WO2007047315A1 - Amifostine dihydrate crystalline composition - Google Patents

Amifostine dihydrate crystalline composition Download PDF

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Publication number
WO2007047315A1
WO2007047315A1 PCT/US2006/039748 US2006039748W WO2007047315A1 WO 2007047315 A1 WO2007047315 A1 WO 2007047315A1 US 2006039748 W US2006039748 W US 2006039748W WO 2007047315 A1 WO2007047315 A1 WO 2007047315A1
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Prior art keywords
amifostine
dihydrate
cancer
pharmaceutical composition
crystalline
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PCT/US2006/039748
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French (fr)
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Edward G. Samsel
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Albemarle Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
    • C07F9/165—Esters of thiophosphoric acids
    • C07F9/1651—Esters of thiophosphoric acids with hydroxyalkyl compounds with further substituents on alkyl

Definitions

  • the invention provides for a hydrate of amifostine and, more specifically, provides for the preparation of amifostine dihydrate.
  • amifostine formerly known as WR (Walter-Reed)-2721
  • WR Wild-Reed
  • amifostine formerly known as WR (Walter-Reed)-2721
  • WR Wild-Reed
  • amifostine is an phosphorylated aminothiol that not only protects hematopoietic progenitor cells from chemotherapy and radiotherapy, but also stimulates normal hematopoiesis.
  • WR Wild-Reed
  • WR-2721 (Ethyofos, Ethyol®, 2-[(3-aminopropyl)amino]ethanethiol dihydrogen phosphate ester) is a non-active prodrug which, following dephosphorylation by the enzyme alkaline phosphatase, is transformed into WR- 1065 (2-[(aminopropyl)amino]ethanethiol), its active metabolite [van der Vijgh, W.J.F., et al, Eur. J. Cancer, 32A: pp. S26-S30 (1996)], as shown below.
  • thiol group primarily a scavenger of free radicals produced by ionizing radiation or chemotherapeutic drugs such as anthracyclines, bleomycins, and bioreductive compounds
  • chemotherapeutic drugs such as anthracyclines, bleomycins, and bioreductive compounds
  • WR-33278 N,N'-(dithiodi-l,2-ethanediyl)bis-l,3- propanediamine
  • WR-1065 produces cytoprotective effects by binding to and detoxifying directly the active forms of chemocytotoxic drugs, scavenging free radicals, and donating hydrogen ions for DNA repair [Foster-Nora, J.A., et ah, Am. J. health-Syst. Pharm., 54: pp. 787-800 (1997); Treskes, M., et al, Biochem. Pharmacol., 43: pp. 1013-1019 (1992)], all of which are thought to be factors of toxicity induced by radiation and some chemocytotoxic drugs.
  • amifostitie trihydrate has demonstrated excellent utility as a chemotherapy-related ionizing radiation radioprotectant and a selective, broad-spectrum chemoprotectant.
  • Experimental evidence has suggested a protective role of amifostine against leukemogenesis and carcinogenesis.
  • amifostine has been shown to prevent the induction of mutations of the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene induced by chemicals such as platinum and nitrosureas [Grdina, DJ., et al., Carcinogenesis, 6: pp.
  • HGPRT hypoxanthine-guanine phosphoribosyl transferase
  • amifostine While the preferred form of amifostine, amifostine trihydrate, has many advantageous properties, numerous difficulties have been encountered while trying to obtain convenient, stable, and sterile dosage formulations.
  • Amifostine has been previously described and sold as a sterile, amorphous, lyophilized powder containing a mixture of the active ingredient and mannitol.
  • the currently available formulation of amifostine is a powder of the trihydrate produced by lyophilization, leading to a thermally unstable product that is shipped and maintained at low temperatures to avoid degradation of the product.
  • U.S. Patent No. 5,424,471 offers a process for the preparation of a crystalline amifostine trihydrate composition
  • U.S. Patent No. 5,591,731 suggests compositions comprising a thermally-stable amifostine trihydrate which is suitable for reconstitution with a pharmaceutically acceptable vehicle into a particulate-free drug product.
  • U.S. Patent Publication No. 2003/0096797 suggests pharmaceutical compositions comprising an aminoalkyl phophorothioate and a surfactant that enhances the biological activity of the composition, as well as a hydrotrope and/or a chelating agent. All of the foregoing references are incorporated herein as if they were each fully recited herein
  • the present invention provides a novel aminoalkyl phosphorothioate form, amifostine dihydrate, and methods of its preparation.
  • Amifostine dihydrate and its pharmaceutically acceptable salts, active metabolites, prodrugs and prodrugs of the active metabolites are contemplated as suitable for use in compositions and formulations as active compounds having therapeutic uses similar to those for conventional forms of amifostine.
  • Pharmaceutical compositions comprising amifostine dihydrate may include additional therapeutic agents that are included in pharmaceutical compositions containing other forms of amifostine.
  • Figure 1 shows the NMR spectrum of amifostine dihydrate according to the present invention in D 2 O.
  • Figure 2 is a differential scanning calorimetry (DSC) thermogram comparative analysis of amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate.
  • Figure 3 is a thermogravimetric analysis (TGA) comparative analysis of amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate.
  • Figure 4 is a chart showing the powder X-ray diffraction (XRD) patterns of the amifostine hydrates, amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate, expressed in terms of 2 theta angles.
  • XRD powder X-ray diffraction
  • Figure 5 is an ORTEP diagram of the amifostine dihydrate x-ray crystal structure, in accordance with the present disclosure. Hydrogen bonds are depicted by the dashed lines.
  • amifostine monohydrate refers to the compound 2-[(3- aminopropyl)amino]ethylphosphorothioic acid monohydrate.
  • Amifostine trihydrate refers to 2-[(3- aminopropyl)amino]ethylphosphorothioic acid trihydrate, equivalently known and referred to by the trade name Ethyol®, and having an orthorhombic crystal structure point group and as described in U.S. Patent No. 5,591,731, having the identifying characteristics described therein.
  • Cytoprotectant refers to compounds which reduce the cytotoxic damage induced by ionizing radiation or by chemotherapeutic drugs to normal tissues or cells by chemical or physical agents, or combinations thereof.
  • the term “compound” includes both the singular and the plural, and includes any single entity or combined entities that have at least the activity disclosed herein and combinations, fragments, analogs or derivatives of such entities. [0016]
  • the terms “individual,” “subject,” “host,” and “patient” equivalently refer to any subject, including but not limited to mammals, for which diagnosis, treatment, or therapy is desired.
  • treatment refers generally to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a subject, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom, but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
  • terapéuticaally effective amount refers to an amount of, for example, amifostine dihydrate as disclosed herein, that is effective for preventing, ameliorating, treating or delaying the onset of a disease or condition.
  • a “prophylactically effective amount” refers to an amount of, for example, amifostine dihydrate as disclosed herein that is effective for preventing a disease or condition.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant, which is useful for delivery of a drug to a subject, such as a mammal or other animal.
  • the compound of the present invention and compositions comprising mixtures containing said compound may be delivered by liposomes.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Liposome formulations, loading of liposomes and administration and delivery of liposomes are known in the art, for example, in Liposomes: A Practical Approach (The Practical Approach Series, Vol. 264); Torchilin, V.P.; Weissig, V., Eds.; Oxford University Press, 2003.
  • the present invention relates to crystalline amifostine dihydrate, as well as a process for the preparation of crystalline compositions comprising amifostine dihydrate.
  • the crystalline amifostine dihydrate disclosed herein has an X-ray powder diffraction (XRD) pattern as shown in Figure 4, the XRD pattern being expressed in terms of 2-theta angles.
  • XRD X-ray powder diffraction
  • crystalline amifostine dihydrate can be characterized by an XRD pattern that comprises 2-theta angles at four or more positions selected from the group consisting of about 10.2 ⁇ 0.1, about 11.2 ⁇ 0.1, about 14.3 ⁇ 0.1, about 20.5 ⁇ 0.1, and about 22.5 ⁇ 0.1 degrees.
  • the present invention also relates to a process for the preparation of crystalline amifostine dihydrate comprising (a) preparing a formulation comprising amifostine monohydrate, alcohol, and water in which the relative amounts of amifostine monohydrate, alcohol and water are such that a particulate-free solution is obtained at a temperature from about 50 °C to about 10 0 C; (b) cooling the formulation to a temperature from about -50 °C to about 10 °C for a period of time sufficient to effect the precipitation of the crystalline dihydrate; (c) isolating the solid by filtration and d) drying the resulting mixture to leave a solid crystalline amifostine dihydrate preparation having enhanced stability.
  • the process can further comprise a step of introducing a sterile inert gas over the preparation after completion of the drying step.
  • Amifostine dihydrate can be depicted as shown in Formula (I) below:
  • Amifostine dihydrate (Formula I) and its pharmaceutically acceptable salts, active metabolites, prodrugs and prodrugs of the active metabolites are contemplated as suitable for use in compositions and formulations as active compounds having therapeutic uses similar to those for other forms of amifostine.
  • amifostine amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention
  • XRD X-ray powder diffraction
  • DSC differential scanning calorimetry
  • TGA thermogravimetric analysis
  • NMR solution and/or solid state nuclear magnetic resonance spectroscopy
  • IR infrared
  • Raman spectra Raman spectra
  • amifostine diliydrate refers to any one or more of: 1) a dihydrate crystalline form of amifostine having substantially the same NMR spectrum as shown in Figure 1, obtained on a spectrometer operating at a frequency of about 400 MHz for 1 H observation at a temperature of about 300 K; 2) a dihydrate crystalline form of amifostine having substantially the same DSC thermogram as shown in Figure 2, obtained on a differential scanning calorimeter operating at a scan rate of about 10 °C/min under nitrogen; 3) a dihydrate crystalline form of amifostine having substantially the same TGA trace as shown in Figure 3, obtained on a thermogravimeteric analysis instrument operating at a scan rate of about 2 °C/min under nitrogen; 4) a dihydrate crystalline form of amifostine having substantially the same powder XRD pattern shown in Figure 4 when measured with a properly aligned diffractometer equipped with a diffracted beam graphite monochromator using copper Ka X-ray radiation;
  • Solution or solid state NMR spectroscopy is a conventional and particularly useful analytical technique suitable for identifying the physical characteristics of a sample of amifostine dihydrate and distinguishing between amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention.
  • solution NMR techniques are used for such differentiation and/or identification, as the solution NMR spectra of each form of amifostine are nearly identical except for the relative integral of the exchangeable protons which is unique.
  • the relative integral of the exchangeable protons refers to the ratio of those protons that exchange on the NMR time scale with deuterons in deuterated wated (D 2 O) to those protons that do not exchange with D 2 O.
  • Certain characteristic chemical shifts can be observed in the solution-phase proton ( 1 H) NMR spectrum of amifostine dihydrate in D 2 O, using a spectrometer operating at a frequency of about 400 MHz for 1 H observation at a temperature of 300 0 K.
  • These characteristic shifts include the following: 2.1 ⁇ 0.1 ppm (a two proton multiplet), 2.9 ⁇ 0.1 ppm (a two proton multiplet), 3.21 ⁇ 0.1 ppm (a four proton multiplet) , 3.34 ⁇ 0.1 ppm (a two proton triplet), and 4.80 ⁇ 0.1 ppm (a broad singlet).
  • the preferred method of determining whether a particular form of amifostine is amifostine dihydrate is to count the waters of hydration from the integral ratio of the 4.8 ppm HOD resonance to the 2.1 ppm CH 2 resonance in highly enriched D 2 O.
  • An appropriate NMR determination will allow adequate relaxation delays within the pulse sequence so that the relative integrals accurately measure the relative number of protons resonating at each frequency.
  • the amifostine molecule itself possesses five exchangeable protons (three N-H and two PO-H) and the remainder of the HOD resonance is comprised of water of hydration.
  • Amifostine dihydrate possesses a relative integral ratio of, ideally, 9:2. Some variation is to be expected, so that an integral ratio of in the range of about 8.6:2 to about 9.4:2 can be expected, corresponding to about 1.8 to about 2.2 waters of hydration.
  • Powder X-ray diffraction (XRD) analysis is yet another conventional analytical technique suitable for identifying the physical characteristics of a sample of amifostine dihydrate and distinguishing between amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention.
  • the X-ray powder diffraction pattern of amifostine dihydrate can be determined using conventional techniques and equipment known to those skilled in the art of analytical chemistry and physical characterization.
  • the diffraction pattern of Figure 4 was obtained with a Scintag Xl diffractometer system equipped with a diffracted beam graphite monochromator using copper Kce X-ray radiation and an automated divergent slit. A xenon proportional counter was used as the detector.
  • the powder sample used to generate the X-ray powder diffraction data was prepared by conventional back filled sample preparation techniques using a 10 mm diameter holder about 1.5 mm thick.
  • the XRD patterns for each crystalline form, as shown in Figure 4, are unique to the particular form, each exhibiting its own set of diffraction peaks which can be expressed in 2 theta angles (°), d-spacings (A), and/or relative peak intensities.
  • 2-Theta diffraction angles and corresponding d-spacing values account for positions of various peaks in the XRD patterns.
  • Identification of the exact crystal form of a compound can be based primarily on observed 2 theta angles or d-spacings with lesser importance placed on relative peak intensities.
  • the certain characteristic 2 theta angle peaks occur at about 10.2 ⁇ 0.1, about 11.2 ⁇ 0.1, about 14.3 ⁇ 0.1, about 20.5 ⁇ 0.1, and about 22.5 ⁇ 0.1 degrees (2 theta angles), or about 8.7 ⁇ 0.1, about 7.9 ⁇ 0.1, about 6.2 ⁇ 0.1, about 4.4 ⁇ 0.1, and about 4.0 ⁇ 0.1 A (d-spacings).
  • amifostine dihydrate from these characteristic 2 theta angle peaks, in some circumstances it may be desirable to rely upon additional 2 theta angles or d-spacings for the identification of amifostine dihydrate.
  • at least five, particularly seven, and more particularly all of the following 2 theta angle peaks can be employed to identify amifostine dihydrate: about 10.2 ⁇ 0.1, about 11.2 ⁇ 0.1, about 14.3 ⁇ 0.1, about 20.5 ⁇ 0.1, about 22.5 ⁇ 0.1, about 23.8 ⁇ 0.1, about 24.8 ⁇ 0.1, about 28.5 ⁇ 0.1, and about 29.2 ⁇ 0.1 degrees.
  • Amifostine dihydrate may also exhibit additional 2 theta angle peaks.
  • amifostine dihydrate can exhibit 2 theta angle peaks at the following positions: 10.2 ⁇ 0.1, 11.2 ⁇ 0.1, 13.6 ⁇ 0.1, 14.3 ⁇ 0.1, 17.1 ⁇ 0.1, 18.1 ⁇ 0.1, 20.5 ⁇ 0.1, 20.8 ⁇ 0.1, 22.5 ⁇ 0.1, 23.1 ⁇ 0.1, 23.8 ⁇ 0.1, 24.0 ⁇ 0.1, 24.8 ⁇ 0.1, 28.5 ⁇ 0.1, 29.2 ⁇ 0.1, .30.8 ⁇ 0.1, 36.2 ⁇ 0.1 and 39.5 ⁇ 0.1 degrees.
  • Some margin of error is present in each of the 2 theta angle assignments and d-spacings reported above.
  • the error in determining d-spacings decreases with increasing diffraction scan angle or decreasing d-spacing.
  • the margin of error in the foregoing 2 theta angles is approximately 0.1 degrees for each of the foregoing peak assignments.
  • the preferred, method of comparing XRD patterns in order to identify the particular form of a sample of amifostine is to overlay the XRD pattern of the unknown sample over the XRD pattern of a known form or forms.
  • the preferred, method of comparing XRD patterns in order to identify the particular form of a sample of amifostine is to overlay the XRD pattern of the unknown sample over the XRD pattern of a known form or forms.
  • one skilled in the art can overlay the XRD pattern for amifostine dihydrate in Figure 4 with an XRD pattern of a sample to be analyzed for amifostine dihydrate.
  • any of the foregoing analytical techniques can be used alone or in combination to identify a particular form of amifostine, especially amifostine dihydrate.
  • other methods of physical characterization can also be employed to identify and characterize amifostine dihydrate.
  • suitable techniques which are known to those skilled in the art to be useful for the physical characterization or identification of a crystalline form or solvate include but are not limited to melting point via differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA), shown in Figures 3 and 4. These techniques can be employed alone or in combination with other techniques to characterize a sample of an unknown form of amifostine, and to distinguish amifostine dihydrate from previously described anhydrous amifostine, amifostine monohydrate and amifostine trihydrate.
  • the present invention includes amifostine dihydrate both in substantially pure form and in admixture with other forms of amifostine, particularly one or both of amifostine monohydrate and anhydrous amifostine.
  • substantially pure it is meant that the composition comprises at least about 90 percent (by weight) of amifostine dihydrate as compared to other forms of amifostine in the composition, more particularly at least about 95 weight percent amifostine dihydrate, and most preferably at least about 97 weight percent amifostine dihydrate. ⁇ . Preparation.
  • amifostine monohydrate, trihydrate, or anhydrous amifostine, and many of their salts, analogues, and derivatives thereof suitable for use in the preparation of amifostine dihydrate of the present invention are commercially available, or can be readily prepared using reported techniques.
  • amifostine monohydrate useful in the preparation of amifostine dihydrate and compositions/formulations of the present invention can be prepared by methods known in the art, such as those described, for example, by Cortese [Organic Syntheses Collection, Vol. II; Blatt, Ed., John Wiley & Sons, Inc., New York, N. Y.: pp. 91-93 (1943)], Piper, et al. [Chem. Ind.
  • crystalline amifostine dihydrate may be prepared by the following process steps: (a) preparing a formulation comprising amifostine monohydrate, alcohol, and water in which the relative amounts of amifostine monohydrate, alcohol and water are such that a particulate-free solution is obtained at a temperature from about 50 °C to about 10 °C; (b) cooling the formulation to a temperature from about -50 °C to about 10 0 C for a period of time sufficient to effect the precipitation of the crystalline dihydrate; (c) isolating the solid by filtration; and d) drying the resulting mixture to leave a solid crystalline amifostine dihydrate preparation having enhanced stability.
  • the process can further comprise a step of introducing a sterile inert gas over the preparation after completion of the drying step.
  • Damp amifostine monohydrate may be used to prepare the dihydrate crystal form of the present invention. Certain factors influence which hydrated crystal form results. These factors include, but are not limited to nucleation, seeding (both active and inadvertent), solvent mediated effects and, critically, water content.
  • the solvent composition and solvent to product ratio is critical for the nucleation of the desired form. Typically seeding can influence the nucleation of the desired form from the solvent mixture. Variation in total water content of the processing solvent can also give rise to unexpected effects.
  • conditions of separation and further processing are selected to produce the crystalline form of the present invention (i.e., amifostine dihydrate).
  • amifostine monohydrate is slurried in an alcohol solvent with water such that a particulate-free solution is obtained.
  • suitable alcohol solvents include but are not limited to C 1 - S alcohols, and mixtures of alcohols.
  • suitable solvents include but are not limited to butanol (e.g., butan-1-ol or butan- 2-ol), propanol (e.g., propan-2-ol or propan-1-ol), methanol, ethanol, absolute ethanol and combinations thereof.
  • Further solvent systems suitable for use herein include alcohol-water systems, such as an admixture of ethanol and water. Additional solvents that may be used in the processes of the present invention can be readily determined by those skilled in the art.
  • the solvent is ethanol, and more preferably, absolute ethanol.
  • the process may further comprise introducing an inert gas over the preparation after the completion of the drying step.
  • the drying step is typically vacuum drying, although those of skill in the art will realize that other steps can be used with equivalent results.
  • the inert gas introduced can be any suitable inert gas, including argon, nitrogen, or helium, or combinations of such gases.
  • the percentage of alcohol (e.g., ethanol) in the crystallization/precipitation mixture ranges from a trace amount of alcohol to about 35 volume % alcohol (e.g., the volume ratio of alcohol-to-water is 1:9).
  • the temperature of the cooling apparatus used to cool the crystallization solution can range from about -50 °C to about 10 0 C.
  • NMR nuclear magnetic resonance
  • ⁇ parts per million
  • D 2 O ⁇ 4.82 (HDO)
  • Proton ( 1 H) NMR information is tabulated in the following format: number of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet; m, multiplet), coupling constant(s) (J) in hertz and, in cases where mixtures are present, assignment as the major or minor isomer, if possible.
  • the prefix "app: is occasionally applied in cases where the true signal multiplicity was unresolved and "br" indicates the signal in question was broadened.
  • the solution was then retained for 2 days in a freezer at about —20 °C, during which time an oil with some crystalline material precipitated.
  • the vial was warmed to about 0 °C, and then cooled back down to about -20 °C with stirring, which resulted in the formation of more crystals.
  • Additional ethanol (0.5 mL) was added, and the solution allowed to stand at + 2 0 C for a period of time, after which additional ethanol (0.5 mL) was added and the solution cooled again to -20 °C, whereupon crystallization was completed. Filtration of the resultant crystals, washing with absolute ethanol, and drying under vacuum gave a small crop of crystals of the dihydrate.
  • amifostine monohydrate (5.0 g) was dissolved in 10% (v/v) ethanol/water (32 mL) at 21 °C and ethanol (9.5 mL), followed by seed crystals of amifostine dihydrate, which were added with stirring.
  • the flask was cooled from about 20 0 C to about 1 °C over a period of about 3 hours using a jacketed beaker connected to a recirculating chiller, which acted as a heat-transfer bath. Upon reaching 1 0 C, the solution was stirred at about 1 °C overnight.
  • the DSC thermogram shown in Figure 2 was obtained on a TA Instruments 2920 Differential Scanning Calorimeter (TA Instruments, New Castle, DE) at a scan rate of 10 0 C per minute, using a sample of amifostine dihydrate in a sealed aluminum crucible.
  • analogous thermograms of amifostine monohydrate and trihydrate are also shown in Figure 2.
  • Thermogravimetric analysis was used to measure changes in weight of a sample with increasing temperature, and to determine both moisture content and presence of volatile species.
  • the thermogravimetric analysis of amifostine dihydrate was carried out on a TA Instruments 2950 High Resolution Thermogravimetric Analyzer (TA Instruments, New Castle, DE) at a scan rate of 2 0 C per minute. Sample size was 15.6480 mg, and measurements were carried out using a platinum crucible, under nitrogen purge, to contain the amifostine dihydrate.
  • the TGA trace is provided at Figure 3.
  • amifostine dihydrate exhibited a weight loss from about 40 0 C to about 100 0 C, corresponding to about 14% (w/w) associated with dehydration.
  • the theoretical loss of two waters from amifostine dihydate gives a weight loss of 14.2% (w/w).
  • analogous TGA traces for amifostine monohydrate and trihydrate are also shown in Figure 3.
  • a colorless, flat plate crystal of C 5 H 15 N 2 O 3 PS.2(H 2 O) having approximate dimensions of 0.25 x 0.10 x 0.03 mm was mounted on a glass fiber. All measurements were made on a KappaCCD diffractometer (Nonnius) equipped with a sealed tube generator with Mo Ka radiation and a rotating anode generator. An Oxford Cryosystems Cryostream Plus Cooler (Oxford, UK) provided low temperature data collection down to about 105 K using a cooled nitrogen gas stream.
  • Symmetry codes (i) 2-x, 1-y, 1-z; (ii) x-1, y, z; (iii) 1-x, y- 1 A , Vz-z; (iv) 1-x, 1-y, 1-z; (v) 2-x, y- 1 A , 1 A-Z. Table 4. Selected Geometric Parameters (A, °).
  • compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and/or processes and in the steps or in the sequence of steps of the methods described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention.

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Abstract

The dihydrate of 2-(3-aminopropylamino)ethyl bromide dihydrobromide (amifostin as well as compositions containing this compound, methods of its production, and uses of th dihydrate of amifostine are described. The present disclosure relates in particular to the dihydrate of amifostine exhibiting radio-protectant, chemo-protectant, and cytoprotective activity, having therapeutic activity, in particular in oncology and cytotoxicology.

Description

AMIFOSTINE DIHYDRATE CRYSTALLINE COMPOSITION
FIELD OF THE INVENTION
[0001] The invention provides for a hydrate of amifostine and, more specifically, provides for the preparation of amifostine dihydrate.
DESCRIPTION OF RELATED ART
[0002] The compound amifostine, formerly known as WR (Walter-Reed)-2721, is an phosphorylated aminothiol that not only protects hematopoietic progenitor cells from chemotherapy and radiotherapy, but also stimulates normal hematopoiesis. Developed originally during the Cold War by the Walter Reed Army Institution as a radioprotectant for use prior to exposure to x-ray or nuclear radiation during military conflicts [McCauley, D.L., et al, Cancer Pract., 5: pp. 189-191 (1997); Valeriote, F., et al, Cancer Res., 42: pp. 4330- 4331 (1982)], the drug was later studied for its potential role in therapeutic radiation therapy and chemotherapy with alkylating agents, organoplatinum agents, and anthracyclines [Tannehill, S.P., et al, Semin. Oncol, 23: pp. S69-S77 (1996)]. WR-2721 (Ethyofos, Ethyol®, 2-[(3-aminopropyl)amino]ethanethiol dihydrogen phosphate ester) is a non-active prodrug which, following dephosphorylation by the enzyme alkaline phosphatase, is transformed into WR- 1065 (2-[(aminopropyl)amino]ethanethiol), its active metabolite [van der Vijgh, W.J.F., et al, Eur. J. Cancer, 32A: pp. S26-S30 (1996)], as shown below.
H2N(CH2)3NH(CH2)2S-PO3H2 *► H2N(CH2)3NH(CH2)2SH
WR-2721 Alkaline phosphatase WR-1065
H2N(CH2)3ΗH(CH2)2SH ► H2N(CH2)3NH(CH2)2S-S-R
WR-1065 Oxidation WR-33278
[0003] Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. The thiol group, primarily a scavenger of free radicals produced by ionizing radiation or chemotherapeutic drugs such as anthracyclines, bleomycins, and bioreductive compounds, is thought to be further metabolized to the disulfide molecule WR-33278 (N,N'-(dithiodi-l,2-ethanediyl)bis-l,3- propanediamine), which can then participate in additional cytoprotective pathways with endogenous thiols and thiol-containing proteins [Koukourakis, M.I., Anti-Cancer Drugs, 13: pp. 181-209 (2002)]. It is known, however, that WR-1065 produces cytoprotective effects by binding to and detoxifying directly the active forms of chemocytotoxic drugs, scavenging free radicals, and donating hydrogen ions for DNA repair [Foster-Nora, J.A., et ah, Am. J. health-Syst. Pharm., 54: pp. 787-800 (1997); Treskes, M., et al, Biochem. Pharmacol., 43: pp. 1013-1019 (1992)], all of which are thought to be factors of toxicity induced by radiation and some chemocytotoxic drugs.
[0004] In addition to its utility as an antiradiation agent, amifostitie trihydrate has demonstrated excellent utility as a chemotherapy-related ionizing radiation radioprotectant and a selective, broad-spectrum chemoprotectant. Experimental evidence has suggested a protective role of amifostine against leukemogenesis and carcinogenesis. For example, amifostine has been shown to prevent the induction of mutations of the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene induced by chemicals such as platinum and nitrosureas [Grdina, DJ., et al., Carcinogenesis, 6: pp. 929-931 (1985)], and reduces the toxicity of alkylating agents without affecting their antineoplastic efficacy [Yuhas, J.M., Cancer Treat. Rep., 63: pp. 971-976 (1979)]. Amifostine has also been indicated to reduce the cumulative renal toxicity associated with the repeated administration of cisplatin and carboplatin in patients with advanced ovarian or non-small cell lung cancer [Santini, V., et al, Haematologica, 84: pp. 1035-1042 (1999); Physicians' Desk Reference, 53rd Ed., pp. 513-515 (1999)].
[0005] Amifostine was approved on December 8, 1995 for the prevention of cisplatin- induced nephrotoxicity in ovarian cancer patients, and in March 1996 was given FDA approval for use in patients with non-small cell lung cancer receiving cisplatin-based chemotherapy. In June 1998, the FDA designated Ethyol® (Medlmmune, Inc., Gaithersburg, MD; the trihydrate form of amifostine) as an orphan drug for reducing the incidence and severity of radiation-induced xerostomia (chronic dry mouth). In June of 1999, the FDA approved amifostine trihydrate for this use in patients undergoing post-operative radiation treatment for head and neck cancer. The crystal structure of the FDA-approved amifostine trihydrate has been reported previously in the literature [Karle, J.M.; Karle, I.L., Acta Cryst., C44: pp. 135-138 (1988)], as well as in U.S. Patent Nos. 5,424,471 and 5,591,731. AU of the foregoing references are incorporated herein as if they were each fully recited herein.
[0006] While the preferred form of amifostine, amifostine trihydrate, has many advantageous properties, numerous difficulties have been encountered while trying to obtain convenient, stable, and sterile dosage formulations. Amifostine has been previously described and sold as a sterile, amorphous, lyophilized powder containing a mixture of the active ingredient and mannitol. However, the currently available formulation of amifostine is a powder of the trihydrate produced by lyophilization, leading to a thermally unstable product that is shipped and maintained at low temperatures to avoid degradation of the product.
[0007] Several attempts to address this problem with stability while maintaining the sterility for formulation have been described previously. U.S. Patent No. 5,424,471 offers a process for the preparation of a crystalline amifostine trihydrate composition, while U.S. Patent No. 5,591,731 suggests compositions comprising a thermally-stable amifostine trihydrate which is suitable for reconstitution with a pharmaceutically acceptable vehicle into a particulate-free drug product. U.S. Patent Publication No. 2003/0096797 suggests pharmaceutical compositions comprising an aminoalkyl phophorothioate and a surfactant that enhances the biological activity of the composition, as well as a hydrotrope and/or a chelating agent. All of the foregoing references are incorporated herein as if they were each fully recited herein
[0008] Thus, there exists a need for stable amifostine forms and compositions that can be manufactured and subsequently formulated into pharmaceutical delivery systems.
SUMMARY OF THE INVENTION
[0009] The present invention provides a novel aminoalkyl phosphorothioate form, amifostine dihydrate, and methods of its preparation. Amifostine dihydrate and its pharmaceutically acceptable salts, active metabolites, prodrugs and prodrugs of the active metabolites are contemplated as suitable for use in compositions and formulations as active compounds having therapeutic uses similar to those for conventional forms of amifostine. Pharmaceutical compositions comprising amifostine dihydrate may include additional therapeutic agents that are included in pharmaceutical compositions containing other forms of amifostine.
DESCRIPTION OF THE FIGURES
[0010] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. Figure 1 shows the NMR spectrum of amifostine dihydrate according to the present invention in D2O.
Figure 2 is a differential scanning calorimetry (DSC) thermogram comparative analysis of amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate.
Figure 3 is a thermogravimetric analysis (TGA) comparative analysis of amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate.
Figure 4 is a chart showing the powder X-ray diffraction (XRD) patterns of the amifostine hydrates, amifostine monohydrate, amifostine dihydrate, and amifostine trihydrate, expressed in terms of 2 theta angles.
Figure 5 is an ORTEP diagram of the amifostine dihydrate x-ray crystal structure, in accordance with the present disclosure. Hydrogen bonds are depicted by the dashed lines.
DEFINITIONS
[0011] The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.
[0012] As used herein, "amifostine monohydrate" refers to the compound 2-[(3- aminopropyl)amino]ethylphosphorothioic acid monohydrate.
[0013] "Amifostine trihydrate", as used herein, refers to 2-[(3- aminopropyl)amino]ethylphosphorothioic acid trihydrate, equivalently known and referred to by the trade name Ethyol®, and having an orthorhombic crystal structure point group and as described in U.S. Patent No. 5,591,731, having the identifying characteristics described therein.
[0014] "Cytoprotectant", as used herein, refers to compounds which reduce the cytotoxic damage induced by ionizing radiation or by chemotherapeutic drugs to normal tissues or cells by chemical or physical agents, or combinations thereof.
[0015] As used herein, the term "compound" includes both the singular and the plural, and includes any single entity or combined entities that have at least the activity disclosed herein and combinations, fragments, analogs or derivatives of such entities. [0016] The terms "individual," "subject," "host," and "patient" equivalently refer to any subject, including but not limited to mammals, for which diagnosis, treatment, or therapy is desired.
[0017] The terms "treatment," "treating," "treat," and the like are used herein to refer generally to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a subject, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom, but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
[0018] The expression "therapeutically effective amount" refers to an amount of, for example, amifostine dihydrate as disclosed herein, that is effective for preventing, ameliorating, treating or delaying the onset of a disease or condition.
[0019] A "prophylactically effective amount" refers to an amount of, for example, amifostine dihydrate as disclosed herein that is effective for preventing a disease or condition.
[0020] A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant, which is useful for delivery of a drug to a subject, such as a mammal or other animal. The compound of the present invention and compositions comprising mixtures containing said compound may be delivered by liposomes. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposome formulations, loading of liposomes and administration and delivery of liposomes are known in the art, for example, in Liposomes: A Practical Approach (The Practical Approach Series, Vol. 264); Torchilin, V.P.; Weissig, V., Eds.; Oxford University Press, 2003.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to crystalline amifostine dihydrate, as well as a process for the preparation of crystalline compositions comprising amifostine dihydrate. As described in detail below, the crystalline amifostine dihydrate disclosed herein has an X-ray powder diffraction (XRD) pattern as shown in Figure 4, the XRD pattern being expressed in terms of 2-theta angles. Alternatively, crystalline amifostine dihydrate can be characterized by an XRD pattern that comprises 2-theta angles at four or more positions selected from the group consisting of about 10.2 ±0.1, about 11.2 ±0.1, about 14.3 ±0.1, about 20.5 ±0.1, and about 22.5 ±0.1 degrees.
[0022] The present invention also relates to a process for the preparation of crystalline amifostine dihydrate comprising (a) preparing a formulation comprising amifostine monohydrate, alcohol, and water in which the relative amounts of amifostine monohydrate, alcohol and water are such that a particulate-free solution is obtained at a temperature from about 50 °C to about 10 0C; (b) cooling the formulation to a temperature from about -50 °C to about 10 °C for a period of time sufficient to effect the precipitation of the crystalline dihydrate; (c) isolating the solid by filtration and d) drying the resulting mixture to leave a solid crystalline amifostine dihydrate preparation having enhanced stability. In accordance with this aspect of the present invention, the process can further comprise a step of introducing a sterile inert gas over the preparation after completion of the drying step.
I. Compound.
[0023] Amifostine dihydrate can be depicted as shown in Formula (I) below:
H H2N (CH2)3-N (CH2)2-S PO3H2 • 2 H2O
(I)
Amifostine dihydrate (Formula I) and its pharmaceutically acceptable salts, active metabolites, prodrugs and prodrugs of the active metabolites are contemplated as suitable for use in compositions and formulations as active compounds having therapeutic uses similar to those for other forms of amifostine.
[0024] The various forms of amifostine — amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention — can be characterized and differentiated using a number of conventional analytical techniques, including but not limited to X-ray powder diffraction (XRD) pattern analysis, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), solution and/or solid state nuclear magnetic resonance spectroscopy (NMR), X-ray crystal structure analysis, infrared (IR) spectra, Raman spectra, and the combinations of such techniques. [0025] As used herein, amifostine diliydrate refers to any one or more of: 1) a dihydrate crystalline form of amifostine having substantially the same NMR spectrum as shown in Figure 1, obtained on a spectrometer operating at a frequency of about 400 MHz for 1H observation at a temperature of about 300 K; 2) a dihydrate crystalline form of amifostine having substantially the same DSC thermogram as shown in Figure 2, obtained on a differential scanning calorimeter operating at a scan rate of about 10 °C/min under nitrogen; 3) a dihydrate crystalline form of amifostine having substantially the same TGA trace as shown in Figure 3, obtained on a thermogravimeteric analysis instrument operating at a scan rate of about 2 °C/min under nitrogen; 4) a dihydrate crystalline form of amifostine having substantially the same powder XRD pattern shown in Figure 4 when measured with a properly aligned diffractometer equipped with a diffracted beam graphite monochromator using copper Ka X-ray radiation; and 5) a dihydrate crystalline form of amifostine having substantially the same X-ray crystal structure shown in Figure 5, obtained from intensity data collected on a Nonius Kappa CCD diffractometer; as well as combinations of any of these characteristics. -Preferred techniques for identification of the amifostine dihydrate of the present invention include powder XRD techniques, NMR spectroscopy, and x-ray crystal structure determination techniques.
[0026] Solution or solid state NMR spectroscopy is a conventional and particularly useful analytical technique suitable for identifying the physical characteristics of a sample of amifostine dihydrate and distinguishing between amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention. Preferably, solution NMR techniques are used for such differentiation and/or identification, as the solution NMR spectra of each form of amifostine are nearly identical except for the relative integral of the exchangeable protons which is unique. The relative integral of the exchangeable protons refers to the ratio of those protons that exchange on the NMR time scale with deuterons in deuterated wated (D2O) to those protons that do not exchange with D2O. Those that do exchange are the protons chemically bonded to nitrogen, those of the two phosphorus OH groups and the protons in water of crystal hydration all of which form the broad HOD peak at about 4.8 ppm. hi very highly enriched D2O solvent that has not been exposed to environmental moisture, the number of waters of hydration can be counted using the relative integral ratio. The solution phase NMR spectrum of the dihydrate form of amifostine according to the present invention is determined using conventional equipment and techniques known to those skilled in the art of analytical chemistry techniques and/or physical characterization. The solution NMR spectrum of amifostine dihydrate in D2O, shown in Figure 1, was obtained on a Bruker spectrometer, operating at a frequency of about 400 MHz for 1H observation at 300 0K (i.e., ambient temperature), a spinning speed of 20 Hz and a recycle delay of about 30 seconds. Chemical shifts were internally referenced by assigning the water (and residual protons from the lock solvent) to 4.8 ppm. Samples were prepared by dissolving amifostine dihydrate in D2O (99.990 atom % D) under nitrogen in a dry glass NMR tube. Chemical shifts are given in ppm (x-axis) and are plotted relative to intensity (y-axis).
[0027] Certain characteristic chemical shifts can be observed in the solution-phase proton (1H) NMR spectrum of amifostine dihydrate in D2O, using a spectrometer operating at a frequency of about 400 MHz for 1H observation at a temperature of 300 0K. These characteristic shifts include the following: 2.1 ±0.1 ppm (a two proton multiplet), 2.9 ±0.1 ppm (a two proton multiplet), 3.21 ±0.1 ppm (a four proton multiplet) , 3.34 ±0.1 ppm (a two proton triplet), and 4.80 ±0.1 ppm (a broad singlet). Slight variations in the observed shifts are expected based on the specific spectrometer employed, the amount of sample, and the analyst's sample preparation techniques. Some margin of error is present in each of the chemical shifts reported above. The margin of error in the foregoing chemical shifts is about ±0.1 ppm.
[0028] The preferred method of determining whether a particular form of amifostine is amifostine dihydrate is to count the waters of hydration from the integral ratio of the 4.8 ppm HOD resonance to the 2.1 ppm CH2 resonance in highly enriched D2O. An appropriate NMR determination, as known to those skilled in the art of analytical chemistry and physical characterization, will allow adequate relaxation delays within the pulse sequence so that the relative integrals accurately measure the relative number of protons resonating at each frequency. The amifostine molecule itself possesses five exchangeable protons (three N-H and two PO-H) and the remainder of the HOD resonance is comprised of water of hydration. Amifostine dihydrate possesses a relative integral ratio of, ideally, 9:2. Some variation is to be expected, so that an integral ratio of in the range of about 8.6:2 to about 9.4:2 can be expected, corresponding to about 1.8 to about 2.2 waters of hydration.
[0029] Powder X-ray diffraction (XRD) analysis is yet another conventional analytical technique suitable for identifying the physical characteristics of a sample of amifostine dihydrate and distinguishing between amifostine monohydrate, amifostine trihydrate, and the amifostine dihydrate of the present invention. The X-ray powder diffraction pattern of amifostine dihydrate can be determined using conventional techniques and equipment known to those skilled in the art of analytical chemistry and physical characterization. The diffraction pattern of Figure 4 was obtained with a Scintag Xl diffractometer system equipped with a diffracted beam graphite monochromator using copper Kce X-ray radiation and an automated divergent slit. A xenon proportional counter was used as the detector. The powder sample used to generate the X-ray powder diffraction data was prepared by conventional back filled sample preparation techniques using a 10 mm diameter holder about 1.5 mm thick.
[0030] A powder sample of amifostine dihydrate, as well as powder samples of amifostine monohydrate and amifostine trihydrate, were used to produce the XRD patterns shown in Figure 4. 2 Theta angles in degrees (x-axis) is plotted against peak intensity in terms of the count rate per seconds (y-axis). The XRD patterns for each crystalline form, as shown in Figure 4, are unique to the particular form, each exhibiting its own set of diffraction peaks which can be expressed in 2 theta angles (°), d-spacings (A), and/or relative peak intensities.
[0031] 2-Theta diffraction angles and corresponding d-spacing values account for positions of various peaks in the XRD patterns. D-spacing values can be calculated with observed 2 theta angles and copper Ka wavelength (λ=1.5406 A) using the Bragg equation (d=λ/2sin0). Slight variations in observed 2 theta angles and d-spacing values and signal intensities are expected based on the specific diffractometer employed and the analyst's sample preparation technique. Typically, and as described herein, the margin of error in the peak assignments is about ±0.1. More variation is expected for the relative peak intensities. Identification of the exact crystal form of a compound can be based primarily on observed 2 theta angles or d-spacings with lesser importance placed on relative peak intensities. To identify amifostine dihydrate, the certain characteristic 2 theta angle peaks occur at about 10.2 ±0.1, about 11.2 ±0.1, about 14.3 ±0.1, about 20.5 ±0.1, and about 22.5 ±0.1 degrees (2 theta angles), or about 8.7 ±0.1, about 7.9 ±0.1, about 6.2 ±0.1, about 4.4 ±0.1, and about 4.0 ±0.1 A (d-spacings).
[0032] Although one skilled in the art can identify amifostine dihydrate from these characteristic 2 theta angle peaks, in some circumstances it may be desirable to rely upon additional 2 theta angles or d-spacings for the identification of amifostine dihydrate. In one embodiment, at least five, particularly seven, and more particularly all of the following 2 theta angle peaks can be employed to identify amifostine dihydrate: about 10.2 ±0.1, about 11.2 ±0.1, about 14.3 ±0.1, about 20.5 ±0.1, about 22.5 ±0.1, about 23.8 ±0.1, about 24.8 ±0.1, about 28.5 ±0.1, and about 29.2 ±0.1 degrees.
[0033] Amifostine dihydrate may also exhibit additional 2 theta angle peaks. For example, amifostine dihydrate can exhibit 2 theta angle peaks at the following positions: 10.2 ±0.1, 11.2 ±0.1, 13.6 ±0.1, 14.3 ±0.1, 17.1 ±0.1, 18.1 ±0.1, 20.5 ±0.1, 20.8 ±0.1, 22.5 ±0.1, 23.1 ±0.1, 23.8 ±0.1, 24.0 ±0.1, 24.8 ±0.1, 28.5 ±0.1, 29.2 ±0.1, .30.8 ±0.1, 36.2 ±0.1 and 39.5 ±0.1 degrees. Some margin of error is present in each of the 2 theta angle assignments and d-spacings reported above. The error in determining d-spacings decreases with increasing diffraction scan angle or decreasing d-spacing. As suggested previously, the margin of error in the foregoing 2 theta angles is approximately 0.1 degrees for each of the foregoing peak assignments.
[0034] Since some margin of error is possible in the assignment of 2 theta angles the preferred, method of comparing XRD patterns in order to identify the particular form of a sample of amifostine is to overlay the XRD pattern of the unknown sample over the XRD pattern of a known form or forms. For example, and as illustrated in Figure 4, one skilled in the art can overlay the XRD pattern for amifostine dihydrate in Figure 4 with an XRD pattern of a sample to be analyzed for amifostine dihydrate.
[0035] Although 2 theta angles and/or d-spacings are the primary method of identifying a particular crystalline form, it may be desirable to compare relative peak intensities. As noted previously, relative peak intensities can vary depending upon the specific diffractometer employed and the analyst's sample preparation technique. The peak intensities, when utilized, are reported as intensities relative to the peak intensity of the strongest peaks. The intensity units on the XRD are counts/second (sec), and the absolute counts (counts/time x count time) is counts/sec x 10 sec.
[0036] Based on the foregoing characteristic features of the XRD pattern of amifostine dihydrate in comparison with amifostine monohydrate and amifostine trihydrate, one skilled in the art can readily identify amifostine dihydrate. It will also be appreciated that the XRD pattern of a sample of amifostine dihydrate, obtained using the methods described herein, can exhibit additional peaks not listed in the table above. The foregoing description and Figure 4 provide the most intense peaks which are characteristic of each of the particular crystalline hydrate forms, and does not represent an exhaustive list of peaks exhibited by amifostine dihydrate or the other amifostine hydrate forms. [0037] Any of the foregoing analytical techniques can be used alone or in combination to identify a particular form of amifostine, especially amifostine dihydrate. In addition to those described in detail above, other methods of physical characterization can also be employed to identify and characterize amifostine dihydrate. Examples of suitable techniques which are known to those skilled in the art to be useful for the physical characterization or identification of a crystalline form or solvate include but are not limited to melting point via differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA), shown in Figures 3 and 4. These techniques can be employed alone or in combination with other techniques to characterize a sample of an unknown form of amifostine, and to distinguish amifostine dihydrate from previously described anhydrous amifostine, amifostine monohydrate and amifostine trihydrate.
[0038] The present invention includes amifostine dihydrate both in substantially pure form and in admixture with other forms of amifostine, particularly one or both of amifostine monohydrate and anhydrous amifostine. By "substantially pure" it is meant that the composition comprises at least about 90 percent (by weight) of amifostine dihydrate as compared to other forms of amifostine in the composition, more particularly at least about 95 weight percent amifostine dihydrate, and most preferably at least about 97 weight percent amifostine dihydrate. π. Preparation.
[0039] Amifostine monohydrate, trihydrate, or anhydrous amifostine, and many of their salts, analogues, and derivatives thereof suitable for use in the preparation of amifostine dihydrate of the present invention are commercially available, or can be readily prepared using reported techniques. For example, amifostine monohydrate useful in the preparation of amifostine dihydrate and compositions/formulations of the present invention can be prepared by methods known in the art, such as those described, for example, by Cortese [Organic Syntheses Collection, Vol. II; Blatt, Ed., John Wiley & Sons, Inc., New York, N. Y.: pp. 91-93 (1943)], Piper, et al. [Chem. Ind. (London), p. 2010 (1966)], and Akerfeldt, et al. [Acta Chem. Scand., 14: p. 1980 (I960)]. Amifostine monohydrate, as well as preparations of this and related aminothiol compounds, are also described in detail U.S. Patent Nos. 3,892,824; 5,424,472; and 5,591,731. AU of the foregoing references are incorporated herein as if they were each fully recited herein. [0040] In accordance with the present invention, crystalline amifostine dihydrate may be prepared by the following process steps: (a) preparing a formulation comprising amifostine monohydrate, alcohol, and water in which the relative amounts of amifostine monohydrate, alcohol and water are such that a particulate-free solution is obtained at a temperature from about 50 °C to about 10 °C; (b) cooling the formulation to a temperature from about -50 °C to about 10 0C for a period of time sufficient to effect the precipitation of the crystalline dihydrate; (c) isolating the solid by filtration; and d) drying the resulting mixture to leave a solid crystalline amifostine dihydrate preparation having enhanced stability. In accordance with this aspect of the present invention, the process can further comprise a step of introducing a sterile inert gas over the preparation after completion of the drying step.
[0041] Damp amifostine monohydrate may be used to prepare the dihydrate crystal form of the present invention. Certain factors influence which hydrated crystal form results. These factors include, but are not limited to nucleation, seeding (both active and inadvertent), solvent mediated effects and, critically, water content. The solvent composition and solvent to product ratio is critical for the nucleation of the desired form. Typically seeding can influence the nucleation of the desired form from the solvent mixture. Variation in total water content of the processing solvent can also give rise to unexpected effects. In the following methods, conditions of separation and further processing are selected to produce the crystalline form of the present invention (i.e., amifostine dihydrate).
[0042] According to the present method, amifostine monohydrate is slurried in an alcohol solvent with water such that a particulate-free solution is obtained. Suitable alcohol solvents include but are not limited to C1-S alcohols, and mixtures of alcohols. Specific examples of suitable solvents include but are not limited to butanol (e.g., butan-1-ol or butan- 2-ol), propanol (e.g., propan-2-ol or propan-1-ol), methanol, ethanol, absolute ethanol and combinations thereof. Further solvent systems suitable for use herein include alcohol-water systems, such as an admixture of ethanol and water. Additional solvents that may be used in the processes of the present invention can be readily determined by those skilled in the art. In one preferred embodiment, the solvent is ethanol, and more preferably, absolute ethanol.
[0043] The process may further comprise introducing an inert gas over the preparation after the completion of the drying step. The drying step is typically vacuum drying, although those of skill in the art will realize that other steps can be used with equivalent results. The inert gas introduced can be any suitable inert gas, including argon, nitrogen, or helium, or combinations of such gases.
[0044] In order to obtain the amifostine dihydrate product, the percentage of alcohol (e.g., ethanol) in the crystallization/precipitation mixture ranges from a trace amount of alcohol to about 35 volume % alcohol (e.g., the volume ratio of alcohol-to-water is 1:9). Similarly, the temperature of the cooling apparatus used to cool the crystallization solution can range from about -50 °C to about 10 0C.
EXAMPLES
[0045] For the following examples, nuclear magnetic resonance (NMR) spectra were recorded on either a Bruker DPX 400 magnetic resonance spectrometer. 1H chemical shifts are given in parts per million (δ) downfield from δ 0.00 using the residual solvent signal (D2O = δ 4.82 (HDO)) as internal standard. Proton (1H) NMR information is tabulated in the following format: number of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet; m, multiplet), coupling constant(s) (J) in hertz and, in cases where mixtures are present, assignment as the major or minor isomer, if possible. The prefix "app: is occasionally applied in cases where the true signal multiplicity was unresolved and "br" indicates the signal in question was broadened.
Example 1: Preparation of Amifostine Dihydrate.
[0046] A sample of amifostine monohydrate (0.25 g) in ACS Reagent Grade water (1.9 mL) at 20 0C was dissolved in a scintillation vial equipped with a Teflon® stir bar. To this solution was added ethanol (0.25 mL, absolute) with stirring. The vial and its contents were then slowly cooled without stirring to about 4 °C, and then cooled further to about -15 °C, without any precipitation evidenced. The solution was warmed to about -5 °C, and an additional aliquot of ethanol (0.25 mL, absolute) was added, and the solution cooled again to about —15 °C, without precipitation. The solution was then retained for 2 days in a freezer at about —20 °C, during which time an oil with some crystalline material precipitated. The vial was warmed to about 0 °C, and then cooled back down to about -20 °C with stirring, which resulted in the formation of more crystals. Additional ethanol (0.5 mL) was added, and the solution allowed to stand at + 2 0C for a period of time, after which additional ethanol (0.5 mL) was added and the solution cooled again to -20 °C, whereupon crystallization was completed. Filtration of the resultant crystals, washing with absolute ethanol, and drying under vacuum gave a small crop of crystals of the dihydrate.
Example 2: Large-Scale Production of Amifostine Dihvdrate
[0047] In a round bottom flask, amifostine monohydrate (5.0 g) was dissolved in 10% (v/v) ethanol/water (32 mL) at 21 °C and ethanol (9.5 mL), followed by seed crystals of amifostine dihydrate, which were added with stirring. The flask was cooled from about 200C to about 1 °C over a period of about 3 hours using a jacketed beaker connected to a recirculating chiller, which acted as a heat-transfer bath. Upon reaching 1 0C, the solution was stirred at about 1 °C overnight. The resultant solid that formed was filtered, washed with ethanol, and dried under vacuum to provide 4.96 g of colorless crystals of amifostine dihydrate. 1H-NMR (D2O): δ 2.1 (m, 2 H), 2.95 (s, 2 H), 3.1 (m, 4 H), 3.35 (t, 2 H), 4.8 (m, 9 H).
[0048] Proton (1H) NMR analysis of the dihydrate product were done using "100 %" D2O (Sigma- Aldrich, St. Louis, MO) that was manipulated in a nitrogen-purged glovebox so as to avoid contamination by atmospheric moisture. As shown in Figure 1, all exchangeable protons form abroad singlet at δ 4.8, consisting of five protons from amifostine and of excess water. Careful integration versus the rnultiplet at δ 2.1 allowed quantification of the waters of hydration to be found, which in this sample was 2.1 waters.
Example 3: Preparation of Single Crystals for X-Ray Structure Determination
[0049] In a scintillation vial, a 0.45 g sample of the dihydrate from Example 2, above, was dissolved in 10% (v/v) ethanol/water (2.85 mL). Seed crystals of the initially isolated crystals from Example 1, above, were added, along with additional ethanol (0.85 mL) with stirring. Using a jacketed beaker having upper and lower hose connections on opposite ends connected to a recirculating chiller (as a heat-transfer bath), the vial was cooled without stirring from a temperature of about 20 0C to a temperature of about 1 °C over a period of about six hours, during which time crystals had formed. The mother liquor was decanted from the vial, and the crystals were rinsed with ethanol and dried under vacuum.
Example 4: Thermal Analysis of Amifostine Dihvdrate
[0050] Thermal analysis of the vacuum dried, crystalline amifostine dihydrate as prepared above was conducted using both differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques [see, Solid State Chemistry of Drugs, 2nd Ed.; Byrn, S.R., et al, Academic Press, 1999]. DSC was used to measure the temperatures and heat flows associated with transitions in materials as a function of time and temperature. Such measurements provide qualitative and quantitative information about physical and chemical changes that involve endothermic and exothermic processes, or changes in heat capacity.
[0051] The DSC thermogram shown in Figure 2 was obtained on a TA Instruments 2920 Differential Scanning Calorimeter (TA Instruments, New Castle, DE) at a scan rate of 10 0C per minute, using a sample of amifostine dihydrate in a sealed aluminum crucible. As shown in Figure 2, amifostine dihydrate exhibited a moderately sharp symmetric endotherm, corresponding to loss of water, at about T= 73 0C and endothermic melting or decomposition at about T= 135 °C, followed by slow exothermic decomposition. For comparison, analogous thermograms of amifostine monohydrate and trihydrate are also shown in Figure 2.
[0052] Thermogravimetric analysis (TGA) was used to measure changes in weight of a sample with increasing temperature, and to determine both moisture content and presence of volatile species. The thermogravimetric analysis of amifostine dihydrate was carried out on a TA Instruments 2950 High Resolution Thermogravimetric Analyzer (TA Instruments, New Castle, DE) at a scan rate of 2 0C per minute. Sample size was 15.6480 mg, and measurements were carried out using a platinum crucible, under nitrogen purge, to contain the amifostine dihydrate. The TGA trace is provided at Figure 3. As can be seen, the amifostine dihydrate exhibited a weight loss from about 40 0C to about 100 0C, corresponding to about 14% (w/w) associated with dehydration. The theoretical loss of two waters from amifostine dihydate gives a weight loss of 14.2% (w/w). For comparison, analogous TGA traces for amifostine monohydrate and trihydrate are also shown in Figure 3.
Example 5: Powder X-Ray Diffraction Analysis of Amifostine Dihvdrate
[0053] A comparison of the structural transformation of amifostine to amifostine dihydrate was studied by powder X-ray diffraction (XRD) analysis using a local X-ray diffraction apparatus under conditions as described below. Specifically, a SCDSfTAG Xl "Advanced Diffraction System" powder x-ray theta-theta diffractometer (Scintag, now Thermo Electron Corporation) using a Cu X-ray tube (Cu Ka radiation, λ=1.5406 A) and run at an accelerating voltage of 45 kV and a tube/filament current of 40 niA. Samples were scanned from 2 to 70° 2Θ at a scan rate of 0.257min. [see Lims 342462]
[0054] The measured XRD patterns of amifostine monohydrate, amifostine trihydrate, and amifostine dihydrate are shown in Figure 4. These XRD powder patterns illustrate two- dimensional structural differences between the varied hydrate forms of amifostine. Characteristic peaks for a monoclinic P2j/c structure were observed in the amifostine dihydrate sample, while these peaks are notably absent in the orthorhombic amifostine trihydrate sample and the amifostine monohydrate samples.
Example 6: Crystal Structure of Amifostine Dihydrate
[0055] The molecular and crystal structure of crystalline amifostine dihydrate was determined. Crystal survey, unit cell determination, and data collection were performed using a Nonnius KappaCCD diffractometer at a temperature of 105 Kelvin (-168.15 0C;
-270.67 0F).
[0056] The structure was solved by direct methods and refined by full-matrix least- squares and difference Fourier transform methods. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms attached to the nitrogen and water oxygen atoms were located from difference Fourier maps and refined isotropically. The positions of the remaining hydrogen atoms were calculated using ideal geometries. These hydrogen atoms were not refined due to the low reflection-to-parameter ratio.
[0057] The dihydrate compound crystallizes in the chiral space group P2i/c. The data presented in this example are from the enantiomeric structure with low R values (R=0.046 and Rw=0.096). A graphic ORTEP depiction [Johnson, C.K., ORTEP. Report ORNL-3794. Oak Ridge National Laboratory, Tennessee, USA] of the molecular and crystal structure of amifostine dihydrate is shown in Figure 5. ORTEP stands for "Oak Ridge Thermal-Ellipsoid Plot program".
Data Collection:
[0058] A colorless, flat plate crystal of C5H15N2O3PS.2(H2O) having approximate dimensions of 0.25 x 0.10 x 0.03 mm was mounted on a glass fiber. All measurements were made on a KappaCCD diffractometer (Nonnius) equipped with a sealed tube generator with Mo Ka radiation and a rotating anode generator. An Oxford Cryosystems Cryostream Plus Cooler (Oxford, UK) provided low temperature data collection down to about 105 K using a cooled nitrogen gas stream.
[0059] Cell constants and an orientation matrix for data collection, obtained from a least-squares refinement corresponded to a monoclinic cell with dimensions: a= 7.350 (4) A; b=17.508 (11) A; c=9.206 (6) A; and V (volume) = 1140.6 (12) A3. For Z = 4 and F.W. = 250.25, the calculated density is 1.457 mg/m3. Based on the successful solution and refinement of the structure, the space group was determined to be P2j/c. The data were collected at a temperature of -168.0 ± 1 0C.
Data Reduction:
[0060] A total of 2029 reflections were collected. The intensities of intensities of three representative reflections which were measured after every 150 reflections remained constant throughout data collection, indicating crystal and electronic stability (no decay correction/extinction correction was applied). The linear absorption coefficient for graphite monochromated Mo Ka is 6.99 cm"1. An empirical absorption correction, based on azimuthal scans of several reflections, was applied which resulted in transmission factors/coefficients ranging from 0.826 (Tmin) to 0.987 (Tmax). The data were corrected for Lorentz and polarization effects. The resulting data is shown in Table 1. Fractional atomic coordinates and equivalent isotropic displacement parameters are shown in Table 2. Table 3, below, shows the hydrogen-bonding geometry of amifostine dihydrate, as described herein, while selected geometric parameters are given in Table 4.
Table 1. Crystal Data and structure refinement of the amifostine dihydrate.
Figure imgf000020_0001
Figure imgf000021_0001
*Otwinoswski, Z. & Minor, W., Methods Enzymol, 276; pp. 307-326 (1997).
Table 2. Fractional atomic coordinates and equivalent isotropic displacement parameters (A2). Ueq is defined as one third of the trace of the orthogonalized Ulj tensor, which can be represented mathematically as:
Figure imgf000022_0001
Atom X y Z Ueq
Sl 0.99721 (10) 0.58881 (4) 0.19378 (7) 0.01542 (19)
Pl 1.20476 (10) 0.63963 (4) 0.37682 (8) 0.01168 (19)
Ol 1.3857 (2) 0.62891 (12) 0.3282 (2) 0.0150 (4)
02 1.2012 (3) 0.59446 (12) 0.5169 (2) 0.0153 (4)
03 1.1503 (3) 0.72208 (11) 0.3860 (2) 0.0150 (4)
Cl 0.8177 (4) 0.56932 (18) 0.2924 (3) 0.0181 (7)
HlA 0.8765 0.5437 0.3896 0.022
HlB 0.7616 0.6180 0.3141 0.022
C2 0.6645 (4) 0.51869 (17) 0.1963 (3) 0.0143 (6)
H2A 0.5892 0.5476 0.1079 0.017
H2B 0.7219 0.4741 0.1594 0.017
N3 0.5399 (3) 0.49240 (15) 0.2914 (3) 0.0129 (5)
H3A 0.621 (4) 0.4628 (18) 0.374 (3) 0.016
H3B 0.491 (4) 0.534 (2) 0.321 (3) 0.016
C4 0.3804 (4) 0.44355 (17) 0.2092 (3) 0.0156 (6)
H4A 0.4292 0.4005 0.1608 0.019
H4B 0.2931 0.4738 0.1291 0.019
C5 0.2748 (4) 0.41266 (18) 0.3177 (3) 0.0157 (6)
H5A 0.3632 0.3843 0.4003 0.019
H5B 0.2210 0.4556 0.3627 0.019
C6 0.1173 (4) 0.35999 (18) 0.2340 (3) 0.0168 (6) Atom x y u, eq
H6A 0.0181 0.3906 0.1642 0.020
H6B 0.1682 0.3234 0.1729 0.020
N7 0.0313 (4) 0.31667 (16) 0.3384 (3) 0.0155 (5)
H7A 0.135 (5) 0.292 (2) 0.411 (4) 0.023
H7B -0.042 (4) 0.276 (2) 0.286 (4) 0.023
H7C -0.041 (5) 0.346 (2) 0.380 (4) 0.023
01W 0.3544 (3) 0.25333 (14) 0.5506 (3) 0.0214 (5)
HlW 0.427 (5) 0.286 (2) 0.593 (4) 0.032
H2W 0.409 (5) 0.223 (2) 0.514 (4) 0.032
O2W 0.6272 (3) 0.15917 (14) 0.4757 (3) 0.0226 (5)
H3W 0.694 (5) 0.197 (2) 0.506 (4) 0.034
H4W 0.625 (5) 0.156 (2) 0.389 (4) 0.034
Figure imgf000023_0001
Symmetry codes: (i) 2-x, 1-y, 1-z; (ii) x-1, y, z; (iii) 1-x, y- 1A , Vz-z; (iv) 1-x, 1-y, 1-z; (v) 2-x, y- 1A , 1A-Z. Table 4. Selected Geometric Parameters (A, °).
Figure imgf000024_0001
[0061] While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and/or processes and in the steps or in the sequence of steps of the methods described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention.

Claims

CLAIMS:
1) Amifostine dihydrate.
2) A pharmaceutical composition comprising amifostine dihydrate, wherein the pharmaceutical composition exhibits a biological activity.
3) The pharmaceutical composition of claim 2, further comprising one or more additional therapeutic agents.
4) The pharmaceutical composition of claim 3, wherein the additional therapeutic agents are cisplatin, carboplatin, gemcitabine, paclitaxel, docetaxel, doxorubicin, bleomycin, BCNU, cyclophosphamide, nitrogen mustard, 5-fluorouracil, mitomycin-C, diaziquone, melphalan, vinblastine, etoposide, vinorlebine, ifosfamide, azidothymidine (AZT), or combinations thereof.
5) The pharmaceutical composition of claim 2, wherein the biological activity is cytoprotection, radioprotection, chemoprotection, or a combination thereof.
6) The pharmaceutical composition of claim 2, wherein the composition is lyophilized or freeze-dried.
7) The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is formulated for use via intravenous administration.
8) The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is formulated for use via subcutaneous administration.
9) The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is formulated for use via oral administration.
10) A method of treating a disease in a mammal in need of such treatment, the method comprising administering a therapeutically effective amount of a composition comprising amifostine dihydrate.
11) The method of claim 10, wherein the disease is cancer, myelodysplastic syndromes, or an HTV-related disease. 12) The method of claim 11 , wherein the cancer is pancreatic cancer, melanoma, breast cancer, prostate cancer, ovarian cancer, endometrial cancer, lung cancer, non-small cell lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, head cancer, neck cancer, or combinations thereof.
13) The method of claim 10, wherein the composition is administered before, during, or after chemotherapy.
14) The method of claim 10, wherein the composition is administered before, during, or after radiation therapy.
15) The method of claim 10, wherein the therapeutically effective amount is from about 200 mg/kg per day to about 1200 mg/kg per day.
16) A process for the preparation of crystalline amifostine dihydrate, the process comprising:
(a) preparing a formulation comprising amifostine monohydrate, alcohol and water in which the relative amounts of amifostine monohydrate, alcohol and water are such that a particulate-free solution is obtained at temperatures ranging from about room temperature to about 10 0C;
(b) cooling the formulation to a temperature below 0 °C for a period of time sufficient to effect the precipitation of the crystalline amifostine dihydrate; and
(c) drying the resulting mixture to leave a solid crystalline amifostine dihydrate preparation having enhanced stability.
17) The process of claim 16 which further comprises introducing a sterile inert gas over the preparation after completion of the drying of step (c).
18) The process of claim 16, wherein the drying is vacuum drying.
19) The process of claim 16 in which the inert gas is argon, nitrogen or helium.
20) Crystalline amifostine dihydrate which is prepared according to the process of claim 16. 21) Crystalline amifostine characterized by substantially the same X-ray powder diffraction (XRD) pattern labeled amifostine dihydrate in Figure 4, wherein the XRD pattern is expressed in terms of 2 theta angles and obtained with a diffractometer equipped with a diffracted beam graphite monochronometer using copper KαX-radiation.
22) Crystalline amifostine characterized by an XRD pattern expressed in terms of 2 theta angles and obtained with a diffractometer equipped with a diffracted beam graphite monochronometer using copper Ka X-radiation, wherein the XRD pattern comprises 2 theta angles at four or more positions selected from the group consisting of about 10.2 ± 0.1, about 11.2 ± 0.1, about 14.3 ± 0.1, about 20.5 ± 0.1, and about 22.5 ± 0.1 degrees.
23) Crystalline amifostine characterized by substantially the same TGA trace labeled amifostine dihydrate in Figure 3, wherein the scan rate is 2 0C per minute under nitrogen.
24) Crystalline amifostine characterized by substantially the same NMR spectrum labeled amifostine dihydrate in Figure 1, wherein the spectrum is obtained on a spectrometer operating at a frequency of about 400 MHz for 1H observation at a temperature of about 300 K.
25) Crystalline amifostine characterized by substantially the same DSC thermogram labeled amifostine dihydrate in Figure 2, wherein the thermogram is obtained on a differential scanning calorimeter operating at a scan rate of about 10 °C/min under nitrogen.
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WO2008118960A2 (en) * 2007-03-26 2008-10-02 William Marsh Rice University Radiation protection using carbon nanotube derivatives
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WO2007096901A1 (en) * 2006-02-24 2007-08-30 Natco Pharma Limited Novel dihydrate form of amifostine and process for its preparation
WO2008118960A2 (en) * 2007-03-26 2008-10-02 William Marsh Rice University Radiation protection using carbon nanotube derivatives
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