HU198079B - Process for producing hydrophobic cis-platinum complexes and incorporating them in liposomes - Google Patents

Abstract

According to the invention, platinum (11) complexes are characterized by the formula r3RaRt2, where and R2 is a carboxylate monoanion containing a hydrophobic group, or a dicarboxylate anion together with a hydrophobic group, R3 and R4 are NH3, allylamino or cycloalkylamino or together vicinal diaminoalkyl or vicinal diaminocycloalkyl. The complex is insoluble in water, soluble in methanol and chloroform and incorporated into phospholipid liposomes. The phospholipid-encapsulated platinum complexes thus obtained are useful for the chemotherapeutic treatment of tumors sensitive to platinum complexes.

Description

The present invention relates to a process for the preparation and incorporation of hydrophobic cis-platinum complexes into a liposome.

Cis-platinum (CDDP) is a high-potency drug for treating various neoplastic diseases in humans [Loeherer et al., Ann. Int. Med. 100, 704-713 (1984)]. However, its use is limited by severe systemic toxicity, in particular nephrotoxicity and neurotoxicilicity (Zwelling et al., 1982, Piatinum Complexes, Pharrheaeological Principles of Cancer Treatment, B.A. Chabner, Saunders, Philadelphia, PA). In order to improve the therapeutic index of CDDP, new derivatives have been synthesized over the last 10 years. However, the development of many promising analogs has been hampered by their low water solubility, which reduces their potential for clinical use (Burchenal et al., 1979, Cancer Treat, Rep. 63, 1493-1947),

Liposomes are lipid vesicles that are formed spontaneously when an aqueous solution is added to an anhydrous lipid film (Mayhew et al., Liposomes, 1983, by Maré J. Ostro, Marccl Dekker, Inc., New York). Liposomes can be used as drug carriers. for hydrophobic or hydrophilic drugs that are enclosed in a hydrophobic or hydrophilic compartment of liposomes. Multilamellar liposomes are multilayer lipid vesicles (MLV) that are particularly suitable for carrying hydrophobic drugs because of their larger hydrophobic compartment than their hydrophilic compartment. For intravenous injection into animals [Kasi et al., Int. J. Nucl. Med. 11, 35-37 (1984) Lopez-Berestein et al. (1) Cancer Drug Deliv. 1, 199-205 (1984)] and intravenous injection into humans [Lopez-Berestein et al. (2) Cancer Slit. 44, 375-378 (1984)] MLV is produced in the liver, spleen and other organs rich in reticuloendothelial cells (RÉS).

Liposomes have so far been used in vitro as chemotherapeutic agents (Mayhew et al., Liposomes Ostro, Marccl Dekker, Inc., New York, 1983), immunomodulators and antifungal agents [Mehta. et al., Immunology 51, 517-527 (1984)] and in in vivo animals (Lopez-Berestein et al. (4) Clin Exp. Metastasis 2: 127-137 (1984) and Lopez-Berestein et al., J. Inf-Dis. 147, 937-945 (1983)] and in humans (Lopez Berestein et al., J. Inf. Dis. 151: 794-710 (1985)].

In our research, we have found that liposomes can reduce the drug-related toxicity of some drugs, such as the cardiotoxicity of doxorubicin [Forssen et al., Proc. Natl. Acad. Sci., 78, 1873-1877 (1981), Olson et al., Eur. J. Cancer Clin. Oncol. 18, 167-176 (1982), Gabizon et al., Cancer Slit. 42, 4734-4739 (1982), Herman et al., Cancer Slit. 43, 5427-5432 (1983)] and nephrotoxicity of CDDP [Friese et al., Arch. Pharmacodynamic Thcrapic 258: 180-192 (1982)]. and may enhance antitumor activity by the slow release mechanism [Mayhew et al., Ann. Ν. Y Acad Sci., 308, 371-386 (1978), Patel et al., Int. 2

J. Cancer 34: 717-723 (1984)], as a result of increased drug uptake by tumor cells or as a result of more selective distribution in organs [Gabizon et al. 43, 4730-4753 (1983), Mayhew et al., Cancer Drug Deliv. 43-59 (1983)]. No. 4,330,534. According to patent U.S. ^N4 -acyl-cytosine arabinoside incorporated into liposomes when administered to animals it has been found to be effective in tumor treatment. Despite these promising results, the clinical use of liposome-encapsulated antitumor agents has not been achieved, mainly due to problems with formulation, drug stability and bulk production.

CDDP has so far been encapsulated in MLV, but the efficiency of encapsulation is very low (7.4%) and stability is poor (75%, kept in 0.9% NaCl for 48 hours) [Friese et al. Arch. Pharmacodynamie Therapie 258: 180-192 (1982)].

No. 4,256,652. U.S. Pat. No. 4,198,195 discloses certain platinum complexes resolved with 1,2-dianino-cyclohexane (DACII). The isomers used are cisDACH, trans-RR-DACH and uans-SS-DACII. The described platinum complexes, in addition to the resolved DACH isomer, contain two hydrophilic platinum ligands, e.g. containing bromide, iodide, nitrate, bromoacetate, sulfate or glucuronate. According to the description, platinum complexes containing trans-RR-DACH are more effective than those containing cis-DACH.

No. 83306726.7. European Patent Application No. 5,198,125 discloses platinum complexes which may contain diaminocyclohexane (ncm-sterically chemically resolved) and contain phosphatidyl groups containing fatty acid linkers. These compounds are said to be highly insoluble in plasma and are preferably used with lipid vesicle carriers. Platinum complex phospholipid vesicles are preferably produced by the ultrasonic oscillator technique, which results primarily in unilaniellar vesicles.

The present invention relates to a process for the preparation of a quaternary co-ordinated platinum (H) complex having the formula

In the formula, R 1 and R ₂ are C 3 -C 7 cycloalkenyl neobonyloxy, C 3 -C 12 alkylcarbonyloxy or (C 8 -C 12 alkyl) imino-bis [(C 1 -C 4 allyl) carboxy] ] R ₃ and R ₄ are as defined

H

I

--- _N ------ r ₅

H

A group of the formula 198,079 wherein R ₅ is hydrogen,

C 1-5 alkyl, such as isopropyl, C 4-8 cycloalkyl, such as cyclohexyl.

R ₃ and R ₄ may also be taken together to form a single group, preferably C 4 -C 8 cycloalkyl-1,2-diamino, C 1 -C 4 (preferably ethyl) alkylene-1,2-diamino. Preferred cycloalkyl-1,2-diamino groups are, for example, 1,2-diaminocyclohexyl, preferably in the form of trans R, R or trans-SS.

In said complex, the carboxylate group at R 1 and R ₂ is preferably a C 5 -C 12 alkyl carboxylate group, more preferably a neodehanocarboxyloxy group,

Cycloalkenyl is preferably C3-C7 cycloalkynyl.

The cycloalkyl-1,2-diamino group present as R ₃ and R ₄ together is preferably C 4 -C 8.

When R 1 and R ₂ together are a dicarboxylate group, it is preferably a cis-dicarboxylate group of the formula

OOC-CH

wherein R ₆ is C 8 -C 12 alkyl, preferably decyl or neodecyl, R ₇ and R _e are hydrogen.

As mentioned above, R ₃ and R ₄ are HI

- _N -R _s

I

H.

When R ₃ and R ₄ together form a group; it may be C4-C8 cycloalkyl-1,2-diamino or C2-C12 alkylene-1,2-diamino. Cycloalkyl-1,2-diamino, preferably cyclohexyl-1,2-diamino, more preferably trans-R, R-cyclohexyl-1,2-diamino or trans-S, S-cyclohexyl-1,2-diamino- group.

This complex, as already mentioned, is soluble in methanol and chloroform, and insoluble in water.

The invention further relates to a process for the preparation of liposomes containing a fat-like substance, such as a phospholipid, optionally cholesterol and a quadruple coordination platinum complex as described above. The liposomes produced by the process of the invention preferably contain platinum complex and phospholipid in a weight ratio of 1:10 to 1:30, more preferably 1:15.

Preferably, the liposomes contain phosphatidylglycerol, phosphatidylcholine, sphingomyelin, phosphatidylic acid or phosphatidyl according to the invention as phosphatidylglycerol, phosphatidylcholine, particularly preferably phosphatidylglycerol, phosphatidylcholine or combinations thereof. The most preferred phosphatidylglycerol consists essentially of dimyristoylphosphatidylglycerol, and the most preferred phosphatidylcholine consists essentially of dimyristoylphosphatidylcholine. When the liposomes produced by the process of the present invention contain diirlyistoylphosphatidylglycerol and dimyristoylphosphatidylcholine, the ratio thereof is preferably 1:10 to 10: 1, more preferably 3: 7.

The liposomes produced by the process of the invention may be multilamellar, unilamellar, or unspecified lamellar. Pharmaceutical compositions containing liposomes and pharmacologically acceptable carriers or diluents can be used to treat diseases such as cancer.

The quadrate coordination platinum (II) complex produced by the method of the invention can be used to treat animals carrying tumor cells sensitive to the presence of platinum (II) complex. In this case, the animal is administered the four-coordination platinum (II) complex or liposome described above, which contains a phospholipid and an amount of platinum complex sufficient to inhibit the tumor cell. Administration is preferably parenteral, intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural or intrathecal injection or topically or orally. Preferably, dosing is repeated at regular intervals, for example twice daily for two weeks. The treatment may be continued until the tumor has regained or disappeared and may be used in combination with other cancerous procedures such as surgery or chemotherapy with various agents.

These antineoplastic treatments can be used to prevent the metastatic spread of tumors, such as reticulosarcoma. The platinum (II) complex is the. preventive treatment with a liposome containing it can be used to preclude the metastatic spread from the outset in a manner similar to that of vaccinization.

Four-coordination platinum (II) complexes were prepared using racemic (non-resolved) DACH, trans-RR-DACH, or transSS-DACH. It has been found that platinum (II) complex containing transRR-DACII or trans-SS-DACII and two hydrophobic ligands, such as cyclopentanecarboxylate groups, can be more efficiently incorporated into llsposomes than the analogue containing racemic DACH complexes.

Liposomes containing such platinum complexes are stable in aqueous media, non-nephrotoxic, and active against L-1210 murine leukemia.

In general terms, the quadrate platinum (II) complex of the present invention has the formula:

R, /

Preferably, R 1 and R 1 are a carboxylate mono anion containing a hydrophobic residue. Rí and

19S 079

R _{2 may also be taken} together to represent a carboxylate dianion, wherein the carboxylate groups are linked by an atom to which the hydrophobic group is then attached. In addition, R ₃ may also be a vicinal diaminoalkane or vicinal diaminoalkene residue. The group R _{3 may also be} formed of two different alkylamines, cycloalkylamines or ammonium residues. These substituents ensure that the compound is highly soluble (usually more than 5.0 mg / mL) in methanol and chloroform at room temperature and substantially insoluble (less than 0.5 mg / mL) in aqueous media at room temperature.

The hydrophobic moiety attached to the carboxyl group attached via an intermediate linking group may be alkyl, alkenyl, cycloalkyl or cycloalkenyl. The hydrophobic residual alkyl or alkenyl group may be straight or branched. Polar groups such as hydroxyl groups in the hydrophobic residue reduce the hydrophobic property and thus reduce the use in the present invention.

In some cases, R! and R ₂ groups may be attached, for example, two acetate or propionate groups may be linked by one nitrogen atom. In this case R ₍ and R ₂₎ represent a carboxylate dianion. A hydrophobic alkyl group

It contains from 8 to 12 carbon atoms, such as n-decane, can be attached to the linking nitrogen atom and the compound so formed can be used in the process of the present invention for the preparation of a four-coordinated platinum (II) complex.

Some experiments using hydrophobic residues present on the carboxylate mono anion (e.g. cyclopentene) have shown that the efficiency of incorporation of platinum complexes into liposomes depends on the characteristics of the R ₃ group. For example, when R _{3 represents a} 1,2-diaminocyclohexane (DACH) group, the amino groups may have different stereochemical configurations, such as cis, trans -RR and trans -SR (a mixture thereof termed "racemic"). . Experiments with platinum (II) complexes containing cyclopentene residues (R 1 and R ₂ ) and various stereochemical variants of DACH have shown that transRR-DACH and trans-SS-DACH complexes can be incorporated more efficiently into phospholipid liposomes. (the term bladder is equivalent to liposomes) as analogs of racemic DACH complexes.

When the hydrophobic residual C 9 alkyl group such as neodecanoate (C ₁₀ H ₂₀ O ₂ ) is present, the incorporation efficiency of the platinum (II) complex is maximal (100%) over racemic DACH or trans-RR-DACH. . Thus, straight or branched C 6 -C 12 alkyl groups possess the DACH-containing platinum (III) coinplcx for incorporation into phospholipid liposomes. with proper properties.

Liposomes containing platinum (II) complex can be made from a variety of materials, including natural and synthetic phospholipids. Many phospholipids can be used to make liposomes and not all are listed, as they are well known in the art. Examples are: lecithin, phosphatidylethanolamine, lysolecithin, lysophatidyl ethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidylic acid, cerebrosides. The most preferred phospholipids for use in the process of the invention are dimyristoyl phosphatidylglycerol (DMPG) and dimyristoyl phosphatidylcholine (DMPC). In combination with phospholipids and platinum (II) complexes, small amounts of less than 1 to 50% by weight of cholesterol can be used in the process of the present invention for the preparation of liposomes, it has been found that DMPG to DMPC 10: 1 ratio. The ratio of platinum (II) complex to phospholipid is generally between J: 10 and 1:30, but 1:15 is preferred.

A liposome containing a unilamellar or multilamellar or other platinum (U) complex may also be used in the process of the invention. Multilamellar liposomes are preferred because the platinum (II) complex produced by the process of the present invention is substantially insoluble in water and appears embedded in bilayer phospholipid bilayers.

According to the process of the invention, platinum (II) complexes are prepared as follows: (1) about 10 mmol of vicinal diaminocycloalkane or aliquot is added to a solution of 3.5 g K ₂ PtCl _{4 in} 50 ml aqueous solution for 6-8 hours. stirring at room temperature. The resulting yellow solid product is cis-bis-dichloro-2-diamino-Pt (II), which can be removed by filtration and washed with, for example, water, methanol or acetone. The solid product was then suspended in about 20 mL of water and ca. 0.75 g of Ag ₂ SO _{4 in} water is added. The mixture was stirred in the dark for 24 hours and the precipitated AgCl was filtered off. The sulfato- (vicinal diamino) -Pt is then ca. Dissolve in 100 ml of water and add ca. 2 mmol of the alkaline earth metal salt of the carboxylate anion produced in situ and then ca. Stir for 30 minutes. After filtration of the alkaline earth metal sulfate, the platinum (III) complex according to the invention is obtained, for example by crystallization or by evaporation of the solvent.

The preparation of each platinum (II) complex and their use in chemotherapy are described in the Examples. The methods can also be used to prepare and use other complexes by replacing the corresponding vicinal diamines or carboxylate mono anions containing a hydrophobic residue.

According to the invention, liposomes (Pt-liposomes) containing the phospholipid and the platinum (II) complex produced by the process are useful for inhibiting the growth and metastatic spread of tumors.

The Pt-liposomes can be administered parenterally, topically or orally. In most cases, oral or parenteral administration will be in the range of 2.5 mg to 25 mg per kilogram body weight. However, the doses used to treat individual human cancer patients will in any case depend on the condition of the subject being treated, the type and size of the tumor, and the toxicity of that Pt-liposonin.

The amount of Pt-liposome to be used in the pharmaceutical compositions of the present invention and the dosage thereof will depend upon the condition of each subject being treated, the nature of the tumor being treated, the antitumor activity, toxicity of the Ptliposome,

It depends on the solubility of 198,079, etc. The Pt-liposome may be administered in combination with other antineoplastic agents in the context of a combined therapeutic diet.

Parenteral administration can be by intraperitoneal, subcutaneous, intrapleural, intrathecal, intraurethral, intravenous, intraarterial, intramuscular or intralymphatic. For such parenteral administration, a Pt-liposome suspension, preferably in a pharmacologically acceptable solution, such as a sterile isotonic aqueous solution, is preferably administered. Such suspensions may be prepared in their complete form or by mixing the prepared components. It is known to those skilled in the art that Pt liposomes can be prepared in the form of pellets or powders. These pellets or powders may be mixed with pharmacologically acceptable solutions to form parenteral suspensions.

Topical application of Pt-liposomes may be in the form of suspensions, creams or ointments which may be prepared in their complete form or from Pt-liposome pellets or powders. Topical application may occur near the cancerous site, such as the cytoplasm or mucosa.

Oral administration of Pt-liposomes can be via an encapsulated Pt-liposome powder or pellet, thereby protecting the Pt-liposome from much of the gastrointestinal digestion before it is released from the capsule.

If desired, Pt-liposomes containing other anti-tumor agent, antioxidant, may also be prepared to ensure the stability of the liposomes.

The use of the complexes produced by the process of the invention, in particular as a component of liposomes, makes it possible to inhibit the growth of tumors and to prevent the metastatic spread of tumors. If a patient is found to have a type of tumor whose growth is generally inhibited by platinum (II) complex, tumor growth can be stopped by administering Pt-containing liposomes produced by the method of the invention.

Similarly, metastatic spread of tumors can be prevented by recognizing that the subject has a type of potentially metastatic tumor that is sensitive to platinum (II) complexes and is treated with Pt-containing liposomes produced by the method of the invention.

The process of the present invention is illustrated by the following non-limiting examples.

Example 1

Materials and methods used

K ₂ was obtained from PtCl ₄ AESAR (Johnson Marrhey, Inc. Seabrook, NH). Cyclopentene carboxylic acid was obtained from Pfaltz and Bauer, Inc., Stamford CT,

1,2-Diaminocyclohexane (DACH) was purchased from Aldrich Chemical Co., Molwaukec, WI., Trans-SSDACH from Moro from Thiokol Inc., Danvers, MO., neodecanoic acid Exxon Chemical Co., Houston. Made by Texas. Elemental analysis of platinum (II) complexes

Integral Microanalitical Laboratories, Inc., Ralcigh, NC and Robertson Laboratory, Inc., Florham Park,

NJ equipment. The infrared spectra of the complexes (in the form of KBr pellets) were measured on a Nicolet 6000 Fourier transform infrared spectrophotometer in the range 600-4000 cm ^-1 .

Chromatographic purity (thin layer chromatography) made by dimiristoylphosphatidylcholine (DMPC) and dimyristoylphosphalidylglycrin (DMPG) from Avinli Polar Lipids (Birmingham, AL). Cholesterol from Sigma Chemical Co., St. Louis, MO.

Example 2

Preparation of Cis-Bis-Cyclopentene Carboxylate-1,2-DACHplatin (II) [Compound (2)]

The preparation was carried out by a multi-step procedure for the preparation of racemic DACH [Khokhar et al. Inorg. Chem. Acta. Bioinorganic Section, 108, 63 (1985)]: 0.96 g of DACH was added to a filtered solution of 3.5 g of K ₂ PtCl _{4 in} 50 ml of water, and the solution was stirred at room temperature for 6-8 hours. The resulting solid yellow cis-bis-dichloro-DACH-Pt (II) was filtered off, washed with water with methanol and then with acetone. The product was dried in vacuo, yield 56%. Then 1.0 g of cis-bis-dichloro-DACH-Pt (II) was suspended in 20 ml of water and

A solution of Ag ₂ SO ₄ (75 g) in water (150 mL) was added to give water-soluble sulfato-DACH-Pt-H ₂ O. The reaction mixture was stirred in the dark for 24 hours and the precipitated AgCl was filtered. The yellow solution was evaporated to dryness at 45-50 ° C under reduced pressure and the resulting product was further dried over P ₂ O ₅ in vacuo. Yield 90% sulfate-DACHPt (II) -H ₂ O. Finally, 0.423 g (1 mmol) of sulfate-DACHPt (II) was dissolved in 100 mL of water and barium cyclopentene carboxylate was prepared locally by 0.3 g. barium hydroxide was added to 0.226 g (2 mmol) of aqueous cyclopentene carboxylic acid. The components were mixed and the reaction mixture was stirred for 30 minutes at room temperature. The precipitated barium sulfate was filtered off and the yellow filtrate was evaporated to dryness under vacuum in a rotary evaporator at 45 ° C. A yellow solid was obtained which was purified from methanol. The product was finally dried in vacuo (70% yield).

Elemental Analysis for C ₁₈ H ₃₀ N ₂ O ₄ Pt: Found: C 40.26%, H5.65% N5, O5%;

Calculated: C 40.67%, H5.65%, N 5.27%.

IR spectrum (KBr pellet): / C = 1632 cm ^-1 and / C-0 1398 cm -1.

Cis-bis-cyclopentene-carboxylate-1,2-DACH-Pt- (II) is good! soluble in methanol and chloroform and only slightly soluble in water (less than 0.5 mg / ml).

By the above procedure, both the transRR and trans-SS isomers of cis-bis-cyclopentene carboxylate-1,2-DACH-Pt (II) were prepared using both trans -RR-DACH and trans -SR-DACH. Analysis of cis-bis-cyclopentene-carboxylate-1,2-tris-RR-DACH-Pt (II) 311 ₂ O:

Found: C, 37.36; H, 5.50; N, 5.12.

Calculated: C 37.93%, H5.82%, N4.80%.

198,079

Example 3

Synthesis of cis-bis-neodecanoato-DA CH-Pt (II)

0.423 g of racemic sulfate-DACH-Pt (II) prepared in Example 2 was dissolved in 10 mL of water. 0.420 g of the potassium salt of neodecanoic acid were added and the reaction mixture was stirred at room temperature for 30 minutes. A gitmium product was obtained, which was extracted with chloroform and the extract was dried over anhydrous magnesium sulfate. The magnesium sulfate was filtered off and the filtrate was evaporated to dryness. An off-white solid was obtained, which was dried in vacuo and dried over phosphorus pentoxide. The product was stored at 0 ° C.

Found: C, 48.30; H, 8.10; N, 3.92.

Calculated: C, 47.93; H, 8.00; N, 4.30.

The cis-bis-peroxyneodecanoate-DACH Pt (II) represented by the formula (3), álról R, ^R? and R may be methyl, ethyl or propyl to give a carboxylate group of C 10 H ₁₉ O ₂ (molecular weight 172).

Cis-bis-neodecanoate-DACH-Pt (H) is very soluble in methanol and chloroform and insoluble in water.

Analysis of cis-bis-neodecanoate trans-RR-DACH-Pt (II):

Found: C, 47.75; H, 8.16; N, 3.98;

Calculated: C, 47.93; H, 8.00; N, 4.30.

Example 4

Preparation of Cis-n-Deeylimino diacetate-DACH-Pt (II)

Sulfato-DACH-Pt (II) prepared in Example 2 (0.423 g) was dissolved in water (10 mL). Sodium salt of n-decyliminodiacetic acid was prepared by adding 0.08 g of sodium hydroxide to a solution of 0.273 g of N-decyliminodiacetic acid in 50 ml of water. The resulting aqueous solution was added to the sulfate-DACHPt (II) solution and the reaction mixture was stirred for 30 minutes at room temperature. The resulting solution was evaporated to dryness in a rotary evaporator at 40 ° C under reduced pressure. The resulting yellow product was dissolved in methanol and filtered through Celite. The yellow filtrate was evaporated to dryness under reduced pressure on a rotary evaporator. A yellow crystalline product was obtained, which was further purified from 1-propanol in 80% yield.

Elemental analysis for C ₁₀ H ₃₉ N ₃ O ₄ Pt · 2H ₂ O found: C 39.28%, H 7.12%, N6.68%;

Calculated: C 38.95%, H7.02%, N6.81%.

IR spectrum (KBr reflection). / C = 0 1580 cin ^_1 , / C -0 1410 cm ^-1 .

Cis-n-decylimino-diaceto-DACH-Pt (II) is represented by formula (4) wherein R is n-decyl or a longer, linear or branched alkyl group.

Cis-n-decylulinodiacyl DACII-Pt (II) is very soluble in methanol and chloroform and insoluble in water.

Preparation was performed using trans-RR-DACH. Analysis of cis-n-decyliminodiacetate-transRR-DACH-Pt (II):

found. C 38.60%, 116.87%, N6.82%; Calculated: C 38.95%, 117.02%, N6.81%,

The prepared compound represents a compound of formula I wherein R 1 and R 1 are the same and the carboxylate mono anion is bonded by an atom on which a hydrophobic alkyl group is present, thus forming a carboxylate dianion.

Example 5

Preparation of Platinum-containing Liposomes (L-PT)

Multilamellar lipid vesicles (MLV) or liposomes containing the platinum complex (PT) described above were prepared according to the procedure described in Lopez Beresicin et al (4) Clin. Exp. Metastasis V2 pp. 127-137; and Lopcz-Bcrestein et al. (1983) 1. Inf. Dis V 147 pp. 937-945. Briefly, chloroform solution of lipids (at the desired molar ratio) and platinum complex were mixed to a ratio of 15: 1 lipid to complex in a rotary evaporator (Buchi, Birkmann Institrumens, Westbury, NY). The aqueous solution of the lipid film containing the dry platinum complex] (0.9% NaCl in water) was dispersed by vigorous shaking. The suspension was then centrifuged at 30,000 g for 45 minutes, the supernatant discarded, and the pellet containing the complex resuspended in water containing 0.9% NaCl.

MLVs or liposomes containing platinum complexes can also be prepared using lyophilized powdered lipid and platinum complexes. The lipid and platinum complex are dissolved in the above weight ratio in tertiary butanol (m.p. 26 ° C) as a hydrophobic solvent. The solution was freeze-dried to give a white powder. MLV containing platinum complex is formed by the addition of 0.9% NaCl solution with gentle shaking.

Example 6

Calculation of encapsulation efficiency (EE) and stability

Elemental platinum was determined in the liposome suspension and pellet by X-ray fluorescence according to Seifert et al. (1979) Proc. Amer. Ass'n. Cancer Slit. V 20 p. 168. The amount of platinum complex in the supernatant was determined by ultraviolet spectrophotometry at 224 µl. The EE was calculated using the following formula:

1. EE = platinum content in the pellet per total platinum content in the initial liposome suspension,

2. EE = (total amount of platinum initially added minus the content of platinum in the prefix) per total amount of platinum initially added).

-611

198,079

Since the results obtained with the two methods were very comparable and the second method requires only ultraviolet spectrophotometric determination of the platinum complex, in most cases the second method was used to determine the EE.

The stability of the various platinum complex liposomes (L-PT) in 0.9% sodium chloride solution at 4 ° C and in 50% human (AB) serum in 0.9% sodium chloride solution at 37 ° C equation:

% EE x. hour _v , _nn braking x. oraban = —-- X 100% EE at hour 0

The EE values used to determine the stability were obtained by measuring the platinum complex content in the supernatant by UV spectrophotometry (0.9% NaCl solution) or X-ray fluorescence (50% human AB serum). Stability in NaCl solution was determined 14 days after preparation. In addition, L-PT formulations were also examined microscopically on day 14 to check for bladder morphology. Stability in 50% human AB serum was assayed 18 hours after preparation, after incubation.

Various cis-platinum complexes were encapsulated in a multilamellar blister of DMPC: DMPG = 7: 3. The encapsulation efficiency of Pt-cyclopentene carboxylator-C-DACH was 66%. The encapsulation efficiency was significantly increased when pure trans-RR-DACII or trans-SSDACH was used (88% and 90%). Table I summarizes the encapsulation efficiency data obtained using the Pt-neodecanoato-racemic-DACH, Pt-neodecanoato-trans-RR-DACH, Pt-n-decyliminodiacetato-racemic-DACH complex.

Table I

Effectiveness of encapsulating platinum-containing liposomes using various analogs

Platinum-containing complex encapsulation efficiency * % Pt-cyclopentene-carboxylate rac DACH mixture 66 Pt-cyclopentene-carboxylato-trans- RR-DACH 88 Pt-cyclopentene-carboxylate trans SS-DACH 90 Pt peroxyneodecanoate-racemic-DACH mixture 99 Pt peroxyneodecanoate-trans-RR-DACH 99 Pt-N-deeiliinino diaccto rac DACL mixture 100 Pt-N-trans- decilimino diacetate RR-DACH 98

* Average of at least three experiments. Liposome composition DMPC: DMPG = 7: 3.

Analogous experiments were performed using DMPC, DMPG, cholesterol alone, and other combinations of DMPG and DMPC alone, with and without cholesterol, which did not show better encapsulation efficiency but significantly worse EE values.

Example 7

Stability of L-PT in aqueous media

The formulations were suspended in 0.9% saline and incubated at 4 ° C for 14 days. Liposomes were examined by light microscopy and the amount of free PT in saline was determined. Liposomes containing DMPG alone as the phospholipid showed significant, microscopically detectable structural changes. Free PT was determined and stability calculated as PT% remaining in the liposome. The results are shown in Table II. are summarized in Table.

II. spreadsheet

Stability of platinum-containing liposomes in platinum-containing liposomes in brine over 14 days *

Platinum complex Stability% Pt carboxylato-cyclopentene-rac DACH 89 Pt-cyclopentene-carboxylato-trans- RR-DACH 94 Pt-cyclopentene-carboxylato-trans- SS-DACH _ Pt-neodecanáto a racemic DACH 100 Pt neodecanáto-trans-RR-DACH 99 Iminodiacetates Pt-racemic-DACH 100 Pt dipicolinates-trans-RR-DACH . 100

* Liposome composition: DMPC: DMPG = 7: 3.

As shown in the results, both Pt-cyclopentene carboxylate trans-RR-DACH and Pt-neodecanoate racemic DACH showed greater stability than Pt-cyclopentene carboxylate racemic DACH.

Example 8

Toxicity studies with L-PT (cyclopentene carboxylate racemic DA CH)

Toxicological studies were conducted on 6- to 8-week-old DC-1 Swiss mice (commercially available from The University of Texas Science Park, Texas, USA). Intraperitoneal (ip) administration of 0.1-0.3 ml of the following formulations: free PT in hydroxypropylcellulose, DMPG, DMPG-PT, DMPC: DMPG 7

-713

Table 198.07: Suspended in 3: DMPC: DMPG = 7: 3-PT. DMPC: DMPG = 7: 3 — PT was also administered intravenously (iv) in a single injection or 3 daily injections. Clinical behavior and survival time were monitored daily. LD _t0 , LD ₅₀ and LD _{90 were} calculated from the mortality occurring within 14 days after dosing.

PT suspended in hydroxypropylcellulose, DMPG-PT and DMPC-DMPG = 7: 3-PT showed similar DLD ₅₀ values in a single ip. injection (91 mg / kg, 86 mg / kg and 75 mg / kg) (Table 1, Table III). DMPG and DMPC: DMPG = 7.3 had higher PT LD ₅₀ values (183 mg / kg and 304 mg / kg) encapsulated in empty liposomes. The DMPC: DMPG = = 7: 3 — PT LD50 of the sheet was similar for a single injection and 3 injections daily (82 mg / kg and 96 mg / kg). Most deaths occurred with both iv and ip dosing between 5 and 10 days after injection. The results are shown in Table III. Table.

III. spreadsheet

Toxicity of various L-PT formulations at IP and IV administration

L-PT preparation Dosage way LDjo mg / kg LD ₅₀ mg / kg LD _W mg / kg Free PT 1pX 1 61 91 125 DMPG PT ipXl 56 86 133 DMPC: DPMG 7: 3-PT ipXl 54 75 94 ivXl - 82 100 arc gdX3 63 86 107 DMPG * ipXl 158 183 228 DMPC: DMPG 7: 3 * ipXl > 304 > 304 > 304

* The encapsulated LDjo, LD and LD _SO values ^ PT values.

Example 9

nephrotoxicity

Blood urea nitrogen (BUN) content was determined in a sample of 22-25 g body weight CD, Swiss mice retroorbital vasculature at 96 hours in CDDP, PT cyclopentene carboxylate racemic-DACH hydroxypropylcellulose, DMPG-PT or DMPG = 7: After injecting 3-PT (single dose) at a dose corresponding to the LD ₅₀ value. L-PT formulations tested for toxicity were prepared on the day of the assay. There was no significant increase in BUN after ip administration of PT LD ₅₀ suspended in hydroxypropyl cellulose as DMPG-PT and DMPC: DMPG = '7: 3-PT (BUN 96 hours 34.4 ± 9.6 mg%). , 30.0 ± 4.6 mg% and 32.0 ± 2.3 mg%). The results are shown in Table IV. are summarized in Table.

Acute nephrotoxicity of CDDP, free PT and L-PT at LD ₅₀ dose in mice

preparation LD ₅ o dose ip single injection (mg / kg) BUN at 96 hours ¹ (mg%) CDDP 17 78.3 + 8.0 Free PT 91 34.4 + 9.6 DMPG PT 86 30.0 ± 4.6 DMPC: DMPG 7: 3 75 32.0 ± 2.3

¹ Normal = 27.2 + mg%.

Example 10

In vivo antitumor activity of cyclopentene carboxylate racemic DACH-Pt against L1210 cells

L1210 leukemia cells were grown in McCoy's medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% horse serum, glutamine, streptomycin and penicillin at 37 ° C, 95% relative humidity, 5% CO ₂ . 4 ml of cell suspension was added to the culture tubes and the appropriate amounts of free PT and L-PT (0.01 micrograms / ml to 10 micrograms / ml final concentration). After 96 h, control cell concentration of experimental cells and cell concentrations of control cultures and experimental cultures were determined with a Coulter Counter (Coulter Electronics, Hialeah FLA) and the percentage inhibition was calculated. The following formulations were tested: free PT, DMPG, DMPC: DMPG = 7: 3, DMPG-PT and DMPC: DMPG = 7: 3-PT. Results are expressed as 50% inhibition (ID _SU ). Results for empty liposomes were expressed as the amount of PT that should have been encapsulated at ID ₅ o.

The ID _SO for free cyclopentene carboxylate racemic DACH was 1.3 in micrograms / ml (Table 1V). Of the two tested L-PT DMPGPT composition was slightly more active than the DMPC: DMPG = = 7: 3-PT _(ID50 average value of three experiments. 0.7 and 1.6 microgram / ml of DMPG in BOD the ID _{50 of the} empty liposomes was 3.7 µg / ml, while the ID ₅₀ of the DMPC: DMPG = 7: 3 empty liposomes was more than 10 µg / ml (Table 1V).

Table V.

In vitro antitumor activity of free PT, L-PT and empty liposomes against L1210 cells

preparation 1 ID _SO (micro g / ml) ¹ 2 3 Average Free PT 1.3 _ __ _ DMPG PT 0.7 1.0 1.4 0.7 DMPG ² 3.0 3.0 5.0 3.7

-815

198,079

Table V (continued)

preparation 1 ID _S0 (micro g / ml) ¹ 2 3 Average DMPC; DMPG 7: 3-PT DMPC: DMPG 1.9 2.0 0.9 1.6 7: 3 ² 10.0 - - -

'Expressed as values of CDDP The ID₅₀ = 0.1 microg / ml, ² ID ₅₀ = PT microg / ml, as far have been encapsulated in the liposome empty 1D concentration of _N5.

Example 11

In vivo antitumor activity of L-PT against murine leukemia L1210, where PT is cyclopentene carboxylator

In vivo anti-tumor activity of CDDP, PT, DMPG, DMPG-PT, DMPC: DMPG = 7: 3 and DMPC: DMPG = 7: 3-PT suspended in hydroxypropylcellulose was investigated in the mouse model L1210-BDFj (25). BDFj mice are commercialized by Charles River (Washington, MA). Groups of 6-8 mice weighing 18-22 g were induced ip with 1 x 10 ⁶ L1210 leukemia cells on day 0. L1210 cells were maintained in DBA ₂ mice, passaged weekly between experiments. L-PT formulations were injected ip in 0.1-0.3 ml 24 hours after tumor inoculation. Dosing was performed in two ways: single injection on day 1 or multiple injections on days 1, 5 and 9. ACDDP was used at a dose that showed maximal antitumor activity in preliminary experiments. The dose of PT, DNPG-PT and DMPC: DMPG = 7: 3-PT ranged from 3.125 to 50 mg / kg (near LD ₁₀ ). Clinical behavior and survival time were determined until all animals had died. Results were expressed as T / C% (mean survival time of treated mice / survival time x 100 of control mice) and expressed as number of long survivors. Mice living for more than 30 and 60 days, respectively, were considered long-term survivors in a single and multiple injection series. L-PT formulations were freshly prepared on the day of the experiment under sterile conditions. In the first set of experiments, the effect of single ip administration of CDDP, free PT, DMPG-PT and DMPC: DMPG = = 7: 3-PT on the treatment of L1210 leukemia was investigated. The T / C% of mice treated with free PT 12.5 mg / kg and 25 mg / kg was comparable to that of mice treated with 10 mg / kg CDDP (162 '17S, mean of three experiments), spreadsheet). Antitumor activity of 12.5 mg / kg, 6.25 mg / kg and 3.125 mg / kg DMPG-PT was comparable to that of free PT and CDDP (mean T / C = 175 for 12.5 mg / kg, 158). to 6.25 mg / kg, 163 to 3.125 mg / kg) (Table 1 VI). DMPG-PT at doses of 25 mg / kg or greater was toxic to L1210 leukemic BDF _t mice. DMPC: DMPG = 7: 3 — PT treated mice at doses of 25 mg / kg, 12.5 mg / kg and 6.25 mg / kg were similar or slightly higher than CDDP treated with free PT and DMPG-PT (mean T / C% = 215 for 25 mg / kg, 178 for 12.5 mg / kg, 200 for 6.25 mg / kg ) (Table 1 VI). DMPC: DMPG = = 7: 3-PT at a dose of 50 mg / kg was toxic to L1210 leukemic BDF mice. Empty liposomes made from DMPG and DMPC: DMPG = 7: 3 showed no antitumor activity when dosed at a similar dose to the active liposomes (T / C% = 105 for DMPG and 93 for DMPC: DMPG). Long-term survivors (one or two of six mice) occurred in groups treated with CDDP, DMPG-PT and DMPC: DMPG = 7: 3 (Table 1 VI).

The highest T / C% for multiple injections was 253 for three 7.5 mg / kg doses of CDDP, 284 for three 12.5 mg / kg doses of free PT, 179 for 6.25 mg / kg three times DMPG-PT, and 403 for a dose of DMPC: DMPG = 7: 3 to PT three times 12.5 mg / kg. Long-term survival (one in six) occurred only in the DMPC: DMPG = 7: 3— PT treated group (Table 1, VII).

VI. spreadsheet

CDDP-free PT and L-PT in vivo antitumor activity against L1210 murine leukemia i.p. dosing (Day 1) (PT Cyclopentene Carboxylator Cream-DACH Complex)

Tube- powder song Preparation ⁴ % T / C Number of survivors 1 ' a 30. 2 ² day 3 ³ First CDDP 10 mg / kg 167 189 (1/6) Second Free PT 25 mg / kg - - 168 Third 12.5 mg / kg 173 136 179 4th DMPG-PT 25 mg / kg 100 - - 5th 12.5 mg / kg 160 209 158 6th 6.25 mg / kg (2.6) 158 7th 3.12 mg / kg - - 163 (2/6) 8th DMPC: DMPG 7: -PT 25 mg / kg 213 200 232 (1/6) 9th 12.5 mg / kg 167 (1/6) 189 (1/6) 10th 6.25 mg / kg (1/6) - 200

¹ control median survival = 7.5 days ² control median survival = 11 days ² control median survival = 9 days ⁴ Each formulation was administered in an amount of 0.1 to 0.3 ml ip after inoculation of L1210 cells (1 · 10 ⁶ ) 24 hours.

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Table VIL

CDDP free PT and L-PT in vivo anti-junior activity against L1210 murine leukemia i.p. dosing (days 1, 5 and 9) (PT cyclopentene carboxylate racemic-DACH complex)

% T / C Group song Preparation ¹ Dose survivors number (60th on the sun) First CDDP 7.5 mg / kg X 3 253 Second Free PT 25 mg / kg X 3 158 Third 12.5 mg / kg X 3 284 4th 6.25 mg / kg X 3 168 5th DMPG PT 12.5 mg / kg X 3 105 6th 6.25 mg / kg X 3 179 7th 3.12 mg / kg X3 168 8th 1.56 mg / kg X 3 115 9- DMPC: DMPG 7: 3-PT 12.5 mg / kg X 3 403 (1/6) 10th 6.25 mg / kg X 3 253 11th 3.125 mg / kg X3 210 (1/6)

'0.1-0.3 µl of each preparation! in quantities i.p. was administered 25 hours, 5 and 9 days after tumor inoculation.

Example 12

In vivo anti-junior activity of various L-PT analogs against L1210 leukemia

The anti-junior activity of the various L-PT analogs was tested against rat L1210 leukemia in the L1210-BDF model (tumor inoculation day: 0, treatment day: 1; i.o.) as previously described. Compared to CDDP (% T / C = 175), the L-PT analogs showed the same or slightly higher antitumor activity, except for L-PT (cyclopentene carboxylate trans-SS-DACH = 155, Ndecyl imino). -diacetacracemic-DACH = 150 and N-decilimino-diacetato-trans-RR-DACH = 162). The results are shown in Table VIII. are summarized in Table.

VIII. spreadsheet

Antitumor effect of cis-platinum and various liposome-platinum analogs against L1210 leukemia ³

Platinum analog ^{% T / C2}

PT-cyclopentene-carboxylates

racemic-DACH 25 213 PT-cyclopentene-carboxylates trans-RR-DACll 25 187 PT-cyclopentene-carboxylates trans-SS-DACH 25 155

Platinum analog The dose is ⁴ mg / kg % t / c ^j PT-peroxyneodecanoate-racemic DACH 50 187 PT-peroxyneodecanoate-trans-RR-DACH 25 175 PT-N-decylimino diacetato-racemic DACH 25 150 PT-N-decilimino díacctáto trans RR-DACH 25 162 Cis-platinum (CDDP) ⁵ 10 175

¹ Liposome composition: DMPC: DMPG = 7: 3, ₂ median survival time of treated mice

Median survival time of 1UU control mice ³ 1X10 ⁶ L1210 cells ip inoculated on day 0, ⁴ Ip treatment on day 1, ⁵ 1mg / ml, dissolved in saline.

Example 13

In vivo antitumor activity of L-PT-neodecanoate racemic-DACH against hepatic metastasis of mouse M5076 reticulosarcoma

Potent antitumor activity of L-PT against liver metastases was tested using L-PT neodecanoato-racemic DACH prepared as in Example 6 and mouse M5076 triculosarcoma, a tumor that causes metastasis to the liver only.

L-PT-neodecanoato-racine-DACH is more active than cis-platinum in M5076-induced liver metastasis, in two tumor inoculations, and in two modes of administration (Tables 1, IX and X). L-PTneodecanoato-racemic-DACH (25 mg / kg in 8, 12 and 10)

Day 16) more than doubled hepatic metastases compared to cis-platinum (7.5 mg / kg on days 8, 12, and 16) on day 30 after 10 ^{s of} mouse M 5076 cell intravenous inoculation (mean liver metastases + SD = 39 + 21 for L-PT versus 114 + 38 for cis-platinum (Table 1 IX). L-PT neodecanoate racemic DACH (25 mg / kg 4,

On days 8 and 12) completely inhibited M5076 liver metastasis on day 45 after inoculation of 10 ⁴ M5076 cells, whereas 4/6 animals treated with cis-platinum (7.5 mg / kg at 4, 8 and 12) days) showed 175 or more liver metastases (Table 1 X).

L-PT neodecanoate racemic DACH was also effective in preventing liver metastases caused by M5076 reticulosarcoma. L-PT-neodecanoato-racemic DACH (37.5 mg / kg sub day) reduced hepatic metastases 5 days after intravenous inoculation of M ⁴ M5076 cells (day 0) compared with cis platinum (10 mg). / kg on day -1) and untreated animals (mean liver metastases ± SD = 52 + 20 for L-PT, 256 + 54 for cis-platinum, and 226 + 21 for control), (Table 1, Table XI).

-1019

198,079

IX. spreadsheet

XI. spreadsheet

Treatment of liver metastases of 76 5076 reticulosarcoma with L-PT-neodecan o-to-racemic -DAC H *

Prevention of hepatic metastasis of M5076 reticulosarcoma with L-PT-neodecanoate racemic DACH *

Treatment Dose (Mg / kg) Dosage time (Sun) Liver metastases at 30 days mean ± SD Cis-platinum 7.5 8,12,16 114 + 38 L-PT-neodeka · Noatak rac DACH 25 8,12,16 39 + 21

A group of 10 C57BL / 6 mice were inoculated with a

Day 0 ΙΟ ^{5 with} M5076 cells intravenously. The animals were treated on days 8, 12 and 16 with cis-platinum and L-PT neodecanoate racemic DACH. The animals had a median survival time of 19 days. Treated animals were sacrificed on day 30, their livers removed, placed in a Bouin's clamp, and liver tumor colonies counted.

Table X.

Treatment of liver metastases of M5076 reticulosarcoma with L-PT-neodecan o-to-racemic-DAC H *

Treatment Dose (Mg / kg) Dosage time (Sun) Liver metastases at 45 days mean + SD Cis-platinum L-PT-neodeca noate racemic 7.5 24,8,12 0.0,175,> 200,> 200,> 200 DACH 25 4,8,12 0,0,0,0,0,1

* 6 C57BL / 6 mice were inoculated on day 0 allo groups ΙΟ ⁴ M5076 cells intravenously. The animals were treated on days 4, 8 and 12 with cis-platinum and L-PT-neodecanoate racemic DACH. The mean survival time of untreated animals was 30 days. Treated animals were sacrificed on day 45, their livers were removed, placed in a Bouin's clamp, and liver tumor colonies were counted. Two of the cis-platinum-treated animals died before day 45, while none of the L-PT-treated animals died.

Example 14

Preparation of cis-bis-neodecanoato-cis-diaminoplatinum (TI)

First, cis-diaminodiodiodatin platinum (II) - (NH ₃ ) ₂ Pt- ₁₂ was prepared by the following procedure:

g K ₂ PtCl ₄ was dissolved in 20 ml water. An aqueous solution of 3 g KI was added to give a dark brown solution. Concentrated aqueous ammonia (2 mL) was added and the mixture was stirred at room temperature for 2-3 hours. The mixture was filtered and the solid phase was

Treatment Dose (Mg / kg) Dosage time (Sun) Liver metastases at 21 days mean ± SD Nothing _ _ 226 + 21 cis-platinum 10 -1 256 ± 54 L-PT-neodeka Noatak rac DACH 37.5 -1 52 ± 20

* Groups of 6 C57BL / 6 mice were treated with the group of -1. days with cis-platinum and L-PT-ncodckanoate racemic DAC11. The animals were inoculated on day 0 intravenously ΙΟ ⁴ M5076 cells. The mean survival time of untreated animals was 21 days. The treated animals were sacrificed on day 21, the liver was prepared, placed in a Bouin's anchor, and tumor colonies were counted.

water, ethanol and ether. The product was dried over phosphorus pentoxide in vacuo. Yield: 4.5 g (77%).

The compound of the present invention was then prepared as follows:

1.0 g of (NH ₃ ) ₂ -Pt-I ₂ was suspended in 50 ml of water and 0.68 g of silver nitrate dissolved in 20 ml of water was added. The reaction mixture was stirred overnight in the dark at room temperature. The precipitated silver iodide precipitate was filtered off and the filtrate was concentrated in a rotary evaporator. To the concentrated solution was added sodium neodecanoate (0.688 g neodecanoic acid in 20 ml methanol and 4 ml 1 N sodium hydroxide). The reaction mixture was stirred overnight at room temperature. The resulting yellow reaction mixture was evaporated to dryness at 40 ° C under reduced pressure in a rotary evaporator. A gummy product was obtained which was extracted with chloroform and the chloroform extract was dried over anhydrous magnesium sulfate. The magnesium sulfate was filtered off and the filtrate was evaporated to dryness under reduced pressure in a rotary evaporator. A cream solid was obtained which was dried over phosphorus pentoxide in vacuo. The final product was stored at 0 ° C.

Elemental Analysis for: C ₂₀ H ₄₄ N ₂₄ Pt Found: C 41.58, H8.03, N4.45;

Calculated: C, 42.00; H, 7.7; N, 4.9.

The structural formula of cis-bis-neodecanoato-bis-diaminoplatinum (II) is as follows.

(11 ₃ N) ₂ -PT-ylC-CR'RRJ ₂ wherein: R, R ', R can be CH ₃ , C ₂ H ₅ or C ₃ H _{2 to give} a carboxylate group of C ₁₀ H _{9 O 2} ( molecular weight: 171).

Cis-bis-neodecanoato-bis-diaminoplatinum (II) is highly soluble in chloroform, methanol and other organic solvents but is insoluble in water.

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198C79

Example 15

Preparation of cis-bis-neodecanoato-bis-cyclohexylaminoplatin (II)

The procedure of Example 14 was followed using cyclohexylamine caseunun instead of ammonia. The resulting complex is very soluble in chloroform, methanol and other organic solvents but insoluble in water.

Elemental Analysis for C32H64N2O4 · 2H ₂ O Calcd: Found: C 49.31%, H8,39% N3,16%;

Calculated: C 49.71%, H 8.80%, N3.62%.

The cis-bis-neodecanoato-bis-cyclohexylaminoplatin (II) has the following structural formula:

(C ₆ H _n NH _{2) 2} -Pt- (OOC CR'RR) ₂ wherein: R, R ', R can be a resulting CII3, _C2 I1 ₅ or C10II19O2 Calculated C3II7 a carboxylate group (molecular weight: 171).

Example 76

Preparation of cis-bis-neodecanoato-ethylene diammo-platinum (II) 11-β

First, cis-diiodoethylenediamine-platinum (II) was prepared as in Example 1 with ethylene-diamine ligand instead of ammonia in 96% yield.

Prepared sulfato-ethylenediamine-platinum (II) · H ₂ O in the following manner

3.9 g of cis-bis-diiodoethylenediaminoplatinum (II) are suspended in 10 ml of water and a solution of 2.2 g of silver sulfate in 200 ml of water is added. The reaction mixture was stirred overnight in the dark at room temperature. The precipitated silver iodide was filtered off and the resulting yellow filtrate was evaporated to dryness under vacuum in a rotary evaporator at 40-45 ° C. The final product was dried over phosphorus pentoxide in vacuo. Yield: 2.35 g (81%).

Subsequently, the compound of the invention was prepared as follows:

Sulfatoethylenediaminoplatinum (II) · II ₂ (0.369 g) was dissolved in water (20 mL) and sodium neodecanoate (0.344 g neodecanoic acid in 20 mL methanol and 2 mL 1 N sodium hydroxide) was added. The reaction mixture was stirred for 2-3 hours, then concentrated to dryness at room temperature and then at 40-45 ° C under reduced pressure in a rotary evaporator. A gummy product was obtained which was extracted with chloroform and the chloroform extract was dried over anhydrous magnesium sulfate. The magnesium sulfate was filtered off and the filtrate was evaporated to dryness under reduced pressure in a rotary evaporator. The resulting final product was dried in vacuo over phosphorus pentoxide and stored at 0 ° C.

Analysis for C ^ N ^ P · _H2 O: Calc: C 42.86%, H 7.79%, N4,54%; Found: C, 43.07%, H7.32%, N4.71%.

Cis-bis-neodecanoatoethylenediaminoplatinum (II) is highly soluble in chloroform, methanol and other organic solvents, but insoluble in water.

The structural formula of eis-bis-neodycanoatoethylenediaminoplatinum (III) is illustrated by formula (16) wherein R, R ', R may be CH ₃ , C ₂ H ₅ or C ₃ H _{7 to} give a C ₁₀ H 19 O 2 carboxylate group (molecular weight: 171).

Example 17

Preparation of cis-bis-neodecanoato-bis-isopropylaminoplatin (II)

First, cis-bis-n-codcanoucto-bis-isopropylaminoplatinum (II) and sulfato-bis-isopropylaminoplatinum (II) were prepared according to the procedure of Example 16, using isopropylamine instead of ethylenediamine. used as a ligand.

Subsequently, the compound of the invention was prepared as follows:

Sulfato-bis-isopropylaminoplatinum (II) (0.427 g) was dissolved in water (50 mL) and sodium neodecanoate (0.344 g neodecanoic acid in 20 mL methanol and 2 mL 1 N sodium hydroxide) was added. The reaction mixture is stirred for 2-3 hours. The reaction mixture was stirred at room temperature and then evaporated to dryness in a rotary evaporator at 40-45 ° C under reduced pressure. A gum was obtained which was extracted with chloroform, the chloroform extract was dried over magnesium sulfate, the magnesium sulfate was filtered off and the filtrate was concentrated to dryness under reduced pressure. The product was dried in vacuo over phosphorus pentoxide and stored at 0 ° C.

Analysis for C _{2 4} 6H56N2O Pt Calcd: Found: C 47.74%, H8,47% N3,93%;

Calculated: C 47.63%, H8.53%, N4.26%.

The cis-bis-neodecanoato-bis-isopropylaminoplatinum (II) has the following structural formula:

[(HaCjj-CH-NH2I2 -Pt- (00C-C R'RR) ₂ wherein R, R ', R may be added to CH 3, C ₂ Hs or a C3H7 C10H19O2 Calculated karboxilátocsoportot (MW. 171).

Cis-bis-neodecanoato-bis-isopropylaminoplatinum (II) is highly soluble in chloroform, methanol and other organic solvents but insoluble in water.

Example 18

Preparation of cis-bis-decanoato-trans-R.R-1,2-diammocyclohexane-platinum (77)

0.423g sulfato-trans-R, R, 2-diaminocyclohexane-platinum (II) H ₂ O dissolved in 20 mg of water and sodium decanoate was prepared on site was added (0.344 g of decanoic acid in 20 ml methanol and 2 ml of 1N sodium hydroxide). The reaction mixture was stirred for 2-3 hours at room temperature and then concentrated to dryness under reduced pressure at 40-45 ° C in a rotary evaporator. The resulting solid product was extracted with chloroform, the chloroform extract was squeezed over anhydrous magnesium sulfate, the magnesium sulfate filtered, and the filtrate was reduced

-1 223

It was evaporated to dryness in a rotary evaporator at 198,079. The product was dried in vacuo over phosphorus pentoxide and stored at 0 ° C.

Analysis CjíHsjNjChPt · H ₂ O Calcd: Found: C 45.94%, H7,87% N4,31%;

Calculated: C 46.59%, H8.06%, N4.18%.

Cis-bis-decanoato-trans-R, R-1,2-diaminocyclohexane-platinum (II) is highly soluble in chloroform and other organic solvents but insoluble in water.

The structural formula (II) for cis-bis-decanoato-trans-R.sub.1-diaminocyclohexane-platinum is shown in formula (18).

Example 19

Preparation of cis-bis-neopentanoato trans-R, R-1,2-diaminocyclohexane platinum (II)

Sulfato-trans-R, R 1, 2-diaminocyclohexane platinum (II) · H ₂ O (0.423 g) was dissolved in water (20 mL) and barium glycopentanoate (0.204 g neopentanoic acid in 5 mL methanol and 0.3 g BafOH) · 8H2O in 50 mL of water). The reaction mixture was evaporated to dryness at 40-45 ° C under reduced pressure in a rotary evaporator, the residue was extracted with methanol, filtered and the filtrate was evaporated to dryness.

The resulting solid product was further extracted with chloroform and the chloroform extract was evaporated to dryness to give a cream product which was dried in vacuo over phosphorus pentoxide.

Analysis Ο Η ^ ^ ^ Ν Ρί · 2H ₂ O Calcd: Found: C 35.16%, H6,17% N5,27%;

Calculated: C 35.00%, H6.57%, N5.11%.

Cis-bis-7-neopentanoato-trans-R, R-1,2-diaminoeocyclohexane platinum (11) is highly soluble in chloroform, methanol and other organic solvents.

The structural formula (II) of cis-bis-neopentanoate trans-R, R-1,2-diaminocyclohexane-platinum (II) is illustrated by the formula (19).

Example 20

Lipofd Cis-Platinum Analogs Encapsulation Effectiveness and Antitumor Activity

14-19. EXAMPLE 6 Compounds prepared according to Example 6b were tested for encapsulation efficiency in liposomes as described in Example 6. The optimum dose and inhibitory efficacy of in vivo tumor growth inhibition of the compounds encapsulated in liposomes were determined as described in Example 12. The results are shown in Table XII. are summarized in Table.

XII. spreadsheet

Encapsulation efficiency and antitumor activity of lipofd cis-platinum analogs incorporated in liposomes

Compound encapsulation efficiency optimum dose (Mg / kg) % T / C L1210 ip / ip single dose Cis-hist-neodecanoato-cis-diaminoplatinum (II) 95% 50 200 Cis-bis-peroxyneodecanoate-bis-cyclohexylamine-platinum (II) 89% 100 125 Cis-bis-peroxyneodecanoate ethylenediaminetetraacetic platma (II) 82% 75 144 Cis-bis-peroxyneodecanoate-bis-isopropyl-amino-platinum (II) 87% 100 150 Cis-bis-decanoate-trans-R, R-l, 2-diaminocyclohexane-platinum (II) 96% 50 187 Cis-bis neopentanoate-trans-R, R-l, 2-Guanidino-cyclohexan-p'attiia (II) 93% 25 170

Example 21

In vitro eitotoxicity against human malignant cell tracts

Cis-bis-neodecanoato-1,2-diaminocyclohexane-platinum (II) was tested as a liposome in three colonic carcinoma human malignant cell lines (L0V0, SW620 and SE403) in a colony inhibition assay. The concentration of 50% inhibition of colony formation (IC50) was 4-8 μιηόΐ. The IC 50 value of cis-platinum for the same cell line was 3-7 pmol.

abbreviations

CDDP cis-platinum

MLV multilayer lipid vesicles

GAP reticuloendothelial cells DACH 1,2-diaminocyclohexane DMPG diinirisztoil phosphatidylcholine Glycerol DMPC dimyristoylphosphatidylcholine phosphatidylcholine PT platinakoniplex EE efficiency of encapsulation L-PT platinum complex containing liposomes SIN blood urea nitrogen

Claims (19)

PATENT CLAIMS 1. Procedure with four-way coordination, -1 325 For the preparation of platinum (II) complexes of the general formula 198,079 wherein R ₁ and R ₂ are a hydrophobic group, i.e. C 3 -C 7 cycloalkenylcarbonyloxy, C 3 -C 12 alkylcarbonyloxy, or the two (8) -12 C 5 -alkyl) iminobis [(C 1 -C 4) -alkylcarbonyloxy], R ₃ and R ₄ are H I 10 - _N - _{R 5} I A group of the formula H wherein R _s is hydrogen, C 1-5 alkyl or C 4-8 cycloalkyl, or R ₃ and R _{4 taken} together are C 4-8 cycloalkyl-1,2-diamino, or C 1-4 alkenyl. - 1,2-diamino-group ²⁰ which is characterized by a complex that is soluble in water and chloroform and insoluble in water, characterized in that a szuifalo-trans-R, R, 2-Dlamini platlna alkane (II) complex above a carboxylato monoanion-alkaline earth metal salt having a hydrophobic group, or ² ^ -alkálifém salt reacted. 2. The method of claim 1, wherein _R5 is appropriate are used as starting material containing 1-5 carbon atoms. The process according to claim 1, wherein the reaction is carried out in an aqueous medium. 4. A process according to claim 1, wherein the alkali metal sulfate or alkaline metal sulfate is separated from the reaction mixture by filtration, and the gg and platinum complex is recovered by crystallization or by evaporation of the solvent. Process according to claim 1, characterized in that the evaporation under reduced pressure for 45- 50 ° C. 40 6. A process according to claim 1, wherein the resulting platinum complex is further dried on a desiccant. 7. The process according to claim 6, wherein the drying agent is phosphorus pentoxide. A process for the preparation of liposomes containing platinum (II) complexes according to claim 1, characterized in that (i) a quaternary coordination platinum (II) complex of the general formula wherein R _{b is} R ₂ , R ₃ and R ₄ mixing according to claim 1 with (i) a phospholipid in a 1:15 ratio by weight in a hydrophobic solvent, (ii) evaporating the hydrophobic solvent to lyophilize a film or mixture of phosphollpid and complex and then (Ill) the resulting film or powder in an aqueous medium dispersed to form a liposome. 9. A process according to claim 8 wherein R _s is a complex having a C 1-5 alkyl group. 10. The process according to claim 8, wherein the phospholipid is phosphatidylglycerol, phosphatidylcholine or a mixture thereof. 11. The process of claim 8, wherein in step (ii) the mixture is further mixed with additional phospholipid and cholesterol. 12. The process of claim 10 wherein the phosphatidylglycerol is dimyristoylphosphatidylglycerol and the phosphatidylcholine is dimyristoylphosphatidylcholine. 13. The process of claim 12, wherein the dimyristoyl phosphatidylglycerol and the dimyristoyl phosphatidylcholine are used in a ratio of 1:10 to 10: 1 by weight. 14. The process of claim 12, wherein the dimyristoyl phosphatidylglycerol and the dimyristoyl phosphatidylcholine are used in a weight ratio of 3: 7. 15. The process of claim 8, wherein the complex and phospholipid are used in a weight ratio of from 1:10 to 1:30. 16. The process of claim 8, wherein the complex and the phospholipid are used in a ratio of 1: 15 by weight, 17. The method of claim 8, wherein the components are spontaneously reacted to produce a multilamellar liposome. 18. The method of claim 8, wherein the liposomes are further converted to nylamers by ultrasound scintillation. The process according to claim 8, wherein the evaporation is carried out in a rotary evaporator.