Indian Patents. 223414:CONJUGATE OF A POLYGLUTAMIC ACID POLYMER COVALENTLY BONDED TO CAPTOTHECIN MOLECULE, CAPABLE OF BEING USED AS MEDICAMENT, AND PROCESS OF ITS PREPARATION

Title of Invention	CONJUGATE OF A POLYGLUTAMIC ACID POLYMER COVALENTLY BONDED TO CAPTOTHECIN MOLECULE, CAPABLE OF BEING USED AS MEDICAMENT, AND PROCESS OF ITS PREPARATION
Abstract	The invention provides new processes for preparing polyglutamic acid-therapeutic agent conjugates for clinical development and pharmaceutical use, and polyglutamic acid- therapeutic agent conjugates prepared by these processes.

Full Text	MANUFACTURE OF POLYGLUTAMATE-THERAPEUTIC AGENT CONJUGATES Field of the Invention This invention relates to a process for scaled-up manufacture of polyglutamate- therapeutic agent conjugates for clinical development. BACKGROUND OF THE INVENTION The antitumor agent paclitaxel shows increased efficacy and decreased toxicity when administered to tumor-bearing hosts as a polygJutamic acid conjugate compared with the unconjugated form of the drug (U.S. Serial No. 08/815,104, allowed; Li et al, Cancer Res. 58:. 2404, 1998). The polyglutamic acid-paclitaxel conjugate shows increased water solubility, a slower clearance from the body, and an increased accumulation in the tumor. Conjugates of polyglutamic acid and various other therapeutic agents are expected to provide clinically useful alternatives to the presently available formulations. For research purposes, the polyglutamic acid-therapeutic agent conjugates can be produced by the method disclosed in Li et al, ibid. In that method, the conjugate is prepared as a sodium salt, dialyzed to remove low molecular weight contaminants and excess salt and then lyophilized. The method is not well-suited for large-scale manufacture of quantities of conjugates for clinical development and use, however. In particular, the use of dialysis to remove impurities is time-consuming and lowers final product yield. In addition, although many pharmaceuticals have more favorable properties when prepared as salts (e.g., improved solubility, storage, and handling), this is not true of the polyglutamate-therapeutic agent conjugates of the present invention. The Attorney Docket No. 1402 salt forms of the conjugates are electrostatic solids, not free flowing powders. They are more difficult to package, more susceptible to dust contamination and more likely to contaminate the workplace with cytotoxic agents than are free flowing powders. Therefore, there is a need for an improved process of manufacture of polyglutamic acid- therapeutic agent conjugates that can be used to produce gram to hundreds of gram quantities of these conjugates in high yields and in a manner that provides for improved materials handling and packaging. SUMMARY OF THE INVENTION The present invention satisfies this need by providing an improved process for preparing a polyglutamic acid-therapeutic agent conjugate that is capable of providing gram to hundreds of gram quantities of pharmaceutical grade conjugate with yields of between 85 and 98%. In one embodiment, the process comprises the steps of: (a) providing the protonated form of a polyglutamic acid polymer and a therapeutic agent for conjugation thereto; (b) covalently linking said agent to said polyglutamic acid polymer in an inert organic solvent to form a polyglutamic acid-therapeutic agent conjugate; (c) precipitating said polyglutamic acid-therapeutic agent conjugate from solution by addition of an excess volume of aqueous salt solution; and (d) collecting said conjugate as a protonated solid- Additional removal of residual low molecular weight contaminants can be carried out between step (c) and step (d) or after step (d). In another embodiment, which is preferred for the large-scale manufacture of polyglutamic acid-2'paclitaxel conjugate, the process comprises the steps of: (a) providing an aqueous solution of the sodium salt of poly-L-glutamic acid; (b) acidifying the solution to a pH of about 2 to 4, thereby converting the sodium salt of poly-L-glutamic acid to the protonated form and precipitating it from solution; (c) collecting the poly-L-glutamic acid precipitate and washing with water; (d) drying the poly-L-glutamic acid to a water content of between 7% and 21% by weight; (e) contacting the poly-L-glutamic acid with paclitaxel under standard coupling conditions for sufficient time to conjugate paclitaxel tothe polyglutamic acid polymer via an ester linkage formed between the 2'-OH group of paclitaxel and a carboxy group of poly-L-glutamic acid; (f) cooling the reaction mixture from between 0°C to 10"C while slowly adding aqueous salt solution to the reaction mixture; (h) acidifying the resulting suspension; (i) collecting the conjugate as a protonated solid; and (j) extracting impurities from the protonated solid. Any polyglutamic acid-therapeutic agent conjugate can be prepared by the processes described herein. In one preferred embodiment, the therapeutic agents are antitumor agents, e.g., paclitaxel, docetaxel, etoposide, teniposide, epothilones, gemcitabine, 20(S)(+) camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl- 10-hydroxycamptothecin, 9-dimethylaminomethyl-10-hydroxycamptothecin, 10,11- methylenedioxycamptothecin, 7-methylpiperizinomethyl-10,l 1- ethylenedioxycamptothecin, flavopiridol, geldanamycin, 17-(allylamino)-17- demethoxygeldanamycin, ecteinascidin 743, phthalascidin, CT-2584 (1-(U- (dodecylaniino)-10-hydroxyundecyl)-3J-dimemylxantirine, CT-4582 (l-(ll-(N-methyl N-dodecyl amino)-10-hydroxyundecyl)-3,7-dimetb.ylxanthine), doxorubicin, adriamycinone, melphalan, fludarabine, daunoraycin, verapamil, 5-fluorouraci], FUDR, cyclosporin, retinoic acids and others. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Exemplary conjugates Fig. 2. Manufacturing Scheme for poly-L-glutamic acid-paclitaxel conjugate Fig. 3. Proton NMR scan of poly-L-glutamic acid paclitaxel conjugate Fig. 4. Preparation of poly-1-glutamic acid-glycyl-20(S)camptothecin Figs. 5-7. Reaction Schemes I-III. DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, "a polyglutamic acid" or "polyglutamic acid polymer " includes poly (1-glutamic acid), poly (d-glutamic acid) and poly (dl-glutamic acid). Preferably the polyglutamic acid polymer comprises at least 50% of its amino acid residues as glutamic acid, and more preferably, 100%. The polyglutamic acid polymer can be substituted up to 50% by naturally occurring or chemically modified amino acids, preferably hydrophilic amino acids, provided that when conjugated to a therapeutic agent, the substituted polyglutamic acid polymer has improved aqueous solubility and/or improved efficacy relative to the unconjugated therapeutic agent, and is preferably nonimmunogenic. The molecular weight of the polyglutamic acid polymer used in the preparation of the conjugate by the methods described herein is typically greater than 5000 daltons, preferably from 20kd to 80kd, more preferably from 30 kd to 60kd (as determined by viscosity). Those skilled in the art will appreciate that the molecular weight values may be different when measured by other methods. These other methods include, for example, gel permeation, low angle light scattering, multiple angle laser light scattering, refractive index and combinations thereof. As used herein, a "polyglutamic acid -therapeutic agent conjugate" refers to a polyglutamic acid polymer that is covalently bonded to the therapeutic agent by a direct linkage between a carboxylic acid residue of the polyglutamic acid and a functional group of the therapeutic agent, or by an indirect linkage via one or more bifunctional spacer groups. Preferred spacer groups are those that are relatively stable to hydrolysis in the circulation, are biodegradable and are nontoxic when cleaved from the conjugate. Of course, it is understood that suitable spacers will not interfere with the antitumor efficacy of the conjugates. Exemplary spacers include amino acids (e.g., glycine, alanine, leucine, isoleucine), hydroxyacids, diols, aminothiols, hydroxythiols, aminoalcohols, and combinations of these. A therapeutic agent can be linked to the polymer or spacer by any linking method that results in a physiologically cleavable bond (i.e., a bond that is cleavable by enzymatic or nonenzymatic mechanisms that pertain to conditions in a living animal organism). Examples of preferred linkages include ester, amide, carbamate, carbonate.acyloxyalkylether.acyloxyalkylthioether, acyloxyalkylester, acyloxyalkylamide, acyloxyalkoxycarbonyl, acyloxyalkylamine, acyloxyalkylamide, acyloxyalkylcarbamate, acyloxyalkylsulfonamide, ketal, acetal, disulfide, thioester, N-acylamide, alkoxycarbonyloxyalkyl, urea, and N-sulfonylimidate. Methods for forming these linkages are well known to those skilled in synthetic organic chemistry, and can be found for example in standard texts such as J. March, Advanced Organic Chemistry, Wiley Interscience, 4th Edition. The degree of loading of therapeutic agent on the polymer (i.e., the" loading density") may be expressed as the number of molecules per polyglutamic acid polymer chain or preferably as a % of total weight of the conjugate ("% loading"). Any desired % loading can be obtained by adjusting the ratios of the therapeutic agent and polymer. The optimal loading density for a given conjugate and given use is determined empirically based on the desired properties of the conjugate (e.g., water solubility, therapeutic efficacy, pharmacokinetic properties, toxicity and dosage requirements). The loading density ranges from between 1% to about 60%, preferably from 5% to 55%, and even more preferably, from 20% to 55% for the conjugates that are specifically described herein. The % loading is typically determined by three methods: (1) calculated weight % (2) uv spectrophotometry; and (3) NMR ratio method. (1). The calculated weight % is based on the known weight of the polyglutamic acid starting material and the weight of the therapeutic agent. For all conjugates, the conversion to conjugate form is 100% complete, as determined by TLC on silica. (2) The uv spectrophotometry method is based on the weight % of the therapeutic agent as measured by uv absorbance, as exemplified for a paclitaxel-polyglutamic acid conjugate. The conjugate is dissolved in deionized water (2.5 or 5 mg/ml), centrifuged at 500g for 15 minutes to remove particulate matter if present, and the clear solution is diluted lOOx to 200x with deionized water. The uv absorption is read against the diluent at 228nm. A solution of the same lot of polyglutamic acid used to prepare the conjugate is dissolved at the same nominal concentration as the conjugate and its absorbance is read against the diluent at 228nm. A linear calibration curve is prepared by measuring the absorbance at 228nm of solutions of known concentrations of the paclitaxel dissolved in methanol. To calculate the percent loading, the absorbance of the polyglutamic acid solution (corrected to account for the theoretical loading of polyglutamic acid in the polyglutamic acid-paclitaxel solution) is subtracted from the polyglutamic acid-paclitaxel absorbance. This corrected absorbance is compared to the paclitaxel standard curve to obtain the paclitaxel concentration (w/v) in the conjugate solution. The percent loading is the ratio of the paclitaxel concentration to the polyglutamic acid-paclitaxel conjugate concentration times 100. (3) The NMR ratio method is based on the weight % of therapeutic agent as measured by the ratio of the peaks in the spectra resulting from the polymer in relation to the peaks from the therapeutic agent. This is illustrated below for polyglutamic acid- paclitaxel conjugate. Attorney Docket No. 1402 The area between 4.5 and 6.5 ppm is totalled and divided by the number of protons (7). This number is then compared to the area between 3.8 to 4.4 ppm for the polymer backbone and is corrected for 2 protons from paclitaxel that overlap. The two areas per proton are compared taking into account the molecular weights of the paclitaxel and the polymer. A= area per proton for polymer -s-area per proton for paclitaxel = 21.36/1.98=10.79. MW paclitaxel =837; MW of polyglutamic acid monomer is 129. % loading = (837 /(10.79 x 129) + 837) x 100 = 37.6%. The processes described herein are generally useful for preparing conjugates of polyglutamic acid with any therapeutic or diagnostic agent that is appropriately functionalized for linking to the polyglutamic acid, as described herein. The conjugates that are exemplified herein are intended to illustrate the invention, but not to limit its scope. In one preferred embodiment, the therapeutic agents comprise drugs that are effective in treating cancerous conditions that are expected to benefit from the unique pharmacokinetic properties of these conjugate (e.g., enhanced permeability and retention in tumor tissue, sustained release of active agent, long biological half life compared with the unconjugated agent, and others). Presently preferred agents include, by way of example, taxanes (e.g., paclitaxel, docetaxel), etoposide, teniposide, epothilones, gemcitabine, 20(S)(+) camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl- 10-hydroxycamptothecin, 9-dimethylaminomethyl-10-hydroxycamptothecin, 10,11- methylenedioxycamptothecin, 7-methylpiperizinomethyl-10,l 1- ethylenedioxycamptothecin, flavopiridol, geldanamycin, 17-(allylamino)-17- demethoxygeldanamycin, ecteinascidin 743, phthalascidin, CT-2584 (1-(11- (dodecylamino)-10-hydroxyundecyl)-3,7-dimethylxanthine, CT-4582 (1-(1 l-(N-methyl N-dodecyl amino)-10-hydroxyundecyl)-3,7-dimethylxanthine), doxorubicin, adriamycinone, melphalan, fludarabine, daunomycin, verapamil, 5-fluorouraci],FUDR, cyclosporin, retinoic acids and others. The therapeutic agent must be capable of attachment to the polymer by means of a functional group that is already present in the native molecule or otherwise can be introduced by well-known procedures in synthetic organic chemistry without altering the activity of the agent, to the examples given herein, the agent is relatively water-insoluble in the unconjugated form and shows greatly improved solubility following conjugation. However, water-soluble drugs are also expected to show advantages following their conjugation to polyglutamic acid (e.g., improved pharmacokinetics and retention at the site of action compared to the unconjugated agent). Reactions performed under "standard coupling conditions" are carried out in an inert solvent (e.g., DMF, DMSO, N-methylpyrrolidone) at a temperature from -20°C to 150°C, preferably from 0°C to 70°C, more preferably from 5°C to 30°C, in the presence of a coupling reagent and a catalyst. Of course, the temperature used will depend on factors such as the stability of the therapeutic agent and the reactivity of the attaching group. Suitable coupling reagents are well-known in synthetic organic chemistry and include, but are not limited to, carbodiimides, alkyl cbloroformate and triethylamine, pyridinium salts-tributyl amine, phenyl dichlorophosphate, 2-choro-l,3,5-trinitrobenzene and pyridine, di-2-pyridyl carbonate, polystyryl diphenylphosphine, (trimethylsilyl)ethoxyacetylene, 1,1' -carbonylbis(3-methylimidazolium)triflate, diethylazodicarboxylate and triphenyl phosphine, N,N' carbonyldiimidazole, methanesulphonyl chloride, pivaloyl chloride, and the like. Suitable catalysts for alcohol coupling include, e.g., 4-N.N dimethylaminopyridine and 4-pyrollidinopyridine. As used herein, the term "inert solvent" means a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF'), dimethylformamide ("DMP'), chloroform ("CHCI3"), methylene chloride (or dichloromethane or "CH2C12"), diethyl ether, ethyl acetate, acetone, methylethyl ketone, dioxane, pyridine, dimethoxyethane, t-butyl methyl Attorney Docket No. 1402 ether, and the like]. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert solvents. If multiple functional groups are present on the therapeutic agent, selective attachment of a particular group of the agent to the polyglutamic acid polymer will require the use of a suitable protecting group. The term "protecting group" or "blocking group" refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group. See, generally, T.W. Greene & P.G.M. Wuts "Protective Groups in Organic Synthesis," 3rd Ed, 1999 , John Wiley and Sons, N.Y. The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl, t-butyldimethylsilyl, triethylsilyl, MOM (methoxymethyl), MEM (2- methoxyethoxymethyl) and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product. Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC) and the like, which can be removed by conventional conditions compatible with the nature of the product. Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild hydrolysis conditions compatible with the nature of the product. Nomenclature Exemplary conjugates prepared according to the embodiments of the invention described herein are shown in Figure 1. The conjugates in the Examples below are named in the same way as the conjugates of Figure 1. Description of Preferred Embodiments In general, the process of manufacturing polyglutamate-therapeutic agent conjugates on a scale that is suitable for clinical development and pharmaceutical use comprises the steps of: (a) providing the protonated form of a polyglutamic acid polymer and a therapeutic agent for conjugation thereto; (b) covalently linking said agent to said polyglutamic acid polymer in an inert organic solvent to form a polyglutamic acid-therapeutic agent conjugate; (c) precipitating said polyglutamic acid-therapeutic agent conjugate from solution by addition of an excess volume of aqueous salt solution; and (d) collecting said conjugate as a protonated solid. The protonated form of the polyglutamic acid polymer in step (a) is obtained by acidifying a solution containing the salt of the polyglutamic acid to be used as a starting material, and converting the salt to its acid form. After separating the solid by centrifugation, the solid is washed with water. (When DMAP is to be used in step (b), it is preferred to wash the solid until the aqueous phase is pH 3 or greater). The polyglutamic acid is then dried, preferably by lyophilization and preferably to a constant weight comprising between 7% and 21% of water, prior to conjugation to a desired therapeutic agent (step (b)). The therapeutic agent of step (b) may require modification prior to conjugation, e.g., the introduction of a new functional group, the modification of a preexisting functional group or the attachment of a spacer molecule. Such modifications may require the use of protecting groups, which are described above. Attorney Docket No. 1402 Reaction schemes I-HI illustrate methods that were used for linking various exemplary therapeutic agents to poly-L-glutamic acid (PG), either directly or through glycine spacer molecules. The conditions shown in these schemes and described in the Examples may be varied, as will be readily appreciated by those skilled in synthetic organic chemistry. As noted previously, the exact conditions that are used for conjugating a particular therapeutic agent to polyglutamic acid may be based on the stability of the therapeutic agent to the reaction conditions, the reactivity of the linking groups, other factors pertinent to the manufacturing process (e.g., safety and regulatory issues), and the like. As described previously, various types of linkages may be used in preparing the conjugates, depending on the functional groups on the therapeutic agent and the spacer molecule, if a spacer is used. Thus the therapeutic agent may be conjugated to polyglutamic acid and/or spacer molecules by linkages other than ester and amide bonds. Spacers other than glycine, and coupling reagents other than those exemplified herein, can also be used. The exact conditions used for preparing the conjugates that illustrate the practice of embodiments of the present invention are described below in the Examples. In step (c), an aqueous salt solution is added to the reaction mixture to precipitate the polyglutamic acid-therapeutic agent conjugate from solution. Any water soluble inorganic salt can be used for this purpose (e.g., NaCl, KC1, NH4C1, sodium sulfate, ammonium sulfate, etc.). Preferably, 10-15% salt solution is used in lx-4x volume. In one preferred embodiment, a 2.5x volume of 10% NaCl is used. The salt solution is added slowly to the reaction mixture, which is cooled during the addition. For optimum yield of conjugate, the temperature is preferably kept between 0°C and 10°C. The precipitation step separates the polyglutamic acid-therapeutic agent conjugate from starting materials and reaction byproducts that are wholly or partially soluble under the conditions used for precipitation of the conjugate. In step (d), the conjugate is collected as the protonated solid. The suspension obtained in step (c) is preferablyacidified. Depending on the stability of the drug Attorney Docket No. 1402 i i molecule to acid conditions, a pH in the range of pH 1- 4 can be used. For preparation of polyglutamic acid-paclitaxel conjugates, however, acidification below pH 2 results in the decomposition of paclitaxel, and acidification is typically carried out at about pH 2.5. Preferably, for the removal of DMAP, HC1 is used in step (d). The suspension can be filtered or centrifuged, preferably filtered, to collect the conjugate. Unreacted starting materials, byproducts and other impurities can be removed prior to, or after acidification to yield the final protonated conjugate (illustrated in Examples 2 and 3 below, and Figs 2 and 4). For example, after addition of the salt solution, the solid can be collected and resolubilized, then either filtered or extracted with an appropriate solvent in which the contaminants are soluble but the conjugate is not (e.g., ethyl acetate). The solution is then acidified and the protonated form of the conjugate is collected as described above. Alternatively, the solid can be lyophilized, then slurried with an appropriate solvent, e.g., acetonitrile(MeCN), to remove impurities from the final protonated conjugate product. In an alternative preferred embodiment, step (c) above is replaced by step (c1), which comprises: (c') separating said polyglutamic acid-therapeutic agent conjugate from unreacted starting materials and byproducts and precipitating said polyglutamic acid-therapeutic agent conjugate from solution by addition of an organic solvent in which unreacted starting materials and byproducts are soluble. In addition to ethyl acetate and acetonitrile, examples of other solvents that can be used to purify the conjugate include chloroform, tetrahydrofuran, dioxane, toluene, 2- butylmethyl ether, and the like. Table 1 shows a representative analysis for poly L-glutamic acid-paclitaxel conjugate prepared as described in Example 3 below. TABLE 1. Analytical data % yield; grams of conjugate; grams paclitaxel/grams conjugate determined by UV method; 'grams paclitaxel/grams conjugate determined by NMR method;' wt % free paclitaxe) relative to conjugate; 'wt % residual acetonitrile relative to conjugate;' wt % residual dimethylformamide relative to conjugate;" wt % diisopropylurea relative to conjugate; wt % residue on ignition. The invention is illustrated by the following examples which should not be regarded as limiting the scope of the invention in any way. EXAMPLES In the following examples, the intermediates in the production of the conjugates were characterized by 'NMR. The molecular weights of the polyglutamic acid (Na salt) used to prepare the conjugates exemplified below ranged from 20kd to 50 kd, as specified by the supplier (Sigma) based on viscosity measurements. The average loading density of the conjugates was 37%. Example 1. Preparation of poly-L-glutamic acid Poly-L-glutamic acid sodium salt (85.9 g) (Sigma Chemical Co., 37kd MW determined by viscosity measurement) was dissolved in USP purified water (534 mL; 6.2 mL/gm), and the solution was cooled to between (0oC- 5° C. Dilute hydrochloric acid solution (1M) was added dropwise with vigorous stirring keeping the temperature Attorney Docket No. 1402 until the pH was between pH 2-2.5. During the addition, the poly-L-glutamic acid separated out of solution. The reaction mixture was warmed to room temperature and stirred for 1 hour. The suspension was centrifuged at 2700 x g for 10 minutes. The upper aqueous layer was removed and the solid was resuspended in 560 ml USP purified water and recentrifuged for 10 minutes. The upper aqueous layer was removed and the pH was measured. Washing was continued if necessary until the pH of the aqueous layer was > 3.0. The wet solid was lyophilized on a Labconco freeze dry system until a constant weight was obtained. The wt % sodium was no greater than 7000 ppm as determined by ICP. Example 2. Preparation of poly-L-glutamic acid-2'-paclitaxel conjugate Poly-L-glutamic acid (16.82 g), prepared as described in Example 1 above, was suspended in anhydrous N,N-dimethylformamide (180 mL), paclitaxel (9.923 g, 11.6 mmol) and N,N-dimethylaminopyridine (283 mg, 2.32 mmol). The reaction mixture was stirred for 30 minutes. A solution of N,N-diisopropylcarbodiimide (1.903 g, 15.08 mmol) in N,N-dimethylformamide (50 mL) was added over a period of 3 hours using a syringe pump. After the addition, the reaction was stirred until complete (about 4 hours at room temperature). The reaction was cooled to 5°C -10 °C and 10% sodium chloride solution (345 mL) was added slowly to precipitate out the poly-L-glutamic acid- paclitaxel conjugate. The precipitate was separated by transferring the mixture to a centrifuge flask and centrifuging it at 1500 g. The wet solid was resuspended in water (150 mL) and 1 M sodium bicarbonate solution (120 mL) was added slowly with vigorous stirring to bring the pH of the solution to pH 7. The reaction was stirred for an hour and filtered through a 0.2 micron filter to remove impurities. The filtrate was cooled to 0°C - 5° C and HC1 (IN) was added slowly with vigorous stirring until the pH of the solution was brought to pH 3. Stirring was continued for 30 minutes. The precipitated solid was centrifuged at 1500 g, and the wet solid was washed twice by suspending in water (150 mL) and centrifuging. The product was lyophilized to yield 24 g of poly-L- glutamic acid-paclitaxel conjugate (90% yield). Attorney Docket No. 1402 In the above procedure, the filtration step can be omitted by washing the solution with ethyl acetate (250 ml, 2x) to remove impurities. Figure 3 shows a representative proton NMR scan for poly-L-glutamic acid-2'- paclitaxel conjugate prepared by same procedure as described above, but having a higher paclitaxel loading (i.e. 55%). Example 3. Preparation of poly-L-glutamic acid-2'-paclitaxel conjugate (manufacturing process) Poly-L-glutamic acid (42 g), prepared as described in Example 1 above, was added to a three-necked three liter round bottom flask equipped with a mechanical stirrer, addition funnel and a thermo probe. N, N-dimethylformamide (350 ml) was added and stirred for 10 minutes. Paclitaxel (24.66 g) and N,N-dimethyIaminopyridine (0.70 g) was added and stirred for 10 minutes. A solution of N, N-diisopropylcarbodiimide (4.73 g) in N, N-dimethylformamide (143 ml) was added at room temperature over a period of 1 hr using the addition funnel and was stirred for four hours. The reaction mixture was cooled to 5cC-10 ° C and a cooled solution of 10% sodium chloride (1.2 L) was added dropwise using the addition funnel and keeping the temperature at 5°C-10°C by cooling the flask in an ice-salt mixture. After the addition of sodium chloride solution, IN solution of hydrochloric acid (35 ml) was added dropwise until the pH of the reaction reached 2.5. The reaction mixture was stirred for 30 minutes at 5°C-10°C and the precipitated poly-L- glutamic acid-paclitaxel conjugate was collected by filtration. The solids were washed three times with water and freeze dried in a lyophilizer for 24 hours. The dried solid was powdered into a fine powder using a mortar and pestle. The finely powdered poly-L- glutamic acid -paclitaxel conjugate was suspended in acetonitrile (1000 ml) and stirred for 2 hrs, then filtered and the solid was washed with 2 x 200 ml of acetonitrile. The solid was dried under vacuum for 24 hrs to give poly-L-glutamic acid-paclitaxel conjugate (60 g). Yield (90%). Example 4, Preparation of poly-L-glutamic acid-glycine-paclitaxel conjugate (Reaction Scheme II) Steps 1 and 2 below were carried out essentially as described in Mathew et al. (Mathew, A.E., Mejillano, M.R., Nath, J.P., Himes, R.H., and Stella, V.J., J. Med. Chem.(1992), 35, 145-151). Step 1. Preparation of 2'-(N-t-BOC-glycyl)paclitaxeI To a solution of N-t-BOC-L-glycine (131 mg, 0.75 mmol) and paclitaxel (640 mg, 0.75 mmol) in dichloromethane (20 ml) was added 1,3-diisopropylcarbodiimide (124 mg, 0.98 mmol) followed by N,N-dimethylaminopyridine (27 mg, 0.23 mmol). After stirring at room temperature for 4 hours, the mixture was concentrated under reduced pressure. The residue was purified by silica gel flash chromatography eluting with 1: l(v/v) ethyl acetate/hexane to yield 2'-(N-t-BOC-glycyl)paclitaxel (720 mg, 95% yield) as a white powder. Step 2. Preparation of 2'-(glycyl)paclitaxel A solution of 2'-(N-t-BOC-glycyl)paclitaxel (245 mg, 0.242 mmol) in formic acid (2 ml) was stirred for 30 minutes. After concentrating under reduced pressure, the residue was suspended in water (15 ml). Cold 0.05 M sodium bicarbonate solution (45 ml) was added and the solution (pH 8.0) was extracted with dichloromethane (2x 40 ml). The combined dichloromethane extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography eluting with 4 % methanol/dichloromethane to yield 2'-(glycyl)paclitaxel (161 mg, 73 % yield) as a white powder. Step 3. Preparation of poly-L-glutamic acid-2'-(glycyl) paclitaxel conjugate To a stirred suspension of poly-L-glutamic acid (275 mg, 1.87 mmol) in anhydrous dimethylformamide (6 ml) was added 2'-(glycyl)paclitaxel (161 mg, 0.177 mmol). A solution of 1,3-diisopropylcarbodimide (29 mg, 0.23 mmol) in Attorney Docket No. 1402 dimethylformamide (1.4 ml) was added to the stirred suspension over a period of 30 minutes. After stirring at room temperature for 3 hours, the mixture was cooled in an ice bath to bring the bath temperature to 0°C - 5°C and then 10 % aqueous sodium chloride solution (7 ml) was added over a period of 30 minutes to precipitate the poly-L-glutamic acid-2'-(glycyl)paclitaxel conjugate. The resulting white suspension was centrifuged at 1500g, 15 minutes. After filtration, the solid was washed twice by suspension in water (10 ml) and centrifugation. The crude product was suspended in water (6 ml) and a 1 M aqueous sodium bicarbonate solution (2.3 ml) was added slowly with stirring to bring the contents of the flask to pH 7.6. After stirring for an additional 2 hours, the aqueous layer was washed with ethyl acetate (3x 6 ml) and then acidified by addition of 1 N hydrochloric acid to pH 2.8. The precipitated solid was separated by centrifugation and washed with water (2x6 ml). The wet solid was lyophilized to give poly-L-glutamic acid-2'(glycyl)paclitaxel conjugate (315 mg, 72 % yield) as a white powder. Using a similar procedure, the above conjugate can be substituted by amino acids other than glycine. Example 5. Preparation of poIy-L-glutamic acid-2'-docetaxel conjugate (Reaction Scheme HI) Step 1. Preparation of 10-deacetylpaclitaxel 10-Deacetylpaclitaxel was prepared essentially as described in Zheng, Q.Y., Darbie, L.G., Chen, X., Murray, C.K., Tetrahedron Letters., 36, (1995), 2001-2004 and U.S. Patent No. 5,629,433. To a solution of paclitaxel (1.0 g, 1.17 mmol) in tetrahydrofuran (20 mi) was added hydrogen peroxide (30%, 20 ml) followed by sodium bicarbonate (1.92 g, 22.85 mmol). After stirring at room temperature for 18 hrs, the mixture was treated with dichloromethane/water (1:1 (v:v), 100 ml). The organic phase was washed with water (2x 30 ml), dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by silica gel flash chromatography eluting with 3% Attorney Docket No. 1402 methanol/dichloromethane to yield 10-deacetylpaclitaxeI (890 mg, 93% yield) as a white powder. Step 2. Preparation of 2%7-bis(triethylsilyl)-10-deacetylpaclitaxel 2',7-Bis(triethylsilyl)-10-deacetylpaclitaxel was prepared as described in U.S. Patent No. 5, 629,433. To a solution of 10-deacetylpacIitaxel (850 mg, 1.05 mmol) in anhydrous pyridinc (20 ml) was added chlorotriethysilane (2.72 ml, 20.1 mmol) at room temperature over a period of 30 minutes under an argon atmosphere. After stirring for 17 hours, the mixture was treated with dichloromethane (75 ml), washed with water (3x 30 ml), with 10% aqueous copper sulfate solution (4x 35 ml), with water (30 ml), and with saturated aqueous sodium chloride solution (30 ml). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to provide 2\7- bis(triethylsilyl)-10-deacetylpaclitaxel (980 mg, 90% yield) as a powder. Step 3. Preparation of 2',7-bis(triethy!sUyI)-10-deacetylpacIitaxel imine 2',7-Bis(triethylsilyl)-10-deacetylpaclitaxelimine was prepared as described in U.S. Patent No. 5, 629,433. To a solution of 2>,7-bis(triethylsilyl)-l0-deacetylpaclitaxel (730 mg, 0.70 mmol) in tetrahydrofuran (7.3 ml) was added zirconocene chloride hydride (543 mg, 2.11 mmol). After stirring at room temperature under an agron atmosphere for 15 hours, the mixture was poured into cold hexanes (75 ml). The precipitated zirconium complexes were removed by filtration. The filtrate was concentrated under reduced pressure to give 2\7- bis(triethyIsilyl)-10-deacetylpaclitaxelimine (636 mg, 92% yield) as a white powder. Step 4. Preparation of 10-deacetylpaclitaxel primary amine 10-Deacetylpaclitaxel primary amine was prepared according to U.S. Patent No. 5,629,433. A solution of 2',7-bjs(triethylsilyl)-10-deacetyipacIitaxel iminc (636 mg, 0.621 mmol) in 1% (w:w) cone, hydrochloric acid/95% cthanol (25 ml) was stirred for 15 hours, treated with water (65 ml), and washed with hexancs (2x 30 ml). The aqueous layer was neutralized (pH 7) by addition of saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (2x 40 m). The combined extracts were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the crude primary amine product (405 mg, 92% yield) as a white powder. This product was used for the next step without further purification. Step 5. Preparation of docetaxel Docetaxel was prepared according to U.S. Patent No. 5,629,433. To a solution of 10-deacetylpaclitaxel primary amine (405 mg, 0.57 mmol) in ethyl acetafe (40 ml) was added saturated aqueous sodiurh bicarbonate solution (40 ml). To this biphasic mixture was added di-(en-butyldicarbonpte (225 mg, 1.03 mmol). After stirring at room temperature for 15 hours, ethyl acetate (75 ml) was added. The organic phase was washed with water (2x 30 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel flash chromatography eluting with 4 % methanol/dichloromethane to provide docetaxel (35\ mg, 76 % yield) as a white powder. Step 6. Preparation of poly-L-glutamic acid-2'-docetaxel conjugate To a suspension of poly-L-glutamic acid (658 mg, 4.47 mmol) in anhydrous dimethylformamide (10 ml) was added docetaxel (385 mg, 0.48 mmol) and N,N- dimethylaminopyridine (12 mg. 0.096 mmol). To this stirred suspension was added a solution of 1,3-diisopropylcarbodiimide (78.8 mg, 0.624 mmol) dimethylformamide (3 Attorney Docket No. 1402 ml) dropwise over 20 minutes. After stirring for 15 hours, the mixture was cooled in an ice bath and 10% aqueous sodium chloride solution (20 ml) was added over 30 minutes. After stirring for an additional 1 hour, the solid was filtered and the filtered cake was washed with water (4x 50 ml). The solid was lyophilized to constant weight and then triturated with acetonitrile (4x 50 ml). Drying under high vacuum for 15 hours provided poly-L-glutamic acid-2'-docetaxel conjugate (890 mg, 87% yield) as a white powder. !H NMR (300 MHz, DMSO-d6): d 12.10 (s, -COOH), 7.05-8.20 (m, aromatic protons), 4.80-6.05 (m), 3.80-4.50 (m), 5.0-5.6 (m, 5-H2,7-H2), 3.70-4.35 (m), 1.20-2.80 (m), 1.00(s). Example 6. Preparation of poly-I-glutamic acid-glycyI-20(S)camptothecin (Reaction Scheme I) Steps 1 and 2 below were carried out as described by Greenwald, R.B., Pendri, A., Conover, CD., Lee, C, Choe, Y.H., Gilbert, C, Martinez, A., Xia, J., Wu, D., and Hsue, M., Bioorg. & Med. Chem. 6, (1998), 551-562. Step 1. Preparation of 20-(N-t-BOC-glycyI)-20(S)camptothedn To a solution of N-t-BOC-glycine (530 mg, 3.0 mmol) in anhydrous dichloromethane (240 ml) was added 1,3-diisopropylcarbodiimide (379 mg, 3.0 mmol), N,N- dimethylaminopyridine (244 mg, 2 mmol), and 20(S)camptothecin (348 mg, 1.0 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature. After stirring for 18 hours, the mixture was washed sequentially with 0.1N aqueous hydrochloric acid solution (2x 50 ml), with water (2x 50 ml), with 0.1M aqueous sodium bicarbonate solution (2x 25 ml), and with water (2 x 50 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was crystallized from methanol (7 ml) to provide 20-(N-t-BOC-glycyl)-20(S)camptothecin (424 mg, 84% yield) as a yellow powder. lH NMR (300 MHz, CDC13): 8 8.35 (s, 1H), 8.22 (d, J = 8.38 Hz 1H), 7.91(d, J = 8.07,1H), 7.76-7.85 (m, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.26 (s, 1H), 5.70 (d, J = 17.25 Hz, 1H), 5.40 (d, J = 17.25 Hz, 1H), 5.25 (s, 2H), Attorney Docket No. 1402 4.95 (br s, 1H), 3.98-4.25 (m, 2H), 2.18-2.26 (m, 2H), 1.38 (s, 9H), 0.95 (t, J = 7.47 Hz, 3H). Step 2. Preparation of 20-GlycyI-20(S)camptothecin trifluoroacetic acid salt A solution of 20-(N-t-BOC-glycyl)-20(S)camptothecin (424 mg, 0.84 mmol) in a 1:1 (v/v) mixture of dichloromethane/trifluoroacetic acid (21 ml) was stirred at room temperature for 1 hour. The solvents were evaporated under reduced pressure. The yellow solid was crystallized from dichloromethane/diethyl ether (3:7 (v/v), 50 ml) to provide 20-glycyl-20(S)camptothecin trifluoroacetic acid salt (361 mg, 83 % yield) as a light yellow powder. !H NMR (300 MHz, DMSO-d6): d 8.78 (s, 1H), 8.45 (br s, 2H), 8.20 (d, J = 8.2 Hz 1H), 7.70-7.95 (m, 2H), 7.30 (s, 1H), 5.55 (s, 2H), 5.30 (s, 2H), 4.35 (d, J = 17.9 Hz, 1H), 4.15 (d, J = 17.9 Hz, 1H), 2.10-2.30 (m, 2H), 1.00 (t, J= 7.4 Hz, 3H). Step 3. Preparation of poly-L-glutamic acid-20-glycyl-20(S)camptothecin conjugate To a stirred suspension of 20-glycyl-20(S)camptothecin trifluoroacetic acid salt (351 mg, 0.68 mmol), poly-L-glutamic acid (465 mg, 3.16 mmol), and N,N- dimethylaminopyridine (249 mg, 2.04 mmol) in anhydrous dimethylformamide (13 ml) was added a solution of 1,3-diisopropylcarbodiimide (111.6 mg, 0.88 mmol) in dimethylformamide (2 ml) over 20 minutes. After stirring under an argon atmosphere for 2 days, the mixture was cooled in an ice bath and 10% aqueous sodium chloride solution (35 ml) was added over 30 minutes. After stirring for an additional 1 hour, the suspension was acidified to pH 2.5 by addition of 1 N aqueous hydrochloric acid solution. The yellow precipitate was collected by filtration, washed with water (5x 25 ml), dried under vacuum overnight, and triturated with acetonitrile (100 ml). After drying under high vacuum for 24 hours, poly-L-glutamic acid-20-glycyl-20(S)camptothecin conjugate (703 mg, 95% yield) was obtained as a yellow powder. 'H NMR (300 MHz, DMSO-de): Attorney Docket No. 1402 d 12.10 (s, -COOH), 7.05-8.74 (m,, 7,9,10,11,12, &14 CH), 5.0-5.6 (m, 5-CH2, 7-CH2), 3.70-4.35 (m, -Gly-CH2, PG-N-CH-), 1.42-2.62 (m, I8-CH2, PG-bCH2, -lCH2), 0.90 (br s, 19-CH3). Example 7. Biological Assay Antitumor activity was assayed in mice implanted subcutaneously with Lewis lung carcinoma cells (LL/2). Tumors were produced in the muscle of the right interscapular region by subcutaneously injecting 2.5 x 105 murine Lewis Lung (LL/2) carcinoma cells (ATTC CRL-1642) in a volume of 0.25 ml PBS + 2% FBS. Test compounds and vehicle control were injected ip 7 days after tumor cell implantation when the tumors had grown to 20 ± 20 mm3 (average of 230 tumors). A single dose of polyglutamic acid-therapeutic agent conjugate in 0.1 N Na2HP04 was administered at a lx-4x the maximum tolerated equivalent dose of the unconjugated agent, which was typically administered in 8.3% cremophore EL/8.3% ethanol in 0.75% saline. Each treatment group consisted of 10 mice randomly allocated to each group. Initially, tumor growth was monitored every 3 to 4 days. When tumor sizes approached the arbitrarily set upper limit of 2500 mm3, tumor sizes were determined daily. Tumor volume was calculated according to the formula (length x width x height)/2. Mice with tumors equal to or greater than 2500 mm3 were euthanized by cervical dislocation. Efficacy of the various treatments was expressed in terms of days for tumor to reach a volume of 2500 mm3 (i.e., TGD, tumor growth delay) compared with maximum tolerated dose of the unconjugated therapeutic agent. The PG-therapeutic agent conjugates described in Examples 2, 3, 5 and 6 above were tested and found to be active in this assay. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt Attorney Docket No. 1402 a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety. CLAIMS What is claimed is: 1. A process for preparing a conjugate of polyglutamic acid and a therapeutic agent comprising the steps of: (a) providing the protonated form of a polyglutamic acid polymer and a therapeutic agent for conjugation thereto; (b) covalently linking said agent to said polyglutamic acid polymer in an inert organic solvent to form a polyglutamic acid-therapeutic agent conjugate; (c)) precipitating said polyglutamic acid-therapeutic agent conjugate from solution by addition of an excess volume of aqueous salt solution; and (d) collecting said conjugate as a protonated solid. 2. The method of claim 1, wherein step (a) further comprises the steps of: (a.l) providing an aqueous solution of the sodium salt of poly-L-glutamic acid; (a.2) acidifying the solution thereby converting the sodium salt of poly L-glutamic acid to the protonated form and precipitating it from solution; and (a.3) collecting the poly-L-gJutamic acid precipitate and washing said precipitate with water. 3. The method of claim 1, step (a) wherein the therapeutic agent is an antitumor agent. 4. The method of claim 3, wherein the antitumor agent is selected from paclitaxel, docetaxel, etoposide, teniposide, epothilones, gemcitabine, 20(S)(+) camptothecin, 9- aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 9- dimethylaminomethyl-10-hydroxycamptothecin, 10,11-methylenedioxycamptothecin, 7- methylpiperizinomethyl-10,ll-etiiylenedioxycamptothecin, flavopiridol, geldanamycin, 17- (allylamino)-17-demethoxygeldanamycm, ecteinascidin 743, phthalascidin, CT-2584 (1-(11- (dodecylamino)-10-hydroxyundecyl)-3,7-dimethylxanthine, CT-4582 (1-(1 l-(N-methyl N- dodecyl amino)-10-hydroxyundecyl)-3,7-dimethylxanthine), doxorubicin or adriamycinone. Attorney Docket No. 1402 5. The method of claim 4, wherein the therapeutic agent is paclitaxel or docetaxel. 6. The method of claim 1, step (a) wherein said polyglutamic acid has a molecular weight of from 20 kd to 80kd as determined by viscosity. 7. The method of claim 1, step (b), wherein said agent is linked directly to a carboxy group of said polyglutamic acid by a physiologically cleavable linkage. 8. The method of claim 7, wherein said linkage is an ester linkage or an amide linkage. 9. The method of claim 8, wherein said linkage is an ester linkage. 10. The method of claim 1, step (b), wherein said agent is indirectly linked to a carboxy group of said polyglutamic acid via a spacer, wherein said spacer is attached to said polyglutamic acid and to said agent through physiologically cleavable linkages. 11. The method of claim 10, wherein said spacer is an amino acid. 12. The method of claim 1, step (b), wherein said polyglutamic acid-therapeutic agent conjugate comprises from about 5% to about 60% by weight of a therapeutic agent. 13. The method of claim 12, wherein said conjugate comprises about 12% to 55% by weight of a therapeutic agent. 15. The method of claim 1, step (c), wherein said aqueous salt solution comprises sodium chloride. 16. The method of claim 15, wherein said aqueous salt solution is added in 1.5x-4x the volume of the reaction mixture solvent. 17. The method of claim 1, step (c), further comprising the step of acidifying the reaction mixture. 18. The method of claim 1, further comprising the act of removing low molecular weight impurities from the conjugate, wherein said removing can be carried out between steps (c) and (d) or after step (d). 19. A process of preparing a poly-L-glutamic acid-2'-paclitaxel conjugate from the sodium salt of poly-L-glutamic acid and paclitaxel, said process comprising the steps of: Attorney Docket No. 1402 (a) providing an aqueous solution of the sodium salt of poly-L-glutamic acid; (b) acidifying the solution to a pH of about 2 to 4, thereby converting the sodium salt of poly-L-glutamic acid to the protonated form and precipitating it from solution; (c) collecting the poly-L-glutamic acid precipitate and washing with water; (d) drying said poly-L-glutamic acid to a water content of between 7% and 21% by weight; (e) contacting said poly-L-glutamic acid with paclitaxel under standard coupling conditions for sufficient time to conjugate said paclitaxel to said polyglutamic acid polymer via an ester linkage formed between the 2'-OH group of paclitaxel and a carboxy group of poly-L-glutamic acid; (f) cooling said reaction mixture from between 0°C to 10° C while slowly adding aqueous salt solution to the reaction mixture; (h) acidifying the resulting suspension; (i) collecting the conjugate as a protonated solid; and (j) extracting impurities from said protonated solid. 20. A polyglutamic acid-therapeutic agent conjugate prepared by the process of claim 1 or claim 19, wherein said conjugate is selected from poly-L-glutamic acid-glycyl- 20(S)camptothecin and poly-L-glutamic acid-CT-4582. The invention provides new processes for preparing polyglutamic acid-therapeutic agent conjugates for clinical development and pharmaceutical use, and polyglutamic acid- therapeutic agent conjugates prepared by these processes.

Full Text

MANUFACTURE OF POLYGLUTAMATE-THERAPEUTIC AGENT
CONJUGATES
Field of the Invention
This invention relates to a process for scaled-up manufacture of polyglutamate-
therapeutic agent conjugates for clinical development.
BACKGROUND OF THE INVENTION
The antitumor agent paclitaxel shows increased efficacy and decreased toxicity
when administered to tumor-bearing hosts as a polygJutamic acid conjugate compared
with the unconjugated form of the drug (U.S. Serial No. 08/815,104, allowed; Li et al,
Cancer Res. 58:. 2404, 1998). The polyglutamic acid-paclitaxel conjugate shows
increased water solubility, a slower clearance from the body, and an increased
accumulation in the tumor. Conjugates of polyglutamic acid and various other therapeutic
agents are expected to provide clinically useful alternatives to the presently available
formulations.
For research purposes, the polyglutamic acid-therapeutic agent conjugates can be
produced by the method disclosed in Li et al, ibid. In that method, the conjugate is
prepared as a sodium salt, dialyzed to remove low molecular weight contaminants and
excess salt and then lyophilized. The method is not well-suited for large-scale
manufacture of quantities of conjugates for clinical development and use, however. In
particular, the use of dialysis to remove impurities is time-consuming and lowers final
product yield. In addition, although many pharmaceuticals have more favorable
properties when prepared as salts (e.g., improved solubility, storage, and handling), this is
not true of the polyglutamate-therapeutic agent conjugates of the present invention. The
Attorney Docket No. 1402
salt forms of the conjugates are electrostatic solids, not free flowing powders. They are
more difficult to package, more susceptible to dust contamination and more likely to
contaminate the workplace with cytotoxic agents than are free flowing powders.
Therefore, there is a need for an improved process of manufacture of polyglutamic acid-
therapeutic agent conjugates that can be used to produce gram to hundreds of gram
quantities of these conjugates in high yields and in a manner that provides for improved
materials handling and packaging.
SUMMARY OF THE INVENTION
The present invention satisfies this need by providing an improved process for
preparing a polyglutamic acid-therapeutic agent conjugate that is capable of providing
gram to hundreds of gram quantities of pharmaceutical grade conjugate with yields of
between 85 and 98%.
In one embodiment, the process comprises the steps of:
(a) providing the protonated form of a polyglutamic acid polymer and a
therapeutic agent for conjugation thereto;
(b) covalently linking said agent to said polyglutamic acid polymer in an inert
organic solvent to form a polyglutamic acid-therapeutic agent conjugate;
(c) precipitating said polyglutamic acid-therapeutic agent conjugate from solution
by addition of an excess volume of aqueous salt solution; and
(d) collecting said conjugate as a protonated solid-
Additional removal of residual low molecular weight contaminants can be carried out
between step (c) and step (d) or after step (d).
In another embodiment, which is preferred for the large-scale manufacture of
polyglutamic acid-2'paclitaxel conjugate, the process comprises the steps of:
(a) providing an aqueous solution of the sodium salt of poly-L-glutamic acid;
(b) acidifying the solution to a pH of about 2 to 4, thereby converting the sodium
salt of poly-L-glutamic acid to the protonated form and precipitating it from
solution;
(c) collecting the poly-L-glutamic acid precipitate and washing with water;
(d) drying the poly-L-glutamic acid to a water content of between 7% and 21%
by weight;
(e) contacting the poly-L-glutamic acid with paclitaxel under standard coupling
conditions for sufficient time to conjugate paclitaxel tothe polyglutamic acid polymer via
an ester linkage formed between the 2'-OH group of paclitaxel and a carboxy group of
poly-L-glutamic acid;
(f) cooling the reaction mixture from between 0°C to 10"C while slowly adding
aqueous salt solution to the reaction mixture;
(h) acidifying the resulting suspension;
(i) collecting the conjugate as a protonated solid; and
(j) extracting impurities from the protonated solid.
Any polyglutamic acid-therapeutic agent conjugate can be prepared by the
processes described herein. In one preferred embodiment, the therapeutic agents are
antitumor agents, e.g., paclitaxel, docetaxel, etoposide, teniposide, epothilones,
gemcitabine, 20(S)(+) camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-
10-hydroxycamptothecin, 9-dimethylaminomethyl-10-hydroxycamptothecin, 10,11-
methylenedioxycamptothecin, 7-methylpiperizinomethyl-10,l 1-
ethylenedioxycamptothecin, flavopiridol, geldanamycin, 17-(allylamino)-17-
demethoxygeldanamycin, ecteinascidin 743, phthalascidin, CT-2584 (1-(U-
(dodecylaniino)-10-hydroxyundecyl)-3J-dimemylxantirine, CT-4582 (l-(ll-(N-methyl
N-dodecyl amino)-10-hydroxyundecyl)-3,7-dimetb.ylxanthine), doxorubicin,
adriamycinone, melphalan, fludarabine, daunoraycin, verapamil, 5-fluorouraci], FUDR,
cyclosporin, retinoic acids and others.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. Exemplary conjugates
Fig. 2. Manufacturing Scheme for poly-L-glutamic acid-paclitaxel conjugate
Fig. 3. Proton NMR scan of poly-L-glutamic acid paclitaxel conjugate
Fig. 4. Preparation of poly-1-glutamic acid-glycyl-20(S)camptothecin
Figs. 5-7. Reaction Schemes I-III.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, "a polyglutamic acid" or "polyglutamic acid polymer " includes
poly (1-glutamic acid), poly (d-glutamic acid) and poly (dl-glutamic acid). Preferably the
polyglutamic acid polymer comprises at least 50% of its amino acid residues as glutamic
acid, and more preferably, 100%. The polyglutamic acid polymer can be substituted up
to 50% by naturally occurring or chemically modified amino acids, preferably
hydrophilic amino acids, provided that when conjugated to a therapeutic agent, the
substituted polyglutamic acid polymer has improved aqueous solubility and/or improved
efficacy relative to the unconjugated therapeutic agent, and is preferably
nonimmunogenic.
The molecular weight of the polyglutamic acid polymer used in the preparation of
the conjugate by the methods described herein is typically greater than 5000 daltons,
preferably from 20kd to 80kd, more preferably from 30 kd to 60kd (as determined by
viscosity). Those skilled in the art will appreciate that the molecular weight values may
be different when measured by other methods. These other methods include, for
example, gel permeation, low angle light scattering, multiple angle laser light scattering,
refractive index and combinations thereof.
As used herein, a "polyglutamic acid -therapeutic agent conjugate" refers to a
polyglutamic acid polymer that is covalently bonded to the therapeutic agent by a direct
linkage between a carboxylic acid residue of the polyglutamic acid and a functional group
of the therapeutic agent, or by an indirect linkage via one or more bifunctional spacer
groups. Preferred spacer groups are those that are relatively stable to hydrolysis in the
circulation, are biodegradable and are nontoxic when cleaved from the conjugate. Of
course, it is understood that suitable spacers will not interfere with the antitumor efficacy
of the conjugates. Exemplary spacers include amino acids (e.g., glycine, alanine, leucine,
isoleucine), hydroxyacids, diols, aminothiols, hydroxythiols, aminoalcohols, and
combinations of these. A therapeutic agent can be linked to the polymer or spacer by any
linking method that results in a physiologically cleavable bond (i.e., a bond that is
cleavable by enzymatic or nonenzymatic mechanisms that pertain to conditions in a living
animal organism). Examples of preferred linkages include ester, amide, carbamate,
carbonate.acyloxyalkylether.acyloxyalkylthioether, acyloxyalkylester, acyloxyalkylamide,
acyloxyalkoxycarbonyl, acyloxyalkylamine, acyloxyalkylamide, acyloxyalkylcarbamate,
acyloxyalkylsulfonamide, ketal, acetal, disulfide, thioester, N-acylamide,
alkoxycarbonyloxyalkyl, urea, and N-sulfonylimidate.
Methods for forming these linkages are well known to those skilled in synthetic
organic chemistry, and can be found for example in standard texts such as J. March,
Advanced Organic Chemistry, Wiley Interscience, 4th Edition.
The degree of loading of therapeutic agent on the polymer (i.e., the" loading
density") may be expressed as the number of molecules per polyglutamic acid polymer
chain or preferably as a % of total weight of the conjugate ("% loading"). Any desired %
loading can be obtained by adjusting the ratios of the therapeutic agent and polymer. The
optimal loading density for a given conjugate and given use is determined empirically
based on the desired properties of the conjugate (e.g., water solubility, therapeutic
efficacy, pharmacokinetic properties, toxicity and dosage requirements). The loading
density ranges from between 1% to about 60%, preferably from 5% to 55%, and even
more preferably, from 20% to 55% for the conjugates that are specifically described
herein.
The % loading is typically determined by three methods: (1) calculated weight %
(2) uv spectrophotometry; and (3) NMR ratio method.
(1). The calculated weight % is based on the known weight of the polyglutamic
acid starting material and the weight of the therapeutic agent. For all conjugates, the
conversion to conjugate form is 100% complete, as determined by TLC on silica.
(2) The uv spectrophotometry method is based on the weight % of the therapeutic
agent as measured by uv absorbance, as exemplified for a paclitaxel-polyglutamic acid
conjugate. The conjugate is dissolved in deionized water (2.5 or 5 mg/ml), centrifuged at
500g for 15 minutes to remove particulate matter if present, and the clear solution is
diluted lOOx to 200x with deionized water. The uv absorption is read against the diluent
at 228nm. A solution of the same lot of polyglutamic acid used to prepare the conjugate
is dissolved at the same nominal concentration as the conjugate and its absorbance is read
against the diluent at 228nm. A linear calibration curve is prepared by measuring the
absorbance at 228nm of solutions of known concentrations of the paclitaxel dissolved in
methanol. To calculate the percent loading, the absorbance of the polyglutamic acid
solution (corrected to account for the theoretical loading of polyglutamic acid in the
polyglutamic acid-paclitaxel solution) is subtracted from the polyglutamic acid-paclitaxel
absorbance. This corrected absorbance is compared to the paclitaxel standard curve to
obtain the paclitaxel concentration (w/v) in the conjugate solution. The percent loading is
the ratio of the paclitaxel concentration to the polyglutamic acid-paclitaxel conjugate
concentration times 100.
(3) The NMR ratio method is based on the weight % of therapeutic agent as
measured by the ratio of the peaks in the spectra resulting from the polymer in relation to
the peaks from the therapeutic agent. This is illustrated below for polyglutamic acid-
paclitaxel conjugate.
Attorney Docket No. 1402
The area between 4.5 and 6.5 ppm is totalled and divided by the number of
protons (7). This number is then compared to the area between 3.8 to 4.4 ppm for the
polymer backbone and is corrected for 2 protons from paclitaxel that overlap.
The two areas per proton are compared taking into account the molecular weights of the
paclitaxel and the polymer.
A= area per proton for polymer -s-area per proton for paclitaxel =
21.36/1.98=10.79.
MW paclitaxel =837; MW of polyglutamic acid monomer is 129.
% loading = (837 /(10.79 x 129) + 837) x 100 = 37.6%.
The processes described herein are generally useful for preparing conjugates of
polyglutamic acid with any therapeutic or diagnostic agent that is appropriately
functionalized for linking to the polyglutamic acid, as described herein. The conjugates
that are exemplified herein are intended to illustrate the invention, but not to limit its
scope.
In one preferred embodiment, the therapeutic agents comprise drugs that are
effective in treating cancerous conditions that are expected to benefit from the unique
pharmacokinetic properties of these conjugate (e.g., enhanced permeability and retention
in tumor tissue, sustained release of active agent, long biological half life compared with
the unconjugated agent, and others). Presently preferred agents include, by way of
example, taxanes (e.g., paclitaxel, docetaxel), etoposide, teniposide, epothilones,
gemcitabine, 20(S)(+) camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-
10-hydroxycamptothecin, 9-dimethylaminomethyl-10-hydroxycamptothecin, 10,11-
methylenedioxycamptothecin, 7-methylpiperizinomethyl-10,l 1-
ethylenedioxycamptothecin, flavopiridol, geldanamycin, 17-(allylamino)-17-
demethoxygeldanamycin, ecteinascidin 743, phthalascidin, CT-2584 (1-(11-
(dodecylamino)-10-hydroxyundecyl)-3,7-dimethylxanthine, CT-4582 (1-(1 l-(N-methyl
N-dodecyl amino)-10-hydroxyundecyl)-3,7-dimethylxanthine), doxorubicin,
adriamycinone, melphalan, fludarabine, daunomycin, verapamil, 5-fluorouraci],FUDR,
cyclosporin, retinoic acids and others.
The therapeutic agent must be capable of attachment to the polymer by means of a
functional group that is already present in the native molecule or otherwise can be
introduced by well-known procedures in synthetic organic chemistry without altering the
activity of the agent, to the examples given herein, the agent is relatively water-insoluble
in the unconjugated form and shows greatly improved solubility following conjugation.
However, water-soluble drugs are also expected to show advantages following their
conjugation to polyglutamic acid (e.g., improved pharmacokinetics and retention at the
site of action compared to the unconjugated agent).
Reactions performed under "standard coupling conditions" are carried out in an
inert solvent (e.g., DMF, DMSO, N-methylpyrrolidone) at a temperature from -20°C to
150°C, preferably from 0°C to 70°C, more preferably from 5°C to 30°C, in the presence
of a coupling reagent and a catalyst. Of course, the temperature used will depend on
factors such as the stability of the therapeutic agent and the reactivity of the attaching
group. Suitable coupling reagents are well-known in synthetic organic chemistry and
include, but are not limited to, carbodiimides, alkyl cbloroformate and triethylamine,
pyridinium salts-tributyl amine, phenyl dichlorophosphate, 2-choro-l,3,5-trinitrobenzene
and pyridine, di-2-pyridyl carbonate, polystyryl diphenylphosphine,
(trimethylsilyl)ethoxyacetylene, 1,1' -carbonylbis(3-methylimidazolium)triflate,
diethylazodicarboxylate and triphenyl phosphine, N,N' carbonyldiimidazole,
methanesulphonyl chloride, pivaloyl chloride, and the like. Suitable catalysts for alcohol
coupling include, e.g., 4-N.N dimethylaminopyridine and 4-pyrollidinopyridine.
As used herein, the term "inert solvent" means a solvent inert under the conditions
of the reaction being described in conjunction therewith [including, for example, benzene,
toluene, acetonitrile, tetrahydrofuran ("THF'), dimethylformamide ("DMP'), chloroform
("CHCI3"), methylene chloride (or dichloromethane or "CH2C12"), diethyl ether, ethyl
acetate, acetone, methylethyl ketone, dioxane, pyridine, dimethoxyethane, t-butyl methyl
Attorney Docket No. 1402
ether, and the like]. Unless specified to the contrary, the solvents used in the reactions of
the present invention are inert solvents.
If multiple functional groups are present on the therapeutic agent, selective
attachment of a particular group of the agent to the polyglutamic acid polymer will
require the use of a suitable protecting group. The term "protecting group" or "blocking
group" refers to any group which when bound to one or more hydroxyl, thiol, amino or
carboxyl groups of the compounds prevents reactions from occurring at these groups and
which protecting group can be removed by conventional chemical or enzymatic steps to
reestablish the hydroxyl, thiol, amino or carboxyl group. See, generally, T.W. Greene &
P.G.M. Wuts "Protective Groups in Organic Synthesis," 3rd Ed, 1999 , John Wiley and
Sons, N.Y.
The particular removable blocking group employed is not critical and preferred
removable hydroxyl blocking groups include conventional substituents such as allyl,
benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl,
t-butyldimethylsilyl, triethylsilyl, MOM (methoxymethyl), MEM (2-
methoxyethoxymethyl) and any other group that can be introduced chemically onto a
hydroxyl functionality and later selectively removed either by chemical or enzymatic
methods in mild conditions compatible with the nature of the product.
Preferred removable amino blocking groups include conventional substituents
such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),
fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC) and the like, which can be
removed by conventional conditions compatible with the nature of the product.
Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl,
t-butyl etc. which can be removed by mild hydrolysis conditions compatible with the
nature of the product.
Nomenclature
Exemplary conjugates prepared according to the embodiments of the invention
described herein are shown in Figure 1. The conjugates in the Examples below are
named in the same way as the conjugates of Figure 1.
Description of Preferred Embodiments
In general, the process of manufacturing polyglutamate-therapeutic agent
conjugates on a scale that is suitable for clinical development and pharmaceutical use
comprises the steps of:
(a) providing the protonated form of a polyglutamic acid polymer and a
therapeutic agent for conjugation thereto;
(b) covalently linking said agent to said polyglutamic acid polymer in an inert
organic solvent to form a polyglutamic acid-therapeutic agent conjugate;
(c) precipitating said polyglutamic acid-therapeutic agent conjugate from solution
by addition of an excess volume of aqueous salt solution; and
(d) collecting said conjugate as a protonated solid.
The protonated form of the polyglutamic acid polymer in step (a) is obtained by
acidifying a solution containing the salt of the polyglutamic acid to be used as a starting
material, and converting the salt to its acid form. After separating the solid by
centrifugation, the solid is washed with water. (When DMAP is to be used in step (b), it
is preferred to wash the solid until the aqueous phase is pH 3 or greater). The
polyglutamic acid is then dried, preferably by lyophilization and preferably to a constant
weight comprising between 7% and 21% of water, prior to conjugation to a desired
therapeutic agent (step (b)).
The therapeutic agent of step (b) may require modification prior to conjugation,
e.g., the introduction of a new functional group, the modification of a preexisting
functional group or the attachment of a spacer molecule. Such modifications may require
the use of protecting groups, which are described above.
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Reaction schemes I-HI illustrate methods that were used for linking various
exemplary therapeutic agents to poly-L-glutamic acid (PG), either directly or through
glycine spacer molecules. The conditions shown in these schemes and described in the
Examples may be varied, as will be readily appreciated by those skilled in synthetic
organic chemistry. As noted previously, the exact conditions that are used for conjugating
a particular therapeutic agent to polyglutamic acid may be based on the stability of the
therapeutic agent to the reaction conditions, the reactivity of the linking groups, other
factors pertinent to the manufacturing process (e.g., safety and regulatory issues), and the
like. As described previously, various types of linkages may be used in preparing the
conjugates, depending on the functional groups on the therapeutic agent and the spacer
molecule, if a spacer is used. Thus the therapeutic agent may be conjugated to
polyglutamic acid and/or spacer molecules by linkages other than ester and amide bonds.
Spacers other than glycine, and coupling reagents other than those exemplified herein,
can also be used. The exact conditions used for preparing the conjugates that illustrate
the practice of embodiments of the present invention are described below in the
Examples.
In step (c), an aqueous salt solution is added to the reaction mixture to precipitate
the polyglutamic acid-therapeutic agent conjugate from solution. Any water soluble
inorganic salt can be used for this purpose (e.g., NaCl, KC1, NH4C1, sodium sulfate,
ammonium sulfate, etc.). Preferably, 10-15% salt solution is used in lx-4x volume. In
one preferred embodiment, a 2.5x volume of 10% NaCl is used. The salt solution is
added slowly to the reaction mixture, which is cooled during the addition. For optimum
yield of conjugate, the temperature is preferably kept between 0°C and 10°C. The
precipitation step separates the polyglutamic acid-therapeutic agent conjugate from
starting materials and reaction byproducts that are wholly or partially soluble under the
conditions used for precipitation of the conjugate.
In step (d), the conjugate is collected as the protonated solid. The suspension
obtained in step (c) is preferablyacidified. Depending on the stability of the drug
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i i
molecule to acid conditions, a pH in the range of pH 1- 4 can be used. For preparation of
polyglutamic acid-paclitaxel conjugates, however, acidification below pH 2 results in the
decomposition of paclitaxel, and acidification is typically carried out at about pH 2.5.
Preferably, for the removal of DMAP, HC1 is used in step (d). The suspension can be
filtered or centrifuged, preferably filtered, to collect the conjugate.
Unreacted starting materials, byproducts and other impurities can be removed
prior to, or after acidification to yield the final protonated conjugate (illustrated in
Examples 2 and 3 below, and Figs 2 and 4). For example, after addition of the salt
solution, the solid can be collected and resolubilized, then either filtered or extracted with
an appropriate solvent in which the contaminants are soluble but the conjugate is not
(e.g., ethyl acetate). The solution is then acidified and the protonated form of the
conjugate is collected as described above.
Alternatively, the solid can be lyophilized, then slurried with an appropriate
solvent, e.g., acetonitrile(MeCN), to remove impurities from the final protonated
conjugate product.
In an alternative preferred embodiment, step (c) above is replaced by step (c1),
which comprises:
(c') separating said polyglutamic acid-therapeutic agent conjugate from unreacted
starting materials and byproducts and precipitating said polyglutamic acid-therapeutic
agent conjugate from solution by addition of an organic solvent in which unreacted
starting materials and byproducts are soluble.
In addition to ethyl acetate and acetonitrile, examples of other solvents that can be
used to purify the conjugate include chloroform, tetrahydrofuran, dioxane, toluene, 2-
butylmethyl ether, and the like.
Table 1 shows a representative analysis for poly L-glutamic acid-paclitaxel
conjugate prepared as described in Example 3 below.
TABLE 1. Analytical data
% yield; grams of conjugate; grams paclitaxel/grams conjugate determined by
UV method; 'grams paclitaxel/grams conjugate determined by NMR method;' wt
% free paclitaxe) relative to conjugate; 'wt % residual acetonitrile relative to
conjugate;' wt % residual dimethylformamide relative to conjugate;" wt %
diisopropylurea relative to conjugate; wt % residue on ignition.
The invention is illustrated by the following examples which should not be
regarded as limiting the scope of the invention in any way.
EXAMPLES
In the following examples, the intermediates in the production of the conjugates
were characterized by 'NMR. The molecular weights of the polyglutamic acid (Na salt)
used to prepare the conjugates exemplified below ranged from 20kd to 50 kd, as specified
by the supplier (Sigma) based on viscosity measurements. The average loading density of
the conjugates was 37%.
Example 1. Preparation of poly-L-glutamic acid
Poly-L-glutamic acid sodium salt (85.9 g) (Sigma Chemical Co., 37kd MW
determined by viscosity measurement) was dissolved in USP purified water (534 mL; 6.2
mL/gm), and the solution was cooled to between (0oC- 5° C. Dilute hydrochloric acid
solution (1M) was added dropwise with vigorous stirring keeping the temperature Attorney Docket No. 1402
until the pH was between pH 2-2.5. During the addition, the poly-L-glutamic acid
separated out of solution. The reaction mixture was warmed to room temperature and
stirred for 1 hour. The suspension was centrifuged at 2700 x g for 10 minutes. The upper
aqueous layer was removed and the solid was resuspended in 560 ml USP purified water
and recentrifuged for 10 minutes. The upper aqueous layer was removed and the pH was
measured. Washing was continued if necessary until the pH of the aqueous layer was >
3.0. The wet solid was lyophilized on a Labconco freeze dry system until a constant
weight was obtained. The wt % sodium was no greater than 7000 ppm as determined by
ICP.
Example 2. Preparation of poly-L-glutamic acid-2'-paclitaxel conjugate
Poly-L-glutamic acid (16.82 g), prepared as described in Example 1 above, was
suspended in anhydrous N,N-dimethylformamide (180 mL), paclitaxel (9.923 g, 11.6
mmol) and N,N-dimethylaminopyridine (283 mg, 2.32 mmol). The reaction mixture was
stirred for 30 minutes. A solution of N,N-diisopropylcarbodiimide (1.903 g, 15.08
mmol) in N,N-dimethylformamide (50 mL) was added over a period of 3 hours using a
syringe pump. After the addition, the reaction was stirred until complete (about 4 hours
at room temperature). The reaction was cooled to 5°C -10 °C and 10% sodium chloride
solution (345 mL) was added slowly to precipitate out the poly-L-glutamic acid-
paclitaxel conjugate. The precipitate was separated by transferring the mixture to a
centrifuge flask and centrifuging it at 1500 g. The wet solid was resuspended in water
(150 mL) and 1 M sodium bicarbonate solution (120 mL) was added slowly with
vigorous stirring to bring the pH of the solution to pH 7. The reaction was stirred for an
hour and filtered through a 0.2 micron filter to remove impurities. The filtrate was cooled
to 0°C - 5° C and HC1 (IN) was added slowly with vigorous stirring until the pH of the
solution was brought to pH 3. Stirring was continued for 30 minutes. The precipitated
solid was centrifuged at 1500 g, and the wet solid was washed twice by suspending in
water (150 mL) and centrifuging. The product was lyophilized to yield 24 g of poly-L-
glutamic acid-paclitaxel conjugate (90% yield).
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In the above procedure, the filtration step can be omitted by washing the solution
with ethyl acetate (250 ml, 2x) to remove impurities.
Figure 3 shows a representative proton NMR scan for poly-L-glutamic acid-2'-
paclitaxel conjugate prepared by same procedure as described above, but having a higher
paclitaxel loading (i.e. 55%).
Example 3. Preparation of poly-L-glutamic acid-2'-paclitaxel conjugate
(manufacturing process)
Poly-L-glutamic acid (42 g), prepared as described in Example 1 above, was
added to a three-necked three liter round bottom flask equipped with a mechanical stirrer,
addition funnel and a thermo probe. N, N-dimethylformamide (350 ml) was added and
stirred for 10 minutes. Paclitaxel (24.66 g) and N,N-dimethyIaminopyridine (0.70 g) was
added and stirred for 10 minutes. A solution of N, N-diisopropylcarbodiimide (4.73 g) in
N, N-dimethylformamide (143 ml) was added at room temperature over a period of 1 hr
using the addition funnel and was stirred for four hours. The reaction mixture was cooled
to 5cC-10 ° C and a cooled solution of 10% sodium chloride (1.2 L) was added dropwise
using the addition funnel and keeping the temperature at 5°C-10°C by cooling the flask in
an ice-salt mixture. After the addition of sodium chloride solution, IN solution of
hydrochloric acid (35 ml) was added dropwise until the pH of the reaction reached 2.5.
The reaction mixture was stirred for 30 minutes at 5°C-10°C and the precipitated poly-L-
glutamic acid-paclitaxel conjugate was collected by filtration. The solids were washed
three times with water and freeze dried in a lyophilizer for 24 hours. The dried solid was
powdered into a fine powder using a mortar and pestle. The finely powdered poly-L-
glutamic acid -paclitaxel conjugate was suspended in acetonitrile (1000 ml) and stirred
for 2 hrs, then filtered and the solid was washed with 2 x 200 ml of acetonitrile. The solid
was dried under vacuum for 24 hrs to give poly-L-glutamic acid-paclitaxel conjugate (60
g). Yield (90%).
Example 4, Preparation of poly-L-glutamic acid-glycine-paclitaxel conjugate
(Reaction Scheme II)
Steps 1 and 2 below were carried out essentially as described in Mathew et al.
(Mathew, A.E., Mejillano, M.R., Nath, J.P., Himes, R.H., and Stella, V.J., J. Med.
Chem.(1992), 35, 145-151).
Step 1. Preparation of 2'-(N-t-BOC-glycyl)paclitaxeI
To a solution of N-t-BOC-L-glycine (131 mg, 0.75 mmol) and paclitaxel (640 mg,
0.75 mmol) in dichloromethane (20 ml) was added 1,3-diisopropylcarbodiimide (124 mg,
0.98 mmol) followed by N,N-dimethylaminopyridine (27 mg, 0.23 mmol). After stirring
at room temperature for 4 hours, the mixture was concentrated under reduced pressure.
The residue was purified by silica gel flash chromatography eluting with 1: l(v/v) ethyl
acetate/hexane to yield 2'-(N-t-BOC-glycyl)paclitaxel (720 mg, 95% yield) as a white
powder.
Step 2. Preparation of 2'-(glycyl)paclitaxel
A solution of 2'-(N-t-BOC-glycyl)paclitaxel (245 mg, 0.242 mmol) in formic acid
(2 ml) was stirred for 30 minutes. After concentrating under reduced pressure, the residue
was suspended in water (15 ml). Cold 0.05 M sodium bicarbonate solution (45 ml) was
added and the solution (pH 8.0) was extracted with dichloromethane (2x 40 ml). The
combined dichloromethane extracts were dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was purified by silica gel flash
chromatography eluting with 4 % methanol/dichloromethane to yield 2'-(glycyl)paclitaxel
(161 mg, 73 % yield) as a white powder.
Step 3. Preparation of poly-L-glutamic acid-2'-(glycyl) paclitaxel conjugate
To a stirred suspension of poly-L-glutamic acid (275 mg, 1.87 mmol) in
anhydrous dimethylformamide (6 ml) was added 2'-(glycyl)paclitaxel (161 mg, 0.177
mmol). A solution of 1,3-diisopropylcarbodimide (29 mg, 0.23 mmol) in
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dimethylformamide (1.4 ml) was added to the stirred suspension over a period of 30
minutes. After stirring at room temperature for 3 hours, the mixture was cooled in an ice
bath to bring the bath temperature to 0°C - 5°C and then 10 % aqueous sodium chloride
solution (7 ml) was added over a period of 30 minutes to precipitate the poly-L-glutamic
acid-2'-(glycyl)paclitaxel conjugate. The resulting white suspension was centrifuged at
1500g, 15 minutes. After filtration, the solid was washed twice by suspension in water
(10 ml) and centrifugation. The crude product was suspended in water (6 ml) and a 1 M
aqueous sodium bicarbonate solution (2.3 ml) was added slowly with stirring to bring the
contents of the flask to pH 7.6. After stirring for an additional 2 hours, the aqueous layer
was washed with ethyl acetate (3x 6 ml) and then acidified by addition of 1 N
hydrochloric acid to pH 2.8. The precipitated solid was separated by centrifugation and
washed with water (2x6 ml). The wet solid was lyophilized to give poly-L-glutamic
acid-2'(glycyl)paclitaxel conjugate (315 mg, 72 % yield) as a white powder.
Using a similar procedure, the above conjugate can be substituted by amino acids
other than glycine.
Example 5. Preparation of poIy-L-glutamic acid-2'-docetaxel conjugate (Reaction
Scheme HI)
Step 1. Preparation of 10-deacetylpaclitaxel
10-Deacetylpaclitaxel was prepared essentially as described in Zheng, Q.Y.,
Darbie, L.G., Chen, X., Murray, C.K., Tetrahedron Letters., 36, (1995), 2001-2004 and
U.S. Patent No. 5,629,433.
To a solution of paclitaxel (1.0 g, 1.17 mmol) in tetrahydrofuran (20 mi) was
added hydrogen peroxide (30%, 20 ml) followed by sodium bicarbonate (1.92 g, 22.85
mmol). After stirring at room temperature for 18 hrs, the mixture was treated with
dichloromethane/water (1:1 (v:v), 100 ml). The organic phase was washed with water
(2x 30 ml), dried over anhydrous magnesium sulfate, and concentrated under vacuum.
The residue was purified by silica gel flash chromatography eluting with 3%
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methanol/dichloromethane to yield 10-deacetylpaclitaxeI (890 mg, 93% yield) as a white
powder.
Step 2. Preparation of 2%7-bis(triethylsilyl)-10-deacetylpaclitaxel
2',7-Bis(triethylsilyl)-10-deacetylpaclitaxel was prepared as described in U.S.
Patent No. 5, 629,433.
To a solution of 10-deacetylpacIitaxel (850 mg, 1.05 mmol) in anhydrous pyridinc
(20 ml) was added chlorotriethysilane (2.72 ml, 20.1 mmol) at room temperature over a
period of 30 minutes under an argon atmosphere. After stirring for 17 hours, the mixture
was treated with dichloromethane (75 ml), washed with water (3x 30 ml), with 10%
aqueous copper sulfate solution (4x 35 ml), with water (30 ml), and with saturated
aqueous sodium chloride solution (30 ml). The organic layer was dried over anhydrous
magnesium sulfate and concentrated under reduced pressure to provide 2\7-
bis(triethylsilyl)-10-deacetylpaclitaxel (980 mg, 90% yield) as a powder.
Step 3. Preparation of 2',7-bis(triethy!sUyI)-10-deacetylpacIitaxel imine
2',7-Bis(triethylsilyl)-10-deacetylpaclitaxelimine was prepared as described in
U.S. Patent No. 5, 629,433.
To a solution of 2>,7-bis(triethylsilyl)-l0-deacetylpaclitaxel (730 mg, 0.70 mmol)
in tetrahydrofuran (7.3 ml) was added zirconocene chloride hydride (543 mg, 2.11 mmol).
After stirring at room temperature under an agron atmosphere for 15 hours, the mixture
was poured into cold hexanes (75 ml). The precipitated zirconium complexes were
removed by filtration. The filtrate was concentrated under reduced pressure to give 2\7-
bis(triethyIsilyl)-10-deacetylpaclitaxelimine (636 mg, 92% yield) as a white powder.
Step 4. Preparation of 10-deacetylpaclitaxel primary amine
10-Deacetylpaclitaxel primary amine was prepared according to U.S. Patent No.
5,629,433.
A solution of 2',7-bjs(triethylsilyl)-10-deacetyipacIitaxel iminc (636 mg, 0.621
mmol) in 1% (w:w) cone, hydrochloric acid/95% cthanol (25 ml) was stirred for 15
hours, treated with water (65 ml), and washed with hexancs (2x 30 ml). The aqueous
layer was neutralized (pH 7) by addition of saturated aqueous sodium bicarbonate
solution and extracted with dichloromethane (2x 40 m). The combined extracts were
dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield
the crude primary amine product (405 mg, 92% yield) as a white powder. This product
was used for the next step without further purification.
Step 5. Preparation of docetaxel
Docetaxel was prepared according to U.S. Patent No. 5,629,433.
To a solution of 10-deacetylpaclitaxel primary amine (405 mg, 0.57 mmol) in
ethyl acetafe (40 ml) was added saturated aqueous sodiurh bicarbonate solution (40 ml).
To this biphasic mixture was added di-(en-butyldicarbonpte (225 mg, 1.03 mmol). After
stirring at room temperature for 15 hours, ethyl acetate (75 ml) was added. The organic
phase was washed with water (2x 30 ml), dried over anhydrous sodium sulfate, and
concentrated under vacuum. The residue was purified by silica gel flash chromatography
eluting with 4 % methanol/dichloromethane to provide docetaxel (35\ mg, 76 % yield) as
a white powder.
Step 6. Preparation of poly-L-glutamic acid-2'-docetaxel conjugate
To a suspension of poly-L-glutamic acid (658 mg, 4.47 mmol) in anhydrous
dimethylformamide (10 ml) was added docetaxel (385 mg, 0.48 mmol) and N,N-
dimethylaminopyridine (12 mg. 0.096 mmol). To this stirred suspension was added a
solution of 1,3-diisopropylcarbodiimide (78.8 mg, 0.624 mmol) dimethylformamide (3
Attorney Docket No. 1402
ml) dropwise over 20 minutes. After stirring for 15 hours, the mixture was cooled in an
ice bath and 10% aqueous sodium chloride solution (20 ml) was added over 30 minutes.
After stirring for an additional 1 hour, the solid was filtered and the filtered cake was
washed with water (4x 50 ml). The solid was lyophilized to constant weight and then
triturated with acetonitrile (4x 50 ml). Drying under high vacuum for 15 hours provided
poly-L-glutamic acid-2'-docetaxel conjugate (890 mg, 87% yield) as a white powder. !H
NMR (300 MHz, DMSO-d6): d 12.10 (s, -COOH), 7.05-8.20 (m, aromatic protons),
4.80-6.05 (m), 3.80-4.50 (m), 5.0-5.6 (m, 5-H2,7-H2), 3.70-4.35 (m), 1.20-2.80 (m),
1.00(s).
Example 6. Preparation of poly-I-glutamic acid-glycyI-20(S)camptothecin (Reaction
Scheme I)
Steps 1 and 2 below were carried out as described by Greenwald, R.B., Pendri, A.,
Conover, CD., Lee, C, Choe, Y.H., Gilbert, C, Martinez, A., Xia, J., Wu, D., and Hsue,
M., Bioorg. & Med. Chem. 6, (1998), 551-562.
Step 1. Preparation of 20-(N-t-BOC-glycyI)-20(S)camptothedn
To a solution of N-t-BOC-glycine (530 mg, 3.0 mmol) in anhydrous dichloromethane
(240 ml) was added 1,3-diisopropylcarbodiimide (379 mg, 3.0 mmol), N,N-
dimethylaminopyridine (244 mg, 2 mmol), and 20(S)camptothecin (348 mg, 1.0 mmol) at
0 °C. The reaction mixture was allowed to warm to room temperature. After stirring for
18 hours, the mixture was washed sequentially with 0.1N aqueous hydrochloric acid
solution (2x 50 ml), with water (2x 50 ml), with 0.1M aqueous sodium bicarbonate
solution (2x 25 ml), and with water (2 x 50 ml). The organic phase was dried over
anhydrous sodium sulfate and concentrated under reduced pressure. The residue was
crystallized from methanol (7 ml) to provide 20-(N-t-BOC-glycyl)-20(S)camptothecin
(424 mg, 84% yield) as a yellow powder. lH NMR (300 MHz, CDC13): 8 8.35 (s, 1H),
8.22 (d, J = 8.38 Hz 1H), 7.91(d, J = 8.07,1H), 7.76-7.85 (m, 1H), 7.65 (t, J = 7.4 Hz,
1H), 7.26 (s, 1H), 5.70 (d, J = 17.25 Hz, 1H), 5.40 (d, J = 17.25 Hz, 1H), 5.25 (s, 2H),
Attorney Docket No. 1402
4.95 (br s, 1H), 3.98-4.25 (m, 2H), 2.18-2.26 (m, 2H), 1.38 (s, 9H), 0.95 (t, J = 7.47 Hz,
3H).
Step 2. Preparation of 20-GlycyI-20(S)camptothecin trifluoroacetic acid salt
A solution of 20-(N-t-BOC-glycyl)-20(S)camptothecin (424 mg, 0.84 mmol) in a 1:1
(v/v) mixture of dichloromethane/trifluoroacetic acid (21 ml) was stirred at room
temperature for 1 hour. The solvents were evaporated under reduced pressure. The
yellow solid was crystallized from dichloromethane/diethyl ether (3:7 (v/v), 50 ml) to
provide 20-glycyl-20(S)camptothecin trifluoroacetic acid salt (361 mg, 83 % yield) as a
light yellow powder. !H NMR (300 MHz, DMSO-d6): d 8.78 (s, 1H), 8.45 (br s, 2H),
8.20 (d, J = 8.2 Hz 1H), 7.70-7.95 (m, 2H), 7.30 (s, 1H), 5.55 (s, 2H), 5.30 (s, 2H), 4.35
(d, J = 17.9 Hz, 1H), 4.15 (d, J = 17.9 Hz, 1H), 2.10-2.30 (m, 2H), 1.00 (t, J= 7.4 Hz,
3H).
Step 3. Preparation of poly-L-glutamic acid-20-glycyl-20(S)camptothecin conjugate
To a stirred suspension of 20-glycyl-20(S)camptothecin trifluoroacetic acid salt
(351 mg, 0.68 mmol), poly-L-glutamic acid (465 mg, 3.16 mmol), and N,N-
dimethylaminopyridine (249 mg, 2.04 mmol) in anhydrous dimethylformamide (13 ml)
was added a solution of 1,3-diisopropylcarbodiimide (111.6 mg, 0.88 mmol) in
dimethylformamide (2 ml) over 20 minutes. After stirring under an argon atmosphere for
2 days, the mixture was cooled in an ice bath and 10% aqueous sodium chloride solution
(35 ml) was added over 30 minutes. After stirring for an additional 1 hour, the
suspension was acidified to pH 2.5 by addition of 1 N aqueous hydrochloric acid solution.
The yellow precipitate was collected by filtration, washed with water (5x 25 ml), dried
under vacuum overnight, and triturated with acetonitrile (100 ml). After drying under
high vacuum for 24 hours, poly-L-glutamic acid-20-glycyl-20(S)camptothecin conjugate
(703 mg, 95% yield) was obtained as a yellow powder. 'H NMR (300 MHz, DMSO-de):
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d 12.10 (s, -COOH), 7.05-8.74 (m,, 7,9,10,11,12, &14 CH), 5.0-5.6 (m, 5-CH2, 7-CH2),
3.70-4.35 (m, -Gly-CH2, PG-N-CH-), 1.42-2.62 (m, I8-CH2, PG-bCH2, -lCH2), 0.90 (br
s, 19-CH3).
Example 7. Biological Assay
Antitumor activity was assayed in mice implanted subcutaneously with Lewis
lung carcinoma cells (LL/2). Tumors were produced in the muscle of the right
interscapular region by subcutaneously injecting 2.5 x 105 murine Lewis Lung (LL/2)
carcinoma cells (ATTC CRL-1642) in a volume of 0.25 ml PBS + 2% FBS. Test
compounds and vehicle control were injected ip 7 days after tumor cell implantation
when the tumors had grown to 20 ± 20 mm3 (average of 230 tumors). A single dose of
polyglutamic acid-therapeutic agent conjugate in 0.1 N Na2HP04 was administered at a
lx-4x the maximum tolerated equivalent dose of the unconjugated agent, which was
typically administered in 8.3% cremophore EL/8.3% ethanol in 0.75% saline. Each
treatment group consisted of 10 mice randomly allocated to each group. Initially, tumor
growth was monitored every 3 to 4 days. When tumor sizes approached the arbitrarily set
upper limit of 2500 mm3, tumor sizes were determined daily. Tumor volume was
calculated according to the formula (length x width x height)/2. Mice with tumors equal
to or greater than 2500 mm3 were euthanized by cervical dislocation. Efficacy of the
various treatments was expressed in terms of days for tumor to reach a volume of 2500
mm3 (i.e., TGD, tumor growth delay) compared with maximum tolerated dose of the
unconjugated therapeutic agent.
The PG-therapeutic agent conjugates described in Examples 2, 3, 5 and 6 above
were tested and found to be active in this assay.
While the present invention has been described with reference to the specific
embodiments thereof, it should be understood by those skilled in the art that various
changes may be made and equivalents may be substituted without departing from the true
spirit and scope of the invention. In addition, many modifications may be made to adapt
Attorney Docket No. 1402
a particular situation, material, composition of matter, process, process step or steps, to
the objective spirit and scope of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.
All of the publications, patent applications and patents cited in this application are
herein incorporated by reference in their entirety to the same extent as if each individual
publication, patent application or patent was specifically and individually indicated to be
incorporated by reference in its entirety.
CLAIMS
What is claimed is:
1. A process for preparing a conjugate of polyglutamic acid and a therapeutic
agent comprising the steps of:
(a) providing the protonated form of a polyglutamic acid polymer and a therapeutic
agent for conjugation thereto;
(b) covalently linking said agent to said polyglutamic acid polymer in an inert
organic solvent to form a polyglutamic acid-therapeutic agent conjugate;
(c)) precipitating said polyglutamic acid-therapeutic agent conjugate from solution by
addition of an excess volume of aqueous salt solution; and
(d) collecting said conjugate as a protonated solid.
2. The method of claim 1, wherein step (a) further comprises the steps of:
(a.l) providing an aqueous solution of the sodium salt of poly-L-glutamic acid;
(a.2) acidifying the solution thereby converting the sodium salt of poly L-glutamic
acid to the protonated form and precipitating it from solution; and
(a.3) collecting the poly-L-gJutamic acid precipitate and washing said precipitate with
water.
3. The method of claim 1, step (a) wherein the therapeutic agent is an antitumor
agent.
4. The method of claim 3, wherein the antitumor agent is selected from paclitaxel,
docetaxel, etoposide, teniposide, epothilones, gemcitabine, 20(S)(+) camptothecin, 9-
aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 9-
dimethylaminomethyl-10-hydroxycamptothecin, 10,11-methylenedioxycamptothecin, 7-
methylpiperizinomethyl-10,ll-etiiylenedioxycamptothecin, flavopiridol, geldanamycin, 17-
(allylamino)-17-demethoxygeldanamycm, ecteinascidin 743, phthalascidin, CT-2584 (1-(11-
(dodecylamino)-10-hydroxyundecyl)-3,7-dimethylxanthine, CT-4582 (1-(1 l-(N-methyl N-
dodecyl amino)-10-hydroxyundecyl)-3,7-dimethylxanthine), doxorubicin or adriamycinone.
Attorney Docket No. 1402
5. The method of claim 4, wherein the therapeutic agent is paclitaxel or docetaxel.
6. The method of claim 1, step (a) wherein said polyglutamic acid has a molecular
weight of from 20 kd to 80kd as determined by viscosity.
7. The method of claim 1, step (b), wherein said agent is linked directly to a carboxy
group of said polyglutamic acid by a physiologically cleavable linkage.
8. The method of claim 7, wherein said linkage is an ester linkage or an amide
linkage.
9. The method of claim 8, wherein said linkage is an ester linkage.
10. The method of claim 1, step (b), wherein said agent is indirectly linked to a
carboxy group of said polyglutamic acid via a spacer, wherein said spacer is attached to said
polyglutamic acid and to said agent through physiologically cleavable linkages.
11. The method of claim 10, wherein said spacer is an amino acid.
12. The method of claim 1, step (b), wherein said polyglutamic acid-therapeutic agent
conjugate comprises from about 5% to about 60% by weight of a therapeutic agent.
13. The method of claim 12, wherein said conjugate comprises about 12% to 55% by
weight of a therapeutic agent.
15. The method of claim 1, step (c), wherein said aqueous salt solution comprises
sodium chloride.
16. The method of claim 15, wherein said aqueous salt solution is added in 1.5x-4x
the volume of the reaction mixture solvent.
17. The method of claim 1, step (c), further comprising the step of acidifying the
reaction mixture.
18. The method of claim 1, further comprising the act of removing low molecular
weight impurities from the conjugate, wherein said removing can be carried out between
steps (c) and (d) or after step (d).
19. A process of preparing a poly-L-glutamic acid-2'-paclitaxel conjugate from the
sodium salt of poly-L-glutamic acid and paclitaxel, said process comprising the steps of:
Attorney Docket No. 1402
(a) providing an aqueous solution of the sodium salt of poly-L-glutamic acid;
(b) acidifying the solution to a pH of about 2 to 4, thereby converting the sodium salt
of poly-L-glutamic acid to the protonated form and precipitating it from solution;
(c) collecting the poly-L-glutamic acid precipitate and washing with water;
(d) drying said poly-L-glutamic acid to a water content of between 7% and 21% by
weight;
(e) contacting said poly-L-glutamic acid with paclitaxel under standard coupling
conditions for sufficient time to conjugate said paclitaxel to said polyglutamic acid polymer
via an ester linkage formed between the 2'-OH group of paclitaxel and a carboxy group of
poly-L-glutamic acid;
(f) cooling said reaction mixture from between 0°C to 10° C while slowly adding
aqueous salt solution to the reaction mixture;
(h) acidifying the resulting suspension;
(i) collecting the conjugate as a protonated solid; and
(j) extracting impurities from said protonated solid.
20. A polyglutamic acid-therapeutic agent conjugate prepared by the process of
claim 1 or claim 19, wherein said conjugate is selected from poly-L-glutamic acid-glycyl-
20(S)camptothecin and poly-L-glutamic acid-CT-4582.
The invention provides new processes for preparing polyglutamic acid-therapeutic
agent conjugates for clinical development and pharmaceutical use, and polyglutamic acid-
therapeutic agent conjugates prepared by these processes.

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Patent Number

223414

Indian Patent Application Number

IN/PCT/2002/00451/KOL

PG Journal Number

37/2008

Publication Date

12-Sep-2008

Grant Date

10-Sep-2008

Date of Filing

09-Apr-2002

Name of Patentee

CELL THERAPEUTICS INC.

Applicant Address

201 ELLIOTT AVENUE, WEST, SUITE 400, SEATTLE, WA 98119

Inventors:

#	Inventor's Name	Inventor's Address
1	BHATT RAMA	1810 NE 175TH STREET, SHORELINE, WA 98155
2	KUMAR ANIL M.	13701-113TH STREET, COURT E PUYALLUP WA 98374
3	KLEIN J. PETER	18822 RIDGE ROAD SW, VASHON, WA 98070
4	VAWTER EDWARD	435-164TH STREET, #W101, LYNNWOOD, WA 98037

PCT International Classification Number

A61K 47/48

PCT International Application Number

PCT/US2000/28109

PCT International Filing date

2000-10-12

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/159,135	1999-10-12	U.S.A.