TECHNICAL FIELD:

The invention relates to novel salt of antitumor prodrug temozolomide with hydrochloric acid in crystalline hydrate form, and to the process of preparation thereof.

BACKGROUND OF INVENTION:

Temozolomide, chemical name 8-carbamoyl-3-methylimidazo[5,l-d]-l,2,3,5-tetrazin-4(3H)-one, (abbreviated as TMZ) is an antitumor prodrug (P. R. Lowe, C. E. Sansom, C. H. Schwalbe, M. F. G. Stevens, and A. S. Clark, J. Med. Chem., 1992, 35, 3377-3382; E Lunt, M. F. G, Stevens, R. Stone, K. R. H. Wooldrdige and E. S. Newlands, US Patent No. 5,260,291, 1993). The chemical structure of TMZ is given in Scheme 1.

H2N^O
II I <
Y CH-
o

TMZ Scheme 1 Chemical structure of Temozlomide (TMZ) Temozolomide belongs to the N-3 substituted carbamoyl imidazo tetrazinone class of drugs (E. S. Newlands, M. F. Stevens, S. R. Wedge, R. T. Wheelhouse, C. Brock, Cancer Treat. Rev. 1997, 23, 35-61). This antitumor prodrug is active against malignant melanoma for the treatment of brain tumor. It is an alkylating agent that acts by the water-assisted tetrazinone ring opening to release 3-methyl-(triazenyl-l-yl)imidazole-4-carboxamide (MTIC) through elimination of H20 and loss of CO2 (Scheme 2). MTIC rapidly degrades to 5-aminoimidazole-4-carboxamide (AIC) and a highly reactive methyldiazonium ion (CH3N2+) (M. J. M. Darkes, G. L. Plosker, B. Jarvis, Am. J. Cancer 2002, 1, 55-80). The mean elimination half-life of temozolomide in plasma concentrations is about 1.8 h (range 1.7-1.9 h) with maximum elimination occurring between 0.33-2 h.

Conversion of TMZ to MTIC and then AIC is pH dependent and irreversible. In aqueous buffers, TMZ is relatively stable at acidic pH < 4, but rapidly hydrolyses to MTIC at pH > 7. In contrast, MTIC is stable at alkaline pH but rapidly breaks down to AIC at pH < 7 (S. D. Baker, M. Wirth, P. Statkevich, P. Reidenberg, K. Alton, S. E. Sartorius, M. Dugan, D. Cutler, V. Batra, L. B. Grochow, R. C. Donchower and E. K. Rowinsky, Clin. Cancer Res., 1999, 5, 309-317). Scheme 2 Mechanism of action for the antitumor prodrug temozolomide by DNA methylation. MTIC = 5-(3-monomethyl-l-triazeno)imidazole-4-carboxamide, AIC = 5-aminoimidazole-4-carboxamide. TMZ has in vitro half life of 1.7-1.9 h in phosphate buffer at 37 °C and pH 7.5 (physiological pH 7.4) whereas MTIC has half life of ~2 min. Temozolomide is completely decomposed at pH 9 buffer. In time-dependent experiments, TMZ started to decompose within 5 min at neutral and alkaline pH solutions, whereas 90% of TMZ was intact in acidic medium for 60 min (T. Kodawara, T. Mizuno, H.

Taue, T. Hashida, I. Yano, T. Katsura and K. Inui, Yakugaku Zasshi, 2009, 129, 353-357). The effect of temperature was also studied. TMZ is stable in acidified human plasma (Ph < 4) for at least 24 h at 25 °C and for at least 30 days at -20 °C (H. Kim, P. Likhari, D. Parker, P. Statkevich, A. Marco, C.-C. Lin and A. A. Nomeir, J. Pharma, Biomed. Anal., 2001, 24, 461-468). Nine polymorphs of TMZ were disclosed in a US patent (I. Adin, C. Iustain, US 2005/0187206 Al, 2005). The monohydrate of Temozolomide was reported (B. Panda, G. C. Maikap, S. K. Agarwal, M. K. Singh, M. Jaggi, A. Nangia, N. J. Babu, S. Aitipamula, L. S. Reddy, WO 111092 Al, 2008). X-ray crystal structures of three TMZ polymorphs and TMZ monohydrate were determined (N. J. Babu, L. S. Reddy, S. Aitipamula, A. Nangia, Chem. Asian J. 2008, 3, 1122-1133). Patent Applications No. WO/2003/072,082 and US/2006/6,987,108 by S. Ugwu, V. Radhakrishnan, P. M. Ihnat, and L. C. Witchey-Lakshmanan, and WO/2008/140,724 by M. Abutarif, and P. Skatkevich relate to pharmaceutical formulation comprising of temozolomide and dissolution enhancing agents such as urea, L-histidine, L-threonine, L-asparagine, L-serine, L-glutamine or mixtures thereof.

Patent Application No. WO/2007/033,374 by C. B. Pickett, W. R. Bishop, and Y. Wang reported pharmaceutical composition consisting of temozolomide or its pharmaceutically acceptable salt in combination with a protein kinase C (PKC) inhibitor. Wang et. al first reported temozolomide hydrochloride by treating the API with 10 M HC1 and characterized the salt by 'H NMR and CHN analysis (Y. Wang, M. F. G. Stevens, J. Org. Chem. 1997, 62, 7288). Temozolomide free base can be obtained by treating temozolomide hydrochloride with a mixture of organic acid, a water miscible organic solvent, and water (O. Etlin, M. Alnabari, Y. Sery, E. Danon, O. Arad, J. Kaspl, US 0183898 Al, 2006). It was suggested that imidazole nitrogen atom is the preferred site of protonation in temozolomide hydrochloride salt structure (Scheme 3).

Temozolomide tablets are marketed under the brand name Temodor® in USA by Schering-Plough-Merck and Temodal® or Temorel® in India (http://www.rxlist.com/temodar-drug.htm., http://www.rellife.com/pdf/temorelpack.pdf). TMZ is obtained as a white precipitate in pure form (S. -C. Kuo, J. L. Mas, and D. Hou, US Patent No. 2002/0095036 Al, 2002). The Temodar® drug leaflet states that the "material is a white to light tan/ light pink powder". The light tan/ pink color is indicative of degradation. An improved storage system for temozolomide was developed (O. Braverman, R. Felnshtein, A. Welsman, and J. Kaspl (US Patent No. US2006/0222792 Al, 2006) to prevent drug decomposition during prolonged storage/ usage. One to three polymeric bags with an optional desiccant in an inert atmosphere were used to keep TMZ as a white solid for 4-6-10 weeks respectively. A sign of TMZ decomposition is change in color from white to pink to traces of tan color. For example, without any protection system, TMZ turned pinkish in 6 weeks but with three layers of sealed package under nitrogen atmosphere and desiccant, the drug remained white at 10 weeks duration. Temozolomide lyophilized material is white to light pink/ light tan powder.

Further, temozolomide rapidly decomposes in aqueous medium or in presence of high relative humidity. A sample containing 1.8% water decomposes faster than samples with 0.1% water over 6 months. White samples of temozolomide stored at 45% RH started to turn pink after 24 hours and the material was distinctly pink after 4 days. In about 2 months, the color of temozolomide (initially pure white) changed to light pink to light tan, suggesting degradation of the API (O. Braverman, R. Felnshtein, A. Welsman, and J. Kaspl, US Patent No. US2006/0222792 Al, 2006). Temozolomide starts to degrade when crystallized from polar protic solvents, e.g. water, methanol, ethanol etc. There is thus an urgent need for an improved solid-state form of temozolomide exhibiting superior drug stability. In view of the apparent tendency of temozolomide to degrade, as is evident by a change in its color, Temozolomide cocrystal with pharmaceutically acceptable carboxylic acids were developed which have higher stability compared to the parent drug (A. Nangia, N. J. Babu, P. Sanphui, Indian patent application No. 2303/CHE/2009.; N. J. Babu, P. Sanphui, A. Nangia, Chem. Asian J. 2012, DOI: 10.1002/asia.201200205).

Salt formation is a usefiil method for isolation and purification of substances. The formation of a pharmaceutical salt can modify the physicochemical as well as the biological properties of an ionizable drug, which cannot be predicted from the properties of the parent drug and of the counter ion. Salt formation influences many properties of pharmaceuticals such as solubility, dissolution rate, melting point, hygroscopicity, chemical stability, solution pH, crystal form, and mechanical properties (R. Banerjee, P. M. Bhatt, N. V. Ravindra, and G. R. Desiraju, Cryst. Growth Des., 2005, 5, 2999; A. Portell, R. Barbas, M. Font-Bardia, P. Dalmases, R. Prohens and C. Puigjaner, CrystEngComm, 2009, 11, 791). It is estimated that half of the drugs in the market are administered in the salt form and hydrochloride is a preferred salt form (G. S. Paulekuhn, J. B. Dressman C. Saal, J. Med. Chem. 2007, 50,6665). Even though TMZ contains ionizable functional groups such as imidazole N and tetrazinone N in the ring structure, only hydrochloride salt is mentioned in publications (E. S. Newlands, M. F. Stevens, S. R. Wedge, R. T.

Wheelhouse, C. Brock, Cancer Treat. Rev. 1997, 23, 35-61). However, the hydrochloride salt of Temozolomide is unstable and dissociated to free temozolomide base (Y. Wang, M. F. G. Stevens, J. Org. Chem. 1997, 62, 7288). Salt forms of APIs are generally preferred in the pharmaceutical industry and drug formulation due to their better stability and solubility and filterability, and furthermore the fact that Temozolomide is stabilized at acidic pH, the present inventors report novel crystalline hydrate form of temozolomide salt, specifically temozolomide hydrochloride.

OBJECT OF THE INVENTION:

It is thus the object of the present invention to provide a novel hydrate form of hydrochloride salt of Temozolomide having improved stability/solubility.

SUMMARY OF THE INVENTION:

In accordance with the above objective a novel hydrate form of Temozolomide specifically temozolomide hydrochloride having improved stability/solubility, is disclosed. In an aspect, the present invention discloses crystallization process for the preparation of novel crystalline hydrate of temozolomide hydrochloride. The novel salt of temozolomide hydrochloride (TMZ HC1) dihydrate is characterized by single crystal X-ray diffraction (SC-XRD), powder X-ray diffraction (PXRD), IR spectroscopy, and stability is assessed by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) etc.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts PXRD of TMZ HC1 dihydrate.

Figure 2 depicts DSC endotherm of TMZ HC1 dihydrate.

Figure 3 depicts TGA of TMZ HC1 dihydrate.

Figure 4 depicts FT-IR spectrum of TMZ HC1 dihydrate.

Figure 5 depicts the (a) ORTEP diagram of TMZ HC1 dihydrate with 35% probability of thermal ellipsoid for heavy atoms and (b) hydrogen bonding interactions in the crystal structure.

Figure 6 depicts overlay of experimental PXRD of TMZ HC1 dihydrate (black trace) on the calculated lines from the X-ray crystal structure (grey trace).

DETAILED DESCRIPTION OF THE INVENTION:

Accordingly, a novel salt of antitumor prodrug temozolomide is disclosed in the present invention, temozolomide hydrochloride in crystalline hydrate form and the process of crystallization thereof. The salt of Temozolomide hydrochloride has dihydrate stoichiometry in the bulk material, i.e. 2 water molecules per TMZ HC1 salt. Temozolomide hydrochloride in crystalline hydrated form is established by single crystal X-ray structure determination (SC-XRD), crystal structure analysis, powder X-ray diffraction (PXRD), infrared spectroscopy (IR), and differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) as detailed herein below.

Typically the novel salt of Temozolomide in crystalline hydrated form is prepared by crystallization of pure Temozolomide from a mixture of water and acid, particularly, in a proportion of 1: 25 w/w drug: cone, hydrochloric acid. In an embodiment, the present invention discloses the preparation of crystalline TMZ HC1 salt dihydrate. Accordingly, 150 mg of freshly purified TMZ (white color) is dissolved in 3 mL cone. HC1 (36.5% by weight in water) by slight sonication in a 100 mL beaker. The mixture is kept for 1 day at room temperature when few plate morphology crystals appear in the flask which is characterized as TMZ HC1 dihydrate.

The TMZ HC1 dihydrate of the present invention is characterized by powder X-ray diffraction depicted in Figure 1 exhibited characteristic peaks at about 26 5.28, 9.69, 14.15, 14.41,18.91,20.40,28.32, 28.61, and 35.57 ±0.2° indicated below in Table 1. Differential scanning calorimetry (DSC) curve of TMZ HC1 dihydrate indicates loss of water from the crystal at 114.6 °C endotherm (Tpeak), followed by sharp exotherm at 184.3 °C (Tpeak) due to decomposition/dissociation of salts as TMZ (Tdecomp 210 °C). The DSC curve is given in Figure 2.

The TMZ HC1 dihydrate is characterized by having a water content ranging from 13 to 14%. The TMZ HC1 dihydrate is further characterized by TGA which indicates the percentage of water content in the sample is 6.57%, which is about half of the calculated water percentage of 13.65% for two water molecules as given in Figure 3. The infrared absorption spectra of TMZ HC1 dihydrate is represented in Figure 4 and in Table 2 below. The characteristic peaks are observed at 3588.6, 3438.5, 3123.1, 1742.1, 1678.9, 1358.0 and 1216.5 cm-1.

The quality of the single crystals TMZ HC1 dihydrate obtained were of poor quality, and confirmed to be twinned by single crystal X-ray diffraction. Reflections were collected on a single crystal of TMZ HC1 dihydrate at 100 K and the structure was solved and refined in triclinic space group P-l with.R-factor 0.1282. All heavy atoms were located from difference electron density Fourier maps. The stoichiometry of TMZ HC1 dihydrate is 2 TMZ, 2 HC1 and 4 H20 molecules in the crystal structure. The asymmetric unit contains one neutral TMZ, one protonated TMZ, one hydronium ion (protonated water), three water molecules and two chloride ions. There are 6 nitrogen atoms in each TMZ molecule and out of which 3 nitrogen atoms can accept protons. In the crystal structure, symmetry independent molecule 2 (TMZ-2) is protonated at imidazole N8 whereas symmetry independent TMZ molecule 1 (TMZ-1) is not protonated. Although the protons on the water molecules could not be located from difference electron density maps, the hydrogen bonding patterns in the X-ray crystal structure suggest that the most probable position for second proton transfer from HC1 is to a water molecule (e.g. 05, 06, 07, or delocalized in the hydrogen bond network of water molecules) rather than N atom in TMZ-1. The single crystal X-ray diffraction pattern is represented in Figure 5.

The bulk material of Temozolomide hydrochloride dihydrate as crystallized from cone. HC1 matches with the single crystal phase confirmed by the excellent match of experimental (black) and calculated (grey) XRD line pattern depicted in Figure 6. In yet another embodiment, there is provided a pharmaceutical composition comprising TMZ hydrochloride salt in crystalline hydrated forms (dihydrate) and pharmaceutically acceptable excipients such as diluents, fillers, disintegrants, binding agents, flavoring agents and the like. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art. The pharmaceutical compositions prepared with the crystalline salt dihydrate of temozolomide are used in the treatment of cancer. Pharmaceutical compositions suitable for use in context of the present invention include compositions where the active ingredients are contained in an amount effective to achieve the intended purpose. The pharmaceutical composition may be administered in the form of oral solid, liquid or in parental dosage forms. The solid oral dosage forms include tablets or capsules. The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1

Preparation And Crystallization Of Novel Dihydrate Salt Of Tmz Hcl Temozolomide was procured from commercial suppliers (GLS Pharma Ltd, New Delhi, India) and used as such. This solid corresponds to essentially pure form I characterized by lH NMR spectroscopy and PXRD match, as described in P. R Lowe, C. E. Sansom, C. H. Schwalbe, M. F. G. Stevens, and A. S. Clark, J. Med. Chem., 1992, 35, 3377. The salt of TMZ was prepared by dissolving 150 mg of fresh TMZ (white color) in 3 mL cone. HC1 (36.5% by weight in water) by slight sonication for 1 min in a 100 mL beaker. Grinding of the slurry and leaving the mixture at room temperature for slow evaporation gave crystals of TMZ HC1 dihydrate.

Example 2

Solid State characterization by powder X-ray diffraction Powder X-ray diffraction is a standard method for the characterization of new solid forms (Selective Polymorph Transformation via Solvent-drop Grinding. A. V. Trask, N. Shan, W. D. S. Motherwell, W. Jones, S. Feng, R. B. H. Tan and K. J. Carpenter, Chem. Commun., 2005, 880-882; Crystal engineering of Organic Cocrystals by the Solid-state Grinding Approach. A. V. Trask and W. Jones, Top. Curr. Chem., 2005, 254, 41-70). The formation of TMZ HC1 dihydrate was monitored by the appearance of new X-ray diffraction peaks. PXRD of the new solid form was recorded on SMART Bruker D8 Advanced Powder X-ray diffractometer using Cu-Ka radiation (X = 1.5406 A) at 40 kV and 30 mA. The powder X-ray diffraction of temozolomide hydrochloride dihydrate (Figure 1) exhibited characteristic peaks at about 29 5.28, 9.69,18.91,20.40,28.32, and 28.61 ±0.2° (Table 1). Table 1 Powder X-ray diffraction data of TMZ hydrochloride dihydrate characterized by 20 angle (°), d values (A), and relative intensity (%).

Example 3 Thermal analysis

Differential scanning calorimetry (DSC) was carried out to investigate the thermal behavior of temozolomide hydrochloride dihydrate. DSC measures the heat changes in the material. It is reflected as an endotherm or exotherm in the DSC scan depending on whether the sample uptakes or releases the heat. Melting phenomenon and water loss are normally indicated by the presence of an endotherm, while the crystallization and decomposition phenomenon are indicated by the presence of exotherm. DSC of new solid phase was carried out on Mettler Toledo DSC 822e module by placing the samples, typically 4-6 mg, in aluminum pans and it was heated in the temperature range of 30-250 °C at the rate of 5 °C /min. DSC of temozolomide hydrochloride dihydrate (Figure 2) indicates loss of water from the crystal at 114.6 °C (Tpeak), followed by sharp exotherm at 184.3 °C (Tpeak) may be because of decomposition/dissociation of TMZ in the salts (Tdecomp 210 °C).

TGA was performed on Mettler Toledo TGA/SDTA 85 le module by placing the sample, typically 9-12 mg, in an open alumina pan and it was heated in the temperature range 30-250 °C at 10 °C /min. TGA of temozolomide hydrochloride dihydrate (Figure 3) was recorded to know the water percentage in the sample. TGA indicates the percentage of water content in the sample is 6.57%, which is about half of the calculated water percentage of 13.65% for two water molecules. Karl Fischer titration also indicated the moisture content in the sample is 6.6%. Both TGA and Karl Fischer titration suggest that there is one free water molecule (H2O) and the second water is bound as hydronium ion (H30+) in the crystal lattice of TMZ hydrochloride dihydrate.

Example 4

FT-IR spectroscopy analysis ■ Infrared spectroscopy (R. M. Silverstein, Spectrometric Identification of Organic Compounds. 6th Ed. John Wiley & Sons, Inc. 2002, pp. 71-143) provides information on the vibrational modes of a compound. This is an absorption phenomenon. IR spectra were recorded on samples dispersed in KBr pellets on a Nicolet 6700 FT-IR spectrometer. In general, IR energy is absorbed by polar functional groups, such as amide carbonyl C=0 stretch at 1679 cm-1, amide N-H stretch at 3421 cm-1 (asymmetric) and 3388 cm-1 (symmetric) for Temozolomide. Broad peak at 3589 cm" 1 corresponds to O-H stretch of water molecule present in salt hydrate. Amide N-H stretch frequency was blue shifted from 3422 (pure TMZ) to 3438 cm-1 due to protonation of imidazole moiety in one of temozolomide molecule. Again amide carbonyl stretching frequency is red shifted from 1758 (pure TMZ) to 1742 cm-1 due to hydrogen bonding with water molecule. FT-IR spectrum of TMZ HC1 dihydrate is shown in Figure 4. The characteristic frequencies of pure TMZ, TMZ monohydrate and TMZ hydrochloride dihydrate are compared in Table 2. Table 2 FT-IR vbar (cm-1) of TMZ and TMZ HC1 salt dihydrate.

Example 5

Single crystal X-ray diffraction Single crystals obtained from cone. HC1 were mounted on the goniometer of Bruker SMART CCD diffractometer equipped with Mo-Ka radiation (k = 0.71073 A) source. Data collection (Bruker SMART), cell refinement (Bruker SMART), data reduction (Bruker SAINT) program(s) distributed by Bruker AXS Inc. were used to solve the crystal structure. Structure refinement was carried out using SHELXS97 (G. M. Sheldrick, 1990; G. M. Sheldrick, 1997) program(s), Molecular graphics were plotted in Bruker SHELXTL, software to prepare material for publication. The quality of single crystals was poor and twinned but the quality of crystallographic data is good enough to confirm the structure as temozolomide hydrochloride dihydrate. Crystal structure refinements were done using F2 intensity of ALL the reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2, conventional R-factors (R) are based on F, with F set to zero for negative F2.

The threshold expression of F2 > 2sigma (F2) is used only for calculating R-factors (gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. The Oakridge thermal ellipsoid plots (ORTEP) were drawn at 35% probability of electron density for the heavy atoms and hydrogen bonding pattern are displayed (Figure 5). Crystallographic data are summarized in Table 3. All estimated standard uncertainties (esu's) were estimated using the full covariance matrix. The cell esu's are taken into account individually in the estimation of esu's for distances, angles and torsion angles; correlations between esu's in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esu's is used for estimating esu's involving l.s. planes.

The bulk material of Temozolomide hydrochloride dihydrate as crystallized from cone. HC1 matches with the single crystal phase confirmed by the excellent match of experimental (black) and calculated (grey) XRD line pattern (Figure 6). Table 3 Crystallographic parameters for Temozolomide hydrochloride dehydrate 2TMZ.2HC1. 4H20 (1:1:2) Empirical formula '2 H atoms of one TMZ NH2, 1 H atom of one HCl, and 4 H atoms of four H20 are excluded' (C6H6N602) (C6H4N602). (HCl) (CI). 4(HO) Formula weight 526.26 Crystal system Triclinic
Mo-Ka X (A) 0.71073
T(K) 100(2)
Space group P-1
a(A) 6.428(13)
b(A) 10.35(2)
c(A) 17.81(4)
a(°) 97.50(3)
P(°) 90.39(3)
y(°) 98.06(3)
V(A0 1162(4)
D (g cm"3) 1.503
u. (mm") 0.344
0 range 2.18 to 23.26
Z 2
range h -7 to +7
range k -llto+11
range 1 -19 to+19
reflections collected 8929
observed reflections 3321
total reflections 2238
R, [I>2CT(I)] 0.1249
wR2 (all) 0.3336
goodness-of-fit 1.172 14

WE CLAIM,

1. Crystalline Temozolomide HC1 dehydrate characterized by XRD having characteristic peaks at about 29 5.28, 9.69, 14.15, 14.41, 18.91, 20.40, 28.32, 28.61, and 35.57 ±0.2°.

2. The crystalline Temozolomide HC1 dehydrate according to claim 1, wherein the differential scanning calorimetric thermogram of Temozolomide HC1 dehydrate exhibits endotherm (Tpeak) at 114.6 °C followed by sharp exotherm at 184.3 °C(Tpeak).

3. The crystalline Temozolomide HC1 dehydrate according to claim 1, characterized by having an infrared spectrum with peaks indicated around at 3588.6, 3438.5, 3123.1, 1742.1, 1678.9,1358.0 and 1216.5 cm-1.

4. The crystalline Temozolomide HC1 dehydrate according to claim 1, characterized by having a water content ranging from 13 to 14%.

5. A process for the preparation of crystalline Temozolomide HC1 dehydrate comprising crystallizing pure Temozolomide from a mixture of water and acid, particularly, in a proportion of 1: 25 w/w drug: cone, hydrochloric acid.

6. The process for the preparation of crystalline Temozolomide HC1 dehydrate according to claim 5, comprising dissolving Temozolomide in cone. HC1 by slight sonication for 1 min followed by grinding of the slurry and leaving the mixture at room temperature for slow evaporation to yield crystals of TMZ HC1 dihydrate.

7. A pharmaceutical composition comprising crystalline Temozolomide HC1 dehydrate according to claim 1, and a pharmaceutically acceptable carrier that may be formulated in the form of oral solid, liquid or in parental dosage forms.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier is selected from diluents, fillers, disintegrants, binding agents, flavoring agents and the like.