WO2005062991A2

WO2005062991A2 - Ten and 20-di-esterification derivatives of camptothecins and methods to treat cancers

Info

Publication number: WO2005062991A2
Application number: PCT/US2004/043978
Authority: WO
Inventors: Zhi-Song Cao; Chunlin Tao
Original assignee: American Bioscience, Inc.
Priority date: 2003-12-23
Filing date: 2004-12-23
Publication date: 2005-07-14
Also published as: WO2005062991A3

Abstract

The present invention is related to lower alkyl 10,20 di-O-ester derivatives of camptothecins and pharmaceutical formulations thereof. The compounds and pharmaceutical formulations of the present invention possess increased biological life span and bioavailablity and reduced toxicity, while maintaining anti-cancer activity.

Description

TEN AND 20-DI-ESTERIFICATION DERIVATIVES OF CAMPTOTHECINS AND METHODS TO TREAT CANCERS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/531,941, filed December 23, 2003, which is incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention is directed to 10,20-di-esterification derivatives of camptothecins.

BACKGROUND OF THE INVENTION

[0003] Camptothecin (CPT) was isolated and purified by Wall and coworkers in 1966 (J. Am. Chem. Soc. 88, 3888, (1966)). This compound was initially tested against the mouse leukemia L 1210 system and found active. The compound was quickly tested in human clinical trials. At this time, unfortunately, inherent anticancer activity of the molecule was not found; instead, severe toxicity was observed for those patients who participated in the trials (Gottlieb et al., Cancer Chemother. Rep. 54, 461, (1970), and 56, 103, (1972), Muggia et al., Cancer Chemother. Rep. 56, 515, (1972), Moertel et al., Cancer Chemother. Rep. 56, 95, (1972), and Schaeppi et al., Cancer Chemother. Rep. 5:25, (1974)). Trials were accordingly discontinued. The reason for the failure of the early trial was later found to be an incorrect drug formulation. Camptothecin is insoluble in water. In order to use the drug for intravenous (iv) administration, camptothecin was converted to its sodium form (CPT sodium carboxylate). This form, although water-soluble, is practically devoid of anticancer activity. For example, a careful evaluation of these agents in animal models made by Wani et al. revealed that the sodium salt is only 10-20% as potent as the parent camptothecin (J Med. Chem. 23, 554, (1980)).

[0004] Important parameters for the anticancer activity of camptothecin derivatives have now been established (Wall et al., Ann. Rev. Pharmacol. Toxicol. 17, 117, (1977)). The intact lactone form with an α-hydroxyl group with (^-configuration at the position 20 of the molecule is essential for antitumor activity. To maintain the molecule as an intact lactone form is critical for success of the treatment. Camptothecin and its derivatives have shown a spectacular activity against a wide spectrum of human tumors grown in xenografts in nude mice (Giovanella et al., Cancer Res. 51, 3052, (1991), and Natelson et al., Annals N. Y. Acad. Sci. 803, 224, (1996)), but much less activity was observed in human clinical trials. This difference in antitumor activity has been associated with the finding that the hydrolysis of lactone to carboxylate of the molecule is much faster in human plasma than in mouse. Burke and coworkers have systematically studied the stability of camptothecin derivatives in human serum (Annals N. Y. Acad. Sci. 803, 29. (1996)).

[0005] Ten-hydroxycamptothecin (10-HCPT) is a derivative of camptothecin, and also a natural occurring compound. This compound was obtained on isolation of camptothecin and can now be synthesized from camptothecin in a number of ways. Currently, two anti-cancer agents directly derived from 10-HCPT are commercially available for treatment. One is topotecan, and the other is irinotecan:

[0006] The molecule, 10-HCPT, is very potent against cancer cells. Unfortunately, 10- HCPT is not useful for cancer treatment because of toxicities. The molecule bears two hydroxyl groups one each at the C-l O and C-20 positions. The C-20 hydroxyl group is adjacent to the carbonyl group of E-ring of the molecule, which constructs a reactive α- hydroxy lactone moiety. This feature of the molecule makes the lactone moiety very sensitive to hydrolysis, and thus the molecule is not stable when circulating in the body. The 10- phenolic hydroxyl group of 10-HCPT is not stable in the process of enzymatic metabolism reactions. It is well known that phenolic hydroxy-containing moiety of an organic compound can be enzymatically oxidized into a semi-quinone or quinone compound during the process of metabolism. The corresponding semi-quinone or quinone metabolite is usually more toxic than the parental phenolic compound.

[0007] Clearly, there is a need to obtain the protected 10-HCPT derivatives having more stability in human and generating longer biological life span.

[0008] Several reports have disclosed the esterification of 10-HCPT at the C-20 position. U.S. patent No. 4,943,579 discloses the preparation of several water-soluble camptothecin esters by the esterification of camptothecin with amino acids as acylating reagents at the C-20 position. U.S. Patent No. 5,646,159 discloses the esterification of 10,11- dioxymethylenecamptothecin with amino acid derivatives as acylating reagents at the position 20 to provide several water-soluble compounds. U.S. Patent No. 5,731,316 discloses preparation of alkyl or alkenyl ester products of camptothecins by the esterification reaction at C-20 position. U.S. Patent No. 6,407,239 Bl discloses the preparation of aromatic ester products of camptothecins by introduction of an aryl group at C-20 position. All of these disclosures are related to single protection of the CPT molecule, meaning that the esterification reaction takes place either at the C-10 position or the C-20 position. The present invention relates to the double protection of 10-HCPT. The present invention simultaneously introduces two acyl groups into the molecules of 10-HCPT. The compounds disclosed by us in this invention significantly increases the biological life span and bioavailability while maintaining the inherent anti-cancer activity and lowering the toxicity. The present invention is the first time to teach the art of making 10,20-diester products of 10- HCPT.

SUMMARY OF THE INVENTION

[0009] Accordingly, it is an object of the present invention to provide new 10-HCPT compounds, which are active against various different types of tumors.

[0010] It is a further object of the present invention to provide 10,20-diester CPT derivatives.

[0011] It is another object of the present invention to provide prodrugs of 10-HCPT.

These prodrugs can release the parent active 10-HCPT compound by an enzymatic cleavage of 10,20-diester chain after reaching the targeting organs.

[0012] It is still another objection of the present invention to provide the methodology of preparing the above-said diester compounds of 10-HCPT.

[0013] It is still a further object of the present invention to provide an improved treatment for certain types of cancers.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Conversion of the compounds disclosed by the present invention to the parental 10-HCPT is mediated by a group of enzymes called esterases. Mammalian carboxylesterases represent a multigene family and are present in a wide variety of organs and tissues of many mammalian species (Satoh, in reviews in Biochemical Toxicology, 8:155-81, New York: Elsevier, (1987); Heymann, in Enzymatic Basis of Detoxication, 2:291-323, New York: Academic, (1980), and in Metabolic Basis of Detoxication, 1:229-45, New York: Academic, (1982)). More information about distribution of carboxylesterases in tissues can be found in a review article written by Satoh et al. (Annu. Rev. Pharmacol. Toxicol. 38, 257, (1998)). Carboxylesterases are known to be responsible for the hydrolysis of many exogenous compounds, the consequences of which include both activation of prodrugs and deactivation of drugs. The compounds disclosed by the present invention are rapidly distributed throughout the body within a short period of time after administration, and the di-ester chain at the positions of C- 10 and C-20 (respectively) are subsequently cleaved to release the active parental compounds by carboxylesterases specifically in organ tissues. [0015] The reaction of 10-HCPT derivatives with the corresponding acylating reagents forms the di-ester products as depicted Scheme 1.

Scheme I

RCO-O-COR/H₂SO₄, heat

[0016] The preparation reaction is carried out in the following way: the starting 10- HCPT, 2 to 10 molar equivalent of the reacting acylating reagents of the general formula RCO-O-COR, and a catalytic amount of concentrated H₂SO₄ are added to a round-bottomed flask equipped with a magnetic st rer. Illustrative acylating reagents are lower alkyl anhydrides, such as, for example, acetic anhydride, propionic anhydride, and the like. The mixture is stirred at elevated temperature (110 ± 10 °C.) under nitrogen gas for 12-48 hours. After cooling to room temperature, the mixture is poured onto a suitable amount of ice- water portion by portion while stirring. The amount of ice- water is governed by the scale of the reaction. Generally, the ratio is about 1:10. For example, the amount of ice-water is 500 mL if the volume of the reaction mixture is 50 mL. The crude product is collected by filtration and then air-dried at room temperature for 12 to 36 hours depending on the moisture in the air. The pure di-ester product is obtained as white or other-color powders after precipitation from petroleum ether. Reaction yields are ranging from 10 to 90%.

[0017] The compounds disclosed in the present invention are prodrugs of 10-HCPT. 10- HCPT is very active against cancers, but very toxic as well. This compound has not been used for treatment as an anti-cancer agent worldwide (except for China where it is used for treatment). The compounds of the present invention inherit the inherent anti-cancer activity and give up most of the toxicity of their parental compound. Thus, the compounds of the present invention can be very effective in the treatment of cancers, including, but not limited to, human cancers of the lung, breast, colon, prostate, melanoma, pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries urinary track, gastrointestinal, and other solid tumors which grow in an anatomical site. Other solid tumors include, but not limited to, colon and rectal cancers. The compounds of the present invention can also be effective in the treatment of the other types of tumors growing in blood stream and blood borne such as leukemia. The compounds of the present invention can be administrated by any acceptable route including, but not limited to, orally, intramuscularly, transdermally, intravenously, through an inhaler or other air borne delivery systems, and the like. Preferably, the compounds and the formulations of the present invention are administrated orally, intramuscularly, or transdermally and most preferably delivered orally.

[0018] For administration orally, the active compound of the present invention could be incorporated into suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

[0019] Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. [002O] Prefened compositions for administration by injection include those comprising a biologically active analog of the present invention as the active ingredient, in association with a surface-active agent (or wetting agent or surfactant) or in the form of an emulsion (as a water-in-oil or oil-in- water emulsion). Suitable surface-active agents include, in particular, nonionic agents, such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85).

[0021] In accordance with the present invention, there are provided compositions and methods useful for in vivo delivery of compounds of this invention, in the form of nanoparticles that are suitable for parenteral administration in aqueous suspension. [0022] It is well known that colloidal nanoparticles or particles <200 nm in size have a widely applications for formulation of various biologies. U. S. Patent Nos. 5,916,596, 6,506,405 and 6,537,579 teach the preparation of nanoparticle from the biocompatible polymers, such as albumin. A large number of conventional pharmacologically active agents circulate in the blood stream bound to carrier proteins (through hydrophobic or ionic interactions) of which the most common example is serum albumin. Thus, the compositions produced thereby provide for a pharmacologically active agent that is "pre-bound" to a protein (through hydrophobic or ionic interactions) prior to administration.

[0023] Thus, in accordance with the present invention, there are provided methods for the formation of nanoparticles of present invention by a solvent evaporation technique from an oil-in- water emulsion prepared under conditions of high shear forces (e.g., sonication, high pressure homogenization, or the like).

[0024] In accordance with the present invention, there are also provided submicron particles in powder form, which can easily be reconstituted in water or saline. The powder is obtained after removal of water by lyophilization. Human serum albumin serves as the structural component of invention nanoparticles, and also as a cryoprotectant and reconstitution aid. The preparation of particles filterable through a 0.22 micron filter according to the invention method as described herein, followed by drying or lyophilization, produces a sterile solid formulation useful for intravenous injection. [0025] A number of biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. Several biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. For example, naturally occurring biocompatible materials such as proteins, polypeptides, oligopeptides, polynucleotides, polysaccharides (e.g., starch, cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, and the like), lipids, and so on, are candidates for such modification. Examples of suitable proteins include albumin, insulin, hemoglobin, lysozyme, immunoglobulins, α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, casein and the like, as well as combinations of any two or more thereof. [0026] Similarly, synthetic polymers are also good candidates for preparation of the drug formulation. Examples include polyalkylene glycols (e.g., linear or branched chain), polyvinyl alcohol, polyacrylates, polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamides, polyisopropyl acrylamides, polyvinyl pyrrolidinone, polylactide/glycolide and the like, and combinations thereof, are good candidates for the biocompatible polymer in the invention formulation.

[0027] In the preparation of invention compositions, one can optionally employ a dispersing agent to suspend or dissolve the compound. Especially prefened combinations of dispersing agents include volatile liquids such as dichloromethane, chloroform, ethyl acetate, benzene, and the like (e.g., solvents that have a high degree of solubility for the pharmacologically active agent, and are soluble in the other dispersing agent employed), along with a less volatile dispersing agent. When added to the other dispersing agent, these volatile additives help to drive the solubility of the pharmacologically active agent into the dispersing agent. Following dissolution, the volatile component may be removed by evaporation (optionally under vacuum).

[0028] Thus, in accordance with the present invention, camptothecin di-O-ester derivative is dissolved in a suitable solvent (e.g., chloroform, methylene chloride, ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide, methyl pyrrolidinone, or the like, as well as mixtures of any two or more thereof). Additional solvents contemplated for use in the practice of the present invention include soybean oil, coconut oil, olive oil, safflower oil, cotton seed oil, sesame oil, orange oil, limonene oil, Q- C₂o alcohols, C₂-C₂o esters, C₃-C₂o ketones, polyethylene glycols, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and combinations thereof. [0029] Unlike conventional methods for nanoparticle formation, a polymer (e.g. polylactic acid) is not dissolved in the solvent. The oil phase employed in the preparation of invention compositions contains only the pharmacologically active agent dissolved in solvent. [0030] Next, a protein (e.g., human serum albumin) is added (into the aqueous phase) to act as a stabilizing agent for the formation of stable nanodroplets. Protein is added at a concentration in the range of about 0.05 to 25% (w/v), more preferably in the range of about 0.5-5% (w/v). Unlike conventional methods for nanoparticle formation, no surfactant (e.g. sodium lauryl sulfate, lecithin, Tween^® 80, Pluronic^® F-68 and the like) is added to the mixture.

[0031] An emulsion is formed by homogenization under high pressure and high shear forces. Such homogenization is conveniently carried out in a high pressure homogenizer, typically operated at pressures in the range of about 3,000 up to 30,000 psi. Preferably, such processes are carried out at pressures in the range of about 6,000 up to 25,000 psi. The resulting emulsion comprises very small nanodroplets of the nonaqueous solvent (containing the dissolved pharmacologically active agent) and very small nanodroplets of the protein stabilizing agent. Acceptable methods of homogenization include processes imparting high shear and cavitation such as high pressure homogenization, high shear mixers, sonication, high shear impellers, and the like.

[0032] Finally, the solvent is evaporated under reduced pressure to yield a colloidal system composed of protein-coated nanoparticles of pharmacologically active agent and protein. Acceptable methods of evaporation include the use of rotary evaporators, falling film evaporators, spray driers, freeze driers, and the like.

[0033] Following evaporation of solvent, the liquid suspension may be dried to obtain a powder containing the camptothecin di-O-ester derivative and protein. The resulting powder can be redispersed at any convenient time into a suitable aqueous medium such as saline, buffered saline, water, buffered aqueous media, solutions of amino acids, solutions of vitamins, solutions of carbohydrates, or the like, as well as combinations of any two or more thereof, to obtain a suspension that can be administered to mammals. Methods contemplated for obtaining this powder include freeze-drying, spray drying, and the like. [0034] A number of biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. As used herein, the term "biocompatible" describes a substance that does not appreciably alter or affect in any adverse way, the biological system into which it is introduced. A presently prefened polymeric for use in the formation of a shell is the protein albumin. Other suitable biocompatible materials maybe utilized in the present formulation and these have been discussed in detail in related applications.

[0035] Several biocompatible materials may be employed in the practice of the present invention for the formation of a polymeric shell. For example, naturally occu ing biocompatible materials such as proteins, polypeptides, oligopeptides, polynucleotides, polysaccharides (e.g., starch, cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, and the like), lipids, and so on, are candidates for such modification.

[0036] Similarly, synthetic polymers are also good candidates for preparation of the drug formulation. Examples include polyalkylene glycols (e.g., linear or branched chain), polyvinyl alcohol, polyacrylates, polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamides, polyisopropyl acrylamides, polyvinyl pynolidinone, polylactide/glycolide and the like, and combinations thereof, are good candidates for the biocompatible polymer in the invention formulation.

[0037] These biocompatible materials may also be employed in several physical forms such as gels, crosslinked or uncrosslinked to provide matrices from which the pharmacologically active ingredient, for example paclitaxel, may be released by diffusion and/or degradation of the matrix. Temperature sensitive materials may also be utilized as the dispersing matrix for the invention formulation. Thus, for example, the camptothecin diester maybe injected in a liquid formulation of the temperature sensitive material (e.g., copolymers of polyacrylamides or copolymers of polyalkylene glycols and polylactide/glycolides) which gel at the tumor site and provide slow release of active drugs.

[0038] The camptothecin diester formulation may be dispersed into a matrix of the above- mentioned biocompatible polymers to provide a controlled release formulation of camptothecin, which through the properties of the camptothecin diester formulation (albumin associated with camptothecin diester) results in lower toxicity to brain tissue as well as lower systemic toxicity. This combination of camptothecin diester or other chemotherapeutic agents formulated similar to camptothecin diester together with a biocompatible polymer matrix may be useful for the controlled local delivery of chemotherapeutic agents for treating solid tumors in the brain and peritoneum (ovarian cancer) and in local applications to other solid tumors. These combination formulations are not limited to the use of camptothecin diester and may be utilized with a wide variety of pharmacologically active ingredients including antiinfectives, immunosuppressives and other chemotherapeutics and the like.

[0039] The compounds and formulations of the present invention are also useful as inhibitors of Topoisomerase I.

[0040] In addition, the compounds and the formulations of the present invention can be used in combination with other drugs and formulations for the treatment of cancers such as

Taxol^®, Taxotere^®, or their derivatives, VP-16, 5-FU, as well as cisplatin and derivatives thereof.

[0041] Other features of the present invention will become apparent in view of the following examples, which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLE 1

[0042] Preparation of 10-Hydroxycamptothecin-10,20-diacetate. To 20 mL acetic anhydride in a 100 mL round-bottomed flask equipped with a magnetic stiner were added 0.5 g of 10-hydroxycamptothecin and a few drops of concentrated sulfuric acid. The mixture was stirred for overnight (~15 h) at 90 ± 10 °C. After cooling down to room temperature, the mixture was poured onto 200 mL ice-water portion by portion while stirring. After stirring for another 20 to 30 min, the suspension was filtrated. The crude product was allowed to be air-dried under room temperature for about a day and was then dissolved into 10 to 15 mL acetone. The acetone solution was poured onto 100 mL petroleum ether portion by portion while stirring. The pure product (0.46 g) was obtained as ^"white powders after precipitation from petroleum ether. Yield 74%.

EXAMPLE 2

[0043] Preparation of 10-Hydroxycamptothecin-10,20-dipropionate. To 20 mL propionic anhydride in a 100 mL round-bottomed flask equipped with a magnetic stiner were added 1.0 g of 10-hydroxycamptothecin and a few drops of concentrated sulfuric acid. The mixture was stined for overnight (~15 h) at 90 ± 10 °C. After cooling down to room temperature, the mixture was poured onto 200 mL ice- water portion by portion while stirring. After stirring for another 20 to 30 min, the suspension was filtrated. The crude product was allowed to be air-dried under room temperature for about a day and was then dissolved into 10 to 15 mL acetone. The acetone solution was poured onto 100 mL petroleum ether portion by portion while stirring. The pure product (1.05 g) was obtained as white powders after precipitation from petroleum ether. Yield 81%. 1H NMR (500 MHz, CDC1₃) B 0.97 (t, J=7.5 Hz, 3H), 1.15 (t, J= 7.5 Hz, 3H), 1.32 (t, J= 7.5 Hz, 3H), 2.15 (dq, J=14.0, 7.5 Hz, 1H), 2.29 (dq, J=14.0, 7.5 Hz, 1H), 2.53 (m, 1H), 2.67 (q., J- 7.5 Hz, 1H), 5.28 (d, J= 3.9 Hz, 2H), 5.41 (d, J=17.2, IH), 5.67 (d, JA7.2, IH), 7.20 (s, IH, 14-H), 7.57 (dd, J=2.5, 9.2 Hz, IH), 7.69 (d, J=A5 Hz, IH), 8.22 (d, J=9.2 Hz, IH), 8.34 (s, IH); Anal. Calcd for (C₂₆H₂₄N₂0₇ + H)⁺ 477. Found: 477.

EXAMPLE 3

[0044] Preparation of 10-Hydroxycamptothecin- 10,20-dipropionate-albumin compositions. 30 mg 10-Hydroxycamptothecin-10,20-dipropionate (as prepared in Example 2) is dissolved in 3.0 mL methylene chloride/methanol (9/1). The solution is then added into 27.0 mL of human serum albumin solution (3% w/v). The mixture is homogenized for 5 minutes at low RPM (Vitris homogenizer model: Tempest I.Q.) in order to form a crude emulsion, and then transfened into a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while recycling the emulsion for at least 5 cycles. The resulting system was transfened into a Rotavap and solvent was rapidly removed at 40 °C, at reduced pressure (30 mm Hg), for 20-30 minutes. The resulting dispersion was translucent and the typical average diameter of the resulting particles was in the range 50-220 ran (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and proportions of drug, solvents, proteins used in this example are not limiting in anyway.

EXAMPLE 4

[0045] Preparation of nanoparticles by sonication. The purpose of this example is to demonstrate the formation of nanoparticles of camptothecin diester by using cavitation and high shear forces during a sonication process. Thus, 20 mg 10-Hydroxycamptothecin- 10,20- dipropionate (as prepared in Example 2) is dissolved in 1.0 mL methylene chloride. The solution was added to 4.0 mL of human serum abumin solution (5% w/v). The mixture was homogenized for 5 minutes at low RPM (Vitris homogenizer, model: Tempest I.Q.) in order to form a crude emulsion, and then transfened into a 40 kHz sonicator cell. The sonicator was performed at 60-90% power at 0 degrees for 1 min (550 Sonic Dismembrator). The mixture was transfened into a Rotary evaporator, and methylene chloride was rapidly removed at 40 °C, at reduced pressure (30 mm Hg), for 20-30 minutes. The typical diameter of the resulting paclitaxel particles was 350-420 n (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 h without adding any cryoprotectant. The resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. EXAMPLE 5

[0046] This example shows the in vitro growth inhibition experiments for 10-hydroxy camptothecin analogs on MX-1 (human breast carcinoma) cells. The cytotoxicity assay was quantitated using the Promega CellTiter Blue Cell Viability Assay. Briefly, cells (5000 cells/well) were plated onto 96-well microtiter plates in RPMI 1640 medium supplemented with 10%) FBS and incubated at 37 °C in a humidified 5% CO₂ atmosphere. After 24 h, cells were exposed to various concentrations of compound and cultured for another 72 h. 100 μl of media were removed and 20 μl of Promega CellTiter Blue reagent were added to each well and shaken to mix. After 4 hours of incubation at 37 °C in a humidified 5% CO₂ atmosphere, the plates were read at 544ex/620em. The fluorescence produced is proportional to the number of viable cells. After plotting fluorescence produced against drug concentration, the IC₅o was calculated as the half-life of the resulting non-linear regression. The data showed in Table 1.

[0047] Table 1 , IC₅₀ of 10-hydroxy camptothecin and its analog

[0048] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0049] Prefened embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those prefened embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A di-ester derivatives of camptothecins having the following general structure:

wherein R is a C_1-2 alkyl group.

2. A pharmaceutical composition, comprising an effective amount of the camptothecin di-ester derivatives of claim 1 and a pharmaceutically acceptable carrier or diluent.

3. The di-ester of claim 1 , wherein R = methyl group.

4. The di-ester of claim 1 , wherein R = ethyl group.

5. A method of inhibiting cancer cells comprising contacting said cancer cells with a compound of claim 1.

The method of claim 5, wherein the cancer cells are breast cancer cells.

7. The method of claim 5, wherein the compound is a compound of claims 3 or 4.

8. A method of inhibiting the enzyme topoisomerase I by contacting said enzyme with a compound of claim 1.