AU2016202982A1 - Crystalline methylthionium chloride (methylene blue) hydrates - Google Patents

Abstract

Abstract Three dihydrate forms B, C and D and a monohydrate form E of methylthioninium chloride are described. Forms B, C, D and E can be prepared under controlled humidity and temperature from methylthioninium chloride with higher water content or conversion of a hydrate. The hydrates can be incorporated in pharmaceutical compositions.

Description

Patents Act 1990 WISTA LABOR TORIES L TD COMPLETE SPECIFICATION STANDARD PATENT Ti/le: Crystaline methyhidoni chloride (methyvene blue) hydrates The following statement is a ful description of this invention including the best method of performing it known to us:- 1A CRYSTALLINE METHYLTHIONINIUM CHLORiDE HYDRATES The present invention relates to crystalne methythioninium chlode hydrates, in particuar 5 methylthioninium chloride dihydrate form B, methlthioninium chlide dihydrate form C, methylthionnium chloride dhydrate form D and methylthoniurn chloride monohydrate form F; the processes for the preparation of forms B, C. D and E; and to preferably pharmaceutica corposions comprising forms B. C, D and E. O Methylthionniurn chloride (MTC) [Methylene Blue: 3.7-bisdimethylaminophenazothionium chloride, C 1 sHoClN 3 S, 319.5 g/mol] was prepared for the first time in 1876 (The Merck index, 13" edition, Merck & Co, Inc, 2001, entry 6085) Various synthesis methods are known and have recently been summarized in WO 2006/032879. WO 2006/032879 also states a number of appKications of methylene blue which include the use as a medical dye, 15 as a redox indicator, an antiseptic, for the treatment and prevention of kidney stones, the treatment of melanoma, malaria, viral infections, and Alzheimer's disease. MTC has also been used as an oxidizing agent and as an antidote in the case of CO, nitrite and aniline poisoning. 20 NMT is known to exist in the form of hydrates For examplethe Fka catalogue states in very general terms that MTC May contain uP to 22% water [luk Caalogue 1997/1998, Fluka Chemie AG, 1997 Structures with from one to fve molecules of water have been formulated in the literature [.O. Warwicker J. Chem Soc. (955) 253 G.F Davidson, J. Textile institute 38 (1947) T408418 ]. he formation of a trihydrate has apparently found 25 widespread acceptance [e, The Merck index ediion, Merck & Co.. Inc. 2001, entry 6085]. However, this claim was aleady disputed more than 80 years ago, and the non specific adsorpton of water by M TCwas proposed instead Wales .A Nelson, 2. Am, Chem. Soc. 45 (1923) 1657; CM. Martin, J.W.G. Neuhaus, F.H. Reuter, Analyst 71 (1946) 29-31]. 30 To date, the only hydrate that has been characterized in detail is a pentahnydrate of MC [-O. Warwie. Chem. Soc (1955) 2531 E Man il J.M. Stewart, M.F. Chiu Aca Crys 829 (1973) 847. For this hydrate, even single crystal X-ray data are avaable. consists o backed columns of mehylthioninium nations that ae arranged in planes 35 perpendicular to the as of the crystal The water molecues and chloride ions are located between these layers, whereby the chloride ions are concentrated in planes almost 2 perpendicular to the water planes and parallel to the axis of the columns. The chloride ions are coordinated with three hydrogen bonds from 3/2 water molecules. Presumably the same structure was earlier attributed to a tetrahydrate [WH Taylor. ZKnst 5 91(1935) 450. A phase mansion between te enahydrate and a second polymorphic form w ,as described to occu near 33Qin aqueou--s suspension ,IS.W. Bodman,: SRP. Kodama, PAC PfeR E. Stevens, J Che. Er. Data 12(1967) 500 The second form was also obtained by vacuum drying of the pentanydrate at room temperature, and itswate cotentas indicated to amount to approximately 1 mol/mol iC The solid state form of a compound is of great iportance for pharmaceutical applications. It may influence the chemical and physical stability of the compound itself and of its formulatons.may have an impact on pharmacokinetics and bioavaabity the case of hydrates, the composidon has also an influence on the correct dosage of the active 15 pharmaceutical ingredient. Methythioium chbride used in pharmaceutical composions is described as a trihydrate (USP Material Safety Data Sheet for Methylene Blue (Catalogue Number 1428008), 2005), wh is thought be methylthioninium chloride pentahydrate admixed with other 20 components he mixture or components in the mature are stable under different crition they may convert to other polymorphic or pseudopolymorphic species and hence change thei composition so that corect dosage is a problem and storage stability may be considered to be insufficient. 25 The present invention provides specific polymorphic forms of methyithioninium chloride hydrates, as well as safe and reproducible processes for their preparation. The present invention also provides specific polymorphic forms of methylthioninium chloride hydrates, which are stable under defined conditions, and which have good solubility and bioavaliability The present invention provides specific polymorphic forms of methylthioninium chloride 30 hydrates. which can be easily metered to arrive at defined contents in pharmaceutical compositions in order to administer exact amounts of the active compound. A first aspect of the present invention is MTC substantially in crystalline Form B of rnethylthioninium chloride dihydrate, 35 3 In some embodiments. Form B is not exactly a dihydrate, but may contain a small amount of water (for example, -0.2-0.3 equivalents) in excess of the dihydrate. However, for convenience it is referred to herein as crystaline Form B of methyithioninium chloride dydra e. Crystalline Form B of methylthioninium chloride dihydrate has a X-ray powder diffraction pattern (wavelength 1.54180 A) containing specific peaks at the following 20 values (t 0.1 ): 5.8 112, 25.3 26.8. 10 Crystalline Form B of methylthioninium chloride dihydrate may also have the following additional peaks in a X-ray powder diffraction pattern at the following 20 values (I 0.1*): 15.6, 169. 23, 283 Crystalline Form B of methylthioninium chloride dehydrate may also be characterized by any 15 combination of three or more peaks selected from the list of 8 peaks above, with a preference given to peaks at low angles. A representative powder XRD pattern of crystalline methylthioninium chloride dihydrate Form B is shown iri Figure 1. Without wishing to be bound by theory, Form B is a thermodynamically metastable form ai room temperature over the whole range of relative humdity PowderX-ray and DSC indicate the crystane character of form B. Thermogravimetry (TG, heating rate 10'C/nin) results in a wateross of 10.6% or of 10.9 to 1,5% between room temperature and 150 O0, 25 corresponding to a water contend of about 2, for example 2.2-2.3 water molecules per molecule methylene blue. The TG analysis enables Form B to be distinguished fromForms A and E, Form B may also be characterized using Differential Scanning Calorimetry (DSC). When 30 subject to DSC, with a heating rate 1004C/min in a gold crucible, Form B has a melting peak at 186*C with a shoulder towards lower temperature. When subject to DSC, with a heating rate of 20*C/min in a gold crucible, Form B exhibits a small endothermic peak near 100 "C and a melting peak at 183C with a shoulder towards lower temperature. The melting peak is immediately followed by decomposition. 35 4 Form B may also be characterized using attenuated totai reflection infrared (ATRiR) spectroscopy. Characteristic IR signals of form B are found at 1068. 877, and 839 cm The crystalline Form B is obtained as a greenish powder. 5 Methylthioninium chloride dehydrate Form B is soluble in methanol and acetic acid and possesses a low to moderate solubiNy in water hydrochlrcacd and other organic solvents. ts solubiity is simar to that of methythioniniurn chloride pentahyd rte Form A. 10 An other aspect of the present invention is a process for te preparation of Form B, which comprises d g of solid Form A methyithioniium chloride pentahydrate at an elevated temperahre and low humidity. The temperature is preferably at least 4U or even 50CC, and may be less han 70C- In preferred embodiments, the temperature is about 60C. The humidity is preferably below40% r h, and is more preferably about 35% r h., or lower. The 15 drying should continue fo.r sufficient time to achieve conversion to Form B. A further aspect of the present invention is a process for the preparation of crystaline methylthioninium chloride dehydrate Form B, which comprises exposing solid methylthioniniunm chloride pentahydrate Form A at ambient temperature to an inert gas flow 10 having a relative humidity from 8 to 15%. The relative humidity is preferably from 9 to 12% at room temperature. Examples for inert gases are air. nitrogen, helium, neon, argon and krypton, or mixtures thereof, The solid methylthioninium chloride pentahydrate Form A is preferably in the form of a crystalline 25 powder, which may be agitated to accelerate the drying operation. The exposure time depends on the amount of methylthioninium chloride pentahydrate Form A and may range from hours to several weeks. Ambient temperature may mean a temperature from 15 to 304C and preferably 20 to 2500. The present inventors have also found that methyithioninium chloride exists in at least two further crystalline dihydrate forms, hereinafter called forms C and D. A further aspect of the present invention is a crystalline Form C of methylthioninium chloride 35 dihydrate.

5 Crystalline Form C of methylthioninium chloride dehydrate has a characteristic X-ray powder diffraction pattern containing specific peaks at the following 20 values (± 0.10): 8.1, 11.1. 17 6, 25.9 27.2. 5 Crystalline Form C of methylthioninium chloride dihydrate may also have the following additional peaks in a X-ray powder diffraction pattern at the following 20 values (± 0.10): 16.2, 17,8, 244 308, 31.3, 33.0. Crystaline Form C of methythioninium chloride dhydrate may also have the folkwng further 0 peaks in a X-ay powder diffraction pattern at the flowing 28 values (± ON 13. 1844 27 295 30., 34.1. ., 367 39.5, 42.7, 45,3: 48.0 Crystalline Form C of methylthioninium chloride dihydrate may also be characterized by any combination of five or more peaks selected from the list of 23 peaks above, with a preference iS given to peaks at low angles. A representave powder XRD pattern of crystalline methylthioninium chloride dihydrate Form Cis shown in Figure 2. O0 V without wishing to be bound by theory, Form C is the thermodynamically stable form at room temperature and a relative humidity of less than 40% and down to about 10%, or possil even dowAn to 4%This broad range of thermodynamic stability (compared to Forms B, D or which n addition broadens at higher temperature, rnakes For C the form of choice for preparatior processes stage or use above temperatures of 25'C, Powder X-ray diffraction 25 and DSC indicate the crystaline character offormi Therrogravimetry (TG), with a heating rate of 0*C/minindicates a watr loss of about 98 to 114% between roomtemperature and 150 "C corresponding to a water content of about 1.9 to 2.3 watermolecules per molecule methylene blue. In preferred embodiments, thermogravimetry with a heating rate 10CImin indicated a water loss of about 98 to 10.% between room temperture and 150 30 C in two steps. The total water oss correspondsto a water contertof almost exactly two water molecules per molecule methylene blue The presence of two steps is characteristic of the TG profile of Form C, The TG analysis enables Fom C to be distinguished fomForms A and E.

3 Form C may also be characterized using Differential Scanning Calorimetry (DSC). When subject to DSC, with a heating rate of 100C/min in a gold crucible, Form C has two endothermic maxima at 151*C and 183*C. $ Form C may also be characterized using attenuated total reflection infrared (ATR-IR) spectroscopy. Characteristic IR signals of form C are found at 1497/1483 (double peak). 1438. 1301, and 1060 cm' The crystalline form C is obtained as a greenish powder with a golden luster. A further aspect of the invention is a process for he preparation of Form Cby re ystallization of water containing meththioninium chloride or specific hydrates from dimethylsuifoxide. 15 Form C can also be prepared by suspension eq uiibration of Forms A or B or other polymorphic forms in acetonitrile or isopropanol in the presence of small amounts of water herefore, another aspect of the present invention is a process fo the preparation of nethylthioninium chloride dehydrate Form C, wherein a water containing methylthioninium chloride or a mixture of various hydrates or a specific hydrate of methyltioninium lr is M0 suspended and stirred at ambient temperature in a solvent selected from the group comprisrig isopropanol 1propano, 1-butano 2-butano tertbutanol, tetrahydrofurane dioxane, acetone, 2-butanone and acetonrle , or mixtures thereof, containing a small amount of water. 25 The selected organic solvents including mixtures of at least two solvent preferably possess a poor solubility for MTC dihydrate Form C at the temperature of isolation of this crystaline product, which is typically at room temperature or below. A solubility of less than 20 g/l and in particular less than 2 g/l at room temperature is preferred. The solvent is miscible with water, and its vapor pressure preferably exceeds the one of water. 30 The amount of hydrates in the suspension may be from i to 70%, preferably from 5 to 60%, more preferably from 5 to 50% and particularly preferred from 10 to 40% by weight, referred to the amount of solvent, Ambient temperature may mean a temperature from 1$ to 300C and preferably 20 to 25 C. 35 The appropriate small amount of water depends on the amount of water already provided by the methylthioninium chloride hydrates added initially, the concentration of methylthioninium chloride in the suspension, and the water activity in the chosen solvent as a function of water content. Aen conducted at roon temperature, the after content at the end of the 5 transformation process has to correspond to a water activity between 0.04 and 0.4, preferably 0.1 and 0. 3 (corresponding to 4 to 40 respectively 10 to 30% relative humidity). The treatment should be long enough for conversion of the other forms into Form C, The treatment time manly depends on the amount of soid in the suspension and the composition .0 of the starting material and may be from hours to several days. Following conversion into Form C the soldmay be isolated Isolating of the solid is carried out by filtton Foowing isolation, solvent may be removed front Form C, Removal of solvent may be carried out in vacuum and at a temperate below 100"C, preferabdy below i5 50%, and most preferred lose to roon temperature Aleratvely a gas flow with a relative umidywhic corresponds to the stability range of the hydrae, maybe passed overthe sample for drying. A further aspect object of the present invention is crystalline Form D of methylthioninium ?0 chloride dehydrate. Crystalline Form D of methylthioninium chloride dihydrate has a X-ray powder diffraction pattern containing specific peaks at the following 29 values (± 0.1: 7.0, 8.5, 12 0, 14.4, 25.3, 25,7, 27.5. 25 Crystalline Form D of methylthioninium chloride dihydrate may also have the following additional peaks in a X-ray powder diffraction pattern at the following 29 values (±0.1 0): 6.0, 10.4, 20.9, 21.1, 21.7 22 3, 23.7, 24.5. 26.9, 28.5. 29.0, 30.4 31.8, 30 Crystalline Form D of methylthioninium chloride dehydrate may also have the following further peaks in a X-ray powder diffraction pattern at the following 29 values ( 01): 9.8, 16.3, 17.1, 18.1, 34.9, 41.5, 46.5. Crystalline Form D of methylthioninium chloride dihydrate may also be characterized by any 35 combination of five or more peaks selected from the list of 27 peaks above, with a preference given to peaks at low angles.

S A representative powder XRD pattern of crystalline methylthioniniurm chloride dihydrate Form D is shown in Figure 3. 5 Without wishing to be bound by theory, Form D is thermodynamically metastable at room temperature and over the whole range of relative humidity. Powder X-ray diffraction and DSC indicate the crystalline character of form D. Thermogravimetry (TG, heating rate 10 C/min) results in a water loss of about 9.3 to 11 .2% between room temperature and 150 *C, corresponding to a water content of about 1.9 to 2.3 water molecules per molecule 0 methylene blue. The TG analysis enables Form D to be distinguished from Forms A and E Fori D may also be characterized using Differential Scanning Calorimetry(DSC) When subject to DS, with a heating rate 10 0 0/min in a gold crucible, Form D has two encothermic peak maxima at 64CC and 18C and a step in the baseline is observed near 5 63"Ce Form D may also be characterized using attenuated total reflection infrared (ATR-IR) spectroscopy. Characteristic IR signals of form D are found at 1181, 1140, 1066, 951, and 831 cm' 1 The crystalline form D is obtained as a grey to violet powder. re form D can be prepared by precipitation processes such as the addition of a solution in a good solvent to a large excess of a non-solvent. Accordngly, a further aspect of the 25 invention is a process for the preparation of methylininuo chloride dihydrate Form D. comprising dissolving methylthioninium chlorde pentahydrate Form A in methanol and combining the soltion with tkbutylmethyl ether enher by adding butyklmethyl ether to the methanoic solution or by adding the methanolic sodui to t-butyl methyl ether. 30 A further aspect of the invention is a process for the preparation of essentially pure methylthioninium chloride dehydrate Form 0, comprising dissolving methylthioninium chloride pentahydrate Form A in acetic acid and combining the solution with toluene, either by adding toluene to the acetic acid solution or by adding the acetic acid solution to toluene, 35 The concentration of Form A in the methano or acetic acid solution may range from 1 to 30% by weight and preferably from 5 to 20% by weight, based on the amount of methanol or 0 acetic acid The amount of t-buty!-methyl ether or toluene may be equal to the volume of methanol or acetic acid, but preferably exceeds this volume by at least a factor of 5, more preferably by a factor of "10 5 After precipitation of Form D in either of the above two methods, the solid may be isolated by filtration. After isolation the solvent may be removed from Form D. The solvent is removed by vacuum drying or in an inert gas flow whereby the relative air humidity in all process steps is less than 50% and preferably less than 40% 0 The present inventors have also found that methylene blue forms a crystalline monohydrate. A further aspect of the present invention is crystaline Form E methylthioinium chloride monohydrate. 15 Crystalline Form E methylthioninium chloride monohydrate has a characteristic X-ray powder diffraction pattern containing specific peaks at the following 20 values (± 0,1*): 9.0, 12.5, 14. 14.4 18 1,232, 24.1 26. Crystalline Form E methylthioninium chloride monohydrate may also have the following 0 additional peaks in a X-ray powder diffraction pattern at the following 26 values (± 0,1*)24 5. 27.2, Crystalline Forr E methylthionrni chloride monohydrate may also have the following further peaks in a Xry powder diffraction pattern at the following 20 values 0. 2 25 221, 284, 29., 32, 393, 417, 41 Crystalline Form E methylthioninium chloride monohydrate may also he characterized by any combination of five or more peaks selected from the list of 18 peaks above, with a preference given to peaks at low angles. 30 A representative powder XRD pattern of crystalline methylthioninium chloride monohydrate Form E is shown in Figure 4. Without wishing to be bound by theory, Form E is thermodynamically stable at room 35 temperature at a relative humidity of less than about 10%, or less than about 4%, and down to about 2%. Povder X-ray diffraction indicates the crystalline character of form E.

10 Thermogravimetry (TG, heating rate 1 0 "Cmin) results in a water loss of 5.1% to 5.4% between room temperature and 1 100C, corresponding to a water content of one water molecule per molecule methylene blue TG can be used to distinguish Form E from forms A, B. C and D. 5 Form E may also be characterized using Differential Scanning Calorimetry (DSC). When subject to DSC, with a heating rate of 1004C/min in a gold crucible, Form E shows no thermal event up to the decomposition temperature near 220C, .0 Form E may also be characterized using attenuated total rejection infrared (ATRR) spectroscopy. Characteristic IR signals of form E are found at 1350, 1323, 1242, 1218, 1175, 1134, and 1035 cm'. The crystalline form E is obtained as an ocher colored powder. 15 Pure forn E can be prepared by suspension equilbration of water containing miethylthioninium chloride or forms A, B, C or D or mixtures thereof under dry conditions in a solvent. Suitable sakents include those used in the suspension equilibralion for the preparation of methylthoninium chloride dihydrate form C namely isopropano. 1prpanol, ?0 '1-butanol, 2butanol, 2nethyh2buAno tetrahydrofurane. dioxane acetone, 2butanone and acetonitre Accordingly another aspect of the present invention is a process for the preparation of crystaline methylthionirnum chloride rmonohydrate form E, wherw water containing methylthoniniur chloride or a mixture of various hydrates ora spefcc hydrate of methyhoninium chloride is suspended and stirred at ambient temperaure in a 25 dry solvent, preferaby isopropano. The amount of hydrates in the suspension may be from 1 to 70% preferably from 5 to 60%, more preferably from 5 to 50% and particularly preferred from 10 to 40% by weight, referred to the amount of non-solvent. Ambient temperature may mean a temperature from 15 to 30 35"Q and preferably 20 to 354C. A temperature cycle from 20 to 3500 within for example 30 minutes may be applied to facilitate water removal. Dry isopropanol means a water content of less than 1% by weight in isopropanol, preferably less than 0.1% by weight. The treatment time shoud be sufficient to lslow for conversion to Form E The treatment 35 timeanaly ,depends on the amount of solid in the suspension and may be from hours to 11 several weeks. After an apropriate equilibration time, the solvent may have to be removed and be replaced by new, dry solvent in order to keep the water content low, After formation of Form E, the solid may be isolated, Isolating of the solid is carried out by 5 filtration. After isolation of the solid, solvent may be removed from Form E. Removal of solvent may be carried out in vacuo and at a temperature below 100*C, preferably below 50'C, and most preferred close to room temperature. Alternatively, a gas flow with a relative humidity, which corresponds to the stability range of the hydrate, may be passed over the sample for drying. Purity In each of the above aspects, methylthioninium chloride is preferably substantially in the Form described. "Substantially in the Form described" means that at least 50% by weight of methylthioninium chloride is in the Form described, preferably at least 70% by weight, 80% 15 or 90% by weight, In some embodiments, at least 95% by weight, 99% by weight or even 99.5% or more by weight may be in the crystalline form described. In each of the above aspects methylthioninium chloride is preferably substantially free from solvent. The term substantially free from solvent" as used herein refers to the form having 20 only insignificant amounts of any solvent e.g. a form with a total of 0.5% by weight or less of any solvent, The total amount of any solvent may be 0.25%, 0.1%, 0.05% or 0.025% by weight or less. Compositons 25 One aspect of the present invention pertains to compositions comprising methylthioninium chloride dehydrate Form B, C or D or methylthioninium chloride monohydrate Form E. as described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable 30 carrier, diluent or excipient, Methods of inactivating pathonens One aspect of the present invention pertains to use of methylthioninium chloride dihydrate Form B. C or D or methylhioninium chloride monohydrate Form E, as described herein, in a 35 method of inactivating a pathogen in a sample (for example a blood or plasma sample), the 12 method comprising introducing the compound into the sample, and exposing the sample to light. Methods of Medical Treatment 5 One aspect of the present invention pertains to a methylthioninium chloride dihydrate Form B, C or D or methylthioninium chloride monohydrate Form E, as described herein, for use in a method of treatment (e.g., of a disease condition) of the human or animal body by therapy, One aspect of the present invention pertains to use of methylthioninus chloride dihydrate 0 Form B, C or D o methylthini chloride monohydrate Form B, as descilbed herein, foi the manufacture of a medicament for use in the treatment of a disease condition One aspect of the present invention perains to use of methylthioninium chlorde dihydrate Form B, C or D or methylMoniniuchlonde monohydrate Form E. as described erein, 15 the treatment of a disease condition. One aspect of the present invention pertains to a method of treatment of a disease condition in a patient, comprising administering to said patient a therapeuticaly-effective amount of methylthioninium chloride dihydrate Form B, C or D or methylthioninium chloride ?0 monohydrate Form E, as described herein. Disease Conditions in one embodiment, the disease condition is a tauopethy. 25 A "tauopathy" is a condition in which tau protein (and aberrant function or processing thereof) plays a role. Aizheimer's Disease is an example of a tauopathy. The pathogenesis of neurodegenerative disorders such as Picks disease and Progressive Supranuclear Palsy (PSP) appears to correlate with an accumulation of pathological truncated tau aggregates in the dentate gyrus and stellate pyramidal cells of the neocortex, respectively. Other 30 dementias include fronto-temporal dementia (FTD); parkinsonism linked to chromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism- amyotrophy complex (DDPAC); palido ponto-nigral degeneration (PPND); Guam-ALS syndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration (CBD) and others (see, e.g, Wichik, C M. Theuring, F. & Harrington, C.R. (2000) The molecular basis of tau protein pathology in Alzheimer's 35 disease and related neurodegenerative dementias. In Neurobiology of Alzheimer's Disease (Eds. D. Dawbarn & S. J. Allen) Oxford University Press, Oxford, 103-206, , especially Table 13 5.1 therein). Each of these diseases, which is characterized primarily or partially by abnormal tau aggregation, is referred to herein as a "tauopathy." In one embodiment the disease condition is Alzheimers disease (AD 5 in one embodiment the disease condion is skAN cancer. in one embodiment thesease condition is melanoma. In one embodiment the disease condition is viral, bacterial or protozoal. In one embodiment he protozaa disease conditins malaria In this emboldimrentreatrmen 0 may be in combination with another animirobial agent eg in combination wih chorquine or atovaquone. in one embodiment, the viral disease condition is caused by Hepatitis CW orWestile Virus, 5 Treatment The term teatmen as used herein in the context of treating a conditia pertains genely to treatment and therapy, whether of a human or an animal (egin veterinary applications) in whih some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition and incldes a reduction in there rate of progress halt in the rate of 0 progress, regression of the condition, arelioration of the condition, and cure of the condition. Treatment as a prophylacic measure (e, prophylaxis, prevention) is also included. The tern 'therapeutcally-effecte amount t as used herein pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound 25 which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefitrisk ratio, when administered in accordance with a desired treatment regimen The term 'Ireatment" includes combation treatments and therapies, whih two or more 30 treatments or theMapes are combined, or example. sequentally or smultaneousy Examples of reatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including eg. drugs, antbodies (eg, as n immotherap prodrugs (g, as in photodynamic therapy DEPT, ADEPT etc sureryradiation 35 therapy; and gene therapy Routes of mnstration Methylthininium chloride dihydrate Form B. C or D or methylthioninium chloride monohydrate form E, or pharmaceutica composition comprising it, may be administered to a subjetpatent by any convenient roue of admiistratio whether systemicallyperpera 5 or topicay (ie. at the sie of desired action. Routes of administation include, but are not limited to, oral (e.g by ingestion buccal sublingua thansdermal (including, eg, by a patch master etc) tranmuosa including , eg. by a patch, plaster, etc intranasal (ag, by nasal spray; ouar e.g, by eyedrops); (0 pulmonary eg by inhaaton or insufflation therapy using, ag., via an aerosol eg. through the mouth or nose); rectal (e~g, by suppository or enema); vaginal eegby pessary); parentera for example, by injection, including subcutaneous, intradernal, intramuscular, ntravenous, intraarteriak intracardiac, intrathecal, intraspinal intracapsular, subcapsular, ntraorbital intraperitoneak intratrachea, subcuticular. intraarticular, subarachnoid, and 15 ntrastemal (including. e.g.. intracatheter injection nto the brain); by implant of a depot or reservoirfor example, subcutaneously or ntramuscularly The Subiect/Patient The subjed/patient may be an animn mamnial a placental mammal a marsupial (e ?0 kangaroo, wombat), a monotreme (eg.. duckbiled platypus), a rodent (e.g. a guinea pi, a hamster a rat, a mouse), urine (e.g. a mouse) a agomorph (eg, a rabbit aMan (eg. a bAd), anine (eg, a dog fine (e.g. a cat), equine ( g a horse), porcine (eg, a pig) ovine (eg. a sheep bovne(ega cow) primate, simi a g., a monkey or ape), a monkey (e g. marmoset baboon), an ape (eg gorlla, chimpazee 25 orangutang, gibbon),or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus, 20 In one preferred embodiment, the subject"patient is a human. Formuaon While it is possible for methythionium chlride dihydrate Form B, C or D or metythioninum chloride monohydrate form E to be used (ag. administered) alne it is 35 often preferable to present i as a composition orormulation.

15 In one embodiment the composition is a phamaceutical composition (e g fomulation preparation, mediament) comprising methylthioiniu chloride dihydrate form B, C Or D or methylthioninium chloide morhydrate form E, as described herein and a pharmaceutically acceptable career diluent, or excipient. 5 In one embodiment the composition is a phannaceutcal composition compriing at least methylthioninium chloride dihydrate Form B. C or D or methylthioninium chloride monohydrate Form E, as described herein, together with one or more other pharnaceutically acceptable ngredietswell known to those skilled in the art including, bt not limited to, 0 pharmaceutically acceptable carrers, diluents, excipients, adjuvants, fillers buffers preservatives, antoxidants, lubricants. st sersolubilisers, surfactants (e.g, wetting agents , masking agents colouring flavouring agents, and sweetening agents In one embodiment, the composition further comprises other active agents, for example. other therapeutic or prophylactic agents. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example. Handbook of Pharmaceutical Additives. 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse information Resources, inc, Endicott, New York, USA), Remington's 0 Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipies, 2nd edon, 1994. Another aspect of the present invention pertains to methods of making a pharmaceutical composition comprising admixing tC]-radiolabelled methyithioninium chloride dihydrate 25 Form B, C or D or methylthioninium chloride monohydrate Form E, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g, carriers, diluents, excipients, etc If formulated as discrete units (e.g, tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound 30 The term pharmaceuticallyy acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g, human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must 35 also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

16 The formulations may be prepared by any methods el nown in he art of phrmacySuch methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general the formations are 5 prepared by uniformly and intimately bringing into association the active cornpound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary. The formulation may be prepared to provide for rapid or slow release; immediate, delayed, 0 timed, or sustained release; or a combination thereof. Formulations suitable for parenteral administration (e.g, by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g, solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or 5 other microparticulate). Such liquids may additional contain other pharmaceutical acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol. vegetable 0 oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 pg/mI, for example from about 10 ng/ml to about 1 pg/ml The formulations may be presented in unit dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in 25 a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Exre of Preferred Formuations 30 One aspect of the present invention pertains to a dosage unk (eg, a pharmaceutical tablet or capsule) comprising 20 to 300 mg of methylthioninium chlorde dihydrate Form B, C or D or methylthioninium chloride monohydrate Form E as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient, 35 in one embodiment, the dosage unit is a tablet. in one embodiment, the dosage unit is a capsule, 1 7 in one embodimenit, the amount is 20 to 200 mg In one embodirment, the antou is about 20 tring In one embodiment, the amount is about mg. 5 In one embodiment, the amount is abou100 mg, in one embodment the amount is about 150 mg. in one embodiment, the amount is about 200 mg in one embodiment, the pharmaceuticaly acceptable carrier, diluent, or excipient is or 0 comprises one or both of a glyceride (e g., Gelucire 44/14 ®; lauroyl macrogol-32 glycerides PhEur USP) and colloidal silicon dioxide (e.g.. 2% Aerosil 200 ;: Colliodal Silicon Dioxide PhEur USP). Dosage 15 I wi be appreciated by one of skill in the art that appropriate dosages of methythioninium chloride dihydrate Form B,. C or D or methylihioniiurn chloride monohydrate Form E, and composions comprsing methythionum chddloride Form B, C or D or nethyithioniiun chloride ronohydrate Form E. can vary from patient to patient. Detemining the optimal dosage will general involve the balancing of the level of therapeuic benefit ?0 against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors inuding, but not mited to, the activity of the particulr compound, the route of administration, the time of administration he rate of excretio of the compound the duration of the treatment, other drugs, compounds and/otrmatenals used in combination, the severity of the condition, and the species, sex, age, weight, condition general health, and 25 pdor edica history of the patient. The amount of compound and route of administration will ultimately be at the discreton of the physician, veterinarian, or clnician although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-ffects. 30 Administration can be effected in one dose continuusyor intermittently e in divided doses at appropriate intervals) throughout the course of teatment. Methods of determining the most effective means and dosage of administration are weil known to those of skill in the art and wiI vary with the formulation used for therapy, the purpose of the therapy the target cells) being treated, and the subject being heated Single or multiple administationscan be 35 carded out with the dose level and pattern being selected by the treating physician, veterinarian, clinician.

18 In general, a suitable dose of methylthioninium chloride dihydrate Form B, C or D or methylthioninium chloride monohydrate Form E is in the range of about 100 ng to about 25 mg (more typically about 1 pg to about 10 mg) per kilogram body weight of the subject per 5 day In one embodiment, methylthioninium chloride dihydrate Form B, C or D or methylthioninium chloride monohydrate Form E is administered to a human patient according to the following dosage regime: about 100 mg. 3 tires daily 10 In one embodiment, methylthioninium chloride dihydrate Form B, C or D or methylthioninium chloride monohydrate Form E is administered to a human patient according to the following dosage regime: about 150 mg, 2 tmes daily. 15 In one embodiment, methylthioninium chloride dehydrate Form B, C or D or methyithioninium chloride monohydrate Form E is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily. Brief Description of Figures ?0 Figure 1 is a characteristXay powder diffraction pattern of the crystanine water containing Form B of nethtioninium chloride; Figure 2 is a charactestic X-ray powder diffraction pattern of the crystaline dihydrate of meth hioninium chloride Form C; Figure 3 is a characteristic a Xay powder diffraction pattern of the crystalline dehydrate of 25 methyithioninium chloride Form D; gure 4 is a chaacterisic Xay powder diffraction pattern of the crystalline monohydrate of methylthioninium chloride Form F The following examples illustrate the prsent invention without limiting he described scope. 30 Experimental: Powder X-ray Diffraction (PXRD): PXRD was performed on a Bruker D8 Advance powder X ray diffractometer using CuKo. radiation. 0-spacings are calculated from the 20 values using the wavelength of 1.54180 A. Generally, 20 values are within an error of 0 1-0.2*. The 35 experimental error on the d-spacing values is therefore dependent on the peak location.

19 Differental Scaning Calorimety (ID): Perkin Elmer DSC measurement perform in gold sample pan heneticay sealed under amilent conditions. A heating rate of either 20/mmn or 100 K/mm was used. All herein gien melng ponts are determined from the peak temperatures of the D0 measurements 5 Thermogravimetry (TG): Perkin Elmer TGS 2, Aluminium crucible (open), N. atmosphere, heating rate 10CC mint range 25~350*C. Thermogravimetric measurements with IR detection (TGFTIR): Netzsch Thermo 10 Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with pinhole, nitrogen atmosphere, heating rate 10 K/min). Hydrate form A Methylthioninium chloride pentahydrate Form A maybe obtained by re-crystallization of the 15 product prepared according to WO 2006/032879 from 0.1 M hydrochloric acid and drying in vacuum at about 60 mbar and room temperature (see Example 17). As a comparison, d values (A) are given in table 1 for form A. Table 1: d-Spacings for crystal Form A I AngOle{201 d-spacing [A] Intensity (qualitative) 5.7 15.5 __ __ __vs__ _ 9.2 96 | vs 96 92 - vs 1088.2 s __ 11 7.8 |jm_ 18,7 4.75 vs . . . .. . . . 19, 4.60 | s 20.4 |4.35 |___ m ____ 21.7 4.10 . m 21.9 4.06m 24.6 3.62 vsm 25613.48 vs_ ___ 26.0 3,43 [s 26.2 | 3.40 vs_______ 26.4 3.38 vs 27.3 3.27 __s 28.0 _ 3.19 s I 28.4 314 s 29.2 |3.06 m 20 The abbreviations irnbracketsrmean: (s)=verystrong intensity (s s tongintensity;(i) medium irntensity;and (w) weak intensity.

20 Characteristic IR signals of form Am i ATR-IR are found at 1491, 1421, 1356, 1225/1215 (double peak), 1177, and 1 151 cm' 5 A) Preparation of polymorph Form B Example A1 150 mg crystalline methythioninium chloride pentahydrate are heated to 600 for 5 days S5% rh. Thermogravimety othe product shows a weight oss of06% up to a temperature of 150Q which coresponds to the presence of two equivalents of water, PXRD 0 revealed a crystallinre sampleThe powder Xray diffracbon pattern is shown in Figure 1 and the characteristic peaks i 20 with the coresponding d-spacing values in A are given in table 2OSO 0*C to 21000, 100 /rmin gold crucible)evealed a melng peak at 166c( vth a shoulder towardslower temperature 5 a e 2; d Spacings for hydrate Forr B Argle [* 20) ---- d spacing [A] intensity (qualitative) 5.8 15.2 9.2 9.6 _ W 11.2 -7.9 1 M - - -_-_--_-_ 1-. ... |..68 WV_ 16.9 5.25 W_______V 20:6 4.31 _________ W 25,3 352 jM ____ -26.8 3$33 9.M........---- .. 28.3 6 8 3 -15 W Example A2: 1 g of crystalline methylthiorninium chloide pentahydrate Form A powder, contaminated with a small mount ofform B, was stored at room temperature for 3 weeks under starring with a 20 smallmagrnetic stirrer under a flow of humidified nitrogen having approximately 9% relative huidity. Dehydration is complete after 3 weeks and yields gquantitative rmethylthioniriurn chloride dihydrate Form B as greenish crystal powder. PXRD corresponds to that of example Al. 25 Example A3: 2 g of crystalline methylthioninium chloride pentahydrate Form A powder, contaminated with a small amount of form B, was stored at room temperature for 4 weeks under stirring with a small magnetic stirrer under a flow of humidified nitrogen having approximately 14% r~h.

21 Dehydration is complete after 3 weeks and yields quantitatively methyithioninium chloride dehydrate Form 8 as greenish crystal powder. PXRD corresponds to that of example Al. B) Preparation of polynorph form C 5 Example 1B: A rmxture of methylhioniniun chlonde pentahydrate Form Aand methylthioninium chloride dehydrate Form B (170 mg) was suspended in 2 ml acetonitrile and stirred at room temperature for 4 days. The solid was filtered off and dried in vacuum at 1 mbar and at room temperature for 15 minutes. 110 mg of methylthioninium chloride dehydrate Form C were 0 obtained as greenish crystal powder, PXRD revealed a crysaine sample. The powder X-ray diffraction patern is shown in Figure 2 and the characteristic peaks in 20 with the correspnding d-spacing values in A are gien in table 3 TG-FTiR revealed a mass loss of about 114% intwo steps between room S temperature and 15000, which corresponds to a water content of 2 equivalents, which is sl hty more than expected for the dhydrate. DSC (00C to 2100 100 I 0/min, gold crucible) revealed two endothermic peaks at 1510C and 183 0C Table 3: d-Spacings for hydrate Form C Angle [ 20) d-spacing [A] Intensity (qualitative) 8.1 10,9 _ vs 11. 8,0 s 13. 6.6 w ____ 16.2 5.47 m [17, 4,98 m 17,6 e,5,04 I .18,4 4.82 w 124 4 3,65 rn_ 23,44 vs 27 2 328 vs 2 -8 1vs 287 3.11 w 29 5 3.03 w ________ 32 96 03 30.8 '2,90 m2__ 3271 m 34,1 2.63 w 36.0 2.49 _ w _____ 36.7 2.45 w 39.5 2.28 1 w 42.7 2.12 _ _ w 45.3 2.00 w 480 1.90 w 22 Example B2: 175 mg of a mixture of methylthioninium chloride pentahydrate Form A and methylthioninium chloride dihydrate Form 8 was dIssolved at about 1000C in 3 ml dimethylsulfoxide (DMSO). 5 The solution was allowed to cool to room temperature and stored overnight in a refrigerator. The cool solid mixture was allowed to warm to room temperature, whereby DMSO meits. The remaining solid was filtered off and dried in vacuo at 1 mbar and at room temperature. This yields 135 mg greenish methyithioninium chloride dihydrate Form C, The PXRD corresponds to that of example B1. Example B3: 2 g of methylthioninium chloride pentahydrate Form A was suspended in 10 mi acetonitrile and stirred at room temperature for 6 days. The solid was filtered off and dried in vacuo at 1 mbar and at room temperature for 15 minutes. This procedure was repeated two times. Pure 5 methylthioninium chloride dihydrate Form C was obtained as greenish crystal powder. The PXRD corresponds to that of example 81. Thermogravimetry revealed a mass loss of 9.8% in two steps between room temperature and 150 *C The total mass loss corresponds almost exactly to a water content of 2 equivalents. O0 Example 84: 100 mg of a mixture comprising methylthioninium chloride pentahydrate Form A, methylthioninium chloride dihydrate Form B. methylthioninium chloride dihydrate Form C and methylthioninium chloride dihydrate Form D were suspended in 2 ni isopropanol containing 20 pI water (corresponding to about 12% relative humidity) The suspension was stirred at 25 room temperature for 5 days. The sold is filtered off and dried in vacuum at I mbar and at room temperature for 5 minutes. This yielded pure methylthioninium chloride dihydrate form C as greenish crystal powder. The PXRD corresponds to that of example 81. Example 85: 30 100 mg of a mixture comprising methylthioninium chloride pentahydrate Form A, methylthioninium chloride dehydrate Form B, methylthioninium chlo ide dihydrate Form C and methylthioninium chloride dihydrate Form D were suspended in 2 ml isopropanol containing 50 pi water (about 28% rh,). The suspension was stirred at room temperature for 6 days. The solid was filtered off and dried under vacuum at 1 mbar and at room temperature for 5 35 minutes, This yielded pure methylthioninium chloride dihydrate form C as greenish crystal powder The PXRD corresponds to that of example 81.

23 100 mg of the powdery product was pressed to a tablet at a pressure of I to/0.5 cm 2 . Form C was retained in the tablet. The PXRD corresponds to that of example B1. 5 Example 86: 500 mg of methylthioninium chloride pentahydrate Form A was suspended in 10 ml isopropanol and stirred for 2 weeks. The solid was filtered off and dried under vacuum at 1 mbar and at room temperature for 5 minutes. This yielded methylthioninium chloride dihydrate form C as greenish crystal powder. The PXRD corresponds to that of example 81. ~0 G) Preparation of hydrate form D Example C1 100 mg of methylthioninium choride dihydrate Form B were dissolved in 2 nil pure acetic acid. The solution was filtered through a 0.2 pm syringe filter and added to 10 ml toluene. A 5 sticky precipitate forms within a short time. The solid was filtered off about 3 minutes after precipitation, washed with toluene and dried under vacuum at 1 mbar and at room temperature for 15 minutes. This yields 70 mg of methylthioninium chloride dihydrate form D as grey to violet crystal powder. PXRD revealed a crystalline sample. The powder X-ray diffraction pattern is shown in Figure 20 3 and the characteristic peaks in 26 with the corresponding d-spacing values in A are given in table 4. TG revealed a mass loss of about 9.3% and TG-FTIR revealed a mass loss of about 11 between room temperature and 1500C, which corresponds to a water content of 2.2 equivalents. which is slightly more than expected for the dihydrate. DSC (-5*C to 210*C, 100 "Cimin, gold crucible) revealed two endothermic peaks at 164*C and 185 *C 25 24 Table 3: d-Spacings for polymorph Form D Angl [* [9 -pcn A] Intensity (qualitative) 6 0 14.7 ___m 7.0 :12.6 s 8.5 10.4 s _______ 98 _ 9.0 w_______ 10,4 8.5 Im 12.0 7.4 a ____ 14.4 __6.2 1s 1625 5.44 w 1L7-5.19 ±w 18.1 -490 w__----- 20.9 4.25 m 21.1 4.21 Em 21.7 4,10 m 22.3 13.99 m _______ 23.7 3,75 m _ .24.5 3.63 m__ _ p25.3 3 ms_ 25,7 _.47 s -26.9 31m--- m --- 27.5 7.24 -~__vs 28.5 '3.13 m____ -------- -------

-

------- --- - 29.0 3108 m _ 30 4 - 2.94 _ m 0 31. .......-- V ....... 5 5_ 2.81 m 34 9 _ I2.57 w 41.5 12.18 1w 46.5 1.95 1w Example 02: 118 mg of methylthioninium chioride pentahydrate Form A were dissolved in 2 nil pure acetic 5 acid, The solution was filtered through a 0.2 pm syringe filter and added to 10 ml toluene, A sticky precipitate forms within a short time. The solid was filtered off about 3 minutes after precipitation. washed with toluene and dried at room temperature for 60 minutes. This yielded methylthioninium chloride dihydrate form D as a grey to violet crystal powder. The PXRD corresponds to that of example 01. Exanmple 03 1g of methylthionirnum chloride pentahydrate Form A weredissolved in 10ml methaol.he solution is filtered through a 0. 2pm syringe filter and added without stirring to 100 nil t-buty. methyl ether (tBME). A precipitate forms withn a short tirme.he solid was filtered off about 3 15 minutes after precipitation, washed with tBME arnd dried in a flow of nitogen for 1 hour. his 25 yeids 850 mg of methylthioninium chloride dihydrate fornn D as grey-violet crystal powder. The PXRD corresponds to that of example Cl. D) Preparation of hydrate Form E 5 Example D1 80 mg of a mixture comprising methylthioninium chloride pentahydrate Form A. methvlthioninium chloride dihydrate Form B, methydthioninium chloride dihydrate Form C and methylthioninium chloride dihydrate Form D were suspended in 2 ml dry isopropanol containing less than 0.1% by weight of water. The suspension was stirred under temperature 10 cycling between 25*C and 35'C for 1 week. The solid was filtered and dried under vacuum at 1 mbar and at room temperature for 5 minutes. This yielded methylthioninium chloride monohydrate form E as ocher crystal powder. PXRD revealed a crystalline sample. The powder X-ray diffraction pattern is shown in Figure 15 4 and the characteristic peaks in 20 with the corresponding d-spacing values in A are given in table 5. TG revealed a mass loss of about 5.1% between room temperature and 125 *C which corresponds to a water content of 1 equivalent. DSC (-50*C to 210*C, 100 *C/min. gold crucible) revealed no thermal events up to the decomposition temperature of about 20 0 C. 20 Table 3: d-Spacings for polymorph Form E Angle [* 201 d-spacing fA] Intensity (qualitative) 9.0 ___9.8 vs 12.5 7. s_ _ _ __ _ _ 14.1 II3 14 s 14. -1 __ _ 21,8 4.08 w 22, .K 4 1 4.02 ~ w _ 23.2 j-3.83 vs . 24.5 |3.63 mt___ 25.1 93.55 ± 26.0 3.43 vs 27.2 3.28 jm 28.4 3.1___..4_________ _w. 29.6 3.02 32.0 2.80 39,6 2.28 w 4 . 2,17 w 4- 7,1 . 1,9 - w--............

26 Example D2: ig of methylhioninium choride pentahydrate Form A was suspended in 20 ml dry isopropanol and stirred at room temperature for 3 days. The soid was filtered off, re suspended in 10 ml dry isopropanoj and stirred foranother 9 days Te solid was filtered off 5 again. When becoming soent-free,the lercake turns to ocher cor. Residual isopropanol is removed under a dry nitrogen flow for 2 hours. This yields 700 mg of methyIthioninium chloride rnonchydrate form E as an ocher crystaline powder. The PXRD corresponds to that of exampleD 10 Exampe 03: 19 of methythioninium chlAide pentahydatde Form A was suspended in 10 rl dry isopropanol and surred at room temperature for 1 day The solid was filtered off, again suspended in 10 mi dryisopropenol and stirred for days, Filtratonre-uspension nd stirringwas repeated once again The solid turns to ocher color. Finally, the ocher solid was 15 filtered off and residual isopropanol is removed under a dry nitrogen flow for 2 hours. This yields 650 mg of methyltioninium chloride monohydrate form E as an ochre crystaline powder The PXRD conesponds to that of example D.

Claims (4)

1. 3. The compound of claim 1 having the folowing characteristic peak s in a powder X-ray diffractior pattern; Peak 20 values (± 01i ) 2 1 , 3 117.6 4 259 5 T 2 7 2 7 178
2. 17. - - -------- 8 24.4 9 30.8 10 4, The compound of any one of claims 1 to 3, which has two endothemic maxma at 151 t 0 and 1S3C when heated at a rate of 100 per minute in Differential Scaning Calorimetry. 15 28 5. A process for the preparation of metyithioninium chlode dihydrate substantiaNy in form C according to any one of climns 1 to 4. wherein a water-containig methyltionnum chloride or a mixture of various hydrates or a specifc hydrate thereof is suspended and stirred at amben temperatre in a solvent selected from the group comprising isopropano. 5 1-propano.t 1-butanol, 2-butanoL tert-butanoi, tetrahydrofurane, dioxane acetone, 2 butanone. and acetonitrile containing a small amount of water, for a time sufficient to generate form C; the solid is then isolated: and the solvent is removed from the solid. 6. Crystalline methyithioninium chide monohyrate as FomE. 10 7 The compound of claim 6 having the following characteristic peaks in a powder X-ray diffraction pattern: Peak 28 values (± 0.1*) 1 9.0 2 12.5 3 14.1
3. 414.4 5 l8 6 232 7 24.1 8 26.0 8 The compound of claim 6 having the following characteritic peaks in a powder X-ray 5 diffraction pattern: Peak 2 values ( 01 ) 1i 9.0 212,5 3 14.1 4 14A 5 18.1 6 23.2 7 124.1 18 126,0 9W 24.5 10 -- ---- -- __ K 7 2 ----------------- 29 9. The compound ofany on of clans 6 to 8, which shows no thermal event up to the decomposition temperature near 2207C when heater at a rate of 100$C per minute in Differential Scanning Calrmetry 5 10. A process for the preparation of methylthiorniim chloride monohydrate substantiall in form E according to any one of claims 6 to 9, wherein: wateeontaining rnethylthionniurm chloride or a mixture of various hydrates or a specific hydrate of metythioninium chlorde is suspended and stirred at ambient temperature in a 10 dry solvent preferably isopropanol for a time sufficient to generate form E; the solid is slated by filtraon and residual soentis removed fom the soid by either vacuum drying or purging wTh dry gas. Crystalline methylthioninium chloride dihydrate as Forni D. 15 12. The compound of claim 11 having the following characteristic peaks in a powder X ray diffraction pattern: Peak2 values (± 01) 1 7.0 28. 3 12.0 4 14,4 5 25.3 6 25,7 -- - -- -- 7----- - ----- -- 5- -- 30 13. The compound of claim 1 having the following characteristic peaks in a powder X ray diffraction pattern: Peak 20 values (± 0.1 1 70 2 85 312.0 5 25.3 6 25.7 7 27,5 9 10.4 10 20.9 11 21.1 12 121.7 113 ~22,3 14 23,7 15 24,5 16 26.9 17. 2. 18 29.0 19 30.4 20 31.8 14. The compound of any one of claims 11 to 13, which has two endothermic peak 53 maxima at 164*0 and 15*CQ and a step in the baseline near 63*C when heated at a rate of 100"C per minute in Differential Scanning Calorimetry. 15, A process for the preparation of methylthioninium chloride dihydrate substantially in form D according to any one of claims 11 t~o 14, comprising: dissolving methylthioninium 10 chloride pentahydrate in acetic acid and combining the solution with toluene, either by adding toluene to the acetic acid solution or by adding the acetic acid solution to toluene; isolating the solid by filtration shortly after precipitation; and removing the solvent by vacuum drying or in an inert gas flow, whereby the relative air humidity in all process steps is less than 50%, 3 1 16 Crystalline methylthioninium chloride dihydrate substantially as Form B 17. The compound of claim 16 having the following characteristic peaks in a powder X ray diffraction pattern: Peak 28 values ( 0.14) 3i 25.3 4 [26.8 _ 5 18. The compound of claim 16 having the following characteristic peaks in a powder X ray drffraction pattern: [Peak 29 values (± 0, 1 15.8 2 11.2 3 ;25.3 4 26.8 5 15,6
4. 616.9 20.3 8 28.3 19. The compound of any one of claims 16 to 18, which has a melting peak at 186C with 10 a shoulder towards lower temperature when heated at a rate of 100C per minute in Differential Scanning Calorimetry. 20 A process for the preparation of methylthioninium chloride dihydrate substantially in forn B, which comprises exposing solid methylthioninium chloride pentahydrate at about 15 room temperature to an inert gas flow having a relative humidity from 8 to 15% for a time sufficient to generate essentially pure form B. 21. A pharmaceutical composition comprising methylthioninium chloride dihydraie form B, C or D or methylthioninium chloride monohydrate form E according to any one of claims 1 to 4. 20 .to i 9, 11 to 14 or 16 to 19, 32 22. A pharmaceutical composition comprising methythioninium chloride dihydrate form B, C or D or methylthioninim chloride monohydrate form E according to any one of claims 1 to 4, 5 to 9 11 to 14 or 16 to 19 and a pharmaceutically acceptable carrier, recipient or diluent 5 23. Methylthioninium chloride dihydrate form B, C or D or methylthioninium chloride monohydrate form E according to to any one of claims I to 4, 5 to 9, 11 to 14 or 16 to 19 for use in a method of treatment of the human or animal body by therapy. 24. Use of methylthioninium chloide dihydrate form 6, C or D or methylthioninium chloride 10 monohydrate form E according to to any one of claims 1 to 4, 5 to 9, 11 to 14 or 16 to 19 in the treatment of a disease condition. 25 Use of methylthioniniur chlorde dihydrate form B, C o D or methininium chloride monohydrate form IE according to to any one of claims I o 45to 59,1 to 14 or 16 to 19 in 15 the treatment of a tauopathy, Aizheimers disease (AD), skin cancer, melanoma, Hepatitis C. HIV or VWest Nile virus. 26, A method of treatment of a tauopathy, Alzheimer's disease (AD), skin cancer, melanoma, Hepatitis C, HIV or West Nile virus in a patient, comprising administering to said patient a 20 therapeuticadly-effective amount of methylthioniniurn chloride dihydrate form B, C or D or methylthioninium chloride monohydrate form E according to to any one of claims 1 to 4, 5 to 9, 11 to 14 or 16 to 19,