DESC:Field of the Invention:
The present invention relates to crystalline hydrated form of anti-HCV compound Sofosbuvir, having the chemical name isopropyl (2S)-2-{[(S)-{[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydro-1(2H)-pyrimidinyl)-4-fluoro-3-hydroxy-4-methyltetrahydro-2-furanyl]methoxy}(phenoxy)phosphoryl]amino}propanoate, is known to be an effective anti-HCV agent, process for its preparation and pharmaceutical composition thereof.
Background of the Invention:
Hepatitis C is recognized as a chronic viral disease of the liver which is characterized by liver disease. Although drugs targeting the liver are in wide use and have shown effectiveness, toxicity and other side effects have limited their usefulness. Inhibitors of hepatitis C virus (HCV) are useful to limit the establishment and progression of infection by HCV as well as in diagnostic assays for HCV.
The hepatitis C virus (HCV) is an RNA virus belonging to the Hepacivirus genus in the Flaviviridae family. The enveloped HCV virion contains a positive stranded RNA genome encoding all known virus-specific proteins in a single, uninterrupted, open reading frame. The open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of about 3000 amino acids. The polyprotein comprises a core protein, envelope proteins El and E2, a membrane bound protein p7, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B.
HCV infection is associated with progressive liver pathology, including cirrhosis and hepatocellular carcinoma. Chronic hepatitis C may be treated with peginterferon-alpha in combination with ribavirin. Substantial limitations to efficacy and tolerability remain as many users suffer from side effects, and viral elimination from the body is often inadequate. Therefore, there is a need for new drugs to treat HCV infection.
Sofosbuvir is described in US 7964580 (‘580), and is the inhibitor of RNA-dependent RNA viral replication and is useful as inhibitor of HCV NS5B polymerase, as inhibitor of HCV replication and for treatment of hepatitis C infection in mammals. Sofosbuvir has the following chemical formula:

Extensive study is carried out in pharmaceutical industry for development of different polymorphs of various drug substances, to obtain suitable polymorphs that possess improved performance characteristics such as aqueous solubility, improved bioavailability, chemical stability, shelf life etc.
Literature survey reveals that Sofosbuvir can exist in different polymorphic forms, which differ from each other in terms of stability, physical properties and pharmacokinetics. The reported polymorphs of Sofosbuvir are incorporated here by way of reference.
US patent 8,618,076 B2 and PCT publication WO2015/099989 A1 describes various polymorphs including amorphous form of Sofosbuvir.
Polymorphs often improve physical and biological characteristics of mother compounds without modifying primary pharmacological activity, based on mechanism of action. Thus there is a continuing need to obtain new polymorphs of Sofosbuvir having improved physical and/or chemical properties.
Description of drawings:
Figure 1: Illustrates X-ray powder diffraction (XRPD) pattern of crystalline monohydrate form of Sofosbuvir.
Figure 2: Illustrates differential scanning calorimetry (DSC) plot of crystalline monohydrate form of sofosbuvir.
Figure 3: Illustrates thermogravimetric analysis (TGA) plot of crystalline monohydrate form of sofosbuvir.
Figure 4: Illustrate FTIR spectra of crystalline monohydrate form of sofosbuvir.
Figure 5: Illustrates Oak Ridge Thermal Ellipsoid Plot (ORTEP) view of crystalline monohydrate of sofosbuvir.

Summary of the Invention:
In one aspect, the present invention provides a stable crystalline hydrated form of Sofosbuvir having enhanced stability, dissolution properties and purity.
In another aspect, the present invention provides a process for the preparation of a stable crystalline hydrated form of Sofosbuvir.
In another aspect, the invention provides a pharmaceutical composition comprising said stable crystalline hydrated form of Sofosbuvir and at least one pharmaceutically acceptable excipient or carrier.

Detail Description of the Invention:
The term "excipient" or “pharmaceutically acceptable excipient” means a component of a pharmaceutical product that is not an active ingredient, and includes but not limited to filler, diluent, disintegrants, glidants, stabilizers, surface active agents etc. The excipients that are useful in preparing a pharmaceutical composition are generally safe, non-toxic and neither biologically nor otherwise undesirable, and are acceptable for veterinary use as well as human pharmaceutical use. One excipient can perform more than one function.
The term "stable" herein means Sofosbuvir that substantially does not convert to any related substances or other solid form after exposure to standard pharmacopeial stability conditions at relative humidity of 60% at 25°C or 75% at 40°C, for a period of at least three months.
The term “hydrated” herein means Sofosbuvir that has water content of 0.2 to 1 mole of water.
In one embodiment of the invention, the present invention provides a stable crystalline hydrated form of sofosbuvir having enhanced flow property, stability, dissolution properties that can be easily formulated into pharmaceutical compositions.
According to the present invention, the stable crystalline hydrated form of Sofosbuvir is substantially free from any other known form of Sofosbuvir.
The present disclosure provides a stable crystalline hydrated forms of Sofosbuvir, may be characterized and distinguished from other solid forms of the same compound using various analytical techniques including, but not limited to, X- ray powder diffraction (XRPD), solid-state nuclear magnetic resonance (NMR, or C SSNMR), Raman spectroscopy, differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), and thermogravimetric analysis (TGA).
In another embodiment, the invention provides a stable crystalline hydrated form of sofosbuvir characterized by an XRPD diffractogram comprising peaks: 7.5, 14.1, 14.5, 15.2, 15.8 and 16.3 degrees two-theta (°2T), ± 0.2° 2T.
In another embodiment, the invention provides a stable crystalline hydrated form of sofosbuvir further characterized by an XRPD diffractogram having additional peaks: 11.4, 20.4, 22.9 and 30.1 degrees two-theta (°2T), ± 0.2° 2T.
The crystalline hydrated form of Sofosbuvir also characterized by a triclinic elementary cell with the parameters a=9.2702(1) Å, b=13.9392(2) Å, c=20.923(3) Å, and cell volume=2703.7(6) Å3 determined by single crystal X-ray structural analysis.
The crystalline hydrated form of Sofosbuvir crystallizes in a triclinic crystallographic system (see Table 1).
Table 1
Chemical formula C22H29O9N3PF. H2O
Formula Weight 547.46
Temperature (K) 298(2)
Crystal lattice Orthorhombic
Space group P212121
a, b, c [Å] 9.2702(1) , 13.9392(2), 20.923(3)
a, ß, ? [°] 90,90,90
V [Å3] 2703.7(6)
Z 4
D(calc) [g/cm3] 1.343
wR2 (all) 0.1785
GOF 1.159
Diffractometer Rigaku Mercury

In another embodiment, the invention provides a process for preparation of stable crystalline hydrated form of Sofosbuvir comprising the steps of:
(i) providing a solution of Sofosbuvir in a solvent;
(ii) adding water;
(iii) isolating crystalline hydrated form of Sofosbuvir.

The isolation of the crystalline hydrated form of Sofosbuvir can be done by substantially removing the solvent or adding anti-solvent to the reaction mixture.

The term "substantially removing" the solvent refers to at least 80%, specifically greater than about 85%, more specifically greater than about 90%, still more specifically greater than about 99%, and most specifically essentially complete (100%), removal of the solvent from the reaction mixture.

The Sofosbuvir employed in step (i) can be crystalline or amorphous obtained by any known methods in the literature.

The solvent employed in step (i) is selected from halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform and carbon tetrachloride; alcohols such as methanol, ethanol, isopropyl alcohol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and t-butyl alcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone, and methyl isobutyl ketone; esters such as ethyl acetate, n-propyl acetate, n-butyl acetate and t-butyl acetate; ethers such as diethyl ether, dimethyl ether, diisopropyl ether, methyl t-butyl ether and 1,4-dioxane; nitriles such as acetonitrile and propionitrile; or mixtures thereof;

The reaction of step (i) is carried out at a temperature of about 20 to about 100°C, preferably at about 50 to about 90°C and more preferably at a temperature of about 25 to about 50°C.

Removal of solvent in step (iii) is accomplished, for example, by filtration, substantially complete evaporation of the solvent, concentrating the solution or distillation of solvent, under inert atmosphere to obtain the stable crystalline form of Sofosbuvir.

In another embodiment, the solvent is removed by evaporation. Evaporation can be achieved at sub-zero temperatures by lyophilisation or freeze-drying techniques. The solution may also be completely evaporated in, for example, a pilot plant Rota vapor, a Vacuum Paddle Dryer or in a conventional reactor under vacuum above about 720 mm Hg by flash evaporation techniques by using an agitated thin film dryer ("ATFD"), or evaporated by spray drying to obtain a dry crystalline powder.

The distillation process can be performed at atmospheric pressure or reduced pressure. Specifically, the solvent is removed at a pressure of about 760 mm Hg or less, more specifically at about 400 mm Hg or less, still more specifically at about 80 mm Hg or less, and most specifically from about 30 to about 80 mm Hg.

Solvents can also be removed by spray-drying, in which a solution comprising of Sofosbuvir and a aerosil is sprayed into the spray drier at the flow rate ranging from about 10 to about 300 ml/hr, specifically about 40 to about 200ml/hr. The air inlet temperature to the spray drier used may range from about 30º C to about 150º C, specifically from about 65 º C to about 110 º C and the outlet air temperature used may range from about 30 º C to about 90 º C.

Another suitable method is vertical agitated thin-film drying (or evaporation). Agitated thin film evaporation technology involves separating the volatile component using indirect heat transfer coupled with mechanical agitation of the flowing film under controlled conditions. In vertical agitated thin-film drying (or evaporation) (ATFD-V), the starting solution is fed from the top into a cylindrical space between a centered rotary agitator and an outside heating jacket. The rotor rotation agitates the downside-flowing solution while the heating jacket heats it.

The stable novel crystalline hydrated form of Sofosbuvir obtained by process disclosed herein may be further dried, preferably spin dried, in, for example, a Vacuum Tray Dryer, a Rotocon Vacuum Dryer, a Vacuum Paddle Dryer or a pilot plant Rota vapor, to further lower residual solvents. Drying can be carried out under reduced pressure until the residual solvent content reduces to the desired amount such as an amount that is within the limits given by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use ("ICH") guidelines.

In another embodiment, the present invention provides a pharmaceutical composition comprising stable crystalline hydrated form of Sofosbuvir and at least one pharmaceutically acceptable excipient or carrier.

The stable crystalline hydrated form of Sofosbuvir can be formulated into various pharmaceutical compositions like powder, granules, capsules, tablets, pellets etc.

The pharmaceutical composition of the invention can be formed by various methods known in the art such as by dry granulation, wet granulation, melt granulation, direct compression, double compression, extrusion spheronization, layering and the like. The composition or formulation may be coated or uncoated. Coating of compositions such as tablets and caplets is well known in the art.

Although for many pharmaceutical compounds oral administration in the form of a tablet or capsule is preferred, some patients, for example elderly and pediatric patients, may have difficulties in swallowing such formulations. Therefore, liquid formulations such as oral solutions may offer a suitable alternative, avoiding the need of swallowing tablets or capsules. An oral solution further provides the possibility of a more flexible dosing regimen. In order to limit the volume of an oral solution it is necessary to have a high concentration of the active ingredient in the solution, which again requires a high solubility of the active ingredient. Hence the superior solubility of novel crystalline hydrated form of Sofosbuvir of the present invention makes this particular solid state form especially suitable for the preparation of liquid pharmaceutical formulations such as oral solutions

Pharmaceutically acceptable excipients may be utilized as required for conversion of the novelcrystaline form of Sofosbuvir into the final pharmaceutical dosage forms and include, for example, any one or more of diluents, binders, stabilizers, lubricants, glidants, disintegrating agents, surfactants, and other additives that are commonly used in solid pharmaceutical dosage form preparations.

The present invention includes administration of an effective amount of stable novel crystalline hydrated form of Sofosbuvir (either alone or as the active component of a pharmaceutical composition), in particular for use in the treatment of hepatitis C virus.

In a further embodiment, the present invention relates to a method for the treatment hepatitis C virus, in a subject in need of such treatment, which method comprises administering to such subject a therapeutically effective amount of stable crystalline hydrated form of Sofosbuvir.

The present invention includes the use of stable crystalline hydrated form of Sofosbuvir in combination with other antiviral agents used in the treatment of Hepatitis C virus.

The diluents, binders, bulking agents, stabilizers, lubricants, glidants, disintegrating agents, surfactants, and other additives that are commonly used in solid pharmaceutical dosage form preparations includes

Diluents:
Various useful fillers or diluents include but are not limited to starches, lactose, mannitol (PearlitolTM SD200), cellulose derivatives, confectioner's sugar and the like. Different grades of lactose include but are not limited to lactose monohydrate, lactose DT (direct tableting), lactose anhydrous, FlowlacTM, PharmatoseTM and others. Different starches include but are not limited to maize starch, potato starch, rice starch, wheat starch, pregelatinized starch and starch 1500, starch 1500 LM grade (low moisture content grade) from Colorcon, fully pregelatinized starch and others. Different cellulose compounds that can be used include crystalline celluloses and powdered celluloses. Examples of crystalline cellulose products include but are not limited to CEOLUSTM KG801, AvicelTM PH101, PH102, PH301, PH302 and PH-F20, PH112 microcrystalline cellulose 114, and microcrystalline cellulose 112. Other useful diluents include but are not limited to carmellose, sugar alcohols such as mannitol (PearlitolTM SD200), sorbitol and xylitol, calcium carbonate, magnesium carbonate, dibasic calcium phosphate, and tribasic calcium phosphate.

Binders:
Various useful binders include but are not limited to hydroxypropylcelluloses, also called HPC (KlucelTM LF, Klucel EXF) and useful in various grades, hydroxypropyl methylcelluloses, also called hypromelloses or HPMC (MethocelTM) and useful in various grades, polyvinylpyrrolidones or povidones (such as grades PVP-K25, PVP-K29, PVP-K30, and PVP-K90), PlasdoneTM S-630 (copovidone), powdered acacia, gelatin, guar gum, carbomers (CarbopolTM), methylcelluloses, polymethacrylates, and starches.

Bulking agents:
Bulking agents are ingredients which may provide bulk to a pharmaceutical composition. Various useful binders include but are not limited to PEGs, mannitol, trehalose, lactose, sucrose, polyvinyl pyrrolidone, sucrose, glycine, cyclodextrins, dextran and derivatives and mixtures thereof.

Disintegrants:
Various useful disintegrants include but are not limited to carmellose calcium, carboxymethylstarch sodium, croscarmellose sodium, crospovidones, examples of commercially available crospovidone products including but not limited to crosslinked povidone, KollidonTM CL, PolyplasdoneTM XL, XI-10, and INF-10 and low-substituted hydroxypropylcelluloses. Examples of low-substituted hydroxypropylcelluloses include but are not limited to low-substituted hydroxypropylcellulose LH11, LH21, LH31, LH22, LH32, LH20, LH30, LH32 and LH33. Other useful disintegrants include sodium starch glycolate, colloidal silicon dioxide, and starches.

Stabilizers:
Various useful stabilizers include basic inorganic salts, such as but not limited to basic inorganic salts of sodium, potassium, magnesium and calcium. Examples of basic inorganic salts of sodium are sodium carbonate, sodium hydrogen carbonate, sodium hydroxide, and the like. Examples of basic inorganic salts of potassium are potassium carbonate, potassium hydrogen carbonate, potassium hydroxide, and the like. Examples of basic inorganic salts of magnesium are heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg6Al2(OH)16.CO3.4H2O], aluminum hydroxide-magnesium [2.5MgO.Al2O3.xH2O], and the like. Examples of basic inorganic salts of calcium include precipitated calcium carbonate, calcium hydroxide, and the like.

Surface-Active Agents:
Useful surface-active agents include non-ionic, cationic and anionic surface-active agents. Useful non-ionic surface-active agents include ethylene glycol stearates, propylene glycol stearates, diethylene glycol stearates, glycerol stearates, sorbitan esters (SPANTM) and polyhydroxyethylenically treated sorbitan esters (TWEENTM), aliphatic alcohols and PEG ethers, phenol and PEG ethers. Useful cationic surface-active agents include quaternary ammonium salts (e.g. cetyltrimethylammonium bromide) and amine salts (e.g. octadecylamine hydrochloride). Useful anionic surface-active agents include sodium stearate, potassium stearate, ammonium stearate, and calcium stearate, triethenolamine stearate, sodium lauryl sulphate, sodium dioctylsulphosuccinate, and sodium dodecylbenzenesulphonate. Natural surface-active agents may also be used, such as for example phospholipids, e.g. diacylphosphatidyl glycerols, diaceylphosphatidyl cholines, and diaceylphosphatidic acids, the precursors and derivatives thereof, such as for example soybean lecithin and egg yolk.

Lubricants:
An effective amount of any pharmaceutically acceptable tableting lubricant can be added to assist with compressing tablets. Useful tablet lubricants include magnesium stearate, glyceryl monostearates, palmitic acid, talc, carnauba wax, calcium stearate sodium, sodium or magnesium lauryl sulfate, calcium soaps, zinc stearate, polyoxyethylene monostearates, calcium silicate, silicon dioxide, hydrogenated vegetable oils and fats, stearic acid and combinations thereof.

Glidants:
One or more glidant materials, which improve the flow of powder blends and minimize dosage form weight variations can be used. Useful glidants include but are not limited to silicone dioxide, talc and combinations thereof.

Coloring Agents:
Coloring agents can be used to color code the compositions, for example, to indicate the type and dosage of the therapeutic agent therein. Suitable coloring agents include, without limitation, natural and/or artificial compounds such as FD&C coloring agents, natural juice concentrates, pigments such as titanium oxide, silicon dioxide, iron oxides, zinc oxide, combinations thereof, and the like.

Useful additives for coatings include but are not limited to plasticizers, antiadherents, opacifiers, solvents, and optionally colorants, lubricants, pigments, antifoam agents, and polishing agents.

Plasticizers:
Various useful plasticizers include but are not limited to substances such as castor oil, diacetylated monoglycerides, dibutyl sebacate, diethyl phthalate, glycerin, polyethylene glycol, propylene glycol, triacetin, and triethyl citrate. Also, mixtures of plasticizers may be utilized. The type of plasticizer depends upon the type of coating agent. An opacifier like titianium dioxide may also be present, typically in an amount ranging from about 10% to about 20% based on the total weight of the coating.

Instrument settings
1) Powder X-Ray Diffraction (PXRD)
The X-ray powder diffraction spectrum (XRPD) was recorded at room temperature using PANalytical X’pert PRO diffractogram with Cu K radiation (?=1.54060 A°), running at 45 kv and 40ma.
2) Thermogravimetric analysis
Thermogravimetric analysis was performed using a Pyris 1 TGA make PERKIN ELMER measurement unit. 2-5 mg samples were placed in open Platinum pans and heated from 25 °C to 300 °C in a nitrogen atmosphere at a heating rate of 10 °C/min.
3) Differential Scanning Calorimetry
Differential Scanning Calorimetry was performed using a DSC 8000 make PERKIN ELMER differential instrument. 2-3 mg samples were placed in 50 µl crimped aluminum pans and heated from 30 °C to 250 °C in a nitrogen atmosphere at a heating rate of 10 °C/minute.
Single Crystal XRD Method for Analysis of crystalline monohydrate of Sofosbuvir:
Data were collected on a Rigaku Mercury 375/M CCD (XtaLAB mini) diffractometer using graphite monochromated Mo K_radiation at 296 K. The data were processed with CrystalClear software (Rigaku, 2009). Structure solution and refinements were executed using SHELX97 (Sheldrick, 2008) and the WinGX (Farrugia, 1999) suite of programs. All non-hydrogen atoms were refined with the full-matrix least-squares method. The hydrogen atoms were located from fourier maps or using a riding model.

Examples
Example 1: Preparation of Crystalline monohydrate of Sofosbuvir
Sofosbuvir (10gm) was charged to reaction flask. Acetonitrile (100ml) and Water (100ml) were added to reaction mixture. The reaction mixture was stirred at 50°C and kept for evaporation at room temperature. The obtained solid was dried under vacuum to afford novel crystalline monohydrate form of sofosbuvir.
Example 2: Preparation of Crystalline monohydrate of Sofosbuvir
Sofosbuvir (5 gm) was charged to reaction flask. Methanol (30ml) was added to reaction mixture. The reaction mixture was stirred to get clear solution. Reaction mass was filtered and solvent was evaporated. Water was added to the reaction mixture, the reaction mixture was stirred overnight. The obtained solid was dried under vacuum to afford crystalline monohydrate form of sofosbuvir.
Example 3: Preparation of Crystalline monohydrate of Sofosbuvir
Sofosbuvir (1gm) was charged in reaction flask. Methanol (5ml) was added to reaction mixture. The reaction mixture was added to a flask containing aqueous sodium chloride solution (5%). The reaction mixture was stirred, the solid was filtered and dried to afford crystalline monohydrate form of sofosbuvir.
,CLAIMS:1. A stable crystalline sofosbuvir hydrate.
2. The stable crystalline sofosbuvir hydrate according to claim 1 is monohydrate.
3. The stable crystalline sofosbuvir hydrate according to claim 1 is characterized by a PXRD pattern having peaks at 2? angle positions 7.5;11.4; 14.1; 14.5; 15.2; 15.8; 16.3; 20.4;22.9 and 30.1 ± 0.2° 2?.
4. The stable crystalline sofosbuvir hydrate according to claim 1 is characterized by a XRPD pattern as shown in Figure 1.
5. The stable crystalline sofosbuvir hydrate according to claim 1 is characterized by a DSC pattern as shown in Figure 2.
6. The stable crystalline sofosbuvir hydrate according to claim 1 is characterized by a TGA pattern as shown in Figure 3.
7. A process for preparation of stable crystalline sofosbuvir hydrate comprising the steps of:
(i) providing a solution of Sofosbuvir in a solvent;
(ii) adding water;
(iii) isolating crystalline sofosbuvir hydrate.

8. The process according to claim 7, wherein the solvent is selected from dichloromethane; 1,2-dichloroethane; chloroform; carbon tetrachloride; methanol; ethanol; isopropyl alcohol; 1-propanol; 2-propanol; 1-butanol; 2-butanol; t-butyl alcohol; acetone; ethyl methyl ketone; diethyl ketone; methyl isobutyl ketone; ethyl acetate; n-propyl acetate; n-butyl acetate; t-butyl acetate; diethyl ether; dimethyl ether; diisopropyl ether; methyl t-butyl ether;1,4-dioxane; acetonitrile and propionitrile or mixtures thereof
9. The process according to claim 8, wherein solvent is methanol.

10. The process according to claim 7, the isolation step is carried by one of the technique selected from filtration, evaporation, concentration of solvent and distillation of solvent.