective dose of the specific therapeutic entity.

The amount of the liposome pharmaceutical composition required to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods common in the drug testing art. See, for example, DB Budman, AH Calvert, EK Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for the various therapeutic entities are well known in the art; and according to the present invention an entity

Therapy administered via the liposome pharmaceutical composition of the present invention provides at least the same activity, or 2 times, 4 times, or 10 times more than the activity obtained by administering the same amount of the therapeutic entity in its non-routine formulation. liposome. Typically, dosages of the liposome pharmaceutical composition of the present invention range from about 0.005 to about 500 mg of the therapeutic entity per kilogram body weight, most often from about 0.1 to about 100 mg of the entity. therapy / kg body weight.

Typically, the pharmaceutical liposome composition of the present invention is prepared as topical or injectable, either as a liquid solution or as a suspension. However, suitable solid forms for solution or suspension may also be prepared in liquid vehicles prior to injection. The composition may also be formulated in an enteric coated tablet or gel capsule according to methods known in the art.

The liposome composition of the present invention may be administered in any form that is medically acceptable, which may depend on the health problem or injury to be treated. Possible routes of administration include parenteral injections such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intraepidural, intrathecal, or otherwise, as well as oral, nasal, ophthalmic, rectal, vaginal, topical or for example by inhalation. For the administration of drugs in liposomes formulated according to the invention to central nervous system tumors, a slow sustained intracranial infusion of the liposomes directly is advantageous.

ΡΕ1746976 in the tumor (an enhanced convection administration, or CED). See Saito, et al., Cancer Research, vol. 64, p. 25722579, 2004; Mamot, et al., J. Neuro-Oncology, vol. 68, p. 19, 2004. The compositions may also be applied directly to fabric surfaces. Also specifically included in the invention is sustained release, pH dependent release, or other release administration mediated by a specific chemical or environmental condition, for example by means such as depot injections or erodable implants.

Examples

The following examples are intended to illustrate but not to limit the invention in any way, form or type, either explicitly or implicitly. While typical of those that could be used, other procedures, methodologies or techniques known to those skilled in the art may alternatively be used.

Example 1. Preparation of substituted ammonium salt solutions.

Trialkyl ammonium and dialkyl ammonium sulfate solutions useful for loading drugs (eg doxorubicin) into liposomes were prepared by diluting sulfuric acid with water to a concentration of 0.25 M and then titrating the sulfuric acid solution. with one of a variety of amines. The substituted amines used in this example were triethylamine, trimethylamine, dimethylamine, diethylamine or diethanolamine. After addition of the amines, the resulting solution was diluted to a final concentration of 0.2 M of the substituted ammonium salt. Osmolarity was

ΡΕ1746976 determined using a dew point osmometer. The properties of the resulting substituted alkylammonium sulfate salt solutions are shown in table 1 below.

Table 1. Properties of various dialkyl ammonium sulfate and trialkyl ammonium solutions

<td>salt</td><td></td><td></td><td>Osmolarity, mmolar / kg</td><td>pH</td>

<td>Sulfate</td><td>in</td><td>dimethylammonium</td><td> 472</td><td> 5, 65</td>

<td>Sulfate</td><td>in</td><td>dime tiletane1ammonium</td><td> 509</td><td> 5,72</td>

<td>Sulfate</td><td>in</td><td>diethylammonium</td><td> 519</td><td> 5, 85</td>

<td>Sulfate</td><td>in</td><td>trimethylammonium</td><td> 497</td><td> 5, 81</td>

<td>Sulfate</td><td>in</td><td>triethylammonium</td><td> 559</td><td> 5,33</td>

<td>Example</td><td> 2</td><td>Preparation of</td><td>liposomes with</td><td>salts of</td>

entrapped dialkylammonium and trialkylammonium, and loading of a substance on these liposomes.

Distearoylphosphatidylcholine (DSPC), cholesterol (Col) and N- (methoxy-poly (ethylene glycol) oxycarbonyl) distearoylphosphatidylethanolamine (PEG-DSPE) (prepared from molar weight poly (ethylene glycol) 2000) were codissolved in chloroform at a molar ratio of 3: 2: 0.015, and chloroform was removed at 55-60 ° C by rotary evaporation. The dried lipid film was then hydrated in a solution of each of the dialkyl or trialkylammonium sulfates listed in example 1 at 60 ° C for 30 min. The lipid suspension was extruded under pressure through two stacked 0.1 µm pore size track-etched polycarbonate membrane filters (Corning Nuclepore). The liposome size determined by the quasi-elastic light scattering method was approximately 110-120 nm. Trialkyl ammonium or dialkyl ammonium salts are not

Encapsulated ΡΕ1746976 were removed from the external medium of the liposomes by gel filtration using a cross-linked dextran gel column (Sephadex G-75, Amersham Pharmacia Biotechnology) eluted with HEPES buffered saline, pH 7.2-7.4, and Liposomes were collected in a column dead volume fraction. 0 USP doxorubicin hydrochloride (lyophilized powder containing 5 parts by weight lactose per 1 part doxorubicin) was added to the liposomes at a concentration of 150 µg liposome phospholipid drug / pmolar. The mixture was incubated at 55 ° C for 45 min., Cooled in ice for 10 min., And unencapsulated drug was removed by gel filtration chromatography using a Sephadex G-75 column eluted with HEPES buffered saline. pH 7.4. The presence of free doxorubicin (characterized by the appearance of a slower moving red band) was visually undetectable. Purified doxorubicin-loaded liposomes were analyzed for phospholipid and doxorubicin according to, respectively, examples 70 and 71 (spectrophotometry method). The resulting drug loading efficiencies are shown in table 2.

<td colspan="2">Table 2. Doxorubicin loading</td><td colspan="2">in liposomes with</td>

<td>trapped solutions</td><td>of salts of</td><td>dialkyl-</td><td>in</td>

<td>trialkylammonium. Reason</td><td colspan="3">drug / phospholipid</td>

<td>150 pg / pmolar.</td><td></td><td></td><td></td>

<td>Salt trapped in</td><td>Reason for</td><td>Efficiency of</td>

<td>liposomes:</td><td>drug /</td><td>imprisonment</td>

<td></td><td>phospholipid liposomes (pg / pmolar)</td><td>in (%)</td>

Trimethylammonium Sulphate

140,74 ± 10,35 93,815,7

ΡΕ1746976

<td>Sulfate</td><td>in</td><td>triethylammonium</td><td> 163,81</td><td> ±</td><td> 16,41</td><td> 109, 2</td><td> ±</td><td> 11, 6</td>

<td>Sulfate</td><td>in</td><td>diethylammonium</td><td> 158,16</td><td> +</td><td> 18,34</td><td> 105,4</td><td> +</td><td> 7,8</td>

<td>Sulfate</td><td>in</td><td></td><td> 155,08</td><td> ±</td><td> 8,51</td><td> 103,4</td><td> ±</td><td> 11, 6</td>

dimethylethanolammonium

<td>Example</td><td>3. Preparation</td><td>of liposomes containing various salts</td><td>in</td>

<td>sulfate</td><td>dialkyl</td><td>, substituted trialkylammonium and</td><td>in</td>

<td>ammonium</td><td>heterocyclic</td><td>replaced and charging</td><td>gives</td>

doxorubicin for these liposomes.

Substituted ammonium sulfate salt solutions were prepared as in example 1, using commercially available hetero-substituted alkyl substituted hydroxyalkyl amines. Liposomes were formed as in example 1, except that instead of the lipid film hydration step, the pure lipids were dissolved in ethanol (approximately 100 μΐ ethanol per 50 pmolar phospholipid) and mixed with the salt solution. of substituted ammonium at 60-65 ° C, so that the resulting lipid dispersion contained about 10% vol. of ethanol.

Doxorubicin loading was performed by adding doxorubicin solution (2 mg / ml in HEPES pH 6.5 buffered saline) to the liposomes at a ratio of 155 pg liposome phospholipid drug / pmolar (PL) and heating to 58 ° C for 45 min. In a hot water bath. The resulting liposomes were separated from any residual unencapsulated doxorubicin and analyzed for drug and lipid content as in the example.

1. Results are shown in table 3.

Table 3. Loading of doxorubicin into liposomes with alkyl, dialkyl, trialkyl ammonium salt solutions

ΡΕ1746976

- Substituted and substituted, encapsulated, substituted heterocyclic ammonium.

Amine used Osmolaralidad Efficiency Load of to prepare salt and, drug, loading, ammonium mg / mmolar% substituted phospholipid

<td>Trimethylamine</td><td> 497</td><td> 149, 4</td><td> ±</td><td> 7, 9</td><td> 96, 4</td><td> ±</td><td> 4, 9</td>

<td>Triethylamine</td><td> 559</td><td> 149, 6</td><td> ±</td><td> 6, 9</td><td> 96, 5</td><td> ±</td><td> 4,3</td>

<td>Dimethylethanolamine</td><td> 509</td><td> 163, 1</td><td> ±</td><td> 6, 6</td><td> 105,3</td><td> ±</td><td> 4,5</td>

<td>Dimethylamine</td><td> 472</td><td> 158, 6</td><td> ±</td><td> 7,4</td><td> 102,3</td><td> ±</td><td> 4, 9</td>

<td>Diethylamine</td><td> 519</td><td> 156,7</td><td> ±</td><td> 13,0</td><td> 101, 1</td><td> ±</td><td> 8,5</td>

<td>Diisopropylamine</td><td> 533</td><td> 159, 9</td><td> ±</td><td> 6,2</td><td> 103,2</td><td> ±</td><td> 4,1</td>

<td>Tris (hydroxymethyl) -</td><td> 423</td><td> 179, 9</td><td> ±</td><td> 15,3</td><td> 116, 1</td><td> ±</td><td> 11,5</td>

<td>minomethane</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1-Piperidineethanol</td><td> 506</td><td> 153, 5</td><td> ±</td><td> 7,1</td><td> 99, 0</td><td> ±</td><td> 4,5</td>

<td>4-Methymorpholine</td><td> 465</td><td> 152,4</td><td> ±</td><td> 9,8</td><td> 98,3</td><td> ±</td><td> 6, 2</td>

<td>Piperidine</td><td> 479</td><td> 158,5</td><td> ±</td><td> 12,5</td><td> 102,3</td><td> ±</td><td> 8,2</td>

<td>1-Methylpyrolidine</td><td> 492</td><td> 153, 6</td><td> ±</td><td> 12,3</td><td> 99, 1</td><td> ±</td><td> 7,8</td>

<td>Dimethylpiperazine</td><td> 378</td><td> 158,0</td><td> ±</td><td> 6, 5</td><td> 101, 9</td><td> ±</td><td> 4,3</td>

Example 4. Preparation of the triethylammonium polyphosphate solution (TEA-Pn).

linear sodium poly (phosphate) having 13-18 phosphate units per molar (phosphate glass; CALGON®, obtained from Sigma Chemical Company) was dissolved in water to a concentration of about 1.3 M phosphate. The solution was passed through a 120 ml column loaded with sulfonated polystyrene divinylbenzene copolymer cation exchange resin microspheres (Dowex 50Wx8-200, Dow Chemical Co.) as hydrogen. The column was pre-equilibrated with 3-3.6 M aqueous HCl to bring the resin to hydrogen form, and washed with deionized water to neutral pH. Fifteen ml of sodium polyphosphate solution was applied to the column, eluting with H₂O

ΡΕ1746976 deionized. The column eluent was monitored using a conductivity detector. The column output flow corresponding to the conductivity peak was titrated with pure triethylamine to pH 5.5-6.0. The solution was analyzed for residual sodium by potentiometry using a sodium sensitive glass electrode and for phosphate content using an inorganic phosphate assay as in example 1. The solution having a residual sodium content of less than 1% was diluted to a final phosphate concentration of 0.55 Μ. The solution usually has a TEA concentration of 0.52-0.55 M, pH 5.5-6.0 and osmolarity of 430480 mmolar / kg.

Example 5. Removal of non-entrapped polyphosphate salts from liposome preparations.

Liposomes (120 nm in size) with entrapped 8-hydroxypyrene trisulfonate fluorescent label were prepared according to Kirpotin, et al., Biochemistry 36: 66-75, 1997, and mixed with sodium polyphosphate solution. The mixture was loaded onto size exclusion columns containing cross-linked dextran microspheres (Sephadex G-75), 6% agarose microspheres (Sepharose 6B-CL) or 4% agarose microspheres (Sepharose 4B-CL). Amersham Pharmacia, and eluted with MESdextrose buffer (pH 5.5). Effluents were analyzed for phosphate content using the Bartlett Phosphate Assay (1959) and for liposome content by spectrofluorometry. Of the gel chromatography vehicles studied, Sepharose CL-6B provided complete separation of polyphosphate from liposomes at a sample volume / column bed ratio of 13.

ΡΕ1746976

Example 6. Preparation of the triethylammonium sucrose octasulfate (TEA-SOS) solution.

Sodium sucrose octasulfate (equivalent weight 144.8) is the sodium salt of the sucrose derivative in which all hydroxyl groups formed esters of sulfuric acid. Sucrose octasulfate (SOS) sodium salt was purchased from Toronto Research Chemicals, Toronto, Canada, p / n S699020. Six grams of sodium sucrose octasulfate were dissolved in 16.57 ml deionized water to give a final concentration of about 2.5 N sulfate groups. The solution was treated by ion exchange as in example 4. The sucrose octasulfuric acid solution obtained as an ion exchange column effluent was then titrated with pure triethylamine to pH 5.7 (neutralization point), and the pH determined. and the osmolarity of the solution. The resulting solution had a calculated triethylammonium concentration of 0.643 M, pH 5.7, and an osmolarity of 530 mmolar / kg. The presence of residual sodium was undetectable by potentiometry (less than 0.1%).

<td>Example 7</td><td>Liposomes</td><td>loaded with irinotecan (CPT-11)</td>

<td>using</td><td>salts of</td><td>substituted ammonium: preparation and</td>

<td>release</td><td>of the drug</td><td>in vitro in the presence of plasma</td>

blood

In this example sulfate, citrate, pyrophosphate, triphosphate and linear polyphosphate (13-18 mer) were studied as anions in the liposome-trapped substituted ammonium salt solutions. Phosphate polymers were chosen because of their biodegradability and because polyphosphates are found naturally in cells, unlike other synthetic polymeric anions (polyacrylate, dextran sulfate and the like). In addition, the viscosity of polyphosphate solutions of

Baixo1746976 The low molar weight was lower than that of other polymers, making polyphosphates more favorable to the process.

The following materials were used for the preparation of saline solutions.

1. Sodium polyphosphate, NaO- [PO3Na] _no-Na, n = 13-18, purchased from Sigma (product no. P-8510, phosphate glass, practical grade, also known as sodium hexametaphosphate or by the brand name CALGON);

2. Pentasodium tripolyphosphate, NasPs10, purchased from Sigma (product no. T-5883);

3 Tetrasodium pyrophosphate decahydrate, Na4P2C&gt; 7 · 1OH2O, purchased from Sigma (product no. P-9146).

4 Dowex 50Wx4 ion exchange resins (4% cross-linked sulfonated polystyrene resin, 100200 mesh) purchased from Sigma (product no. 50X4-200) or Dowex

HCR-W2 (8% cross-linked sulfonated polystyrene resin, 50-100 mesh) purchased from Sigma (product no. 1-8880) was used interchangeably. The resins were washed by decantation in the following order: three times with deionized water, twice with 1 N HCl (3x excess resin by volume), three times with water, twice with 1 N NaOH, three times with water, three times with 1 N HCl and three times with water. After decantation, the resins were in the form of H⁺.

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5 Trimethylamine (TMA), 40% aqueous solution, from Aldrich Chemical Co. (product no. 43, 326-8). The concentration was established by acid titration to be about 5.9 N.

<td>6. Triethylamine (TEA),</td><td>99% grade</td><td>in</td><td>HPLC from</td><td>Fisher</td>

<td>(product no. 04884). THE</td><td>concentration</td><td>per</td><td>titration</td><td>acidic</td>

<td>was 6.9-7.1 N.</td><td></td><td></td><td></td><td></td>

Water was purified by reverse osmosis, ion exchange and organic removal to achieve an organic free quality 16-18 MOhm.

Aqueous solutions of pyrophosphate, triphosphate and polyphosphate salts were prepared by the ion exchange method. Sodium polyphosphate (3 g in 25 ml water), pyrophosphate (4 g in 27 ml water) or polyphosphate (6.7 g in 30 ml water) solutions were loaded onto the column containing 100 ml (volume of bed) of the ion exchange resin prepared as above. The column was eluted with water and fractions were collected. Fractions with acid pH (pH &lt;3) were pooled. Triplicates of 0.5 ml aliquots of the combined phosphate acid containing fraction were diluted with 20 ml water and titrated with 0.100 N NaOH to pH 4.5-

<td>5.0 (solution</td><td>analytical</td><td>Fisher) to</td><td>to determine</td><td>The</td>

<td>normality. At</td><td>fractions</td><td>combined after</td><td colspan="2">ion exchange</td>

<td>were titled</td><td colspan="2">with trimethylamine (to</td><td>get salts</td><td>in</td>

<td>trimethylammonium)</td><td>up to pH</td><td>5.4-5.5. After</td><td>titration,</td><td>at</td>

<td>solutions were</td><td>diluted,</td><td colspan="2">if necessary to obtain</td><td>an</td>

final concentration of trimethylammonium close to 0.5 N.

The trimethylammonium sulfates prepared by diluting 1.39 concentrate (17.9 M) with 80 ml and triethylammonium were ml of water sulfuric acid, and the solution

Diluted ΡΕ1746976 was titrated with pure triethylamine or aqueous trimethylamine under the control of a pH meter to the equivalence point (pH 5.1-5.5). The volume was adjusted to 100 ml with water.

The trimethylammonium citrate solution was prepared by dissolving 1.572 g of Sigma ACS citric acid monohydrate (product no. C-1909) in 20 ml of water, and the solution was titrated to aqueous trimethylamine equivalence. The volume was adjusted to 25 ml with water.

The solutions were filtered through a 0.2 µm cellulose acetate filter using positive pressure. The osmolarity and pH of the solutions were measured using, respectively, a vapor pressure osmometer and a glass-calomel electrode pH meter. The normality of the anion in phosphate solutions was determined by the blue phosphomolaribdate spectrophotometric assay (see Example 70) after acid hydrolysis (5 min. at 100 ° C, 3 N H 2 SO 4) · The normality of the anion took into account only acid functional groups which are substantially ionized at pH 5.5. Cation normality was determined based on the trialkylammonium base added. The solutions obtained had the following properties (Table 4):

Table 4. Properties of substituted ammonium salt solutions for loading CPT-11 into liposomes.

<td>salt</td><td></td><td>cation normality</td><td>anion normality</td><td>pH</td><td>Osmolaralide in (mmolar / kg)</td>

<td>Citrate of</td><td>TMA</td><td> 0,58</td><td> 0, 60</td><td> 5, 1</td><td> 791</td>

<td>Sulphate</td><td>TMA</td><td> 0,50</td><td> 0,50</td><td> 5,4</td><td> 625</td>

<td>Pyrophosphate</td><td>in</td><td>TMA 0.44</td><td> 0,54</td><td> 5,4</td><td> 651</td>

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<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 Sulphate</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. PEG-DSPE (PEG molar weight 2000) was from Shearwater Polimers, Huntsville, AL, USA. DSPC, cholesterol and PEG-DSPE at a 3: 1: 0.1 weight ratio (approximately 3: 2: 0.03 molar ratio) were dissolved in chloroform (Fisher; Optimum grade, stabilized with amylene) to 60 mg / ml DSPC. The solution was dispensed into 30 mg DSPC Pyrex tubes (0.5 ml) per tube and slowly evaporated under reduced pressure using a rotary evaporator at 60 ° C. The lipid films were dried under vacuum (100 microns mercury, oil pump) for 30-60 minutes at room temperature.

The dried lipid films were hydrated by gentle stirring in the above aqueous saline solutions at 60 ° C for 15-20 minutes. The lipids formed a milky suspension (multilamellar vesicles). This milky suspension was subjected to five freezing cycles in the mixture of dry ice and isopropanol (-80 ° C, 3 minutes), and thawed in a 60 ° C water bath for 3 minutes. Then, the lipid suspension was extruded 10 times (double screw) through two stacked polycarbonate membrane filters (Nucleopore, Whatman, pore size 0.1 pm) using a manually operated reciprocating extruder (Avanti Polar Lipids). ) heated to 60 ° C.

The extruded liposomes were kept at 60 ° C for five minutes and quenched in ice water (0-4 ° C) for

ΡΕ1746976 five minutes. The liposomes were then separated from the gradient-forming salt solution to the MES-dextrose loading buffer (50 g / l ACS dextrose, 0.975 g / l 2- (N-morpholino) ethanesulfonic acid (MES ), and sufficient 5 M NaOH to bring the pH to 6.4) by Sephadex G-75 gel chromatography. Liposomes appear in the dead volume fraction (approximately 30% of the column bed volume).

The CPT-11 (irinotecan hydrochloride) preparation containing 0.860 mg of CPT-11 base per 1 mg of solid was dissolved in 0.001 N HCl to make a 16.5 mg / ml stock solution of CPT-base. 11 This solution was mixed with the liposomes in MES-dextrose buffer to achieve the 150 pg ratio of CPT-11 per 1 pmolar liposome phospholipids. The mixture was incubated at 55 ° C in a water bath with occasional gentle shaking (approximately every five minutes) for 30 minutes and then quenched in ice water (0-4 ° C). Liposomes were separated from unencapsulated drug by Sephadex G-75 gel chromatography using MES-dextrose as eluent. Encapsulated drug was determined by a spectrophotometric assay (Example 71), and phospholipids were determined using an extraction assay (Example 70).

In vitro drug release of CPT-11 loaded liposomes in the presence of 50% human plasma was studied 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 cellulose acetate syringe filter. CPT-11 loaded liposome preparations were sterilized by passing through a sterile acetate syringe filter

ΡΕ1746976 of 0.2 pm surfactant-free cellulose (SFCA). 0.5 ml of the liposomes were mixed with 0.5 ml of plasma in sterile 1.5 ml copolymer Eppendorf tubes, which were sealed and incubated on a shaking platform at 37 ° C for 24 hours. The blank sample contained 0.5 ml of sterile MES dextrose instead of liposomes. Liposomes were isolated by gel chromatography on a 2% cross-linked agarose microsphere gel (Sepharose CL-2B, Pharmacia; 10 ml bed volume) using 144 mM NaCl, 5 mM HEPES-Na buffer pH 7 .4 (HBS-5). Liposomes appeared in the dead volume fraction, while plasma proteins and released drug (if present) were retarded by the gel. Liposome fractions were tested for CPT-11 and phospholipids, and the drug / phospholipid ratio (exit ratio) was determined. Blank sample readings (plasma only) were subtracted from liposome containing readings. The percent drug remaining in the liposomes after incubation was determined by dividing the drug / lipid exit ratio by the drug / lipid entry ratio (drug / lipid ratio prior to plasma incubation). Loading and release data are summarized in table 5.

Table 5. Loading of CPT-11 into liposomes with salts of

<td colspan="5">tertiary alkylammonium and drug release in vitro in the</td>

<td>presence of</td><td colspan="4">human plasma.</td>

<td>Salt solution</td><td>Before plasma</td><td>incubation with</td><td colspan="2">After plasma incubation</td>

<td>imprisoned</td><td>reason of</td><td>efficiency</td><td>reason of</td><td>drug that</td>

<td></td><td>drug /</td><td>in</td><td>drug /</td><td>remains</td>

<td></td><td>lipid</td><td colspan="2">lipid encapsulation (%)</td><td>encapsulated (%)</td>

ΡΕ1746976

<td>Sulphate TMA</td><td> 127,2</td><td> ±</td><td> 5, 6</td><td> 84,</td><td> 8</td><td> ±</td><td> 3, 8</td><td> 132,1</td><td> ±</td><td> 6,9103,8 ± 10,0</td>

<td>TMA pyrophosphate</td><td> 136, 2</td><td> ±</td><td> 9, 0</td><td> 90,</td><td> 8</td><td> ±</td><td> 6, 0</td><td> 132,3</td><td> ±</td><td> 5, 0 97,1 ± 10, 1</td>

<td>TMA triphosphate</td><td> 132, 9</td><td></td><td></td><td> 88,</td><td> 6</td><td></td><td></td><td> 129, 2</td><td></td><td> 97,3</td>

<td>TMA-Pn</td><td> 134,4</td><td> ±</td><td> 9,3</td><td> 89,</td><td> 6</td><td> ±</td><td> 6, 2</td><td> 135, 0</td><td> ±</td><td> 7,4 100,4 ±12,4</td>

<td>Sulphate TEA</td><td> 131,1</td><td> ±</td><td> 6, 5</td><td> 87,</td><td> 4</td><td> ±</td><td> 4,4</td><td> 125,2</td><td> ±</td><td> 5, 0 95,5 ± 8,6</td>

Example 8. Stability of CPT-11 loaded in vivo liposomes using trialkyl ammonium pyrophosphate, triphosphate, polyphosphate, citrate and sulfate salts.

Although camptothecin liposomes may show acceptable drug leakage in blood plasma in vitro, the drug may release more rapidly into the bloodstream in vivo. Therefore, a panel of CPT-11 liposome formulations were screened for drug stability in blood circulation in vivo using a single time point assay in mice.

Liposomes were prepared and loaded with CPT-11 as described in example 6 with the following modifications. Instead of using Shearwater Polimers PEG-DSPE, we used similar PEG-DSPE from Avanti Polar Lipids. For quantification of the liposome lipid matrix in blood / tissue samples, a non-exchangeable radioactive marker was added, [³H] -hexadecyl cholesteryl ether ([³H] -CHE; (Amersham, USA) to the chloroform solution of lipids in an amount of 0.25 mCi / mmolar phospholipids. Lipid solutions were dispensed into 12 mg DSPC Pyrex tubes / tube, and lipid films were formed by rotary evaporation / vacuum drying. Lipid films were hydrated in 0.7 ml of ammonium salt solutions.

ΡΕ1746976 replaced gradient former. Lipid concentration in liposomes with trapped phosphate-containing salts was determined by scintillation radioactivity counting. Salt-free preparations containing trapped phosphate were also tested for phospholipids without extraction, as described in example 70, and were used as lipid radioactivity standards. Portions of the liposome-drug mixtures prepared for loading were stored and analyzed to confirm the preload ratio of CPT-11 added to the liposome lipid prior to loading (entry ratio). The mean mean volume and standard deviation of the quasi-elastic light scattering liposome size distribution (QELS) were determined using the Gaussian model. The properties of these liposomes are summarized in table 6.

Table 6. Characterization of CPT-11 loading in

<td>liposomes</td><td>marked</td><td>with</td><td> [³H] -CHE for the study of</td>

<td>stability</td><td>in vivo</td><td></td><td></td>

Liposome size efficiency, loading (mean ± (%) SD) nm

Drug / drug / entrapped lipid ratio solution before lipid after loading loading

<td>Citrate of</td><td> 159,</td><td> 2</td><td> ±</td><td> 3,</td><td> 5</td><td> 156,</td><td> 7</td><td> ±</td><td> 3,</td><td> 6</td><td> 98,</td><td> 5</td><td> ±</td><td> 4,4</td><td> 122, 1</td><td> ±</td>

<td>TMA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 25, 3</td><td></td>

<td>Sulphate</td><td> 156,</td><td> 1</td><td> ±</td><td> 2,</td><td> 5</td><td> 156,</td><td> 1</td><td> ±</td><td> 3,</td><td> 1</td><td> 100</td><td>r</td><td> 0 ±</td><td> 3, 6</td><td> 122,2</td><td> ±</td>

<td>TMA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 28,4</td><td></td>

<td>Pyrophosphate</td><td> 164,</td><td> 6</td><td> ±</td><td> 5,</td><td> 8</td><td> 156,</td><td> 6</td><td> ±</td><td> 4,</td><td> 3</td><td> 95,</td><td> 2</td><td> ±</td><td> 6, 0</td><td> 121, 1</td><td> ±</td>

<td>from TMA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 19, 9</td><td></td>

<td>Triphosphate</td><td> 163,</td><td> 6</td><td> ±</td><td> 5,</td><td> 7</td><td> 156,</td><td> 0</td><td> ±</td><td> 3,</td><td> 2</td><td> 95,</td><td> 3</td><td> ±</td><td> 5,3</td><td> 122,4</td><td> ±</td>

<td>from TMA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 12, 9</td><td></td>

<td>Polyphosphate</td><td> 170,</td><td> 5</td><td> ±</td><td> 8,</td><td> 0</td><td> 162,</td><td> 4</td><td> ±</td><td> 4,</td><td> 0</td><td> 95,</td><td> 3</td><td> ±</td><td> 6, 8</td><td> 123, 0</td><td> ±</td>

<td>from TMA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 12,7</td><td></td>

<td>Sulphate</td><td> 153.</td><td colspan="2"> +</td><td> 3, 3</td><td></td><td> 154,</td><td> 9</td><td> +</td><td> 4,</td><td> 9</td><td> 101</td><td>r</td><td> 0 ±</td><td> 5,3</td><td> 121, 1</td><td> +</td>

ΡΕ1746976

TEA

18,0

Six-week female CD-I (Charles River) mice received, in duplicate, tail vein injections of these liposomal CPT-11 formulations at a dose of 10 mg / kg (0.2 mg CPT-11 / mouse). Eight hours later, the mice were anesthetized and bled by cardiac puncture. Blood was collected in heparinized syringes (10-20 μΐ 1000 U / ml USP heparin) and transferred to heavy tubes containing 0.4 ml phosphate buffered saline (PBS) containing 0.04% EDTA (Gibco BRL), kept on ice. Tubes were weighed to determine the weight of blood samples, blood cells were separated by centrifugation at 9,000 g for 5 minutes, and supernatants containing PBS diluted plasma were stored for drug and liposome lipid testing. CPT-11 was guaranteed by fluorometric assay (Example 71). Liposome lipid was guantified by extinction-corrected scintillation radioactivity counting. Liposome and phospholipid radioactivity patterns were counted in parallel with plasma samples. The percent drug that remained encapsulated was calculated by dividing the drug / radioactivity ratio in plasma samples by the drug / radioactivity ratio of the injected liposomes. Due to the rapid clearance of free blood CPT-11 (see Example 69) and the known stability of [³H] -CHE against lipid exchange, test readings were considered indicative of the blood content of liposome CPT-11 and lipid. The percentage of injected lipid dose (% DI) remaining in the circulation was calculated assuming 100% of the injected bolus entered the circulation; being the volume

ΡΕ1746976

79 blood 6.3% of the body weight of the mouse, and the hematocrit 45%. The results are summarized in table 7.

Table 7. In vivo stability of CPT-11 encapsulation and

<td>longevity</td><td>of circulation</td><td>From</td><td>liposomes</td><td>loaded with</td>

<td>CPT-11 on</td><td>mice in a</td><td>single</td><td>point (8</td><td>hours) after</td>

injection. % DI,% injected dose.

Salt trapped in lipid ratio of drug / lipid liposomes,% liposome,%

<td></td><td></td><td></td><td>value</td><td>gives</td><td>pre-injection</td><td>D. I</td><td> •</td><td>nc</td><td>&gt; blood</td>

<td>Citrate of</td><td>TMA</td><td></td><td> 80,2 ±</td><td> 7,</td><td> 8</td><td> 18,</td><td> 8</td><td> ±</td><td> 3,4</td>

<td>Sulphate</td><td>TMA</td><td></td><td> 70,1 ±</td><td> 4,</td><td> 8</td><td> 23,</td><td> 6</td><td> ±</td><td> 1,8</td>

<td>Pyrophosphate</td><td>in</td><td>TMA</td><td> 67,3 ±</td><td> 9,</td><td> 2</td><td> 23,</td><td> 2</td><td> ±</td><td> 3,1</td>

<td>Triphosphate</td><td>in ^r</td><td>ΓΜΑ</td><td> 70, 6 ±</td><td> 6,</td><td> 0</td><td> 24,</td><td> 9</td><td> ±</td><td> 8,2</td>

<td>Polyphosphate</td><td>in</td><td>TMA</td><td> 107,5</td><td> ± 8</td><td> , 9</td><td> 24,</td><td> 3</td><td> ±</td><td> 3,4</td>

<td>Sulphate</td><td>TEA</td><td></td><td> 76, 6 ±</td><td> 13</td><td> ,1</td><td> 23,</td><td> 6</td><td> +</td><td> 0,1</td>

The preparations all showed the drug encapsulation level after 8 hours in the blood in vivo at 70-80% of the pre-injection level, while the liposomes containing the polyphosphate were the most stable (drug encapsulation remains at about 100%).

Example 9. Pharmacokinetics of blood CPT-11 liposomes prepared using triethylammonium polyphosphate.

The liposomal CPT-11 formulation using the triethylammonium polyphosphate salt was prepared as described in example 3. Lipids - DSPC, cholesterol and N- (methoxypoly (ethylene glycol) (PM 2000) -oxycarbonyl) -DSPE (PEG-DSPE) (all from Avanti Polar Lipids, Inc.) - were combined as dry powders at 3: 2: 0.015 molar ratio, and dissolved in 100% ethanol (USP grade, approx. 0.15 ml / 100 mg of lipids)

41746976 at 62-65 ° C. For pharmacokinetic studies, an additional³Cholesteryl hexadecyl H-ether (³H-CHE, obtained from Amersham Pharmacia) to lipids in the amount of 0.5 mCi / mmolar phospholipids as a non-exchangeable radioactive lipid marker. TEA-Pn aqueous solution (0.5 M triethylammonium, pH 5.7-6.2) was prepared as in example 4. TEA-Pn solution (10 times the volume of ethanol added) was mixed with lipid solution at 60-65 ° C, and stirred at this temperature until a homogeneous milky suspension of multilamellar vesicles was formed. This suspension was extruded 15 times through 2 stacked track-etched polycarbonate filters (Corning Nuclepore) with a pore size of 100 nm using an argon pressure extruder (Lipex Biomembranes) at 60-65 ° C, and liposomes. The resulting unilamellar were rapidly cooled on ice and then allowed to reach room temperature. Ethanol and unincorporated polyphosphate salt were removed by gel column chromatography of Sepharose CL-4B eluted with MES-dextrose buffer (5 mM MES, 50 g / 1 dextrose, pH adjusted to 6.5 with NaOH) .

A stock solution of CPT-11 (irinotecan hydrochloride) containing 20 mg / ml irinotecan base in water was added to the liposomes at a drug / lipid ratio of 150-200 mg / mmole phospholipids, and the mixture was incubated. with occasional stirring for 45-60 minutes at 60-62 ° C. The incubation mixture was rapidly cooled and incubated for 10 minutes at 0 ° C, and then allowed to reach room temperature. 1/20 of the 2.88 M volume of NaCl was added to adjust physiological ionic strength and improve the removal of membrane bound CPT-11 (as opposed to encapsulated drug within the liposome). Unencapsulated drug was removed by

ΡΕ1746976 Sephadex G-25 or G-75 column gel chromatography (Amersham Pharmacia) eluted with HBS-6.5 (5 mM 2- (4- (2-hydroxyethyl) piperazine) ethylsulfonic acid (HEPES) buffer ), 144 mM NaCl, pH 6.5). The dead volume eluted liposome fractions were pooled, sterilized by 0.2 micron filtration, and stored at 4-6 ° C prior to use. Liposomes were characterized by lipid concentration, drug concentration, and particle size, as in example 7. Liposomes had an average size of 108 nm and a CPT-11 content of 139 ± 18 mg CPT-1 base per mmole phospholipids.

The longevity of liposome lipid and blood liposome drug and liposome drug release characteristics in vivo were studied in female Sprague-Dawley rats (190-210 g) with implanted central venous catheters. Rats were injected with a bolus of 0.2-0.3 ml irinotecan liposomes labeled with³HCHE (0.05 mmole phospholipids or 7-8 mg CPT-11 per kg body weight). Blood samples (0.2-0.3 ml) were collected at various times after injection using a heparin-treated syringe. The volume of blood drawn was renewed with phosphate buffered saline. Blood samples were diluted with 0.3 ml ice cold PBS containing 0.04% EDTA, weighed, and blood cells separated by centrifugation. Supernatant fluids were collected and tested for CPT11 using the fluorometric procedure of example 71, and for liposome lipid marker by scintillation radioactivity counting using standard methods. Liposome preparations with known drug and the concentration of³H-CHE-lipid were used as standards. The patterns of radioactivity

ΡΕ1746976 contain an equal amount of diluted rat plasma to account for extinction. The amount of CPT-11 and liposome lipid in the blood was calculated assuming the blood volume in ml as 6.5% body weight in grams and the hematocrit 40%. The total amount of lipid and drug in the blood was expressed as% of the injected dose (% DI,% DI) and plotted against 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 of the injected liposomes (taken as 100%). Since the graphs generally showed good agreement with monoexponential kinetics (semi-logarithmic scale linearity), blood drug, lipid and drug release half-lives were calculated from the best fit of the data to monoexponential decline equation using the TREND option of the Microsoft EXCEL computer program (Microsoft Corp., USA). The results are shown in figure 1. From the best-fit parameters, blood lipid and drug half-lives were 16.4 hours and 6.61 hours, respectively. Under these conditions, free CPT-11 exits circulation very quickly (see Example 69).

The blood drug / lipid ratio revealed a biphasic release character of CPT-11 from liposomes (Figure 2). For the first 24 hours, the drug release followed was linear over time (R = 0.992), showing

<td>kinetics of</td><td>release</td><td>in</td><td>order</td><td>zero.</td><td colspan="2">The liberation</td>

<td>subsequent only</td><td>it became</td><td>no</td><td>linear</td><td>after</td><td>fence</td><td>75%</td>

<td>of the drug be</td><td>released</td><td>at the</td><td>Score</td><td>of time</td><td>from 24</td><td>hours</td>

For 24 hours, the liposomes released the drug at a constant rate of about 3.6% of the initial charge / hour. Of that

Thus, over the period of approximately 14 hours, 50% of the drug was released. Zero-order drug release is an attractive quality in sustained release formulations since the drug release rate remains constant over time.

Example 10. Antitumor efficacy of CPT-11 liposomes prepared using triethylammonium polyphosphate against breast cancer xenografts in nude mice

The antitumor efficacy of CPT-11 liposomes was studied in the BT-474 human breast carcinoma model, an estrogen-dependent ductal adenocarcinoma with C-ErbB2 receptor overexpression (HER2). BT-474 cells were obtained from the American Type Culture Collection (Rockville, MD). A BT-474 subline with a higher tumor growth rate was established from a rapidly growing xenograft tumor node created as described below. Cells were propagated in vitro in RPMI-1460 medium with 10% fetal bovine serum, 0.1 mg / ml streptomycin sulfate and 100 U / ml penicillin G in T-150 flasks, and divided each week at 1: 3 Nu / nu female NCR mice (4-6 weeks old; Taconic Farms) were implanted subcutaneously (at the base of the tail) with 0.72 mg of 60 day sustained release 17 βestradiol granules (Innovative Research of America, Inc .), and within two days were inoculated subcutaneously in the upper back area with 0.1 ml suspension containing 2x10⁷ BT-474 cells in cell growth medium. Tumor progression was monitored by palpation and compass measurements of the tumors along the major (length) and minor (width) axis twice a week. Tumor sizes were determined twice weekly from

41746976 bar measurements using the formula (Geran, RI, et al., 1972 Cancer Chemother. Rep. 3: 1-88):

tumor volume = [(length) x (width)²] / 2

On day 13 after inoculation, the tumor reached an average size of 200 mm³ and the animals were randomly divided into three groups of 13-15 animals.

Liposomal CPT-11 was prepared as in example 8 (drug / phospholipid ratio 192 mg / mmolar; mean liposome size 86.8 nm). Free and liposomal CPT-11 were diluted with MES-dextrose vehicle at 5 mg / ml CPT11 base. Animals were injected into the tail vein with free CPT-11, liposomal CPT-11 or vehicle only on days 14, 18, 21 and 25 after tumor inoculation. Drug-containing formulations were given at a dose of 50 mg CPT-11 / kg per injection, which is the mean of the doses reported in the literature for CPT-11 studies in rodent tumor models.

To assess treatment-related toxicity, animals were also weighed twice a week. Observations were made until day 60 after inoculation, at which time the estrogen supplementation granule was depleted. Mean tumor volumes between groups were plotted together and compared over time. As shown in Figure 3, although free CPT-11 reduced tumor growth rate, in the group receiving liposomal treatment tumors regressed dramatically. While on day 36 in the control group the tumors reached the maximum allowable size of 3.50 mm³, and on day 46 in the free drug group the tumors averaged about 1,000 mm³,

At the same time point, none of the animals in the liposome-treated group had a palpable tumor.

Treatment-related toxicity was assessed by the body weight dynamics of the animals (Figure 4). None of the groups revealed any significant toxicity. The weight of animals in the control group increased steadily. On the day of the last treatment, there was a slight decrease, about 3.3%, in the average body weight of animals receiving liposome CPT-11. However, this weight loss was reversed and the animals reached the expected weight. This decrease in mean body weight was not statistically significant by Student's t-test compared with pretreatment weight (p = 0.274). Thus, the treatments were all tolerated without significant toxicity.

Thus, the CPT-11 liposome formulation obtained by loading the drug through the sterically hindered substituted ammonium salt (triethylammonium) pre-trapped with a biodegradable, polyanionic (polyphosphate) polymer exhibited prolonged blood life, sustained release characteristics and increased antitumor activity in the tumor model studied, with a significant increase in toxicity.

<td>Example 11. Evaluation</td><td>comparative</td><td>in</td><td>liposomes</td><td>loaded</td>

<td>with CPT-11 prepared</td><td colspan="2">using salts</td><td colspan="2">triethylammonium</td>

<td>trapped: effect</td><td>of size</td><td>of</td><td>liposome,</td><td>reason of</td>

drug / lipid, and the nature of the pre-trapped anion.

Two prototype formulations of CPT-11 loaded liposomes were prepared, one using pre-entrapped TEA-Pn liposomes and the other with pre-entrapped TEA-SOS. THE

The preparation of these liposomes included the following manufacturing steps.

1) Combination of lipids by codissolution in ethanol. The lipid matrix composition consisted of 1,2-distearoylSN-phosphatidylcholine (DSPC) (molar weight. 790), 3 molar parts (59.8 molar%); cholesterol (Col) (molar weight. 387), 2 molar parts (39.9 mol%); and N- (omega-methoxypoly (ethylene glycol) -oxycarbonyl) -1,2-distearoylphosphatidyl ethanolamine (molar weight. 2787) (PEG-DSPE), 0.015 molar parts (approx. 0.3 mol%). DSPC and PEG-DSPE were acquired from Avanti Polar Lipids, Birmingham, Alabama. Cholesterol (highest degree of purity) was purchased from Calbiochem. The dried lipids were weighed to within ± 0.1 mg in a borosilicate glass vessel and combined with absolute ethanol in the appropriate ratio for the lipid dispersion step below. Due to the high transition temperature of DSPC (55 ° C), dissolution was usually performed at 55-60 ° C until a clear solution was obtained.

2) Preparation of TEA-Pn and TEA-SOS solutions. Sodium polyphosphate (n = 13-18) was purchased from Sigma

Chemical Co., p / n P 8510. Sodium sucrose octasulfate 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 or Dowex HCR-W2 anion exchangers in H + form (available from Sigma) were used to convert the sodium salts. in free acids. Prior to first use, the resins were washed by stirring with 3 volumes of the following solutions, followed by decantation: (1) 1.0-1.2 M aqueous HCl 2 times; (2) water, 2 times;

ΡΕ1746976 (3) 1.0-1.2 M aqueous NaOH, 2 times; (4) water, 2 times;

(5) 1.0-1.2 M aqueous HCl 2 times. The water-washed resin suspension was compacted under gravity flow on an appropriately sized chromatography column to have at least 8 ml of the resin compacted per ml of sodium salt solutions. The resin was then equilibrated by passing 2 column volumes of 3.03.6 M aqueous HCl, followed by 5 column volumes of water, or until the eluate conductivity dropped below micro-S. After use, the columns were regenerated by sequential passage of: 1, -1.2 M HCl - 3 column volumes; 3.0-3.6 M HCl - 2 column volumes; water - at least 5 column volumes, or until the conductivity of the eluate dropped below 1 pS, and were stored under 0.2 µm filtered water at room temperature. Pn and SOS sodium salt solutions were applied to the drained column surface (approximately 1 ml per 4 ml volume of the compacted resin) and allowed to flow by gravity at a rate of about 1-2 ml / min to The resin bed size is 75-150 ml. The column was eluted. The eluate was tested for

If more than the most concentrated polyacid solutions needed were collected, the collection may start at 20-50 mS, but to the detriment of a slightly higher loss of the gradient-forming salt. In the case of polyphosphoric acid, the collected solution is kept refrigerated (0-4 ° C) until the amine titration step due to the hydrolytic instability of the phosphodiester bond at low pH. The collected eluates would have a pH of less than 0.7 (usually about 0.4) and conductivity of about 120-200 mS. Optionally, the amine titration step is performed immediately eluting with conductivity conductivity to the 10 mS fractions. If they are

ΡΕ1746976 due to polyphosphate stability at low pH. Fisher-HPLC grade triethylamine (99.5+% purity), p / n 04884, was used to titrate the acid solutions obtained from ion exchange. The normality of pure TEA was determined by potentiometric titration. Aliquots of 0.100 ml of TEA (0.100 ml) in 20 ml of water were collected in triplicate. Aliquots are titrated with 0.1 N HCl standard solution to pH (glass electrode) end point 5.5-6.0. The calculated normality (7.07 N) was close to the theoretical 7.17 N. A measured volume of polyphosphoric acid (Pn) or sucrose octasulfuric acid (SOS) solution was titrated with pure TEA under the control of pH electrode (glass). Careful agitation was required to disperse the amine. The endpoint of the titration was pH 5.6-6.2. The volume of TEA added was accurately recorded. The volume of the titrated solution was measured, and the TEA concentration was calculated based on the added TEA volume and normality. Water was added as needed to adjust the TEA concentration to 0.55 ± 0.05 N or 0.65 ± 0.03 N as indicated below. The amount of residual sodium in the TEA-Pn or TEA-SOS solutions obtained was determined by potentiometry using a sodium selective glass electrode (Corning). One ml of the solution was diluted in 19 ml of water, and the sodium concentration was determined using the increment method according to the electrode manufacturer's manual. The amount of residual sodium was less than 1 mM, usually less than 0.5 mM. The obtained TEA-Pn or TEA-SOS solutions were passed through a sterile 0.2 µm cellulose acetate filter using a positive pressure flow. The final pH and osmolarity of the solutions were measured and recorded. For pH measurements we use an all glass micro electrode combined with

ΡΕ1746976 calomel, and for osmolarity measurements we used a vapor pressure / dew point osmometer. The solutions were stored in the refrigerator until use.

3) Preparation of lipid dispersion in the gradient-forming buffer by mixing the ethanolic solution of the lipids with the gradient-forming buffer. The lipids were dispersed in the gradient-forming salt solution using the ethanol blending method. The steps were all performed at 60-65 ° C. The lipids were dissolved in 100% USP ethanol at a concentration of about 0.50.6 M DSPC in a chemical resistant glass pear-shaped flask or tube. The gradient-forming salt solution (TEA-Pn or TEA-SOS) was pre-warmed to 60-65 ° C and added immediately to the ethanolic lipid solution, and the components were carefully mixed by circular and / or vortex stirring. The final amount of ethanol was about 10% by volume. For preparations on the scale above 0.1 mmole phospholipid, the resulting suspension was placed in a rotary evaporator at 60-65 ° C and vacuum rotated until ethanol evolution stopped, as manifested by the end of foaming. For the scale equal to or less than 0.1 mmole phospholipid, ethanol was not removed from the lipid dispersion in this step. The resulting lipid suspensions were kept at 60-65 ° C and used immediately for the extrusion step.

4) Sequential extrusion of lipid dispersion through defined pore membranes. For lipid suspension volumes up to 1 ml we used a manually operated reciprocating extruder supplied by Avanti Polar Lipids. The extruder is loaded with track-etched filter membranes of

ΡΕ1746976 mm and thermoregulated by a metal heating block. For volumes of 1 to 10 ml, we used a one-way, gas pressure operated, extruder, thermoregulated Lipex Biomembranes. The extruder is loaded with 25 mm filter membranes. Lipid suspensions were repeatedly extruded at 60-65 ° C by hand feed or argon gas pressure, as appropriate, through a series of 2 stacked polycarbonate membrane filters (Corning-Nuclepore filters were equally suitable). and Osmonics Corp.) having a nominal pore size of 100 nm, 80 nm or 50 nm. When the effect of liposome size was of interest, extrusion was stopped at step 100 nm, 80 nm or 50 nm. The exact type of filters used and the number of extrusions are given below for each experiment. The extruded liposomes were kept at 60-65 ° C for about 15 min., And cooled rapidly to 2-4 ° C in an ice bath. After about 15 min. In the ice bath, the liposomes were allowed to reach room temperature.

5) Removal of extraliposomal gradient-forming buffer and transfer of liposomes to a drug loading buffer. Unencapsulated gradient-forming salt was removed, and liposomes were transferred to drug loading buffer using size exclusion chromatography (SEC). In scale-up fabrication tangential flow filtration, hollow fiber dialysis or another scalable step may be used. To ensure complete removal of the extraliposomal polyanion it is advantageous to treat the liposomes with an anion exchange resin (e.g. Dowex-1 or Dowex-2 quaternary ammonium cross-linked polystyrene microspheres). The drug loading buffer contained 50 g / l USP anhydrous dextrose, and 5 mM

HEPES ΡΕ1746976 certified for tissue culture in water adjusted to pH 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 drug loading buffer. Liposomes appeared in the dead volume fraction and were collected, based on turbidity of the eluate, at about 2x volume applied. Eluted liposomes were tested for the concentration of

<td>phospholipids</td><td>in</td><td>according to</td><td>example</td><td> 70,</td><td>size</td><td>in</td>

<td>particle by</td><td colspan="2">QELS, and stored</td><td>at 4-6 ° C.</td><td></td><td></td><td></td>

<td>6) Incubation</td><td>From</td><td>liposomes with the</td><td>drug.</td><td>Was</td><td>ready</td><td>an</td>

<td colspan="2">stock solution</td><td colspan="2">of CPT-11 (hydrochloride</td><td>in</td><td colspan="2">irinotecan)</td>

<td>immediately</td><td colspan="2">before mixing</td><td>with</td><td colspan="2">liposomes,</td><td>per</td>

dissolving irinotecan hydrochloride in water to reach a concentration of 20 mg / ml of the drug base. The pH of the solution was between 4.0 and 5.0. The drug solution was filtered through a 0.2 micron sterile polyethersulfone (PES) filter using positive pressure. Aliquots of the liposomes in the drug loading buffer produced in step 5 above were mixed at room temperature with the irinotecan stock solution to achieve the drug / lipid entry ratio in the range of 0.15-0.55 g of the drug per mmole of the liposome phospholipid. Specific drug / lipid entry ratios are given below, as appropriate. The pH of the mixtures was adjusted to 6.5 with 1 M NaOH, the glass vial mixtures were incubated in the thermoregulated water bath at 58-62 ° C, with slow stirring for 30-45 min., Cooled rapidly in the bath. of ice water (0-2 ° C), and left at this temperature for 15 min. The liposomes were then allowed to warm to room temperature for the next step (removal of the

ΡΕ1746976 unencapsulated drug and transfer to storage buffer). This step resulted in encapsulation efficiency of more than 95%, typically 98-100% over the full range of drug / lipid ratios studied.

7) Removal of unencapsulated CPT-11, transfer of liposomes to storage buffer, final filtration and storage. Unencapsulated drug was removed, and liposomes were transferred to storage buffer using size exclusion chromatography. The storage buffer contained 20 mM HEPES, 135 mM NaCl, pH 6.5 (adjusted with NaOH) in water, and was vacuum filtered at 0.2 microns before use. Gel chromatography on Sephadex G-75 (Amersham Pharmacia Biotech) was performed essentially as described in step 2 above. Column eluted CPT-11 liposomes (dead volume fraction) were tested for liposome phospholipid and CPT-11 (by spectrophotometry, see examples 70 and 71), and the volume-weighted average particle size by QELS. The drug concentration was adjusted, if necessary, to be in the range of 2.0 mg / ml. Liposomes were filtered through sterile 0.2 micron polyethersulfone filters and dispensed into sterile polypropylene vials (Corning Cryovials) or 4 ml screw-capped borosilicate glass vials to approximately 70-80% of the volume of the bottle. The vials were asset-sealed (in air), identified and stored at 4-6 ° C.

Example 12. Effect of drug / lipid ratio on drug loading efficiency and drug retention in vivo of TEA-Pn-containing liposomes

ΡΕ1746976

Liposomes with 0.65 N aqueous trapped TEA-Pn solution, pH 6.1, 531 mmolar osmolarity / kg were prepared following the procedure of example 11. Lipid dispersion was extruded ten times through two stacked polycarbonate filters pore size 100 nm. The liposome lipid matrix also included [³H] -CHE at 0.5 mCi / mmolar phospholipid. The liposome size before drug loading was 98.5 ± 34.3 nm. Liposomes were loaded at initial ratios of pharmacophospholipid 200, 300, 400 and 500 mg of phospholipid CPT-11 / mmole. The amounts of drug and phospholipid in the liposomes were determined, respectively, by spectrophotometry according to example 71, and by the phosphomolaribdate blue phospholipid extraction-digestion test of example 72.

To assess the rate of drug release in vivo, the method of example 8 was followed. Liposomes were injected into the tail vein of 6-week-old Swiss Webster female mice (body weight 18-22 g) at a dose of 5 mg of CPT11 / kg. At 8 and 24 hours after injection, mice in groups of 3 were anesthetized and bled by cardiac puncture. Blood was mixed with 0.4 ml 0.04% ice cold EDTA in PBS, blood cells were separated by centrifugation, and plasma CPT-11 concentration was measured by spectrofluorometry as described in example 71. Lipid was determined. measuring the amount of [³H] -CHE using extinction-corrected liquid scintillation counting, and the amount of drug retained in the liposomes was calculated by dividing the calculated drug / lipid ratio by the drug / lipid ratio in the injected liposomes. Due to the rapid clearance of free CPT-11 in the blood,

ΡΕ1746976 resulting in a low blood level, we assumed that all drugs tested were in liposomal form.

Results are shown in Table 8. Differences between drug retention between groups were not statistically significant. As a result of these studies, we conclude that increasing drug loading up to 500 mg / mmolar will not negatively affect drug loading or stability in vivo. This reason was adopted for further studies.

Table 8. The effect of drug / lipid ratio on drug loading and drug retention in vivo in TEA-Pn irinotecan liposomes (mean ± standard deviation).

<td colspan="3">Drug / lipid ratio, mg / mmolar phospholipid</td><td colspan="2">Drug remaining in liposomes,% of preinjection value</td>

<td>Input</td><td>Output</td><td>% loaded</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> 6 6,8 ± 19,6</td><td> 13,47 ± 1,44</td>

Example 13. Drug loading efficiency of CPT-11 loading in TEA-SOS-containing liposomes: effect of liposome size and in vivo drug retention in mice.

Liposomes with entrapped solutions prepared as in Example 11, using a gradient forming solution having 0.643 N TEA-SOS, pH 5.7, 530 mmolar osmolarality / kg. The lipid dispersion was extruded ten times through two stacked polycarbonate filters with a pore size of 50 nm, 80 nm or 100 nm. The matrix of

ΡΕ1746976 liposome lipid also included [³H] -CHE at 1.5 mCi / mmolar liposome phospholipid. Liposome size was determined by dynamic light scattering. Liposomes were loaded with CPT-11 at initial drug-to-phospholipid ratios of approximately 550 mg irinotecan / mmolar phospholipid. Drug-loaded liposomes were sized by QELS and tested as described in examples 70 and 71.

Female Swiss Webster mice (8-10 weeks, mean 27-30 grams) were injected into the tail vein with these CPT-11 liposome formulations at a drug dose of 10 mg / kg. Mice were sacrificed at 24 h and blood was collected and tested for CPT-11 and liposome lipids as in example 11. Results are summarized in table 9.

Table 9. Irinotecan loading and drug retention in vivo on TEA-SOS liposomes.

<td>Size of</td><td>Size of</td><td>Charge of</td><td>Drug that</td>

<td>pore of</td><td>liposome,</td><td>drug, mg of</td><td>remains in</td>

<td>membrane of</td><td>nm average SD</td><td>irinotecan / mmol</td><td>liposomes after</td>

<td>extrusion, nm</td><td></td><td>air of phospholipid</td><td>24 hours in mice,% of pre-injection value</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>

Surprisingly, the liposomes with the triethylammonium salt of sucrose octasulfate, a non-polymeric, polyanionized hydroxylated organic compound (sugar),

ΡΕ1746976

<td>provided</td><td>a hold</td><td>of</td>

<td>dramatically</td><td>; much better</td><td> (4-5</td>

<td>liposomes</td><td>similar</td><td>with</td>

(polyphosphate).

Example 14. Pharmacokinetics drug in vivo in liposome folds) compared to a polyanionic polymer of CPT-11 loaded SOS-TEA liposomes in rats.

Liposomes (extrusion membrane pore size 100 nm) were prepared as described in Example 12. Liposomes were administered intravenously at a dose of 10 mg CPT11 / kg to two female Sprague Dawley (Harlan) rats of nine weeks (body weight about 200 g) with a central venous catheter implanted at a dose of 10 mg CPT-11 / kg (17.6 pmolar phospholipids / kg). Blood samples were collected at defined time points and analyzed for drug and liposome lipid content, as in example 9. Data were expressed as% injected lipid dose / ml plasma and% drug retained within the liposome at each time point, plotted against time after injection, and lipid lipid half-lives, as well as half-lives for drug release from liposomes, were calculated by best fit to a monoexponential kinetics model (Fig. 5). The drug release half-life of CPT-11 loaded TEA-SOS liposomes was 56.8 hours, much longer than similar TEA-Pn liposomes.

Example 15. Antitumor activity of free CPT-11 and CPT-

<td>11 encapsulated</td><td>in liposomes containing TEA-</td><td>Pn and TEA-SOS, in</td>

<td>naked mice</td><td>atimicos carriers of</td><td>xenografts</td>

human subcutaneous colon carcinoma (HT-29).

ΡΕ1746976

Liposomes were prepared as in Example 11, using a 0.65 M TEA-Pn TEA-Pn solution, pH 6.1 and 531 mmolar / kg osmolarity, or a 0.643 M TEA-SOS TEA-SOS solution. , pH 5.7 and osmolarity of 530 mmolar / kg. The extrusion included 10 passes through two stacked 100 nm pore size polycarbonate membranes. The resulting TEA-Pn and TEA-SOS liposomes were, respectively, size 112.3 ± 15.5 nm and 120.5 ± 42.5 nm (mean ± SD of size distribution). Liposomes were loaded with CPT-11 at the drug / phospholipid entry rate of 500 mg / mmolar. The resulting liposomes had a drug content of 465.6 ± 26.5 (93% loading efficiency) and 499.9 ± 22.5 mg (100% loading efficiency) of phospholipid CPT-11 / mmolar for, respectively, the formulations of TEA-Pn and TEA-SOS.

HT-29 cells were obtained from the American Type Culture Collection, Rockville, MD, and propagated in DMEM medium supplemented with 10% fetal bovine serum, 50 U / ml penicillin G and 50 pg / ml streptomycin sulfate at 37 ° C. C, 5% CO₂as recommended by the supplier. Homogeneous nude, homozygous NCR nu / nu male mice (6 weeks, weight of at least 16 g) were obtained from Charles River. Mice were inoculated subcutaneously in the right flank with 0.1 ml of the suspension containing 5 x 10⁶ cells suspended in growth medium without antibiotics. Eleven days later, animals bearing tumors between 150 mm in size³ and 350 mm³ were assigned to treatment groups according to the following method. The animals were classified according to tumor size, and divided into 6 categories of decreasing tumor size. Six treatment groups of 11 animals / group were formed, randomly selecting one animal from each category of

ΡΕ1746976 size, so that in each treatment group all tumor sizes were equally represented. Starting on day 13, the animals received four tail vein injections at 4-day intervals of the following preparations: 1) control (HEPES buffered saline, pH 6.5); 2) free CPT-11, 50 mg / kg, freshly prepared 5 mg / ml solution in non-buffered physiological saline solution; 3) liposome CPT-11 in TEA-Pn at 25 mg / kg by injection; 4) liposome CPT-11 in TEA-Pn at 50 mg / kg by injection; 5) liposome CPT-11 in TEA-SOS at 25 mg / kg by injection; 6) Liposomal CPT-11 in TEA-SOS at 50 mg / kg by injection. Animal weight and tumor size were monitored twice a week as described in Example 10. Tumor weight was subtracted from animal weighing results to obtain animal body weight. The animals were observed for 60 days after tumor inoculation. When tumors in the group reached 20% of the body weight of the mouse, the animals in the group were sacrificed. There were complete regressions of the tumor in some groups, with no signs of tumor growth at the end of the study. Tissue inoculation site tissues from these animals were collected and preserved for pathological analysis of residual tumor cells.

The results of this study are shown in Figures 6 and 7. Free CPT-11 only had a small effect on tumor growth. Liposomes all had a pronounced effect resulting in tumor regression, followed by regrowth in most animals. In both CPT-11 TEA-Pn and TEA-SOS liposomes, the 50 mg / kg dose was more effective than the 25 mg / kg dose. The average doubling times tumor calculated from tumor size data (Fig. 7) were: control - 4.2 days; drug free,

ΡΕ1746976 mg / kg - 4.8 days; liposomal drug TEA-Pn, 25 mg / mg 43.6 days; liposomal drug TEA-Pn, 50 mg / kg - 47.5 days; TEA-SOS liposome drug at 25 mg / kg - 48.2 days and TEA-SOS liposome drug at 50 mg / kg - more than 56 days (no doubling time was reached). Thus, liposome CPT-11 prepared in accordance with the present invention was at least about 10 times more active than free drug administered at the same dose and time. Unexpectedly, CPT-11 TEA-SOS liposomes were manifestly more effective in reducing tumor growth than CPT-11 TEA-Pn liposomes administered at the same dose. While in the groups treated with free drug and TES-Pn liposomal drug at 50 mg / kg per injection there were no animals without tumor regrowth, in the groups receiving 25 mg / kg of each liposomal formulation, one animal (9.1 %) was tumor free at the end of the study, and in the group receiving 50 mg / kg of the TEA-SOS liposomal CPT-11 formulation, 4 animals (36.4%) were tumor free at the end of the study with no signs of regrowth.

The drug manifested some toxicity. Animals receiving free but not liposomal CPT-11 experienced temporary morbidity (loss of attention, stooped posture, wrinkled skin, decreased mobility) for about one hour after injection of the drug. Animals receiving free CPT-11 experienced a permanent loss of about 6% weight during treatment and did not recover. Animals receiving both liposomal CPT-11 formulations experienced a temporary weight loss between the second and third injections, giving an average of about 5% (at 25 mg / kg) or about 9% (at 50 mg / kg). kg) of the pre-treatment value, and finally reached normal weight. Therefore, the toxicity of the liposome drug does not

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100 was greater than that of the free (non-liposomal) drug, while the efficacy of the liposomal drug was substantially higher. Weight loss was reversed when drug treatment was terminated, and all animals regained their weight without terminal morbidity or toxic deaths. Later, the animals gained weight concomitantly with tumor regressions. In the saline control group, animals that developed large tumors experienced a weight loss due, of course, to tumor-related morbidity. In general, the liposome drug formulation, in which the drug was loaded into liposomes having pre-entrapped polyanionized sugar (sucrose octasulfate), proved to be the most effective, having less toxicity than the non-liposomal drug.

Example 16. Toxicity of free and liposomal CPT-11 in mice.

The acute toxicities of free CPT-11 and liposome-encapsulated CPT-11 prepared according to the present invention were compared by determining the maximum tolerated dose (DMT) following a single iv injection in normal (immunocompetent) mice.

The following materials were used:

1) Preparation of CPT-11 irinotecan hydrochloride 7.6%. In this study, those prepared on a correction basis for irinotecan content.

(irinotecan hydrochloride) having 98.9% by HPLC, and moisture to drug formulations were as shown, without moisture or the content of the base.

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101

2) Liposomal CPT-11 (Ls-CPT-11) was prepared as in Example 11 using the 200 molar part DSPC lipid matrix, 133 molar part cholesterol, 1 molar PEG-DSPE; trapped TEA-SOS solution having 0.65 M TEA, pH 6.4; liposome-loaded drug in 5 mM HEPES buffer, 5% dextrose, pH 6.5, at 60 ° C for 30 min., at drug / lipid entry rate of 500 mg drug / mmole phospholipid. Charging efficiency was&gt; 99%. Liposome size (mean mean volume ± standard deviation by QELS): 101 ± 37 nm. Liposomes were formulated in the vehicle, 20 mM HEPES-Na, 135 mM NaCl, pH 6.5. Drug concentrations in the injected formulations were as indicated in the tables below.

3) Free CPT-11 solution. The free drug stock solution was prepared by dissolving irinotecan hydrochloride in 5% aqueous dextrose at 22 mg / ml, and was sterile filtered by 0.2 pm. This stock solution was diluted with 5% sterile dextrose prior to injection.

4) Animals. Female Swiss Webster mice, 6-8 Harlan, USA.

weeks of

The determination of DMT was generally followed by the United States National Cancer Institute Therapeutics Program, and included the following three steps:

the deveiopmental protocol

The protocol

Step 1): Range search step with dose increase factor of 1.8. Groups of two animals were injected into the tail vein with increasing doses of free or liposomal irinotecan, starting at a dose of 60 mg / kg and continuing with a dose-increasing factor of 1.8 until

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102 acute mortality or terminal morbidity (within&gt; 1 day after injection) was observed in either animal. The dose is recorded one step below the terminal mortality / morbidity dose.

Step 2): Range search step with dose increase factor of 1.15. Groups of two animals were injected into the tail vein with increasing doses of free or liposomal irinotecan, starting with the dose recorded in step 1 and continuing with the dose increase factor of 1.15 until acute mortality or terminal morbidity was observed ( &gt; 1 day after injection) in any of the animals. The preliminary DMT dose is recorded one step below the terminal mortality / morbidity dose.

Step 3): Validation Step. The group of 5 animals is injected iv (tail vein) with free or liposomal irinotecan in the preliminary DMT determined in step 2. The animals are followed for 7 days, the animal's body weight is recorded twice a week and compared with the weight. pre-injection. The general state of health of the animals is observed (vigilance, hygiene, feeding, excrement, skin, fur and mucous membrane conditions, ambulation, breathing, posture). If during the observation period there is no mortality, progressive morbidity or weight loss greater than 15% of pre-injection body weight, the dose is considered to be validated as a single acute DMT injection. If any of these effects occur, the experiment is repeated at the next lowest dose by a factor of 1.15.

For validation statistics, it was followed to surviving animals until

<td>additional to</td><td>the step</td><td>in</td>

<td>weight dynamics</td><td>body</td><td>From</td>

<td>11 days after</td><td>injection.</td><td>Was</td>

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103

<td>impossible</td><td>administer the</td><td>dose</td><td>superior than</td><td>324 mg / kg</td><td>in</td>

<td>irinotecan</td><td>liposomal</td><td>per</td><td>cause of</td><td>limitations</td><td>gives</td>

<td>concentration</td><td>and volume</td><td>in</td><td>injection. The</td><td>results</td><td>are</td>

<td>presented</td><td>in table 10.</td><td></td><td></td><td></td><td></td>

Table 10. DMT search study of CPT11 formulations in mice.

RESULTS

Step 1. Dose increase by a factor of 1.8

Animal body weight, day after injection:

<td colspan="2">drug Dose inj. (mg / kg)</td><td>conc. drug (mg / ml)</td><td>Volume inj. (μΐ)</td><td>O no little mouse</td><td>0 (g /</td><td>1 2 4 5 6 7 11 &gt; (g) (g) (g) (g) (g) (g) (g)</td>

<td>Ls-</td><td> 60</td><td> 8</td><td> 150</td><td> 1</td><td> 19,</td><td>218.0 nd 20.3 20, 6 20, 6 20.0 19.7</td>

<td>CPTll</td><td></td><td></td><td></td><td> 2</td><td> 19,</td><td>719.3 na 20,620,419,619,720.7</td>

<td></td><td> 100</td><td> 12</td><td> 165</td><td> 1</td><td> 19,</td><td>518.6 nd 19.6 20.0 20.1 19.4 19.9</td>

<td></td><td></td><td></td><td></td><td> 2</td><td> 20,</td><td>118.9 nd 20.2 21.5 22.2 21.8 22.5</td>

<td></td><td> 180</td><td> 22</td><td> 165</td><td> 1</td><td> 19,</td><td>418.4 nd 18, 9 19, 7 20.5 19.5 20.5</td>

<td></td><td></td><td></td><td></td><td> 2</td><td> 20,</td><td>019.3 nd 19.6 20.6 6 21.4 21.6 6 21.7</td>

<td></td><td> 324</td><td> 30, 6</td><td> 210</td><td> 1</td><td> 21,</td><td>8 21.2 21.2 na 20.2 na 20.2 na</td>

<td></td><td></td><td></td><td></td><td> 2</td><td> 21,</td><td>6 20.4 21.3 na 20.8 na 21.4 na</td>

<td>CPT11</td><td> 60</td><td> 8</td><td> 150</td><td> 1</td><td> 20,</td><td>6 20.4 nd 2 2.1 22.1 22.2 22.0 22.5</td>

<td>free</td><td></td><td></td><td></td><td> 2</td><td> 19,</td><td>519.1 nd 20.120,320,420,521.1</td>

<td></td><td> 100</td><td> 12</td><td> 165</td><td> 1</td><td> 19,</td><td>3died 1-2 min. after injection</td>

<td></td><td></td><td></td><td></td><td> 2</td><td> 20,</td><td>1 died 1-2 min. after injection</td>

<td></td><td></td><td></td><td></td><td> 3</td><td> 19,</td><td>9died 1-2 min. after injection</td>

After injection, all free CPT11-treated mice became sick, breathless for about 1 h and then recovered.

After injection, all Ls-CPT11 treated mice were normal.

Step 2. Dose increase by a factor of 1.15

Animal body weight, day after injection:

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<td>drug</td><td>Injection dose (mg / kg)</td><td>conc. drug (mg / ml)</td><td>Volume inj. (μΐ)</td><td>_no ° little mouse</td><td>0 (g)</td><td>1 (g)</td><td>2 (g)</td><td>5th (g)</td><td>7th (g)</td>

<td>CPT11</td><td> 60</td><td> 8</td><td> 150</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>free</td><td></td><td></td><td></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></td><td> 70</td><td> 8</td><td> 175</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></td><td></td><td></td><td></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></td><td> 80</td><td> 8</td><td> 200</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></td><td></td><td></td><td></td><td> 8</td><td> 20, 6</td><td> 20, 8</td><td> 21,1</td><td> 20, 7</td><td> 21,4</td>

<td></td><td> 90</td><td> 12</td><td> 150</td><td> 9</td><td>died</td><td> 1-2</td><td colspan="3">min after injection</td>

<td></td><td></td><td></td><td></td><td> 10</td><td>died</td><td> 1-2</td><td colspan="3">min after injection</td>

<td></td><td></td><td> 8</td><td> 225</td><td> 11</td><td>died</td><td> 1-2</td><td colspan="3">min after injection</td>

Step 3. Validation

<td rowspan="3">drug</td><td rowspan="3">Injection dose (mg / kg)</td><td rowspan="3">conc. drug (mg / ml)</td><td rowspan="3">Volume inj. (μΐ)</td><td rowspan="3">no ° little mouse</td><td colspan="3">Animal body weight per day</td><td rowspan="3">of after 7th (g)</td>

<td rowspan="2">0 (g)</td><td colspan="2">the injection:</td>

<td>3 (g)</td><td>5th (g)</td>

<td>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>free</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></td><td></td><td></td><td></td><td> 5</td><td> 21, 9</td><td> 21, 9</td><td> 21, 6</td><td> 21,5</td>

<td>Ls-CPTll</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, while the free CPT-11 DMT was 80 mg / kg, the liposomal CPT-11 DMT was surprisingly not achieved even at the highest administered dose of 324 mg / kg. Therefore, CPT-11 encapsulation in the

ΡΕ1746976

105 Liposome according to the present invention reduced drug toxicity at least 4-fold.

Example 17. Storage stability of CPT-11 loaded TEA-SOS liposomes against drug leakage. Five batches of liposome CPT-11 were prepared using the TEA-SOS method (Example 11) at the phospholipid 500-550 mg / mmole drug / lipid entry ratio. 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. Liposomes were sterilized with a 0.2 pm 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 released drug was removed by gel chromatography on Sephadex G-75 using the storage buffer as eluent. Drug and phospholipid concentrations in the liposomes before and after gel chromatography were tested using, respectively, the spectrophotometry method and the acid phospho-molybdibate digestion method as described in Examples 70 and 71. CPT-11 liposomes prepared according to with the present invention were very stable. CPT-11 leakage from these liposomes during storage was less than 5% over 6 months (Table 10).

Table 11. Encapsulation stability of CPT11 liposomes during storage (data are mean + SE).

Batch of Size pore liposomes no. ° of extrusion,

Concentration Time% CPT-11 drug, storage, which remains mg / ml encapsulated months

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106 nm

<td> 1</td><td> 80</td><td> 3,44</td><td> ±</td><td>O,</td><td> 06</td><td> 6</td><td> 99, 02</td><td> ±</td><td> 3,77</td>

<td> 2</td><td> 80</td><td> 7,88</td><td> ±</td><td> 0,</td><td> 19</td><td> 6</td><td> 102,38</td><td> ±</td><td> 4,78</td>

<td> 3</td><td> 100</td><td> 4,57</td><td> ±</td><td> 0,</td><td> 06</td><td> 6</td><td> 96, 38</td><td> ±</td><td> 4,69</td>

<td> 4</td><td> 100</td><td> 4,62</td><td> +</td><td> 0,</td><td> 11</td><td> 6</td><td> 95,72</td><td> ±</td><td> 4,36</td>

<td> 5</td><td> 80</td><td> 14,52</td><td> ±</td><td> 0</td><td> ,42</td><td> 3</td><td> 103, 4</td><td> ±</td><td> 5, 92</td>

Example 18. Topotecan-loaded liposomes.

Liposomes with entrapped TEA-Pn solution and TEA-SOS solution were prepared as in Example 11. Topotecan hydrochloride stock solution (GlaxoSmithKline, PA, USA) was prepared immediately prior to mixing with the liposomes by dissolving the hydrochloride hydrochloride. topotecan in water at 15-20 mg / ml with the actual topotecan HCl The pH was adjusted to 3.0 with 1 N HCl. The drug solution was filtered through a 0.2 micron sterile polyethersulfone (PES) filter using positive pressure. Aliquots of the TEA-Pn or TEA-SOS-containing liposomes in the drug loading buffer were mixed at room temperature with topotecan HCl stock solution to achieve the drug / lipid entry ratio in the range of 0.15-0.45 g / mmolar liposome phospholipid. The preferred ratio was 0.35 g of topotecan HCl per mmole of liposome phospholipid. The mixtures in glass containers were incubated in the thermoregulated water bath at 55-62 ° C with slow shaking for 3060 min., Cooled rapidly in ice water bath (02 ° C) and left at this temperature for 5-15 min. . This step produced an encapsulation efficiency of 89-90% (TEA-Pn gradient) or 97-100% (TEA-SOS gradient). Unencapsulated topotecan was removed, and liposomes were transferred to storage buffer using size exclusion column chromatography. Before

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107 application to the column, the ionic strength of the liposome preparation was increased by mixing with 1/20 vol. 2.88 M aqueous sodium chloride, and the mixture was incubated for about 15 min. We unexpectedly found that adjusting the ionic strength of the liposome medium from the low value during loading (typically equivalent to less than 20 mM NaCl) to the highest value above 20 mM NaCl and preferably to 50 mM NaCl and above improved the removal of unencapsulated drug and increased the stability of topotecan-loaded liposomes against aggregation, possibly facilitating the removal of membrane-bound topotecan, unlike the drug encapsulated within the liposome. The rest of the procedure followed example 11, step 7. For results, see table 12 below.

Example 19. Preparation of topotecan anti-HER2 immunoliposomal formulations.

Topotecan immunoliposomes specifically internalized by HER2 tyrosine kinase surface receptor oncoprotein (C-ErbB-2) overexpressed cancer cells were prepared by conjugating topotecan liposomes with the anti-HER2 human single chain Fv antibody fragment. F5, selected from the phage display library for its high internalization in HER2 overexpressed cells (Poul, et al., 2000, J. Molarecular Biology, v. 301, p. 1149-1161). F5 is a 27-kDa protein that binds to the extracellular domain of the HER2 receptor with an affinity of about 150 nM, causing rapid internalization (Neve, et al., 2001, Biophys. Biochem. Res. Commun. V. 280 , pp. 274-279). For liposome conjugation, the method generally followed was

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108 U.S. Patent No. 6,210,707 and to Nielsen, et al. (2002), Biochim. Biophys. Minute, v. 1591, p. 109-118. A hydrophilic F5 lipopolymer conjugate was first prepared. The C-terminal of the F5 amino acid chain had an added terminal cysteine ​​terminal group (F5Cys). The F5Cys construct was expressed in E. coli and isolated from bacterial lysate by protein A column chromatography. Protein A eluted fractions were adsorbed on the anion exchange resin to remove pyrogens and host DNA, and treated with a thiol reducing agent to release the thiol group from the terminal cysteine. Reduced F5Cys was further purified by ion exchange chromatography using purified SP Sepharose Fast Flow (Amersham Pharmacia) conjugated with reactive poly (ethylene glycol)

N- (3- (In a thiol binder, maleimidate) propionylamido) -poly (oxyethylene) oxycarbonyl) 1,2-distearoylphosphatidyl ethanolamine protein (Mal-PEG-DSPE), a 2,000 molar weight PEG derivative Figure 4.1), commercially produced by Avanti Polar Lipids, Inc., Alabama, USA. The protein and binder were incubated in an aqueous buffer solution at a 1: 4 molar ratio, and the unreacted binder was quenched with 1 mM cysteine. During the reaction, the F5Cys terminal cysteine ​​is covalently linked to the binder maleitus group. The resulting F5-PEG-DSPE conjugate was water-soluble as micelles, having high apparent molar weight (500-850 kDa), and was separated from 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 binder was tested using a spectrophotometric method identical to that used for the

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109 quantification of phospholipids (see Example 70). Purified F5-PEG-DSPE conjugate was water stable, fully immunoreactive, and was stable against denaturation and loss of reactivity for at least 1 hour at 65 ° C, and for at least 3 months at 37 ° C.

To prepare the anti-HER2 immunoliposomal topotecan, the topotecan-loaded liposomes from Example 18 were mixed with F5-PEG-DSPE in the aqueous saline buffer at a rate of 15 micrograms protein per 1 micromolar phospholipid (about 45 copies of F5 per liposome). The mixture was incubated for 40 min. at 60 ° C, chilled on ice, and chromatographed on a column with Sepharose CL-4B (4% crosslinked agarose microspheres, Amersham Pharmacia) to remove residual micellar conjugate, unconjugated protein and any traces of extraliposomal drug that may have released during incubation. The membrane-embedded F5-PEG-DSPE liposomes were eluted with 5 mM buffer of HEPES-144 mM NaCl, pH 7.4, collected in the column dead volume, filter sterilized and dispensed for storage (4-6 ° C ). The amount of F5 incorporated into liposomes was typically&gt; 80% of the added conjugate.

It was determined by SDS-PAGE of the Coomassie-stained F5 band quantified liposomes by densitometry. Drug and lipid concentrations in immunoliposome preparations were determined similarly to non-directed liposomes. The properties of topotecan liposomes and F5 immunoliposomes (Examples 18-19) are summarized in Table 12.

Table 12. Characteristics of topotecan liposomes and immunoliposomes.

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<td rowspan="2">salt imprisoned in liposomes</td><td rowspan="2">F5 scFv connection:</td><td>Reason for drug / lipid, enc g / molar phospholipid</td><td rowspan="2">% in psulation</td><td rowspan="2">Liposome size, mean ± SD, nm</td>

<td>Entrance exit</td>

<td>TEA-Pn</td><td>No</td><td> 173,6 155,2 ±5,9 89,4</td><td> ± 3,4%</td><td> 96,4 ± 38,7</td>

<td>TEA-Pn</td><td>Yes</td><td> 173,6 156,2 ± 5,2 90,0</td><td> ± 3,0%</td><td> 96,2 ± 33,8</td>

<td>TEA-SOS</td><td>No</td><td> 347,2 340, 8 ± 14,7 98,2</td><td> ± 4,2%</td><td> 99,1 ± 32,6</td>

<td>Example 20.</td><td>It is made</td><td colspan="3">loading buffer pH and ratio</td>

of drug / lipid in topotecan loading in liposomes.

Liposomes (DSPC / Col / PEG-DSPE, 3: 2: 0.015 molar ratio) with 0.5 N entrapped TEA-Pn, pH 6.2, 413 mmolar / kg osmolarity, were prepared using the injection method. Ethanol (Example 18), extruded 5 times through two stacked 100 nm pore size stacked filters and 10 times the 50 nm pore size. The loading buffer was 5 mM MES, 50 g / 1 dextrose, adjusted to various pH in the 5.06.5 range. Liposome size was 73.1 ± 21.3 nm by QELS. Liposomes were loaded by mixing a stock solution of topotecan (20 mg / ml) with the liposomes in the loading buffer at 100 mg / mmolar drug-paraphospholipid entry ratio, incubating the mixture at 60 ° C for 45 min. ., extinction on ice for 15 min. and removing unencapsulated drug using a Sephadex G-75 column eluted with 20 mM HEPES, 135 mM NaCl, pH 6.5. Topotecan and phospholipid were quantified by spectrophotometry (Examples 70 and 71). The results (Table 13) indicated that topotecan loading was almost quantitative in the pH 5.5-6.5 range.

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Table 13. Effect of loading buffer pH on% of

<td>encapsulation</td><td>in</td><td>topotecan</td><td>in TEA-Pn liposomes</td>

<td colspan="4">imprisoned.</td>

<td>buffer pH</td><td>in</td><td>charge</td><td>Encapsulation%</td>

<td> 5, 0</td><td></td><td></td><td> 50,1 ±2,1</td>

<td> 5,5</td><td></td><td></td><td> 97,2 ±8,1</td>

<td> 6, 0</td><td></td><td></td><td> 115,5 ± 15,0</td>

<td> 6, 5</td><td></td><td></td><td> 102,1 ±8,1</td>

The effect of drug to lipid ratio (0.15-0.45 mg / mmolar phospholipid) on loading efficiency was also studied. The trapped TEA-Pn liposomes (0.5 M TEA, pH 5.8, 480 mmolar / kg osmolarity) were prepared as above, except for the final extrusion step which was ten times through two stacked polycarbonate filters 0.08 pm. Charging was at pH 6.5. Liposome size was 93.1 ± 15.1 nm by QELS. The results (Table 14) showed that drug loading efficiency was above 85% over the entire range of drug / lipid ratios studied.

Table 14. Effect of drug / lipid ratio on topotecan encapsulation efficiency in TEA-Pn-containing liposomes.

Topotecan / phospholipid ratio,% encapsulation mg / mmolar (mean ± SE)

Input Ratio Output Ratio (after upload)

<td> 168,2</td><td> 166, 9</td><td> ±</td><td> 11,</td><td> 1</td><td> 99, 2</td><td> ±</td><td> 6, 6</td>

<td> 224,4</td><td> 232,5</td><td> ±</td><td> 47,</td><td> 6</td><td> 103,7</td><td> ±</td><td> 21,2</td>

<td> 280,3</td><td> 253,5</td><td> ±</td><td> 19,</td><td> 8</td><td> 90,4</td><td> ±</td><td> 7,0</td>

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<td> 336, 4</td><td> 298,3</td><td> ±</td><td> 18,0</td><td> 88,</td><td> 7</td><td> ±</td><td> 5,3</td>

<td> 392,5</td><td> 361,2</td><td> +</td><td> 36, 8</td><td> 92,</td><td> 0</td><td> +</td><td> 9, 4</td>

<td> 448,5</td><td> 394,9</td><td> ±</td><td> 29, 5</td><td> 88,</td><td> 0</td><td> ±</td><td> 6, 6</td>

Example 21. Stability of topotecan liposomes in vitro in the presence of plasma.

Liposomes (DSPC / hol / PEG-DSPE, 3: 2: 0.015 molar ratio) with 0.5 N entrapped TEA-Pn, pH 6.2, 413 mmolar osmolarality / kg, were prepared as described in Example 18. Liposomes of 96.4 ± 29.3 nm size were produced by extrusion ten times through two stacked 100 nm pore size polycarbonate filters. For the quantification of plasma liposome lipid, [³H] -CHE in the 0.5 pCi / pmolar lipid solution of DSPC. Topotecan was charged at pH 6.0.58 ° C for 45 min. at a drug / phospholipid ratio of 150 mg / mmolar. The loading efficiency was 148.48 ± 10.26 pg topotecan / pmolar phospholipid (99.0 ± 6.8%).

Liposomes were incubated with 50% human plasma in a multiwell microdialysis device (Spectra-Por

<td>MicroDialyzer</td><td>10 wells,</td><td>Spectrum, USA)</td><td> . 0</td><td>plasma of</td><td>donor</td>

<td colspan="3">human was diluted with equal volume</td><td>in</td><td>solution</td><td>saline</td>

<td>buffered with</td><td>HEPES (20</td><td>mM HEPES,</td><td> 135</td><td colspan="2">mM NaCl), pH</td>

<td>6.5 containing</td><td>0.02% of</td><td>sodium azide</td><td>and</td><td>loaded</td><td>to</td>

<td>reservoir</td><td>bottom</td><td>of dialyzer</td><td> (32</td><td>ml). The</td><td>wells</td>

(0.4 ml) were separated from the reservoir by a 30 nm pore size polycarbonate membrane to provide free passage of plasma proteins and small but not liposome molecules. Liposomes were mixed with calculated amounts of plasma and HEPES-buffered saline to achieve a concentration of 2.5 mM phospholipid and 50% vol.

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113 plasma. The device was incubated at 37 ° C, and the contents of the reservoir were shaken slowly. After 8 hours of incubation, the contents of the lower reservoir were changed to 50% fresh plasma. At the indicated time points (see below), 50 µl aliquots were taken from the wells, and chromatographed on columns containing 2.2-2.4 ml Sepharose CL-2B, HEPES-buffered saline eluent, to separate liposomes from plasma and free drug proteins. Liposomes were collected in the dead volume fractions. Topotecan was quantified by fluorometry using excitation at 384 nm and emission at 524 nm after solubilization of plasma samples in 90% aqueous isopropanol-0.1 N HCl, and lipid was quantified by scintillation counting of [³H] -CHE (corrected extinction). The determined drug-to-lipid ratio over time was compared to the initial preincubation ratio to obtain the% topotecan that remained encapsulated at each time point. After 8 hours of incubation, the amount of drug remaining in the liposome was about 55% of its initial value (Table 15).

Table 15. In vitro release of topotecan from

<td>liposomes</td><td>loaded</td><td>by TEA-Pn gradient at 50% of</td>

<td colspan="2">human plasma at 37 ° C.</td><td></td>

<td>Time to</td><td>incubation,</td><td>hours% of drug remaining encapsulated</td>

<td> 1</td><td> 95,5</td><td> ±</td><td> 5,4</td>

<td> 4</td><td> 76, 8</td><td> +</td><td> 7,3</td>

<td> 8</td><td> 55, 9</td><td> ±</td><td> 4,1</td>

<td> 24</td><td> 55,4</td><td> ±</td><td> 16, 8</td>

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114

Example 22. TEAPn-gradient topotecan liposomes entrapped at various drug / lipid ratios: in vivo drug retention and circulation longevity in mice.

Liposomes (DSPC / Col / PEG-DSPE at 3: 2: 0.015 molar ratio, containing [³0.5 mCi / mmolar H] -CHE of DSPC) with encapsulated gradient-forming salt solution (0.5 N TEA-Pn, pH 6.2, 413 mmolar / kg osmolarity) were prepared as in example 18 using 12-fold extrusion through two 100 nm pore size stacked polycarbonate filters. Liposome size was 107.7 ± 19.1 nm by QELS. Liposomes at 5 mM of

HEPES, 50 g / l dextrose, pH 6.5 were mixed with the aqueous stock solution of topotecan (20 mg / ml) at drug / phospholipid ratios in the range 130-360 ugpg / umolar, followed by incubation of the mixture 58 ° C for 45 min., Put on ice for 15 min. and removal of the unencapsulated drug by Sephadex G-75 chromatography. Twelve-week female FvB mice were injected with tail vein liposomes at a dose of 5 mg topotecan per kg body weight (approx. 0.2 mg topotecan / animal) in triplicate. At the indicated times, usually 8 hours or 24 hours after injection, mice were anesthetized, bled, and blood samples were tested for drug and liposome lipid as in Example 8. Results are shown in Table 16. After 24 hours, about 6-32% of the initial drug load remained encapsulated. Higher drug loads (&gt; 200 mg / mmole phospholipid) resulted in longer drug retention.

Table 16. In vivo drug retention and circulation longevity of prototype topotecan liposomes

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115 loaded using the TEA-Pn gradient method for different drug / lipid ratios.

Drug ratio Lipid remaining encapsulated / phospholipid, circulating mg / mmolar,% injected dose

Topotecan remains encapsulated,% of initial charge

After After After After 24 hours 24 hours 8 hours 24 hours

<td> 127,2</td><td> ±</td><td> 10, 9</td><td> 36, 1</td><td> ±</td><td> 2,0</td><td> 18</td><td>U7</td><td> ±</td><td> 8,1 51,7</td><td> ±</td><td> 7,</td><td> 1</td><td> 6, 72</td><td> ±</td><td> 2,5</td>

<td> 207,2</td><td> ±</td><td> 21, 6</td><td> 32,1</td><td> ±</td><td> 5,2</td><td> 9,</td><td> 84</td><td> ±</td><td> 1, 88 75,6</td><td> ±</td><td> 13</td><td> ,0</td><td> 13, 8</td><td> ±</td><td> 3, 5</td>

<td> 301,3</td><td> +</td><td> 24,5</td><td> 34,4</td><td> +</td><td> 3,2</td><td> 8,</td><td> 04</td><td> +</td><td> 4,25 79,2</td><td> +</td><td> 4,</td><td> 2</td><td> 25, 6</td><td> +</td><td> 4,4</td>

<td> 360,3</td><td> ±</td><td> 35, 6</td><td> 33, 6</td><td> ±</td><td> 2,4</td><td> 8,</td><td> 68</td><td> ±</td><td> 4, 96 73,5</td><td> ±</td><td> 7,</td><td> 0</td><td> 32,3</td><td> ±</td><td> 9,8</td>

Example 23. In vivo drug retention and longevity of circulation of charged topotecan liposomes using different trapped ammonium and triethylammonium salts.

Liposomes composed of DSPE, cholesterol and PEG-DSPE (3: 1: 0.1 by weight), also containing [³H] -CHE at 0.22 mCi / mmolar DSPE were prepared as in example 18, except for the extrusion step which included 10 passes through 2 stacked 200 nm pore filters, 10 passes through 2 stacked pore filters. 100 nm and 20 passes through 2 stacked 50 nm pore filters. The liposomes contained the following saline solutions:

A 0.5 N ammonium dextran sulfate (ADS) solution was prepared from sodium dextran sulfate (PM 5000), purchased from Sigma, and converted to the ammonium salt by the similar ion exchange procedure as in the example. 4. The dextran sulfuric acid solution was immediately titrated with 12.4 M aqueous ammonia. The A-DS solution has pH 5.66 and osmolarity of 208 mmolar / kg.

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A 0.48 N solution of ammonium sucrose octasulfate (A-SOS) was prepared similarly to Example 6, but ammonium hydroxide was used for titration. The solution had pH 6.27 and osmolarity of 258 mmolar / kg.

A 0.47 M solution of triethylammonium sucrose octasulfate (TEA-SOS) was prepared as in example 6. The solution has pH 6.6 and osmolarity of 297 mmolar / kg.

Topotecan was loaded into the liposomes in 10 mM aqueous solution of MES-Na, 50 g / l dextrose, pH 6.5, by incubation of the liposomes with the drug at 61-62 ° C and at a drug entry rate. / phospholipid 346 ± 1 mg / mmolar for 40 min, followed by incubation on ice for 10 min. Liposomes were purified from unencapsulated drug by Sephadex G-25 chromatography, eluent - 2 mM aqueous histidine, 144 mM NaCl, pH 6.6 (HCl).

Female seven to nine week old Swiss Webster mice were injected into the tail vein with these liposomal topotecan formulations at a dose of 5 mg topotecan per kg body weight (approx. 0.2 mg topotecan / animal) in triplicate. After 8 hours or 24 hours after injection, blood was collected and analyzed for topotecan and liposome lipid, as in example 22.

Results are presented in table 17 below. Although the three liposome formulations demonstrated very close liposome circulation longevity, with about 23-28% of the injected dose remaining in the blood 24 hours after injection, unexpectedly drug retention in TEA-SOS liposomes and A-SOS liposomes have been

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117 better than in A-DS liposomes, both in terms of magnitude (about 2-fold improvement in drug retention) and statistical significance (statistical significance at 95% confidence level by 2-tailed unpaired Student's t-test respectively, p = 0.0257 and p = 0.00995 (and by Mann's U test the difference was significant with α = 0.01). Drug retention in topotecan-containing TEA-SOS liposomes was better than in topotecan-containing A-SOS liposomes.

Table 17. Drug retention in vivo and persistence of

<td>circulation</td><td>of the liposomes of</td><td colspan="2">topotecan</td><td>prepared</td>

<td>using</td><td>TEA-SOS, ammonium-SOS</td><td>(ALONE)</td><td>and</td><td>sulfate</td>

<td>dextran of</td><td>ammonium (A-DS).</td><td></td><td></td><td></td>

Gradient Efficiency RatioLipid Size Topotecan drug / loading, liposome, remains in remains phospholipid,% nm circulation,% encapsulated,% mg / mmolar injected dose of initial load

<td>After</td><td>After</td><td>After</td><td>After</td>

<td>8 hours</td><td>24 hours</td><td>8 hours</td><td>24 hours</td>

<td>A-DS</td><td> 288, 1</td><td> ±</td><td> 20, 683, 3 ± 6,0</td><td> 76, 9</td><td> ±</td><td> 43,7</td><td> ±</td><td> 27, 7 ±</td><td> 43,6 ±</td><td> 18,7</td><td> ±</td>

<td></td><td></td><td></td><td></td><td> 22,7</td><td></td><td> 1,2</td><td></td><td> 1,5</td><td> 6, 8</td><td> 1,5</td><td></td>

<td>ALONE</td><td> 346, 2</td><td> ±</td><td> 14,3100,0 ± 4,1</td><td> 99,7</td><td> ±</td><td> 42,3</td><td> ±</td><td> 23,4 ±</td><td> 53,3 ±</td><td> 31,3</td><td> ±</td>

<td></td><td></td><td></td><td></td><td> 28, 9</td><td></td><td> 2,2</td><td></td><td> 2,0</td><td> 0, 8</td><td> 3,2</td><td></td>

<td>TEA-SOS</td><td> 340, 8</td><td> ±</td><td> 14,7 98, 5 ± 4,2</td><td> 99,1</td><td> ±</td><td> 42,1</td><td> ±</td><td> 23,0 ±</td><td> 57,0 ±</td><td> 38,1</td><td> ±</td>

<td></td><td></td><td></td><td></td><td> 32,6</td><td></td><td> 2,3</td><td></td><td> 2,9</td><td> 5, 6</td><td> 6,1</td><td></td>

<td>Example</td><td> 24.</td><td></td><td colspan="2">Pharmacokinetics</td><td>of</td><td colspan="3">drug and</td><td colspan="2">of lipid</td><td>of</td>

plasma liposomal topotecan in rats

Circulation longevity and topotecan release parameters were evaluated in rats. Liposomes (DSPC / Cholesterol / PEG-DSPE, 3: 2: 0.015 molar ratio) were prepared by the ethanol / extrusion mixing method and loaded with topotecan using either the TEAPn gradient or the sucrose TEA-octasulfate gradient (TEA). -SOS), as described in example 18, and loaded for various reasons

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118 of drug / lipid (15-450 mg / mmolar phospholipid). For guantification of the lipid matrix, the liposome lipid contained [3 H] -CHE at 0.5-1.5 mCi / mmolar DSPC. Female Sprague Dawley rats (6-8 weeks; body weight about 200 g) with an implanted central venous catheter were injected iv (through the catheter) with topotecan liposomes at a dose of 4-5 mg / kg body weight. The catheter was flushed with saline. At selected times (up to 48 hours after injection), blood samples (0.2-0.3 ml) were collected through the catheter in heparinized syringes, mixed with 0.4 ml phosphate-buffered cold saline with 0.04% EDTA, blood cells were separated by centrifugation, and supernatants (PBS-diluted plasma) were tested for lipid by radioactivity counting. ³H-CHE (corrected extinction) and for topotecan by fluorometry (Example 71). Test results were corrected for plasma dilution, calculated from the weight of the blood sample obtained and assuming a hematocrit of 40%. The total blood dose of the drug and lipid was calculated from the blood volume calculated as 6.5% of body weight. The percentage of topotecan retained in the liposomes was calculated by comparing the drug / lipid ratio at a given point in time with the drug / lipid ratio of the injected liposomes. Table 18 below summarizes blood lipid and drug half-lives and drug release half-lives, as well as other liposome properties shown in drug / lipid)

The pharmacokinetic figure 8A (PK) curves are (lipid) and 8B (ratio of

In summary, blood PK curves for the drug and lipid fit well with the single exponent model (R² 0,984-0,999). Despite its size of 90-100 nm and the very low amount of PEGylated lipid (0.3 mol%),

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119 liposomes showed, plasma lipid circulation longevity was slower release hours) was observed with the TEA-SOS methods.

unexpectedly good (component half-lives in the 11-16 hour range). Topotecan (22.9 times liposomes loaded using

Table 18. Circulation half-life (ti /₂) of lipid, drug, and half-time for drug release from prototype topotecan liposomes in rats.

<td rowspan="2">salt imprisoned, and concentration</td><td rowspan="2">Topotecan loading, mg / mmolar phospholipid</td><td colspan="2">Serving Size</td><td rowspan="2">ti / 2 of lipid, hours</td><td rowspan="2">ti / 2 of drug, hours</td><td rowspan="2">ti / 2 da release of drug, hours</td><td rowspan="2">No. of: animals per group</td>

<td>liposome, nm (mean ± SD)</td><td>injected, mg / kg</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 0.643 N</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>

<td colspan="6">Example 25. Stability of the drug against</td><td>escape</td><td>during</td>

the storage of topotecan liposomes

Samples of various prototype formulations prepared for the studies described above were stored at 4-6 ° C at various times to assess the storage stability of encapsulated topotecan against liposome drug leakage. Liposome samples were passed through Sephadex G-75 columns, eluted with 20 mM HEPES, 135 mM NaCl, pH 6.5, to remove extraliposomal drug, and analyzed for spectrophotometry and lipid content by [3H] -CHE radioactivity count. Results (Table 19) indicate good topotecan retention in liposomes during storage.

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120

Table 19. Retention of drug in prototype topotecan liposomes during storage.

<td>Salt forming</td><td>in</td><td>Size of</td><td>Initial charge</td><td>Time to</td><td>Charge of</td>

<td>gradient in</td><td>liposome</td><td>liposome,</td><td>of drug, mg</td><td>storage,</td><td>drug after</td>

<td></td><td></td><td>mean ± SD,</td><td>of drug /</td><td>months</td><td>storage</td>

<td></td><td></td><td>nm</td><td>mmolar of</td><td></td><td>as% of</td>

<td></td><td></td><td></td><td>phospholipid</td><td></td><td>initial</td>

<td>TEA-Pn</td><td> 0, 500</td><td>N</td><td>pH</td><td> 6,2</td><td> 96, 4</td><td> ±</td><td> 29,3</td><td> 148, 5</td><td> ±</td><td> 10,3</td><td> 8</td><td> 101, 6</td><td> ±5,5</td>

<td>TEA-Pn</td><td> 0, 500</td><td>N</td><td>pH</td><td> 6,2</td><td> 107,7</td><td> +</td><td> 19,1</td><td> 127,2</td><td> +</td><td> 10, 9</td><td> 6</td><td> 94,6 ±</td><td> 6,2</td>

<td>TEA-Pn</td><td> 0, 500</td><td>N</td><td>pH</td><td> 6,2</td><td> 107,7</td><td> ±</td><td> 19,1</td><td> 207,2</td><td> ±</td><td> 21,6</td><td> 6</td><td> 113, 9</td><td> ±9,4</td>

<td>TEA-Pn</td><td> 0, 500</td><td>N</td><td>pH</td><td> 6,2</td><td> 107,7</td><td> +</td><td> 19,1</td><td> 301, 3</td><td> +</td><td> 24,5</td><td> 6</td><td> 112, 9</td><td> ± 9,3</td>

<td>TEA-SOS</td><td> 0, 643</td><td colspan="2">: N pH</td><td> 5,6</td><td> 108,8</td><td> ±</td><td> 13, 4</td><td> 439, 2</td><td> ±</td><td> 15, 9</td><td> 2</td><td> 97,8 ±</td><td> 9,4</td>

<td>Example 26. Assimilation</td><td>in vitro</td><td>of liposomal topotecan and</td>

<td>immunoliposome by</td><td>cells</td><td>carcinogens with</td>

HER2 expression.

This study addressed the ability of topotecan-loaded antiHER2 immunoliposomes prepared according to the disclosure to specifically administer topotecan to HER2 overexpressed cells in cell culture. Immunos (liposomes) were prepared and loaded with topotecan using the TEA-Pn method of example 19. HER-2 overexpressed human breast carcinoma cells (SKBr-3, ATCC) were grown to confluence in modified McCoy's 5A medium (without tricine) supplemented with 10% fetal bovine serum, 50 pg / ml streptomycin sulfate and 50 U / ml penicillin G (complete growth medium) in T-75 flasks at 37 ° C, 5% CO2. Cells were harvested by trypsinization, inoculated into 24-well cell culture plates at 150,000 cells / well in 0.5 ml complete growth medium, and allowed to acclimate overnight. The medium was replaced with 0.5 ml complete growth medium containing the topotecan formulations at the concentration chosen in the range 0.01-0.1 mM phospholipid. For

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121 In each condition triplicate wells were used. Control wells were incubated in the absence of drug and / or liposomes (to obtain baseline drug test readings). The plates were incubated with slow shaking at 37 ° C, 5% CO 2 for 4-8 hours. Media was aspirated and cells were washed 4 times with 1 ml portions of cold Hank's balanced saline containing Ca and Mg salts. Cells were solubilized by the addition of 0.1 ml of 1% Triton X-100 in water, and the amount of drug in the cell lysates was determined by fluorometry (Example 71). The standard curve was obtained in the range 10-2500 ng topotecan / well, and adjusted to the second order polynomial (to account for self-extinction at a higher drug concentration) after subtracting the basal cell autofluorescence. When a microplate fluorimeter was used, the filter selection was 400/30 nm for excitation and 530/25 nm for emission. The cuvette and microplate fluorometers gave the same results.

The results of two experiments are summarized in table 20 below. There was prominent cellular uptake of the HER2-directed liposomal drug (50-300 times greater than that of the non-targeted liposomal topotecan). Interestingly, uptake of free topotecan was also significantly lower than that of HER2-directed immunoliposome topotecan. This can be explained by the rapid hydrolysis of the camptothecin lactone ring of topotecan molar molecule in the cell growth medium in the presence of serum, generating the carboxylate form of the drug, which may have lower cell permeability and lower cytotoxicity. In summary, the ability of immunoliposomes has been confirmed.

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122 internalized ligand conjugates directed to cells to administer topotecan intracellularly.

Table 20. In vitro cellular assimilation of liposomes from

<td>topotecan and anti-HER2 immunoliposomes containing</td><td>TEA-Pn</td><td>(na</td>

<td>not determined). See table 12</td><td>for</td><td>at</td>

features of the liposome.

<td rowspan="2">Liposome concentration, mM of phospholipid</td><td rowspan="2">Topotecan concentration, pg / ml</td><td rowspan="2">Time to exposure, hours</td><td colspan="3">Topotecan assimilation by SK-Br-3 cells, ng / 100,000 cells</td>

<td>Non-Targeted Liposomes</td><td>Immunoliposomes -F5</td><td>Drug free</td>

<td> 0,1</td><td> 15, 5</td><td> 4</td><td> 1,45 ± 0,09</td><td> 163 ± 5,7</td><td>na</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>na</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. Cytotoxicity of liposomal and immunoliposomal topotecan against HER2 overexpressed cancer cells in vitro.

Once the ability of anti-HER2 topotecan immunoliposomes to administer intracellular drug to HER2 overexpressed cancer cells (Example 26) was established, it was important to ensure that internalized liposomes can release the drug in its active form. To this end, the in vitro cytotoxicity of free topotecan (i.e., topotecan formulated as one of liposomal topotecan and topotecan) was formulated. Liposomal topotecan formulations were prepared, and SKBr-3 cells were grown and harvested as described in Example 26. Cells were inoculated, in triplicate, into 96-well cell culture plates at 5,000 cells in 0.1 ml of complete growth medium, and allowed to acclimate overnight. The rows and columns of the plate ends were anti-HER2 immunoliposome solution left empty. Sterile preparations were diluted with

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123 topotecan liposomes, immunoliposomes or free drug (freshly prepared by diluting the stock of topotecan 20 mg / ml, pH 3, in 2 mg / ml non-buffered saline) with complete drug medium to reach concentrations from 90, 30 or 10 pg / ml and diluted serially in the medium by a factor of 3. Medium in the wells was replaced with 0.2 ml drug / liposome dilutions, and incubated at 37 ° C, 5% CO2 for the specified time (4-6 hours). One well in each row was incubated with drug-free medium to serve as an untreated control. Drug-containing media was aspirated from the wells, cells were washed with 0.2 ml of drug-free medium, and 0.2 ml of drug-free fresh medium was added to all wells. The plates were incubated for 4 days at 37 ° C, 5% CO 2. Without changing the medium, 0.03 ml of 2 mg / ml solution of a tetrazolium dye (thiazolyl blue, MTT) (Sigma Chemical Co.) in serum-free medium was added to each well. The plates were incubated for a further 2-3 hours at 37 ° C, 5% CO 2. The media was aspirated and the wells were filled with 0.2 ml 70% by volume aqueous isopropanol, 0.075 N HCl, and gently shaken until formazan dye dissolved (15-30 min). The optical density of the formazan solutions was determined using a 540 nm microplate photometer. Cell viability, as% of untreated control, was calculated as the ratio between the optical density in the experimental wells and the base-corrected optical density in the wells containing untreated cells. Data were plotted against drug concentration, and the IC50 dose was estimated graphically from the intersection of the viability-concentration curve with the 50% viability line.

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The results are shown in Figure 9. The drug dose resulting in 50% growth inhibition (ICso) for free topotecan or undirected liposomal topotecan was above 30 pg / ml; for F5 immunoliposomal topotecan, 0.15 pg / ml. These results are consistent with targeted drug assimilation data.

Example 28. Comparative and pharmacokinetic stability of plasma liposomal and immunoliposomal F-topotecan in mice.

Topotecan liposomes containing radioactive lipid marker [³Phospholipid H] -CHE at 1.5 mCi / mmole were prepared according to Examples 11 and 19 using a lipid solution and ethanol extrusion mixing procedure under the following conditions: gradient-forming salt solution: 0.643 N triethylammonium sucrose octasulfate; polycarbonate membrane extrusion: 15 passes through 2 stacked PCTE filters, pore size 80 nm; topotecan loading: 350 mg / mmolar drug / phospholipid entry ratio (calculated for topotecan free base); F5 scFv conjugation was performed as described in example 19. Liposomes had the following characteristics:

Size by QELS: average weight 101.2 nm; standard deviation, 20.1 nm.

Drug Encapsulation: Topotecan Liposomes (Topo-Ls) 359.3 ± 27.4 mg / mmolar phospholipid; topotecan F5scFv immunoliposomes (Topo-F5-ILs) 326.3 ± 15.9 mg / mmolar phospholipid.

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125 The study was generally performed as in Example 22. Groups of nine male Swiss Webster mice (8-10 weeks, 24-27 g) were injected into the tail vein with Topo-Ls, TopoF5ILs or freshly prepared topotecan at 1 mg. / ml in unbuffered saline at a dose of 5 mg base topotecan per kg body weight (equivalent to 14-16 pmolar phospholipid lipid dose / kg body weight). At 1 hour, 8 hours or 24 hours after injection, 3 animals were bled for each time point by cardiac puncture under ketamine / xylazine anesthesia, blood was collected in tubes containing PBS-EDTA, and tested for topotecan (fluorometry). ) and liposome lipid (by scintillation radioactivity counting). The amounts of drug and lipid dose remaining in the blood at certain time points were calculated by the administered dose taken as 100%, assuming the amount of blood per animal as 6.3% of body weight, and the cell fraction. 45% blood pressure. The amount of drug that remains encapsulated in the liposomes at each time point was calculated for each individual animal by comparing the drug / lipid radioactivity ratio of the plasma samples to that of the injected liposomes. The amount of free topotecan in plasma samples collected 1 hour after injection was less than 1% of the injected dose (in fact, it was below the detection limit of our test method); therefore, no time points of the free topotecan group were studied. Due to the rapid clearance of blood and the low blood levels of free topotecan, we assume that virtually all topotecan found in the blood at all time points represents liposome-encapsulated topotecan.

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The results are summarized in table 21 below. Notably, the liposomes prepared according to the disclosure retained 79-85% of the original drug load, even 24 hours after injection, into the animals' bloodstream. The differences between the mean lipid or drug plasma values ​​between the liposome and immunoliposome groups were in the range of 1.8-13.6%, and were close to or within the range of the assay error. The odds of the null hypothesis between the liposome and immunoliposome group with respect to drug or lipid values ​​at each time point, calculated by Student's t-test, were in the range 0.543-0.938. We concluded that the differences in drug or lipid residual blood levels between the two preparations were insignificant and statistically indistinguishable.

<td>Table 21</td><td>Quantities of</td><td colspan="3">liposome lipid, topotecan</td>

<td colspan="5">and topotecan that remains encapsulated in liposomes</td>

<td>in plasma</td><td>of mice in</td><td>several</td><td>points</td><td>of time after</td>

<td>injection i.</td><td>v.</td><td></td><td></td><td></td>

<td>Time after</td><td>to Lipid,% of</td><td>Drug,</td><td colspan="2">% of Drug / Lipid,% of</td>

<td>injection</td><td>injected dose</td><td colspan="3">injected dose pre-injection value</td>

<td colspan="2">Liposomal Topotecan</td><td>conjured</td><td>with F5</td><td>(Topo-F5ILs):</td>

<td>1 hour</td><td> 57,58 ± 4,95</td><td> 55,45 ±</td><td> 7,23</td><td> 96,14 ± 7,32</td>

<td>8 hours</td><td> 35,37 ± 3,84</td><td> 34,18 ±</td><td> 5, 87</td><td> 96,31 ± 11,92</td>

<td>24 hours</td><td> 15,51 ± 11,84</td><td> 12,30 ±</td><td> 9, 02</td><td> 79,36 ± 8,03</td>

<td colspan="4">Liposomal Topotecan (unconjugated)</td><td>(Topo-Ls):</td>

<td>1 hour</td><td> 58,88 ± 9,51</td><td> 57,63 ±</td><td> 9, 45</td><td> 97,90 ± 5,29</td>

<td>8 hours</td><td> 39, 61 ± 1,99</td><td> 38,82 ±</td><td> 1,49</td><td> 98,06 ± 4,44</td>

<td>24 hours</td><td> 15,84 ± 3,85</td><td> 13,45 ±</td><td> 2, 64</td><td> 85,25 ± 7,03</td>

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127

Example 29. Antitumor efficacy of anti-HER2 liposomal and immunoliposomal topotecan in the BT-474 xenograft model.

In this study, we used the first topotecan immunoliposome prototypes that use the triethylammonium polyphosphate gradient for drug entrapment. Liposomes were generally prepared following the methods of examples 11 and 19. The lipid matrix components - DSPC (Avanti Polar Lipids; 3 molar parts.), Cholesterol (Calbiochem, 98.3%; 2 molar parts.) And methoxyPEG (2000) -DSPE (Avanti Polar Lipids, 0.015 molar parts.) were combined with 100% USP ethanol to produce the solution containing 0.5 mM phospholipid at 60 ° C. The lipid ethanol solution was diluted at 60 ° C with the aqueous triethylammonium polyphosphate solution (0.608 M triethylamine, 0.65 N phosphate, pH 6.1, 531 mmolar osmolarity / kg), mixed thoroughly, and extruded. 10 times through 2 stacked 100 nm pore size polycarbonate membranes (Nuclepore, Corning) using a thermoregulated gas pressure extruder (Lipex Biomembranes) at 60 ° C. The extruded liposomes were chilled on ice, and unencapsulated triethylammonium polyphosphate was removed by gel chromatography on Sepharose CL-4B using 5% dextrose-5 mM HEPES-Na buffer, pH 6.5 as eluent. Liposome size was 103.8 ± 35.1 nm by QELS. The liposomes in this buffer were incubated with topotecan hydrochloride at 60 ° C for 30 min., In the ratio of 0.35 mg topotecan base per pmolar phospholipid. At the end of incubation, the liposomes were chilled on ice and chromatographed on Sephadex G-75, 20 mM HEPESNa eluent, 135 mM NaCl, pH 6.5, to remove any unencapsulated drug. The drug content was determined by fluorometry, and the lipid content by phosphate assay as

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128 previously reported. The liposomal topotecan thus obtained has 365.4 ± 23.1 mg of topotecan base per mmole phospholipid. To prepare HER2-directed topotecan immunoliposomes, a portion of this liposomal topotecan preparation was incubated with the purified scFv F5 anti-HER2 conjugate and maleimido-PEGDSPE binder in general as described in Example 19. Briefly, the F5-PEG-DSPE conjugate in 10% aqueous solution of 10 mM Na citrate sucrose, pH 6.5, was combined with topotecan liposomes in the ratio of 15 mg protein per mmole liposome phospholipid , and incubated at 60 ° C for 30 min. The incubation mixture was ice-cooled and chromatographed on Sepharose CL-4B, 20 mM HEPES-Na eluent, 135 mM NaCl, pH 6.5, to remove any unincorporated scFv conjugate. The drug-paralipid ratio decreased by 14% after this additional incubation.

Topotecan and immunoliposome liposome formulations containing 1-2 mg / ml topotecan were passed through a 0.2 micron sterile syringe filter, dispensed into polypropylene vials and stored at 4-6 ° C for up to 1 month prior. of use.

Free topotecan was freshly prepared by dissolving powdered topotecan hydrochloride at 2 mg / ml in 5% dextrose, and sterilized by passing through a 0.2 micron syringe filter.

A HER2 overexpressed human breast adenocarcinoma xenograft model was established as described in example 10. On day 13 after tumor inoculation, animals with tumors in the 120350 mm cubic range were selected and divided. randomly

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129 in 3 treatment groups and 1 control group each with 12 animals. On days 14, 18 and 21 post tumor inoculation, mice were treated with iv (tail vein) injections of topotecan formulations, either by injection of 5 mg / kg body weight or with an equal volume of saline. physiological. The general health status of the animals was monitored daily. 0 Tumor size and body weights were monitored twice weekly until day 53 after tumor inoculation. Animals whose tumors reached 20% of body weight, or those with progressive weight loss of 20% or more were sacrificed.

Figures 11 and 12 show, respectively, tumor growth and animal body weight data. Liposomal topotecan formulations were more active in suppressing tumor growth than free drug, and F5-directed liposomal formulation was more active than non-targeted. Mean tumor size at the end of the observation period was significantly different between treatment groups (p-values ​​by the 2-tailed unpaired Student's t-test were 1.2 x 10 “⁶ for free drug v. immunoliposomal drug, 0.000114 for free drug v. liposomal drug, and 0.00718 for liposomal drug v. immunoliposomal drug). Thus, liposome-encapsulated topotecan was more active than free drug, and anti-HER2 immunoliposomal topotecan was more active than non-targeted liposomal drug. In the liposomal and immunoliposomal group, after an initial regression, the tumor recurred within 10 days of the last treatment. There was no tumor regression in the free drug group. Topotecan liposome formulations at a given dose were found to be more toxic than

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130 free drug. There was gastrointestinal toxicity. Animals receiving liposomal topotecan developed diarrhea and experienced average body weight loss of about 14% at its peak. While in the non-targeted liposomal group the animals recovered except one (12.5%) who had persistent weight loss of 15% at the end of the study, in the F5-targeted group five animals (41.6%) developed terminal morbidity. and died, and two others (16.7%) had persistent weight loss of about 15%. In the control group and the free drug group there was no weight loss or treatment-related morbidity.

Example 30. Maximum tolerated dose (DMT) of free and liposomal topotecan in mice receiving 3 weekly iv injections.

This study used a liposome topotecan formulation prepared as in Example 29, except that the triethylammonium polyphosphate solution was replaced with the triethylammonium sucrose octasulfate solution having 0.65 M triethylammonium, pH 6.2; and for extrusion 80 nm polycarbonate membrane filters were used instead of 100 nm. The volume-weighted liposome size determined by the Gaussian approximation elastic scattering light (QELS) method was 95.1 ± 19.6 nm (mean ± SD); The drug / lipid ratio was 369.1 ± 18.3 mg / mmolar phospholipid. Female Swiss-Webster mice at five to six weeks (18-20 g) in groups of two received three iv injections. free or liposomal topotecan (tail vein) on a once weekly basis from the dose of 2 mg / kg base topotecan per injection and increasing for each subsequent group by a factor of 1.8 to the dose of 37.8 mg / kg. The topotecan

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131 Immunoliposome was not included in this study. Animal body weight and general health were monitored daily. Progressive weight loss of more than 20% or natural death at any time in either animal in a group within ten days of initiation of treatment was considered indicative of the toxic dose. According to animal mortality and weight data, DMT was determined within the range of 11.7-21 mg / kg for free topotecan and 2.0-3.6 mg / kg for liposomal topotecan (prototype 2) . In the second study, mice received injections of free, liposomal or F-immunoliposome topotecan (prepared from the liposomal topotecan of this example as described in example 29) at doses of 2.0 mg / kg liposomal / immunoliposome) or 12 mg / kg kg (topotecan (free topotecan), which were increased for each subsequent group

<td>from 1.15,</td><td>up until</td><td>what</td><td>was</td><td>achieved</td><td>the next dose</td>

<td>higher</td><td>of</td><td colspan="2">interval</td><td>from DMT</td><td>established. THE</td>

<td>high</td><td>what</td><td>no</td><td colspan="2">resulted in</td><td>mortality or</td>

was terminal morbidity in any of the animals considered a DMT and was found to be 18.4 mg / kg for free topotecan, 3.0 mg / kg for liposomal and immunoliposomal topotecan

3.0 mg / kg for topotecan Thus, liposomal topotecan showed higher toxicity than free drug

Example 31. Antitumor efficacy of liposomal topotecan in the BT-474 xenograft model in the range 0.1251, OxDMT

Example 30 topotecan liposomes and F5 immunoliposomes were used in this study. BT-474 subcutaneous xenografts were bred in

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132 nude mice as in example 29. On day 18 following tumor cell inoculation, the tumor animals (105-345 cubic mm, mean about 200 cubic mm) were randomized into treatment groups of 6 animals / group, and one 8 animals control group / group. Animals received free or liposomal topotecan in lxDMT, 0.5xDMT, 0.25xDMT or 0.125xDMT in three iv (tail vein) injections on days 19, 23 and 27 post tumor inoculation. The control group received injections of physiological saline. Tumor size and animal body weight were monitored as in the example

29 To obtain measurements of animal body weight, tumor weight (calculated from tumor size assuming tumor density as 1.0) was subtracted from measurements of total animal weight. All drug formulations in DMT had antitumor activity (Figures 13A13D). There was no significant difference in efficacy between the free and liposomal drug administered in its respective DMT or in identical fractions (1/2, 1/4 or 1/8) thereof. Thus, drug encapsulation in liposomes using the TEA-SOS gradient resulted in an approximately 6-fold increase in antitumor activity, but also a similar increase in drug toxicity. The dynamics of the animals' body weight revealed that all treatments were non-toxic except free topotecan treatment in DMT, which showed a temporary decrease in body weight (about 15% of pretreatment value) that later resolved (Figure 14).

Example 32. Preparation and in vitro directed cytotoxicity of topotecan liposomes prepared using the triethylammonium sucrose octasulfate entrapment method.

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133 Liposomal topotecan was generally prepared following the procedure of example 18 using the entrapped TEA-SOS solution having 643 mM TEA, pH 5.7, osmolarality of 530 mmolar / kg, and 170 mg drug / phospholipid ratio / mmolar. Liposomes had 155 mg of drug / mmole phospholipid, 90% charge efficiency and 105 nm particle size. These liposomes were incubated with the F5-PEGDSPE conjugate micellar solution at about 30 scFv per liposome (15 mg antibody / mmole phospholipid) at 60 ° C for 1 hour,

<td>generally,</td><td>how</td><td>described</td><td>at the</td><td>Example 19.</td><td>The</td><td>liposomes</td>

<td>conjugates</td><td>with</td><td>antibody</td><td colspan="2">were separated</td><td>per</td><td>SEC using</td>

<td>Sepharose</td><td>CL-4B</td><td colspan="2">and formulated</td><td>at HBS- 6.5</td><td colspan="2">saline solution</td>

<td>buffered</td><td>with</td><td>HEPES.</td><td>No</td><td colspan="2">there was any</td><td>change</td>

detectable drug / lipid ratio during anti-HER2 (F5) scFv binding.

The uptake of topotecan formulations by cancer cells was determined as follows. HER2-overexpressed human breast adenocarcinoma cells (SK-Br-3, ATCC HTB-30) were plated in 24-well cell culture plates at 150,000 cells / well, and allowed to acclimate overnight. Cells were incubated (in triplicate) with F5-directed and non-directed liposomal topotecan in complete growth medium at 0.1 mM and 0.01 mM liposome concentrations for 4 hours at 37 ° C. The cells were washed 4 times with Hank's Ebalanced Saline, solubilized in 0.1% Triton X-100 - 70% 1:10 acidified isopropanol mixture, and the amount of cell-associated topotecan per well was determined by fluorometry. . Results (mean ± standard error) are summarized in table 22. Liposomes

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134 targeted drugs administered 100-300 times more drug to targeted cells than non-targeted liposomes.

Table 22. Assimilation of liposomal topotecan by SK-Br-3 breast carcinoma cells.

<td>Formulation</td><td>Assimilation of 0.1mM phospholipid topotecan, ng / well</td><td>Uptake of 0.01 mM phospholipid topotecan, ng / well</td>

<td>Non-Targeted Liposome</td><td> 4,76 ± 0,24</td><td> 0,607 ± 0,088</td>

<td>HER2 targeted liposome</td><td> 533,8 ± 13,7</td><td> 197,0 ±4,6</td>

<td>Reason: Targeted / Untargeted</td><td> 112,1 ± 8,6</td><td> 324 ± 55</td>

The cytotoxicity of these topotecan formulations against SKBr-3 breast cancer cells was determined as described in Example 27. SKBr-3 cells were inoculated into 96-well plates at 5,000 cells / well, allowed to acclimate overnight, and incubated with increasing concentrations (0.004-30 pg / ml) of free, liposomal or immunoliposome-F5 topotecan in cell growth medium for 4 hours at 37 ° C. Drug-containing medium was removed and cells were allowed to grow in drug-free medium for 72 hours. The amount of viable cells per well was determined using the thiazolyl blue tetrazolium (MTT) assay, and expressed as% control (untreated) cells. The results are shown in Figure 10. Topotecan immunoliposomes were more cytotoxic (ICso 0.15-0.5 pg / ml) than

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135 untargeted topotecan liposomes (ICso at 3.1 pg / ml) and free topotecan liposomes (ICso at 2.3 pg / ml).

Example 33. In vivo stability of topotecan liposomes of different size.

Lip ossomas containing TEA-Pn were prepared as in Example 22 using extrusion 12 times through pore size polycarbonate membranes of 100 nm or additionally 12 times through 50 nm pore size polycarbonate membranes. Topotecan (TPT) was added at a ratio of 150 pg / pmolar phospholipid. Charging was completed at 58 ° C for 45 minutes in a hot water bath, followed by extinction on ice. The loading efficiency for the extruded liposome at 50 nm and 100 nm was respectively 126, 80 ± 19.24 pg TPT / pmolar PL (84.5 ± 12.8%) and 148.48 ± 10.26. pg TPT / pmolar PL (99.0 ± 6.8%). Female Swiss Webster mice, in groups of three,

<td>were injected intravenously</td><td>with</td><td colspan="2">an</td><td colspan="2">of the two</td>

<td>Ls-TPT formulations at a dose of</td><td> 5</td><td>mg</td><td>in</td><td>TPT / kg.</td><td>The</td>

<td>mice were sacrificed after 6</td><td>H</td><td>and</td><td>O</td><td>blood</td><td>was</td>

<td colspan="2">collected. Plasma was analyzed for</td><td>TPT</td><td>and</td><td>the lipid</td><td>of</td>

<td>liposome as described in the example presented in table 23.</td><td> 22.</td><td>The</td><td colspan="2">results</td><td>are</td>

<td>Table 23. In vivo stability of the</td><td>Ls-</td><td>TPT</td><td>in</td><td colspan="2">many different</td>

<td>sizes loaded using the method</td><td>in</td><td colspan="3">imprisonment</td><td>in</td>

TEA-Pn.

<td>Size of</td><td>Drug in</td><td>Lipid's</td><td></td><td>Reason for</td>

<td>liposome,</td><td>nmplasma,% of</td><td>liposome</td><td>at the</td><td>drug / lipid,%</td>

<td></td><td>injected dose</td><td>plasma,%</td><td>gives</td><td>of the value of the</td>

<td></td><td></td><td colspan="2">injected dose</td><td>injection</td>

74,2 ± 21,6 32,93 ± 1,97 45,7 ± 2,2 72,06 ± 5,51

96,4 ± 29, 3 33,26 ± 3,56 37, 6 ± 5,3 88,41 ± 15,68

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Example 34. Synthesis and liposome encapsulation of 6- (3aminopropyl) elipticine (6-APE).

6- (3-Aminopropyl) elipticine was prepared from ellipticine in a two-step method based on the procedure of Werbel et al., J. Med. Chem. 1986, v. 29, p. 1321-1322. 501.4 mg ellipticine base (NSC 71795) (Aldrich Chemical Co.) was stirred with approximately 100 mg sodium hydride (Sigma; washed with anhydrous petroleum ether) in 5 ml dry dimethylformamide (DMF) at room temperature. room for 30 min. To this mixture was added dropwise a solution of 678 mg of N-bromopropylphthalimide (Aldrich) in 2 ml of dry DMF. The purple reaction mixture was stirred under argon overnight, treated with 1 ml water, and poured into 60 ml water. The mixture was extracted twice with 25 ml of methylene chloride, the extract was dried over anhydrous sodium sulfate and passed through a neutral alumina layer. The alumina layer was washed twice with 10 ml of methylene chloride, and the filtrate and the combined washes were brought to dryness in vacuo. The product was stirred overnight with 20 ml absolute ethanol and 2 ml anhydrous hydrazine at room temperature. The obtained aqueous slurry was vacuum filtered, a 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 brought to dryness in vacuo. The crude product (408 mg yield) was chromatographed on a silica 60 column eluted isocratically with the chloroform-methanol mixture (7: 3, by volume), saturated with dry trimethylamine. Fractions which were eluted in a second yellow band following the unreacted ellipticine were shown to contain the desired compound in approximately 30% yield. THE

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137 structure was confirmed by ¹1 H NMR. TLC: Rf 0.29-0.31 (silica 60; CHCl 3 -MeOH 7: 3, by volume, saturated with trimethylamine). Ellipticine, Rf 0.81-0.83. The obtained compound was converted to the dihydrochloride salt by dissolving in anhydrous ethanol and titrating with 6 N HCl solution in dry isopropanol. The orange crystals of 6-APE dihydrochloride (NSC 176328) were filtered, washed with ether and dried in vacuo. Dihydrochloride yield 86%.

Liposomes were prepared by hydrating the pure lipid film of DSPC, cholesterol and PEG (MW 2,000) -DSPE (3: 2: 0.015 molar ratio) in a solution of trimethylammonium polyphosphate (TMA-Pn) in 0.5 M of TMA, pH 5.6, at 60 ° C, followed by six cycles of rapid freezing (78 ° C) and thawing (60 ° C), and extruding ten times through two 50 nm pore size stacked polycarbonate filters . Unencapsulated TMA-Pn was removed using a HEPESdextrose-eluted Sepharose CL-4B column (5 mM HEPES, 5% dextrose, pH 5.5). The liposome size was 85.7 ± 32.1 nm.

Concentrated 6-APE solution (10 mg / ml) was added to TMA-Pn-containing liposomes at a drug-paraphospholipid ratio of 100 pg phospholipid APE / pmolar, the mixture was incubated at 58 ° C for 45 min. and cooled rapidly on ice for 15 min. Unencapsulated drug was removed by gel chromatography on a Sephadex G-75 column eluted with HEPES-dextrose buffer (5 mM HEPES-Na, 5% dextrose, pH 6.5). The liposome entrapped APE was then quantified by spectrophotometry as in example 71, and the liposome phospholipid was determined using the extraction assay.

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138 Example 70. The drug encapsulation was practically quantitative.

Example

35. Immunoliposome 6-EPA preparation

<td>directed to HER2</td><td>and</td><td>cytotoxicity</td><td>of</td><td>formulations of</td>

<td>6-APE against cells</td><td>in</td><td>breast cancer</td><td>BT-</td><td>474 with over-</td>

HER2 expression in vitro.

Encapsulated 6-APE liposomes (Ls-APE) were prepared as in example 34 above. Anti-encapsulated 6-APE-HER2 immunoliposomes (F5-ILs-APE) were prepared from Ls-APE by the method of example 19. An MTT-based cell viability assay from example 27 was used to determine the cytotoxicity of 6-APE administered as a solution, as Ls-APE or as HER2-directed F5-ILsAPE against HER2 overexpressed human breast carcinoma cells (BT-474). Cells were exposed to drug containing medium for 6 hours, and postincubated in drug free medium for 3 days. The results are shown in Figure 15. The IC50 for free APE was 0.26 pg APE / ml, for F5-ILs-APE was 0.756 pg APE / ml and for non-directed Ls-APE 51.0 pg APE / ml. There was a 67.5-fold difference in activity between targeted and non-directed liposomal 6-APE, indicating a considerable effect of targeted administration.

Example 36. EGFR-targeted immunoliposomal 6-APE formulations and cytotoxicity against cancer cells in vitro.

6-APE loaded liposomes were prepared as described in example 34. EGFR-directed immunoliposomes were prepared by ligating EGFR-specific Fab 'antibody fragments as follows. IgG MAb C225 (cetuximab, ERBITUX ™,

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139

EGFR-specific ImClone Systems) was digested with pepsin to produce (Fab ') 2 fragments. Purified (Fab ') 2 fragments were reduced by treatment with 10-20 mM 2-mercaptoethylamine for 15 min. at 37 ° C, and Fab 'fragments were purified by gel filtration using Sephadex G-25. The presence of reactive thiol groups was usually about 0.9 thiol groups per protein molarecule (guantified using Ellmann's reagent). C225Fab ​​'were covalently conjugated to a Mal-PEG-DSPE amphiphilic binder (Avanti Polar Lipids, AL) in aqueous solution at pH 6.2-6.5 and protein-binder molar ratio of 1: 4 for 2-4 hours at room temperature, or overnight at 4-6 ° C, to produce the C225Fab'-PEG-DSPE conjugate in 30-50% protein yield. This micelle-forming conjugate was separated from unreacted protein by size exclusion column chromatography on 3% agarose - 4% polyacrylamide microsphere gel (Ultrogel AcA34, obtained from Sigma Chemical Co.), eluted with buffer HBS-6.5. The conjugate was recovered in dead volume fractions. Immunoliposomal 6-APE was formed by incubating these liposomes with C225 Fab'-PEG-DSPE with drug-loaded liposomes in the ratio of 30 mg liposome phospholipid C225 protein / mmolar for 30 min. at 60 ° C, extinction on ice for 15 min., and purification of immunoliposomes by gel chromatography on a Sepharose CL-4B column also eluted with HBS-6.5 buffer (liposomes appear in or near column dead volume).

MDA-MB-468 EGFR overexpressed human breast cancer cells and MCFR-7 poorly expressed EGFR human breast cancer cells (ATCC, Rockville, MD) were grown in their growth medium recommended by the

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140 anti-EGFR free, liposomal and immunoliposomal 6-APE cytotoxicity against these cells were studied according to the method of Example 27. The cells were incubated with drug-containing medium for 6 hours, followed by 3 days. post incubation in drug free medium. The results are shown in Figure 16. In MDA-MB-468 cells, the ICso for free 6-APE was about 0.1 pg / ml and for C225-ILs-APE was about 0.9 pg / ml . In MCF-7 cells, ICso was about 0.1 for free 6-APE was about 0.5 pg / ml and for C225-ILs-APE was about 14 pg / ml. Lso-APE ICso in both cell lines was&gt; 30 pg / ml. Thus, EGFR-directed 6-APE-loaded immunoliposomes demonstrated antigen-specific cytotoxic activity in EGFR overexpressed MDA-MB-468 breast cancer cells, but not in MCF-7 breast cancer cells that do not overexpress EGFR. In MCF-7 cells, targeted and non-directed 6-APE liposomes were equally active.

Example 37. Pharmacokinetics of liposomal 6-APE in rats. Liposomes with entrapped TEA-Pn solution (557 mM phosphate groups, 500 mM TEA, pH 5.8, 480 mmolar osmolarity / kg) and lipid composition of DSPC, cholesterol and PEG-DSPE (3 molar ratio : 2: 0.015) were prepared as in example 11 above. The ethanolic lipid solution was combined at 60 ° C with 10 volumes of the aqueous TEA-Pn solution, and extruded ten times through two stacked 80 nm pore size polycarbonate membranes. Unencapsulated TEA-Pn was removed using a MES-dextrose eluted Sepharose CL-4B column (5 mM MESNa, 5% dextrose, pH 5.5). Liposome size was 92.3 ± 23.3 nm by QELS. A non-exchangeable radioactive lipid marker [³H] -CHE was included in the lipid matrix a

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0.5 mCi / mmolar phospholipid. Liposomes were loaded with 6-APE as described in example 34.

The pharmacokinetic study followed the protocol of example 9. Female Sim Albino mice (9 weeks, 200 g) were injected iv with a 10 mg dose of 6-APE / kg. Blood was collected at the chosen time points, and plasma was analyzed for 6-APE by fluorometry. Plasma aliquots (0.05-0.2 ml) were mixed with 1-2 ml 90% aqueous isopropanol-0.1 N HCl, and 6-APE was quantitated by fluorescence as in Example 71. Lipid was quantified by scintillation radioactivity counting of [³H] -CHE.

The results are shown in Figure 17. The blood half-life of the drug (ti / 2) was 13.7 hours and the liposome lipid was 16.6 hours (panel A). The half-life of liposome drug release was 77.9 hours, demonstrating remarkable encapsulation stability (panel B).

Example 38. Synthesis and liposomal encapsulation of 2- (2. (N, N-diethylamino) ethyl) ellipticinium (2-DAE).

2- (2- (N, N-Diethylamino) ethyl ellipticinium chloride (NSC 359449) is an anticancer derivative of ellipticine, which is prepared by alkylation of ellipticine with 2- (N, Ndiethylamino) ethylchloride in methanol in the presence of triethylamine (see Werbel, LM, Angelo, M., Fry, DM, and Worth, DFJ Med. Chem. 1986, 29: 1321-1322.) The entrapped TEA-Pn-containing liposomes were prepared as described in Example 37. 2-DAE.2HCl was incubated with TEA-Pn liposomes in 5 mM HEPES-Na, 5% dextrose, pH 7.4, at a ratio of 2-DAE-to-phospholipid.

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100 pg / pmolar. The amount of drug loaded was 88.2 pg APE / pmolar PL (efficiency 88.2%).

Example 39. Pharmacokinetics of liposomal 2-DAE in rats. The pharmacokinetics of liposomal 2-DAE (Example 38) in rat blood were studied, as in Example 37. The 2 / DAE ti / 2 was 17.8 h and the liposome lipid matrix was 18.2 h (A ). The half-life of drug release of liposomes in blood was t1 / 2 = 677 h (B). Thus, these liposomes were extremely stable against drug leakage into the bloodstream.

Example 40. Loading of vinorelbine into liposomes using the TEA-Pn method. The effect of pH.

Liposomes were prepared by the ethanol injection method, as in example 11, using a 0.608 M TEAPn solution of TEA, 0.65 M phosphate groups, pH 6.1 and 531 mmolar / kg osmolarity, and extrusion of the liposomes. lipid suspension 15 times across two stacked 100 nm pore size polycarbonate membranes. The resulting liposome size was 108.3 ± 17.1 nm by QELS. Vinorelbine (VRB), as a 10 mg / ml USP vinorelbine bitartrate solution, was added to the liposomes in 5 mM aqueous HEPES-Na, 5% dextrose, pH 6.5, at a ratio of 350 µg / pmolar drug-to-phospholipid, the pH was adjusted to the desired value using 1-5 N NaOH, and the mixture was incubated at 58 ± 2 ° C for 30 min. The mixture was then ice-cooled for 15 minutes, and the unencapsulated drug was removed by Sephadex G75 gel filtration chromatography, eluting with HBS-6.5 buffer (20 mM HEPES-Na, 135 mM NaCl, pH 6.5 ). Aliquots of purified liposomes were then solubilized in acid isopropanol and

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143 analyzed for vinorelbine using spectrophotometry at

270 nm. Liposome phospholipid was quantified using Bartlett's phosphate assay (1959) after extraction of methanol-chloroform.

The calculated drug-to-lipid ratios after loading were as shown in Table 24. Vinorelbine loading was quantitative (ie, practically 100%) and independent of pH in the range studied.

Table 24. Loading of vinorelbine into TEA-Pn liposomes trapped at various pH values ​​of external buffer

<td>pH</td><td>Drug-paraphospholipid ratio (pg / molar)</td><td>Charging 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> 355,2 ± 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>

<td>Example</td><td>41. Liposomal vinorelbine</td><td>prepared by the method</td>

<td>of TEA-</td><td colspan="2">Pn at various drug / lipid ratios: efficiency of</td>

encapsulation and stability in vivo in mice.

Liposomes with entrapped TEA-Pn solution were prepared according to example 40, except that [³H] -CHE in the 1.5 mCi / mmolar lipid matrix of phospholipid. Liposome size was 98.5 ± 34.3 nm by QELS. Liposomes were mixed with USP vinorelbine bitartrate in 5 mM aqueous buffer of HEPES-Na, 5% dextrose, pH 6.5, in a drug-to-phospholipid ratio of 150-450 mg VRB / mmolar, and incubated. at 58 ± 2 ° C for 30 min. No pH adjustment was made after drug addition. The

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144 Vinorelbine-loaded liposomes (Ls-VRB) were isolated and analyzed for the drug and phospholipid as in example 40.

Female Swiss Webster mice (Harlan Bioresearch) five to six weeks in groups of three were injected intravenously with Ls-VRB-Pn at a dose of 5 mg VRB / kg. The dose of lipid varied according to the degree of loading and can be determined from the above drug-to-lipid ratios. At 8 hours or 24 hours after injection, animals were anesthetized, bled, and blood collected on ice in heavy tubes containing known amounts of PBS with 0.04% EDTA. Blood cells were separated by centrifugation, and supernatants were analyzed for liposome lipid by scintillation radioactivity counting [³H] -CHE and for vinorelbine using HPLC as follows. The samples were enriched with vinblastine (internal standard), extracted with diethyl ether, evaporated, and the residues were dissolved in the mobile phase consisting of 50 mM aqueous triethylammonium acetate (pH 5.5) and acetonitrile (58:42 in volume). Samples were loaded onto a Cys reverse phase silica column (Supelco C-18 column, 250 mm x 4 mm id, 5 pm particle size) preceded by a C-18 pre-column. The column was isocratically eluted with 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 respectively 9.1 min and 7.8 min.

The results are shown in table 25. Charge efficiency decreased with increasing drug / lipid ratio from

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145 approximately 100% at 150 mg / mmolar to about 66% at

450 mg / mmolar. The addition of vinorelbine bitartrate at ratios greater than 250 mg vinorelbine per mmole phospholipid was found to substantially acidify the liposome suspension (pH &lt;4.0), which led to a reduction in loading efficiency. Thus, the need for pH control was established during the drug loading step. The liposome matrix quantities detected in the blood after 8 hours were in the range of 30.4 ± 6.6% of the injected dose (% ID) to 38.6 ± 5.2% DI, with no apparent relationship to the absolute amount of injected lipid. After 24 hours there was still 6.4% DI to 14.8% DI of the detectable lipid matrix in the blood. The amount of drug that remained encapsulated after 8 hours ranged from 37% to 63%. However, 24 hours after injection, drug levels were below the detection limit of the analytical method used.

Table 25. In vivo drug encapsulation and retention efficiency of liposomal vinorelbine prepared at different drug / lipid ratios using the TEA-Pn method (no pH adjustment of loading buffer). Drug retention data are the mean ± SD (N = 3).

Vinorelbine / phospholipid ratio% drug that remains encapsulated 8 hours after injection

Input, mg / mmolar r

calculated

Output, mg / mm encapsulation efficiency,% r, measured

<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>

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146

<td> 450 299</td><td> 66, 4</td><td colspan="2"> 63, 0 ±</td><td> 4,1</td>

<td>Example 42.</td><td>Loading</td><td>gives</td><td>vinorelbine in</td><td>liposomes</td>

<td>using the</td><td>method of</td><td>TEA-</td><td>To many</td><td>reasons for</td>

<td>drug / lipid</td><td></td><td></td><td></td><td></td>

<td>Liposomes</td><td>from TEA-SOS</td><td>for</td><td>the shipment</td><td>of drug</td>

were prepared as in example 40, except that the 0.65 M TEA-SOS solution with TEA pH 5.4, 521 mmolar / kg osmolarity was used instead of the TEAPn solution, and the liposomes were extruded through 80 nm pore size polycarbonate membranes. Liposome size was 86.6 ± 12.9 nm by QELS. VRB was added to the liposomes in 5 mM aqueous HEPES-Na, 5% dextrose, pH 6.5, at various drug-paraphospholipid ratios, and the mixture was subsequently incubated at 60 ° C for 30 min. VRB-loaded liposomes were then isolated and analyzed as in example 40.

The calculated drug-to-lipid ratios in VRB liposomes are shown in Table 26. Notably, in contrast to assisted loading of the polymeric anion, the loading of vinorelbine on the polyanionized sugar (sucrose octasulfate) liposomes was virtually quantitative, regardless drug / lipid ratio up to 450 mg VRB / mmolar phospholipid, and only slightly lower (88%) to 550 mg VRB / mmolar phospholipid.

Table 26. Dependence of liposome loading of vinorelbine on drug-to-lipid ratio

Vinorelbine / phospholipid ratio, mg / mmolar

Charging Efficiency (%)

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Total Encapsulated in Liposomes

<td> 150</td><td> 159,</td><td> 9</td><td> ±</td><td> 11,5</td><td> 106, 6</td><td> ±</td><td> 8, 1</td>

<td> 250</td><td> 255</td><td></td><td> ±</td><td> 12,4</td><td> 102,2</td><td> ±</td><td> 5, 1</td>

<td> 350</td><td> 381,</td><td> 8</td><td> ±</td><td> 16, 3</td><td> 109, 1</td><td> ±</td><td> 5, 1</td>

<td> 450</td><td> 456,</td><td> 1</td><td> +</td><td> 29, 5</td><td> 101,4</td><td> +</td><td> 6, 6</td>

<td> 550</td><td> 486,</td><td> 2</td><td> ±</td><td> 26, 0</td><td> 88,4</td><td> ±</td><td> 4,2</td>

Example 43. Preparation of vinorelbine-loaded HER2-directed immunoliposomes by the TEAPn method, and comparative pharmacokinetics in the blood of HER2-directed and non-HER2-directed vinorelbine liposomes in rats.

scFv F5-PEG-DSPE anti-HER2 conjugate was prepared as in example 19. HER2-directed vinorelbine immunoliposomes were prepared by incubation of non-targeted vinorelbine liposomes (Example 41, in a drug / phospholipid ratio of 350 with F5-PEG-DSPE conjugate (Example 19) in 20 mM HEPES-Na aqueous buffer, 135 mM NaCl, pH 6.5, at 15 mg / mmole protein / phospholipid ratio at 60 ° C for 30 min. Unincorporated F5 conjugate was removed by gel chromatography on a Sepharose 4B column eluted with the same buffer. Untargeted (Ls-Pn-VRB) and HER2-directed liposomes (F5-ILs-Pn-VRB) were administered iv to albino female rats (8-9 weeks; 200 g) at a dose of 5 mg VRB / kg At various time points, blood was collected as described in example 9, and analyzed for VRB and liposome lipid as in example

<td> 41.</td><td>The half life</td><td>of lipids</td><td>of</td><td>liposome</td>

<td>in</td><td>release</td><td>50%</td><td>of</td><td>drug</td>

<td colspan="2">respectively</td><td>starting</td><td>From</td><td>graphics</td>

were calculated, either the time lipid concentration or the time drug / lipid ratio graphs found the best fit for the kinetics

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148 using the MICROSOFT spreadsheet

EXCEL (Microsoft Corp.), TREND function. The results (Figure

18) indicated that both targeted vinorelbine liposomes had identical lipid e, with lipid half-life of about 12.1 hours and 50% drug release time of about 4.3 hours.

non-pharmacokinetic drugs

Example 44. Preparation and comparative in vivo stability of vinorelbine liposomes prepared using ammonium and substituted ammonium salts.

Ammonium dextran sulfate solution (DS-A) pH 5.8, 0.65 M NHG, osmolarity 390 mmolar / kg, and triethylammonium dextran sulfate solution (DS-TEA) pH 6 0.65 M NHG, 465 mmolar / kg osmolarity were prepared from 10,000 molar dextran sulfate (Sigma Chemical Co.) according to the method of Example 4 using titration with, respectively, 12.4 M aqueous ammonia or pure triethylamine. The 325 mM aqueous ammonium sulfate (SA) solution, pH 5.1, 703 mmolar / kg osmolarity, was prepared from analytical grade ammonium sulfate. All three solutions contained less than 1% Na⁺ of the total cation content. Liposomes trapping these solutions were prepared using the ethanol-extrusion mixing method of example 41 (DSPC / Cholesterol / PEGDSPE, 3: 2: 0.015 molar ratio). Radioactive lipid marker [³H] -CHE in the 1.5 mCi / mmole lipid matrix of phospholipid. The extrusion step consisted of 10 passes through two stacked 0.1 pm polycarbonate membranes. VRB was added to the liposomes in 5 mM HEPES-Na, 5% dextrose, pH 6.5, at a drug-to-phospholipid ratio of 350 mg / mmolar,

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149 pH was adjusted to 6.5 using 1 N NaOH, and the mixture was incubated at 58-60 ° C for 30 min. The reaction was then cooled on ice for 15 min, and the unencapsulated drug was removed by Sephadex G-75 gel filtration chromatography, eluting with 20 mM aqueous HEPES-Na, 135 mM NaCl, pH 6.5. Purified vinorelbine-loaded liposomes were analyzed spectrophotometrically for VRB and phospholipid using Bartlett's phosphate assay (1959) (see Examples 70, 71). The pharmacokinetics of liposome lipid and blood drug in rats were studied, as in example 43.

Results are shown in Figures 19-20 and Table 27. Liposomes loaded with triethylammonium dextransulfate were compared with those loaded using dextran sulfate ammonium salt. Unexpectedly, those loaded using the triethylammonium salt were considerably more stable than those loaded using the ammonium salt. The pharmacokinetics of the liposomal vehicle itself were similar with the three different formulations, and was thus mainly dependent on the lipid composition employed. Vinorelbine leakage of LsVRB charged using triethylammonium dextran sulfate was about three times slower than those loaded using ammonium dextransulfate. Liposomes loaded using ammonium sulfate had the fastest drug leakage rate.

Table 27. Compared in vivo stability of liposome drug encapsulation using entrapped ammonium and substituted ammonium salt.

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Formulation, trapped salt

Liposome size, nm, mean ± half-life of lipid matrix in the

Time to release 50% of drug into the

<td>in liposomes</td><td>SD (for</td><td>QELS)</td><td>blood,</td><td colspan="2">blood hours,</td><td colspan="2">hours</td>

<td>DS-TEA</td><td> 120,8 ±</td><td> 28,5</td><td> 9,5 ±</td><td> 3,3</td><td> 66, 3</td><td> ±</td><td> 13,4</td>

<td>DS-A</td><td> 107,8 ±</td><td> 15,4</td><td> 11,2 ±</td><td> 0, 6</td><td> 22, 9</td><td> +</td><td> 1,7</td>

<td>SA</td><td> 114,5 ±</td><td> 15, 6</td><td> 10,7 ±</td><td> 0,2</td><td> 1,77</td><td> ±</td><td> 0,16</td>

Example 45. Preparation and in vivo stability of vinorelbine-loaded liposomes of various sizes.

Liposomes labeled with [³H] -CHE (1.5 mCi / mmolar phospholipid), with entrapped solution of triethylammonium sucrose octasulfate (0.65 M TEA, pH 6.4, 502 mmolar osmolarity / kg) were prepared by the mixture method of ethanol-extrusion of example 11. The extrusion step contained 15 passes through two stacked polycarbonate membranes, with a pore size of 0.05, 0.08 or 0.1 gm. Vinorelbine loading, isolation of vinorelbine liposomes and characterization of liposomes followed the method of example 40. Female albino rats (8-9 weeks; 200 g) were used to study liposome stability in vivo. The pharmacokinetics of liposome lipid and drug were studied in rats as in example 43.

Results are shown in figures 21, 22 and table 28 below. Extruded liposomes through 0.05, 0.08 and 0.1 gm polycarbonate filters were compared and shown to have similar pharmacokinetics of the drug and liposome vehicle as well as a similar degree of content leakage. Drug release of liposomes in the blood was characterized by 50% release times in the range of approximately 40-80 hours, well above 24 hours.

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Table 28. Characterization of vinorelbine liposomes.

<td rowspan="2">Liposome size, nm, mean ± SD QELS)</td><td rowspan="2">Drug loading, mg / mmolar phospholipid</td><td rowspan="2">Charging efficiency,</td><td rowspan="2">half life % of blood lipid matrix, hours</td><td colspan="2">Time for liberation</td>

<td>50% drug blood, hours</td><td>in at the</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 ±</td><td> 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 +</td><td> 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 +</td><td> 1,4</td>

<td>Example 46.</td><td>Preparation</td><td>in</td><td>liposomes of</td><td colspan="2">vinorelbine</td>

<td>targeted</td><td>for HER2</td><td></td><td>using the</td><td>method</td><td>in</td>

<td>imprisonment</td><td>from TEA-SOS,</td><td>and</td><td>pharmacokinetics of</td><td colspan="2">vinorelbine</td>

<td colspan="3">targeted immunoliposomal</td><td colspan="2">and not directed to</td><td>HER2</td>

scFv in mice.

Liposomes were prepared, loaded with vinorelbine at a drug-phospholipid ratio of 350 mg / mmolar, and analyzed as described in example 43, except that the TEA-SOS solution from example 45 was replaced with the TEA-Pn solution. The extrusion step included 15 passes through 0.08 µm pore size polycarbonate filters. Liposome size was 95.0 ± 26.0 nm by QELS. F5scFv-linked anti-HER2 vinorelbine immunoliposomes were prepared from these vinorelbine liposomes, and the pharmacokinetics in blood of liposome lipid and HER2-directed and liposomal vinorelbine drug were studied in rats as described in Example 43. . Liposome lipid circulation half-life was 11.4 hours and 10.3 hours, and the 50% drug release time was 30.9 hours and 30.3 hours for respectively F5-ILs-VRB and LsVRB. Thus, the pharmacokinetics of lipid and drug of Ls-VRB and F5-ILs-VRB were very close, indicating that the

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152 The introduction of scFv-PEG-DSPE conjugate did not affect the clearance of the vehicle itself nor did it result in increased leakage of the drug from the vehicle while in circulation (Figs. 23, 24).

Example 47. Preparation and pharmacokinetic properties of vinorelbine liposomes comprising nonionic lipid derivatives of poly (ethylene glycol).

Methoxy-PEG derivative (molar weight, 2000) of synthetic C20-ceramide (PEG-ceramide) was obtained from Northern Lipids, Inc., Canada. Methoxy-PEG (molar weight. 2000) distearoylglycerol (PEG-DSG) (SUNBRIGHT GS20) was from NOF Corp., Japan.

Liposomes having the composition of DSPC, cholesterol and PEG-lipid lipid (PEG-ceramide or PEG-DSG) in the 3: 2: 0.3 molar ratio and the entrapped TEA-SOS solution (0.65 M TEA pH 6.4, osmolarity of 502 mmolar / kg) were prepared by the ethanol / extrusion blending method of Example 11. The extrusion step included two passes through two stacked polycarbonate membrane filters, 2 times the size of 0.2 pm pore and 10 times with the 0.08 pm pore size. Liposomes were loaded with vinorelbine at a drug / phospholipid ratio of 350 mg / mmolar, characterized by size, drug and lipid concentration, and their pharmacokinetics were studied in rats as in example 46. Both formulations showed prolonged circulation time. lipid matrix and slow drug release in vivo, with at least 50% of the drug remaining encapsulated after 24 hours in blood in vivo, as shown in table 29 below.

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<td>Table</td><td> 29.</td><td>Characterization of vinorelbine liposomes with</td>

<td>several</td><td>Peg-</td><td>-lipids.</td>

<td rowspan="2">Peg- lipid</td><td rowspan="2">Liposome size, nm, mean d SD (by QELS)</td><td rowspan="2">Drug charge, : mg / mmolar of phospholipid</td><td colspan="2">Matrix half loading efficiency</td><td rowspan="2">Time to release 50% of drug into the blood, hours</td>

<td>O 0</td><td>of lipid in blood, hours</td>

<td>Peg-</td><td> 103,3 ± 30,</td><td> 9 291,4 ± 18,0</td><td> 83,26 ± 5,14</td><td> 14,0</td><td> 102,7</td>

<td>ceramide</td><td></td><td></td><td></td><td></td><td></td>

<td>PEG-DSG</td><td> 101,3 ± 20,</td><td> 1 359,3 ± 7,2</td><td> 102,7 ±2,1</td><td> 15,1</td><td> 24, 6</td>

Surprisingly, the increase in PEGylation of these liposomes (PEG lipid content of about 5.7 mole% of total lipid) had virtually no effect on blood circulation longevity of liposomes compared with similarly sized liposomes. having low pegylation of about 0.3 molar. % total lipid (Example 45, 109, 6 nm, ti / 2 =

14.3 hours; 98.5 nm, t1 / 2 = 13.0 hours).

Example 48. Preparation of HER2-directed liposomal vinorelbine and cytotoxicity of free and non-HER2-directed liposomal vinorelbine against MDA-MB-453 cells in vitro.

Vinorelbine-loaded liposomes (Ls-VRB) were prepared as in example 42 (without [³H] -CHE) using the

350 pg of vinorelbine formed by loading the drug to pH 6.0 and vinorelbine / pmolar phospholipid. Anti-HER2 immunoliposome (F5-ILs-VRB) was incubation of these liposomes with F5-PEG-DSPE conjugate as described in examples 19 and 42 above, except that no [³H] -CHE. Free vinorelbine was

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154 prepared by diluting vinorelbine bitartrate, 10 mg / ml USP solution, in the cell culture medium.

MDA-MB-453 are human breast adenocarcinoma cells (American Type Culture Collection, Rockville, MD) with moderate HER2 receptor overexpression (about 3x10⁴ at lx10⁵ copies / cell). The cytotoxicity of VRB administered as free drug, as non-targeting liposomal vinorelbine or as immunoliposome- (F5) -ororated vinorelbine against MDA-MB-453 cells was determined as described in Example 27, except that the cells were plated 96-well microtiter assay under supplier recommended growth conditions (Leibowitz L-15 with 10% fetal bovine serum, without CO2 supplementation) at a density of 10,000 cells / well, and drug formulations were added in a series of 1: 3 gradual dilutions starting at 0.03-0.1 mg / ml. Cell viability data were plotted against drug concentration (Figure 25), and the drug concentrations required to reduce cell viability to 50% (IC50) were estimated from the graphs. THE

IC50 of the F5-directed vinorelbine liposome (0.06 pg / ml) was close to that of the free drug (0.07 pg / ml) and substantially smaller than that of non-directed liposomes (2.2 pg / ml). This represents a 37-fold improvement in activity as a result of drug delivery targeted to the specific cancer cell.

Example 49. Cytotoxicity of free, liposomal targeted and non-HER2 directed vinorelbine against CaLu-3 cells in vitro.

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The liposomes and methods from the previous example (Example 48) were used to study the cytotoxicity of free vinorelbine, Ls-VRB and F5-ILs-VRB in CaLu-3 human non-small cell lung carcinoma cells (American Type Culture Collection , Rockville, MD). Cells were grown in RPMI-1460 medium with 10% fetal bovine 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 for undirected LsVRB. This represents a 5-fold improvement in liposome-encapsulated drug activity as a function of cell-directed administration.

Example 50. Cytotoxicity of free, non-HER2-directed liposomal vinorelbine against SKBr-3 cells in vitro.

The liposomes and methods of Example 48 were used to study the cytotoxicity of free vinorelbine, Ls-VRB and F5-ILs-VRB in HER2 overexpressed SKBr-3 human breast carcinoma cells (American Type Culture Collection, Rockville , MD), except that the cells were grown in McCoy 5A medium modified with 10% fetal bovine serum in the presence of 5% CO2, plated at a density of 5,000 cells / well, and the drug was incubated with the cells for 6 h.

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 for non-directed Ls-VRB. . This represents a 4.7-fold improvement in drug activity as a function of targeted administration.

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Example 51. In vivo antitumor efficacy of liposomal vinorelbine in human HT29 colon cancer xenografts in mice.

Small unilamellar vesicle liposomes (93.2 ±

26.4 nm by QELS) were prepared from distearoylphosphatidylcholine, cholesterol and PEG-DSPE (3: 2: 0.045 molar ratio) by hydration of an ethanolic solution concentrated in an aqueous solution of triethylammonium sucroseoctasulfate (0.6 M triethylammonium pH 5.7-6.2), followed by repeated extrusion through polycarbonate membranes (100 nm pore size), removal of the extraliposomal polyanionic salt, and vinorelbine loading by incubation with the liposomes in iso-osmotic buffer at pH 6.5, 325 mg VRB / mmolar phospholipid drug / lipid ratio at 60 ° C as described in example 42.

Homozygous BALB / c nude female mice (6-8 weeks, weighing 17-20 g) were injected subcutaneously into the flank area with 1 x 10⁶ HT29 human colon carcinoma cells (American Type Culture Collection, Rockville, MD). Starting 16 days after tumor inoculation, when the average tumor diameter reached 5-8 mm, mice were randomly divided into three groups of six animals each, and treated with free or liposomal vinorelbine at a dose of 5 mg / kg through tail vein every three days for a total of four injections. For the control group, mice were treated with an equal volume of saline. The tumor size of each mouse was measured using a bar, and the tumor volume was calculated using the formula: (tumor length) x (tumor width)²/2. To assess treatment-related toxicity, animals were also weighed twice a week. Liposomal vinorelbine has been shown to be

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157 considerably more effective in suppressing HT-29 tumor growth than free vinorelbine, causing tumor regression, while in the free drug group tumors continued to grow (Figure 28). There were no significant changes in animal body weight during the course of treatment, indicating that treatment was well tolerated and liposomalization did not increase drug toxicity (Figure 29).

Example 52. In vivo antitumor efficacy of liposomal vinorelbine against C-26 syngeneic murine colon cancer tumors.

Liposomal vinorelbine and free vinorelbine were prepared as in example 48. Male BALB / c mice (68 weeks, weighing 17-20 g) were inoculated subcutaneously with 2x100.⁵ C-26 murine colon carcinoma cells. On day 17 after inoculation, when the mean tumor diameter reached 5-8 mm, the mice were randomly divided into six treatment groups of five animals / group. Tumor-bearing mice were injected into the tail vein with either 6 mg / kg, 8 mg / kg or 12 mg / kg free vinorelbine, and 4 mg / kg or 6 mg / kg liposomal vinorelbine every three days for one week. total of four injections. For the control group, mice were injected with an equal volume of normal saline. Tumor size and animal body weight were followed as in example 51. Liposomal vinorelbine, even at 4 mg / kg, was considerably more effective in reducing tumor growth than free drug at 12 mg / kg (Figure 30). The body weight of the animals in the course of treatment showed little change (&lt;10% reduction), indicating that the toxicity of liposomal vinorelbine did not increase compared with that of free drug (Figure 31).

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Example 53. In vivo antitumor efficacy of HER2-directed liposomal vinorelbine against BT-474 human breast cancer xenograft tumors in mice: effect of contraction loading.

VRB-loaded liposomes, 99.5 ± 10.2 nm in size, were prepared, respectively, by the TEA-Pn method of example 41 and the TEA-SOS method of example 42, except that it was not added. [³H] -CHE. VRB was loaded at the drug / phospholipid ratio of 350 mg / mmolar. HER2-targeted liposomal vinorelbine was formed by incubating these liposomes with the F5-PEGDSPE conjugate (see Example 19) as described in Example 43. HER2 overexpressed BT-474 human breast carcinoma xenografts were grown in homozygous nude mice as in example 10. On day 25 after tumor cell inoculation, when tumors reached about 200 mm³ in size (range 144-309 mm³), mice were randomly assigned to four groups of eight animals / group, and treated iv with 5 mg / kg free VRB, F5ILs-VRB with Pn as contraction or F5-ILs-VRB with SOS as contraction at a dose of 5 mg / kg. mg / kg per week for a total of three injections. The control group received equal volume of normal saline. Tumors and body weight of the animals were monitored as in example 10. HER2-targeted liposomal vinorelbine loaded using sucrose octasulfate was noticeably more effective in reducing tumor growth than the same poly-phosphate-loaded targeting construct, and both immunoliposomal preparations were considerably more effective than administered free vinorelbine when administered. at a dose of 5 mg VRB / kg (Figure 32). Drug-treated mice demonstrated little change in weight, indicating that treatment was well tolerated (Figure 33).

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Example 54. In vivo antitumor efficacy of HER2-directed liposomal vinorelbine against BT-474 human breast cancer xenograft tumors in mice: effect of PEGylation.

DSPC liposomes and cholesterol at the 3: 2 molar ratio were prepared according to example 48 by hydration of the 2,000 molar weight PEG DSPC, cholesterol and PEGdistearoylglycerol lipid matrix (GS-20, NOF Corp., Japan) at a molar ratio of 3: 2: 0.015 (0.5% PEG) or 3: 2: 0.3 (10% PEG) by the aqueous triethylammonium sucroseoctasulfate ethanolic solution followed by membrane extrusion according to example 48. VRB was loaded onto the liposomes at a drug / phospholipid ratio of 350 mg / mmolar. Immunoliposomal vinorelbine F5 was formed by incubating these liposomes with the F5-PEG-DSPE conjugate (Example 19) as described in example 43. Bare-xenograft nude mice were raised and treated iv with free VRB, F5- ILs-VRB-0.5% PEG or F5-ILs-VRB-10% PEG at 5 mg / kg, as in example 53. As shown in Figure 34, the higher PEGylation F5-ILs-VRB provided with a nonionic lipid PEG-derived PEGDSG was noticeably more effective in reducing tumor growth than the lower amount of PEG-DSG, although both preparations were more active than the free drug.

Example 55. In vivo antitumor efficacy of vinorelbine

<td>liposomal targeted</td><td>for</td><td>EGFR</td><td>against</td><td>tumors</td>

<td colspan="2">cancer xenografts</td><td>brain</td><td colspan="2">human U87 in</td>

<td>mice.</td><td></td><td></td><td></td><td></td>

<td>Liposomes (size of</td><td> 86, 6</td><td colspan="2">± 12.9 nm per</td><td>QELS) with</td>

0.65 M encapsulated TEA-SOS solution was prepared and loaded with VRB according to example 42. VRB

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160 Anti-EGFR immunoliposomal (C225Fab'-ILs-VRB) was prepared by incubating VRB liposomes with Fab 'PEG-DSPE fragment conjugate of an anti-EGFR antibody as described in Example 36.

Nu / nu male NCR mice (5-6 weeks, weighing 17-20 g) were injected subcutaneously into the flank area with 1x10⁷ U87 human glioblastoma (ATCC) cells suspended in the growth medium in a total volume of 150 μΐ. When the tumor reached an average size of 250 mm³, mice were randomly divided into four groups of 10-12 animals. Mice were treated with three weekly iv injections of free VRB, untargeted Ls-VRB or C225Fab'-ILs-VRB at a dose of 5 mg VRB / kg. The control group received an equal volume of saline. Tumor size and animal body weight were monitored as in example 10. In C225-Fab'-ILs-VRB there was noticeably more suppression of growth of EGFR overexpressed human brain cancer xenograft tumors than either non-targeted liposomal vinorelbine or free vinorelbine (Figure 35).

equal dose, effective in

Example 56. Preparation and pharmacokinetics of liposome encapsulated doxorubicin using the triethylammonium sulfate method.

Liposomes with various lipid matrix compositions (as indicated in the table below) were formed as described in Example 2. N-Glutaryl-DSPE (Glu-DSPE) was from Avanti Polar Lipids, AL, USA. A pure lipid film was formed from the chloroform lipid solution using rotary evaporation, volatile traces were removed under vacuum (90 pm Hg, 2 hours), the

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161 The lipid was hydrated in a solution of triethylammonium sulfate (TEA-SCd) (0.65 N TEA), subjected to six freeze-thaw cycles, and extruded ten times through two 0-pore size stacked polycarbonate filters. , 1 pm and ten times through two 0.05 pm pore size stacked polycarbonate filters. For quantification of the lipid matrix in blood samples, [³H] -CHE in the 0.5-1.5 mCi / mmole lipid matrix of phospholipid. Liposomes with entrapped TEA-SO4 solution were loaded with doxorubicin according to example 2. Liposomes in HEPES-buffered saline (20 mM HEPES-Na, 135 mM NaCl, pH 6.5) were incubated with doxorubicin (drug / phospholipid ratios 140-170 mg / mmolar) at 60 ° C for 45 min, followed by ice extinction and removal of non-encapsulated doxorubicin by gel chromatography. Doxorubicin was tested by spectrophotometry (Example 71) and phospholipid was tested by Bartlett's method (Example 70). The properties of the resulting liposomes are summarized in table 30 below.

Table 30. Properties of liposomal doxorubicin in various lipid compositions.

Lipid composition Drug / phospholipid size (molar ratio) liposome, nm (mg / mmolar) (mean ± SD by QELS)

<td>DSPC / Col / PEG-DSPE</td><td> 81,</td><td> 8</td><td> ±</td><td> 27,3</td><td> 163,</td><td> 6</td><td> ±</td><td> 4,4</td>

<td> (3:2:0,015)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>DSPC / Col (3: 2)</td><td> 79,</td><td> 1</td><td> ±</td><td> 27, 9</td><td> 137,</td><td> 0</td><td> ±</td><td> 17,5</td>

<td>DSPC / Col / Glu-DSPE</td><td> 83,</td><td> 6</td><td> ±</td><td> 27,2</td><td> 141,</td><td> 7</td><td> ±</td><td> 10,4</td>

<td> (2,85:2:0,15)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>DSPC / Col / PEG-DSPE</td><td> 83,</td><td> 7</td><td> ±</td><td> 23, 1</td><td> 175,</td><td> 0</td><td> ±</td><td> 6, 8</td>

(2,7:2:0,3)

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The pharmacokinetics of these doxorubicin-containing blood liposomes having the lipid composition of DSPC / Col / PEG-DSPE 2.7: 2: 0.3 was studied in rats with a single iv dose of 5 mg doxorubicin / kg as described. in example 9. Liposomes had a long circulation (about 28 hours half-life) (Figure 36). The stable doxorubicin-to-phospholipid ratio indicated that the formulation was remarkably stable against circulating drug leakage, losing less than 25% of the drug over a 48 hour time period.

<td>Example 57. Loaded Liposomes</td><td>with</td><td>doxorubicin and</td>

<td>anti-HER2 immunoliposomes prepared</td><td>fur</td><td>TEA method</td>

<td colspan="2">sulfate: preparation and antitumor efficacy</td><td>in vivo against</td>

<td>breast cancer xenografts</td><td colspan="2">human with</td>

HER2 expression.

Doxorubicin-loaded liposomes having various lipid compositions and properties (listed in the table below) were prepared as described in Example 56. Doxorubicin-loaded anti-HER2 immunoliposomes were prepared from doxorubicin-loaded liposomes by coincubation with the scFv conjugate F5PEG-DSPE anti-HER2 (approx. 30 scFv / liposome) as described in example 19. Nu / nu NCR mice carrying human breast tumor subcutaneous xenograft (BT474) were raised, treated (in groups of 10-12 animals) with antiHER2 liposomal or immunoliposomal doxorubicin at a dose of 5 mg / kg once weekly. for a total of three weeks so that the tumors reached an average size of 200 mm³, and tumor progression and animal body weight were monitored as described

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163 in Example 29. For the non-targeting doxorubicin liposome formulations, non-PEG-DSPE-containing lipid compositions containing 0.5 molar% PEG-DSPE or 10 molar PEGDSPE were studied; For F5-immunoliposomal doxorubicin, the formulations with 0.5 mol% PEG-DSPE and 10 mol% PEG-DSPE formulations (here the amount of PEG-DSPE is expressed as molar% liposome phospholipid) . The results (Figure 37, Table 31) demonstrated that all doxorubicin treatments were effective in retarding tumor growth. Based on tumor size on day 53 post-inoculation, differences in tumor growth inhibition between the three groups of non-targeting liposomes did not increase statistical significance (ANOVA, p = 0.081), but immunoliposomal doxorubicin was significantly more effective. than non-targeted liposomal doxorubicin (ANOVA p = 5.5 x 10 “¹⁰), with 10% PEG-DSPE being more effective than 0.5% PEG-DSPE (Student's t-test, p = 0.027). In the F5-ILs 10% PEG-DSPE group, tumors regressed to 1 mm³ or less in 67% of the animals, although in the F5-ILs group 0.5% PEG-DSPE only in 9%. In the control group (saline treatment) the tumors exceeded the acceptable size limit of 15% of body weight on day 38-43.

Table 31. In vivo antitumor efficacy study of liposomal doxorubicin: liposome characteristics and treatment results.

<td>Composition of</td><td>Size of</td><td>reason of</td><td>Average size</td>

<td>lipid</td><td>liposome, nm</td><td>drug / phosphate</td><td>of the tumor in</td>

<td></td><td>(mean ± SD)</td><td>lipid,</td><td>day 58, mm³</td>

<td></td><td></td><td>mg / mmolar</td><td>(mean ± SD)</td>

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164

<td colspan="6">(mean ±</td><td colspan="4">SD)</td>

<td>DSPC / Col / PEG-DSPE (3: 2: 0.015)</td><td> 83,</td><td> 4 ±</td><td> 23,3</td><td> 136, 7</td><td> ±</td><td> 6,7</td><td> 490</td><td> ±</td><td> 74</td>

<td>DSPC / Col (3: 2)</td><td>CO O</td><td> 5 ±</td><td> 26, 6</td><td> 151,2</td><td> +</td><td> 1, 9</td><td> 587</td><td> +</td><td> 61</td>

<td>DSPC / Col / PEG-DSPE (2.7: 2: 0.3)</td><td> 81,</td><td> 0 ±</td><td> 24,7</td><td> 140, 1</td><td> ±</td><td> 4,2</td><td> 365</td><td> ±</td><td> 60</td>

<td>DSPC / Col / PEG-DSPE</td><td>no</td><td colspan="2">measured</td><td> 140,7</td><td> ±</td><td> 2,8</td><td> 119</td><td> ±</td><td> 39</td>

<td>(3: 2: 0.015) + F5 scFv-PEG-DSPE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>DSPC / Col / PEG-DSPE</td><td>no</td><td colspan="2">measured</td><td> 132, 9</td><td> ±</td><td> 2,2</td><td> 15,</td><td> 5 ±</td><td> 7, 6</td>

<td>(2.7: 2: 0.3) + F5 scFv-PEG-DSPE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Example 58.</td><td>Vinblastine Preparation</td><td>liposomal and</td>

<td colspan="2">pharmacokinetics of liposomal vinblastine</td><td>in the blood in</td>

rats .

Liposomes with entrapped aqueous TEA-SOS solution (0.65 M TEA, pH 6.4, 502 mmolar / kg osmolarity) and size 99.5 ± 10.2 nm (mean ± SD by QELS) were prepared. by the method of example 11, using 2 times extrusion through two stacked 0.2 µm polycarbonate membranes and ten times through two stacked 0.08 µm polycarbonate membranes. Vinblastine (VBL) as USP vinblastine sulfate was added at a drug-to-phospholipid ratio of 150 mg / mmolar. The pH of the drug-liposome mixture was adjusted to 6.5 using 1 N NaOH, and the mixture was subsequently incubated at 60 ° C for 30 min. The reaction was then cooled on ice for 15 min. and the unencapsulated drug was removed using Sephadex G-75 gel filtration chromatography, eluting with 5 mM HEPESNa, 135 mM NaCl, pH 6.5. Purified liposomes were then analyzed spectrophotometrically for VBL and phospholipid by Bartlett's method as in the examples.

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165 and 71. It was included [³H] -CHE in the formulation at a ratio of

1.5 mCi / mmolar phospholipid. Liposomal vinblastine had 152.4 ± 12.0 mg VBL / mmolar phospholipid (quantitative encapsulation).

The pharmacokinetics of blood liposomal vinblastine in albino female rats (8-9 weeks; 200 g) were studied at a dose of 5 mg VBL / kg as described in example 9. Vinblastine was quantified in blood plasma samples, as described in example 41 (using vinorelbine as internal standard). Vinblastine liposomes showed good circulation longevity (plasma lipid component half life 12.8 ± 0.04 hours) (Figure

38) and very good stability against drug leakage from the liposomes, with over 70% of the initial vinblastine loading remaining encapsulated after 24 h (Figure

39). Post-injection time to achieve 50% release of encapsulated drug was 40.6 ± 1.2 hours.

Example 59. Preparation of vincristine-loaded liposomes using the TEA-SOS method and the effect of pH on loading efficiency.

Liposomes of 86.6 ± 12.9 nm size (by QELS), DSPC / Col / PEG-DSPE lipid composition at 3: 2: 0.015 molar ratio and encapsulated TEA-SOS aqueous solution (0.65 TEA (pH 5.4, osmolarity of 521 mmolar / kg) were prepared by the method of example 11 using the 15-pass extrusion step through two stacked 0.08 µm pore size polycarbonate membranes. Vincristine (VCR) was added to the liposomes in 5 mM HEPES-Na 5% aqueous dextrose buffer pH 6.5 as vincristine sulfate at a 350 pg vincristine / pmolar phospholipid-phospholipid ratio ,

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166 pH was adjusted to the indicated ratio using 1 N NaOH, the mixture was incubated at 60 ° C for 30 min, ice-cooled for 15 min, and the liposomes were separated from unencapsulated drug using gel filtration chromatography. Sephadex G-75, eluting with HBS-6.5 (20 mM HEPES, 135 mM NaCl, pH 6.5). Purified liposomes were then analyzed for vincristine by spectrophotometry using absorbance at 265 nm after solubilization in acid isopropanol, and for phospholipid content using Bartlett's phosphate assay (1959).

Table 32. O in the pH range 5.0-7.5. At pH liposomes after loading

The results are shown below at drug loading was above 90% 4.5-7.5, and practically quantitative at pH 3.5, which is the pH observed in the drug addition mixture, but without pH adjustment. , was considerably inferior.

Table 32. Vincristine loading pH dependence

<td colspan="3">in liposomes with entrapped TEA-SOS.</td>

<td colspan="2"></td><td></td>

<td>pH</td><td>Reason for drug / phospholipid, pg / pmolar</td><td>Charging Efficiency (%)</td>

<td> 3,5</td><td> 39, 7</td><td> ±</td><td> 4, 9</td><td> 11,3</td><td> ±</td><td> 0,2</td>

<td> 4,5</td><td> 327,2</td><td> ±</td><td> 20, 6</td><td> 93,5</td><td> ±</td><td> 5,4</td>

<td> 5, 0</td><td> 360, 6</td><td> ±</td><td> 5, 8</td><td> 103, 0</td><td> ±</td><td> 1,7</td>

<td> 5,5</td><td> 371,2</td><td> ±</td><td> 30,2</td><td> 106, 1</td><td> ±</td><td> 9,1</td>

<td> 6, 0</td><td> 347,7</td><td> ±</td><td> 20,4</td><td> 99, 3</td><td> ±</td><td> 5, 8</td>

<td> 6, 5</td><td> 347,7</td><td> +</td><td> 20, 9</td><td> 99, 4</td><td> +</td><td> 5, 9</td>

<td> 7,0</td><td> 377,3</td><td> ±</td><td> 22,2</td><td> 107,8</td><td> ±</td><td> 6, 8</td>

<td> 7,5</td><td> 371,5</td><td> ±</td><td> 24, 9</td><td> 106, 1</td><td> ±</td><td> 7, 6</td>

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Example 60. Preparation of vincristine-loaded liposomes using the TEA-SOS method: effect of drug / lipid ratio on loading efficiency.

SOS-TEA-containing liposomes were prepared as in Example 59 and loaded with vincristine sulfate at a drug-to-phospholipid ratio of 150-550 pg vincristine / pmolar phospholipid at pH 6.5 according to the procedure of the example. 59. Purified non-encapsulated drug liposomes were then analyzed for VCR by spectrophotometry and for liposome phospholipid using Bartlett's (1959) assay. Drug loading efficiency was above 90% across the studied range of drug / lipid ratios, and was nearly quantitative between 150-450 pg vincristine / pmolar phospholipid (Table 33).

Table 33. Vincristine loading into TEA-SOS-containing liposomes at different drug-to-lipid ratios.

<td>Phospholipid er entry drug (pg / umolar)</td><td>Drug encapsulated-paraphospholipid (pg / umolar)</td><td>Charging 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> 9 9,4 ± 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>

<td>Example 61. Preparation</td><td>of vincristine</td><td>immunoliposome and</td>

<td>cytotoxicity of</td><td>vincristine</td><td>liposomal and</td>

<td>immunoliposome against</td><td colspan="2">cancer cells in vitro.</td>

Liposomal vincristine (Ls-VCR) was prepared as described in example 59 using the ratio of

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168 350 mg / mmolar drug / phospholipid. HER2-specific immunoliposomal F5-vincristine (F5-ILs-VCR) was prepared from liposomal vincristine by coincubation with the anti-HER2 sc5 FF-PEG-DSPE conjugate as described in Example 19. The free vincristine solution ( VCR) was prepared by diluting USP vincristine sulfate in water, followed by sterile filtration. Cytotoxicity of VCR, Ls-VCR and F5-ILs-VCR against HER2-overexpressed SKBr-3 human breast carcinoma cells (ATCC) was determined by the MTT-based cell viability assay using the procedure of example 27 , where cells were inoculated into 96-well microtiter plates at 5,000 cells / well, acclimated overnight, and incubated with drug-containing medium for 4 hours, followed by post-incubation in drug-free medium for 3 days. The results are shown in Figure 40. The ICso was 75 ng / ml for free VCR, 11 ng / ml for F5-ILa-VCR and 3 pg / ml for Ls-VCR. Targeted liposomal vincristine, prepared according to the disclosure, was 6.8 times more active than free drug, and 273 times more active than non-targeted liposomal drug, showing a substantial improvement in anticancer activity as a function of administration. cell-specific drug

Example 62. Pharmacokinetics of Ls-VCR in blood in rats.

Liposomes with entrapped SOS-TEA solution (0.65 M TEA, pH 5.8, 530 mmolar / kg osmolarity) and DSPC / Col / PEG-DSPE lipid composition (3: 2: 0.015 molar ratio ), also containing [³H] -CHE at 1.5 mCi / mmolar phospholipid were prepared by the method of example 11 using the 10-pass extrusion step through two stacked polycarbonate membranes the size of

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169 80 nm or 100 nm portion. Liposomes were loaded with VCR at pH 6.5, 350 mg / mmolar drug / phospholipid ratio as described in Example 59. VCR-loaded liposomes were administered iv to albino female rats (180-220 g) in a 5 mg VCR / kg, and the pharmacokinetics of the drug and blood liposome lipid were studied as described in 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. Normal retention time for CRV was 8.8 min. Results are shown in Figure 41 and Table 34. Both preparations had long circulation longevity (12-17 hour blood half-lives). Liposomal vincristine was remarkably stable against drug leakage in both preparations (half-release time above 120 hours) (Figure 42).

Table 34. Characteristics of liposomes loaded with

<td colspan="3">vincristine at 350 mg / mmolar of</td><td colspan="2">phospholipid</td><td colspan="2">using the</td>

<td>method of</td><td>TEA-SOS.</td><td></td><td></td><td></td><td></td><td></td>

<td>Size of</td><td>Size of</td><td>Charge of</td><td>tl / 23 of</td><td>tl / 2p</td><td>gives</td><td>ti / 2 da</td>

<td>pore of</td><td>liposome,</td><td>drug,</td><td>lipid,</td><td>VCR,</td><td></td><td>release</td>

<td>extrusion, nm</td><td>nm (mean ± SD)</td><td>mg / mmolar in phospholipid</td><td>hours</td><td colspan="2">hours</td><td>VCR hours</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> ±</td><td> &gt; 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> ±</td><td>No detectable</td>

<td>Example 63</td><td colspan="2">. Pharmacokinetics of Ls</td><td>; -VCR in</td><td colspan="2">blood</td><td>in rats</td>

various drug / lipid ratios.

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Liposomes with entrapped SOS-TEA solution (0.65 M TEA, pH 6.4, 485 mmolar / kg osmolarity) and DSPC / Col / PEG-DSPE lipid composition (3: 2: 0.015 molar ratio ), also containing [³H] -CHE at 1.5 mCi / mmolar phospholipid were prepared by the method of example 11 using the 10-pass extrusion step through two stacked polycarbonate membranes with a pore size of 50 nm or 80 nm. Liposomes were loaded with VCR at pH 6.5 as described in Example 59 by the addition of an aqueous styrene solution at 20 mg / ml VCR sulfate with calculated drug / lipid ratios at 100, 200 or 350 mg. / mmole phospholipid. Drug loading efficiency was above 96% for all preparations. VCR-loaded liposomes were administered iv to albino female rats (8-9 weeks, 190-220 g) at a dose of 5 mg VCR / kg, and the pharmacokinetics of the drug and blood liposome lipid were studied as described in example 62. The results are shown in table 35. Liposomal vincristine had good circulation longevity (blood drug half-life of about 20-30 hours) and was exceptionally stable at all sizes and studied drug-to-lipid ratios (drug release half-life more than 93 hours ).

Table 35. Characteristics of vincristine-loaded liposomes using the TEA-SOS method at various drug / lipid ratios.

<td colspan="2">Size Size</td><td rowspan="2">VCR, mg / mmolar phospholipid</td><td rowspan="2">lipid ti / 2,</td><td colspan="2">ti / 2 da. ti / 2 da</td>

<td>pore of</td><td>liposome,</td><td>VCR,</td><td>release</td>

<td>extrusion,</td><td>nm (mean +</td><td rowspan="2">added encapsulated</td><td>hours</td><td>hours</td><td>in</td>

<td rowspan="2">nm</td><td rowspan="2">SD)</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2">drug, hours</td>

<td></td>

96,1 ± 3,0 35, 6 ± 30,3 ± 227 ± 96

2,7 4,0

76,8 ± 27,2 100

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200 193,3 ± 3,9 20,8 ± 18,4 ±244 ± 130

2,2 0,7

350 375,2 ± 10,024,8 ± 19,6 ±93,2 ± 6,7

0,9 0,9

<td> 80 101,6 ± 25,3100</td><td> 104,5 ± 2,1 33,0 ± 26,8 ±153 ± 10 7,6 4,8</td>

<td>Example 64. Preparation</td><td>of liposomal vincristine</td>

<td>directed to HER2</td><td>and antitumor efficacy of</td>

<td>non-liposomal vincristine</td><td>directed and directed to</td>

<td colspan="2">HER2 against human breast cancer xenografts with</td>

<td colspan="2">HER2 overexpression in mice.</td>

<td>Liposomes loaded with</td><td>vincristine (Ls-VCR-SOS) using</td>

ο TEA-SOS method was prepared according to example 63 (without component [³H] -CHE) using 50 nm pore size membrane extrusion and drug loading at 100 mg / mmolar drug / phospholipid ratio. Immunoliposomal vincristine F5 (F5-ILs-VCR) was formed by incubating Ls-VCR-SOS with the anti-HER2 F5-PEG-DSPE scFv conjugate (Example 19) as described in Example 43. Vincristine-loaded liposomes using TEA-citrate (Ls-VCRCit) were prepared similarly to Ls-VCRSOS liposomes, except that triethylammonium citrate solution (prepared by titration of pure citric acid with triethylamine to pH 5, 1, and concentration adjustment to 0.65 M triethylamine) was replaced by the TEASOS solution. The treatment study design followed the method of example 10. Subcutaneous human breast carcinoma xenograft tumors BT-474 were raised in nude mice, and when the tumors reached the size of 250 mm³(range 144-309 mm³) mice in groups eight to nine were treated with free VCR, Ls-VCR or F5-ILs-VCR at a weekly iv dose of 2 mg VCR / kg for a total of three weeks, starting on day 19 after tumor inoculation. O

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172 Tumor size and animal body weight were monitored as described in Example 10. For the control group, mice were treated with an equal volume of saline. Differences in tumor size between treatment groups were statistically evaluated using the Mann-Whitney test on day 63 after tumor inoculation. The dynamics of the average tumor size in the groups is shown in figure 43. F5-ILs-VCR demonstrated maximum efficacy compared to Ls-VCR or free VCR, causing complete tumor regressions on day 63 in six of eight animals (75%). Ls-VCR-Cit was also effective, causing complete tumor regressions still observable on day 63 in two of nine animals (22%), however, was less effective than F5-ILs-VCR (p &lt;0.005) . Ls-VCR-SOS and free VCR were equally effective (p&gt; 0.2) and less effective than F5-ILs-VCR or Ls-VCR-Cit. Thus, surprisingly, with cell-directed administration, an encapsulated liposomal drug using a polyvalent anion of the present disclosure has proven to be more effective than liposome encapsulated drug via a non-binding anion. The dynamics of the animal's body weight showed that all liposomal VCR preparations were less toxic than free VCR, causing less body weight loss during treatment (Figure 44).

Example 65. Preparation of Liposomal Vincristine

<td>targeted</td><td>for EGFR and antitumor efficacy</td><td>gives</td>

<td>vincristine</td><td>directed and liposomal liposome</td><td>for</td>

<td>EGFR against</td><td colspan="2">human brain cancer xenografts</td>

with EGFR overexpression in mice.

Vincristine-loaded liposomes (Ls-VCR) were prepared using the TEA-SOS method as in example 64. EGFR-directed immunoliposome vincristine was

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173 prepared by coincubating the liposomes with the anti-HER2 Fab 'C225Fab-PEG-DSPE Fab conjugate as described in example 36.

Nu / nu male NCR mice (5-6 weeks, weighing 17-20 g) were injected subcutaneously into the flank area with 0.15 ml of cell growth medium containing 1 x 10 6⁷U87 human glioblastoma cells that stably express the epidermal growth factor (HER1) receptor mutant EGFRvIII. On day 11, when the average tumor size reached 300-400 mm³, mice were randomly divided into four groups of 10-12 animals / group. Treatments with free VCR (vincristine sulfate, 1 mg / ml in saline), Ls-VCR or C225FabILs-VCR at the iv dose of 1.5 mg / kg were administered on days 11, 18 and 25 after tumor. Mice in the control group were similarly injected with an equal volume of normal saline. Tumor size and mouse body weight were monitored as in example 10. Results are shown in Figure 45. All animals treated with VCR formulations showed tumor growth retardation compared to control animals. There was no significant difference between the groups treated with free VCR and Ls-VCR. EGFR-targeted C225Fab-ILs-VCR was more effective than free or non-targeted liposomal VCR.

Example 66. Preparation of liposomes with a solution of triethylammonium inositol hexaphosphate (TEA-IHP).

A polyanionized polyol, dodecasodium inositol hexaphosphate (IHP) salt, was obtained from Sigma (St. Louis, MO). An aqueous solution containing 0.65 M triethylammonium and 0.681 M phosphate groups, pH 6.5, and

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174 osmolarity of 718 mmolar / kg by ion exchange on the Dowex 50Wx8200 cross-linked sulfonated polystyrene resin, followed by titration with pure TEA and dilution with water according to the procedure of example 4. The residual sodium content was less than 1% of sum of cations. The dried lipids (150 pmolar DSPC, 100 pmolar Col, 0.75 pmolar PEG-DSPE) were dissolved in 0.5 ml 100% USP ethanol at 60 ° C, and mixed with 4.5 ml solution. of triethylammonium inositol hexaphosphate, preheated to the same temperature. Ethanol was partially removed by rotary evaporation at 30-40 mm Hg and 40-45 ° C until the mixture showed no bubbling. The lipid suspension was then extruded 15 times at 60-65 ° C through two stacked 0.1 µm pore size polycarbonate membranes. The resulting liposomes were 104.3 ± 39.0 nm in size by QELS. Unencapsulated triethylammonium IHP was removed by gel chromatography on a Sepharose 4B column, eluted with 5 mM HEPES-Na buffer, 5% dextrose, pH 6.5, and liposomes were quantified by phospholipid concentration using the Bartlett, with extraction according to example 70.

Example 67. Liposome drug loading with a trapped TEA-IHP solution.

Liposomes from Example 67 were loaded with CPT11 or vinorelbine. Vinorelbine was charged at a drug-to-phospholipid ratio of 175 or 350 g / molar, and CPT11 at a rate of 250 or 500 g / molar. Drugs were added to the liposomes in HEPES dextrose buffer (Example 67) at drug / phospholipid entry ratios indicated below (see Table 36). If necessary, the pH was adjusted to 6.5-6.8 using 1N NaOH. The mixtures were incubated at 60 ° C for 30 min., Chilled on ice for 15 min., And

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175 chromatographed on a Sephadex G25 gel filtration column, eluted with 5 mM HEPES-Na, 145 mM NaCl, pH 6.5. Aliquots of purified liposomes were solubilized in acidified methanol and analyzed by spectrophotometry (Example 71). The phospholipid was guantified by Bartlett's (1959) method with extraction (Example 70). Both drugs charged (ie, practically 100%) on the liposomes, as shown below in table 36.

Table 36. Properties of liposome-loaded drugs with entrapped inositol hexaphosphate.

<td>Drug</td><td>Reason for input drug / lipid, g / molar phospholipid</td><td>Reason for drug encapsulated / lipid, g / molar phospholipid</td><td>Charging efficiency, O 0</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>

<td>Example 68.</td><td>Stability</td><td>CPT chemistry</td><td>-11 free or</td>

liposome in the presence of mouse plasma in vitro.

In the body, CPT-11, which is a prodrug, undergoes a chemical transformation to form an active drug metabolite known as SN-38. Both SN-38 and CPT-11 are converted from their active lactone forms to inactive products known as SN-38 or CPT11 carboxylates. In this example, the effect of CPT-11 liposomalization according to the present invention on the chemical conversion of CPT-11 in these products in the presence of blood plasma was studied. Liposomes with entrapped triethylammonium sucrose octasulfate (0.65 M TEA, pH 6.4,

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176 osmolarality of 485 mmolar / kg) and lipid composition of

DSPC, cholesterol and PEG-DSPE in a molar ratio of 3: 2: 0.015 were prepared according to example 11 using ten-fold extrusion through two 0.08 pm stacked polycarbonate filters. Liposomes had 87.4 ±

19.2 nm in size by QELS. CPT-11 was charged to approximately 500 mg base of liposome phospholipid CPT-11 / mmolar base by incubation in 5 mM aqueous HEPES-Na, 5% dextrose, pH 6.5, at 60 ° C for 30 min. ., followed by extinction on ice for 15 min. CPT-11 loaded liposomes were then purified on a Sephadex G-75 column eluted with HEPES-buffered saline (5 mM HEPES, 145 mM NaCl, pH 6.5). The resulting CPT-11 liposomes had 536.5 ± 20.1 mg of phospholipid CPT-11 / mmolar. The free CPT-11 solution was freshly prepared by dissolving 1 mg / ml irinotecan hydrochloride USP in 144 mM aqueous NaCl, acidified to pH 3 with dilute HCl. Ten μΐ aliquots of free or liposomal CPT-11 or free CPT-11 were mixed with 90 μΐ of heparin-stabilized mouse plasma (Harlan Bioproducts, USA), and incubated at 37 ° C in a shaking water bath. At a given point in time, triplicate liposome samples were chromatographed on Sepharose CL-4B size exclusion columns (2 ml bed volume), eluted with HBS-6.5, and drug containing fractions. were detected by fluorescence. The first (dead volume) and second (posterior) drug-containing peaks were collected and considered as the liposome-encapsulated and released drug fractions. Samples were extracted with 400 μΐ of cold methanol vortexing for 10 s, followed by centrifugation at 14,100xg for 5 min. Supernatants were analyzed for CPT-11 and its conversion products by HPLC using the

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177 modification of a method of Warner and Burke, J Chromatogr., Ser. B Biomed. Know. Appl. 1997, vol. 691, p. 161-71. The mobile phase consisted of 3% triethylammonium acetate, pH 5.5 (solution A) and acetonitrile (solution B) administered at 1.0 ml / min in a linear gradient of 20% by volume from B to 50%. by volume B for 14 min. Eluted products were detected by fluorescence with an excitation at 375 nm and emission at 500 nm. Retention times were 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) indicated that although free CPT-11 and liposome-released CPT-11 were converted, intraliposomal CPT-11 was quite stable.

Table 37. Conversion of free and liposome CPT-11 to SN-38

<td>and carboxylate forms in the</td><td>mouse plasma in</td><td>vitro.</td>

<td></td><td></td><td></td>

<td>Sample Time, CPT-11,% hours</td><td>SN-38,%</td><td></td>

<td>lactone</td><td>lactone carboxylate</td><td>carboxylate</td>

<td>Free CPT-11</td><td> 2</td><td> 1, 9 ± 0</td><td> ,4 35,2 ±</td><td> 1,9</td><td> 4,4 ±</td><td> 0,1</td><td> 58,4 ±</td><td> 2,1</td>

<td></td><td> 12</td><td> &lt; 0,1</td><td> 11,5 ±</td><td> 0,9</td><td> 9,9 ±</td><td> 0,8</td><td> 78, 6 ±</td><td> 1,3</td>

<td></td><td> 24</td><td> &lt; 0,1</td><td> &lt; 0,1</td><td></td><td> 22,5 :</td><td> ± 9,</td><td> 8 77,5 ±</td><td> 9,8</td>

<td>Ls-CPT-11</td><td> 12</td><td> 97,7 ±</td><td> 0,1 &lt; 0,1</td><td></td><td> 2,3 ±</td><td> 0,1</td><td> &lt; 0,1</td><td></td>

<td>(encapsulated)</td><td> 24</td><td> 97,7 ±</td><td> 0,1 &lt; 0,1</td><td></td><td> 2,3 ±</td><td> 0,1</td><td> &lt; 0,1</td><td></td>

<td>Ls-CPT-11</td><td> 12</td><td> 60,5 ±</td><td> 10, 4 25,0 ±</td><td> 7,1</td><td> 5,0 ±</td><td> 0,3</td><td> 9,5 ± 3</td><td> ,0</td>

<td>(released)</td><td> 24</td><td> 78,3 ±</td><td> 6,7 14,0 ±</td><td> 5,2</td><td> 6,5 ±</td><td> 0,5</td><td> 1,2 ± 1</td><td> ,7</td>

<td>Example 69.</td><td colspan="3">Chemical stability</td><td>in</td><td>I saw you</td><td>of</td><td>CPT-11</td><td>free</td>

or liposomal in rats.

Liposomal CPT-11 was prepared as in example 68 using triethylammonium sucrose octasulfate having 0.65 M TEA, pH 6.4 and 502 mmolar / kg osmolarity. The liposome size was 98.5 ± 18.4 nm, and the

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178 The encapsulation of CPT-11 was 510.1 ± 16.5 mg of phospholipid CPT11 / mmolar. Free liposome CPT-11 was administered intravenously at a dose of 25 mg / kg in albino female rats (180-220 g) with implanted central venous catheter, and blood samples were taken at intervals over a period of 48 hours. hours Blood samples were mixed with ice cold PBS containing 0.04% EDTA and rapidly centrifuged to remove blood cells. Aliquots of supernatant fluids were tested by HPLC for CPT-11, SN-38 and their carboxylate forms as in example 68 above. The results are shown in Figures 46 and 47. Although free CPT-11 was cleared very rapidly and was undetectable after 30 min., Liposomal CPT-11 was persistent in circulation (15.2 hours ti / 2) at 37 ° C. , 8% of the drug in the blood at 24 h, and approximately 10% of the drug still in circulation after 48 h. There was no detectable conversion of the liposome form of CPT-11 to either SN-38 or the carboxylate form of CPT-11. Free CPT-11, i.e. administered as a solution, was cleared of circulation quite rapidly (about 16 min. Half-life), and there was considerable conversion to the carboxylate form of the drug.

Example 70. Quantification of liposome phospholipid. Modified acid digestion - Method I of blue phosphomolaribdate. This method is modified from Bartlett (1959). 10-20 ml aliquots of liposomes are placed in heat-resistant glass tubes, heat-digested with 0.5 ml 10 N sulfuric acid for 2 hours at 110-130 ° C, mineralized by adding 50 ml 9 % hydrogen peroxide, and heated for 30 min.

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179 until no hydrogen peroxide is detected by an indicator paper strip. Samples digested at room temperature are diluted with 1 ml 0.2% aqueous ammonium molaridate, mixed with 0.1 ml 5% aqueous ascorbic acid, and incubated in a boiling water bath for 10 min. The absorbance of the reduced phosphomolaribdate complex is measured at 800 nm and compared to a standard curve produced simultaneously using standard inorganic phosphate solutions.

Modified acid digestion - method II of blue phosphomolaribdate. This method is a modification of Morrison's (1964) method. Aliquots of 5 μΐ liposomes having 1-10 mM phospholipid with 60 μΐ concentrated sulfuric acid 30 μΐ hydrogen peroxide are mixed in heat-resistant glass tubes. The mixtures are heated at 200-220 ° C for 10 min., Diluted with 0,7 μΐ deionized water, mixed with 10 μΐ 10% aqueous sodium sulfite, incubated in a boiling water bath for 5 min. and cooled to room temperature. 200 μΐ of 2% aqueous ammonium molaribdate and 10 μΐ aqueous ascorbic acid are added, and the samples are incubated in a boiling water bath for 10 min. The samples are rapidly cooled to room temperature, and the absorbance of the reduced phosphomolaribdate complex at 825 nm against the blank sample is determined. The amount of phospholipid is determined from the standard curve obtained in the same series using standard solutions having 2, 4, 6, 8 and 10 mM potassium dihydrogen phosphate.

Extraction Method 25-100 μΐ aliquots of liposomes are extracted 3 times with 200 μΐ portions of a mixture of

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180 methanol-chloroform (1: 2 by volume). The organic phases are combined in a heat resistant glass tube, and the solvents removed under vacuum. Residues are treated with 10 N sulfuric acid and further tested for phosphorus according to method I above.

Unless otherwise indicated, analytical data are presented as the mean ± standard error of the triplicate series.

Example 71. Quantification of drugs in liposomes. Spectrophotometric quantification. Liposome aliquots (10-50 μΐ) are mixed with 1 ml of 70% by volume of aqueous isopropanol containing 0.075-0.1 N HCl, and the absorbance against the blank sample at wavelengths a is measured. following: doxorubicin, 485 nm; CPT11 and topotecan, 372 nm; ellipticines, 306 nm, vinorelbine, 270 nm; vincristine and vinblastine, 265 nm. The amount of drug is determined by comparison with a standard curve run simultaneously.

Fluorometric quantification. Aliquots of liposome-containing samples (eg, blood plasma) are diluted with acidified isopropanol (0.020.1 ml aliquots: 1 ml 70% isopropanol-0.075 N HCl;&gt; 0.1 ml aliquots: 90% isopropanol-0.1 N HCl to 1 ml). If protein precipitation occurs, the samples are incubated on ice for 1-2 hours and clarified by centrifugation for 10 min. at 12,100xg. The fluorescence of the supernatants is measured at the following wavelengths: CPT-11, excitation 370 nm, emission 423-425 nm;

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181 topotecan, excitation 380-385 nm, excitation 520-525 nm;

ellipticines, excitation 306 nm, emission 520 nm. The amount of drug is calculated from standard curves performed simultaneously after subtraction of white fluorescence.

Example 72. Effect of lipopolymers on vinorelbine loading efficiency in liposomes.

Liposomes composed of DSPC, 200 molar parts, cholesterol, 133 molar parts, and PEG-DSPE lipids (1-20 molar parts) or PEG-DSG (20 molar parts) derived from poly (ethylene glycol) (molar weight 2,000), and containing 0.65 M encapsulated TEA-SOS solution were prepared according to the method of example 11, using a pore size membrane of 80 nm for the extrusion step. Liposomes were loaded with vinorelbine at the drug / phospholipid ratio of 350 mg / mmolar and purified from unencapsulated drug according to the method of example 40. Liposomes were tested for drug and lipid content as described in examples 70, 71, and for QELS liposome size using a volume-weighted Gaussian approximation. The results (Table 38) indicated that while the anionic PEG derivative, PEG-DSPE, in the amount of more than 1 mol% liposome phospholipid (0.3 mol% total lipid), had a negative effect on Drug loading efficiency, the neutral derivative, PEG-DSG, surprisingly, did not affect the loading efficiency, even at 9.1 mole% liposome phospholipid (5.7 mole% total lipid).

Table 38. Properties of vinorelbine liposomes prepared by the TEA-SOS method with various amounts of PEG-lipid derivatives.

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182

PEG-lipid Amount of molar PEG-lipid. % total lipid

Liposome Load Size, drug, nm (mean ± mg / mmolar SD) phospholipid

Loading efficiency,% encapsulation

<td>PEG-DSPE</td><td> 0,3</td><td> 108</td><td> ± 32</td><td> 359, 5</td><td> +</td><td> 17,8</td><td> 102</td><td> ,7</td><td> ± 5,2</td>

<td>PEG-DSPE</td><td> 0, 6</td><td> 110</td><td> ± 18</td><td> 346, 6</td><td> ±</td><td> 14,5</td><td> 99,</td><td> 0 ±</td><td> 4,1</td>

<td>PEG-DSPE</td><td> 1,8</td><td> 104</td><td> ± 35</td><td> 332,0</td><td> ±</td><td> 14,0</td><td> 94,</td><td> 9 ±</td><td> 3, 8</td>

<td>PEG-DSPE</td><td> 2, 9</td><td> 94</td><td> ± 33</td><td> 259, 8</td><td> ±</td><td> 9,5</td><td> 74,</td><td> 2 ±</td><td> 2,0</td>

<td>PEG-DSPE</td><td> 4,0</td><td> 100</td><td> ± 36</td><td> 155,4</td><td> ±</td><td> 7,0</td><td> 44,</td><td> 4 ±</td><td> 0, 9</td>

<td>PEG-DSPE</td><td> 5,7</td><td> 103</td><td> ± 31</td><td> 61,2</td><td> ± 5</td><td> ,2</td><td> 17,</td><td> 5 ±</td><td> 0,3</td>

<td>PEG-DSG</td><td> 5,7</td><td> 97</td><td> ± 36</td><td> 362,7</td><td> ±</td><td> 14,2</td><td> 103</td><td> , 6</td><td> ± 4,2</td>

<td>Example 73.</td><td>It is made</td><td colspan="2">of agent</td><td colspan="4">intraliposomal &lt;</td><td>in</td><td>catch</td>

of drug on CPT-11 longevity in blood in mice.

Liposomes with triethylammonium trapped 0.65 N solutions (TEA) or inositol hexaphosphate triethanolammonium (TEOA) salts (IHP, phytic acid) or sucrose octasulfate were prepared and loaded with CPT-11 at 500 g / molar of phospholipid following 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, the mice were anesthetized and bled by cardiac puncture. Blood was collected, analyzed by HPLC for blood plasma CPT-11 content as described in Example 68, and the amount of drug was expressed as% of the injected dose remaining in the blood (% DI). TEOA-IHP was less effective at improving drug longevity in the blood than TEA-IHP, TEOA-SOAS, and TEA-SOS (Table 39).

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183

<td colspan="2">Table 39. CPT-11 remaining in blood 24 hours after</td>

<td>intravenous administration</td><td>of CPT-11 liposomes in</td>

<td colspan="2">mice.</td>

<td>Intraliposomal agent of</td><td>% Dl remaining in blood</td>

<td>drug capture</td><td></td>

<td>TEOA-IHP</td><td> 2,74 ± 0,54</td>

<td>TEA-IHP</td><td> 5,86 ± 0,20</td>

<td>TEOA SOS</td><td> 7,03 ± 0,17</td>

<td>TEA-SOS</td><td> 11,32 ± 0,46</td>

<td>Example 74. Loading the</td><td>liposome-containing drug</td>

1.05 N Diethylammonium sucrose octasulfate

The 1.05 N aqueous solution of diethylammonium sucrose octasulfate (DEA-SOS), pH 6.0, osmolarality of 727 mmolar / kg, was prepared using the ion exchange / titration method of Example 6 using pure diethylamine (99 , 5% purity). The 3 molar parts lipid matrix of DSPC, 2 molar parts of cholesterol and 0.015 molar parts of PEG2000-DSPE was formulated in liposomes (92.4 nm volume weighted average size) in the presence of DEA-SOS solution, and the CPT-11 was loaded onto liposomes at various drug / lipid entry ratios using the method of example 11. Unencapsulated drug was removed by gel chromatography, and the amount of encapsulated drug per lipid unit (drug / lipid output ratio) was determined. Encapsulation efficiency was calculated as% of the drug / lipid output ratio to the input ratio. Results are shown in table 40. The loading achieved is the maximum level of about 1.76 molar drug per molar phospholipid (1.67-1.70 molar drug / g total lipid), which is in good agreement with the amount (1 .78 molar of

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184 diethylammonium / phospholipid molar) based on the diethylammonium content of liposomes, assuming a stoichiometric exchange of intraliposomal diethylammonium ions by the drug molar molecules, and estimated trapped intraliposomal volume of approximately 1.71 / molar phospholipid.

Table 40. Loading of CPT-11 into liposomes from

DSPC / Col / PEG-DSPE containing 1.05 N DEA-SOS.

<td>Drug / lipid entry ratio, molar / g</td><td>Drug / lipid exit ratio, molar / g</td><td>Encapsulation efficiency,%</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> 6 9,6 ± 4,3</td>

Unless otherwise indicated, analytical data are presented as the mean ± standard error of the triplicate series. Pharmacokinetic data in rat plasma are the mean ± standard error of the duplicate series.

Lisbon, April 11, 2017