US20020077279A1 - Manufacture of polyglutamate-therapeutic agent conjugates - Google Patents

Manufacture of polyglutamate-therapeutic agent conjugates Download PDF

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US20020077279A1
US20020077279A1 US09/971,657 US97165701A US2002077279A1 US 20020077279 A1 US20020077279 A1 US 20020077279A1 US 97165701 A US97165701 A US 97165701A US 2002077279 A1 US2002077279 A1 US 2002077279A1
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conjugate
acid
therapeutic agent
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polyglutamic acid
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Anil Kumar
J. Klein
Rama Bhatt
Edward Vawter
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to a process for scaled-up manufacture of polyglutamate-therapeutic agent conjugates for clinical development.
  • the antitumor agent paclitaxel shows increased efficacy and decreased toxicity when administered to tumor-bearing hosts as a polyglutamic acid conjugate compared with the unconjugated form of the drug (U.S. Pat. No. 5,977,163; 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.
  • the polyglutamic acid-therapeutic agent conjugates can be produced by the method disclosed in Li et al., ibid.
  • 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.
  • the use of dialysis to remove impurities is time-consuming and lowers final product yield.
  • 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 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.
  • 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 kilogram quantities of pharmaceutical grade conjugate with yields of between 85% and 98% or between about 85% to about 98%.
  • the process comprises:
  • step (c) Additional removal of residual low molecular weight contaminants can be carried out between step (c) and step (d) or after step (d).
  • in situ generation of a protonated polyglutamic acid-therapeutic agent conjugate is carried out by a process comprising:
  • the process for the large-scale manufacture of polyglutamic acid-2′ paclitaxel conjugate comprises:
  • any polyglutamic acid-therapeutic agent conjugate can be prepared by the processes described herein.
  • the therapeutic agents are antitumor agents, e.g., paclitaxel; docetaxel; etoposide; teniposide; epothilones, such as epothilone A, epothilone B, epothilone C, epothilone, epothilone F and 12,13-disoxyepothilone F; gemcitabine; 20(S)(+) camptothecin; 9-aminocamptothecin; 9-nitrocamptothecin; 7-ethyl-i 0-hydroxycamptothecin; 9-dimethylaminomethyl-10-hydroxycamptothecin; 10,11-methylenedioxycamptothecin; 7-methylpiperizinomethyl-10,11-ethylenedioxycamptothecin; flavopiridol; geldanamycin; 17-(
  • FIG. 1 Exemplary conjugates
  • FIG. 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-L-glutamic acid-glycyl-20(S)camptothecin
  • FIGS. 5 - 7 Reaction Schemes I-iHI.
  • a polyglutamic acid or “polyglutamc acid polymer” includes poly Q1-glutamic acid), poly (d-glutamic acid) and poly (di-glutamic acid).
  • the polyglutamifc acid polymer comprises at least 50% of its amino acid residues as glutamic acid, and more preferably, 100%.
  • the polyglutarnic 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 1 Skd to 80 kd, more preferably 20 kd to 80 kd, even more preferably from 20 kd to 60 kd, and most preferably from 30 kd to 60 kd (as determined by viscosity).
  • the polyglutarmic acid polymers of this invention have a molecular weight of about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, to about 30,000 daltons.
  • the polyglutamic acid polymers of this invention have a molecular weight of about 31,000, about 32,000, about 33,000, about 34,000, about 35,000, about 36,000, about 37,000, about 38,000, about 39,000, about 40,000, about 41,000, about 42.000, about 43,000, about 44,000, about 45,000, about 46,000, about 47,000, about 48,000, about 49,000, about 50,000, about 51,000, about 52,000, about 53,000, about 54,000, about 55,000, about 56,000, about 57,000, about 58,000, about 59,000, about 60,000, about 61,000, about 62,000, about 63,000, about 64,000, about 65,000, about 66,000, about 67,000, about 68,000, about 69,000, about 70,000, about 71,000, about 72,000, about 73,000, about 74,000, about 75,000, about 76,000, about 77,000, about 78,000, about 79,000, to about 80,000 daltons.
  • the molecular weight values may be different when measured by other methods. These other methods.
  • 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 linkers.
  • Preferred linkers 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 linkers will not interfere with the antitumor efficacy of the conjugates.
  • linkers include amino acids (e.g., glycine, alanine, leucine, isoleucine), hydroxyacids (e.g., y-hydroxybutyric acid), diols, aminothiols, hydroxythiols, aminoalcohols, and combinations of these.
  • a therapeutic agent can be linked to the polymer or linker 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, acyloxyalkylarnide, acyloxyalkylcarbamate, acyloxyalkylsulfonamide, ketal, acetal, disulfide, thioester, N-acylamide, alkoxycarbonyloxyalkyl, urea, and N-sulfonylimidate.
  • the degree of loading of bioactive, therapeutic or diagnostic agent on the 15 polymer may be expressed as the number of molecules or average number of molecules per polyglutamic acid polymer chain or preferably as a percent (%) of total weight of the conjugate (“% loading”).
  • % loading may be obtained by adjusting the ratios of the therapeutic agent and polymer, and optimizing other reagents as necessary.
  • 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% or from about 1% to about 60%, preferably from 5% to 55% or from about 5% to about 55%, and more preferably from 10% to 45% or from about 10% to about 45% for the conjugates that are specifically described herein.
  • the % loading is typically determined by four methods: (1) calculated weight % (2) spectrophotometry, preferably UV spectrophotometry; (3) NMR ratio method; and (4) hydrolysis method.
  • 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.
  • the spectrophotometry method is based on the weight % of the therapeutic agent as measured by absorbance at an appropriate wavelength (e.g., UV absorbance), or fluorescence, as exemplified for a paclitaxel-polyglutamic acid conjugate.
  • the conjugate is dissolved in deionized water (2.5 or 5 mg/mnL), centrifuged at 500 g for 15 minutes to remove particulate matter if present, and the clear solution is diluted 100 ⁇ to 200 ⁇ with deionized water.
  • the absorbance is read against the diluent at a specified wavelength, e.g., UV absorption is read against the diluent at 228nm or 260 nm.
  • 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, e.g., at 228 nm or 260 nm.
  • a linear calibration curve is prepared by measuring the absorbance, e.g., at 228 nm or 260 nm, of solutions of known concentrations of the paclitaxel dissolved in methanol.
  • 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.
  • 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.
  • the two areas per proton are compared taking into account the molecular weights of the paclitaxel and the polymer.
  • 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, such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone F and 12,13-disoxyepothilone F; gemcitabine; 20(S)(+) camptothecin; 9-aminocamptothecin; 9-nitrocamptothecin; 7-ethyl-10-hydroxycamptothecin; 9-dimethylaminomethyl-10-hydroxycamptothecin; 10,11-methylenedioxycamptothecin; 7-methylpiperizinomethyl-10,11-ethylenedioxycamptothecin; flavopiridol; geldanamycin; 17-(allylarnino)-17-demethoxygeldanamycin; ecteinascidin 7
  • 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 M.; chemistry without altering the activity of the agent.
  • the agent is relatively water-insoluble in the unconjugated form and shows greatly improved solubility following conjugation.
  • 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. or from about ⁇ 20° C. to about 150° C., preferably from 0C. to 70° C, or from about 0C., to about 70° C., more preferably from 5° C. to 30° C. or from about 5° C. to about 30° C., in the presence of a coupling reagent and a catalyst.
  • an inert solvent e.g., DMF, DMSO, N-methylpyrrolidone
  • Suitable coupling reagents are well-known in synthetic organic chemistry and include, but are not limited to, carbodiimides, alkyl chloroformate and triethylamine, pyridinium salts-tributyl amine, phenyl dichlorophosphate, 2-choro-1,3,5-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, bis(2-oxo-3-oxazolidinyl)phosphinic acid (“BOP-Cr”), 2-chloromethylpyridinium iodide (“CMPI”)
  • 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 (“DMF”), chloroform (“CHCl 3 ”), methylene chloride (or dichloromethane or “CH 2 Cl 2 ”), diethyl ether, ethyl acetate, acetone, methylethyl ketone, dioxane, pyridine, dimethoxyethane, t-butyl methyl ether, and the like].
  • the solvents used in the reactions of the present invention are inert solvents.
  • protecting 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.
  • 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), t-BOC (tert-butyloxycarbonyl), CBZ (benzyloxycarbonyl) 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.
  • substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl,
  • Preferred removable amino blocking groups include conventional substituents, such as t-butyloxycarbonyl (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.
  • t-BOC t-butyloxycarbonyl
  • CBZ benzyloxycarbonyl
  • FMOC fluorenylmethoxycarbonyl
  • ALOC allyloxycarbonyl
  • pyro-derivatized amino blocking groups such as pyroglutamic acid
  • the pyroglutamic acid may or may not be removed.
  • Preferred carboxyl protecting groups include esters, preferably esters containing alkyl groups such as methyl, ethyl, propyl, t-butyl etc., which can be removed by mild hydrolysis conditions compatible with the nature of the product.
  • FIG. 1 Exemplary conjugates prepared according to the embodiments of the invention described herein are shown in FIG. 1.
  • the conjugates in the Examples below are named in the same way as the conjugates of FIG. 1.
  • the process of manufacturing polyglutamate-therapeutic agent conjugates on a scale that is suitable for clinical development and pharmaceutical use comprises the steps of:
  • 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 dimethylaminopyridine (“DMAP”) is to be used in step (b), it is preferred to wash the solid until the aqueous phase is pH 3 or greater).
  • DMAP dimethylaminopyridine
  • the polyglutamic acid is then dried, preferably by lyophilization and preferably to a constant weight comprising between about 2% to about 21% water, preferably between about 7% to about 21% water, more preferably 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.
  • Reaction schemes I-III 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.
  • 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.
  • various types of linkages may be used in preparing the conjugates, depending on the available fuinctional groups on the therapeutic agent and the linker molecule, if a linker is used.
  • the therapeutic agent may be conjugated to polyglutamic acid and/or linker molecules by linkages other than ester and amide bonds.
  • Linkers 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.
  • 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, such as salts of sodium, potassium and ammonium, as well as halide and sulfate salts (e.g., NaCl, KCI, NH 4 Cl, sodium sulfate, ammonium sulfate, etc.).
  • 10-15% salt solution is used in 1 ⁇ -4 ⁇ volume.
  • a 2.5 ⁇ 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 kept between about 0C. to about 10C., preferably 0C. and 10C.
  • 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.
  • step (d) the conjugate is collected as the protonated solid.
  • the suspension obtained in step (c) is preferably acidified.
  • a pH in the range of about pH 1 to about pH 4, preferably pH 1-4 can be used.
  • acidification below pH 2 results in the decomposition of paclitaxel, and acidification is typically carried out at about pH 2.5.
  • acid such as hydrochloric acid (HCl)
  • 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).
  • 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, methylene chloride, chloroform, hexanes, heptane, diethyl ether and dioxane).
  • the solution is then acidified and the protonated form of the conjugate is collected as described above.
  • the solid can be lyophilized, then slurried with an appropriate solvent or mixtures thereof, e.g., acetonitrile (MeCN); ethers, such as diethyl ether, dioxane, tetrahydrofuran; halogenated solvents, such as choloform, methylene chloride; ketones, such as acetone and methylethyl ketone (MEK); Cl to CIO alcohols, such as tert-butyl alcohol, isopropyl alcohol, ethyl alcohol or methanol; to remove impurities from the final protonated conjugate product.
  • an appropriate solvent or mixtures thereof e.g., acetonitrile (MeCN); ethers, such as diethyl ether, dioxane, tetrahydrofuran; halogenated solvents, such as choloform, methylene chloride; ketones, such as acetone and methylethyl ketone
  • step (c) above is replaced by step (c′), which comprises:
  • examples of other solvents that can be used to purify the conjugate include chloroform, tetrahydrofuran, dioxane, toluene, 2-butylmethyl ether, and the like.
  • the in situ procedure eliminates multiple steps in preparing the protonated PG polymer and reduces the overall process time by up to a week.
  • the product appears to dissolve in aqueous solutions more rapidly when produced by the in situ procedure in comparison with the other methods disclosed herein.
  • any anhydrous acid may be used in step (b) above provided that the salt of the conjugate base is soluble in the organic solvent selected for use in the procedure.
  • suitable acids include trifluoroacetic acid, chloroacetic acid, bromobenzoic acid, chlorobenzoic acid, chlorophenoxyacetic acid, chlorophenylacetic acid, cyanoacetic acid, cyanobutyric acid, cyanophenoxyacetic acid, cyanopropionic acid, dichloroacetic acid, acetoacetic acid, fumaric acid, hippuric acid, iodoacetic acid, lactic acid, malonic acid, mesaconic acid, naphthoic acid, nitrobenzoic acid, phthalic acid, methane sulfonic acid, BBr, HCI, and HI.
  • Steps (c), (d) and (e) are carried out as described above for the general procedures.
  • Table 1 shows a representative analysis for poly L-glutamic acid-paclitaxel conjugate prepared as described in Example 3 below.
  • Table 2 shows a representative analysis for poly L-glutamic acid-paclitaxel conjugate prepared in situ as described in Example 7 below.
  • TABLE 1 Analytical data total % out- % % free % % mass put b loading loading pacli- residual residual DIP % a (g) (UV) c (NMR) d taxel e MeCN f DMF g U h ROI i 93.6 87.80 42.0 34.0 0.128 0.15 0.27 0.160 0.87
  • the intermediates in the production of the conjugates were characterized by 1 HNMR.
  • the molecular weights of the polyglutamic acid (Na salt) used to prepare the conjugates exemplified below range from 20 kd to 50 kd, as specified by the supplier (Sigma Chemical Co., Milwaukee, WI) based on viscosity measurements.
  • the average loading density of the conjugates was 37%
  • Poly-L-glutamic acid sodium salt (85.9 g) (Sigma Chemical Co., 37 kd MW determined by viscosity measurement) was dissolved in USP purified water (534 mL; 6.2 mL/g), and the solution was cooled to between 0C.-5° C. Dilute hydrochloric acid solution (IM) was added dropwise with vigorous stirring keeping the temperature ⁇ 10° C. until the pH was between pH 2 to 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 ⁇ g for 10 minutes.
  • IM hydrochloric acid solution
  • 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 LABCONCOT freeze dry system until a constant weight was obtained. The wt % sodium was no greater than 7000 ppm as determined by ICP.
  • the reaction was cooled to 5° C.-10C. 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 niL) 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 0C.-5° C. and HCI (IN) was added slowly with vigorous stirring until the pH of the solution was brought to pH 3.
  • the filtration step can be omitted by washing the solution with ethyl acetate (250 mL, 2x) to remove impurities.
  • FIG. 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%).
  • the reaction mixture was cooled to 5° C.-0C. and a cooled solution of 10% sodium chloride (1.2 L) was added dropwise using an addition funnel and keeping the temperature at 5° C.-10C. by cooling the flask in an ice-salt mixture.
  • IN solution of hydrochloric acid 3 5 mL was added dropwise until the pH of the reaction reached 2.5.
  • the reaction mixture was stirred for 30 minutes at 5° C.-10C. 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 ⁇ 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%).
  • 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., 35:145-151, 1992).
  • Step 3 Preparation of poly-L-glutamic acid-2′-(glycyl) paclitaxel conjugate
  • 10-Deacetylpaclitaxel was prepared essentially as described in Zheng, Q. Y., Darbie, L. G., Chen, X., Murray, C. K., Tetrahedron Letters., 36:2001-2004, 1995 and U.S. Pat. No. 5,629,433.
  • Step 2 Preparation of 2′7-bis(triethylsilyl)-10-deacetylpaclitaxel 2′,7-Bis(triethylsilyl)-10-deacetylpaclitaxel was prepared as described in U.S. Pat. No. 5, 629,433.
  • Step 3 Preparation of 2′, 7 -bis(triethylsilyl)-10-deacetylpaclitaxel imine 2′,7-Bis(triethylsilyl)-10-deacetylpaclitaxelimine was prepared as described in U.S. Pat. No. 5, 629,433.
  • Docetaxel was prepared according to U.S. Pat. No. 5,629,433.
  • Steps 1 and 2 below were carried out as described by Greenwald, R. B., Pendri, A., Conover, C. D., Lee, C., Choe, Y. H-, Gilbert, C., Martinez, A., Xia, J., Wu, D., and Hsue, M., Bioorg. Med. Chem., 6:551-562, 1998.
  • Paclitaxel (170 mg, 0.199 mol, 1.0 eq) was added as a solid, followed by 4-(N,N-dimethylamino)pyridine (10 mg, 0.082 mmol, 0.4 eq) and diisopropylcarbodiimide (40 lL, 0.259 mmol, 1.3 eq).
  • the solution was stirred at room temperature for 18 hours and was then cooled to 0° C with an ice bath.
  • a solution of 10 wt % aqueous sodium chloride was added slowly with vigorous stirring, resulting in precipitation of a fine white solid.
  • the pH was adjusted to 2.5 with dilute hydrochloric acid and the suspension transferred to a 50 muL centrifuge tube.
  • 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 ⁇ 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 mm′ (average of 230 tumors). A single dose of polyglutamic acid-therapeutic agent conjugate in 0.
  • Efficacy of the various treatments was expressed in terms of days for tumor to reach a volume of 2500 mm 3 (i.e., TGD, tumor growth delay) compared with maximum tolerated dose of the unconjugated therapeutic agent.
  • TGD tumor growth delay
  • the PG-therapeutic agent conjugates described in Examples 2, 3, 5 and 6 above were tested and found to be active in this assay.
  • the reaction was cooled to 5° C-10IC and 10% sodium chloride solution (200 mL) was added slowly to precipitate out the poly-L-glutamic acid CT 2584 conjugate.
  • the precipitate was collected by centrifuging at 1500 ⁇ g.
  • the wet solid was washed twice by suspending in water (150 mnL) and centrifuging.
  • the product was characterized by hu 1 H NMR, which showed a singlet at 3.9 ppm and 3.4 ppm corresponding to methyl group at N3 and N7 and a broad singlet at 1.24 ppm corresponding to the alkyl protons and a broad peak at 0.85 ppm for the terminal methyl group of CT 2584.
  • NMR showed multiplets between 1.5 ppm-3.0 ppm and 3.5 ppm-4.5 ppm corresponding to poly-L-glutamic acid backbone.

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US7259156B2 (en) 2004-05-20 2007-08-21 Kosan Biosciences Incorporated Geldanamycin compounds and method of use
US7378407B2 (en) 2004-05-20 2008-05-27 Kosan Biosciences Incorporated Geldanamycin compounds and method of use
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US8048891B2 (en) 2006-02-09 2011-11-01 Enzon Pharmaceuticals, Inc. Treatment of non-hodgkin's lymphomas with multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin
US8299089B2 (en) 2006-02-09 2012-10-30 Enzon Pharmaceuticals, Inc. Multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin for treatment of breast, colorectal, pancreatic, ovarian and lung cancers
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US20100204261A1 (en) * 2006-02-09 2010-08-12 Enzon Pharmaceuticals, Inc. Multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin for treatment of breast, colorectal, pancreatic, ovarian and lung cancers
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US20100098654A1 (en) * 2008-10-21 2010-04-22 Fabio Pastorino Treatment of neuroblastoma with multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin
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