EA040073B1 - PHARMACEUTICAL COMPOSITIONS BASED ON TROPOMYOSIN RELATED KINASE (TRK) INHIBITORS - Google Patents

Description

Background of the invention

The field of technology to which the invention belongs

The invention relates to the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b] pyridine-2-amine and pharmaceutical compositions based on it. 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-III-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- amine is an inhibitor of tropomyosin-related kinases (Trk inhibitor). The monohydrate form has desirable properties that facilitate the preparation of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine in pharmaceutical formulations.

Trk inhibitor, 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Himidazo[4,5-b]pyridine- 2-amine and methods for producing a Trk inhibitor are disclosed in International Patent Application PCT/US14/69469 and US Patent Application No. 14/564773, each of which is called tropomyosin-related kinase (TRK) inhibitors, which are incorporated herein by links.

Brief description of the invention

The first aspect of the present invention relates to a compound represented by the following structural formula:

The second aspect of the present invention relates to crystalline form I of the above compound, the powder X-ray diffraction (XRPD) which contains the following peaks at 2θ, measured using CuKa radiation: 3.6, 7.1, 8.9, 10.4, 10 .7, 12.4, 12.7 and 14.3.

The third aspect of the present invention relates to a pharmaceutical composition having an inhibitory effect on tropomyosin-related kinases (Trk) or cellular receptor for colony stimulating factor-1 (c-FMS), containing the above crystalline Form I and a pharmaceutically acceptable excipient.

The fourth aspect of the present invention relates to a pharmaceutical composition having a Trk or c-FMS inhibitory action, containing the above compound and a pharmaceutically acceptable excipient.

Brief description of graphics images

Fig. 1 - powder X-ray diffraction (XRPD) of the monohydrate form of 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4 ,5-b]pyridine-2-amine, 4-40 at °2θ. XRPD of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2 -amine shows unique peaks at 7.14, 8.89, 10.22, 12.42, 12.73 and 14.31 at 2θ measured using CuKa radiation.

Fig. 2 is a differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) thermogram of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazole-4- yl)-3H-imidazo[4,5-b]pyridine-2-amine heated at 10°C/min. The DSC thermogram shows three thermal events at 76.72, 160.13 and 195.78°C, the TGA thermogram shows a weight loss of 3.7% from 25 to 100°C.

Fig. 3 - crystal structure of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine. The crystal structure of the monohydrate form of the Trk inhibitor is shown.

Fig. 4 is an overlap of the XRPD diffractogram obtained from the experiments and the XRPD diffraction pattern calculated from the single crystal structure. This figure shows the overlap of the powder X-ray diffraction pattern obtained from the experiments with that calculated from the single-crystal structure. The high degree of agreement indicates that the single crystal

- 1 040073 structure is typical for bulk material.

Fig. 5 - API load effect on IVR profile - microspheres 12% API/9:1 R202H:752H, 16%

API/9:1 R202H:752H and 20% API/9:1 R202H:752H. This figure shows a comparison of the load effect of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b] pyridine-2-amine on the in vitro release profile of microspheres with different compositions.

Fig. 6 - thermograms of DSC microspheres 16% API/9:1 R202H:752H and 20% API/9:1 R202H:752H. The DSC thermogram indicates that 20% of the microspheres are characterized by endothermic melting between 130-150°C, which confirms the presence of a drug on the surface of the crystals.

Fig. 7 is a scanning electron microscope (SEM) image of 16% API/9:1 R202H:752H microspheres (1500X). Microspheres loaded with 16% drug show no drug crystals, indicating that the drug is amorphous.

Fig. 8 is a scanning electron microscope (SEM) image of 20% API/9:1 R202H:752H microspheres (1500X). Microspheres loaded with a drug by 20% are characterized by the presence of drug crystals on the surface.

Fig. 9 - Effect of API loading on IVR profile - 15% API/R202H, 17% API/R202H and 19% API/R202H microspheres. This figure shows a comparison of the loading effect of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine on the in vitro release profile of microspheres with different compositions.

Fig. 10 - API load effect on IVR profile - microspheres 16% API/9.5:0.5 R202H:752H, 18% API/9.5:0.5 R202H:752H and 2o% API/9.5:0, 5 R202H:752H. This figure shows a comparison of the load effect of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5b ]pyridine-2-amine on the in vitro release profile of microspheres with different compositions.

Fig. 11 - Effect of polymer blend on the IVR profile of 16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16% API/9:1 R202H:RG752H microspheres. This figure shows the effect of the polymer blend on the in vitro release profile of microspheres with different compositions.

Fig. 12 - Effect of polymer blend on the IVR profile of 16% API/R202H, 16% API/9.5:0.5 R202H:RG502H and 16% API/9:I R202H:RG502H microspheres. This figure shows the effect of the polymer blend on the in vitro release profile of microspheres with different compositions.

Fig. 13 - formulations showing a zero order IVR profile over 180 days - 15% API/9:1 R203H:RG752H and 16% API/9.5:0.5 R202H:RG502H microspheres. Microspheres prepared using 15% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Himidazo[4,5-b ]pyridine-2-amine in polymers R203H:752H at a ratio of 9:1 and 16% 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole-4 -yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202H:RG502H polymers at a ratio of 9.5:0.5 show a pseudo-zero order release profile over 6 months.

Fig. 14 - effect of auxiliary solvent systems used in preparation on the IVR profile - microspheres 16% API/9:1 R203H:RG752H - 9:1 DCM:MeOH, 9:0.5:0.5 DCM:MeOH:BA, 9 .5:0.5 DCM:BA, 9:1 DCM:BA. This figure shows a comparison of the co-solvent system used in the preparation of the microspheres against the in vitro release profile of microspheres with 16% drug loading and a 9:1 polymer blend of R203H:RG752H.

Fig. 15 - the effect of the DCM:BA co-solvent system to increase the API load on the IVR profile - microspheres 16% API / 9:1 R202H: RG752H, 25% API / 9: 1 R202H: RG752H, 30% API / R202H, 40% API / R202H, 25% API/R203H, 30% API/R203H, 40% API/R203H, 50% API/R203H. This figure shows a comparison of the effect of the excipient system used in the preparation of microspheres to increase drug loading on the in vitro release profile of microspheres with different polymer blends.

Fig. 16 is a scanning electron microscope (SEM) image of microspheres obtained using a fine suspension microencapsulation process (1500X). Microspheres obtained by encapsulating a suspension of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine were spherical in shape with a rough surface texture due to the presence of drug crystals embedded in the surface.

Fig. 17 - effect of microencapsulation process of fine suspension on IVR profile - microspheres 25% API/9:1 R202H:RG752H 39 ml 5% PVA+2.6 ml EA, 25% API/9:1 R202H:RG752H 39 ml 5% PVA+ 3.25 ml EA, 25% API/R203H 39 ml 5% PVA+2.0 ml EA, 30% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA+2.0 ml EA, 40% microfluidized API /9:1 R202H:RG752H 39 ml 5% PVA+2.0 ml EA, 30% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA+2.0 ml EA, 30% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA + 2.3 ml EA, 30% microfluidized API / R202S 39 ml 5% PVA + 2.0 ml EA, 30% microfluidized API / R203S 39 ml 5% PVA + 2.0 ml. This figure shows a comparison of the effect of the fine suspension microencapsulation process used in the preparation of microspheres on the in vitro release profile of microspheres with different drug loadings.

Fig. 18 Scanning Electron Microscope (SEM) image of 16% API/9:1 microspheres

R202H:RG752H, solvent extraction (1000X). The microspheres obtained by solvent extraction were spherical in shape with a smooth surface structure.

Fig. 19 is a scanning electron microscope (SEM) image of 16% API/9:1 R202H:RG752H microspheres, spray dried (1000X). The microspheres obtained by spray drying were spherical in shape with a peculiar surface structure.

Fig. 20 - Effect of the microencapsulation process on the IVR profile of 16% API/9:1 R202H:RG752H microspheres obtained by solvent extraction (OW), 16% API/9:1 R202H:RG752H microspheres obtained by spray drying (20 and 22 .5% polymer), and 16% API/9:1 R203H:RG752H microspheres obtained by spray drying (22.5% polymer). This figure shows a comparison of the in vitro release profile of 9:1 R202H:RG752H microspheres loaded with 16% drug obtained by solvent extraction and spray drying and the effect of the microencapsulation process on the in vitro release profile of microspheres with different compositions.

Fig. 21 is a scanning electron microscope (SEM) image of 16% API/1:1 R202H:R203H/no additives microspheres, spray dried (1000X). Microspheres present are 16% API/1:1 R202H:R203H/no additives obtained by spray drying.

Fig. 22 is a scanning electron microscope (SEM) image of 16% API/1:1 R202H:R203H/31.25 mg PEG microspheres, spray dried (1000X). 16% API/1:1 R202H:R203H/31.25 mg PEG microspheres are present, obtained by spray drying.

Fig. 23 is a scanning electron microscope (SEM) image of 16% API/1:1 R202H:R203H/31.25 mg poloxamer 407 microspheres, spray dried (1000X). 16% API/1:1 R202H:R203H/31.25 mg poloxamer 407 microspheres are present, obtained by spray drying.

Fig. 24 - Effect of % 10 kDa PEG or 1% Poloxamer 407 on IVR profile - 16% API/1:1 R202H:R203H/no additives, 16% API/1:1 R202H:R203H/31.25 mg PEG, 16 % API/1:1 R202H:R203H/31.25 mg poloxamer 407. This figure shows a comparison of the in vitro release profile of microspheres 16% API/1:1 R202H:R203H/no additives, microspheres 16% API/1:1 R202H :R203H/31.25 mg PEG and microspheres 16% API/1:1 R202H:R203H/31.25 mg poloxamer 407 obtained by spray drying.

Fig. 25 - IVR profile (rat) in vivo - microspheres 16% API/9:I 202H:RG502H and 15% API/9:1 R203H:RG752H. This figure shows the nearly zero order release profile in vivo over about 3-4 months and 5-6 months for 16% API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H microspheres.

Fig. 26 - retention over time [ ¹⁴ C] 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4, 5-b]pyridine-2-amine (rat knee) microspheres 16% API/9:1 202H:RG502H and 15% API/9:1 203H:RG752H. After intra-articular injection into the rat knee joints, microspheres 16% API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H showed drug release within 5-6 months; 16% API/9:1 202:H:RG502H shows 12% joint residue after 5 months and 15% API/9:1 R203H:RG752H shows 30% drug residue after 6 months.

Fig. 27 - concentration-time profiles for 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4, 5-b]pyridine-2-amine (rat blood) microspheres 16% API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H. This figure shows the blood drug concentration-time profile after intra-articular injection of microspheres loaded with 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole- 4-yl)-3Himidazo[4,5-b]pyridine-2-amine.

Fig. 28 - IVR profile - microspheres 16% API/9:1 2O2:H:RG752H, 15% API/9:1 R203H:RG752H and 40% API/203H.

Fig. 29 - concentration-time profiles for 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b] pyridine-2-amine microspheres 16% API/9:1 202:H:RG752H, 15% API/9:1 R203H:RG752H and 40% API/203H.

Fig. 30 Measured or simulated XRPD of forms 1-4 (anhydrous and hydrated forms). XRPD of anhydrous and hydrated forms of 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine.

Fig. 31 - XRPD form 5 (ethanol). XRPD of form 5 (ethanol) 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amines.

Fig. 32 - XRPD form 9 (acetone). XRPD of form 9 (acetone) 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amines.

Fig. 33 Comparison of XRPD Form 10 (acetone) and Form 9 (acetone). This figure shows a comparison of two forms of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Himidazo[4,5-b ]pyridine-2-amine obtained in acetone.

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Fig. 34 - XRPD form 11 (acetonitrile). XRPD of form 11 (acetonitrile) 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-III-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine.

Fig. 35 - solid crystalline phases -2-amines. This figure shows a comparison of ten crystalline forms of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-III-pyrazol-4yl)-3H-imidazo[4,5- b]pyridine-2-amine obtained from experiments.

Detailed description of the invention

The present invention relates to a compound represented by the following structural formula:

In addition, the present invention also relates to crystalline form I of the above compound, the X-ray powder diffraction pattern (XRPD) of which contains the following peaks at 2θ measured using CuKa radiation: 3.6, 7.1, 8.9, 10.4, 10.7, 12.4, 12.7 and 14.3.

The present invention also relates to a pharmaceutical composition having an inhibitory effect on tropomyosin-related kinases (Trk) or cellular receptor for colony stimulating factor-1 (c-FMS), containing the above crystalline Form I and a pharmaceutically acceptable excipient.

Said pharmaceutical composition is intended for the treatment of osteoarthritis, pain, and pain associated with osteoarthritis.

In a preferred embodiment of the present invention, the proposed pharmaceutical composition contains a pharmaceutically acceptable excipient, which includes a diluent, a suspending agent and a buffering agent.

Preferably, the diluent is sorbitol, the suspending agent is povidone, and the buffering agent is phosphoric acid.

The present invention also relates to a pharmaceutical composition having a Trk or cFMS inhibitory effect, containing (a.) the above compound and (b.) a pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutically acceptable excipient comprises a diluent.

In a preferred embodiment, the diluent is selected from: maltitol, sunflower oil, ammonium alginate, calcium carbonate, calcium lactate, anhydrous dibasic calcium phosphate, dibasic dihydrate, tribasic, calcium silicate, calcium sulfate, cellulose, cellulose acetate, compressible sugar, confectionery sugar, corn starch and pregelatinized starch, dextrates, dextrin, dextrose, erythritol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, lactose for inhalation, isomalt, kaolin, lactitol, lactose, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, medium chain triglycerides, microcrystalline cellulose, polydextrose, polymethacrylates, simethicone, sodium alginate, sodium chloride, sorbitol, starch, sucrose, sugar spheres, beta-cyclodextrin sulfobutyl ether, talc, tragacanth, trehalose, xylitol.

In a preferred embodiment, the pharmaceutically acceptable excipient comprises a suspending agent.

In a preferred embodiment, the suspending agent is selected from: gum arabic, agar, alginic acid, bentonite, calcium stearate, carbomer, carboxymethylcellulose, carrageenan, cellulose, colloidal silicon dioxide, dextrin, gelatin, guar gum, hectorite, hydrophobic colloidal silicon dioxide, hydroxyethylcellulose, hydroxymethylcellulose , hydroxypropylcellulose, hypromellose, kaolin, magnesium aluminosilicate, maltitol solutions, medium chain triglycerides, methylcellulose, phenylmercurborate, phospholipids, polycarbophil, polyethylene glycol, polyoxyethylene sorbitan fatty acid esters, povidone (polyvinylpyrrolidone), propylene glycol alginate, sesame oil, saponite, sodium alginate, esters of sorbitol, sucrose, tragacanth, vitamin E polyethylene glycol succinate and xanthan gum.

In a preferred embodiment, the pharmaceutically acceptable excipient comprises a buffering agent.

In a preferred embodiment, the buffering agent is selected from: adipic acid, ammonia solution, boric acid, calcium carbonate, calcium hydroxide, calcium lactate, calcium phosphate, tribasic, citric acid monohydrate, dibasic sodium phosphate, diethanolamine, glycine, maleic acid, malic acid, methionine , sodium phosphate monobasic, monoethanolamine, sodium monoglutamate, phosphoric acid, potassium citrate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate dihydrate, sodium hydroxide, sodium lactate, or triethanolamine.

The above pharmaceutical composition is for the treatment of osteoarthritis, pain and pain associated with osteoarthritis.

In a preferred embodiment, the pharmaceutical composition contains a pharmaceutically acceptable excipient, which includes a diluent, a suspending agent and a buffering agent.

Preferably, the diluent is sorbitol, the suspending agent is povidone, and the buffering agent is phosphoric acid.

Pharmaceutical formulations containing a Trk inhibitor of the present invention inhibit tropomyosin related kinase A (TrkA), tropomyosin related kinase B (TrkB), tropomyosin related kinase C (TrkC) or c-FMS (cellular receptor for colony stimulating factor-1 (CSF -1)). Tropomyosin-related kinases (Trks) are high-affinity receptors activated by soluble growth factors called neutrophins (NTs). TrkA, also known as neurotrophic tyrosine kinase type 1 receptor, is activated by nerve growth factor (NGF). TrkB is activated by brain growth factor and NT-4/5. TrkC is activated by NT3. Activation of Trk leads to the activation of downstream kinases that are involved in cell signaling, including proliferation, survival, angiogenesis, and metastasis. Trk has been implicated in a number of diseases, including OA.

Pharmaceutical formulations containing a Trk inhibitor of the present invention can also inhibit c-FMS (cellular receptor for colony stimulating factor-1 (CSF-1)). C-FMS has a role in the regulation of macrophage function and is thought to play a role in inflammatory diseases, autoimmune disease, bone metabolism disorders, and cancer (Burns and Wilks, 2011, Informa Healthcare).

OA is a widespread and severe joint disease characterized by chronic pain and destruction of articular cartilage. Recent clinical trials have confirmed the value of NGF blocking in OA knee pain, demonstrating significant pain relief and high treatment response rates in patients treated with intravenous infusion of anti-NGF blocking antibodies (Lane, 2010, N Engl J Med). However, this technique may lead to an increased risk of side effects due to systemic inhibition of the NGF signaling pathway (FDA Arthritis Advisory Committee Meeting to Discuss Safety Issues Related to the Anti-Nerve Growth Factor Agents; http://www.fda.gov/AdvisoryCommittees/Calendar /ucm286556.htm). Accordingly, novel approaches have been taken to target pain in NGF-mediated OA through the development of Trk inhibitors, namely TrkA inhibitors, the high affinity receptor for NGF (Nicol, 2007, Molecular Interv). The Trk inhibitors of the present invention are delivered locally and thus avoid the systemic distribution seen with intravenous administration of anti-NGF antibodies. This treatment strategy offers the advantage of higher doses, as well as greater safety by allowing the preservation of the physiologically essential NGF signaling pathway (ie preservation of sensory/sympathetic nerves, angiogenesis) at sites other than local application sites.

Pharmaceutical compositions containing Trk inhibitors can be administered in multiple dosage forms, including injections for local delivery of both microcrystalline suspension and sustained release formulations. In pharmaceutical compositions containing a Trk inhibitor, the Trk inhibitor is the active pharmaceutical ingredient. The Trk inhibitor may also be co-administered and/or formulated with other active ingredients for the treatment of a disease, including the treatment of pain, OA, and pain associated with OA.

3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- amine is also known as GZ389988.

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Formula (I) -2-amine (GZ3 89988).

The problem solved in the present invention lies in the difficulty of formulating compositions containing 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- 3H-imidazo[4,5-b]pyridine-2-amine. The solution to this problem is to reveal that the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo [4,5-b]pyridine-2-amine is characterized by improved physicochemical properties compared to the anhydride form, including improved physical stability and slower dissolution in water. The anhydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- amine is relatively unstable in solution, which leads to problems in its formulation into a pharmaceutical composition. The anhydrate form exhibits various changes in solid form under certain conditions, including conversion to a hydrate. Monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- amine provides additional stability and is essential to avoid polymorphism during long-term storage and processing, and also helps to achieve improved physical stability. In addition, slower dissolution in aqueous solution was observed when using the monohydrate form. Monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Himidazo[4,5-b]pyridine-2- amine, also known as GZ389988A, is represented by the following formula (II).

Formula (II): 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b] monohydrate form ]pyridine-2-amine (GZ389988A).

Molecular Weight of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)3H-imidazo[4,5-b]pyridine-2 -amine is 470.54 g/mol, and its chemical formula is C ₂₆ H ₂₆ N ₆ O ₃ . Monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- amine has the appearance of a white with a tinge of powder, which under a microscope under polarized light looks like thin needles or fibrous particles.

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The monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4yl)-3H-imidazo[4,5-b]pyridine-2 -amine is obtained in accordance with example 1.

Pharmaceutical formulations containing a Trk inhibitor may contain one or more pharmaceutically acceptable excipients. Routes of administration of pharmaceutical compositions containing a Trk inhibitor include oral, sublingual, intravenous, subcutaneous, intramuscular, intraarticular, transdermal, rectal, inhalation, intrathecal/intraventricular and topical applications. Accordingly, pharmaceutical formulations containing a Trk inhibitor may be formulated, for example, as capsules, tablets, powder, solution, suspension, emulsion, lyophilized powder, or sustained release formulation containing injectable microcapsules.

Excipients used in pharmaceutical formulations containing a Trk inhibitor will depend on the route of administration for which the pharmaceutical formulation containing the Trk inhibitor is intended.

Suitable excipients include, without limitation, inorganic or organic materials such as diluents, solvents, gelatin, albumin, lactose, starch, stabilizers, dissolving agents, emulsifying agents, suspending agents, salts, and buffer solutions. Suitable pharmaceutically acceptable excipients for intra-articular formulations such as solutions or suspensions include, without limitation, commercially available inert gels or liquids.

Taking into account the low solubility of the Trk inhibitor, 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine, and the monohydrate form of 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo [4,5-b]pyridine-2-amine, a suspending agent is needed to prepare a microcrystalline suspension formulation for intra-articular injection containing a Trk inhibitor as an active pharmaceutical ingredient. Commonly used pharmaceutically acceptable suspending agents include: gum arabic, agar, alginic acid, bentonite, calcium stearate, carbomer, carboxymethyl cellulose (calcium and sodium), carrageenan, cellulose (microcrystalline, microcrystalline and sodium carboxymethyl cellulose, powdered), colloidal silicon dioxide, dextrin , gelatin, guar gum, hectorite, hydrophobic colloidal silicon dioxide, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hypromellose, kaolin, magnesium aluminum silicate, maltitol solutions, medium chain triglycerides, methylcellulose, phenylmercurborate, phospholipids, polycarbophil, polyethylene glycol, polyoxyethylene sorbitan fatty acid esters, povidone (polyvinylpyrrolidone), propylene glycol alginate, saponite, sesame oil, sodium alginate, sorbitol esters, sucrose, tragacanth, vitamin E polyethylene glycol succinate, and xanthan gum (Handbook of Pharmaceutical Excipients, 6th edition).

As part of a solution for intra-articular administration, the active ingredient in which is the monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- 3Himidazo[4,5-b]pyridine-2-amine, a Trk inhibitor, buffer agents are also used. Pharmaceutically acceptable buffer agents include: adipic acid, ammonia solution, boric acid, calcium carbonate, calcium hydroxide, calcium lactate, calcium phosphate, tribasic, citric acid monohydrate, sodium phosphate dibasic, diethanolamine, glycine, maleic acid, malic acid, methionine , sodium phosphate monobasic, monoethanolamine, sodium monoglutamate, phosphoric acid, potassium citrate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate dihydrate, sodium hydroxide, sodium lactate, and triethanolamine (Handbook of Pharmaceutical Excipients, 6th edition) .

Since the pharmaceutical compositions containing the Trk inhibitor of the present invention are compositions for intra-articular administration, they may also contain solvents. Suitable diluents for uses as in the present invention include: maltitol, sunflower oil, ammonium alginate, calcium carbonate, calcium lactate, anhydrous dibasic calcium phosphate, dibasic dihydrate, tribasic, calcium silicate, calcium sulfate, cellulose (powdered, siliconized microcrystalline), cellulose acetate, compressible sugar, confectionery sugar, corn starch and pregelatinized starch, dextrates, dextrin, dextrose, erythritol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, lactose for inhalation preparations, isomalt, kaolin, lactitol, lactose (anhydrous, monohydrate and corn starch, spray-dried monohydrate and microcrystalline cellulose), magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, medium chain triglycerides, microcrystalline cellulose, polydextrose, polymethacrylates, simethicone, sodium alginate, sodium chloride, sorbitol, starch (pregelatinized,stabilized maize), sucrose, sugar spheres, beta-cyclodextrin sulfobutyl ether, talc, tragacanth, trehalose, xylitol (Handbook of Pharmaceutical Excipients, 6th edition).

Pharmaceutical formulations in the form of a microcrystalline suspension containing as an active ingredient a Trk inhibitor with a monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)

- 7 040073 hydroxy)benzyl)-6-(1-methyl-III-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine are described in example 2.

Method for preparing the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine- 2-amine provides:

a. mixing 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1 H-pyrazol-4-yl)-3Himidazo[4,5-b]pyridin-2- amine with a solvent to form a suspension;

b. stirring the suspension;

c. collecting solids from the slurry by filtration; And

d. drying of solids.

In this method, the solvent used to obtain the suspension may be a mixture of acetone and water. In addition, the suspension can be stirred overnight and the collected solids can be air dried.

The following non-limiting examples illustrate various embodiments of the present invention, including methods for preparing the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-III-pyrazole-4- yl)-3H-imidazo[4,5-b]pyridine-2-amine, pharmaceutical formulations containing the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl- 1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine, sustained release pharmaceutical formulations containing 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl )-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine, and methods for making such sustained release formulations.

Examples

Example 1 3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridine monohydrate form -2-amines.

Example 1-1. Synthesis of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2 -amine.

To a stirred solution of 3-methoxy-4-(4-methoxybenzyloxy)phenyl)methanamine (2.00 g, 7.32 mmol) and 5-bromo-2-chloro-3-nitropyridine (1.66 g, 6.97 mmol ) in acetonitrile (50 mL) was added N,N-diisopropylethylamine (1.13 g, 8.71 mmol). The resulting mixture was heated to reflux temperature and allowed to stir. After 64 hours the reaction mixture was allowed to cool to room temperature and diluted with water. The mixture was extracted twice with dichloromethane. The combined organic phases were dried over magnesium sulfate, filtered and concentrated to give 3.34 g (>100%) 5-bromo-N-(3-methoxy-4-(4-methoxybenzyloxy)benzyl)-3-nitropyridine-2-amine as a yellow-brown solid.

To a stirred solution of 5-bromo-N-(3-methoxy-4-(4-methoxybenzyloxy)benzyl)-3nitropyridine-2-amine in tetrahydrofuran (40 ml), ethanol (40 ml) and water (40 ml) was added sodium hydrosulfite (6.09 g, 34.99 mmol). The resulting mixture was heated to reflux temperature and allowed to stir. After 4 hours the reaction mixture was allowed to cool to room temperature and diluted with water. The yellow mixture was extracted three times with dichloromethane. The combined organic phases were washed with brine, dried (magnesium sulfate), filtered and concentrated to give 3.10 g of a tan solid. Purification by chromatography (Combi-Flash 40 g SiO ₂ , gold column, 1-2.5% methanol/dichloromethane) gave 1.28 g (51%) 5-bromo-M ² -(3-methoxy-4-( 4-methoxybenzyloxy)benzyl)pyridine-2,3-diamine as a yellow solid.

To a stirred solution of 5-bromo-N ² -(3-methoxy-4-(4-methoxybenzuloxy)benzyl)-pyridine-2,3diamine (0.850 g, 1.91 mmol) in dichloromethane (30 ml) and methanol (30 ml) cyanogen bromide (5.0 M in acetonitrile, 573 μl, 2.87 mmol) was added. The resulting solution was allowed to stir at room temperature. After 24 hours, a second aliquot of cyanogen bromide solution (600 μl) was added and stirring was continued. After 48 hours, a third aliquot of cyanogen bromide solution (600 μl) was added and stirring was continued. After a total of 72 hours, the reaction mixture was concentrated and the residue was taken up in dichloromethane. The solution was washed with 1 N. sodium hydroxide solution, dried over magnesium sulfate, filtered and concentrated to give 1.17 g of a brown solid. Chromatographic purification (Combi-Flash, 40 g SiO ₂ , gold column, 1-10% 2 M ammonia in methanol/dichloromethane) gave 0.28 g (32%) 6-bromo-3-(3-methoxy- 4-(4methoxybenzyloxy)benzyl)-3H-imidazo[4,5-b]pyridine-2-amine as a brown solid.

To a stirred solution of 6-bromo-3-(3-methoxy-4-(4-methoxybenzyloxy)benzyl)-3Himidazo[4,5-b]pyridine-2-amine (0.25 g, 0.53 mmol) in 1 ,4-dioxane (10 ml) and water (4 ml) was added 1methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.14 g, 0.66 mmol), tribasic potassium phosphate (0.39 g, 1.84 mmol), tricyclohexylphosphine (0.015 g, 0.052 mmol), palladium(II) acetate (0.005 g, 0.026 mmol). The reaction mixture was heated to 125°C in a microwave reactor. After 15 min the reaction mixture was allowed to cool to room temperature and diluted with water. The mixture was extracted twice with ethyl acetate. The combined organic phases were washed with brine, dried over magnesium sulfate, filtered and concentrated to give 0.36 g of a greenish brown solid. Purification by chromatography (Combi-Flash, 12 g SiO ₂ , gold column, 1-10% 2 M ammonia in methanol/dichloromethane) gave 0.10 g (41%) of the product as a light green solid substances.

3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2- the amine was added to a solution of acetonitrile (2 ml) and water (0.1 ml). This suspension was stirred using magnetic stirring at room temperature overnight. The solids were collected by filtration and air dried. The structure was confirmed by proton NMR. The differential scanning calorimetry (DSC) thermogram showed three thermal events at 76.72, 160.13 and 195.78°C, the thermal gravimetric analysis (TGA) thermogram showed a 3.7% weight loss from 25 to 100°C, and analysis with using X-ray powder diffractometry (XRPD) showed unique peaks at 3.6, 7.1, 8.9, 10.4, 10.7, 12.4, 12.7 and 14.3 at 2θ measured using CuKa- radiation (accuracy ±0.2°).

Example 1-2. Identification of the crystal structure of the monohydrate form of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine -2-amines.

The crystal structure of the monohydrate form of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amines. Crystal of the monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Nimidazo[4,5-b]pyridine-2 -amine was recrystallized from a mixture of acetone/water. The 0.20x0.08x0.03 mm crystal was made to fit the diffraction gratings when placed on top of the MiTeGen MicroMount. X-ray diffraction data were collected using a Bruker/AXS three-circle diffractometer equipped with a SMART APEX area detector, a low temperature device (model LT 2) and a copper-K microfocus generator operating at 45 kV/650 µA, and Montel multilayer optics for focusing. beam with a focusing spot diameter at the second focus of ~250 µm (Wiesmann et al., 2007). Data were collected using the SMART software package ver. 5.628 (Bruker AXS, 2001) and processed using SAINT+final version 6.45 (Bruker AXS, 2003). This analysis showed 2459 reflections (9 _min = 1.78, R _max = 50.21; 0<h<24, 4<k<0, -19<l<19), of which all 2459 reflections were unique (R _int =na, _RCT =0.1583). Cell parameters were refined using 1405 reflections. An empirical absorbance correction was applied and the phase problem was solved using the structure-solution module of the APEX2 kit.

The structure was refined using least squares ((F _o ² -Fc ² ) ² minimization) using the XL module of the APEX2 kit (Bruker AXS, 2011). The positions of all ^{hydrogen atoms were calculated S k} -criterion ^1.0 8 ^{5, R} of all data ^{0.1329 (R} ob. data ^{0.0920 for} R 1614 reflections with ^|F ob α ob ^| > ^4σ, wR ² of all data = 0.2710, wR ² _{obs =} 0.2377). The largest unidentified peaks on the difference map corresponded to -0.348 versus +0.386 electrons per A ³ . The mean standard deviation (esd) obtained for the C-C bond was 0.009 A, the O-C bond was 0.009 A and the NC bond was 0.009 A. The mean esd of the C-C-C bond angles was 0.7 and the C-C- C-C - 1.004°.

Crystal structure of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine -2-amine is shown in FIG. 3. In FIG. 4 shows the overlap of the powder X-ray diffraction obtained as a result of the experiments with that calculated from the single-crystal structure. A high degree of coincidence indicates that the single-crystal structure is characteristic of the bulk material.

Example 2 Microcrystalline suspension formulations of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5] monohydrate -b]pyridine-2-amine.

Pharmaceutical formulations have been developed in the form of a microcrystalline suspension of the Trk inhibitor, in which the monohydrate form of 3-(3methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole-4-) is used as the active ingredient. yl)-3H-imidazo[4,5-b]pyridine-2amine. These formulations were developed as a suspension of particles of insoluble 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine for long-term delivery of a Trk inhibitor to the body over time. This approach is based on the low solubility of the active ingredient and the size of the dose to control the duration of release; it also demonstrated sustained in vivo delivery of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine for about 1 month.

Example 2-1. Microcrystalline suspension for injection containing 20 mg/ml 3(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[monohydrate 4,5-b]pyridine-2amine.

A pharmaceutical composition was prepared containing 20 mg of the monohydrate form of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4.5 -b]pyridine-2-amine per ml of solution. The composition of the microcrystalline suspension of 20 mg/ml is given in table. 1 below.

- 9 040073

Table 1, Composition in the form of microcrystalline suspension 20 mg/ml

Component Composition per Dose Function Monohydrate form of 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3Nimidazo[4,5-b]pyridine-2-amine 72 mg Active Ingredient Sorbitol 162 mg Diluent Povidone K17 _g pyrogen-free 72 mg Suspending agent Phosphate buffer (10 mM, pH 7.4) 3.6 ml+ Buffer

t - 10% overflow applied.

Example 2-2. Method for preparing a composition in the form of a microcrystalline suspension containing 20 mg/ml of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- monohydrate 3nimidazo[4,5-b]pyridine-2-amine.

A 20 mg/ml solution of the monohydrate form 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl 1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine was prepared according to the following steps.

1) Phosphate buffer 10 mM, pH 7.4, was prepared by combining appropriate amounts of water for injection (WFI) and sodium dihydrogen phosphate, stirring to complete dissolution, and then adjusting the pH to 7.4 (±0.2) s using 1 and. sodium hydroxide solution.

2) Vehicle was prepared by combining appropriate amounts of sorbitol and pyrogenic povidone K17, adding 10 mM phosphate buffer pH 7.4 and stirring until complete dissolution.

3) Thereafter, the resulting carrier was filtered through a 0.22 μm PVDF [polyvinylidene fluoride] hydrophilic filter.

4) A concentrated suspension was obtained by mixing the monohydrate form of 3-(3methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4, 5-b]pyridine-2amine with a suitable carrier to obtain a 30 mg/ml concentrated suspension.

5) The concentrated suspension was filtered with agitation through a 75 µm stainless steel sieve.

6) With continued agitation, the pre-filtered slurry was filtered through a 40 µm stainless steel sieve to obtain a concentrated slurry.

7) The concentration of the concentrated suspension was adjusted using a filtered vehicle to obtain a suspension of 20 mg/ml.

8) The finished suspension was filled into sterile and apyrogenic type I colorless glass vials until the volume was filled to 3.6 ml. The vials were stoppered with sterile pyrogen-free bromobutyl stoppers coated with ETFE. The stoppers were crimped with sterile aluminum caps and sterile white plastic protective caps were put on the vials.

9) Filled vials were sterilized on autoclaving equipment.

Example 2-3. Clinical doses of the composition in the form of a microcrystalline suspension containing 20 mg/ml of monohydrate 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- 3nimidazo[4,5-b]pyridine-2-amine.

Microcrystalline suspension of 20 mg/ml monohydrate form of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine was further modified to achieve a dose and volume sufficient to administer to the patient in the final drug configurations. If necessary, the microcrystalline suspension of 20 mg/ml was processed into target doses. The final characteristics of various dosage formats and volumes are given in table. 2.

Table 2. Clinical presentations of microcrystalline suspension containing 20 mg/ml 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-monohydrate 3H-imidazo[4,5-b]pyridine-2-amine

Target dose (mg) Total volume of reconstitute vial (ml) Final Vial Concentration (mg/mL) Volume administered to the patient (ml) 3 3.47 0.98 3.2 10 3.95 3.29 3.2 thirty 6, 60 10 3.2 60 Not applicable ¹ 20 3.2 100 Not applicable ¹ 20 5.4*

t - to achieve the required final concentration, there is no need for re-dissolution, t - 2 vials of 2.7 ml.

- 10040073

Example 3 Extended Release Formulations Containing 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4.5 -b]pyridine-2-amine.

In some cases, it is required to extend the duration of drug release, for example to an exposure time of more than 3 months. Accordingly, sustained release formulations containing as active ingredient a Trk inhibitor, 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H -imidazo[4,5-b]pyridine-2-amine is prepared and formulated using either the anhydride or monohydrate form of the compound. An extended release formulation approach involves the use of poly(D,L-lactide) (PLA) polymers, D,L-lactideα-glycolide copolymer (PLGA), or a combination of PLA-PLGA polymers to encapsulate 3-(3-methoxy-4- ((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-n-pyrazol-4yl)-3H-imidazo[4,5-b]pyridine-2-amine (active pharmaceutical ingredient [API] or drug agent [DS]) to obtain a drug solution [DP] of injectable microcapsules. Such formulations are capable of providing prolonged, or over 3 months, release of the drug into the body. A brief description of the various polymers used in the preparation of the sustained release formulations of this example is shown in Table 1. 3.

Table 3. Polymers used in sustained release formulations containing 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H________pyrazol-4-yl)-3H- monohydrate imidazo[4,5-b]pyridine-2-amine________

Reference code from Evonik catalog Polymer name General structure Intrinsic VISCOSITY* (g/mol) end group R202H Poly(D,Llactide) [PLA] 0.16-0.24 Acid R202S Poly(D,Llactide) [PLA] V-^k 0.16-0.24 Ester R203H Poly(D,Llactide) [PLA] V^k 0.25-0.35 Acid R203S Poly(D,Llactide) [PLA] V^k 0.25-0.35 Ester RG502H Copolymer of P,L-lactide and glycolide 50:50 [PLGA] 0 0.16-0.24 Acid RG752H Copolymer of P,L-lactide and glycolide 75:25 [PLGA] 0 0.14-0.22 Acid

* - intrinsic viscosity of the solution was measured at a concentration of 0.1% w/v. in CHCl ₃ at 25°C using a 0c Ubbelohde glass capillary viscometer.

Source: http://www.resomer.com/product/biodegradable-polymers/en/pharmapolymers/products/pages/ bioresorbable-polymer.aspx.

In sustained release pharmaceutical formulations containing a Trk inhibitor using 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)6-(1-methyl-1H-pyrazole-4) as the active ingredient -yl)-3H-imidazo[4,5-b]pyridine-2-amine, use biodegradable polymers, poly(D,L-lactide) and/or D,L-lactide-glycolide copolymer to modify the release control of the active ingredient in the result of the dissolution of the drug particles in relation to the hydrolysis of polymers. By using the appropriate combination of polymers and active ingredient loading, the drug release rate can be controlled to provide an effect of 3 months or more. Specific combinations of PLGA/PLA polymers and 3-(3methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrαzol-4-yl)-3H-imidazo[4,5-b ]pyridine-2- 11 040073 amine is prepared in such a way as to extend the duration of drug release to more than 3 months; such compositions are further described in this example.

Example 3-1. Influence of load amine on the in vitro release profile.

Pharmaceutical compositions containing 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine was obtained by solvent extraction. Amounts of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4yl)-3H-imidazo[4,5-b]pyridine-2-amine and the masses of PLA/PGLA polymers used to obtain each batch are given in table. 4. The in vitro release kinetics (IVR) of these various formulations were studied.

Table 4. Microsphere compositions - 12% API/9:1 R202H:752H,

16% API/9:1 R202H:752H and 20% API/9:1 R202H:752H

Batch number Load GZ3899881 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg)* 1 12% 36 9:1 R202H:752H 234:26 2 16% 50 9:1 R202H:752H 234:26 3 20% 65 9:1 R202H:752H 234:26

J - obtained by solvent extraction, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-[1-methyl1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine, * - total amount of polymers = 260 mg.

3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5b]pyridine-2-amine was dissolved in 2 ml of a solution of dichloromethane (DCM): methanol (MeOH) 9:1 with low heating (~30 min). The polymer was added and dissolved for 30 minutes. Separately, 39 ml of cold, sterile filtered polyvinylamine (PVA) solution (5% w/v in water) was placed in a 250 ml beaker equipped with a 7.9x38.1 mm Teflon magnetic stirrer. The beaker was placed in an ice bath on an IKA RCT magnetic stirrer platform set at 500 rpm. The polymer/drug solution was filtered into the PVA solution using a 0.2 µm Pall Acrodisc PTFE syringe filter attached to a 5 cc Hamilton glass syringe. After addition, an emulsion was obtained from the polymer/drug solution. After 1 min, 160 ml of cold deionized water was added. After 5 minutes, the stirring speed was reduced to 300 rpm. Microspheres were obtained by solvent extraction for 3 hours. Microspheres were sieved through 75 and 38 μm structured sieves using a cold 0.1% solution of Kolliphor P 407 in deionized water; a 38-75 µm fraction was collected and excess wash solution was removed. The microspheres were frozen at -80°C and lyophilized.

IVR kinetics for the release of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b]pyridin- 2-amines from the microsphere formulations were determined by preparing a 2% (w/v) aqueous solution of the microspheres in 0.2% hyaluronic acid + 0.2% Kolliphor P 407 to mimic the intra-articular space of the knee. In three repetitions, the volumes of suspensions with a theoretical load of 500 μg 5-b]pyridine-2-amine was pipetted into separate 50 ml glass centrifuge tubes containing release medium containing 0.5% sodium dodecyl sulfate in PBS, pH 7.4. The tubes were placed on their side in a reciprocating shuttle incubator at 37°C. At each time point, the microspheres were allowed to settle and 1 ml release media samples were removed and replaced. To determine the actual total mass of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3Nimidazo[4,5-b]pyridine -2-amine in IVR samples, the same volumes of suspensions were taken as used for the release study (three times) and 10 ml of dimethyl sulfoxide was added. The samples were sonicated, slightly heated and placed on a shaker to dissolve at room temperature. These samples were analyzed for the total content of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine by HPLC-UV. The cumulative IVR profile was constructed as a percentage of the actual load of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4, 5-b]pyridine-2-amine from time to time.

Efficiency of encapsulation amine was determined by dividing the actual drug load by the theoretical load. To determine the actual load, the mass of accurately weighed microspheres loaded with 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo [4,5-b]pyridine-2-amine, dissolved in dimethyl sulfoxide and measured the concentration of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H- 12 040073 pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine by HPLC-UV.

The IVR profiles showed drug loading levels above 16% (w/w), demonstrating explosive release (FIG. 5). The reason for this explosive behavior was determined using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). DSC analysis showed that at drug loading levels above 16% (w/w), crystallization occurred in the microspheres, as evidenced by the melting endotherm at ISO-150°C (FIG. 6). Drug crystallization was also detected by SEM, where drug loading levels above 16% showed drug crystals on microsphere surfaces (FIG. 7).

Fig. 6 shows that 20% of the microspheres showed that a melting endotherm between 130-150° C. confirmed the presence of drug crystals. In FIG. 7 and 8 show SEM images of microspheres loaded with 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hn-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine, at 1500X magnification. In FIG. 7 microspheres loaded with 16% drug did not show the presence of drug crystals (drug was amorphous); in fig. 8, microspheres loaded with 20% drug showed drug crystals on the surface.

The effect of drug crystallization on IVR has also been demonstrated using appropriate microsphere formulations. In FIG. 9 shows the IVR of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]niridine-2 -amine from microspheres obtained using polymer R202H. In FIG. 10 shows IVR 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2 -amine from microspheres obtained using the ratio of polymers R202H and RG752H 9.5:0.5. In these examples, at drug loading levels above 16% (w/w), explosive release was demonstrated; wherein the duration of the explosive release of the drug is directly related to the level of drug loading.

Example 3-2. Effect of polymer blends of polylactide and lactide-glycolide copolymer on the in vitro release profile of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4yl)-3H- imidazo[4,5-b]pyridine-2-amine.

Example 3-2-1. Microspheres 16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16% API/9:1 R202H:752H.

Received pharmaceutical compositions containing 16% 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]niridine-2-amine in R202H, 9.0:0.5 R202H:RG752H and 9:1 R202H:752H, 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6 -(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202H, 9.0:0.5 R202H:RG502H and 9:1 R202H:RG502H, with to evaluate the effect of various polymer blends on the in vitro release kinetics of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-n-pyrazol-4-yl)-3H-imidazo [4,5-b]pyridine-2-amine. In table. 5 lists the weights of drug and polymer used to make each batch. The method used to obtain these compositions is described in example 3-1.

Table 5. Compositions of microspheres* containing 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo [4, 5-b]pyridine-2-amine, and polymer blends of polylactide and lactide-glycolide copolymer (16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16% API/9:1 R202H: RG752H).

Batch number Load GZ3899881 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg)* 4 16% 50 mg R202H 260 5 16% 50 mg 9.5:0.5 R202H:RG752H 247:13 6 16% 50 mg 9:1 R202H:RG752H 234:26

* - obtained by solvent extraction, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine, * - total amount of polymers = 260 mg.

Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. The encapsulation efficiency for formulations 4, 5 and 6 was 96.1±2.6, 87.8±6.3 and 77.7±8.6%, respectively.

Microspheres prepared using 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine, in R202H polymer showed a biphasic release rate with a slow initial release rate of 0-70 days followed by an accelerated secondary release rate of 70-110 days (FIG. 11). Blending the more hydrophilic polymer RG752H (75:25 PLGA) into PLA microsphere formulations increased the initial release rate. This PLGA polymer allowed accelerated water uptake by the microspheres resulting in increased speed. With the ratio

- 13 040073

R202H:RG752H 9:1 initial release matched the secondary release rate, providing a pseudo zero degree release profile over 3 months (FIG. 11).

Example 3-2-2. Microspheres 16% API/R202H, 16% API/9.5:0.5 R202H:RG502H and 16% API/9:1

R202H:RG502H.

Received pharmaceutical compositions containing 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine in R202H, 9.0:0.5 R202H:RG502H and 9:1 R202H:RG502H, in order to evaluate the effect of various polymer blends on the in vitro release kinetics of 3-(3-methoxy-4- ((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrαzol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine. In table. 6 shows the weights of drug and polymer used to make each lot. The method used to obtain such compositions is described in example 3-1.

Table 6 Microsphere Compositions ^J - 16% API/R202H, 16% API/9.5:0.5 R202H:RG502H and 16% API/9:1 R202H:RG502H

Batch number Load GZ3899881 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg)* 7 16% 50 mg R202H 260 8 16% 50 mg 9.5:0.5 R202H:RG502H 247:13 9 16% 50 mg 9:1 R202H:RG502H 234:26

J - obtained by solvent extraction, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine, * - total polymers = 260 mg.

Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. The encapsulation efficiency for formulations 7, 8 and 9 was 96.1±2.6, 91.7±3.3 and 94.2±2.5%, respectively.

Microspheres prepared using 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine, the R202H polymer exhibited a biphasic release rate with a slow initial release rate of 0-70 days followed by an accelerated secondary release rate of 70-110 days (FIG. 12). Blending the more hydrophilic polymer 50:50 PLGA (RG752H) into PLA microsphere formulations increased the initial release rate. The RG752H allowed for accelerated water uptake by the microspheres resulting in increased speed. At a ratio of R202H:RG502H of 9.5:0.5, the initial release matched the secondary release rate, providing a pseudo zero degree release profile over 6 months (FIG. 12).

Example 3-3. Preparation and characterization of microsphere formulations demonstrating zero degree in vitro release kinetics over 180 days.

Received pharmaceutical compositions containing 15% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amineα in 9:1 R203H:RG752H and 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole-4 -yl)-3H-imidazo[4,5-b]pyridine-2amine in 9.0:0.5 R202H:RG502H and analyzed for in vitro release kinetics. In table. 7 shows the weights of drug and polymer used to make each lot.

Table 7. Compositions of microspheres ^J - 15% API/9:1 R203H:RG502H and ___________________16%API/9.5:0.5R202H:RG502H)___________________

Batch number Load GZ389988+ Weight GZ389988 (mg) Polymer ratio Number of polymers (mg)* 10 15% 4 6 mg 9:1 R203H:RG752H 234:26 eleven 16% 50 mg 9.5:0.5 R202H:RG502H 247:13

J - obtained by solvent extraction, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine, * - total polymers = 260 mg.

Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. The encapsulation efficiency for formulations 10 and 11 was 89.7±2.1% and 91.7±3.3%, respectively.

Microspheres prepared using 15% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine in polymers R203H:752H at a ratio of 9:1 and 16% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole -4-yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202H:RG502H polymers at a ratio of 9.5:0.5 showed a pseudo-zero order release profile over 6 months (Fig. 13) .

Example 3-4. Influence of co-solvent systems used in preparation on the in vitro release profile of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol4-yl)-3H- imidazo[4,5-b]pyridine-2-amine.

- 14 040073

Example 3-4-1. 16% API/9:1 R202H:RG752H as prepared for 9:1 DCM:MeOH, 9:0.5:0.5

DCM:MeOH:BA, 9:5 DCM:BA and 9:1 DCM:BA.

Pharmaceutical compositions containing 16% microspheres loaded with 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine in 9:1 R202H:RG752H was prepared using different co-solvent solutions with different solvent ratios of dichloromethane (DCM), methanol (MeOH) and benzyl alcohol (BA). The ratios used were 9:1 DCM:MeOH, 9:0.5:0.5 DCM:MeOH:BA, 9.5:0.5 DCM:BA and 9:1 DCM:BA. For information about the medicinal product, polymers and auxiliary solvents used to obtain each batch, see table. 8. The method used to obtain such compositions is described in example 3-1.

Table 8. Compositions of microspheres obtained using systems of various co-solvents - 16% API/9:1 R203H:RG752H

Batch number Load GZ3899881 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg) * Auxiliary Solvent System 12 16% 50 mg 9:1 R202H:RG752H 234:26 9:1 DCM:MeOH 13 16% 50 mg 9:1 R202H:RG752H 234:26 9:0.5:0.5 DCM:MeOH:BA 14 16% 50 mg 9:1 R202H:RG752H 234:26 9.5:0.5 DCM:BA 15 16% 50 mg 9:1 R202H:RG752H 234:26 9:1 DCM:BA

J - obtained by solvent extraction (systems of various auxiliary solvents), t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl )-3H-imidazo [4,5-b]pyridine-2-amine, * - total amount of polymers = 260 mg.

Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. Encapsulation efficiency for lots 12, 13, 14 and 15 were 77.7±8.6, 94.9±2.0, 90.5±2.6 and 94.4±1.5%, respectively. Benzyl alcohol was chosen to be included in the auxiliary solvent systems due to its increased ability to dissolve 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl) -3H-imidazo[4,5-b]pyridine-2-amine. In FIG. 14 and 15 show that benzyl alcohol can be used as part of a co-solvent system along with dichloromethane or dichloromethane and methanol to form microspheres without affecting release rates. This can be used to reduce the possible recrystallization of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine in the preparation of microspheres.

Example 3-4-2. Using DCM:BA to Boost 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine.

Received pharmaceutical compositions containing 16 and 25% 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrαzol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amineα in 9:1 R202H:RG752H, 30% and 40% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H- pyrazol-4yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202H, and 25, 30, 40 and 50% 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)- 6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineα in R203H using varying volumes of dichloromethane (DCM) and benzyl alcohol (BA). Information about the masses of the drug and polymer, the volume of solvents used to obtain each batch, see table. 9. The method used to obtain these compositions is described in example 3-1.

- 15 040073

Table 9. Compositions of microspheres^ obtained using the co-solvent system DCM:BA - 16% API/9:1 R202H:RG752H,

25% API/9:1 R202H:RG752H, 30% API/R202H, 40% API/R202H, 25%

API/R203H, 30% API/R203H, 40% API/R203H, 50% API/R203H

Batch number Load GZ389988 1 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg) * Auxiliary solvent system [solvent volume + solvent volume (ml)] 16 16% 50 mg 9 : 1 R202H:RG752 H 234:26 0.2 ml VA + 1.8 ml DCM 17 25% 8 4 mg 9:1 R202H:RG752 H 234:26 0.2 ml VA + 1.8 ml DCM 18 thirty% 112 mg R202H 260 0.26 ml VA + 1.2 ml DCM 19 40% 17 4 mg R202H 260 0.4 ml VA + 1.2 ml DCM 20 40% 17 4 mg R202H 260 0.5 ml VA + 1.2 ml DCM 21 25% 8 4 mg R203H 260 0.2 ml VA + 1.8 ml DCM 22 thirty% 112 mg R203H 260 0.2 ml VA + 1.8 ml DCM 23 40% 17 4 mg R203H 260 0.4 ml VA + 1.2 ml DCM 24 40% 17 4 mg R203H 260 0.5 ml VA + 1.2 ml DCM 25 50% 2 60 mg R203H 260 0.5 ml VA + 1.2 ml DCM 26 50% 2 6 mg R203H 260 0.61 ml VA + 1.2 ml DCM

J - obtained by solvent extraction, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine, * - total amount of polymers = 260 mg.

Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. Encapsulation Efficiency of Formulations Produced Using Varying Volumes of Dichloromethane

Benzyl alcohol was chosen to be included in the auxiliary solvent systems due to its increased ability to dissolve 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1H-pyrazol-4-yl)- 3H-imidazo[4,5-b]pyridine-2-amine. In FIG. 10 shows that benzyl alcohol can be used as part of a co-solvent system along with dichloromethane to produce microspheres with loading up to 50% (w/w). Some release profiles are shown, characterized by minimal explosive release and kinetics, which made it possible to achieve a duration of 3-6 months. The volume of benzyl alcohol used during the preparation affected the explosive release of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1 H-pyrazol-4-yl)- 3Nimidazo[4,5-b]pyridine-2-amine. Reducing the amount of benzyl alcohol in the solvent system while maintaining the same solubility imidazo[4,5-b]pyridine-2-amine, in a solution of polymers, reduced the explosive effect. Increasing the API loading provided the benefit of reducing the amount of polymer administered to the patient. This is expected to improve the biocompatibility of the implant and reduce possible accumulation

- 16 040073 polymer after repeated injections. In addition, the increased API load should translate into lower unit production costs.

Example 3-5. The influence of the process of microencapsulation of a fine suspension on the release profile in vitro 5-b]pyridine-2-amine.

Pharmaceutical compositions containing 25% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b ]pyridine-2-amine (monohydrate form) in 9:1 R202H:RG752H, 30% and 40% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl- 1H-pyrazol-4-yl)3H-imidazo[4,5-b]pyridin-2-amine in 9:1 R203H:RG752H, 25% 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202H, 30% 3-(3-methoxy-4-(( 4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine in R202S and 30% 3-(3-methoxy- 4-((4-Methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine in R203S was prepared using monohydrate form of 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2- amine. For information on the masses of drug and polymer used to obtain each batch, see table. 10. Lots 27, 28 and 29 were prepared using 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1H-pyrazol-4-yl)-3H-imidazo[4 ,5-b]pyridine-2-amine from production lots, while lots 30, 31, 32, 33, 34 and 35 were made using 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl) -6-(1-methyl1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-2-amine, finely ground using a microfluidization method to ensure that the particle size is less than 10 µm in diameter . This example compares the differences in microspheres obtained by dispersing the polymer in a solution of polyvinyl alcohol (PVA) with or without ethyl acetate (EA), as well as loading API, polymer composition and polymer solutions of various concentrations (by varying the volume of EA).

Table 10. Compositions of microspheres obtained using the fine suspension microencapsulation process - 16% API/9:1 R202H:RG752H, 25% API/9:1 R202H:RG752H, 30% API/R202H, 40% API/R202H, 25 % API/R203H, 30% API/R203H, 40% API/R203H, 50% API/R203H

Batch number Load GZ389988 1 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg) * Volume and composition of the dispersion solution (ml) Ethyl acetate volume (ml) 27 25% 8 4 mg 9:1 R202H:RG752 H 234:26 3 9 ml 5% PVA 2.6 28 25% 8 4 mg 9:1 R202H:RG752 H 234:26 3 9 ml 5% PVA 3.25 29 25% 8 4 mg R203H 260 39 ml 5% PVA 2.0 thirty thirty% 112 mg 9:1 R203H:RG752 H 234:26 3 9 ml 5% PVA+2.5% ethyl acetate 2.0 31 40% 17 4 mg 9:1 R203H:RG752 H 234:26 39 ml 5% PVA+2.5% ethyl acetate 2.0 32 thirty% 112 mg 9:1 R203H:RG752 H 234:26 39 ml 5% PVA+2.5% ethyl acetate 2.0 33 thirty% 112 mg 9:1 R203H:RG752 H 234:26 3 9 ml 5% PVA+2.5% ethyl acetate 2.3 34 thirty% 112 mg R202S 260 39 ml 5% PVA+2.5% ethyl acetate 2.0 35 thirty% 112 mg R203S 260 3 9 ml 5% PVA+2.5% ethyl acetate 2.0

$ - obtained by solvent extraction, t - GZ38998 8=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-( 1 methyl-1H-pyrazol-4-yl)-3H- imidazo[4,5-b]pyridine-2-amine, * - total amount of polymers = 260 mg.

SEM analysis showed that microspheres obtained by encapsulating a suspension of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- 3H-imidazo[4,5-b]pyridine-2-amine were spherical in shape with a rough surface structure due to the presence of drug crystals embedded in the surface (see FIG. 16). Encapsulation performance and IVR assays were performed as described in Example 3-1. The encapsulation efficiency for lot numbers 30, 31, 32, 33, 34 and 35 was 103.3±3.8, 101.6±5.3, 94.7±4.0, respectively,

97.1±0.2, 35.6±0.8 and 62.5±3.0%. Encapsulation performance for Lots 27, 28, or 29 was not evaluated.

The IVR profiles showed an explosive release ranging from 25% to 47% followed by an insufficient release after 4 days (see FIG. 17). The batches were evaluated for 21 days.

Example 3-6. Effect of the microencapsulation process on the in vitro release profile of 3-(3-methoxy4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine.

Received pharmaceutical compositions containing 16 and 25% 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine in 9:1 R202H:RG752H, 25 and 30% 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-III-pyrazole -4-yl)3H-imidazo[4,5-b]pyridine-2-amine in R203H using dichloromethane (DCM):benzyl alcohol 9:1 (BA). For information on the masses of drug and polymer used to obtain each batch, see table. 11. The method used to prepare Lot 23 is described in Example 3-1 for Lot 2. Lot 24 was prepared by dissolving 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl -1H-pyrazol-4-yl)-3H-imidaz[4,5-b]pyridine-2-amine in 5.016 g of 9:1 DCM/MeOH with mild sonication. Then two polymers were added and dissolved.

Table 11. Compositions of microspheres obtained by solvent extraction and spray drying 16% API/9:1 R202H:RG752H and no API/9:1 R202H:RG752H

Batch number Load GZ389988 1 Weight GZ389988 (mg) Polymer ratio Number of polymers (mg) * Microencapsulation process 36 16% 50 mg 9:1 R202H:RG752 H 234:26 Solvent extraction 37 16% 238.8 mg 9 : 1 R202H:RG752 H 1140:114 Spray Drying 38 16% 238.8 mg 9:1 R202H:RG752 H 1140:114 Spray Drying 39 16% 238.8 mg 9:1 R203H:RG752 H 1140:114 Spray Drying

t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine- 2-amine, * - total polymer = 260 mg for lot 23 and 1254 mg for lot 24.

Spray drying was applied using a ProCepT, 4M8 block equipped with a 0.4 mm fluid nozzle. Spray drying conditions and parameters are listed below:

Batch number 37 38 39 Load GZ389988 (% w/w) 16 16 16 Solvent (methylene chloride: methanol (v/v) 90:10 90:10 90:10 PLGA Solution Concentration (% w/v): 20 22.5 22.5 Nebulization rate (ml/min.): 0.7 0.7 0.7 Nitrogen consumption for fine spraying (l/min.): 4 4 4 Spray quantity (g): 6.4 5, 8 5.8 Inlet temperature (°C) : 50 50 50 Chamber temperature (°C) : 41-43 40.7 40.0 Outlet temperature (°C) : 27.4 22.0 22.1 Nitrogen flow (m ³ /min.): 0.35 0.35 0.35 Transfer line pressure (mbar): 31-32 31-32 32 Yield (% w/w) 72.8 76.7 51.6

Analysis by SEM showed that the microspheres obtained by solvent extraction were spherical with a smooth surface structure, and the microspheres obtained by spray drying were spherical with a peculiar surface structure (see FIGS. 18 and 19). Encapsulation efficiency and IVR assays were performed as described in Example 3-1. The encapsulation efficiency of Lot 36 obtained by solvent extraction and Lots 37 and 38 obtained by spray drying were 107.4±11.6%, 91.4±4.3% and 92.6±3.6%, respectively.

- 18 040073

These two microencapsulation processes provided microspheres with a nearly zero order release profile. In FIG. 18 and 19 show SEM images of microspheres loaded with 3-(3methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2amine, at 1000X magnification. The IVR profiles showed different release rates (FIG. 20); the formulation obtained by spray drying achieved the required delivery time of 3-6 months.

Example 3-7. Effect of 1% 10 kDa PEG or 1% poloxamer 407 on the in vitro release profile of spray-dried microspheres containing 16% (w/w) 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)- 6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.

This experiment was carried out in order to determine whether the addition of a hydrophilic additive can be used to increase the in vitro release rate of GZ389988 from PLA/PGA microspheres. In this experiment, microspheres (without additives) were compared with microspheres prepared using either 1% 10 kDa PEG or 1% poloxamer 407. 12 shows the weights of drug and polymer used to make each lot.

Table 12. Compositions of microspheres ¹ obtained using

1% 10 kDa PEG or 1% poloxamer 407-16% API/1:1 R202H:R203H/no additives, 16% API/1:1 R202H:R203H/31.25mg PEG, 16% ._______ API/1: 1 R202H:R203H/31.25 mg poloxamer 407____________

Batch number Load GZ389988 t Weight GZ389988 (mg) Polymer ratio Number of polymers (mg:mg) Additive quantity (mg) 40 16% 500 mg 1:1 R202H:R203H 1310:1310 Not applicable 41 16% 500 mg 1:1 R202H:R203H 1297:1297 31.25 10 kDa PEG 42 16% 500 mg 1:1 R202H:R203H 1297:1297 31.25 Poloxamer 407

J - obtained by spray drying, t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine.

Spray drying was applied using a ProCepT, 4M8 block equipped with a dual 0.4 mm fluid nozzle. Spray drying conditions and parameters are listed below:

Batch number 40 41 42 Load GZ389988 (% w/w) 16 16 16 PLGA solution concentration (% w/v): 22.5 22.5 22.5 Nebulization rate (ml/min.): 0.7 0.7 0.7 Air consumption (l/min.): 0.35 0.35 0.35 Nitrogen consumption for fine spraying (l/min.): 4 4 4 Cooler temperature (°C) : -4 -4 -4 Inlet temperature (°C) : 50 50 50 Chamber temperature (°C) : 40 40 40 Outlet temperature (°C) : 29 29 29 Pressure in front of the cyclone (mbar): 27-30 27-30 27-30

Analysis by SEM showed that the microspheres obtained by spray drying with or without hydrophilic additives showed the same size distribution of substances and the same surface structure; while all the microspheres obtained by spray drying were spherical in shape with a peculiar surface structure (see Fig. 21, 22 and 23). Encapsulation efficiency and IVR analyzes were performed as described in Example 3-1. The encapsulation efficiency for lot numbers 40, 41 and 42 obtained by spray drying were 102.0±16.3, 101.7±16.3 and 100.5±16.1%, respectively.

Microspheres made without additives showed a slow release profile of GZ389988, almost zero order, with about 20% release of the active compound within 35 days (see Fig. 24). The addition of either 1% 10 kDa PEG or 1% poloxamer 407 increased the release rate; while approximately 42% of the active compound was released in 35 days. This example demonstrates that the inclusion of a hydrophilic excipient accelerates the in vitro release of GZ389988 from PLA/PGA microspheres.

- 19 040073

Example 4 In vivo studies.

Example 4-1. In vivo clinical performance of microspheres loaded with [ ¹⁴ C]-3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[ 4,5-b]pyridine-2-amine after intra-articular injection into the rat knee joint.

To compare in vitro release kinetics with in vivo drug persistence in the rat knee joint, two formulations were selected. The compositions are shown in table. 13.

In vitro release testing was performed using the method described in Example 3-1. In vivo drug stability testing was performed using [ ¹⁴ C]-3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)- 3H-imidazo[4,5-b]pyridine-2-amine and unlabeled 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol4-yl) -3H-imidazo[4,5-b]pyridine-2-amine; moreover, these two forms of the drug during the microencapsulation process were dissolved together, in order to ensure the same distribution of the drug in the microspheres. The total amount of radioactivity received by the joint of each rat was ~1.2 MBq.

The amount of drug remaining in the rat knee joint was assessed by killing 2-3 rats at each time point, cryogenically grinding the knee joint, and extracting 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)- 6-(1-methyl-1H-pyrazol-4-yl)-3Himidazo[4,5-b]pyridin-2-amine from crushed tissues.

Quantification was performed using liquid scintillation detection. The amount of radioactivity recovered in the joint was calculated by dividing the radioactivity counter reading at each time point by the radioactivity counter reading for rats euthanized 0.1 hour after the start of treatment. The concentration of radioactive drug in rat blood (expressed in units of neq/g) was plotted against time and compared with the known IC ₅₀ value for the compound.

Table 13 Compositions of in vivo tested microspheres 16% API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H

Batch number 43 Load GZ389988t+[ 14C] GZ389988 16% Weight GZ389988 (mg) 50 mg Polymer ratio 9:1 R202H:RG502H Number of polymers (mg)* 234:26 44 15% 4 7 mg 9:1 R203H:RG752H 234:26

t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine- 2-amine, * - total amount of polymers=260 mg.

The IVR profiles for batches 40 and 41 showed a near zero order release profile for about 3-4 months and 5-6 months, respectively (see FIG. 25). Following intra-articular injection into the rat knee joint, batches 43 and 44 showed drug release within 5-6 months; Lot 43 showed 12% residual in the joint after 5 months and Lot 44 showed 30% residual after 6 months (see FIG. 26). The in vivo drug release rate was slightly slower than the IVR rate, presumably due to the location of the microspheres in the synovial membrane.

In FIG. 27 shows the drug concentration-time profile in the blood. After intra-articular administration of microspheres loaded with 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazol-4-yl)-3H-imidazo[4,5-b] pyridine-2-amine, concentration of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5 -b]pyridine-2-amine in the system compartment slightly exceeded the EC ₅₀ value (cell based) during the first week, but then fell below the EC ₅₀ value for the duration of the experiment (5-6 months). This experiment indicates that microspheres loaded with 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4, 5-b]pyridine-2-amine provide long-term local drug delivery to the knee joint with low systemic (ie, sub-therapeutic) drug exposure.

Example 4-2. Evaluation of the pharmacokinetics of GZ389988 after a single intra-articular injection of three sustained-release microsphere formulations loaded with 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H -imidazo[4,5-b]pyridine-2-amine.

Three formulations were selected to compare in vitro and in vivo pharmacokinetics in the systemic compartment after intra-articular injection into rat knee joints. The compositions are shown in table. 14.

In vitro release testing was performed using the method described in Example 3-1. In vivo clinical performance was measured by injecting 3 male Wistar rats with the given microsphere formulation delivering either 0.1 mg or 1 mg of GZ389988 into the knee joint. At each time point, blood samples (~0.25 ml) were taken from the jugular vein using a catheter and placed in chilled tubes containing K2EDTA as an anticoagulant, mixed and kept for

- 20 040073 on ice prior to centrifugation. Samples were centrifuged within 1 hour after collection at a temperature

4°C, at 3000xg for 5-10 min. After centrifugation of blood samples, plasma was collected in polypropylene tubes. Plasma samples were frozen on dry ice and stored frozen at -60 to -80°C until LC-MS/MS analysis was performed. Rat plasma concentration of GZ389988 (in units of ng/mL) was plotted against time (28 days in total).

Table 14 Compositions and dosages of microspheres tested in the _________ PK Evaluation of GZ389988____________

Compound Batch number Load GZ3899881 Polymer ratio Administered dose (mg) A 45 16% 9:1 R202H:RG752 H 0.1 46 16% 9:1 R202H:RG752 H 1 IN 47 15% 9 : 1 R203H:RG752 H 0.1 48 15% 9:1 R203H:RG752 H 1 WITH 49 40% R203H 0.1 50 40% R203H 1

t - GZ389988=3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-61-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2 -amine.

The IVR profiles for Formulation and Formulation B showed a slow release, with a release of about 10% of the active compound over 28 days. Composition C showed a slightly higher initial release during the first 5 days, followed by a decrease in release rate over the remaining 23 days; within 28 days, approximately 22% of the active compound was released.

Following intra-articular injection into the rat knee joints, formulations A and B showed similar plasma drug exposure profiles over 28 days; while both compositions showed T _max values at the level of 1-1.5 h with stable plasma levels throughout the experiment (respectively 0.08 and 0.8 ng/ml for doses of 0.1 and 1.0 mg). Compared to Formulations A and B, Formulation C exhibited higher _Cmax and higher stable plasma levels throughout the experiment (1.2 to 12 ng/mL for 0.1 and 1.0 mg doses, respectively) ( see Fig. 29). These results are in good agreement with the results of the IVR experiments.

Example 4-3. Clinical study to evaluate the stability, tolerability and pharmacokinetics of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1 H-pyrazol-4-yl)-3H-imidazo [ 4,5-b] pyridine-2-amine in patients with painful osteoarthritis of the knee.

Safety and efficacy of 3-(3-methoxy-4-((4-methoxybenzyl)oxy) benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b] monohydrate form ]pyridine-2-amine in humans was tested in a two-part, double-blind, placebo-controlled clinical trial to evaluate the safety, tolerability, and pharmacokinetics with increasing single intra-articular doses, followed by evaluating the efficacy, safety, tolerability, and pharmacokinetics of a single intra-articular dose in patients with painful knee osteoarthritis.

The first part of the study tested a single intra-articular injection into the knee at various doses of a microcrystalline suspension of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl) monohydrate )-3H-imidazo[4,5-b]pyridine-2-amine prepared according to Example 2 above compared to placebo doses of 3 and 5 ml.

Adult males and females diagnosed with primary osteoarthritis of the knee were eligible to participate in the study. Patients had been symptomatic for more than 6 months and signed a written informed consent prior to any procedure regarding the study.

Efficacy was assessed based on safety and tolerability (adverse events, physical examination, body weight, body temperature, clinical laboratory tests, blood pressure,

- 21 040073 heart rate, 12-channel electrocardiogram, local tolerance) at 12 weeks after injection. In addition, pharmacokinetics (in plasma and, if possible, in synovial fluid) and pharmacodynamics were evaluated.

Patients were followed up for 84 ± 7 days after administration of study drug or placebo, with the possibility of continuing a long-term observational safety study without additional administration of study drug to assess long-term safety and efficacy.

Example 5 Investigation of polymorphism 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl1 H-pyrazol-4-yl)-3 H-imidazo[4,5-b ]pyridine-2-amines.

To identify the crystalline forms of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine- 2-amines in pure solvents and controlled mixtures of solvent and water, a GZ389988 polymorphism study was performed. Techniques used as a tool to characterize polymorphisms included X-ray powder diffractometry (XRPD), high resolution X-ray powder diffractometry (HR-XRPD) and single crystal X-ray diffractometry (XRSCD), thermogravimetric analysis (TGA) together with infrared spectroscopy (FT-IR ) or mass spectroscopy (MS), dynamic vapor sorption (DVS) of water and solvents, and optical microscopy (OM). These techniques are described in more detail below.

High Resolution X-Ray Powder Diffraction (HR-XRPD).

High resolution diagrams were recorded under ambient conditions on a Panalytical X'Pert Pro MPD powder diffractometer using Bragg-Brentano parafocus (vertical configuration type 0-20) along with an X'Celerator detector. An x-ray tube with a sealed copper anode was used, operating at 45 kV and 40 mA. An incident beam monochromator (Johansson type: symmetrically cut bent germanium (111) crystal) emitted pure CuK _a 1 radiation (λ=1.54060 A).

For each series of experiments, a thin layer of the product was deposited on the surface of a sample holder coated with a silicon substrate one crystal thick. The substrate was cut in accordance with the Si (510) crystallographic orientation so that any Bragg reflection from silicon was hindered by systematic extinction. The available angle range spanned from 2° to 50° at 2θ in 0.017° increments at 2θ. The counter time was changed from 100 to 2500 s per step.

X-ray powder diffractometry (XRPD).

Other XRPD analyzes were performed on a Siemens-Brucker D8 Advance powder diffractometer, also using a Bragg-Brentano parafocus (θ-θ vertical configuration) and an Anton-Paar TTK450 temperature chamber. A thin layer of the product was deposited on a silicone substrate with a thickness of one crystal, cut out in accordance with the crystallographic orientation of Si (510) so that any Bragg reflection from the substrate was hindered by systematic extinction. A sealed copper anode x-ray tube was used, operating at 40 kV and 35 mA. As a rule, two lines were emitted: CuK _a 1 (λ=1.5405 A) and CuK _a 2 (λ= 1.5443 A). The nickel β-filter located between the detector and the sample did not generally affect the CuK radiation (λ=1.3922 A), which still contributed approximately 1% of the diffracted beam in the detector (according to the manufacturer). To increase the parallelism, the beam was directed through the Soller slits. Variable divergence slits maintained the illumination in the sample area at a constant level. The collimator limited diffusion between the tube and the sample. The LynxEye line detector completed the installation. It had a 3.5° wide detection window at a 2θ angle. Charts were recorded under the following conditions: at ambient temperature, scans from 2 to 40° at 2θ. The charge accumulation time depended on the experimental conditions. The study of development and most of the scans were obtained using a time counter with a resolution of 0.1 s per step at 2θ. A longer signal accumulation time (up to 5 s) could be used to characterize stable forms.

X-ray single crystal diffractometry (XRSCD, ALSO KNOWN AS SCXRD).

XRSCD data were collected on a Bruker Smart Apex single crystal diffractometer. A molybdenum microfocus IμS X-ray source was used, operating at 50 kV and 0.6 mA, emitting Mo-Cs radiation (λ=0.710731 A). The semiconductor light receiver area detector (CCD chip: 4K, 62 mm) was set to 6.0 cm. An Oxford Cryosystems nitrogen cryostat (Cryostream Plus) allowed XRSCD experiments at 100 K.

Both crystals were mounted with a drop of Paratone N™ oil on a MiTeGen low background Mylar loop. A complete sphere of Ewald reflections was collected (3 omega scans of 680 frames with a frame width of 0.3°). The accumulation time depended on the crystal.

The orientation matrix and the base cell were installed using the Arex2 software package (version 2014.11-0). A three-dimensional reflection profile and embedding of all reflections was obtained using the SAINT program (version 8.34A). The SADABS program was used to correct for the deflection of electrons under the action of the Lorentz force and polarization effects, as well as for absorption by the sample.

- 22 040073 (version 2014/5). The supposed symmetry group of the crystal lattice was determined using the XPREP program (version 2014/2). The SHELXTL software package (version 2014/7) was used to determine the structure using the internal phasing method and to improve the obtained solution using the least squares technique in the full matrix approximation in ^F2 .

Detection and characterization of polymorphism.

Crystalline forms of 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazole-4-ul)-3H-imidazo[4,5- b]pyridine-2-amine. Such shapes, XRPD peaks at 2θ measured using CuK _α radiation, and preparation conditions are shown in Table 1 below. 15.

Table 15 Forms pyridine-2-amine

Anhydrous, hydrate or solvate Polymorph number Form number XRPD peaks at 2Θ measured using SpK _a radiation Terms of receipt Anhydrous 1 Form 2 3.8, 7.6, 11.0, 11.5, 13.3, 13.9 and 15.3 The form was obtained by dehydration of monohydrate 1 Anhydrous 1 Form 3 6.0, 6.9, 10.9, 12.4, 12.7, 13.7 and 15.3 Prepared at high temperature (160°C for monohydrate 1, ~120°C for solvates) Anhydrous 3 Form 6/7 5.6, 8.1, 12.1, 14.0, 16.2 and 20.7 Form obtained by desolvation of monohydrate 2 and solvates 2 of ethanol, acetonitrile, and acetone Monohydrate 1 Form 1 3.6, 7.1, 8.9, 10.4, 10.7, 12.4, 12.7 and 14.3 See example 1 Monohydrate 2 Form 8 5.5, 6.4, 8.2, 12.5, 12.8 and 16.5 The form was obtained from solvates 2 of ethanol, acetonitrile and acetone (as a result of the cycle of pairs N ₂ /H ₂ O) Anhydrous, hydrate or solvate One and a half drat Ethanol solvate Acetone solvate Acetone solvate Polymorph number 1 2 Form number Form 4 Form 5 Form 9 Form 10 XRPD peaks at 2Θ measured using SpK _a radiation 3.5, 7.1, 9.3, 10.3, 10.6, 12.4, 13.0 and 14.1 5.3, 6.3 , 7.5, 11.4, 12.6, 12.8 and 15.1 6.1, 8.9, 12.1, 15.3, 16.4, 18.2 and 21.2 5.4 , 6.2, 7.4, 11.4, 12.6 and 14.8 Preparation Conditions The form was obtained by crystallization from acetone/water solution The form was obtained by recrystallization of monohydrate 1 in ethanol or by ethanolic solvation anhydrous form 3 Form was obtained by recrystallization of monohydrate 1 in pure acetone and solution acetone/water 99/1 The form was obtained by recrystallization of monohydrate 1 in pure acetone or by acetone solvation of anhydrous forms 3 Acetonitrile solvate Form 11 5.3, 6.4, 7.7, 11.6, 12.8, 13.2 and 15.5 The form was obtained by recrystallization of monohydrate 1 in acetonitrile or acetonitrile solvation anhydrous forms 3

In FIG. 30 plots together measured HR-XRPD plots of monohydrate form 1, anhydrous forms 2 and 3, and a simulated XRPD plot of sesquihydrate form 4.

A 20 mg/ml suspension of the monohydrate form GZ389988A in a mixture with an ethanol/water solvent (99/1) was heated to 80° C. for 1 hour. The suspension was then filtered. Next, the solution was kept overnight at room temperature. Overnight, the crystals were examined and then identified as corresponding to the ethanol solvate form (labeled Form 5). The physical quality of the crystal obtained by slow evaporation of a mixture of ethanol/water (99/1) was suitable for analysis by X-ray single crystal diffraction. The measurements were carried out at 100 K on crystals 5x50x200 μm ³ in size, with an accumulation time of 300 s per frame. Diffractograms

- 23 040073 confirmed the crystal structure of the analyzed substances. In FIG. 31 shows an HRXRPD plot of ethanol solvate powder (bottom) measured at room temperature compared to an XRPD plot simulated from data measured for XRSCD at 100 K (top).

A 20 mg/ml suspension of GZ389988A monohydrate Form 1 (Form 1) was prepared in a solvent mixture of acetone/water (99/1) or in pure acetone and heated to 80° C. for 1 hour until complete dissolution was achieved. Next, the suspension was filtered. The solution was kept at room temperature or at 5°C. After one night, the crystals were visually inspected with the naked eye, then identified as corresponding to the acetone solvate form (labeled Form 9). The physical quality of the crystal obtained by slow evaporation of a mixture of acetone/water (99/1) was suitable for analysis by X-ray single crystal diffraction. The measurements were carried out at 100 K on crystals 100x200x2000 μm ³ in size, with an accumulation time of 30 s per frame. In FIG. 32 shows an XRPD plot of acetone solvate powder measured at room temperature (bottom) and simulated from XRSCD data at 100 K (top).

In another experiment, a 20 mg/ml suspension of GZ389988A monohydrate Form 1 (Form 1) was prepared in pure acetone and heated at 80° C. for 1 hour. The suspension was then filtered, then directly cooled to 5° C. and stored at this temperature. After one night, the crystals were visually inspected with the naked eye, then identified as corresponding to those obtained for acetone solvate 1, form 9. After grinding in suspension in a mortar, they were identified as corresponding to another acetone solvate, form 10. FIG. 33 shows an XRPD comparison of two forms of acetone solvate, form 9 (bottom) and form 10 (top).

A 20 mg/ml suspension of the monohydrate form of GZ389988A in acetonitrile (ACN) was heated at 80° C. for 4 hours. The suspension was then filtered. Next, the solution was kept at 40°C, then left for 2 h to cool to room temperature. After a night, crystal clusters formed at the bottom of the flask. An HR-XRPD diagram of the solvate form of acetonitrile (ACN) (Form 11) is shown in FIG. 34.

Identification of the solid crystalline phase formed in mixed solvents acetone/water and acetonitrile/water.

50 mg of monohydrate 1 GZ389988A was supplemented with a mixture of solvent in a volume of 2 ml (acetone or acetonitrile) and demineralized water in three different weight ratios: 50/50, 80/20 and 95/5. With the help of acetone, mixtures were additionally studied at ratios of 99/1 and 98/2. After 2 hours at 80°C, the samples were filtered through a PTFE syringe filter with a nominal pore size of 0.45 μm, and again after filtration, they were kept at 80°C for 15 minutes.

The samples were then allowed to cool overnight at 40°C and again kept at room temperature for 24 h.

The samples were then analyzed by XRPD in a chamber saturated with the appropriate solvent. If necessary, large crystals were crushed in the vial with a spatula into a fine powder. The wet powder sample was then placed on a sample holder as horizontal as possible. The results of these analyzes are presented in table. 16 below.

Table 16. Solid crystalline phase of GZ389988A in acetone/water and acetonitrile/water mixtures

Solvent Weight ratio solvent/water Crystalline phase(s) Acetone 99/1 Acetone solvate form 9 98/2 Monohydrate 1 97/3 Monohydrate 1 95/5 Monohydrate 1 80/20 Monohydrate 1 50/50 Monohydrate 1 Acetonitrile 95/5 Monohydrate 1 80/20 Monohydrate 1 50/50 Monohydrate 1

The formation of solvates and hydrates upon crystallization of monohydrate 1 in ethanol, acetone, acetonitrile and acetone/water and acetonitrile/water mixtures was studied and is presented in this example. Ten crystalline phases have been identified:

anhydrous phase 1 (form 2), anhydrous phase 2 (form 3), anhydrous phase 3 (form 6/7), monohydrate 1 (form 1), monohydrate 2 (form 8), sesquihydrate (form 4),

- 24 040073 ethanol solvate (form 5), acetone solvate 1 (form 9), acetone solvate 2 (form 10), acetonitrile solvate (form 11).

The corresponding diffractograms are plotted together in Fig. 35.

As a result of polymorphism studies performed on ethanol, acetone, and acetonitrile, as well as on solvent mixtures (acetone/water and acetonitrile/water), several conclusions can be drawn. All three pure solvents lead to the formation of solid crystalline solvate phases. Recrystallization of GZ389988A in ethanol and acetonitrile separately leads to one solvate form. Upon recrystallization in acetone, two solvate forms can be obtained. Crystals formed in acetone/water mixed solvent systems with weight ratios of 98:2, 97:3 95:5, 80:20 and 50:50 all belonged to the monohydrate crystalline phase 1. Crystals formed in acetonitrile/water mixed solvent systems with weight ratios of 95:5, 80:20 and 50:50 also all belonged to the crystalline phase of monohydrate 1. It was observed that upon dissolution of solvate 1 of acetone passes into a more amorphous solid state. With an increase in molecular mobility due to the presence of vapors of mixed acetone and water, both initial states, crystals of acetone solvate 1 and amorphous solid particles, rearrange into monohydrate 1. Ethanol, acetonitrile, and acetone solvate 1 desolvated under the influence of a temperature of 120 the same anhydrous crystalline phases 2. For solvate forms 2 of ethanol, acetonitrile and acetone, polymorphism was observed (confirmation by single crystal X-ray diffraction required). They were observed to reversibly desolvate into the same anhydrous phase 3. Anhydrous phase 3 was hydrated into the monohydrate form, monohydrate 2, different from monohydrate 1.

Claims (20)

CLAIM 1. A compound represented by the following structural formula: 2. Crystal form I of the compound according to claim 1, the powder X-ray diffraction (XRPD) of which contains the following peaks at 2θ, measured using CuKa radiation: 3.6, 7.1, 8.9, 10.4, 10.7 , 12.4, 12.7 and 14.3. 3. Pharmaceutical composition having an inhibitory effect on tropomyosin-related kinases (Trk) or cell receptor for colony stimulating factor-1 (c-FMS), containing crystalline form I according to claim 2 and a pharmaceutically acceptable excipient. 4. Pharmaceutical composition according to claim 3, intended for the treatment of osteoarthritis. 5. Pharmaceutical composition according to claim 3 for the treatment of pain. 6. Pharmaceutical composition according to claim 3 for the treatment of pain associated with osteoarthritis. 7. A pharmaceutical formulation according to any one of claims 3 to 6, wherein the pharmaceutically acceptable excipient comprises a diluent, a suspending agent, and a buffering agent. 8. The pharmaceutical composition of claim 7 wherein the diluent is sorbitol, the suspending agent is povidone and the buffering agent is phosphoric acid. 9. Pharmaceutical composition having Trk or c-FMS inhibitory action, containing a. connection according to claim 1; And b. pharmaceutically acceptable excipient. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipient comprises a diluent. - 25 040073 11. Pharmaceutical composition according to claim 10, where the diluent is selected from: maltitol, sunflower oil, ammonium alginate, calcium carbonate, calcium lactate, anhydrous dibasic calcium phosphate, calcium silicate, calcium sulfate, cellulose, cellulose acetate, compressible sugar, confectionery sugar, corn starch and pregelatinized starch, dextrates, dextrin, dextrose, erythritol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, lactose for inhalation, isomalt, kaolin, lactitol, lactose, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, medium chain triglycerides , microcrystalline cellulose, polydextrose, polymethacrylates, simethicone, sodium alginate, sodium chloride, sorbitol, starch, sucrose, sugar spheres, beta-cyclodextrin sulfobutyl ether, talc, tragacanth, trehalose, xylitol. 12. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipient comprises a suspending agent. 13. The pharmaceutical composition according to claim 12, where the suspending agent is selected from: gum arabic, agar, alginic acid, bentonite, calcium stearate, carbomer, carboxymethyl cellulose, carrageenan, cellulose, colloidal silicon dioxide, dextrin, gelatin, guar gum, hectorite, hydrophobic colloidal silicon dioxide, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hypromellose, kaolin, magnesium aluminum silicate, maltitol solutions, medium chain triglycerides, methylcellulose, phenylmercurborate, phospholipids, polycarbophil, polyethylene glycol, polyoxyethylene sorbitan fatty acid esters, povidone (polyvinyl pyrrolidone), propylene glycol alginate, sesame oil, sodium alginate, esters of sorbitol, sucrose, tragacanth, vitamin E polyethylene glycol succinate and xanthan gum. 14. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipient comprises a buffering agent. 15. The pharmaceutical composition according to claim 14, where the buffer agent is selected from: adipic acid, ammonia solution, boric acid, calcium carbonate, calcium hydroxide, calcium lactate, calcium phosphate, citric acid monohydrate, dibasic sodium phosphate, diethanolamine, glycine, maleic acid , malic acid, methionine, sodium phosphate monobasic, monoethanolamine, sodium monoglutamate, phosphoric acid, potassium citrate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate dihydrate, sodium hydroxide, sodium lactate, or triethanolamine. 16. Pharmaceutical composition according to any one of claims 9 to 15, intended for the treatment of osteoarthritis. 17. Pharmaceutical composition according to any one of claims 9 to 15, intended for the treatment of pain. 18. A pharmaceutical composition according to any one of claims 9 to 15 for the treatment of pain associated with osteoarthritis. 19. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipient comprises a diluent, a suspending agent, and a buffering agent. 20. The pharmaceutical composition of claim 19, wherein the diluent is sorbitol, the suspending agent is povidone, and the buffering agent is phosphoric acid. powder X-ray diffraction pattern (XRPD) of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5- b]pyridine-2-amine, 4-40 at °20 Position [°2 Theta] (copper (Ci)) Fig. 1 - 26 040073 differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) thermogram of the monohydrate form of 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1Hpyrazole-4- yl)-3H-imidazo[4,5-b]pyridine-2-amine heated at 10°C/min. I em of the pen η ra ( ^_ C) Fig. 2 crystal structure of the monohydrate form 3-(3-methoxy-4((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5b]pyridine- 2-amine Fig. 3 is the overlap of the XRPD diffraction pattern obtained as a result of the experiments and the XRPD diffraction pattern. calculated from the single crystal structure And shereno on < ΐΙΪΝ/ΥΜΓ. Fig. 4 - 27 040073 API loading effect on IVR microsphere profile 12% AP1/9:1 R202H:752H, 16% API/9:1 R202H:752H and 20% API/9:1 R202H:752H 140 120 20% GZ389988 at 9:1... 16% GZ389988 at 9:1... T ^T T T 100 N _t T ¹ i ^{1 1 1} I T . About 50 100 150 200 Time (days) Fig. 5 thermograms of DSC microspheres 16% API/9:1 R202H:752H and 20% API/9:1 R202H:752H 16% GZ389988 at 9:1 202 H752H O 50 100 150 200 250 Temperature (°C) Fig. 6 Scanning Electron Microscope (SEM) image of 16% AP1/9:1 R202H:752H microspheres (1500X) Fig. 7 - 28 040073 Scanning Electron Microscope (SEM) image of microspheres 20% API/9:1 R202H:752H (1500X) Fig. 8 API loading effect on IVR microsphere profile 15% API/R202H, 17% API/R202H and 19% API/R202H Time (days) Fig. 9 influence of API load on the profile of IVR microspheres 16% API/9.5:0.5R202H:H752.18% API/9.5:0.5R202H:H752 and 20% API/9.5:0.5R202H:H752 140-, O 20 40 60 80 100 120 140 160 180 Time (days) Fig. 10 - 29 040073 Influence of the polymer blend on the IVR profile - microspheres 16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16% API/9:1 R202H:RG752H io О 20 40 60 80 100 120 AI 160 180 Time (days) Fig. 11 Effect of Polymer Blend on IVR Microsphere Profile 16% API/R202H, 16% API/9.5:0.5 R202H:RG502H and 16% API/9:! R202H:RG502H 16% GZ389988 _Β ι 202H 16% GZ389988 in 202H 502H (9.5:0.5) 16% GZ389988 bj 202N 502N (9 P 100 120 140 160 180 200 Time (days) Fig. 12 formulations showing zero order IVR profile over 180 days - microspheres 15% API/9:1 R203H:RG752H and 16% API/9.5:0.5 R202H:RG502H 120 Time (days) Fig. 13 - 30 040073 influence of auxiliary solvent systems used in preparation on the IVR profile - microspheres 16% AP1/9:1 R203H:RG752H - 9:1 DCM:MeOH, 9:0.5:0.5 DCM:MeOH:BA, 9.5:0.05 DCM:BA, 9:1 DCM:BA. Time (days) Fig. 14 Effect of DCM:BA co-solvent system to increase API filling on IVR profile - microspheres 16% AP1/9:1 R202H:RG752H, 25% API/9:1 R202H:RG752H, 30% API/R202H, 40% API/R202H , 25% API/R203H, 30% API/R203H, 40% API/R203H, 50% API/R203H. Fig. 15 Scanning Electron Microscope (SEM) images of microspheres obtained using a fine suspension microencapsulation process (1500X). Fig. 16 - 31 040073 effect of the microencapsulation process of the fine suspension on the profile of the IVR microsphere 25% API/9:1 R202H:RG752H 39 ml 5% PVA + 2.6 ml EA, 25% API/9:1 R202H:RG752H 39 ml 5% PVA + 3.25 ml EA, 25% API/R203H 39 ml 5% PVA + 2.0 ml EA, 30% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA + 2.0 ml EA, 40% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA + 2.0 ml EA, 30% microfluidized API/9:1 R202H:RG752H 39 ml 5% PVA + 2.0 ml EA, 30% microfluidized API/9: 1 R202H:RG752H 39 ml 5% PVA + 2.3 ml EA, 30% microfluidized API/R202S 39 ml 5% PVA + 2.0 ml EA, 30% microfluidized API/R203S 39 ml 5% PVA + 2.0 ml . - *-25% GZ389988 at 202H:752H (9:1) - I 25% GZ389988 at 202H:752H (9:1) 25% GZ389988 in 203H „„„30% GZ389988 in 203H:752H (9:1) microfluidized API. - -40% GZ389988 in 203H:752H (9:1) microfluidized API -c- -30% GZ389988 in 203H:752H (9:1) microfluidized API - 30% GZ389988 in 203H:752H (9:1) Microfluidized API™,™ 30% GZ389988 in 202S Microfluidized API - 30% GZ389988 in 203S Microfluidized API Time (days) Fig. 17 Scanning Electron Microscope (SEM) image of 16% AP1/9:1 R202H:RG752H microspheres, solvent extraction (1000X) Fig. 18 Scanning Electron Microscope (SEM) image of 16% AP1/9:1 R202H:RG752H microspheres, spray dried (1000X) Fig. 19 - 32 040073 effect of the microencapsulation process on the IVR profile - microcapsules 16% API/9:1 R202H:RG752H obtained by solvent extraction (OW), microspheres 16% API/9:1 R202H:RG752H obtained by spray drying (20% and 22.5% polymer), and 16% AP1/9:1 R203H:RG752H spray-dried microspheres (22.5% polymer) Time (days) Fig. 20 Scanning Electron Microscope (SEM) image Fig. 21 Scanning Electron Microscope (SEM) images Fig. 22 - 33 040073 Scanning Electron Microscope (SEM) image of microspheres 16% API/1:1 R202H:R203H/31.25 mg poloxamer 407, spray dried (1000X) Fig. 23 Effect of 10 kDa PEG or 1% poloxamer 407 on IVR profile - 16% API/1:1 R202H:R203H/6e3 supplementation, 16% API/1:1 R202H:R203H/31.25 mg PEG, 16% ART/1 :1 R202H:R203H/31.25 mg poloxamer 407 100 16% GZ389988 at 50:50 202H:203H 16% GZ389988 at 49.5:49.5:1 202H:203H:10kDa PEG 16% GZ389988 at 49.5:49.5:1 202H:203H: poloxamer 407 Time (days) Fig. 24 Profile (rat) IVR/and vivo- microspheres 16% AP1/9:1 202H:RG502H and 15% API/9:1 R203H:RG752H Fig. 25 - 34 040073 preservation of [ ¹⁴ C] 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6 methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b] pyridine-2-amine over time (rat knee joint) - 16% API/9:1 202H:RG502H microspheres and 15% API/9:1 203H:RG752H microspheres 100 o 0 1 2 3 4 5 6 Time (months) Fig. 26 concentration-time profiles for 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5b]pyridine -2-amines (rat blood) - microspheres 16% API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H X 16% GZ389988 in 9:1 202N:752N □ 15% GZ389988 in 9:1 202N:752N 1 2 3 4 5 Time (months) - represents the ECso value (in relation to cells) for 3-(3-mstoxy-4-((4mstoxybsnzyl)oxy)bsnzyl)-6-(1-mstyl-111-pyrazol-4-yl)-311 -imidazo|4.5-b|pyridine-2-amine at a concentration of 1.4 ng/ml) Fig. 27 profile IVR microspheres 16% API/9:1 202:H:RG752H, 15% AP1/9:1 R203H:RG752H and 40% API/203H — Composition A: 16% GZ389988A in 9:1 202H752H Composition: 15% GZ389988A at 9:1203H752H Composition C: 40% GZ389988A in 203H Time (days) Fig. 28 - 35 040073 concentration-time profiles for 3-(3-methoxy-4-((4methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5b ]pyridine-2-amine microspheres 16% API/9:1 202:H:RG752H, 15% AP1/9:1 R203H:RG752H and 40% API/203H K 0.0 about « 0 200 400 600 800 Time (h) Fig. 29 measured or plotted XRPD forms 1-4 (anhydrous and hydrated forms) Form I Form 2 Form 3 Form 4 _ Scale 2 theta [T] Monohydrate 1 (Form 1) [D Anhydrous 1 (Form 2) □ Anhydrous 2 (Form 3) Plotted XRPD for sesquihydrate (Form 4) Fig. thirty XRPD form 5 (ethanol) Scale 2 theta III Solvate - EtOH - File: br 1136.raw And built by XRSCD Ethanol solvate bar chart by XRPD curve indexing Y Ethanol solvate bar chart by XRSCD Fig. 31 - 36 040073 XRPD form 9 (acetone) Scale 2 theta Acetone isolvate 1 - File: XA1089.raw ^Builded from XRSCD XRSCD acetone solvate bar chart Fig. 32 comparison of XRPD form 10 (acetone) and form 9 (acetone) Fig. 33 XRPD form 11 (acetonitrile) Scale 2 theta Acetonitrile solvate - File: GZ38_ACN_PDl.raw SH Bar chart for acetonitrile solvate by indexing Fig. 34 - 37 040073 solid crystalline phases -2-amine Solvate 2 acetone Acetonitrile solvate Anhydrous monohydrate 2 Monohydrate 2 Acetone solvate I Ethanol solvate Phase I Anhydrous monohydrate I Polz torahydrate (built) Monohydrate I Scale 2 theta Fig. 35 Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2