CROSS REFERENCE TO PRIORITY APPLICATION

[0001] This patent application claims the benefit of U. S . Provisional Application No. 61/409,263 filed November 2, 2010, the disclosures of which are herein incorporated by reference.

FIELD OF INVENTION

[0002] The present invention encompasses solid state forms of Pralatrexate, processes for preparation thereof, and pharmaceutical compositions thereof.

BACKGROUND OF THE INVENTION

[0003] Pralatrexate, (25 -2-[[4-[(li?5)-l-[(2,4-diaminopteridin-6-yl)methyl]but-3- ynyl]benzoyl]amino]pentandioic acid is an anti-cancer drug having the following formula:

Pralatrexate is approved for a treatment for patients with relapsed or refractory peripheral T-cell lymphoma. It is an antifolate and acts as an inhibitor of

dihydrofolate reductase.

[0004] Pralatrexate is disclosed in several documents such as DeGraw et al. (J Med. Chem, 1993, 36, 2228, US 5,354,751, US 6,028,071 B2 and EP 0944389 Bl.

[0005] Polymorphism, the occurrence of different crystal forms, is a property of some molecules and molecular complexes. A single molecule, like Pralatrexate, may give rise to a variety of polymorphs having distinct crystal structures and physical properties like melting point, thermal behaviors (e.g. measured by thermogravimetric analysis - "TGA", or differential scanning calorimetry - "DSC"), X-ray powder diffraction (XRPD) pattern, infrared absorption fingerprint, and solid state nuclear magnetic resonance (NMR) spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

[0006] Discovering polymorphic forms and solvates of a pharmaceutical product can provide materials having desirable processing properties, such as ease of handling, ease of processing, storage stability, and ease of purification or as desirable intermediate crystal forms that facilitate conversion to other polymorphic forms. New polymorphic forms and solvates of a pharmaceutically useful compound can also provide an opportunity to improve the performance characteristics of a

pharmaceutical product. It enlarges the repertoire of materials that a formulation scientist has available for formulation optimization, for example by providing a product with different properties, e.g., better processing or handling characteristics, improved dissolution profile, or improved shelf-life. For at least these reasons, there is a need for crystalline forms of Pralatrexate.

SUMMARY OF THE INVENTION

[0007] The present invention provides solid state forms of Pralatrexate and pharmaceutical compositions comprising said solid state forms and at least one pharmaceutically acceptable excipient.

[0008] The present invention also encompasses the use of any of the above solid- state forms for the preparation of a pharmaceutical composition.

[0009] The present invention further encompasses the use of any of the above solid-state forms and pharmaceutical compositions, as a medicament, particularly for the treatment for patients with relapsed or refractory peripheral T-cell lymphoma.

[0010] The present invention also provides a method of anti-cancer therapy, comprising administering a therapeutically effective amount of at least one of the Pralatrexate solid-state forms of the present invention, or at least one of the above pharmaceutical compositions to a person with relapsed or refractory peripheral T-cell lymphoma. BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 shows an X-ray powder diffractogram of Pralatrexate form A.

[0012] Figure 2 shows an X-ray powder diffractogram of Pralatrexate form B.

[0013] Figure 3 shows an X-ray powder diffractogram of Pralatrexate form C.

[0014] Figure 4 shows an X-ray powder diffractogram of Amorphous Pralatrexate.

[0015] Figure 5 shows a ¹³ C NMR spectrum of Pralatrexate form A between 0-200 ppm.

[0016] Figure 6 shows a ¹³ C NMR spectrum of Pralatrexate form B between 0-200 ppm.

[0017] Figure 7 shows a ¹³ C NMR spectrum of Pralatrexate form C between 0-200 ppm.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention addresses a need in the art by providing Pralatrexate in a crystalline form, which is believed to provide significant advantages for making pharmaceutical compositions, as compared to the amorphous form disclosed in the prior art. Particularly, important factors include chemical stability, solid state stability and "shelf life" of the drug substance. Ideally, the drug substance, and compositions containing it, should be capable of being stored over appreciable periods of time, under conditions such as high humidity without exhibiting a significant change in the active component's physico-chemical characteristics (e.g. its chemical composition, density, hygroscopicity, solubility and rate of dissolution). Crystalline forms of Pralatrexate, such as the crystalline forms of the present invention (namely, Forms A, B and C) are believed to provide advantages over the prior art amorphous Pralatrexate in this regard.

[0019] In some embodiments, solid state forms of Pralatrexate of the invention are substantially free of any other polymorphic forms of Pralatrexate, or of specified polymorphic forms of Pralatrexate, respectively. In any embodiment of the present invention, by "substantially free" is meant that the forms of the present invention contain 20% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, 2% (w/w) or less, particularly 1% (w/w) or less, more particularly 0.5% (w/w) or less, and most particularly 0.2% (w/w) or less of any other polymorphs of Pralatrexate, or of a specified polymorph of Pralatrexate. In other embodiments, the polymorphs of Pralatrexate of the invention contain from 1% to 20% (w/w), from 5% to 20% (w/w), or from 5% to 10% (w/w)) of any other polymorphs or of a specified polymorph of Pralatrexate or of an amorphous form of Pralatrexate.

[0020] The present invention addresses a need in the art by providing new crystalline forms of Pralatrexate that have advantageous properties selected from at least one of: chemical purity, flowability, solubility, dissolution rate, morphology or crystal habit, stability- such as thermal and mechanical stability to polymorphic conversion, stability to dehydration and/or storage stability, low content of residual solvent, a lower degree of hygroscopicity, flowability, and advantageous processing and handling characteristics such as compressibility, and bulk density. [0021] A crystal form may be referred to herein as being characterized by graphical data "as shown in," or "as depicted in" a Figure. Such data include, for example, powder X-ray diffractograms and solid state NMR spectra. The skilled person will understand that such graphical representations of data may be subject to small variations, e.g., in peak relative intensities and peak positions due to factors such as variations in instrument response and variations in sample concentration and purity, which are well known to the skilled person. Nonetheless, the skilled person would readily be capable of comparing the graphical data in the Figures herein with graphical data generated for an unknown crystal form, and confirm whether the two sets of data are characterizing the same crystal form or two different crystal forms. A crystal form of Pralatrexate referred to herein as being characterized by graphical data "as shown in," or "as depicted in" a Figure will thus be understood to include any crystal forms of the Pralatrexate characterized with the graphical data having such small variations, as are well known to the skilled person, in comparison with the Figure.

[0022] As used herein, the term "isolated" in reference to any of the Pralatrexate crystalline forms of the present invention corresponds to Pralatrexate crystalline form that is physically separated from the reaction mixture, wherein it was formed. [0023] As used herein, unless stated otherwise, the XRPD measurements are taken using copper Ka radiation wavelength 1.54184 A.

[0024] A thing, e.g., a reaction mixture, may be characterized herein as being at, or allowed to come to "room temperature (often abbreviated "RT"). This means that the temperature of the thing is close to, or the same as, that of the space, e.g., the room or fume hood, in which the thing is located. Typically, room temperature is from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25°C.

[0025] A process or step may be referred to herein as being carried out

"overnight." This refers to a time interval, e.g., for the process or step, that spans the time during the night, when that process or step may not be actively observed. This time interval is from about 8 to about 20 hours, or about 10-18 hours, typically about 16 hours.

[0026] As used herein, the term "reduced pressure" refers to a pressure of about 10 mbar to about 50 mbar.

[0027] As used herein, and unless stated otherwise, the term "anhydrous" in relation to crystalline Pralatrexate relates to a crystalline Pralatrexate which contains not more than 1.5% (w/w), or not more than 1% (w/w) of either water or organic solvents (bound and unbound) as measured by TGA or by Karl Fisher titration, for example, Pralatrexate which contains between about 0% to about 1.5% (w/w) or between about 0% to about 1% (w/w) of either water or organic solvents as measured by TGA or by Karl Fisher titration.

[0028] As used herein, the terms "vol." or "volume" can be used to refer to ml per gram of the corresponding Pralatrexate. For example, a statement that 0.5 g of Pralatrexate is dissolved in ten volumes of a Solvent X would be understood to mean that the 0.5 g of Pralatrexate was dissolved in 5 ml of Solvent X.

[0029] The present invention comprises a crystalline form of Pralatrexate, designated as Form A. Form A can be characterized by data selected from: an X-ray powder diffraction (XRPD) pattern having peaks at 8.7, 12.3, 15.2, 19.2 and 21.9° 2Θ ± 0.2° 20; an X-ray powder diffraction (XRPD) pattern having peaks at 8.7, 12.3, 14.3, 16.0 and 21.9° 20 ± 0.2° 20; an XRPD pattern substantially as depicted in figure 1; a solid state ¹³ C NMR spectrum having signals at 25.1, 35.9, 53.2, 122.0 and 146.8 ± 0.2 ppm; a solid state C NMR spectrum as depicted in Figure 5; and combinations thereof. Crystalline Form A of Pralatrexate can be further characterized by data selected from: an XRPD pattern having any one, two, three, four or five additional peaks selected from: 10.9, 14.3, 16.0, 23.3 and 24.3° 2Θ ± 0.2° 2Θ; and an XRPD pattern having any one, two, three, four or five additional peaks selected from: 10.9, 15.2, 19.2, 23.3 and 24.3° 2Θ ± 0.2° 2Θ.

[0030] The present invention comprises a crystalline form of Pralatrexate, designated as Form B. Form B can be characterized by data selected from: an XRPD pattern having peaks at 8.9, 11.0, 13.8 and 15.4° 2Θ + 0.2° 2Θ; an XRPD pattern substantially as depicted in figure 2; a solid state ¹³ C NMR spectrum having signals at 27.8, 35.4, 68.6, 128.8 and 157.4 ± 0.2 ppm; a solid state ¹³ C NMR spectrum as depicted in Figure 6; and combinations thereof. Crystalline Form B of Pralatrexate can be further characterized by an X-ray powder diffraction (XRPD) pattern having any one, two, three or four additional peaks selected from: 12.3, 19.2, 21.9 and 24.7° 2Θ ± 0.2° 2Θ.

[0031] The present invention comprises a crystalline form of Pralatrexate, designated as Form C. Form C can be characterized by data selected from: an XRPD pattern having peaks at 8.5, 12.5, 13.1, 22.2, and 24.0° 2Θ ± 0.2° 20; an XRPD pattern having peaks at 8.5, 12.5, 13.1, 22.2, and 29.3° 2Θ ± 0.2° 20; an XRPD pattern substantially as depicted in figure 3; a solid state ¹³ C NMR spectrum having signals at 42.0, 44.6, 132.5, 153.1 and 180.1 ± 0.2 ppm; a solid state ¹³ C NMR spectrum as depicted in Figure 7; and combinations thereof. Crystalline Form C of Pralatrexate can be further characterized by data selected from: an XRPD pattern having any one, two, three, four or five additional peaks selected from: 10.8, 15.1, 19.1, 28.5 and 29.3° 2Θ + 0.2° 2Θ; and an XRPD pattern having any one, two, three, four or five additional peaks selected from: 10.8, 15.1, 19.1, 24.0 and 28.5 ° 20 ± 0.2° 20.

[0032] Typically, the crystalline forms of the present invention (namely, Form A, Form B and Form C) are anhydrous forms.

[0033] The present invention also describes an amorphous form of Pralatrexate. The amorphous form of Pralatrexate can be characterized by an XRPD pattern as depicted in figure 4. [0034] The above described forms of Pralatrexate can be used to prepare pharmaceutical compositions by any method known in the art.

[0035] The present invention further encompasses 1) a pharmaceutical composition comprising at least one of the solid state forms of Pralatrexate as described above, and at least one pharmaceutically acceptable excipient; 2) the use of any one or combination of the above-described solid state forms of Pralatrexate in the manufacture of a pharmaceutical composition, and 3) a method of treating relapsed or refractory peripheral T-cell lymphoma, said method comprising administering a pharmaceutically effective amount of at least one of the solid state forms of

Pralatrexate as described above to a subject, e.g., a patient, in need of the treatment. The pharmaceutical composition can be useful for preparing a medicament. The present invention also provides at least one of the solid state forms of Pralatrexate as described above for use as a medicament. The pharmaceutical composition may be in a form of a solution.

[0036] Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those in the art can appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The Examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to limit its scope in any way.

X-ray powder diffraction (XRPD method

[0037] XRPD was performed on a Philips X'Pert PRO powder diffractometer equipped with an X'Celerator detector (active length 2Θ = 2.022°), CuKa radiation, λ = 1.54184 A at a laboratory temperature 22-25°C. Prior to analysis the samples were gently ground using a mortar and pestle in order to obtain a fine powder and the powder was applied directly on silicon zero background holder. The scanning parameters were: range: 3-40 degrees two-theta; scan mode: continuous scan; step size: 0.0167°; and time per step: 37 sec.

Solid state ¹³ C NMR Instrumentation and Method:

[0038] NMR spectra of solid samples were recorded on a Varian NMR System 600 MHz NMR spectrometer equipped with a 3.2 mm NB Double Resonance HX MAS Solids Probe. Larmor frequency of carbon nuclei was 150.79 MHz. The C CP- MAS NMR spectra were externally referenced to the methyl groups of

hexamethylbenzene (6=17.3 ppm). Samples were spun at the magic angle with 10 kHz during ¹³ C measurement.

[0039] The pulse sequence used for acquiring spectra was a standard cross-polarization MAS pulse sequence with high-power proton decoupling during acquisition. Repetition delay was 5 s and the number of scans was between 3200 and 9000.

EXAMPLES

Example 1: Preparation of Pralatrexate according to U.S. Patent No. 6,028,071, example 1

[0040] FIG. 4 of U.S. Patent No. 6,028,071 shows a synthetic scheme useful in preparing pralatrexate.

Example 1A: Compound 2 of Figure 4 of US 6,028,071

[0041] A mixture of 60% NaH in oil dispersion (1.06 g, 26.5 mmol) in 18 mL of sieve-dried THF was cooled to 0°C. The cold mixture was treated with a solution of homoterephthalic acid dimethyl ester (5.0 g, 24 mmol.; compound 1 in FIG. 4 of US 6,0280,71) in dry THF (7 mL), and the mixture was stirred for 1 hour at 0°C. Propargyl bromide (26.4 mmol) was added, and the mixture was stirred at 0°C for an additional 1 hour, and then at room temperature for 16 hours. The resulting mixture was treated with 2.4 mL of 50% acetic acid and then poured into 240 mL of water. The mixture was extracted with ether (2 times; 150 mL). The ether extracts were combined, dried over Na ₂ S04, and concentrated to an orange-yellow oil.

Chromatography on silica gel (600 mL of 230-400 mesh) eluting with cyclohexane- EtOAc (8:1) gave the product alpha-propargylhomoterephthalic acid dimethyl ester (compound 2) as a white solid (4.66 g) which appeared by TLC (cyclohexane-EtOAc, 3:1) to be homogeneous. Mass spectral data on this product, however, showed it to be a mixture of the desired product 2, and the dipropargylated compound. No starting material 1 was detected. HPLC showed the ratio of mono- to di-propargylated products to be about 3:1. Since the dipropargylated product, unlike compound 1, cannot produce an unwanted coproduct in the next step of the reaction, this material was suitable for conversion to compound 3. Absence of starting compound 1 in the product used to proceed in the synthesis is very important in order to avoid the sequential formation of 10-dAM during the transformations leading to the final product, because complete removal from 10-dAM from 10-propargyl-l-dAM is very difficult.

Example IB: 10-Propargyl-10-carbomemoxy-4-deoxy-4-arnino-10-deazapteroic acid methyl ester (compound 3 in Figure 4 of US 6,028,071)

[0042] A mixture was formed by combining 0.36 g of NaH (9 mmol, 60% dispersion in oil) with 10 mL of dry DMF, and cooling the mixture to 0-5°C. The cold mixture was treated dropwise with a solution of the product of the first reaction (compound 2 from Example 1 A) (2.94 g, 12 mmol) in 10 mL dry DMF, and then stirring this mixture at 0°C for 30 minutes. After cooling the mixture to -25°C, a solution of 2,4,diamino-6-(bromomethyl)pteridine hydrobromide-0.2 2-propanol (1.00 g, 2.9 mmol) in 10 mL dry DMF was added dropwise while the temperature was maintained near -25°C. The temperature of the stirred mixture was then allowed to rise to -10°C. over aperiod of 2 hours. After an additional 2 hours at -10°C, the temperature was allowed to rise to 20°C. Stirring at room temperature was continued for 2 hours longer. The reaction was then adjusted to pH 7 by addition of solid C0 ₂ . After concentration in vacuo to remove the solvent, the residue was stirred with diethyl ether. The ether insoluble material was collected by filtration, washed with water, and dried in vacuo to give 1.49 g of a crude product. This crude product was dissolved in CHCl ₃ ~MeOH (10: 1) and applied to a silica gel column. Elution by the same solvent system afforded 10-propargyl-10-carbomethoxy-4-deoxy-4-amino-10- deazapteroic acid methyl ester (compound 3 of Figure 4 of US 6,028,071) which was homogenous by TLC in 40% yield (485 mg).

Example 1C: Compound 4 of Figure 4 of US 6,028,071

[0043] A stirred suspension of compound 3 from Example IB (400 mg, 0.95 mmol) in 2-methoxyethanol (5 mL) was treated with water (5 mL), and then 10% aqueous sodium hydroxide solution (3.9 mL). The resulting mixture was stirred at room temperature for 4 hours, during which time a solution was formed. The solution was adjusted to pH 8 with acetic acid and concentrated under high vacuum. The resulting residue was dissolved in 15 mL of water and acidified to pH 5.5-5.8 resulting in formation of a precipitate. The precipitate was collected, washed with water and dried in vacuo to recover 340 mg of compound 4 (of Figure 4 of US 6,028,071) (91% yield). HPLC analysis indicated a product purity of 90%.

Example ID: 10-Propargyl-4-deoxy-4-amino-10-deazapteroic acid (compound 5 of Figure 4 of US 6,028,071)

[0044] Compound 4 from Example 1C (330 mg) was decarboxylated by heating in 15 mL DMSO at 115-120°C. for 10 minutes. HPLC analysis after 10 minutes confirmed that the conversion was essentially complete. The DMSO was removed by distillation in vacuo (bath at 40°C). The resulting residue was stirred with 0.5 N NaOH to give a clear solution. Acidification to pH 5.0 with 1 N HC1 gave 10-propargyl-4-deoxy-4- amino-10-deazapteroic acid (compound 5 of Figure 4 of US 6,028,071) as a yellow solid in 70% yield. HPLC indicated that the product purity at this stage was 90%.

Example IE: 10-Propargyl-lO-deazaaminopterin dimethyl ester (compound 6 of Figure 4 of US 6,028,071

[0045] Compound 5 from Example ID (225 mg, 0.65 mmol) was coupled with dimethyl L-glutamate hydrochloride (137 mg, 0.65 mmol) using benzotriazole-1- yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) reagent (287 mg, 0.65 mmol, Aldrich Chemical Co.) in DMF (10 mL) containing triethylamine (148 mg, 1.46 mmol). The mixture was stirred for 3 hours at 20-25°C. and then evaporated to dryness. The residue was stirred with water, and the water-insoluble crude product was collected and dried in vacuo. The crude product (350 mg) was purified by silica gel chromatography eluting with CHCl ₃ ~MeOH (10:1) containing triethylamine (0.25% by volume) to recover 165 mg of 10-propargyl-lO-deazaaminopterin dimethyl ester (compound 6 of Figure 4 of US 6,028,071, 50% yield), which was homogeneous by TLC (CHCb-MeOH 5:1).

Example IF: 10-Propargyl-lO-deazaaminopterin

[0046] Compound 6 from Example IE (165 mg, 0.326 mmol) was suspended in 10 mL stirred MeOH to which 0.72 mL (0.72 meq) 1 N NaOH was added. Stirring at room temperature was continued until solution occurred after a few hours. The solution was kept at 20-25°C. for 8 hours, and then diluted with 10 mL water.

Evaporation under reduced pressure removed the methanol, and the concentrated aqueous solution was left at 20-25°C for another 24 hours. HPLC then showed the ester hydrolysis to be complete. The clear aqueous solution was acidified with acetic acid to pH 4.0 to precipitate 10-propargyl-lO-deazaaminopterin as a pale yellow solid. The collected, water washed and dried in vacuo product weighed 122 mg (79% yield). Assay by elemental analysis, proton NMR and mass spectroscopy were entirely consistent with the assigned structure. HPLC analysis indicated purity of 98% and established the product to be free of 10-deazaaminopterin. The obtained product was confirmed to be in amorphous form by XRPD analysis.

Example 2: Preparation of crystalline Pralatrexate Form A:

[0047] Amorphous pralatrexate (400 mg, purity 96%) was dissolved in formamide at room temperature (1 mL, 2.5 Vol.). The solution was poured into water at room temperature (10 mL, 25 Vol.), resulting in the formation of a precipitate. The precipitate was filtered off and dried in a vacuum drying oven at 35°C under vacuum for 18 h, furmshing pralatrexate Form A (200 mg, 50% yield, 96% purity).

Example 3: Preparation of crystalline Pralatrexate Form B:

[0048] Amorphous pralatrexate (200 mg, purity 96% %) was suspended in MeOH (2 mL, 10 Vol.), and the slurry was stirred at room temperature. After 24 h of stirring, the solid was filtered off and dried in a vacuum drying oven at 35°C under vacuum for 18 h, furrmhing pralatrexate Form B (118 mg, 59% yield, 96% purity).

Example 4: Preparation of crystalline Pralatrexate Form B:

[0049] Amorphous pralatrexate (200 mg, purity 96%) was suspended in MeOH (2 mL, 10 Vol.) and the slurry was heated up to 50 °C. After 24 h the solid was filtered off and dried in a vacuum drying oven at 35°C under vacuum, furnishing pralatrexate Form B (96 mg, 48% yield, 96% purity).

Example 5: Preparation of Amorphous Pralatrexate

[0050] A solution of pralatrexate in water was prepared as described in example IF above (according to U.S. Patent No. 6,028,071, example 1). Pralatrexate was precipitated by addition of aq. HC1 10 % to adjust to pH 4.5. The solid was filtered off and dried in a vacuum drying oven at 35°C under vacuum, furnishing pralatrexate as a pale yellow solid. Example 6: Preparation of crystalline Pralatrexate Form A:

[0051] Amorphous pralatrexate (400 mg, purity 98.5%) was suspended in water (4 mL, 10 Vol.) pre-heated at 50 °C, and the slurry was stirred for 4 h at 50°C. The solid was then filtered off and dried in a vacuum drying oven at 45 °C under vacuum for 18h, furnishing pralatrexate form A (305 mg, 76% yield, 98.5% purity).

Example 7: Preparation of crystalline Pralatrexate Form B:

[0052] Amorphous pralatrexate (400 mg, purity 98.5%) was suspended in MeOH (4 mL, 10 Vol.) pre-heated at 50 °C, and the slurry was stirred for 16 h at 50 °C. The solid was then filtered off and dried in a vacuum drying oven at 45 °C under vacuum for 18 h, furnishing pralatrexate form B (250 mg, 63% yield, 97% purity).

Example 8: Preparation of crystalline Pralatrexate Form C:

[0053] Amorphous pralatrexate (400 mg, purity 98.5%) was suspended in a mixture of water (2 mL, 5 Vol.) and MeOH (2 mL, 5 Vol.) pre-heated at 50 °C, and the slurry was stirred for 2 h at 50 °C. The solid was filtered off and dried in a vacuum drying oven at 45 °C under vacuum for 18 h, furnishing pralatrexate form C (320 mg, 80% yield, 98.5% purity).

Example 9: Preparation of crystalline Pralatrexate Form C:

[0054] Amorphous pralatrexate (200 mg, purity 96%) was suspended in MeOH (2 mL, 10 Vol.) Upon heating at 50 °C, water was added (2 mL, 10 Vol.) and the resulting slurry was kept at 50 °C for 24 h. The solid was then filtered off and dried in a vacuum drying oven at 35°C under vacuum, furnishing pralatrexate Form C (96 mg, 48% yield, 96% purity).