WO2019025600A1

WO2019025600A1 - Sofosbuvir hydrate

Info

Publication number: WO2019025600A1
Application number: PCT/EP2018/071156
Authority: WO
Inventors: Manuel ALBERTINI; Veronika WERNER; Anke Schuster
Original assignee: Sandoz Ag
Priority date: 2017-08-03
Filing date: 2018-08-03
Publication date: 2019-02-07
Also published as: EP3661944A1

Abstract

The present invention relates to a hydrate of sofosbuvir, more precisely to a monohydrate of sofosbuvir, and to a process for its preparation. Furthermore, the invention relates to a pharmaceutical composition comprising the sofosbuvir hydrate and at least one pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be used as a medicament, in particular for the treatment of viral hepatitis C infections.

Description

SOFOSBUVIR HYDRATE

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION Sofosbuvir is an orally available nucleotide analog NS5B polymerase inhibitor of hepatitis C virus (HCV) and acts as a prodrug, which is converted through a series of in vivo transformations to an active triphosphate metabolite. It is indicated for the treatment of hepatitis C virus infections, either alone (Sovaldi^®), or in combination e.g. with ledipasvir (Harvoni^®), velpatasvir (Epclusa^®) or velpatasvir and voxilaprevir (Vosevi^®). Sofosbuvir has the IUPAC name (5)-isopropyl 2-(((5)-(((2i?,3i?,4i?,5i?)-5-(2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (phenoxy)phosphoryl)-amino)propanoate and can be represented by the chemical structure according to formula (I)

(I)-

Sofosbuvir and its preparation are described in WO 2008/121634 A2. WO 2010/135569 Al discloses amorphous sofosbuvir and crystalline forms 1 to 5 as well as processes for their preparation. According to WO 2010/135569 Al, crystalline form 1 is an anhydrate, whereas crystalline forms 2 and 3 are solvates with dichloromethane and chloroform, respectively. However, no sufficient data could be collected to determine, whether crystalline forms 4 and 5 are unsolvated, hydrated or solvated forms of sofosbuvir. It is further mentioned that all crystalline forms transform to crystalline form 1 upon isolation and that crystalline form 1 liquefies when exposed to elevated humidity levels. WO 2011/123645 Al discloses an additional crystalline form, form 6, of sofosbuvir. According to example 21 of WO 2011/123645 Al, crystalline form 6 can be prepared in two different ways. On the one hand, crystalline form 6 is obtained by exposing crystalline form 1 to atmospheric humidity for 6 to 10 weeks, whereby a solidified gum is formed which needs to be ground prior to further storage in order to obtain crystalline form 6. On the other hand, crystalline form 6 is prepared by stirring a mixture of crystalline form 1 in water at a concentration of 5-50 mg/mL.

WO 2015/099989 A2 discloses two additional crystalline forms of sofosbuvir designated as form 7 and form 8. The DSC and gravimetric moisture sorption curves provided in Figures 4- 5 and 9-10 of said application indicate that both forms are of anhydrous nature. Crystalline forms of sofosbuvir are also subject-matter for example in patent applications WO 2015/126995 A2, WO 2015/191945 A2, WO 2016/008461 Al, WO 2016/016327 Al, WO 2016/023906 Al, WO 2016/035006 Al, WO 2016/038542 A2, CN104130302A, CN104974205A, CN105732751A and CN105985394. However, none of these applications indicate the presence of hydrated forms of sofosbuvir. WO 2016/070569 Al discloses besides solvates with methanol (Form H2), ethanol (Form H3) and diphenyl ether (Form H4) a monohydrate form of sofosbuvir, which is denominated "form HI" in said application.

Different solid forms of an active pharmaceutical ingredient often possess different physical and chemical properties. For example, the presence of water molecules in the crystal lattice influences intermolecular interactions and confers unique physical properties to hydrates. Consequently, solubility and dissolution rate of a hydrate usually differ from those of the corresponding anhydrous phase, which increases the repertoire of materials available for a formulation scientist and may allow for the development of more customized formulations such as extended release formulations. In general, hydrates are thermodynamically more stable than their anhydrous counterparts in aqueous media. Therefore, hydrates are of special interest because drug product manufacturing often uses water-based processes such as wet-granulation and aqueous film- coating, which may lead to solid form transitions of anhydrous forms e.g. due to hydrate formation. The sudden appearance or disappearance of a solid form of an active pharmaceutical ingredient can pose a problem in process development. Similarly, serious pharmaceutical consequences can arise if transformation occurs in a dosage form. Due to their stability, hydrates may therefore allow for formulations with improved stability and shelf- life and thus for the preparation of safe and efficacious drug products. However, according to WO 2016/070569 Al, the therein disclosed monohydrate form HI shows partial deliquescence when exposed to an atmosphere of 25 °C/60% RH for 72 hours and completely liquefies when subjected to an atmosphere of 40 °C/75% RH for 72 hours, which poses a huge problem since drug substances may be exposed to water and water vapor during storage e.g. when the finished dosage form is an aqueous suspension, when the drug substance is subjected to an atmosphere containing water vapor or the drug substance is part of a dosage form consisting of materials that contain water and are capable of transferring it to other ingredients. It is further mentioned in WO 2016/070569 Al that the monohydrate form HI is obtained as colorless needles. Needle-shaped crystals are less preferred by formulation scientists, since said morphology usually translates into poor powder properties such as unfavorable filterability, low bulk density, poor compressibility and powder flow.

It is thus an objective of the present invention to provide an improved hydrate form of sofosbuvir, in particular a hydrate of sofosbuvir which is physically stable and preserves its crystal structure regardless the relative humidity of the surrounding atmosphere. A further objective of the present invention is the provision of a hydrate of sofosbuvir with improved powder characteristics such as flowability, bulk density and compressability.

SUMMARY OF THE INVENTION

The present invention relates to a hydrate of sofosbuvir, more precisely to a monohydrate of sofosbuvir, according to formula (II)

ηΗ₂0 (Π), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example n is 1.0, characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (7.6 ± 0.2)°, (12.7 ± 0.2)° and (17.0 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm.

The inventors of the present invention have surprisingly found, that in contrast to the monohydrate form HI of WO 2016/070569 Al, the hydrate of the present invention does not liquefy but preserves its crystal structure when subjected to atmospheres of 40 °C/ 75% RH or 25 °C/ 60% RH, at least for 72 hours. It was further found that the hydrate of the present invention shows almost no interaction with water vapor during a GMS experiment over the whole relative humidity range of from 0 to 90% relative humidity, when measured at (25.0 ± 0.1) °C. In addition, the lath-like morphology of the crystalline material confers excellent powder properties to the hydrate of the present invention, such as good flowability, high bulk density and good compressibility.

Abbreviations

PXRD powder x-ray diffraction

FTIR Fourier transform infrared

DSC differential scanning calorimetry GMS gravimetric moisture sorption

RH relative humidity

SXRD single crystal x-ray diffraction TGA thermogravimetric analysis

DFMU 2 ' -Deoxy-2 ' - fluoro-2 ' -methyluridine

SEM scanning electron microscopy

NMR nuclear magnetic resonance API active pharmaceutical ingredient

DMSO Dimethylsulfoxide

ACNL Acetonitrile

FCT film-coated tablet

Definitions The term "sofosbuvir" as used herein refers to (5)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy) (phenoxy)phosphoryl)-amino)propanoate according to formula (I) disclosed herein above.

The term "sofosbuvir form 1" as used herein, refers to the crystalline form of sofosbuvir, which is disclosed in WO 2010/135569 Al and can be characterized by having a powder X- ray diffractogram comprising reflections at 2-theta angles of (5.0 ± 0.2)°, (7.3 ± 0.2)°, (9.4 ± 0.2)° and (18.1 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai _j2 radiation having a wavelength of 0.15419 nm.

The term "sofosbuvir form 6" as used herein, refers to the crystalline form of sofosbuvir, which is disclosed in WO 201 1/123645 Al and can be characterized by having a powder X- ray diffractogram comprising reflections at 2-theta angles of (6.1 ± 0.2)°, (8.2 ± 0.2)°, (10.4 ± 0.2)°, (12.7 ± 0.2)°, (17.2 ± 0.2)°, (17.7 ± 0.2)°, (18.0 ± 0.2)°, (18.8 ± 0.2)°, (19.4 ± 0.2)°, (19.8 ± 0.2)°, (20.1 ± 0.2)°, (20.8 ± 0.2)° (21.8 ± 0.2)° and (23.3 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm. The term "sofosbuvir form 7" as used herein, refers to the crystalline form of sofosbuvir, which is disclosed in WO 2015/099989 Al and can be characterized by having a powder X- ray diffractogram comprising reflections at 2-theta angles of (12.6 ± 0.2)°, (13.5 ± 0.2)°, (16.9 ± 0.2)° and (17.3 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm.

The term "sofosbuvir form 8" as used herein, refers to the crystalline form of sofosbuvir, which is disclosed in WO 2015/099989 Al and can be characterized by having a powder X- ray diffractogram comprising reflections at 2-theta angles of (8.6 ± 0.2)°, (9.2 ± 0.2)°, (14.2 ± 0.2)°, (15.6 ± 0.2)°, (16.0 ± 0.2)°, (17.1 ± 0.2)°, (17.5 ± 0.2)°, (18.1 ± 0.2)°, (19.8 ± 0.2)° and (25.6 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with Cu- Kalphai _j2 radiation having a wavelength of 0.15419 nm.

As used herein, the term "measured at a temperature in the range of from 20 to 30 °C" refers to a measurement under standard conditions. Typically, standard conditions mean a temperature in the range of from 20 to 30 °C, i.e. at room temperature. Standard conditions can mean a temperature of about 22 °C. Typically, standard conditions can additionally mean a measurement under 20-80% relative humidity, preferably 30-70% relative humidity, more preferably 40-60% relative humidity and most preferably 50% relative humidity.

The term "reflection" with regards to powder X-ray diffraction as used herein, means peaks in an X-ray diffractogram, which are caused at certain diffraction angles (Bragg angles) by constructive interference from X-rays scattered by parallel planes of atoms in solid material, which are distributed in an ordered and repetitive pattern in a long-range positional order. Such a solid material is classified as crystalline material, whereas amorphous material is defined as solid material, which lacks long-range order and only displays short-range order, thus resulting in broad scattering. According to literature, long-range order e.g. extends over approximately 100 to 1000 atoms, whereas short-range order is over a few atoms only (see "Fundamentals of Powder Diffraction and Structural Characterization of Materials " by Vitalij K. Pecharsky and Peter Y. Zavalij, Kluwer Academic Publishers, 2003, page 3).

As used herein, the term "amorphous" refers to a solid form of a compound that is not crystalline. An amorphous compound possesses no long-range order and does not display a definitive X-ray diffraction pattern with reflections. The term "essentially the same" with reference to powder X-ray diffraction means that variabilities in reflection positions and relative intensities of the reflections are to be taken into account. For example, a typical precision of the 2-theta values is in the range of ± 0.2° 2- theta, preferably in the range of ± 0.1° 2-theta. Thus, a reflection that usually appears at 7.6° 2-theta for example can appear between 7.4° and 7.8° 2-theta, preferably between 7.5 and 7.7° 2-theta on most X-ray diffractometers under standard conditions. Furthermore, one skilled in the art will appreciate that relative reflection intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, sample preparation and other factors known to those skilled in the art and should be taken as qualitative measure only.

The term "essentially the same" with reference to Fourier infrared spectrometry means that variabilities in peak positions and relative intensities of the peaks are to be taken into account. For example, a typical precision of the wavenumber values is in the range of ± 2 cm^"1. Thus, a peak at 1740 cm^"1 for example can appear in the range of from 1738 to 1742 cm^"1 on most infrared spectrometers under standard conditions. Differences in relative intensities are typically smaller compared to X-ray diffraction. However, one skilled in the art will appreciate that small differences in peak intensities due to degree of crystallinity, sample preparation and other factors can also occur in infrared spectroscopy. Relative peak intensities should therefore be taken as qualitative measure only. The term "physical form" as used herein refers to any crystalline and/or amorphous phase of a compound.

The term "hydrate" as used herein refers to a crystalline solid, in which either water is cooperated in or accommodated by the crystal structure e.g. is part of the crystal structure or entrapped into the crystal (water inclusions). Thereby, water can be present in a stoichiometric or non-stoichiometric amount.

The term "non- hygroscopic" as used herein refers to compounds showing a mass change of less than 2 weight%, preferably of less than 1 weight% and most preferably of less than 0.5 weight% in the sorption cycle from 0 to 90% relative humidity, when measured with gravimetric moisture sorption at a temperature of (25.0 ± 0.1) °C, based on the weight of the compound. The terms "laths" or "lath-shaped" as used herein with regard to particle shape refers to elongated, thin and blade-like crystals.

As used herein the term "needles" describes acicular, thin and highly elongated crystals having similar width and breadth. As used herein, the term "essentially pure" with reference to the sofosbuvir hydrate of the present invention, means that the sofosbuvir hydrate includes less than about 20 weight%, preferably less than about 10 weight%, more preferably less than about 5 weight%, even more preferably less than about 2 weight% and most preferably less than about 1 weight% of any other physical form of sofosbuvir, in particular any of form 1, form 6, form 7 form 8, amorphous sofosbuvir or mixtures of two or more thereof as defined herein.

Crystalline forms of sofosbuvir may be referred to herein as being characterized by a powder X-ray diffractogram or a Fourier transform infrared spectrum "as shown in" a figure. The person skilled in the art understands that factors such as variations in instrument type, response and variations in sample directionality, sample concentration, sample purity, sample history and sample preparation may lead to variations, for example relating to the exact reflection or peak positions and intensities. However, a comparison of the graphical data in the figures herein with the graphical data generated for an unknown physical form and the confirmation that two sets of graphical data relate to the same crystal form is well within the knowledge of a person skilled in the art. As used herein, the term "mother liquor" refers to the solution remaining after crystallization of a solid from said solution.

A "predetermined amount" as used herein with regard to sofosbuvir of the present invention refers to the initial amount of sofosbuvir used for the preparation of a pharmaceutical composition having a desired dosage strength of sofosbuvir. The term "effective amount" as used herein with regard to sofosbuvir of the present invention encompasses an amount of sofosbuvir which causes the desired therapeutic effect.

As used herein, the term "about" means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, typically within 10%, more typically within 5%, even more typically within 1% and most typically within 0.1% of the indicated value or range. Sometimes, such a range can lie within the experimental error, typical of standard methods used for the measurement and/or determination of a given value or range.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: illustrates a representative powder X-ray diffractogram of the hydrate of sofosbuvir according to the present invention. The x-axis shows the scattering angle in °2-theta, the y- axis shows the intensity of the scattered X-ray beam in counts of detected photons.

Figure 2: illustrates a further representative powder X-ray diffractogram of the hydrate of sofosbuvir according to the present invention. Again, the x-axis shows the scattering angle in °2-theta and the y-axis shows the intensity of the scattered X-ray beam in counts of detected photons.

Figure 3: illustrates a representative Fourier transform infrared spectrum of the hydrate of sofosbuvir according to the present invention. The x-axis shows the wavenumbers in cm^"1, the y-axis shows the relative intensity in percent transmittance.

Figure 4: illustrates a representative differential scanning calorimetry curve of the hydrate of sofosbuvir according to the present invention. The x-axis shows the temperature in degree Celsius (°C), the y-axis shows the heat flow rate in Watt per gram (W/g) with endothermic peaks going up.

Figure 5: illustrates a representative photomicrographic image of lath-shaped sofosbuvir hydrate crystals of the present invention under a light microscope (scale-bar 100 micrometer). Figure 6: illustrates the dissolution rate in phosphate buffer (900 mL, 75 rpm, 37°C) of 400mg tablets sofosbuvir hydrate (Hydrate 1, 2 and 3) and form 6 (Sovaldi^® 1, 2 and 3) according to example 14.

Figure 7: illustrates the dissolution rate measured with a discriminatory dissolution method (110 mL HC1 0.1 N, 150 rpm, 37°C) of two 400mg tablets containing sofosbuvir hydrate (Hydrate 1, 2 and 3) and form 6 (Sovaldi^® 1, 2 and 3) according to example 14.

Figure 8: illustrates the recovery percentage of sofosbuvir hydrate and form 6 at 0 to 6 months (hydrate) and 0 to 3 months (form 6) at the stress conditions 1) 25°C/60% RH and 2) 40°C/75% RH according to example 12. Figure 9: illustrates the recovery percentage of sofosbuvir hydrate and form 6 (Sovaldi ) in tablets subjected to stress conditions of 25°C/60% RH in climate chamber at 0 to 9 months according to example 13.

Figure 10: illustrates the dissolution rate measured with a discriminatory dissolution method (110 mL HCl 0.1 N, 150 rpm, 37°C) of two film-coated 400mg tablets containing of sofosbuvir hydrate and form 6 (Sovaldi^®) after 9 months stress test (25°C/60% RH) wherein the dissolution is expressed as concentration (mg/mL) per minutes according to example 13.

Figure 11: illustrates the sofosbuvir hydrate and the form 6 plasma level in male Beagle dogs following oral administration (mean value (n=10)-semi log scale) according to example 15. Figure 12: illustrates the sofosbuvir hydrate and the form 6 plasma level in female Beagle dogs following oral administration (mean value (n=10)-semi log scale) according to example 15.

Figure 13: illustrates the metabolite DMFU plasma level in male Beagle dogs following oral administration of a tablet comprising sofosbuvir hydrate or form 6 (mean value (n=10)-semi log scale) according to example 15.

Figure 14: illustrates the metabolite DMFU plasma level in female Beagle dogs following oral administration of a tablet comprising sofosbuvir hydrate or form 6 (mean value (n=10)- semi log scale) according to example 15.

Figure 15: illustrates the SEM image of sofosbuvir hydrate of the invention. Figure 16: illustrates the NMR spectrum of the sofosbuvir hydrate of the invention. Figure 17: illustrates the SRDX of the sofosbuvir hydrate of the invention. DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a hydrate of sofosbuvir, more precisely to a monohydrate of sofosbuvir. The sofosbuvir hydrate of the present invention is physically stable e.g. preserves its crystal structure and does not liquefy for at least 72 hours when exposed to atmospheres of 25 °C/60% RH and 40 °C/75% RH, respectively. Furthermore, the sofosbuvir hydrate of the present invention is physically stable e.g. preserves its crystal structure and does not liquefy for at least 6 months when exposed to atmospheres of 25 °C/60% RH and 40 °C/75% RH, respectively (see example 12). Furthermore, the hydrate of the present invention shows almost no interaction with water vapor during a GMS experiment over the whole relative humidity range of from 0 to 90%, when measured at (25.0 ± 0.1) °C and therefore can be assigned to be non-hygroscopic. Moreover, the hydrate of the present invention is thermodynamically stable when stirred in water. In addition, the lath-like morphology of the crystalline material confers excellent powder properties to the hydrate of the present invention, such as good flowability, high bulk density and good compressibility. All in all, these favorable properties allow for a straightforward formulation of drug products containing the hydrate of the present invention by using standard formulation processes and without the need for controlled atmospheres during production and storage. The provision of a physically stable hydrate of sofosbuvir, like the hydrate of the present invention, is highly desirable because the risk of phase transitions, which may occur during manufacture and/or storage of a drug product, such as solid form conversions and/or deliquescence, can be excluded. Such transitions can in general have serious consequences for the pharmaceutical product with regard to safety and efficacy. Hence, the hydrate of the present invention is the favored hydrate form of sofosbuvir, to be used for the preparation of a pharmaceutical drug product. This is because the usage of the sofosbuvir hydrate of the present invention ensures a reliable safety and efficacy profile of a drug product containing said hydrate during the whole shelf-life of the product.

The hydrate of sofosbuvir of the present invention may be characterized by analytical methods well known in the field of the pharmaceutical industry for characterizing crystalline solids. Such methods comprise but are not limited to PXRD, SXRD, FTIR, DSC, TGA, GMS and microscopy. The hydrate of the present invention may be characterized by one of the aforementioned analytical methods or by combining two or more of them. In particular, the hydrate of the present invention may be characterized by any one of the following embodiments or by combining two or more of the following embodiments.

Hence, in a first embodiment, the invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II)

ηΗ₂0 (Π), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of:

(7.6 ± 0.2)°, (12.7 ± 0.2)° and (17.0 ± 0.2)°,

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (12.7 ± 0.2)° and (17.0 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.7 ± 0.2)° and (17.0 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.7 ± 0.2)°, (14.2 ± 0.2)° and (17.0 ± 0.2)°;

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (1 1.5 ± 0.2)°, (12.7 ± 0.2)°, (14.2 ± 0.2)°, (17.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (14.2 ± 0.2)°, (17.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (13.4 ± 0.2)°, (14.2 ± 0.2)°, (17.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (13.4 ± 0.2)°, (14.2 ± 0.2)°, (15.9 ± 0.2)°, (17.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (13.4 ± 0.2)°, (14.2 ± 0.2)°, (15.9 ± 0.2)°, (16.5 ± 0.2)°, (17.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (13.4 ± 0.2)°, (14.2 ± 0.2)°, (15.9 ± 0.2)°, (16.5 ± 0.2)°, (17.0 ± 0.2)°, (18.0 ± 0.2)° and (20.5 ± 0.2)°; or

(7.6 ± 0.2)°, (10.4 ± 0.2)°, (11.5 ± 0.2)°, (12.2 ± 0.2)°, (12.7 ± 0.2)°, (13.4 ± 0.2)°, (14.2 ± 0.2)°, (15.9 ± 0.2)°, (16.5 ± 0.2)°, (17.0 ± 0.2)°, (18.0 ± 0.2)°, (19.6 ± 0.2)° and (20.5 ± 0.2)°; when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai_i2 radiation having a wavelength of 0.15419 nm. In another embodiment, the invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of:

(7.6 ± 0.1)°, (12.7 ± 0.1)° and (17.0 ± 0.1)°,

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (12.7 ± 0.1)° and (17.0 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.7 ± 0.1)° and (17.0 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.7 ± 0.1)°, (14.2 ± 0.1)° and (17.0 ± 0.1)°;

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (1 1.5 ± 0.1)°, (12.7 ± 0.1)°, (14.2 ± 0.1)°, (17.0 ± 0.1)° and (20.5 ±

0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (14.2 ± 0.1)°, (17.0 ± 0.1)° and (20.5 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (13.4 ± 0.1)°, (14.2 ± 0.1)°, (17.0 ± 0.1)° and (20.5 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (13.4 ± 0.1)°, (14.2 ± 0.1)°, (15.9 ± 0.1)°, (17.0 ± 0.1)° and (20.5 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (13.4 ± 0.1)°, (14.2 ± 0.1)°, (15.9 ± 0.1)°, (16.5 ± 0.1)°, (17.0 ± 0.1)° and (20.5 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (13.4 ± 0.1)°, (14.2 ± 0.1)°, (15.9 ± 0.1)°, (16.5 ± 0.1)°, (17.0 ± 0.1)°, (18.0 ± 0.1)° and (20.5 ± 0.1)°; or

(7.6 ± 0.1)°, (10.4 ± 0.1)°, (11.5 ± 0.1)°, (12.2 ± 0.1)°, (12.7 ± 0.1)°, (13.4 ± 0.1)°, (14.2 ± 0.1)°, (15.9 ± 0.1)°, (16.5 ± 0.1)°, (17.0 ± 0.1)°, (18.0 ± 0.1)°, (19.6 ± 0.1)° and (20.5 ± 0.1)°; when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm.

In yet another embodiment, the present invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a powder X-ray diffractogram essentially the same as shown in figure 1 or figure 2 of the present invention, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm.

The powder X-ray diffractogram of the sofosbuvir hydrate of the present invention can be clearly distinguished from sofosbuvir hydrate form HI of WO 2016/070569 Al . A reflection list of the hydrate of the present invention is provided in Table 2 hereinafter and a reflection list of form HI is provided in Table 2 of WO 2016/070569 Al . The hydrate of the present invention shows for example a reflection at (12.7 ± 0.2)° 2-theta, whereas Form HI shows no reflection in the same range. On the other hand, the hydrate of the present invention for example shows no reflection in the range of from 2.0 to 7.3° 2-theta, whereas according to the disclosure of WO 2016/070569 Al, form HI possesses several characteristic reflections in this range, present at 4.68°, 4.93°, 5.20°, 6.64° and 7.12° 2-theta.

Thus, in a further embodiment, the invention relates to a hydrate, preferably a monohydrate of sofosbuvir, as defined above, characterized by having a powder X-ray diffractogram comprising no reflections in the range of from 2.0 to 7.3° 2-theta, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm.

In a further embodiment, the invention relates to a hydrate, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a powder X-ray diffractogram comprising no reflections in the range of from 2.0 to 7.3° 2-theta, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm.

In another embodiment, the present invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a Fourier transform infrared spectrum comprising peaks at wavenumbers of:

(3512 ± 2) cm^"1, (1740 ± 2) cm^"1 and (1673 ± 2) cm^"1; or

(3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1 and (1673 ± 2) cm^"1; or

(3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1 and (946 ± 2) cm^"1; or (3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1, (946 ± 2) cm^"1 and (760 ± 2) cm^"1 ; or

(3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1, (1250 ± 2) cm^"1, (946 ± 2) cm^{" 1} and (760 ± 2) cm^"1 ; or (3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1, (1458 ± 2) cm^"1, (1250 ±2) cm^" (946 ± 2) cm^"1 and (760 ± 2) cm^"1; or

(3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1, (1458 ± 2) cm^"1, (1250 ± 2) cm^{" J}, (1082 ± 2) cm^"1, (946 ± 2) cm^"1 and (760 ± 2) cm^"1; or

(3512 ± 2) cm^"1, (3402 ± 2) cm^"1, (1740 ± 2) cm^"1, (1673 ± 2) cm^"1, (1458 ± 2) cm^"1, (1250 ± 2) cm^{" J}, (1208 ± 2) cm^"1, (1082 ± 2) cm^"1, (946 ± 2) cm^"1 and (760 ± 2) cm^"1;

when measured at a temperature in the range of from 20 to 30 °C with a diamond ATR cell.

In yet another embodiment, the invention relates to hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by having a Fourier transform infrared spectrum essentially the same as shown in figure 3 of the present invention, when measured at a temperature in the range of from 20 to 30 °C with a diamond ATR cell.

In another embodiment, the present invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by a differential scanning calorimetry curve comprising an endothermic peak having an onset temperature in the range of from 73 to 77 °C, when measured with DSC at a heating rate of 10 K/min. In still another embodiment, the present invention relates to a hydrate of sofosbuvir, preferably a monohydrate of sofosbuvir, according to formula (II), wherein n is in the range of from 0.7 to 1.2, preferably of from 0.9 to 1.1, even more preferably of from 0.95 to 1.05, for example wherein n is 1.0, characterized by a differential scanning calorimetry curve comprising an endothermic peak having a peak temperature in the range of from 77 to 80 °C, when measured with DSC at a heating rate of 10 K/min.

In one aspect, the present invention relates to a composition comprising the hydrate of sofosbuvir of the present invention as defined in any of the embodiments described above, said composition being essentially free of any other physical form of sofosbuvir. For example, a composition comprising the hydrate of sofosbuvir of the present invention comprises at most 20 weight%, preferably at most 10 weight%, more preferably at most 5 weight%, even more preferably at most 2 weight% and most preferably at most 1 weight% of any other physical form of sofosbuvir, based on the weight of the composition. Preferably, the any other physical form of sofosbuvir is form 1 of WO 2010/135569 Al, form 6 of WO 2011/123645 A2, form 7 of WO 2015/099989 A2, form 8 of WO 2015/099989 A2, amorphous sofosbuvir or a combination of any two or more thereof. In a further aspect, the present invention relates to a process for the preparation of the sofosbuvir hydrate of the present invention or the composition comprising the sofosbuvir hydrate as defined in any of the embodiments described above comprising:

(a) providing sofosbuvir in crystalline form 1, crystalline form 8, amorphous form or mixtures of two or more thereof;

(b) suspending the sofosbuvir provided in (a) in water, wherein the sofosbuvir provided in (a) is present in the suspension in an amount in the range of from 100 to 700 g/L;

(c) slurrying the suspension provided in (b);

(d) optionally, seeding the suspension obtained in (c) with sofosbuvir hydrate crystals according to the present invention;

(e) optionally, separating at least a part of the crystals obtained in (c) or (d) from the mother liquor;

(f) optionally, washing the isolated crystals obtained in (e); and

(g) optionally, drying the crystals obtained in any one of steps (c) to (f).

Crystalline form 1 of sofosbuvir can be prepared according to example 10 of WO 2011/123645 Al . Crystalline form 8 of sofosbuvir can be prepared according to example 4 of WO 2015/099989 A2. Said procedure requires form 6 seed crystals, which may be prepared either by exposing crystalline form 1 to atmospheric humidity for 6 to 10 weeks, whereby a solidified gum is formed which needs to be ground prior to further storage in order to obtain crystalline form 6 or by stirring a mixture of crystalline form 1 in water at a concentration of 5-50 mg/mL as described in WO 2011/123645 Al . Amorphous sofosbuvir can be prepared by lyophilization of an aqueous ethanol solution comprising sofosbuvir e.g. according to reference example 1 herein.

Sofosbuvir form 1, form 8, amorphous sofosbuvir or mixtures of two or more thereof are suspended in water e.g. deionized water, wherein the sofosbuvir is present in the suspension in an amount in the range of from 100 to 700 g/L, more preferably of from about 100 to 300 g/L, for example the concentration is about 100 g/L. The suspension is slurried, preferably at room temperature but depending on the applied concentration slurrying may also be conducted at elevated temperature for example at a temperature in the range of from about 40 to 80 °C, preferably from about 40 to 60 °C. Slurrying encompasses any kind of movement of the solid material suspended in water caused by, but not limited to e.g. agitation, stirring, mixing, shaking, vibration, sonication, wet milling and the like.

Slurrying is conducted for a time sufficient that at least a substantial part, preferably all of the form 1, form 8 and/or amorphous sofosbuvir starting material has converted to the hydrate of the present invention. Preferably slurrying is performed for a period in the range of from several hours to several days. Slurrying may for example be performed for a period in the range of from 30 min to 7 days or longer, preferably of from 2 hours to 7 days, more preferably of from 1 hour to 5 days, most preferably of from 2 hours to 3 days. The skilled person may monitor the conversion of sofosbuvir form 1, form 8 and/or amorphous sofosbuvir to the hydrate of the present invention by withdrawing samples from the slurry and analyzing the samples by e.g. powder X-ray diffraction. Optionally, sofosbuvir hydrate seed crystals may be added in order to accelerate the formation of the hydrate of the present invention. The amount of seed crystals employed may range from about 1 to 20 weight%, preferably from about 1 to 10 weight% and most preferably from about 1 to 5 weight%, based on the weight of applied sofosbuvir starting material. Seed crystals may be prepared according to steps (a) to (c) of the above described procedure. Once the sofosbuvir hydrate of the present invention is obtained or preferably obtained in essentially pure form, at least a part of the crystals may be optionally separated from the mother liquor. Preferably, the crystals are separated from their mother liquor by any conventional method such as filtration, centrifugation, solvent evaporation or decantation, more preferably by filtration or centrifugation and most preferably by filtration. Optionally, in a further step the isolated crystals are washed with water e.g. with deionized water.

The obtained crystals may then optionally be dried. Drying may be performed at a temperature in the range of from about 20 to 50 °C, preferably in the range of from about 20 to 40 °C and most preferably in the range of from about 20 to 30 °C. Drying may be performed for a period in the range of from about 1 to 72 hours, preferably of from about 2 to 48 hours, more preferably of from about 4 to 24 hours and most preferably of from about 6 to 18 hours. Drying may be performed at ambient pressure and/ or under reduced pressure. Preferably, drying is performed at a pressure of about 100 mbar or less, more preferably of about 50 mbar or less and most preferably of about 30 mbar or less, for example a vacuum of about 20 mbar or less such as about 1 mbar.

As shown in the above described process, form 1, form 8 and amorphous sofosbuvir each undergo phase transformations to the hydrate of the present invention when slurried for a sufficiently long period of time in water at a concentration of at least 100 g/L. This indicates that the hydrate of sofosbuvir of the present invention is thermodynamically more stable in aqueous systems e.g. in water compared to forms 1 , 8 and amorphous sofosbuvir.

Moreover, the sofosbuvir hydrate of the present invention is physically more stable compared to the monohydrate form HI of WO 2016/070569 Al, since it preserves its crystal structure and does not liquify for at least 72 hours when exposed to atmospheres of 25 °C/60% RH and 40 °C/75% RH, respectively (see example 6 herein). In contrast, according to WO 2016/070569 Al, form HI shows partial deliquescence when exposed to an atmosphere of 25 °C/60% RH for 72 hours and completely liquifies when subjected to an atmosphere of 40 °C/75% RH for 72 hours.

Furthermore, the hydrate of the present invention almost shows no interaction with water vapor during a GMS experiment over the whole relative humidity range of from 0 to 90%, when measured at (25.0 ± 0.1) °C and therefore can be assigned to be non- hygroscopic.

In addition, its lath-like morphology (see figure 5 herein) confers excellent powder properties to the sofosbuvir hydrate of the present invention, such as good flowability, high bulk density and good compressibility. In contrast thereto, it is mentioned in WO 2016/070569 Al that the monohydrate form HI is obtained as colorless needles. Needle-shaped crystals are less preferred by formulation scientists, since said morphology usually translates into poor powder properties such as unfavorable filterability, low bulk density, poor compressibility and powder flow.

The fact that the hydrate of the present invention can be prepared from water alone without the need of organic solvents is an additional advantage, since organic solvents may be expensive and hazardous to health and the environment. According to WO 2016/070569 Al the preparation of the therein disclosed monohydrate requires the usage of hazardous organic solvents such as anisole and an ether solvent selected from dibutyl ether or isopropyl ether.

In a further aspect, the present invention relates to the use of the sofosbuvir hydrate of the present invention as defined in any of the embodiments described above for the preparation of a pharmaceutical composition.

The pharmaceutical composition of the present invention can be prepared by wet or dry processing methods. In certain embodiments the pharmaceutical composition is prepared by wet processing methods, such as, but not limited to, wet granulation methods. Suitable wet granulation methods comprise high-shear granulation or fluid-bed granulation. In another embodiment the pharmaceutical composition is prepared by dry processing methods, such as, but not limited to, direct compression or dry granulation methods. An example of dry granulation is roller compaction. The pharmaceutical composition obtained by dry or wet processing methods may be compressed into tablets, encapsulated or metered into sachets.

In a further aspect, the present invention relates to a pharmaceutical composition comprising the sofosbuvir hydrate of the present invention or the composition comprising the hydrate as defined in any of the embodiments described above, preferably in an effective and/or predetermined amount, and at least one pharmaceutically acceptable excipient and optionally one or more additional active pharmaceutical ingredient(s). Most preferably, the pharmaceutical composition of the present invention is an oral solid dosage form, such as a tablet or a capsule. Preferably, the pharmaceutical composition of the present invention is a tablet. In a preferred embodiment, the tablet is film-coated with a coating material comprising one or more pharmaceutically acceptable excipients selected from the group consisting of polyvinyl alcohol, talc, macrogol, polyethylene glycol, titanium dioxide and a colorant. In a particular embodiment, the coloring agent of the film coating is selected from the group consisting of iron oxide (e.g. yellow, red), ferrosoferric oxide and sunset yellow FCF aluminium lake.

The at least one pharmaceutically acceptable excipient, which is comprised in the pharmaceutical composition of the present invention, is preferably selected from the group consisting of carriers, fillers, diluents, lubricants, sweeteners, stabilizing agents, solubilizing agents, antioxidants and preservatives, flavouring agents, binders, colorants, osmotic agents, buffers, surfactants, disintegrants, granulating agents, coating materials and combinations thereof.

In a preferred embodiment, the at least one pharmaceutically acceptable excipient is selected from the group consisting of mannitol, microcrystalline cellulose, croscarmellose sodium, colloidal anhydrous silica and magnesium stearate. In a preferred embodiment, all of these pharmaceutically acceptable excipients are comprised by the pharmaceutical composition of the present invention.

In another preferred embodiment, the at least one pharmaceutically acceptable excipient is selected from the group consisting of copovidione, lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, colloidal anhydrous silica and magnesium stearate. In a preferred embodiment, all of these pharmaceutically acceptable excipients are comprised by the pharmaceutical composition of the present invention.

In still another preferred embodiment, the at least one pharmaceutically acceptable excipient is selected from the group consisting of colloidal silicon dioxide, copovidone, croscarmellose sodium, lactose monohydrate, magnesium stearate and microcrystalline cellulose. In a preferred embodiment, all of these pharmaceutically acceptable excipients are comprised by the pharmaceutical composition of the present invention.

In still another preferred embodiment, the at least one pharmaceutically acceptable excipient is selected from the group consisting of copovidone, croscarmellose sodium, magnesium stearate and microcrystalline cellulose. In a preferred embodiment, all of these pharmaceutically acceptable excipients are comprised by the pharmaceutical composition of the present invention.

In another preferred embodiment, the one or more additional active pharmaceutical ingredient(s) is/are selected from the group consisting of daclatasvir, dasabuvir, elbasvir, grazoprevir, ledipasvir, ombitasvir, paritaprevir, ribavirin, simeprevir, velpatasvir and voxilaprevir. Most preferably, the one or more additional active pharmaceutical ingredient(s) is/are selected from the group consisting of ledipasvir, velpatasvir and voxilaprevir.

Preferably, the present invention relates to a pharmaceutical composition as described above, wherein the predetermined and/or effective amount of the sofosbuvir hydrate of the present invention is 400 mg calculated as water-free sofosbuvir. Preferably, the present invention relates to a pharmaceutical composition as described above, wherein the pharmaceutical composition is to be administered once-daily.

In a further aspect, the present invention relates to the hydrate of sofosbuvir or the pharmaceutical composition as described above for use as a medicament. In yet another aspect, the invention relates to the hydrate of sofosbuvir of the present invention or the pharmaceutical composition as described above for use in the treatment of viral hepatitis C infections.

In another aspect the present invention relates to the hydrate of sofosbuvir of the present invention or the pharmaceutical composition as described above intended for the treatment of viral hepatitis C infections in combination with one or more additional active pharmaceutical ingredient(s) selected from the group consisting of daclatasvir, dasabuvir, elbasvir, grazoprevir, ledipasvir, ombitasvir, paritaprevir, ribavirin, simeprevir, velpatasvir, and voxilaprevir. Preferably, the additional active pharmaceutical ingredient(s) is ledipasvir or velpatasvir or velpatasvir and voxilaprevir. A treatment in combination with one or more additional active pharmaceutical ingredient(s) can mean the administration of a pharmaceutical dosage form comprising the hydrate of sofosbuvir of the present invention and the one or more additional active pharmaceutical ingredient(s) in the same dosage form, for example as a fixed-dose combination.

Alternatively, a treatment in combination with one or more additional active pharmaceutical ingredient(s) can mean the administration of separate pharmaceutical dosage forms, one comprising the sofosbuvir hydrate of the present invention, and the other(s) comprising the one or more additional active pharmaceutical ingredient(s) in separate dosage form(s). Typically in such a combination treatment instructions are provided that the pharmaceutical dosage form comprising the sofosbuvir hydrate of the present invention is to be administered in combination with said separate dosage form(s) for the effective treatment of a viral infection, such as hepatitis C infection.

EXAMPLES

The following non-limiting examples are illustrative for the disclosure and are not to be construed as to be in any way limiting for the scope of the invention. Example 1

Example la: Preparation of the sofosbuvir hydrate of the present invention

A round bottom flask equipped with a magnetic stirrer was charged with deionized water and sofosbuvir, either in form 1 of WO 2010/135569 Al, form 8 of WO 2015/099989 A2 or in amorphous form at concentrations in the range of from 100 to 700 g/L. The obtained suspensions were stirred at room temperature for a period in the range of from 5 to 60 h before the solids were collected by filtration and dried under vacuum (1 mbar) for 72 hours. Table la below provides further experimental details with regard to applied amounts and physical forms of the starting material as well as stirring time. PXRD confirmed that all experiments listed in Table la yielded the hydrate of the present invention.

Table la

Table 1 a: Preparation of the hydrate of the present invention - experimental details

Example lb: Preparation of the sofosbuvir hydrate of the present invention

In a 250 mL round bottom flask, equipped with a magnetic stirrer, approximately 10 g Sofosbuvir (form 1) was suspended in 100 mL of deionized H₂0 and stirred (220 rpm) at RT for 60 h. A sample was taken and analyzed by PXRD. The PXRD confirmed that the experiment yielded the hydrate of the present invention. Example lc (1 to 8): Preparation of the sofosbuvir hydrate of the present invention

In a 20 mL glass flask equipped with a mechanical stirrer, 200 mg Sofosbuvir (form 1) was suspended in 6 mL aqueous media (Table lc) and stirred (260 rpm) at 37°C for 24 h. A sample of the suspension was then taken, filtered and the solid was analyzed by PXRD. The PXRD confirmed that all experiments listed in Table lb yielded the hydrate of the present invention.

Table lb: Different aqueous media tested:

FeSSIF = Fed State Simulated Intestinal Fluid

*FaSSIF = Fasted State Simulated Intestinal Fluid

Example Id (A to F): Preparation of the sofosbuvir hydrate of the present invention

In a lOOmL flask, equipped with a magnetic stirrer, 2.0 g of Sofosbuvir (see Table Id for the starting material) was suspended in 20mL of deionized water (100 g/L) and the mixture was stirred for 5h. After lh and 2h, a small sample was taken for PXRD analysis. After 5h, the suspension was filtered and the solid was dried at RT under vacuum for 72 h. The formation of the hydrate was confirmed by PXRD for all samples. Table lc: Conditions for the preparation of sofosbuvir hydrate of the invention:

Example le: Attempt to transform sofosbuvir hydrate into form 6

Sofosbuvir hydrate according to the invention was dried under vacuum at 50 °C for 24h. A sample was analyzed by PXRD and resulted in the sofosbuvir hydrate according to the invention (no reaction occurred). The hydrate is therefore stable under these conditions.

Example 2: Powder X-ray diffraction (PXRD)

The sofosbuvir hydrate according to the present invention was investigated by powder X-ray diffraction. PXRD was performed with a PANalytical X'Pert PRO diffractometer equipped with a theta/theta coupled goniometer in transmission geometry,

radiation (wavelength 0.15419 nm) with a focusing mirror and a solid state PIXcel detector. Diffractograms were recorded at a tube voltage of 45 kV and a tube current of 40 mA, applying a stepsize of 0.013° 2-theta with 40s per step (255 channels) in the angular range of 2° to 40° 2-theta at ambient conditions. A typical precision of the 2-theta values is in the range of ± 0.2° 2-theta, preferably of ± 0.1° 2-theta. Thus, the diffraction peak of the sofosbuvir hydrate of the present invention that appears for example at 7.6° 2-theta can appear in the range of from 7.4 to 7.8° 2-theta, preferably in the range of from 7.5 to 7.7° 2-theta on most X-ray diffractometers under standard conditions. Representative diffracto grams of the hydrate of sofosbuvir according to the present invention are displayed herein in figure 1 and figure 2, respectively. The reflection list corresponding to the diffractogram presented in figure 1 is provided in Table 2 below.

Table 2

of from 2 to 30° 2-theta; A typical precision of the 2-theta values is in the range of ± 0.2° 2-theta, preferably of ± 0.1° 2-theta.

Example 3: Fourier transform infrared (FTIR) spectroscopy

The sofosbuvir hydrate according to the present invention was investigated by FTIR spectroscopy. The FTIR spectrum was recorded (obtained) on an MKII Golden Gate™ Single Reflection Diamond ATR cell with a Bruker Tensor 27 FTIR spectrometer with 4 cm^"1 resolution at RT. To record a spectrum a spatula tip of a sample was applied to the surface of the diamond in powder form. Then the sample was pressed onto the diamond with a sapphire anvil and the spectrum was recorded. A spectrum of the clean diamond was used as background spectrum. A typical precision of the wavenumber values is in the range of about ± 2 cm^"1. Thus, the infrared peak of the sofosbuvir hydrate according to the present invention that appears at 1673 cm^"1 can appear between 1671 and 1675 cm^"1 on most infrared spectrometers under standard conditions. A representative FTIR spectrum of the hydrate of sofosbuvir according to the present invention is displayed in figure 3 and the corresponding peak list from 4000 to 600 cm^"1 is provided in Table 3 below.

Table 3

Wavenumber Wavenumber Wavenumber Wavenumber

[cm ¹] [cm ¹] [cm ¹] [cm ¹]

3512 1673 1208 858

3402 1490 1152 839

3269 1458 1082 817

3066 1382 1024 800

2988 1311 971 760

2941 1271 946 721

1740 1250 887 688

Table 3: FTIR peak list of hydrate of sofosbuvir according to the present invention between 4000 and 600 cm^"1; a typical precision of the wavenumbers is in the range of ± 2 cm^"1

Example 4: Differential scanning calorimetry (DSC)

The sofosbuvir hydrate according to the present invention was investigated by DSC, which was performed on a Mettler Polymer DSC R instrument. Different sofosbuvir hydrate samples (from 3.5 mg to 4.7 mg) were heated in a 40 microL aluminium pan with a pierced aluminium lid from 25 to 200 °C at a rate of 10° K/min. Nitrogen (purge rate 50 mL/min) was used as purge gas. The DSC curves show an endothermic peak with an onset temperature in the range of from about 73 to 77 °C and a peak temperature in the range of about 77 to 80 °C.

Example 5: Light microscopy The photomicrographic image displayed in figure 5 was collected with a Keyence VHX-5000 microscope and a Keyence VHX-5020 camera.

Example 6: Stress test

The sofosbuvir hydrate of the present invention was subjected to different stress conditions according to Table 4 below. The occurrence or absence of deliquescence was observed visually, while the occurrence or absence of crystal structure changes was investigated by means of PXRD. The results are summarized in Table 4 below.

Table 4

Table 4: Stress conditions and results In contrast to the monohydrate form HI disclosed in WO 2016/070569 Al, which is reported to liquify under the above applied stress conditions, the sofosbuvir hydrate of the present invention not only remained solid but also preserved its crystal structure, indicating its excellent physical stability even under accelerated stress conditions.

Example 7: HPLC

HPLC: Agilent 1200 Series/1260 Infinity Series

Column: HALO CI 8, 150 x 4.6 mm, 2.7 μιη

Oven temperature: 27 °C

Injection volume: 2.0 μΐ.

Flow: 1.3 mL/min

Posttime: 7 min

Detector: 254 nm

Stock A: 1.200 g KH₂P0₄ add 1.0 L H₂0 (pH ~ 4.8)

Stock B: 730 mL ACNL + 270 mL Methanol

Eluent A: 900 mL Stock A + 100 mL ACNL Eluent B: 880 mL Stock B + 120 mL H₂0

Setting (pump): t [min] 0 3 12 25 28 28.1 35

% Eluent B 3 25 40 100 100 3 3 Sample preparation: appr. 0.8 mg/mL in H₂0/ACNL (70:30) Example 8: Karl Fischer titration

The water content was determined by coulometric Karl Fischer titration with a Metrohm 832 KF Thermoprep oven and a Metrohm 831 KF Coulometer using a generator electrode without diaphragm and Hydranal-Coulomat AG Oven analyte solution. The sample was heated to 110°C in the oven and the released water transferred to the titration cell for analysis, using dry air as a carrier gas. Samples were analyzed in triplets.

Example 9: NMR

NMR spectra were recorded on a Bruker AVANCE III HD 400 nano spectrometer equipped with a Prodigy Cryoprobe head. 1H and ¹³C spectra were recorded in DMSO-d6 at 298 K. Chemical shifts are reported as δ-values in ppm relative to the residual solvent peak of DMSO-d6 (5H: 2.50; 5c: 39.5). For the characterization of the observed signal multiplicities the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sept (septet), m (multiplet) as well as br (broad).

Quantitative NMR (qNMR) analysis was performed using the internal standard 1,2,4,5- tetrachloro-3 -nitrobenzene (TCNB) in DMSO-d6 with a chemical shift of 8.5 ppm (1H- NMR). The spectrum is shown in figure 16.

Example 10: Scanning Electron Microscopy (SEM)

The SEM images were recorded with a TM3030Plus from Hitachi. The image is shown in figure 15. Reference Example 1: Preparation of amorphous sofosbuvir

To crystalline form 1 of sofosbuvir (400 mg, prepared according to WO 2011/123645 Al, Example 10) were added 3.5 mL ethanol, followed by 12 mL of deionized water. The mixture was subjected to sonication (2 to 5 min at room temperature in a VWR Ultrasonic Cleaner apparatus) to accelerate the dissolution of the solid material. The homogeneous solution was frozen in a bath of liquid nitrogen and lyophilized at -36 °C at a pressure of from 0 to 2 mbar, yielding amorphous sofosbuvir.

Reference Example 2: Tableting

All components of the tablet mixture were sieved through a 1000 μιη mesh screen and thoroughly mixed using a TURBULA® T2F three dimensional shaker-mixer (WAB) for 5 min (level 34).

Tablets were pressed on a single punch tablet press Flexitab S (Roltgen Marking System) with a 9 x 20 mm die (lower punch height: 12 mm) using 280 bar compaction pressures (return time: 3 sec). Reference Example 3: Dissolution Test Apparatus

Dissolution was performed on a paddle apparatus (Agilent) according to BP/EP/USP II, consisting of a waterbath (708-DS) for eight identical vessels (lOOOmL or 200mL) connected to a variable wavelength UV analytical detector.

Example 11: Comparison of the solubility the hydrate of the invention and sofosbuvir form 6 The solubility in water of the sofosbuvir hydrate of the invention and sofosbuvir form 6 has been tested (1.5g of API in 20 mL of water at 37°C over 24h) . The results are reported in the Table 5 herein below:

Table 5 time concentration

[h] [mg/mL]

Form 6 hydrate

0 2.2 2.4 4 1.9 2.2

24 2.1 2.4

Average 2.1 2.3

RSD [%] 7.4 4.9

Example 12: Long-term stability tests of sofosbuvir hydrate

A long-term stability tests of the sofosbuvir hydrate of the invention in climate chambers under two conditions were performed (25 °C/60% RH and 40 °C/75% RH; both open flask). The same conditions have been used by Gilead to test the water absorption of form 6.

The following analytical measurements were performed within these stress tests:

Water content

- HPLC assay (content of API)

PXRD (polymorphic stability)

- SEM images

Example 12a: Water content

The water content of the samples was measured according to the method disclosed in Example 8. The results are reported in Tables 6 and 7:

Table 6: Water content of the hydrate of the invention over time during storage

Stress initial 3 1 3 6

2h 4h 24h

conditions value days month months months

25 °C/60%

2.99% 2.79% 2.85% 2.84% 2.86% 2.73% 2.90% 2.67% RH

40 °C/75%

2.99% 2.90% 2.84% 2.91% 2.83% 3.03% 2.79% 2.66% RH Table 7: Water content of Form 6 over time during storage

No increased water content was observed over the 6 months of stress conditions. The initial value of about 3% of sofosbuvir hydrate matches the water content of a monohydrate form (18 g/mol / 547.5 g/mol = 0.0329, that is * 100% = 3.29%).

Example 12b: HPLC Assay

The HPLC was performed according to Example 7. The HPLC assay revealed that no degradation of the Sofosbuvir hydrate occurs under this stress conditions as specified herein below. The conditions and the results are reported in Table 8 herein below and in figure 8.

Table 8

Sofosbuvir hydrate

Storage time

Storage conditions (content Sofosbuvir)

[months]

[%]

25 °C/60% RH 0.1 98.3

25 °C/60% RH 1 97.5

25 °C/60% RH 3 101.2

25 °C/60% RH 6 101.0

40 °C/75% RH 0.1 100.9

40 °C/75% RH 1 99.8

40 °C/75% RH 3 99.6

40 °C/75% RH 6 100.5

Storage conditions Storage time Form 6 [month] (content Sofosbuvir)

[%]

25 °C/60% RH 0.1 104.3

25 °C/60% RH 0.5 101.5

25 °C/60% RH 1 101.2

25 °C/60% RH 3 99.8

40 °C/75% RH 0.1 98.7

40 °C/75% RH 0.5 103.4

40 °C/75% RH 1 100.3

40 °C/75% RH 3 97.7

Example 12c: PXRD Analysis

Samples for the PXRD analysis were collected. The PXRD analysis revealed that sofosbuvir hydrate is stable during 6 months of stress as no transformation of the crystalline structure was observed. The conditions and the results are reported in Tables 9 and 10:

Table 9

Sofosbuvir

25°C / 60% RH 40°C / 75% RH

hydrate

Storage time Results Results

Oh (Start) Sofosbuvir hydrate Sofosbuvir hydrate

2h Sofosbuvir hydrate Sofosbuvir hydrate

4h Sofosbuvir hydrate Sofosbuvir hydrate

24h Sofosbuvir hydrate Sofosbuvir hydrate

72h Sofosbuvir hydrate Sofosbuvir hydrate

1 month Sofosbuvir hydrate Sofosbuvir hydrate 3 months Sofosbuvir hydrate Sofosbuvir hydrate

6 months Sofosbuvir hydrate Sofosbuvir hydrate

Table 10

Example 12d: SEM imaging The SEM image of sofosbuvir hydrate is shown in figure 15.

Example 13: Long-term stability (up to 9 months) tests of tablets comprising sofosbuvir hydrate

Tablets comprising sofosbuvir hydrate were subjected to a long term stability test. The test was carried out in a climate chambers at 25 °C/60% RH. The tablets were sealed in Aluminium Compound Foil packages. To make better predictions of the API- and polymorph stability, the same tests was carried out with the tablets of Sovaldi^® (400 mg FCT) comprising form 6.

The following analytical measurements were performed within these stress tests:

Water content

- HPLC assay (content of API)

PXRD (polymorphic stability) Dissolution

The tested tablets had the following composition:

Table 11

400mg of sofosbuvir hydrate corresponding to 386.8mg of sofosbuvir Example 13a: Water content

The tablets as disclosed above were kept under stress conditions as reported in the tables herein below. The water content of the samples was measured according to the Karl Fischer method disclosed in Example 8. The water concentration of the tablets stressed at 25°C/60% RH is of 3.1wt% (after 9 month) The tablets of the commercial product Sovaldi^® were also tested with an increase of water content of 2.6 wt.%. The results are reported in Table 12a.

Table 12a

Tablets Stress Duration Average weight Water content conditions [months] [g] [%]

hydrate

1 tablet 25 °C/60% RH 1 1.200 1 tablet 25 °C/60% RH 3 1.215

4 tablets 25 °C/60% RH 9 1.216 3.1

Sovaldi^®

1 tablet 25 °C/60% RH 1 1.2497

1 tablet 25 °C/60% RH 3 1.2469

4 tablets 25 °C/60% RH 9 1.2436 2.6

Example 13b: HPLC

The tablets were analyzed with respect to stability of the API via HPLC. The results are reported in the below Table 12b and in figure 9.

Table 12b

The HPLC assay revealed an API recoveries >94%, accordingly no degradation of the API occurred under these stress conditions within the formulation. Example 13c: PXRD

PXRD analysis revealed following outcome: sofosbuvir hydrate was stable during the tested stress conditions at 25 °C/60% RH

Example 13d: Dissolution Two tablets (9 month stressed at 25 °C/60% RH) containing sofosbuvir hydrate were tested using the discriminatory dissolution method and compared with two tablets (9 months stressed) containing form 6 (Sovaldi^®). The dissolutions profiles are reported in figure 10 expressed as drug concentration (mg/mL) per minutes.

Example 14: Preparation of tablets comprising 400 mg of sofosbuvir hydrate of the invention

Film coated tablet comprising each 400 mg of sofosbuvir hydrate were prepared for the dog- pharmacokinetic study using the below formulation.

Ingredients 1 to 5 of Table 13 were sieved through a 800μιη mesh screen and thoroughly mixed using a Heidolph Reax 20 at level 16 for 15 min and additionally 5 min after the addition of ingredient 6. This part of the mixture was dry compacted via a TFC Lab Mikro, Freund Vector with a 100 bar pressure, granulated thereafter and sieved through a 840μιη and a 250μιη mesh sieve. The fraction between those screens was further processed.

Ingredients 7 to 9 of Table 13 were added to the granulate and thoroughly mixed using a Heidolph Reax 20 at level 16 for 15 min and additionally 5 min after the addition of ingredient 10. The tablets were produced using a FlexiTab single-punch tablet press (Roltgen Marking System) with a 19.3 x 8.4 mm die, applying 13kN compaction force.

The tablets were coated with ingredient 11 dispersed in water.

Table 13

Number Component Content [%]

1 Sofosbuvir hydrate 32.9

2 Mannitol 28.6 3 Microcrystalline Cellulose 24.0

4 Croscarmellose Sodium 2.4

5 Colloidal Silicon Dioxide 0.44

6 Magnesium Stearate 0.73

7 Microcrystalline Cellulose 4.9

8 Croscarmellose Sodium 2.4

9 Colloidal Silicon Dioxide 0.05

10 Magnesium Stearate 0.73

11 Polyvinyl Alcohol Coating 2.9

The dissolution test was performed with the apparatus according to Reference Example 3. The dissolution test was performed in phosphate buffer (900 mL, 75 rpm, 37 °C). The dissolution profiles of the sofosbuvir hydrate tablet and the commercial product Sovaldi^® was compared (figure 6). The dissolution test was repeated using the 400mg tablets in 110 mL HC1 0.1 N, 150 rpm, 37°C (figure 7).

Examples 15: Pharmacokinetic studies in Beagle dogs.

Pharmacokinetic studies were carried out on Beagle dogs comparing 400mg film-coated tablets comprising sofosbuvir hydrate and prepared according to Example 14 and Solvaldi (form 6). The tablets were orally administered. In this study bio equivalence (BE) with the reference Sovaldi^® was shown. The blood plasma levels for the parent compound sofosbuvir (see figures 11 and 12) as well as for the inactive metabolite (=DFMU, figures 13 and 14) were very well aligned.

Cited prior art: -WO 2008/121634 A2 -WO 2010/135569 Al

-WO 2011/123645 Al

-WO 2015/099989 A2 -WO 2016/008461 Al -WO 2016/016327 Al -WO 2016/023906 Al -WO 2016/035006 Al -WO 2016/038542 A2 -CN104130302A -CN 104974205 A -CN105732751A -CN105985394 -WO 2016/070569 Al

Claims

1) A hydrate of sofosbuvir according to formula (II)

nH₂0 (Π), wherein n is in the range of from 0.9 to 1.1, characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (7.6 ± 0.2)°, (12.7 ± 0.2)° and (17.0 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai_,2 radiation having a wavelength of 0.15419 nm.

The hydrate of claim 1 characterized by having a powder X-ray diffractogram comprising additional reflections at 2-theta angles of (10.4 ± 0.2)° and (11.5 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm.

The hydrate of claim 1 or 2 characterized by having a powder X-ray diffractogram comprising no reflection at 2-theta angles in the range of from 2.0 to 7.3°, when measured at a temperature in the range of from 20 to 30 °C with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm.

The hydrate according to any one of the preceding claims characterized by having a Fourier transform infrared spectrum comprising peaks at wavenumbers of (3512 ± 2) cm^"1, (1740 ± 2) cm^"1 and (1673 ± 2) cm^"1, when measured at a temperature in the range of from 20 to 30 °C with a diamond ATR cell.

The hydrate according to claim 4 characterized by having a Fourier transform infrared spectrum comprising additional peaks at wavenumbers of (3402 ± 2) cm^"1 and (946 ± 2) cm^"1, when measured at a temperature in the range of from 20 to 30 °C with a diamond ATR cell.

The hydrate according to any one of the preceding claims characterized by having a differential scanning calorimetry curve comprising an endotherm with a peak temperature in the range of from 77 to 80 °C, when measured at a heating rate of 10 K/min.

7) A composition comprising the hydrate according to any one of the preceding claims and at most 20 weight%, 10 weight%, 5 weight%, 2 weight% or 1 weight% of any other physical form of sofosbuvir, based on the weight of the composition.

8) A process for the preparation of the sofosbuvir hydrate as defined in any one of claims 1 to 6 or the composition as defined in claim 7 comprising:

(a) providing sofosbuvir in crystalline form 1 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (5.0 ± 0.2)°, (7.3 ± 0.2)°, (9.4 ± 0.2)° and (18.1 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm, in crystalline form 8 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (8.6 ± 0.2)°, (9.2 ± 0.2)°, (14.2 ± 0.2)°, (15.6 ± 0.2)°, (16.0 ± 0.2)°, (17.1 ± 0.2)°, (17.5 ± 0.2)°, (18.1 ± 0.2)°, (19.8 ± 0.2)° and (25.6 ± 0.2)°, when measured at a temperature in the range of from 20 to 30 °C with

radiation having a wavelength of 0.15419 nm and/or in amorphous form;

(c) slurrying the suspension provided in step (b).

9) Use of the hydrate as defined in any one of claims 1 to 6 or the composition as defined in claim 7 for the preparation of a pharmaceutical composition.

10) A pharmaceutical composition comprising the hydrate as defined in any one of claims 1 to 6 or the composition as defined in claim 7 and at least one pharmaceutically acceptable excipient.

11) The pharmaceutical composition of claim 10, further comprising one or more additional active pharmaceutical ingredient(s).

12) The pharmaceutical composition of claim 11, wherein the one or more additional active pharmaceutical ingredient(s) is/are selected from the group consisting of ledipasvir, velpatasvir and voxilaprevir.

13) The pharmaceutical composition according to any one of claims 10 to 12, wherein the pharmaceutical composition is an oral solid dosage form. 14) The pharmaceutical composition of claim 13, wherein the oral solid dosage form is a tablet.

15) The pharmaceutical composition according to anyone of claims 10 to 14 comprising a predetermined and/or effective amount of the sofosbuvir hydrate according to any one of claims 1 to 6 wherein the predetermined and/or effective amount of the sofosbuvir hydrate is 400 mg calculated as water-free sofosbuvir.

16) The hydrate as defined in any one of claims 1 to 6 or the pharmaceutical composition according to any one of claims 10 to 15 for use in the treatment of viral hepatitis C infections.