Esters of l-carnitine or alkanoyl l-carnitines useful as cationic lipids for the intracellular delivery of pharmacologically active compounds.

Description

The invention described herein refers to the class of novel esters of L-carnitine and acyl L-carnitine and their use as cationic lipids which are suitable for supporting intracellular transport of pharmaceutically active compounds, by using them through membrane transfer, or to promote their interaction with certain cell sites (receptors).

The invention described herein also relates to other known esters of L-carnitine and acyl L-carnitine, which are useful for the same purpose as the novel compounds mentioned above.

What is meant by the term "intracellular transport" is the transmission of the genes with a gene or a combination of natural origin or alteration, given by therapeutic activity (gene transfer) or the introduction of drugs or immunogenic peptides into the cells.

Many of the medicinal products, such as, for example, polypeptides and proteins or drugs generally need to enter (the cells to apply their function at the subcellular or molecular level. For these molecules, the cell membrane consists of a selective inhibitor. The cell membrane effectively performs protection , which prevents the entry of potentially toxic substances, but also leads to drug-bearing compounds. The complex composition of the cell membrane comprises phospholipid, glycolipid and protein; its effects are influenced by factors such as Ca⁺⁺ and other ions, ATP, microorganisms, microbes, enzymes and proteins that bind Ca⁺⁺. The interaction between the structural and peripheral factors of the cell and the response to external signals is responsible for the selectivity shown and among many different cellularities. The hemispheric effect of the membranes can be overcome by combining material in complexes with lipid formulations that reconstruct the composition of membrane membranes that occur in nature. These lipids have the ability to unite with the heavens and release the substances that are combined with them into the cells. The lipid complexes not only compensate for intracellular delivery through the membrane, but also minimize load loss between the molecules that need to penetrate into the cell. Dissoluble lipids, such as the phospholipid membrane, form lipids or lipids in the aqueous systems.

Liposomes are bleeds where the volume of water is completely closed with one or more membranes composed of lipid molecules, usually phospholipids. Phospholipids, consisting of hydrophobic head and pair of carbon chains (hydrophobic halves), are the main components of biological membranes. in the aqueous solution connect to the hydrophobic halves to exclude water, while the hydrophobic headers interact with the medium, which suddenly form a number of different diameter leaves. The lipids are usually bi-ionic, neutral or anionic. These blisters can be used as ferrous drugs, small molecules, proteins, nuclei and plasmids.

In recent years, cationic liposomes, a category of positively charged cysts made from synthetic lipids, have been used extensively for the introduction of genetic material into the cells. Negative loading DNA can interact with positive cationic lipid ligands, forming a stable DNA-lipid complexion. The simplicity and versatility of this technology has made liposomes an important ferry for the distribution of genes for gene therapy in human subjects. Today, most of the genotypes used for gene therapy, and approved by NIH's recombinant advisory committee, for virus systems and ready-made systems.

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Viral infection involves a series of complex processes in order to assault a particular cell and carry the DNA into the nucleus. The basic prerequisite for the use of viral viruses is based on the possibility of replacing the viruses with gene coding for the treatment mode without removing the virus's ability to infect cells. Viral therapy limits contain those viral factors that can be immunogenic, cytokine-dependent and cause rearrangement.

High hopes are attached to the use of cationic lipids in gene therapy. These genetic features have great potential compared to those of biological origin, as they are much safer, less toxic and also have the ability to implement large-scale genes.

However, as compared to biotype genotypes, they still have low yield on intracellular gene recoding. However, it should be remembered that the use of such genome switching systems is at the initial stage of research. Cationic lipids play a very important role in the formation of the DNA lipid complex, in cellular interaction, in merging with the membrane, in the release of DNA within the cell and in transcription.

There are important examples of the use of cationic lipids in living bodies. The first clinical trial of genetic therapy was performed by implementing an expression vector containing the human liposome-complex HLA-B7 gene for melanoma melanoma. Another important use refers to the treatment of pulmonary fibrosis in the lungs through a respiratory tract or as a nasal spray on the liposome-complexed expression vector SV-40C-FTR. Other clinical studies involving the use of liposomes in cancer treatment are currently underway.

Four basic elements are usually identified by the construction of cationic lipids: the positively charged cationic head, the spacer, the festulipid and the linkage.

The cationic head is responsible for the interaction between cationic lipids and DNA, in between

DNA liposome complex and cell membrane and other parts of the cell. It consists of single or multicandionic groups (which depends on the number of loads) that can be varied.

The spacecraft is part of the molecule that separates the cationic head from the hydrophobic tail and contributes to ensuring the maximum connection between the cationic haze and the negative charge of the DNA phosphates.

The festulipid is the nonpolar hydrocarbon component and determines the physical properties of the double lipid layer, such as the rigidity and rate of interchange of membrane glycol. What is meant by a "linker" is the connection between the hydrocarbon chain and the leaving of the molecule. This interface determines chemical stability and the biological degradability of cationic lipids.

In recent years, the use of liposomes has increased steadily in the cosmetics industry. The success of the liposomes in this field is due to the fact that these compounds are well tolerated by the skin. They are used both as ferries for active ingredients and as compounds that support later absorption.

Published science and patent materials are full of references to the production and use of liposomes; However, there are very few references describing the use of carnitine derivatives useful in gene distribution, as there are no documents dealing with known forces in the proliferation of compounds that resemble slightly those consistent with the invention described. is here.

Patent Application EP 0 279 887 describes the use of a derivative of carnitine, i.e. phosphatidyl carnitine, optionally in combination with other phospholipids and lipids (cholesterol, phosphatidylkolfni, phosphatidylserin), in the production of liposomes.

In the examples that relate to liposuction, lipoproteins of phosphatidyl carnitine are produced as propranolol prodrug, medicines known to be potent as hypertensive agents, cardiovascular and cardiac arrhythmias. The carnitine derivative is used here because of the apparent cardiac dysfunction of carnitine. This tendency makes it possible for the force of the liposomes to be broken down rather than power reach the desired destination.

Viflurist phosphatidyl carnitine also results in a large amount of power giving the liposomes by mouth, the pairs of poles against the liposome parma.

In J. Med. Chem. 1998 Jun 18; 41 (13): 2207-15, there are described the esters of L-carnitines which are used for gene distribution, but no disclosure enhancing enhancers for drug distribution are disclosed.

EP 559 625 B1 discloses a wide range of esters of L-carnitine and acyl carnitine which possess the functional interleukin activity of the gastrointestinal tract.

WO 96/39193 discloses innovative targeting agents which direct power into a catalytic converter through carnitine-acyl carnitine tract phase keratin, but does not indicate the usefulness of potentiophageal leukocytes.

As previously mentioned, catechologic liposomes are widely used in the intracellular distribution of drug-containing compounds, including the use of transfer through the membrane enhancing the effect of the tumor effect on a particular cellular membrane (VFL).

On the swing of drug distribution, there is a strong sense of pflrf for stable, repeatable seat-specific systems that are also active after a very long period of time.

Now, there has been evidence that the force of cationic lipids which are highly active agents enhances intracellular distribution of drug-containing compounds consisting of the use of esters of L-carnitine and the acyl L-carnitine of formula (II) for the propagation of a drug for the transport of drugs .

These new compounds are stable and highly selective because of the importance of reaching the target organ.

This feature makes the pau very useful for transferring active compounds directly to the pile of pairs that can be used by their pharmacist.

The compounds of general formula (II) are now known as other types of use (EP 559 625 as indicated above).

The compounds of Formula (II) are L-carnitine esters, which are useful in liposomes that possess fl uffic activity of drug distribution, and show signs of hypotension and selectivity of vasculitis.

These compounds have general formula (II):

<img file="IS2539B_D0002.tif" />

where:

R₃ is a saturated straight or branched chain alkyl group having 4-26 carbon atoms;

R₄ is a saturated or unsaturated, straight or branched alkyl chain, with 4-26 carbon atoms; and X is an anion of pharmaceutically acceptable acid.

An ideal example of R₃ are nonanoyl, dodecanoyl, myristoyl, palmitoyl or streroyl. An ideal example of R₄ are nonyl, undecyl, tetradecyl, hexadecyl or oleyl.

Examples of specific compounds of formula (II), according to the invention described herein are:

- palmitoyl L-carnitine chloride undecyl ester (ST 983);

- streroyl L-carnitine chloride undecyl ester (ST 1055);

- slerenoyl L-carnitine chloride tetradecyl ester (ST 1351);

palmitoyl L-carnitine chloride tetradekyl ester (ST 1379);

- Mititol L-carnitine chloride tetradecyl ester (ST 1380);

- palmitoyl L-carnitine bromide hexadecyl ester (ST 1390);

What is meant by anion of pharmaceutically acceptable acid is any anion an acid that does not cause undesirable toxicity or side effects.

These acids are well-known pharmacists and experts in the field of pharmaceutical technology.

The case of these anions, but not only those listed herein, are: chloride; bromide; iodide; aspartate; sýruastpartat; citrate; acid citrate; acid tartrate; phosphate; acid phosphate; fumarate; glycerophosphate; glycerophosphate; glucose phosphate; lactate; maleate; acid maleate; mucate; orotate; oxalate; sýruoxalat; sulfate; trichloroacetate; trichloroacetate; methanesulfonate; pamate and syrupamate.

The liposomes containing the compound of formula (II) are prepared using conventional methods, well-known persons of ordinary skill in the art; see, for example, Allen TM Drugs 56, 747-56 (1998). Lipomycin according to the present invention may also be prepared by using other factors well known in the art of lipoprotein technology. In one embodiment of the present invention, the liposomes may contain a helpful lipid, a term well known in the art. A case of helper lipid are cholesterol, 1-palmitoyl-2-oleoyl phosphatidylcholine, edioethylphosphatidylcholine.

The liposomes according to the present invention are in a yielding manner as a combination. In the embodiment depicting the distribution of drug-containing compounds, the compositions are separated as pharmacologically, which may comprise pharmaceutical vehicles and / or auxiliaries.

Preferred compounds of formula (II), namely ST 1380 and ST 1390 are known and described in the above-referenced article.

J. Med. Chem. 1998 Jun 18; 41 (13): 2207-15, which utilizes a proliferation of liposomes for genital mutilation, which has never been described as useful mutations for the proliferation of liposomes for administration.

A skilled person with experience in the field of pharmaceutical formulations clearly identifies the inheritance that arises from the production of drug libraries with liposomes. In fact, it is impossible to determine whether the liposome used for gene delivery is useful for drug distribution due to the number of problems that pea power overcomes to power getting lipids that have been forced to form a drug with drugs and which will spread to the organs that produce a peel force show their leakage activity.

Compounds of formula (II), in the form of liposomes, are useful in the distribution of drugs, such as, for example, anticancer drugs, antivirals, antivirals, antibiotics, fungicides, protozoa drugs, enhancing drugs that are useful in cardiovascular surgery. and vascular diseases, enhancing immunogenic peptides, and many drugs that are effective in migraine.

The mentioned liposomes may consist of auxiliary lipids.

Lipids composed of compounds of formula (II) may be administered by oral, intravenous, intravenous, subcutaneous, intradermal, nasal enhancers.

EXAMPLE 1

7-benzyloxyiminomethyl-cocotothecin (OPT 172)

500 mg (1.33 mmol) of 7-formyl camptothecin are dissolved in 100 ml of ethanol. 15 ml of pyridine and 638 mg (4 mmol) of O-benzylhydroxylamine hydrochloride is added. The solution is refluxed for 5 hours. The solvent is an evaporator in a ventilator and the resulting residue is purified by flash chromatography on silica gel using power using 4: 6 volumes of hexane / ethyl acetate as a ferrite phase.

Yield: 65% bm: 200-205 ° C degrees.

The resulting product comprises about 8: 2 of two syn and anti stereoisomers (stereoisomeric A: rt 0.32, stereoisomer B, rt 0.19, Merck 60 F254 silica gel; Ferfla phase: hexane: ethyl acetate 3: 7) .

HPLC: The analysis was performed on a device that was equipped with HP 1050 with a Rheodyne Injection Valve (20 μl loop) and Diode Voltage (HP 1050) drive with HPLC-ChemStation software. The collection of chromatography was measured from 200 to 600 nm and the chromatography registry recorded 360 and 400 nm.

C18 reverse phase column (Rainin C18; 25x0.4 cm, Varian) was used with an RP18 compound. The analysis was performed by linear elution level starting from acetonitrile water 30:70 to acetonitrile 100% in 20 minutes at a flow rate of 1 ml / min. The time ranges were: 12.51 min for ambient B and 14.48 mm for ambient A

¹1 H-NMR (300 MHz; DMSO-d₆): δ: 0.88 (t, H₃-18A + H₃-18B), 1.87 8m, (H₂-19A + H₂-19B), 5.18 (s, H₂-5B), 5.21 (8s, H₂-Ph B), 5.30 (H₂-Ph A), 5.40 (s, H₂-5A), 5.45 (s, H₂-17 + H₂-17B), 6.53 (s, -OH A + -OH B), 7.3-7.6 (m, ArA + Ar B, H-14A, H-14B), 7.75 (m, H-11A + H-11 B), 7.85-7.95 (m, H-10A + H-1OB), 7.98 (dd, H-12B), 8.18-8.27 (m, H-12A + H9-B), 8.45 (s, CH = NB), 8.59 (dd, H-9A), 9.38 (s, CH = NA).

Mass m / z 481 (M⁺ 100) 374 (30) 330 (70) 300 (30) 273 (20) 243 (20) 91 (34).

EXAMPLE 2

7-butoxyiminomethyl combocothecin (CPT 184)

400 mg (1.06 mmol) of 7-formylkamocytes are dissolved in 80 ml of ethanol. 12 ml of pyridine and 400 mg (3.18 mmol) of Ot-butylhydroxylamine hydrochloride are charged. The solution is refluxed for 4 hours. The solvent is concentrated in vacuo and the resulting residue is purified by flash chromatography on silica gel using a 4: 6 mixture of hexane / ethyl acetate as a mobile phase.

322 mg (0.72 mmol) of yellow solid are obtained.

Yield: 68% bm: 250 ° C degrees.

The product obtained consists of approximately 8: 2 mixture of two syn and anti-stereoisomers (stereoisomer A: rt 0.31, stereoisomer B, 0.24, Merck 60 F254 silica gel; Tripod:

hexane: ethyl acetate 3: 7).

HPLC: The assay was performed by a four-dose (HP 1050) vasodilator with a Rheodyne injection valve (20 μl loop) and the diode fillers (HP 1050) run with the HPLC-ChemStation software. Spectrum collection was made from 200 to 600 nm and chromatographic chromatography was recorded

360 and 400 nm.

C18 reverse phase column (Rainin C18; 25x0.4 cm, Varian) was used with an RP18 compound. The analysis was performed by linear elution level starting from acetonitrile: water 30:70 to acetonitrile 100% in 20 minutes at a flow rate of 1 ml / min. The rhythm times were: 12.92 min for ambient B and 14.61 mm for stereochemistry A.

¹1 H-NMR (300 MHz; DMSO-d₆): δ: 0.88 (t, H₃-18A + H₃-18B), 1.30 (s, t-but.B), 1.47 (s, t-but.A), 1.87 (m, H₂-19A + H₂-19B), 5.18 (s, H₂-5 B), 5.37 (H₂-5 A), 5.42 (a, H₂-17 + H₂-17B), 6.54 (s, -OH A + -OH B), 7.35 (s, H-14A), 7.36 (s, H-14B), 7.69-7.83 (m, H-11A + H-11B), 7.85-7.98 (m, H-10A + H-10B), 8.07 (dd, H-9B), 8.16-8.27 (m, H -9A + H-12B) 8.40 (s, CH B), 8.62 (dd, H-12A), 9.31 (s, CH A).

Mass m / z 448 (M⁺ 28) 391 (40) 374 (100) 362 (40) 330 (34) 57 (17).

PRODUCTION ON FITUKORNUM

The known compounds can be used to prepare multifarious nitrous oxide (MLV) and monofilament (SUV), both in the form of dry powder and as aqueous suspension.

The compounds are used to prepare liposomes according to the following method. The dissolved amount of the compound is dissolved in chloroform, the solution is concentrated in vacuo to rotate in a rotary evaporator until liposuction is obtained. The liposuction is purrkud in the presence of the air until the clearest residue of solvent has been removed and then dissolved in tert-butyl alcohol or with water. The resulting solution is frostbite, which gives a soft dry powder.

Dust time is extracted with a suitable amount of aqueous solution, which gives the fat of the compound used, which is then complexed with the polynucleotide or with the desired drug.

Another method of manufacturing liposomes consists of collecting a lipid film comprising a solvent according to the invention in a solvent, having inactive adjuvants such as sorbitol, mannitol, and pharmaceutically acceptable carbohydrates. The mixture is dried at an aerodynamics, which gives a substance that can last easily and very well before use.

Combinations in the form of dry dust show the benefits of being stable over a long period of time, and are in use.

Further, the compounds according to the invention may be used to prepare lipids contained in the desired drug, in the form of dry powder, according to the following behaviors. A compound of the invention is dissolved in tert-butyl alcohol with water; The resulting solution is mixed with a driped drug and is a mixture of freeze-dried powder, a complex of complexes that can be defined as anti-corrosive drugs, in the form of a soft, powdered powder.

Powdery dust can be used for the preparation of pharmaceutical formulations that can be administered by air cushion, which can then be reconstituted with water with a suitable buffer solution, which can be administered by mouth by mouth.

Solid carcasses can also be obtained with the behavior of lipid pooling on inert solids such as sorbitol, mannitol or other carbohydrates using the above-described behaviors.

Test for the formation of lipids

The formation of the liposomes was tested by photoconductivity using a water-soluble pigment, according to the following behavior. Aqueous solution of water-soluble Arsenazo III was obtained (molar mass = 776.37: 2.3 mg / mL).

This solution was used in the city of water for adipose lipids, which came from the production mentioned above.

A dose of suspension containing fitukomide with the incorporated pigment was painful

10O-fold with water.

Two mL of liposomal suspension were used to obtain the first reading intensity at 660 nm; the reading was obtained in combination with an equivalent sample, which was defined as a zero sample. 200 μl of CaCl₂ solution (15 mg / mL; 100 mM) was added to the first sample and the optical density measured at 660 nm to the zero sample, which was loaded with 200 μl of water. The resulting digestion time was considered as reading 2. Continue to add 100 μl of a solution of Triton X-100 (5% v / v, 0.26% final concentration) and 200 μl of water in the blank sample; The optical density flux at 660 nm gave the light density determined as reading 3. For the calculation of the percentage of embedded dyes, the following formula was used:

_. ,. , Reading3-reading2x100% of color that is internal reading = reading 3

The percentage of ink dyes gives a measurement of the formation of the liposomes and has an average of about 40%: the concentration of liposomes was performed by using a laser dissolution with a positive outcome.

EXAMPLES OF PRODUCTION ON FITUKORNUM

Production of palmitoyl L-carnitine chloride undecyl ester (ST 983) in the form of:

a) lyophilised powder

65 mg, 0.11 mmol of palmitoyl L-carnitine chloride undecyl ester were dissolved in 20 mL of chloroform, in a 100 mL flask.

The solution was evaporated until lipid was obtained which was dried in vacuo for 3 hours. The thus obtained was dissolved in tert-butyl alcohol and this solution was rapidly cooled at -70 ° C with liquid curing agent and freeze drying for 24 hours.

A fungus duty white solid was obtained.

b) Ásogad dufts

143 mg, 0.231 mmol of palmitoyl L-carnitine chloride undecyl ester were dissolved in 10 mL of chloroform. The resulting solution was poured into small volumes of 100 mL flask containing 750 mg sorbitol. At the end of different bulk chloroform solutions, the chloroform was evaporated.

The resulting solid was dried at room temperature for 3 hours.

893 mg of white solid product was obtained.

Before use, vdtnud product is supplied with a suitable volume of water to obtain an isotonic solution.

c) MLV suspension mg, 0.11 mmol of palmitoyl L-carnitine chloride undecyl ester were dissolved in 20 mL of chloroform, 100 mL flask.

The resulting solution was evaporated until the lipid layer was evaporated which was then dried in vacuo for 3 hours.

The lipid skin was watered with 10 mL of water at 30 ° C for 3 hours to give MLV suspension.

The MLV suspension, diluted appropriately, was administered by drug and used in biological analysis.

a) SUV suspensions mg, 0.11 mmol of palmitoyl L-carnitine chloride undecyl ester were dissolved in 20 mL of chloroform, 100 mL flask.

The resulting solution was evaporated until lipid was obtained which was then dried at ambient temperature for 3 hours.

The lipid coating was watered with 10 mL of water at 30 ° C for 3 hours to give MLV suspension.

The MLV suspension was pressed 10 times through a polycarbonate filter with a mesh size of 200 nm. The single-layered liposomal suspension thus obtained was placed in a drug drug and used in biological analysis.

b) Test of physical stability of the liposomes

The etchological stability of the liposomal suspension was tested using a turbidity analysis over a period of 30 days.

Aperture measurement at 600 nm for certain time points was calculated for each suspension to be tested. Medalgleypnigidid was measured at time 0 was stable for all combinations tested.

The compounds contemplated showed a residual value over the period of thought.

MLV and SUV liposomal solutions can be prepared by formulating compounds of the invention with helper lipids such as cholesterol, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) edoleyl dioleyl phosphatidyl carbon (DOPE).

The compounds are assembled with help lipids for the purpose of obtaining liposomes with resistant membranes. Hereinafter, the section describing the progression of liposomes is provided by a construct, wherein the compound according to the invention is assembled with helper lipid such as POPC.

EXAMPLE OF MANUFACTURING PRODUCTS FOR SUPPLIERS

EXAMPLE 3

Frontalization of taxol-ST 983 MLV fatty acids (1:40) mg, 0.0234 mmol of taxol and 556 mg, 0.9417 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was concentrated until liposuction was obtained on the surface of a glass bottle.

After removal of the final residues of chloroform with the aid of a vacuum pump, 20 mL of tertbutyl alcohol was added to the lipid film and the resulting solution was divided into 19 doses which were immediately frozen at -70 ° C with flashy nitrogen and lyophilized for 24 hours. Each dose of solid contained taxol (1.05 mg) and ST 983 (29.2 mg).

To obtain a complete liposomal suspension, the lyophilised product was watered for use with water (450 L) or other saline solutions, stirred for 10 min and allowed to wait for 30 minutes to allow the swelling process to run out.

MLV liposomes were obtained.

Test of physico-chemical stability of production

Physical stability of the production was tested using turbidity by collecting TDC (operating time curve) at 800 nm at 20 ° C for 20 hours.

Continuous turbidity, indicating production stability, was obtained, with no precipitation phenomenon.

Test of chemical stability of taxol in production

Chemical stability of taxol was tested by HPLC.

Color separation conditions were as follows:

Sula: pBondapack C-18 Tripod: acetonitrile water 70:30 Nemi UV-VIS 227 nm Flip rate: 1 mL / min

Reaction time: 4.5 mm

Taxol concentration, determined against standard, was 2.13 mg / mL.

The percentage of incorporated taxol was 98%.

Example 4

Proliferation of taxol-ST 983 SUV liposomes mg, 0.0234 mmol of taxol and 556 mg, 0.9417 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was a reinforced pair of lipid skin obtained on the surface of a glass bottle.

After removal of the final residues of chloroform with the aid of vacuum pump, 20 mL of tert-butyl alcohol was added to the lipid film and the solution obtained was divided into 19 doses frozen immediately at -70 ° C with liquid nitrogen and frost pads for 24 hours. Each dose of solid contained taxol (1.05 mg) and ST 983 (29.2 mg).

To get a completed SUV liposomal suspension, watered with PBS solution (1 mL), it was heard for 20 minutes at 0 ° C.

The operation was carried out at 400 nm to remove distant residues of titanium released from the sound source.

Test of editable performance of the production

The editable performance of the production was tested using a turbidity measurement with TDC (operating time curve) at 800 nm at 20 ° C, 20 hours.

Adequate turmoil, indicating the supportability of the production, was obtained, with no exclusionary phenomenon.

Test of chemical stability of taxol in the production

The chemical stability of taxol was tested by HPLC.

Color separation contrasts were as follows:

Sula: pBondapack C-18 Ferdaphase: acetonitrile water 70:30 Nemi UV VIS 227 nm Flow rate: 1 mL? Min Ratchet time: 4.5 mm

HPLC analysis of the SUV liposomal suspension gave the same nidastöd and the corresponding MLV liposomal suspension, and in this case, also, the percentage of incorporated taxol was 98%.

The HPLC analysis repeated after 24 hours showed no new ADRA levels than the taxol peak, indicating the supportability of the active ingredient.

EXAMPLE 5

Proliferation of taxol-ST 983-cholesterol liposomes (1:15)

These fungus flakes were prepared to get a stick with a constant sky.

mg, 0.0101 mmol of taxol, 62.2 mg, 0.105 mmol of ST 983 and 40 mg of cholesterol were dissolved in 10 mL of chloroform.

The solution obtained was strengthened pair to lipid skin obtained on the surface of a glass vial.

After removal of the final residues of chloroform with the aid of vacuum pump, 6.3 mL of tertbutyl alcohol was added to the lipid film and the solution obtained was divided doses immediately frozen at -70 ° C with flashing nitrogen and freeze-dried for 24 hours. Each dose of solid contained taxol (1.2 mg) and ST 983 (12.44 mg) and cholesterol (8 mg).

To obtain liposomal suspension, the lyophilised product was watered for 10-minute use (1000 μί) or other saline solutions, stirred for 10 minutes and allowed to wait for 30 minutes to allow the swelling process to run out.

MLV liposomes were obtained.

Test of physical stability of survival

The physical stability of the survival was tested using turbidity measurement by collecting TDC (drift time curve) at 800 nm at 20 ° C for 6 hours.

Continuous sluggishness, indicating stability of the survival, was obtained, with no precipitation phenomenon.

EXAMPLE 6

Proliferation of CPT 83-ST 983 MLV liposomes (1:40)

6.3 mg, 0.0168 mmol of CPT 83 (7-carbonitrile camptothecin, described in WO 97/31003) and 400 mg, 0.667 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was a reinforced pair to a lipid coating obtained on the surface of a glass bottle.

After removal of the final residues of chloroform with the aid of vacuum pump, 26 mL of tert-butyl alcohol was added to the lipid film and the solution obtained was divided 12 doses immediately frozen at -70 ° C with flash chromatography and freeze-dried for 24 hours. Each dose of solid contained CPT 83 (0.525 mg) and ST 983 (33.33 mg).

In order to obtain a liposomal suspension, the freeze-dried apricot was extracted with a large amount of water for use with water (1000 L), stirring several saline solutions and stirring for 10 min.

MLV lipstick was obtained.

Test of physical stability of survival

The physical stability of the survival test was tested by force using turbidity measurement using TDC (drift time curve) torque 800 nm, vial 20 ° C, for 6 hours.

Continuous sluggishness, indicating stability of the production, was obtained, with no precipitation phenomenon.

Test for chemical stability of CPT 83 in the production.

Chemical stability of CPT 83 was tested by HPLC.

Color separation contrasts were as follows:

Sula: Supelcosil LC-ABZ

Ferdaphase: Phosphate Stud Powder 20 mM: Methanol 40:60, pH = 7.3 Nuclear UV VIS: 360 nm Flow Rate: 1 mL / min Fasting Time: 4.033 min

CPT 83 The concentration, determined against the state, was 0.502 mg / mL.

The percentage of internal CPT 83 was 99%.

EXAMPLE 7

Production of CPT 83-ST 983 SUV lipstick (1:40)

6.3 mg, 0.0168 mmol of CPT 83 and 400 mg, 0.667 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was concentrated until lipid coating was obtained on the surface of a glass bottle.

After removal of the final residues of chloroform with the aid of a vacuum pump, 26 mL of tert-butyl alcohol was added to the lipid film and the resulting solution was divided into 12 doses frozen immediately at -70 ° C with liquid nitrogen and freeze drying for 24 hours. Each dose of solid contained CPT 83 (0.525 mg) and ST 983 (33.33 mg).

To get a complete liposomal suspension, watered with water (1000 pL), she was listening for 20 minutes at 0 ° C.

Then a 400 nm filter was applied to the removable left-handed titanium residue.

Test for chemical stability of CPT 83 in the production

Chemical stability of CPT 1B4 was tested by HPLC

Color separation contrasts were as follows:

Sula: Supelcosil LC-ABZ

Ferdaphase: phosphate buffer buffer 20 mM: methanol 40:60, pH 7.3

Nemi UV VIS: 360 nm Flow Rate: 1 mL / min Runtime: 4.033 min

The CPT 83 concentration, as opposed to standard, was 0.3 mg / mL.

The percentage of CPT 83 incorporated was 59%.

HPLC analysis repeated after 24 hours showed no new peaks other than the peak for OPT 83, indicating stability of the active ingredient.

Test of physical stability of the production

The physical stability of the production was tested using turbidity measurement by collecting TDC (operating time curve) at 600 nm at 20 ° C for 6 hours.

Continuous turbidity, indicating production stability, was obtained, with no precipitation phenomenon.

EXAMPLE 8

Production of CPT 184-ST 983 MLV liposomes (1:40)

7.29 mg, 0.0168 mmol of CPT 184 and 400 mg, 0.677 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was concentrated until lipid skin was obtained on an overboard glass bottle.

After removal of the last residues of chloroform with the aid of vacuum pump, 26 mL of tert-butyl alcohol was added to the lipid film and the resulting solution was divided into 12 doses frozen immediately at -70 ° C with liquid nitrogen and freeze drying for 24 hours. Each dose of solid contained CPT 184 (0.607 mg) and ST 983 (33.33 mg).

To obtain a liposomal suspension, the freeze drying was evaporated on use with water (1000 mL) and salted saline solution for 10 minutes.

MLV lipstick was obtained.

Test of physical stability of the production

Physicochemical stability of the production was tested by using turbidity measurement by collecting TDC (operating time curve) at 600 nm at 20 ° C for 6 hours.

Adequate turmoil, indicating the sustainability of the production, was obtained, with no precipitation phenomenon.

Test for chemical stability of CPT 184 in the production.

Chemical stability of CPT 184 was tested by HPLC.

Color separation conditions were as follows:

Sula: Supelcosil LC-ABZ

Fermentation: phosphate buffer 20 mM: methanol 40:60, pH = 7.3 Nemi UV VIS: 360 nm Flow rate: 1 mL / min Reaction time: 25.5 min

The CPT 184 concentration, determined versus standard, was 0.600 mg / mL.

The percentage of CPT 184 incorporated was 99%.

EXAMPLE 9

Production of CPT 184-ST 983 SUV Fits (1:40)

7.29 mg, 0.0168 mmol of CPT 184 and 400 mg, 0.677 mmol of ST 983 were dissolved in 20 mL of chloroform.

The solution was concentrated until liposuction was obtained on the surface of a glass bottle.

After removal of the final residues of chloroform with the aid of a vacuum pump, 26 mL of tert-butyl alcohol was added to the lipid film and the resulting solution was divided into 12 doses frozen immediately at -70 ° C with liquid nitrogen and lyophilized for 24 hours. Each dose of solid contained CPT 184 (0.607 mg) and ST 983 (33.33 mg).

To obtain a liposomal suspension, the lyophilised product was watered for use with water (1000 pL).), It was allowed to stand for 40 minutes at 0 ° C.

Filtration was then performed on a 400 nm filter to remove residual titanium released by the silencer.

Test for chemical stability of CPT 184 in production

Chemical stability of CPT 184 was tested by HPLC.

Color separation conditions were as follows:

Sula: Supelcosil LC-ABZ

Fermentation: phosphate buffer 20 mM: methanol 40:60, pH = 7.3 Nemi UV VIS: 360 nm Flow rate: 1 mL / min Operating time: 25.5 min

The CPT 184 concentration, determined versus standard, was 0.36 mg / mL.

The percentage of CPT 184 incorporated was 70%.

HPLC analysis repeated after 24 hours showed no new peaks other than the peak for OPT 83, indicating stability of the active ingredient.

Test of physico-chemical stability of production

Physical stability of the production was tested using turbidity by collecting TDC (operating time curve) at 600 nm at 20 ° C for 6 hours.

Continuous turbidity, indicating production stability, was obtained, with no precipitation phenomenon.

In the following examples, lipoproteins were prepared using helper lipid and / or lymphocytes.

DÆM110

Production of CPT 184-ST 983 liposomes in 2 bottles, 100 ml of methyl chloroform was added to 20 mg CPT 184 and 600 mg of ST 983 and the mixture was heated slightly to obtain full resolution. The resulting solution was concentrated in a rotary evaporator until liposuction was obtained, which was dried by a high-vacuum pump. The lipid skin was watered with a lactose solution (6 g / 300 ml water) at 45 ° C and separated by stirring in the precipitate for about 2 hours. The suspension was then sounded for 2 hours, each cycle lasting for half an hour. Afterwards, the solution was filtered through the 200 nm filter and the freeze drier.

Test for chemical stability of CPT184 in production

Chemical stability of CPT 184 was assayed by HPLC. The product was stable during a 24-hour test.

Test of ecological stability of production

The physical stability of the production was tested using turbidity measurement. The product was stable during a 24-hour test. The particle size was also stable (mean 100 nm).

EXAMPLE 11

Production of CPT 184-ST 983 liposomes

For 1 ml liposomal composition (POPC-1-palmitoyl-2-oleoyl phosphatidylcholine 5mM; ST 40 983 1.25 mM; CPT 184 0.25 and threalose 150 mM), the following procedure was used:

0.11 mg, 0.25 μl of CPT 184 were dissolved in 250 μl of ethyl acetate, 3.79 mg, 4.89 μmol of PCPC were dissolved in 100 μL ethanol and 0.74 mg, 1.25 μmol of ST 983 were dissolved 100 pL of ethanol. The three solutions were mixed with ring blending. The solvents were embedded with a rotary evaporator at room temperature, 80 mbar. The lipid skin was dried for two hours in the dark. The lipid skin was dissolved in 1 ml of 150 mM D (+) - threose dihydrate (Fluka, HPLC 99%) solution, sterilized through 0.22 nm and stirred for two minutes.

The suspension was pressed 21 times through 200 nm polycarbonate filters. The extruded liposomal suspension was brothed in liquid nitrogen and lyophilized for 2 nights. White solid was obtained.

DÆM112

Production of CPT 184-ST 983 liposomes

The same procedure as in Example 11 was used, except that 500 mM was used.

EFFECT ST 983 FITNUTS ON CERTAIN CRAZY CIRCUMSTANCES

As can be seen below, the ST 983 liposome has demonstrated controlling pooling of the lungs. This characteristic set-up technology has its use for the use of a mouse model for cancer of the lungs.

The cancer drug used in this experiment was taxol.

In order to cause tumors, unbreakable Balb / c mice received an injection of 3x10 '⁵lung cancer cells of mice M109 in 0.1 ml RPMI-i 640 (Sigma) in the femoral vein on the right back.

Ten days after tumor transplantation, liposome taxol was diluted diluted with phosphate-doped saline (PBS, SIGMA, P-4417) and injected intravenously at a concentration of 2.5 mg / mL of ST 983 and 75 μg / mL of taxol.

Taxol (paclitaxel INDENA) used as a solution was dissolved in a Cergelophor EL (BASF) ferry with a concentration of 20 mg / mL and stored at + 4 ° C for the next 24 24 hours, protected by light. At the time of use, it was diluted with phosphate-buffered saline (PBS, SIGMA) and injected intravenously with the same volume and concentration conditions as described for taxol distributed with the ST 983 liposome.

Chromophore was prepared by diluting 1: 1 with ethyl alcohol.

Administration was given for 7 adjoining days beginning on day 10 from the tumor saturation. The animals were monitored until day 17 after sowing and sacrificed by a rupture, and their lungs were removed to determine the number of sperm count. Coloring of the lungs to find the sperm was performed by incubating the lungs for 10 days in 5 ml of Bouin solution, which consists of 71% saturated acetic acid solution, 4.8% glacial acetic acid (Merck), and 24% 10% formaldehyde (Fluka). At the end of the incubation run in the Bouin solution, the number of sperm count was considered.

As compared to untreated standard mice, corophor-transported taxol showed no reductive effect on the number of pulmonary arteries, although they were less than those in unreacted norms, on the contrary, taxol in complex relationship with ST 983 showed a significant reduction in both the number and size of the pulmonary artery .

Statistical analysis of the number of pulmonary arteries was performed using Mann-Whitney non-randomized test for unpaired data.

The results obtained are shown in Table 1 below.

TABLE 1 Pulmonary artery on day 17 after M109 seeding in BALB / c mice after treatment with 10 taxol and taxol / fitukom ST983.

<td>group</td><td>sadmet Mean ± sf</td><td>sadmet size</td>

<td>benchmarks</td><td>21 ± 12</td><td>M</td>

<td>taxol</td><td>22 ± 9</td><td>S</td>

<td>Taxol-ST983</td><td>10 ± 2</td><td>S</td>

<td>M = mean (1-2 mm in diameter)</td><td></td><td></td>

<td>S = small (1-2 mm in diameter)</td><td></td><td></td>

Initial test of test tubes

Efficacy assays were performed on HeLa and M 109 cells in 96-well plates. On the day after placement, the cells were treated with the molecules that were to be tested for the next 48 hours. The cells were washed with PBS and left in normal growth conditions for 48 hours. After removal of the growth medium, the cells were incubated on ice with 16% TCA, washed 3 times in H₂O treated for 30 minutes with sulfurodamine BB (SRB) in 1% acetic acid, washed 3 times only in acetic acid, incubated for 20 minutes in TRIS 10 mM pH 10.5 and, finally, reading was taken at 540 nm.

Cell test with CPT 83

Cell cytotoxicity tests with CPT 83 were performed to evaluate the cytotoxicity of the anticancer drug with lipoprotein, as transient indications of efficacy.

In order to evaluate the lipotomy capability of moving CPT 83 into the test ί M109 cells, the sulfurodamine B test was used above.

In addition, cytotoxicity effects CPT 83 dissolved in dimethylsulphoxide (DMSO) were also evaluated for comparison with the same molecule of the ST 983 liposome.

Fitukorns CPT 83 complex was used in the cytotoxicity assays with the concentrations indicated in Tables 2.1, 2.2 and 2.3 below in both the SUV and MLV installations. The molar ratio of liposome. CPT 83 40: 1 was used.

The mean proliferative levels of ST 983-CPT 83 complexes in both SUV and MLV installations given in Tables 2.1.2.2 and 2.3 below indicate that the ST 983 liposome is capable of transferring CPT 83 in a similar manner to DMSO , show cell counts of the same magnitude.

TABLE 2.1 Efficacy (SRB) ST 983 SUV-CPT 83: Strength in μm

<td>benchmarks</td><td>1.3</td><td>12:13</td><td>0013</td><td>00013</td>

<td>0911</td><td>0294</td><td>0705</td><td>0908</td><td>0911</td>

<td>0745</td><td>0198</td><td>0525</td><td>0821</td><td>0.83</td>

<td>0884</td><td>0204</td><td>0801</td><td>0906</td><td>0.91</td>

<td>0833</td><td>12:25</td><td>0748</td><td>0856</td><td>0853</td>

<td>0854</td><td>0254</td><td>0778</td><td>0867</td><td>0873</td>

<td>0793</td><td>0231</td><td>0739</td><td>0802</td><td>0803</td>

<td>0792</td><td>0193</td><td>0602</td><td>0827</td><td>0829</td>

<td>0901</td><td>0248</td><td>0.69</td><td>0904</td><td>0.89</td>

<td>0.839125</td><td>0.352444</td><td>0.635333</td><td>0.767111</td><td>0.7667</td>

<td>0.060645</td><td>0.034513</td><td>0.092925</td><td>0.042054</td><td>0.040149</td>

The values ​​given in the table show in light density at 540 nm.

TABLE 2.2 Eitrogen Effects (SRB) ST 983 MLV-CPT 83: Strength ί μΜ

<td>benchmarks</td><td>1.3</td><td>12:13</td><td>0013</td><td>0.0013</td>

<td>0895</td><td>0.038</td><td>12:04</td><td>0095</td><td>0088</td>

<td>0.82</td><td>0.038</td><td>0046</td><td>0109</td><td>0124</td>

<td>0896</td><td>0041</td><td>0049</td><td>0128</td><td>0127</td>

<td>0847</td><td>0041</td><td>0042</td><td>0105</td><td>0115</td>

<td>0863</td><td>0041</td><td>0053</td><td>0111</td><td>0107</td>

<td>0794</td><td>0043</td><td>0041</td><td>0073</td><td>0095</td>

<td>0829</td><td>0039</td><td>0044</td><td>12:08</td><td>0085</td>

<td>0893</td><td>0041</td><td>0044</td><td>0064</td><td>0065</td>

<td>0.854625</td><td>0.04025</td><td>0.044875</td><td>0.095625</td><td>0.10075</td>

<td>0.038682</td><td>0.001753</td><td>0.004357</td><td>0.021738</td><td>0.021359</td>

The values ​​given in the table show in light density at 540 nm.

TABLE 2.3 Eitropic effect (SRB) DMSO-CPT 83: Strength ί μΜ

<td></td><td>viflmifl</td><td>13</td><td>1.3</td><td>12:13</td><td>0013</td><td>0.0013</td>

<td></td><td>0898</td><td>0281</td><td>12:33</td><td>.406</td><td>0.8</td><td>0809</td>

<td></td><td>0774</td><td>0267</td><td>0302</td><td>.407</td><td>0804</td><td>0816</td>

<td></td><td>0857</td><td>0.3</td><td>0285</td><td>12:57</td><td>0863</td><td>0886</td>

<td></td><td>0787</td><td>0286</td><td>0287</td><td>0383</td><td>0836</td><td>0841</td>

<td></td><td>0808</td><td>0285</td><td>0318</td><td>0474</td><td>0851</td><td>0863</td>

<td></td><td>0745</td><td>0288</td><td>0317</td><td>0467</td><td>0.79</td><td>0795</td>

<td></td><td>0775</td><td>0312</td><td>0328</td><td>0429</td><td>0806</td><td>0831</td>

<td></td><td>0864</td><td>0318</td><td>0305</td><td>0421</td><td>0.81</td><td>0878</td>

<td>Average</td><td>0.8135</td><td>0.292125</td><td>0309</td><td>0.444625</td><td>0.82</td><td>0.839875</td>

<td>sf</td><td>0.053519</td><td>0.016848</td><td>0.017205</td><td>0.059269</td><td>0.026506</td><td>0.033237</td>

The values ​​given in the table show in light density at 540 nm.

Lifestyle ST 983 liposome-CPT 184 fleece

The liver function of liposomes 10 (hereinafter referred to as liposomes A) and 12 of the body (for the aforementioned liposome B) were tested.

Digestive effect in healthy mouse

Liposomes A and B were given by intravenous administration to free CPT 184, with a dose of 1.2 mg / kg according to the q4dx4 schedule. The liposome two pillow does not significantly affect the body's body weight, lung, spleen and kidney. Liposuction B, intravenously, and liposome A, administered by mouth, affected the cervical gland in a similar manner to free CPT 184. Liposome A in a vein had only minimal effect. Blood disease levels did not show significant changes after 24 hours, for both liposomes. Liposuction A, Fifl In accordance with the qd5 scheme, toxicity was similar to those seen by free CPT 184.

Lung naleitni liposomes

Liposomes A and B showed a dominant pooling of the lungs. This characteristic set-up technology has supported its use in a mouse model for cancer of the lungs. Lipokornin was given in the blue-eye 1.2 mg / kg. Free CPT 184 was administered at about 1.2 mg / kg in DMSO. Animals, healthy mice were sacrificed 24 hours after the last administration. The lungs were removed from the bodies and frozen in fluorescent stimulants. After dilution, the organs were pooled and homogenized in 0.1% acetic acid / acetonitrile 1: 5. The equation was divided into three doses, of which two were CPT 184 for calculation of recovery. The three samples were forced for 16,000 g for 5 minutes.

The supernatant was collected and extracted with dichloromethane. The organic phase was dried with an evaporator and the residue was redissolved in acetonitrile to allow the amount to correspond to one animal to 50 L for loading on HPLC. HPLC was run on Waters Symmetry C18 3.5 (4.6x7.5 mm). Merck fluorescence meter was the sensor at 370 nm induction and 510 nm radios. The turf was water / acetonitrile 60:40, icecream. The volume of the sample was 50 μL. CPT 184 recovery was approximately 70%. Both lipids A and B gave a sampling rate for CPT 184 haer than for free CPT 184 in DMSO, as shown in Figure 1.