HK40004663B - Liquid formulations of a compound - Google Patents

Description

A liquid formulation of a compound

Cross-references to related applications

This application claims priority to U.S. Provisional Application Serial No. 62/318,041, filed April 4, 2016; U.S. Provisional Application Serial No. 62/323,452, filed April 15, 2016; and U.S. Provisional Application Serial No. 62/329,561, filed April 29, 2016, the entire contents of which are incorporated herein by reference.

Technical Field

This disclosure relates to liquid formulations of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (Formula I), pharmaceutically acceptable salts thereof, or combinations thereof, and the use of such liquid formulations in the treatment of pain, inflammation, cancer, and certain infectious diseases.

Background Technology

Trks are high-affinity receptor tyrosine kinases activated by a group of soluble growth factors called neurotrophic factors (NTs). The Trk receptor family has three members—TrkA, TrkB, and TrkC. Neurotrophic factors include (i) nerve growth factor (NGF) which activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activate TrkB, and (iii) NT3 which activates TrkC. Trks are widely expressed in neuronal tissues and are involved in neuronal cell maintenance, signal transduction, and survival (Patapoutian, A. et al., Current Opinion in Neurobiology, 2001, 11, 272-280).

Recent literature has shown that overexpression, activation, amplification, and/or mutation of Trk are associated with many cancers, including neuroblastoma (Brodeur, G.M., Nat. Rev. Cancer 2003, 3, 203-216), ovarian cancer (Davidson., B. et al., Clin. Cancer Res. 2003, 9, 2248-2259), breast cancer (Kruettgen et al., Brain Pathology 2006, 16: 304-310), and prostate cancer (Dionne et al., Clin. Cancer Res. 1998, 4(8): ). 1887-1898), pancreatic cancer (Dang et al., Journal of Gastroenterology and Hepatology 2006, 21(5):850-858), multiple myeloma (Hu et al., Cancer Genetics and Cytogenetics 2007, 178:1-10), astrocytoma and medulloblastoma (Kruettgen et al., Brain Pathology 2006, 16:304-310), glioma (Hansen et al., Journal of N Eurochemistry 2007, 103:259–275), melanoma (Nakagawara, A. (2001) Cancer Letters 169:107-114; Meyer, J. et al. (2007) Leukemia, 1–10; Pierottia, M.A. and Greco A., (2006) Cancer Letters 232:90–98; Eric Adriaenssens, E. et al. Cancer Res (2008) 68:(2)346-351), thyroid cancer (Brz Perez-Pinera et al., Neuroendocrinology Letters 2007, 28(3), 221-229), lung adenocarcinoma (Perez-Pinera et al., Molecular and Cellular Biochemistry 2007, 295(1&2), 19-26), large cell neuroendocrine tumor (Marchetti et al., Human Mutation 2008, 29(5), 609-616), and colorectal cancer (Bardelli, A., Science 2003, 300, 949). In preclinical models of cancer, Trk inhibitors were effective in inhibiting tumor growth and preventing tumor metastasis. In particular, non-selective small molecule inhibitors of TrkA, TrkB, TrkC, and Trk/Fc chimeras are effective in inhibiting tumor growth and preventing tumor metastasis (Nakagawara, A. (2001) Cancer Letters 169:107-114; Meyer, J. et al. (2007) Leukemia, 1–10; Pierottia, M.A. and Greco A., (2006) Cancer Letters 232:90–98; Eric Adriaenssens, E. et al. Cancer Res (2008) 68:(2)346-351). Therefore, inhibitors of the Trk kinase family are expected to be used for cancer treatment.

Furthermore, inhibitors of the Trk/neurotrophic pathway have been shown to be effective in many preclinical animal models of pain. For example, antagonistic NGF and TrkA antibodies (e.g., RN-624) have been shown to be effective in animal models of inflammation and neurotrophic pain and in human clinical trials (Woolf, C.J. et al. (1994) Neuroscience 62, 327–331; Zahn, P.K. et al. (2004) J. Pain 5, 157–163; McMahon, S.B. et al. (1995) Nat. Med. 1, 774–780; Ma, Q.P. and Woolf, C.J. (1) 997) Neuroreport 8,807–810; Shelton, D.L. et al. (2005) Pain 116,8–16; Delafoy, L. et al. (2003) Pain 105,489–497; Lamb, K. et al. (2003) Neurogastroenterol. Motil. 15,355–361; Jaggar, S.I. et al. (1999) Br.J. Anaesth. 83,442–448. Furthermore, recent literature indicates that BDNF levels and TrkB signaling increase in dorsal root ganglia after inflammation (Cho, L. et al. Rain Research 1997,749,358), and some studies have shown that antibody pathways that reduce BDNF/TrkB signaling inhibit neuronal hypersensitivity and related pain (Chang-Qi, L. et al. Molecular Pain 2008,4:27).

It has been shown that NGF secreted by tumor cells and tumor-invading macrophages directly stimulates TrkA located on peripheral pain fibers. Using various tumor models in mice and rats, it has been demonstrated that neutralizing NGF with monoclonal antibodies inhibits cancer-related pain to a degree similar to or higher than the highest tolerated dose of morphine. Furthermore, activation of the BDNF/TrkB pathway has been implicated as a modulator of various types of pain, including inflammatory pain (Matayoshi, S., J. Physiol. 2005, 569:685-95), neuropathic pain (Thompson, S.W., Proc. Natl. Acad. Sci. USA 1999, 96:7714-18), and surgical pain (Li, C.-Q. et al. Molecular Pain, 2008, 4(28), 1-11). Because TrkA and TrkB kinases can act as mediators of NGF-driven biological responses, inhibitors of TrkA and/or other Trk kinases could provide effective treatment for chronic pain states.

Current treatments for pain disorders utilize several classes of compounds. Opioids (such as morphine) have several drawbacks, including emetic, constipation, and negative respiratory effects, as well as the potential for addiction. Nonsteroidal anti-inflammatory drugs (NSAIDs, such as COX-1 or COX-2 types) also have disadvantages, including insufficient efficacy in treating severe pain. Furthermore, COX-1 inhibitors can cause mucosal ulcers. Therefore, there is a constant need for new and more effective treatments to relieve pain, especially chronic pain.

Furthermore, inhibition of the neurotrophin/Trk pathway has been shown to be effective in treating preclinical models of inflammatory diseases. For example, inhibition of the neurotrophin/Trk pathway has been applied to preclinical models of inflammatory lung diseases, including asthma (Freund-Michel, V; Frossard, N.; Pharmacology & Therapeutics (2008), 117(1), 52-76), interstitial cystitis (Hu Vivian Y; et al. The Journal of Urology (2005), 173(3), 1016-21), and inflammatory bowel diseases, including ulcerative colitis and Crohn's disease (Di... Mola, F.F, et al., Gut (2000), 46(5), 670-678) and inflammatory skin diseases such as atopic dermatitis (Dou, Y.-C. et al., Archives of Dermatological Research (2006), 298(1), 31-37), eczema and psoriasis (Raychaudhuri, S.P. et al., Journal of Investigative Dermatology (2004), 122(3), 812-819).

The neurotrophic factor/Trk pathway, particularly BDNF/TrkB, is also involved in the etiology of neurodegenerative diseases, including multiple sclerosis, Parkinson's disease, and Alzheimer's disease (Sohrabji, Farida; Lewis, Danielle K. Frontiers in Neuroendocrinology (2006), 27(4), 404-414). Regulation of the neutrophil/Trk pathway could be used to treat these and related diseases.

The TrkA receptor is also considered crucial for the infection process of Trypanosoma cruzi (Chagas disease) in human hosts (de Melo-Jorge, M. et al., Cell Host & Microbe (2007), 1(4), 251-261). Therefore, TrkA inhibition could be used to treat Chagas disease and related protozoan infections.

Trk inhibitors can also be used to treat diseases associated with dysregulation of bone remodeling, such as osteoporosis, rheumatoid arthritis, and bone metastases. Bone metastases are a common complication of cancer, occurring in up to 70% of patients with advanced breast or prostate cancer and approximately 15%–30% of patients with lung, colon, stomach, bladder, uterine, rectal, thyroid, or kidney cancer. Osteolytic metastases can cause severe pain, pathological fractures, life-threatening hypercalcemia, spinal cord compression, and other nerve compression syndromes. For these reasons, bone metastases are a serious and costly complication of cancer. Therefore, agents that can induce apoptosis in proliferating osteoblasts would be highly advantageous. The expression of TrkA and TrkC receptors has been observed in bone-forming regions in fracture mouse models (K. Asaumi et al., Bone (2000) 26(6) 625–633). Furthermore, NGF localization has been observed in almost all osteoblasts (K. Asaumi et al.). Recently, it has been demonstrated that pan-Trk inhibitors suppress tyrosine signaling activated by all three Trk receptors in neurotrophic factor-bound human hFOB osteoblasts (J. Pinski, et al. (2002) 62, 986-989). These data support the basic principle of using Trk inhibitors to treat bone remodeling disorders, such as bone metastases in cancer patients.

Several classes of small molecule inhibitors of Trk kinase are known to be used to treat pain or cancer (Expert Opin. Ther. Patents (2009) 19(3)).

International patent applications WO2006/115452 and WO2006/087538 describe several classes of small molecules that are claimed to be inhibitors of Trk kinase, which could be used to treat pain or cancer.

Pyrazolo[1,5-a]pyrimidine compounds are known. For example, International Patent Application Publication WO2008/037477 discloses pyrazolo[1,5-a]pyrimidine compounds carrying an alkyl, aryl, or heterocyclic group at the 3-position. These compounds are considered to be inhibitors of PI3K and/or mTOR lipid kinases.

PCT patent publication number WO2008/058126 discloses pyrazolo[1,5-a]pyrimidine compounds carrying a phenyl group at the 3-position. These compounds are considered to be Pim-kinase inhibitors.

US Patent Publication No. 2006/0094699 discloses pyrazolo[1,5-a]pyrimidine compounds carrying a -C(=O)NH-phenyl, -C(=O)(4-methylpiperidinyl) or -C(=O)NMe( _CH2 -trimethylpyrazolyl) group at the 3-position for use in combination therapy with glucocorticoid receptor agonists.

PCT patent publication numbers WO2010/033941, WO2010/048314, WO2011/006074 and WO2011/146336 disclose compounds exhibiting inhibition of Trk family protein tyrosine kinases, which can be used to treat pain, cancer, inflammation, neurodegenerative diseases and certain infectious diseases.

WO2010/048314 discloses the hydrogen sulfate of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide in Example 14A. When prepared according to the method of Example 14A in that document, WO2010/048314 does not disclose the specific form of the hydrogen sulfate described herein. In particular, WO2010/048314 does not disclose the crystalline form (I-HS) as described below.

All references cited in this disclosure, including scientific articles, patent publications and applications, are incorporated herein by reference in their entirety.

Summary of the Invention

This document provides a liquid formulation comprising a solubilizer and (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide having formula (I):

Its pharmaceutically acceptable salt, or a combination thereof.

In some embodiments, the presence of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, is from about 0.5% by weight to about 7% by weight. For example, a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, may be present in a liquid formulation in an amount from about 1.5% by weight to about 2.5% by weight.

In some embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, has a concentration of about 5 mg/mL to about 50 mg/mL in the liquid formulation. For example, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, may have a concentration of about 15 mg/mL to about 35 mg/mL in the liquid formulation. In some embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, has a concentration of about 20 mg/mL in the liquid formulation.

The solubilizer may be selected from the group consisting of cyclodextrins, ethylene glycol, glycerol, and combinations thereof. In some embodiments, the solubilizer includes cyclodextrins. For example, the solubilizer may be selected from the group consisting of β-cyclodextrin derivatives, γ-cyclodextrins, and combinations thereof. In some embodiments, the solubilizer includes hydroxyalkyl-γ-cyclodextrin. The solubilizer may include β-cyclodextrins selected from the group consisting of hydroxyalkyl-β-cyclodextrins, sulfonyl ether-β-cyclodextrins, and combinations thereof. In some embodiments, the solubilizer includes hydroxypropyl-β-cyclodextrin.

In some embodiments, the solubilizer is present in the liquid formulation in an amount of about 5% to about 35% by weight. For example, the solubilizer may be present in the liquid formulation in an amount of about 13% to about 17% by weight.

Liquid formulations may further include buffers. In some embodiments, the buffer includes at least one of citrate buffers, lactate buffers, phosphate buffers, maleate buffers, tartrate buffers, succinate buffers, or acetate buffers. In some embodiments, the buffer includes at least one of lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, sodium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, or calcium acetate. The buffer may be a citrate buffer. Citrate buffers may include at least one of lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate. In some embodiments, the buffer includes at least one of sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate.

In some implementations, the buffer includes sodium citrate dihydrate.

The buffer may be present in the liquid formulation in amounts of about 0.1% by weight to about 5% by weight.

In some embodiments, the formulation has a pH of about 2 to about 7. For example, the formulation may have a pH of about 3 to about 4. In some embodiments, the formulation has a pH of about 3.5.

In some embodiments, the pH of the liquid formulation is adjusted. In some such embodiments, the formulation includes a base. For example, the base may include one or more of citrate, lactate, phosphate, maleate, tartrate, succinate, acetate, carbonate, and hydroxide. In some embodiments, the formulation includes at least one of lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate, sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, or combinations thereof. In some embodiments, the base includes a citrate. Citrates may include at least one of lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate. In some embodiments, the liquid formulation includes at least one of sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate.

In some embodiments, the base includes sodium citrate dihydrate.

In some embodiments, the formulation contains about 0.1% to about 5% by weight of a base, such as a citrate (e.g., sodium citrate dihydrate).

Liquid formulations may further include sweeteners. In some embodiments, the sweetener includes sugar. Sugar may include sucrose. In some embodiments, the sweetener includes a strong sweetener. A strong sweetener may include sucralose.

In some embodiments, the sweetener is present in the liquid formulation at an amount of about 30% to about 70% by weight. For example, the sweetener may be present in the liquid formulation at an amount of about 45% to about 55% by weight.

The liquid formulation may further include a bitterness masking agent. In some embodiments, the bitterness masking agent is present in the liquid formulation in an amount of about 0.01% by weight to about 2% by weight. For example, the bitterness masking agent may be present in the liquid formulation in an amount of about 0.2% by weight to about 0.5% by weight.

The liquid formulation may further include a flavoring agent. The flavoring agent may include at least one of natural flavoring agents, natural fruit flavoring agents, artificial flavoring agents, artificial fruit flavoring agents, or flavor enhancers. In some embodiments, the flavoring agent is present in the liquid formulation at an amount of about 0.01% by weight to about 2% by weight. For example, the amount of flavoring agent present in the liquid formulation may be about 0.01% by weight to about 0.1% by weight.

In some embodiments, the liquid formulation also includes a colorant.

In some embodiments, the liquid formulation is prepared from a pharmaceutically acceptable salt of a compound of formula (I). For example, the liquid formulation may be prepared from a hydrogen sulfate salt of a compound of formula (I).

In some embodiments, the liquid formulation is prepared from the crystalline form of a compound of formula (I). In some embodiments, the crystalline form has the formula (I-HS):

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salt, or a combination thereof. The liquid formulation also contains a solubilizer and a buffer. The pH of the liquid formulation is from about 2.5 to about 5.5. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof in the liquid formulation is from about 15 mg/mL to about 35 mg/mL.

In some embodiments, the liquid formulation has a pH of about 3 to about 4.

In some implementations, the buffer includes sodium citrate dihydrate.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The liquid formulation contains a pharmaceutically acceptable salt of the compound (I), or a combination thereof. The liquid formulation also contains a solubilizer and a base. The pH of the liquid formulation is from about 2.5 to about 5.5. In some embodiments, the base includes a citrate (e.g., sodium citrate). The concentration of the compound (I), a pharmaceutically acceptable salt of the compound, or a combination thereof, in the liquid formulation is from about 15 mg/mL to about 35 mg/mL.

In some embodiments, the liquid formulation has a pH of about 3 to about 4.

In some embodiments, the base includes sodium citrate dihydrate.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salt, or a combination thereof. The liquid formulation also contains solubilizers, buffers, sweeteners, bitterness masking agents, and flavoring agents. The pH of the liquid formulation is from about 3 to about 4. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is from about 15 mg/mL to about 35 mg/mL.

In some implementations, the buffer includes sodium citrate dihydrate.

In some implementations, the sweetener includes sucrose.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The liquid formulation contains a pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation also contains a solubilizer, a base, a sweetener, a bitterness masking agent, and a flavoring agent. The pH of the liquid formulation is from about 3 to about 4. In some embodiments, the base includes a citrate (e.g., sodium citrate). The concentration of the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, in the liquid formulation is from about 15 mg/mL to about 35 mg/mL.

In some embodiments, the base includes sodium citrate dihydrate.

In some implementations, the sweetener includes sucrose.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The pharmaceutically acceptable salt of the compound (I), or a combination thereof, is present in the liquid formulation in an amount of about 5% to about 35% by weight of a solubilizer and in an amount of about 0.1% to about 5% by weight of a buffer. The pH of the liquid formulation is about 2.5 to about 5.5. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation also contains, in an amount of about 5% to about 35% by weight, a solubilizer; in an amount of about 0.1% to about 5% by weight, a buffer; in an amount of about 30% to about 70% by weight, a sweetener; in an amount of about 0.2% to about 0.5% by weight, a bitterness masking agent; and in an amount of about 0.01% to about 2% by weight, a flavoring agent. The pH of the liquid formulation is about 2.5 to about 5.5. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation also contains, in an amount of about 5% to about 35% by weight, a solubilizer; in an amount of about 0.1% to about 5% by weight, a base; in an amount of about 30% to about 70% by weight, a sweetener; in an amount of about 0.2% to about 0.5% by weight, a bitterness masking agent; and in an amount of about 0.01% to about 2% by weight, a flavoring agent. The pH of the liquid formulation is about 2.5 to about 5.5. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The liquid formulation contains a pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation contains a solubilizer present in an amount of about 5% to about 35% by weight. The liquid formulation also contains a buffer containing sodium citrate dihydrate present in an amount of about 0.1% to about 5% by weight. The liquid formulation also contains a sweetener containing sucrose present in an amount of about 30% to about 70% by weight. The liquid formulation also contains a bitterness masking agent present in an amount of about 0.2% to about 0.5% by weight. The liquid formulation also contains a flavoring agent present in an amount of about 0.01% to about 2% by weight. The pH of the liquid formulation is about 3 to about 4. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The liquid formulation contains a pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation contains a solubilizer present in an amount of about 5% to about 35% by weight. The liquid formulation also contains a base comprising sodium citrate dihydrate present in an amount of about 0.1% to about 5% by weight. The liquid formulation also contains a sweetener comprising sucrose present in an amount of about 30% to about 70% by weight. The liquid formulation also contains a bitterness masking agent present in an amount of about 0.2% to about 0.5% by weight. The liquid formulation also contains a flavoring agent present in an amount of about 0.01% to about 2% by weight. The pH of the liquid formulation is about 3 to about 4. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

This document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The liquid formulation contains a pharmaceutically acceptable salt thereof, or a combination thereof. The liquid formulation contains a solubilizer present in an amount of about 5% to about 35% by weight. The liquid formulation also contains sodium citrate dihydrate present in an amount of about 0.1% to about 5% by weight. The liquid formulation also contains a sweetener comprising sucrose present in an amount of about 30% to about 70% by weight. The liquid formulation also contains a bitterness masking agent present in an amount of about 0.2% to about 0.5% by weight. The liquid formulation also contains a flavoring agent present in an amount of about 0.01% to about 2% by weight. The pH of the liquid formulation is about 3 to about 4. The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof, in the liquid formulation is about 20 mg/mL to about 30 mg/mL.

The liquid formulation provided herein can be prepared from the crystalline form of a compound of formula (I), which has the formula (I-HS):

In some embodiments, the crystalline form may be characterized by having XRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2, 27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

This article also provides a method for treating cancer in patients in need. This method involves identifying patients with dysphagia who require treatment and administering a therapeutically effective amount of the liquid formulation provided herein.

This article also provides a method for treating cancer in patients in need, comprising identifying patients with dysphagia, determining whether the cancer is mediated by Trk kinase, and, if the cancer is determined to be mediated by Trk kinase, administering a therapeutically effective amount of the liquid formulation provided herein to the patient.

This article also provides methods for treating cancer in patients in need, which include administering a therapeutically effective amount of the liquid formulation provided herein to the patient.

In some implementations, the cancer is selected from the group consisting of head and neck cancer, pharyngeal cancer, esophageal cancer, or combinations thereof.

In some implementations, the patient is an infant, child, adolescent, or elderly patient.

This article also provides a method for treating cancer in a subject in need. The method includes determining whether the cancer is associated with and/or exhibits one or more of overexpression, activation, amplification, and mutation of Trk kinases, and if the cancer is determined to be associated with and/or exhibits one or more of overexpression, activation, amplification, and mutation of Trk kinases, administering a therapeutically effective amount of the liquid formulation provided herein to the subject.

This article also provides a method for treating cancer in a subject in need, comprising determining whether the cancer is mediated by Trk kinase, and if the cancer is determined to be mediated by Trk kinase, administering to the subject a therapeutically effective amount of the liquid formulation provided herein. This article also provides a method for treating cancer in a subject in need, comprising identifying cancer mediated by Trk kinase and administering to the subject a therapeutically effective amount of the liquid formulation provided herein.

This article also provides a method for treating subjects. The method includes measuring samples obtained from the subjects to determine whether the subjects have dysregulation of the NTRK gene, Trk protein, or their expression or level. The method also includes administering a therapeutically effective amount of the liquid formulation provided herein to subjects determined to have dysregulation of the NTRK gene, Trk protein, or their expression, activity, or level.

In some implementations, dysregulation of the NTRK gene, the Trk protein, or their expression or level is a chromosomal translation that leads to the translation of the Trk fusion protein. Trk fusion proteins can be selected from the following groups: TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C20ORF112-TrkA, DNER-TrkA, ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA6-TrkA, PEAR1-TrkA. A, MRPL24-TrkA, MDM4-TrkA, LRRC71-TrkA, GRIPAP1-TrkA, EPS15-TrkA, DYNC2H1-TrkA, CEL-TrkA, EPHB2-TrkA, TGF-TrkA, NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, SQ STM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB, DAB2IP-TrkB, ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC and TEL-TrkC.

In some embodiments, dysregulation of the NTRK gene, Trk protein, or their expression or activity is one or more point mutations in the gene. The NTRK gene may be the NTRK1 gene, and one or more point mutations in the NTRK1 gene may result in the translation of a TrkA protein with substitutions at one or more of the following amino acid positions: 33, 336, 337, 324, 420, 444, 517, 538, 649, 682, 683, 702, and 1879.

In some implementations, one or more point mutations in the NTRK1 gene result in the translation of a TrkA protein with one or more of the following amino acid substitutions: R33W, A336E, A337T, R324Q, R324W, V420M, R444Q, R444W, G517R, G517V, K538A, R649W, R649L, R682S, V683G, R702C, and C1879T.

The features and advantages described in the present invention and the following detailed description are not exhaustive. Many additional features and advantages will be apparent to those skilled in the art from the accompanying drawings, specification, and claims.

Attached Figure Description

Figure 1 shows an X-ray powder diffraction (XRPD) pattern of the crystalline form (I-HS) prepared according to Example 2 according to one embodiment.

Figure 2 shows the simultaneous thermogravimetric/differential calorimetry (TG/DTA) curves of the crystalline form (I-HS) prepared according to Example 2 according to one embodiment.

Figure 3 shows the differential scanning calorimetry (DSC) curve of the crystalline form (I-HS) prepared according to Example 2 according to one embodiment.

Figures 4A and 4B show polarized light microscopy (PLM) images of the crystalline form (I-HS) prepared under (A) unpolarized and (B) polarized light according to Example 2 according to one embodiment.

Figure 5 shows the dynamic vapor adsorption (DVS) isotherm of the crystalline form (I-HS) prepared according to Example 2 according to one embodiment.

Figure 6 shows the infrared (IR) spectrum of the crystalline form (I-HS) prepared according to Example 2 according to one embodiment.

Figure 7 shows an XRPD diagram of the amorphous free base form of the compound of formula I according to one embodiment.

Figure 8 shows the X-ray powder diffraction (XRPD) pattern of the crystalline form (I-HS).

Figure 9 is a pictogram of the instructions for use of pediatric solution formulations in crystalline form (I-HS).

Figure 10 is a set of six MR images showing the brain in the neck of a patient diagnosed with infantile fibrosarcoma. (A) and (B) are MR images of the brain and neck showing a 20 mm × 19 mm × 18 mm hyperenhancing mass, including the base of the skull in the middle cranial fossa, located anterior and inferior to the structures of the inner ear, five weeks after surgical resection. (C) and (D) are MR images of the brain and neck showing a significant reduction in size and an increase in mass of over 90% from baseline at the end of cycle 1 (day 28) of administration of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide BID. (E) and (F) are MR images of the brain and neck taken at the end of cycle 2, confirming the reduction in size and showing a continued decrease in enhancement, confirming a partial response.

Figure 11 is a sequence listing of an exemplary wild-type TrkA polypeptide (SEQ ID NO: 1).

Figure 12 is a sequence listing of an exemplary wild-type TrkA polypeptide (SEQ ID NO: 2).

Figure 13 is a sequence listing of an exemplary wild-type TrkA polypeptide (SEQ ID NO: 3).

Detailed Implementation

This disclosure relates to liquid formulations of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, pharmaceutically acceptable salts thereof, or combinations thereof, and the use of such liquid formulations in the treatment of pain, inflammation, cancer, and certain infectious diseases.

This invention provides a liquid formulation comprising a solubilizer and (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide:

Its pharmaceutically acceptable salt, or a combination thereof.

In some embodiments, the compound of formula (I), its pharmaceutically acceptable salts, or combinations thereof may be present in the liquid formulation in amounts of about 0.5% by weight to about 7%, about 1% by weight to about 3% by weight, or about 1.5% by weight to about 2.5% by weight. For example, the compound of formula (I), its pharmaceutically acceptable salts, or combinations thereof may be present in the liquid formulation in amounts of about 0.5% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, or about 7% by weight. In some embodiments, the compound of formula (I), its pharmaceutically acceptable salts, or combinations thereof may be present in the liquid formulation in an amount of about 2% by weight.

In some embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, is present in a liquid formulation at a concentration of about 5 mg/mL to about 50 mg/mL, about 15 mg/mL to about 35 mg/mL, or about 20 mg/mL to about 30 mg/mL. For example, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, may be present in a liquid formulation at a concentration of about 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or about 50 mg/mL. In some embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof, may be present in a liquid formulation at a concentration of about 20 mg/mL.

The formulations described herein may include solubilizers that increase the solubility of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof. A solubilizer is a polar organic compound having one or more hydroxyl groups. The solubilizer also enables the attainment of higher concentrations of the compound of formula (I) (e.g., free base) in aqueous solutions compared to the aqueous solubility of the compound of formula (I) within a similar pH range without a solubilizer. Solubilizers may include, for example, cyclodextrins, ethylene glycol, glycerol, polyethylene glycol, self-emulsifying drug delivery systems (SEDDS), or combinations thereof.

In some embodiments, the cyclodextrin may include α-cyclodextrin, β-cyclodextrin derivatives, δ-cyclodextrin derivatives, γ-cyclodextrin, or combinations thereof. For example, the solubilizer may include cyclodextrin. The solubilizer may include β-cyclodextrin derivatives, γ-cyclodextrin, or mixtures thereof. For example, the solubilizer may include hydroxyalkyl-γ-cyclodextrin. In some embodiments, the solubilizer includes β-cyclodextrin, comprising at least one of hydroxyalkyl-β-cyclodextrin (e.g., hydroxypropyl-β-cyclodextrin) or sulfonyl ether-β-cyclodextrin (e.g., sulfobutyl ether-β-cyclodextrin). For example, the liquid solubilizer may include hydroxypropyl-β-cyclodextrin. In some embodiments, the cyclodextrin is W7 HP (hydroxypropyl-β-cyclodextrin). In some embodiments, the cyclodextrin is HP (hydroxypropyl-β-cyclodextrin). In some embodiments, the cyclodextrin is W7 (β-cyclodextrin). In some embodiments, the cyclodextrin is (sulfonyl ether-β-cyclodextrin). In some embodiments, the cyclodextrin is W7M (methyl-β-cyclodextrin). In some embodiments, the cyclodextrin is W8 HP (hydroxypropyl-γ-cyclodextrin). In some embodiments, the cyclodextrin is W8 (γ-cyclodextrin). In some embodiments, the cyclodextrin is W6 (α-cyclodextrin).

SEDDS are isotropic mixtures of oils, surfactants, solvents, and co-solvents/surfactants that can be used to improve the oral absorption of highly lipophilic drug compounds. See, for example, Tarate, B. et al., Recent Patents on Drug Delivery & Formulation (2014) Vol. 8.

In some embodiments, the poly(ethylene glycol) molecule is a linear polymer. The molecular weight of linear PEG can be from about 1,000 Da to about 100,000 Da. For example, the linear PEG used herein can have molecular weights of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, or 1,000 Da. In some embodiments, the molecular weight of linear PEG is from about 1000 Da to about 50000 Da. In some embodiments, the molecular weight of linear PEG is from about 1000 Da to about 40000 Da. In some embodiments, the molecular weight of linear PEG is from about 5000 Da to about 40000 Da. In some embodiments, the molecular weight of linear PEG is from about 5000 Da to about 20000 Da.

In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG can be from about 1,000 Da to about 100,000 Da. For example, the branched PEG used herein can have molecular weights of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, or 1,000 Da. In some embodiments, the molecular weight of branched PEG is from about 1000 Da to about 50000 Da. In some embodiments, the molecular weight of branched PEG is from about 1000 Da to about 40000 Da. In some embodiments, the molecular weight of branched PEG is from about 5000 Da to about 40000 Da. In some embodiments, the molecular weight of branched PEG is from about 5000 Da to about 20000 Da.

In some embodiments, the solubilizer may be present in the liquid formulation in amounts of about 5% to about 35% by weight, about 10% to about 25% by weight, about 10% to about 20% by weight, or about 13% to about 17% by weight. For example, the solubilizer may be present in amounts of about 5% by weight, 7% by weight, 10% by weight, 13% by weight, 15% by weight, 17% by weight, 20% by weight, 23% by weight, 26% by weight, 30% by weight, or about 35% by weight. In some embodiments, the solubilizer is present in the liquid formulation at 15% by weight.

A buffer can be added to a liquid formulation to adjust the pH of the formulation to the desired pH. In some embodiments, a buffer can be added in an amount that adjusts the pH of the formulation to about 2 to about 7, about 2.5 to about 5.5, or about 3 to about 4. For example, a buffer can be added in an amount that adjusts the pH of the formulation to about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, or about 7.0. In some embodiments, a buffer can be added in an amount that adjusts the pH of the formulation to about 3.5. In some embodiments, the buffer includes citrate buffers, lactate buffers, phosphate buffers, maleate buffers, tartrate buffers, succinate buffers, acetate buffers, or combinations thereof. In some embodiments, the buffer comprises lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate, or combinations thereof. In some embodiments, the buffer is a citrate buffer. For example, citrate buffers may include at least one of lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, calcium citrate heptahydrate, or mixtures thereof. Buffers may include sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate. In some embodiments, the buffer includes sodium citrate dihydrate.

In some embodiments, the buffer is present in the liquid formulation in amounts of about 0.1 wt% to about 5 wt%, about 0.3 wt% to about 4 wt%, about 0.5 wt% to about 3.5 wt%, about 0.6 wt% to about 3 wt%, 0.7 wt% to about 2.5 wt%, about 0.7 wt% to about 2.0 wt%, or about 0.7 wt% to about 1.5 wt%. For example, the buffer may be present in the liquid formulation in amounts of about 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, 0.9 wt%, 1.1 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, or about 5 wt%. In some embodiments, the buffer is present in the liquid formulation at about 0.9 wt%.

The pH of the liquid formulation can be adjusted to a desired pH. In some embodiments, the pH of the formulation can be adjusted to about 2 to about 7, about 2.5 to about 5.5, or about 3 to about 4. For example, the pH of the formulation can be adjusted to about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, or about 7.0. In some embodiments, the pH of the formulation is adjusted to about 3.5. In some such embodiments, when the pH of the liquid formulation is adjusted to the desired pH, the liquid formulation includes a base. In some embodiments, the base is selected from citrate, lactate, phosphate, maleate, tartrate, succinate, acetate, carbonate, hydroxide, or combinations thereof. In some embodiments, the base includes lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate, sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, or combinations thereof. In some embodiments, the base includes citrate. For example, citric acid may include at least one of lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, citric acid hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, calcium citrate heptahydrate, or a mixture thereof. The base may include sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, or calcium citrate heptahydrate. In some embodiments, the base includes sodium citrate dihydrate.

In some embodiments, the base is present in the liquid formulation in amounts of about 0.1 wt% to about 5 wt%, about 0.3 wt% to about 4 wt%, about 0.5 wt% to about 3.5 wt%, about 0.6 wt% to about 3 wt%, 0.7 wt% to about 2.5 wt%, about 0.7 wt% to about 2.0 wt%, or about 0.7 wt% to about 1.5 wt%. For example, the base may be present in the liquid formulation in amounts of about 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, 0.9 wt%, 1.1 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, or about 5 wt%. In some embodiments, the base is present in the liquid formulation in an amount of about 0.9 wt%. For example, citrate is present in the liquid formulation in amounts of about 0.1 wt% to about 5 wt%, about 0.3 wt% to about 4 wt%, about 0.5 wt% to about 3.5 wt%, about 0.6 wt% to about 3 wt%, 0.7 wt% to about 2.5 wt%, about 0.7 wt% to about 2.0 wt%, or about 0.7 wt% to about 1.5 wt%. In some embodiments, citrate may be present in the liquid formulation in amounts of about 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, 0.9 wt%, 1.1 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, or about 5 wt%. For example, the amount of citrate present in the liquid formulation is about 0.9 wt%.

Liquid formulations may have a pH of about 2 to about 8, about 2.5 to about 6, about 3 to about 4, or about 3 to about 4. For example, liquid formulations may have a pH of about 2, 2.5, 3.0, 3.5, 4.0, 4.5, or about 5. In some embodiments, the formulation may have a pH of about 3.5.

Sweeteners can be added to liquid formulations to make them less bitter or more palatable, or both. Suitable sweeteners to be included in formulations can include natural and artificial sweeteners. In some embodiments, the sweetener is an artificial sweetener and may include strong or high-intensity sweeteners. Strong sweeteners are often used as sugar substitutes or alternatives because they are sweeter than sugar but contribute only a small amount to no calories when added to food. Exemplary strong sweeteners include sorbitol, sucrose, saccharin such as sodium saccharin, cyclohexylsulfamic acid salts such as sodium cyclohexylsulfamic acid, aspartame, sucralose, kiwifruit protein, and acesulfame potassium. In some embodiments, the sweetener is a natural sugar. For example, sugars such as monosaccharides, disaccharides, and polysaccharides may be used in the liquid formulations provided herein. Sugars may include xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, partially hydrolyzed starch or corn syrup, and sugar alcohols such as sorbitol, xylitol, mannitol, glycerol, and combinations thereof. In some embodiments, the liquid formulation also includes a sweetener. Sweeteners may include sugars. For example, sweeteners may include sucrose. For example, a sweetener may be a sweetener ORA—comprising purified water, sucrose, glycerol, sorbitol, and a flavoring agent; buffered with citric acid and sodium phosphate; and preserved with methylparaben and potassium sorbate. Sweeteners may also include strong sweeteners. Strong sweeteners may include sucralose. For example, the sweetener can be a sugar-free sweetener ORA - comprising purified water, glycerin, sorbitol, sodium saccharin, xanthan gum and flavoring agent; buffered with citric acid and sodium citrate; and preserved with methylparaben (0.03%), potassium sorbate (0.1%) and propylparaben (0.008%).

In some embodiments, the sweetener includes one or more of sucrose, glycerol, sorbitol, and flavoring agents. In some such embodiments, the sweetener also includes citric acid and sodium phosphate. In some such embodiments, the sweetener may include preservatives such as methylparaben and potassium sorbate. For example, the sweetener includes sucrose, glycerol, sorbitol, flavoring agents, citric acid, sodium phosphate, methylparaben, and potassium sorbate. In some embodiments, the sweetener includes one or more of glycerol, sorbitol, sodium saccharin, xanthan gum, and flavoring agents. In some such embodiments, the sweetener also contains citric acid and sodium citrate. In some such embodiments, the sweetener includes preservatives such as methylparaben, potassium sorbate, and propylparaben. For example, sweeteners may include glycerol, sorbitol, sodium saccharin, xanthan gum, flavoring agents, citric acid and sodium citrate, methylparaben (0.03%), potassium sorbate (0.1%) and propylparaben (0.008%).

In some embodiments, the sweetener is present in the liquid formulation in an amount of about 30% to about 70% by weight, about 35% to about 65% by weight, about 40% to about 60% by weight, or about 45% to about 55% by weight. For example, the sweetener may be present in the liquid formulation in an amount of about 30% by weight, 35% by weight, 40% by weight, 45% by weight, 50% by weight, 55% by weight, 60% by weight, 65% by weight, or about 70% by weight. In some embodiments, the sweetener is present in the liquid formulation in an amount of about 50% by weight.

In some embodiments, the liquid formulation also includes a bitterness masking agent. The bitterness masking agent may include 231a12 natural masking flavoring, 231a39 natural bitterness masking flavoring (natural), and FINATECH flavor improver flavoring (natural).

The bitterness masking agent may be present in the liquid formulation in amounts from about 0.01 wt% to about 2 wt%, from about 0.1 wt% to about 1.0 wt%, or from about 0.2 wt% to about 0.5 wt%. For example, the bitterness masking agent may be present in the liquid formulation in amounts from about 0.01 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.7 wt%, 1.0 wt%, 1.5 wt%, or 2.0 wt%. In some embodiments, the bitterness masking agent is present in the liquid formulation in an amount of about 0.4 wt%.

Flavoring agents may be included in liquid formulations to give the final formulation a substantially non-bitter and palatable taste. Flavoring agents may include at least one of natural flavorings, natural fruit flavorings, artificial flavorings, artificial fruit flavorings, flavor enhancers, or mixtures thereof. Exemplary flavorings can be found, for example, in US CFR 21 §172.515 (April 1, 2015), the entire contents of which are incorporated herein by reference. Examples include cinnamon, raspberry, orange, maple, caramel, licorice (Eurasian licorice) syrup, fruit, berry, vanilla, acacia syrup, coca, chocolate mint, wild cherry, walnut, sage, bubble gum, grapefruit, lime, marshmallow, melon, coffee, peach, lemon, anise, apricot, honey, mint, wintergreen, and cherry. In some embodiments, the flavoring agent may include natural flavorings. The flavoring agent is present in the liquid formulation in amounts from about 0.01% by weight to about 2% by weight, from about 0.01% by weight to about 0.1% by weight, or from about 0.2% by weight to about 0.5% by weight. For example, the flavoring agent may be present in amounts of about 0.01 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.7 wt%, 1.0 wt%, 1.5 wt%, or 2.0 wt%. In some embodiments, the flavoring agent may be present in the liquid formulation in an amount of about 0.5 wt%.

Liquid formulations may also contain colorants.

The liquid formulations provided herein can be prepared from the crystalline form of compounds of formula (I). The crystalline form can be of formula (I-HS):

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Pharmaceutically acceptable salts of formula (I), or combinations thereof, solubilizers, and buffers. In some embodiments, the formulation has a pH of about 2.5 to about 5.5. In some embodiments, the compound of formula (I) has a concentration of about 15 mg/mL to about 35 mg/mL. In some embodiments, the formulation has a pH of about 3 to about 4, and the compound of formula (I), or a pharmaceutically acceptable salt of formula (I), or a combination thereof, is present in the liquid formulation at a concentration of about 15 mg/mL to about 35 mg/mL. Buffers may include sodium citrate dihydrate.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The formulation may contain a pharmaceutically acceptable salt thereof, or a combination thereof, a solubilizer, and a base. In some embodiments, the formulation has a pH of about 2.5 to about 5.5. In some embodiments, the compound of formula (I) has a concentration of about 15 mg/mL to about 35 mg/mL. In some embodiments, the formulation has a pH of about 3 to about 4, and the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a combination thereof, is present in the liquid formulation at a concentration of about 15 mg/mL to about 35 mg/mL. The base may include sodium citrate dihydrate.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The compound (I) may contain a pharmaceutically acceptable salt thereof, or a combination thereof, a solubilizer, a buffer, a sweetener, a bitterness masking agent, or a flavoring agent. In some embodiments, the formulation has a pH of about 3 to about 4, and the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a combination thereof, is present in the liquid formulation at a concentration of about 15 mg/mL to about 35 mg/mL. In some embodiments, the buffer comprises sodium citrate dihydrate. In some embodiments, the sweetener comprises sucrose.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

The compound (I) may contain a pharmaceutically acceptable salt thereof, or a combination thereof, a solubilizer, a base, a sweetener, a bitterness masking agent, or a flavoring agent. In some embodiments, the formulation has a pH of about 3 to about 4, and the compound (I) or a pharmaceutically acceptable salt thereof, or a combination thereof, is present in the liquid formulation at a concentration of about 15 mg/mL to about 35 mg/mL. In some embodiments, the base includes sodium citrate dihydrate. In some embodiments, the sweetener includes sucrose.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salt, or a combination thereof, solubilizer, buffer, sweetener, bitterness masking agent, and flavoring agent, wherein the formulation has a pH of about 3 to about 4.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof, solubilizers, bases, sweeteners, bitterness masking agents, and flavoring agents, wherein the formulation has a pH of about 3 to about 4.

The document also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salt, or a combination thereof, solubilizer, buffer, sweetener, bitterness masking agent and flavoring agent, wherein the compound of formula (I) has a concentration of about 15 mg/mL to about 35 mg/mL in a liquid formulation.

This article also provides a liquid formulation comprising (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salt, or combination thereof, solubilizer, base, sweetener, bitterness masking agent and flavoring agent, wherein the compound of formula (I) has a concentration of about 15 mg/mL to about 35 mg/mL in a liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) A solubilizer, present in an amount of about 5% by weight to about 35% by weight; and

(c) A buffer, present in an amount of about 0.1% by weight to about 5% by weight. In some embodiments, the buffer comprises sodium citrate dehydrate. In some embodiments, the formulation further comprises a sweetener, present in an amount of about 30% by weight to about 70% by weight. In some embodiments, the sweetener comprises sucrose. In some embodiments, the formulation further comprises a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight. In some embodiments, the formulation further comprises a flavoring agent, present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) A solubilizer, present in an amount of about 5% by weight to about 35% by weight; and

(c) A base, present in an amount of about 0.1% by weight to about 5% by weight. In some embodiments, the base comprises a dehydrated sodium citrate. In some embodiments, the formulation further comprises a sweetener, present in an amount of about 30% by weight to about 70% by weight. In some embodiments, the sweetener comprises sucrose. In some embodiments, the formulation further comprises a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight. In some embodiments, the formulation further comprises a flavoring agent, present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) A solubilizer (e.g., cyclodextrin, such as hydroxypropyl-β-cyclodextrin), present in an amount of about 5% by weight to about 35% by weight; and

(c) A buffer (e.g., a citrate buffer, such as sodium citrate) is present in an amount of about 0.1% by weight to about 5% by weight;

(d) A sweetener (e.g., a sweetener containing sucrose or a strong sweetener) present in an amount of about 30% by weight to about 70% by weight;

(e) a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight; and

(f) A flavoring agent present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) A solubilizer (e.g., cyclodextrin, such as hydroxypropyl-β-cyclodextrin), present in an amount of about 5% by weight to about 35% by weight; and

(c) A base (e.g., a citrate, such as sodium citrate), present in an amount of about 0.1% by weight to about 5% by weight;

(d) A sweetener (e.g., a sweetener containing sucrose or a strong sweetener) present in an amount of about 30% by weight to about 70% by weight;

(e) a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight; and

(f) A flavoring agent present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) Hydroxypropyl-β-cyclodextrin, present in amounts from about 5% by weight to about 35% by weight; and

(c) Sodium citrate, present in an amount of about 0.1% by weight to about 5% by weight;

(d) Sucrose or strong sweetener, present in an amount of about 30% to about 70% by weight;

(e) a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight; and

(f) A flavoring agent present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

This article also provides a liquid formulation comprising: (a)(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, having formula (I):

Its pharmaceutically acceptable salts, or combinations thereof;

(b) Hydroxypropyl-β-cyclodextrin, present in amounts from about 5% by weight to about 35% by weight; and

(c) Sodium citrate dihydrate, present in an amount of about 0.1% by weight to about 5% by weight;

(d) Sucrose or strong sweetener, present in an amount of about 30% to about 70% by weight;

(e) a bitterness masking agent, present in an amount of about 0.2% by weight to about 0.5% by weight; and

(f) A flavoring agent present in an amount of about 0.01% by weight to about 2% by weight. In some embodiments, the formulation has a pH of about 3 to about 4. In some embodiments, the compound of formula (I) has a concentration of about 20 mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation is prepared from a pharmaceutically acceptable salt of a compound of formula (I). For example, a pharmaceutically acceptable salt is a hydrogen sulfate. In some embodiments, the liquid formulation is prepared from the crystalline form of a compound of formula (I). For example, the crystalline form of a compound of formula (I) may have the formula (I-HS):

In some embodiments, the crystalline form is characterized by XRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form is characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2, 27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

In some embodiments, the crystalline form (I-HS) has an XRPD pattern that is substantially as shown in Figure 1 or Figure 8.

In some embodiments, the crystalline form exhibits an initial temperature of approximately 193 to approximately 205°C, as measured by differential scanning calorimetry. In some embodiments, the crystalline form (I-HS) exhibits a heat of fusion of approximately 2.415 mW, as measured by differential scanning calorimetry.

This article also provides a method for treating cancer in patients in need. This method involves administering a therapeutically effective amount of the liquid formulation provided herein to the patient.

In some implementations, cancer causes difficulty or difficulty swallowing. For example, the cancer may be head and neck cancer, oral cancer, throat cancer, or esophageal cancer. In some implementations, patients with cancer experience difficulty swallowing due to one or more of the following: fibrosis of the throat, esophagus, or mouth; infection of the mouth or esophagus (e.g., from radiation therapy or chemotherapy); swelling or narrowing of the throat or esophagus (e.g., from radiation therapy or surgery); physical changes in the mouth, jaw, throat, or esophagus after surgery; mucositis; pain, discomfort, or inflammation of the throat, esophagus, or mouth; or xerostomia, commonly referred to as dry mouth (e.g., from radiation therapy or chemotherapy).

In some implementations, the patient is an infant, child, adolescent, or elderly patient.

In some implementations, the patient suffers from dysphagia. Dysphagia can be oropharyngeal dysphagia. Oropharyngeal dysphagia can be caused by cancer (such as certain cancers and some cancer treatments such as radiation that can cause dysphagia), neurological disorders (such as certain diseases such as multiple sclerosis, muscular dystrophy, and Parkinson's disease that can cause dysphagia), nerve damage (such as sudden nerve damage such as from a stroke or brain or spinal cord injury that affects a person's ability to swallow), and pharyngeal diverticula.

In some implementations, the patient has a neurological disorder (such as certain disorders like multiple sclerosis, muscular dystrophy, and Parkinson's disease that can cause difficulty swallowing), nerve damage (such as sudden nerve damage from a stroke or brain or spinal cord injury that affects a person's ability to swallow), and a pharyngeal diverticulum.

This article also provides a method for treating cancer patients with dysphagia (e.g., difficulty swallowing) who require treatment. The method includes identifying patients with dysphagia who require treatment. The method also includes administering a therapeutically effective amount of the liquid formulation described herein to the patient.

In some implementations, dysphagia is oropharyngeal dysphagia.

This article also provides a method for treating cancer in patients with dysphagia who require treatment. The method includes identifying patients with dysphagia who require treatment. The method also includes determining whether the cancer is mediated by Trk kinase. If the cancer is determined to be mediated by Trk kinase, a therapeutically effective amount of the liquid formulation described herein is administered to the patient. This article also provides a method for treating cancer in patients with dysphagia who require treatment. The method includes identifying patients with dysphagia who require treatment. The method also includes identifying cancer mediated by Trk kinase and administering a therapeutically effective amount of the liquid formulation described herein to the patient.

In some implementations, dysphagia is oropharyngeal dysphagia. Oropharyngeal dysphagia can be caused by cancer (e.g., certain cancers and some cancer treatments such as radiation can cause dysphagia), neurological disorders (e.g., certain diseases such as multiple sclerosis, muscular dystrophy, and Parkinson's disease can cause dysphagia), nerve damage (e.g., sudden nerve damage such as from a stroke or brain or spinal cord injury that affects a person's ability to swallow), and pharyngeal diverticula.

Crystallization form of compound (I)

As discussed herein, liquid formulations can be prepared from crystalline forms of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, its pharmaceutically acceptable salts, or combinations thereof. In some embodiments, the crystalline form is the crystalline form (I-HS).

As shown in Figure 1, in some embodiments, the crystalline form (I-HS) can be characterized by its X-ray powder diffraction (XRPD) pattern. XRPD was performed on a D5000 X-ray diffractometer using a finely focused sealed tube source from Siemens (CuKα1, 0.1540562 nm long) and by scanning the sample between 3 and 40°2-θ (step size 0.0200°2-θ, 1 second per step). The effective scan rate was 0.0200°/s, the instrument voltage was 40 kV, and the current was set to 40 mA. The sample was analyzed using a 2 mm diverging slit in reflection mode under the following experimental conditions.

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least 20 characteristic peaks (2θ degrees ± 0.3), as listed in Table 1.

Table 1. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least eight characteristic peaks (2θ degrees ± 0.3), which include peaks with a relative intensity of more than or equal to about 15%, as listed in Table 2.

Table 2. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least five characteristic peaks (2θ degrees ± 0.3), which include peaks with a relative intensity of more than or equal to about 25%, as listed in Table 3.

Table 3. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least four characteristic peaks (2θ degrees ± 0.3), each containing a peak with a relative intensity greater than or equal to about 30%, as listed in Table 4.

Table 4. XRPD peaks of crystalline form (I-HS)

In some implementations, the crystalline form (I-HS) has an XRPD diagram that is substantially the same as the XRPD diagram shown in Figure 1.

In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 18.4, 20.6, 23.0, and 24.0. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 10.6, 18.4, 20.6, 23.0, and 24.0. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 10.6, 18.4, 19.1, 20.2, 20.6, 21.5, 23.0, and 24.0. In some embodiments, the crystalline form (I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at approximately 10.6, 15.3, 16.4, 18.4, 19.1, 19.8, 20.2, 20.6, 21.5, 22.0, 23.0, 24.0, 24.4, 25.6, 26.5, 27.5, 28.2, 28.6, 30.8, and 38.5.

In some implementations, the crystalline form (I-HS) has a substantially identical XRPD pattern to the XRPD pattern shown in FIG8.

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least 20 characteristic peaks (2θ degrees ± 0.3), as listed in Table 1.

Table 5. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least eight characteristic peaks (2θ degrees ± 0.3), which include peaks with a relative intensity of more than or equal to about 15%, as listed in Table 6.

Table 6. XRPD peaks of crystalline form (I-HS)

Position (°2θ) Relative strength (%) 10.76 29.85 18.50 48.07 19.22 22.92 20.26 30.80 20.74 100.00 21.56 23.78 23.16 32.52 24.10 33.89

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least five characteristic peaks (2θ degrees ± 0.3), which include peaks with a relative intensity of more than or equal to about 25%, as listed in Table 7.

Table 7. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) has an XRPD plot with at least four characteristic peaks (2θ degrees ± 0.3), each containing a peak with a relative intensity greater than or equal to about 30%, as listed in Table 8.

Table 8. XRPD peaks of crystalline form (I-HS)

Position (°2θ) Relative strength (%) 18.50 48.07 20.74 100.00 23.16 32.52 24.10 33.89

In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 18.5, 20.7, 23.2, and 24.1. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 10.8, 18.5, 20.7, 23.2, and 24.1. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at approximately 10.8, 18.5, 19.2, 20.3, 20.7, 21.6, 23.2, and 24.1. In some embodiments, the crystalline form (I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at approximately 10.8, 15.4, 16.5, 18.5, 19.2, 19.9, 20.3, 20.7, 21.6, 22.2, 23.2, 24.1, 24.5, 25.7, 26.5, 27.6, 28.3, 28.7, 30.9, and 38.6.

In some implementations, given the XRPD patterns provided in Figures 1 and 8, the crystalline form (I-HS) is characterized by having XRPD peaks (2θ degrees), as shown in Table 9.

Table 9. XRPD peaks of crystalline form (I-HS)

In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form (I-HS) is characterized by XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystalline form (I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2, 27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

It should be understood that the 2-θ values of X-ray powder diffraction patterns for the crystalline form (I-HS) can vary slightly from instrument to instrument and also depend on variations in sample preparation and batch-to-batch variability; therefore, the cited values should not be interpreted as absolute values. It should also be understood that the relative intensity of peaks can vary due to orientation effects, making the intensities shown in the XRPD traces included herein illustrative and not intended for absolute comparison. Therefore, it should be understood that the phrase “substantially identical XRPD pattern to that shown in Figure 1 or Figure 8” indicates, for comparative purposes, the presence of at least 90% of the peaks shown in Figure 1 or Figure 8. It should be understood that relative peak positions can vary by ±0.3 degrees from the peak positions shown in Figure 1 or Figure 8. It should be further understood that some variations in peak intensity compared to those shown in Figures 1 and 8 are permissible for comparative purposes.

Figure 2 shows the simultaneous thermogravimetric/differential calorimetry (TG/DTA) curves of the crystalline form (I-HS) according to one embodiment. For analysis, approximately 5 mg of the crystalline form (I-HS) was weighed into an open aluminum pan and placed in a simultaneous thermogravimetric/differential calorimetry (TG/DTA) instrument and held at room temperature. The sample was then heated from 25 °C to 300 °C at a rate of 10 °C/min, during which the change in sample weight and any differential thermal events were recorded. Nitrogen was used as the purge gas at a flow rate of 100 ^cm³ /min. The TG/DTA curves of the crystalline form (I-HS) show an initial weight loss of 0.8% between 27.4 °C and 182.4 °C, followed by a weight loss of 4.9% in the TG curve between 182.4 °C and 225.0 °C, which is also considered an endothermic agent in the DTA curve. These weight losses may be due to material decomposition.

Figure 3 shows the differential scanning calorimetry (DSC) curve of the crystalline form (I-HS) according to one embodiment. DSC analysis of the sample was performed using a Seiko DSC6200 differential scanning calorimeter (equipped with a cooler). Approximately 5 mg of the crystalline form (I-HS) was weighed into an aluminum DSC pan and loosely sealed with a perforated aluminum cap. The sample pan was then placed in the Seiko DSC6200 (equipped with a cooler), cooled, and maintained at 25°C. Once a stable heat flow response was obtained, the sample and reference were heated to 270°C at a scan rate of 10°C/min while monitoring the resulting heat flow response. In some embodiments, the crystalline form (I-HS) has a DSC thermal analysis curve substantially as shown in Figure 3. As used herein, “substantially as shown in Figure 3” means that the temperature of the endothermic event shown in Figure 3 can vary by approximately ±5°C.

As shown in Figure 3, the DSC thermal analysis of the crystalline form (I-HS) indicates a small endothermic change along the baseline between 122.9 °C and 152.8 °C, followed by a sharp endothermic reaction corresponding to the melting of the crystalline form (I-HS). At the melting initiation temperature of 190.8 °C, the peak melting temperature is 197.9 °C, with a heat of fusion of 2.415 mW. The transition after melting endothermation is likely caused by the decomposition of the molten crystalline form (I-HS).

Figures 4A and 4B show polarized light microscopy (PLM) images of the crystalline form (I-HS) under (A) and (B) unpolarized light, according to some embodiments. The presence of crystallinity (birefringence) was determined using an Olympus BX50 polarized light microscope equipped with a Motic camera and image capture software (MoticImages Plus 2.0). All images were recorded using a 20x objective. When examined under polarized light, the crystalline form (I-HS) exhibited birefringence without showing a defined morphology or agglomerates.

Figure 5 shows the dynamic vapor adsorption (DVS) isotherm of the crystalline form (I-HS) according to one embodiment. For DVS measurements, the hygroscopicity of the crystalline form sample (I-HS) was determined by cycling it under varying humidity conditions. The sample was analyzed using the DVS-1 dynamic vapor adsorption system for surface measurement. Approximately 10 mg of the crystalline form (I-HS) was placed in a mesh vapor adsorption balance pan and loaded into the dynamic vapor adsorption balance as part of the surface measurement system. Data were collected at 1-minute intervals. Nitrogen was used as the carrier gas. The sampled crystalline form (I-HS) was subjected to a temperature ramp from 20-90% relative humidity (RH) in 10% increments, with the sample held at each step until a stable weight was reached (99.5% step completion). After the adsorption cycle was complete, the sample was dried using the same procedure, but up to 0% RH and eventually back to the starting point of 20% RH. The weight change during the adsorption/desorption cycle was plotted to determine the hygroscopic properties of the sample.

As shown in Figure 5, the crystalline form (I-HS) appears to be non-hygroscopic. A small increase in mass of approximately 1.7% was observed between 0% and 90% RH during the adsorption cycle. Furthermore, a very small hysteresis was observed between the adsorption and desorption cycles. The XRPD plot of the crystalline form (I-HS) after DVS analysis (not shown) is similar to its pre-DVS XRPD plot shown in Figure 1 or Figure 8. Figure 8 indicates that no change in the crystalline form (I-HS) occurred during DVS.

Figure 6 shows the infrared (IR) spectrum of the crystalline form (I-HS) of the compound of formula I according to one embodiment. IR spectra were performed on a Bruker ALPHA P spectrometer. Sufficient crystalline material (I-HS) was placed at the center of the spectrometer plate, and transmission spectra were obtained at a resolution of 4 ^cm⁻¹ , with 16 background scans and 16 sample scans, and data were collected from 4000 ^cm⁻¹ to 400 ^cm⁻¹ . The observed IR spectra of the crystalline form (I-HS) are shown in Figure 6.

The crystalline form (I-HS) possesses numerous properties that unexpectedly outperform the amorphous form (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate (AM(HS)). For example, the crystalline form (I-HS) exhibits properties that contribute to its manufacturability and the production of commercial products. As shown in Example 8, the crystalline form (I-HS) demonstrates better flow properties compared to the amorphous API (AM(HS)), as evidenced by the Carr's and Hausner indices. For example, the crystalline form (I-HS) exhibits a Carr index value greater than 20%. In some embodiments, the crystalline form (I-HS) exhibits a Hausner ratio of less than 1.35 (e.g., a value between about 1.26 and about 1.34). This difference in flow properties can make the development of solid oral dosage forms of the amorphous API more difficult than that of the crystalline API.

The crystalline form (I-HS) also demonstrated better stability in a five-week accelerated stability study conducted in LDPE bags at 40°C/75% RH. While neither AM(HS) nor the crystalline form (I-HS) exhibited significant changes in chemical impurity levels during the study, the study did reveal stable physicochemical properties in the crystalline form (I-HS). On the other hand, the amorphous API was transformed into a crystalline form substantially similar to the crystalline form (I-HS) by XRPD, DSC, TGA, KF, and polarized light microscopy. Furthermore, during stability testing, the amorphous API transformed into an agglomerated powder with reduced flowability. Such changes in the physical properties of the compound during storage, including the shift from amorphous energy to crystalline material and/or agglomerated powder with reduced flowability, would make it virtually impossible to manufacture solid oral dosage forms for patients based on amorphous compounds. However, the properties observed for the crystalline form (I-HS) are consistent with those required for commercial products, including a stable physical and chemical structure.

As previously stated, the crystalline form (I-HS) is non-hygroscopic. As used herein, “non-hygroscopic” means a compound that exhibits less than a 2% weight increase at 25°C and 80% RH after 24 to 48 hours (see, for example, Example 10). However, AM(HS) compounds have been found to deliquesce upon exposure to moisture. Given this tendency, extensive handling precautions are required during storage and manufacturing of AM(HS) compounds to prevent such changes in form, whereas such precautions are not required during the manufacture of the crystalline form (I-HS). This moisture stability is expected to extend to any solid oral dosage products prepared using the crystalline form (I-HS).

Compared to amorphous APIs, crystalline forms (e.g., I-HS) can provide improved impurity distribution. The ability to control impurity distribution is important for patient safety, developing reproducible manufacturing processes, and meeting regulatory requirements before use in humans. Further properties and characteristics of polymorphs can be found in U.S. Patent Application Serial No. 14/943,014, the entire contents of which are incorporated herein by reference.

The liquid formulation described herein contains (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (Formula I), which exhibits inhibition of Trk family protein tyrosine kinases and can be used to treat pain, inflammation, cancer, and certain infectious diseases.

Some embodiments include using the liquid formulation provided herein to treat conditions and diseases that can be treated by inhibiting TrkA, TrkB, and/or TrkC kinases, such as TrkA, TrkB, and/or TrkC-mediated conditions, including one or more conditions described herein, including Trk-related cancers. In some embodiments, the liquid formulation can be used to treat pain, including chronic and acute pain. In some embodiments, the liquid formulation can be used to treat multiple types of pain, including inflammatory pain, neuropathic pain, surgical pain, and pain associated with cancer, surgery, and fractures. Furthermore, the liquid formulation can be used to treat inflammatory, active, and chronic neurodegenerative diseases and certain infectious diseases.

The ability of compounds of formula (I), in their pharmaceutically acceptable salt or crystalline form (I-HS), to act as inhibitors of TrkA, TrkB, and/or TrkC can be demonstrated by the assays described in Examples A and B. As disclosed in U.S. Patent No. 8,513,263, published August 20, 2013, which is incorporated herein by reference.

In some embodiments, this document provides a method for treating a patient diagnosed with TRK-related cancers, comprising administering to the patient a therapeutically effective amount of the liquid formulation provided herein. The Trk family of neurotrophic receptors, TrkA, TrkB, and TrkC (encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively), and their neurotrophic ligands regulate neuronal growth, differentiation, and survival. Dysregulation of NTRK genes, Trk proteins, or their expression, activity, or levels, such as translocations involving NTRK kinase domains, mutations involving TRK ligand binding sites, NTRK gene amplification, Trk mRNA splicing variants, and Trk autocrine/paracrine signaling, is described in a number of tumor types and may contribute to tumorigenesis. Recently, NTRK1 fusion ² was described in a group of adenocarcinoma lung cancer patients. The translocations in NTRK1, NTRK2, and NTRK3 that result in constitutively active TrkA, TrkB, and TrkC fusion proteins are oncogenic and prevalent in a variety of tumor types, including lung adenocarcinoma, thyroid cancer, head and neck cancer, glioblastoma, and others.

In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level, includes overexpression of wild-type TrkA, TrkB, or TrkC (e.g., leading to autocrine activation). In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level, includes overexpression, activation, amplification, or mutation in a chromosomal segment containing the NTRK1, NTRK2, or NTRK3 gene, or a portion thereof. In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level, includes one or more chromosomal translocations or inversions that respectively lead to fusions of the NTRK1, NTRK2, or NTRK3 genes. In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level, is the result of a genetic translocation, wherein the expressed protein is a fusion protein containing residues from a non-TrkA chaperone protein and TrkA, a non-TrkB chaperone protein and TrkB, or a non-TrkC chaperone protein and TrkC protein, respectively including minimally functional TrkA, TrkB, or TrkC kinase domains.

In some embodiments, the TrkA fusion protein is one of the TrkA fusion proteins shown in Table 10, wherein:

Table 10. Exemplary Trk fusion proteins and cancer

^A Créancier et al., Cancer Lett. 365(1): 107-111, 2015.J

^US Patent Application Publication No. 2015/0315657.

^C US Patent Application Publication No. 2015/0283132.

^D Egren et al., Cancer Research. 75(Suppl. 15): 4793, 2015.

^E US Patent Application Publication No. 2015/0073036.

^F PCT patent application publication number WO2015184443A1.

^G Haller et al., The Journal of Pathology 238.5 (2016): 700-710.

^H Wong et al., J Natl Cancer Inst 2016;108:djv307.

^I Haller et al., J. Pathol. 238(5): 700-10.

^J Wu et al., Mod Pathol. 2016 Apr; 29(4): 359-69.

^K Konicek et al., Cancer Research, Vol. 76, No. 14, Supplementary. Abstract number: 2647; ^107th Annual Meeting of the American Association for Cancer Research, AACR 2016, New Orleans, Louisiana; April 16-20, 2016.

^L. Drilon et al., Cancer Research, Vol. 76, No. 14, Supplementary. Abstract number: CT007; 107th Annual Meeting of the American Association for Cancer Research, AACR 2016, New Orleans, Louisiana; April 16-20, 2016.

In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level includes one or more deletions, insertions, or point mutations in the TrkA protein. In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level includes the deletion of one or more residues from the TrkA protein, resulting in constitutive activity of the TrkA kinase domain. In some embodiments, the deletion includes the deletion of amino acids 303-377 in TrkA isotype 2.

In some embodiments, dysregulation of the NTRK gene, the Trk protein, or its expression or activity or level includes at least one point mutation in the NTRK1 gene that results in a TrkA protein having one or more amino acid substitutions (see, for example, point mutations listed in Table 11) compared to the wild-type TrkA protein. An exemplary wild-type TrkA polypeptide is SEQ ID NO: 1, an exemplary wild-type TrkB polypeptide is SEQ ID NO: 2, and an exemplary TrkC polypeptide is SEQ ID NO: 3.

Table 11. Activating TrkA point mutation ^A

^The reference TrkA sequence is UniProtKB/Swiss-Prot:P04629.4, and can be found at the URL: www.ncbi.nlm.nih.gov/protein/94730402?report=genbank&log$=protalign&blast_rank=0&RID=0 (SEQ ID NO.1)

^B Zhang et al. Blood 124(21):1682,2014. Mutations were found in T-cell prolymphocytic leukemia.

^C Park et al. Proc. Natl. Acad. Sci. USA 112(40): 12492-12497, 2015. Found mutations in colorectal cancer.

^D Russo et al. Cancer Discov. Jan; 6(1):36-44, 2016.

^EPPCT application number WO2016196141A1.

^F www.ncbi.nlm.nih.gov/protein/56118210? report=genbank&log$=protalign&blast_rank=3&RID=0

^G www.ncbi.nlm.nih.gov/protein/59889558

In some embodiments, dysregulation of the NTRK gene, the Trk protein, or its expression, activity, or level includes splicing variations in TrkA mRNA that result in the expressed protein being an alternative splice variant of TrkA with at least one residue deletion (compared to wild-type TrkA protein), leading to constitutive activity of the TrkA kinase domain. In some embodiments, the constitutively active alternative splice form of TrkA has deletions of exons 8, 9, and 11, resulting in the expressed protein having deletions of residues 192-284 and 393-398 relative to TrkA isotype 2, having deletions of exon 10 of TrkA, or having a deletion in the NTRK1 gene, which encodes a TrkA protein with a 75-amino acid deletion in its transmembrane domain (Reuther et al., Mol. Cell Biol. 20:8655-8666, 2000).

Cancers identified as having dysregulation of the NTRK gene, Trk protein, or their expression or activity or level (see references cited in this article and the websites www.cancer.gov and www.nccn.org) include:

(A) Cancers in which dysregulation of the NTRK gene, Trk protein, or their expression or activity or level includes one or more chromosomal translocations or inversions that produce the TrkA fusion protein, such as:

(B) Cancers in which dysregulation of the NTRK gene, Trk protein, or its expression or activity or level includes one or more deletions, insertions, or mutations in the TrkA protein, such as:

(C) Cancers in which dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level includes overexpression (autocrine activation) of wild-type TrkA, for example including:

In some implementations, dysregulation of the NTRK gene, Trk protein, or their expression or activity or level includes translocations that lead to the expression of TrkB fusion proteins, such as one of the TrkB fusion proteins shown in Table 12.

Table 12. Exemplary TrkB fusion proteins and cancer

^A PCT patent application publication number WO 2015/183836A1

^B Drilon et al., Ann Oncol. May 2016; 27(5): 920-6.

^C Yuzugullu et al., Cell Discov. 2:16030, 2016.

^D Ni et al., Neuro Oncol. January 2017 (Jan); 19(1): 22-30.

^E Lin et al., Neuro-Oncol, Vol. 18, Supplement 3, p. iii58, Abstract number: HG-48; ^17th International Symposium on Pediatric Neuro-Oncology, ISPNO 2016, Liverpool, UK, 12-15 June 2016.

In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity or level includes at least one point mutation in the NTRK1 gene that results in the production of a TrkB protein with one or more amino acid substitutions compared to the wild-type TrkB protein (see, for example, point mutations listed in Table 13).

Table 13. Activating TrkB point mutation ^A

^The reference TrkB sequence is UniProtKB/Swiss-Prot:Q16620.1, and can be found at the URL: www.ncbi.nlm.nih.gov/protein/2497560?report=genbank&log$=protalign&blast_rank=0&RID=0(SEQ ID NO.2)

^B PCT application number WO2016196141A1.

^C Bonanno et al., Journal of Thoracic Oncology, Vol. 11, No. 4, Supplement 1, p. 67. Abstract: 28 pages; ^6th European Lung Cancer Conference, ELCC 2016, Geneva, Switzerland.

^D www.ncbi.nlm.nih.gov/protein/NP_006171.2

In some implementations, dysregulation of the NTRK gene, Trk protein, or their expression or activity or level includes translocations that lead to the expression of TrkC fusion proteins, such as one of the TrkC fusion proteins shown in Table 14.

Table 14. Exemplary TrkC fusion proteins and cancer

^A Tannenbaum et al., Cold Spring Harb. Mol. Case Stud. 1:a000471, 2015.

^US Patent Application Publication No. 2015/0315657.

^C Yeh et al., J Pathol. 240(3): 282-90, 2016

^D Montalli et al., JOralPatholMed.doi:10.1111/jop.12491, 2016

^E Alassiri et al., Am J Surg Pathol., Aug; 40(8): 1051-61, 2016.

^F Nagasubramanian et al., Pediatr Blood Cancer, August 2016; 63(8): 1468-70, 2016.

^G Chintakuntlawar et al. Oral Surg Oral Med Oral Pathol Oral Radiol. 2016 May; 121(5): 542-549.e1.

^H US Patent No. US9511050B2.

^I. US Patent No. US9447135B2.

^J Skalova et al., Modern Pathology 30, S27-S43, 2017.

^K Hyrcza et al., Vol. 469, Supplement 1, p. 17. Abstract: OFP-1997-7; 31st International Congress of the International Academy of Pathology and ^{the 28th} Congress of the European Society of Pathology, Cologne, Germany. 25-29 September 2016. ^L Sims et al., Journal of Immunotherapy of Cancer, Vol. 4, Supplement 1; Abstract: p. 280; 31st Annual Meeting and Related Programs of the Society for Immunotherapy of Cancer, SITC 2016, National Harbor, Maryland; 9-13 November 2016.

^K Roberts et al., Blood, Vol. 128, No. 22, Abstract No.: 278, 58th Annual Meeting of the American Society of Hematology, ASH 2016, San Diego, California, USA. December 3-6, 2016.

^M Pavlick et al., Pediatric Blood Cance, doi: 10.1002/pbc.26433, 2017.

In some embodiments, dysregulation of the NTRK gene, Trk protein, or its expression or activity or level includes at least one point mutation in the NTRK1 gene that results in a TrkC protein with one or more amino acid substitutions compared to the wild-type TrkC protein (see, for example, point mutations listed in Table 15).

Table 15. Activating TrkC point mutation ^A

^A reference TrkC sequence is UniProtKB/Swiss-Prot:Q16288.2, and can be found at the URL: www.ncbi.nlm.nih.gov/protein/134035335?report=genbank&log$=protalign&blast_rank=0&RID=0 (SEQ ID NO.3)

^B Drilon et al. Ann Oncol. 2016 May; 27(5):920-6. doi:10.1093/annonc/mdw042. Epub 2016 Feb 15.

^PCT application number WO2016196141A1.

^D www.ncbi.nlm.nih.gov/protein/NP_001007157

In some implementations, TRK-related cancers have been identified as having one or more TRK inhibitor resistance mutations (which lead to increased resistance to TRK inhibitors). Non-limiting examples of TRK inhibitor resistance mutations are listed in Tables 17-19.

Table 17. Exemplary TrkA Resistance Mutations

Amino acid position 517 (e.g., G517R) Amino acid position 542 (e.g., A542V) Amino acid position 568 (e.g., Q568x) Amino acid position 573 (e.g., V573M) Amino acid position 589 (e.g., F589L, F589C) <![CDATA[amino acid position 595 (e.g., G595S, G595R¹)]]> Amino acid position 599 (e.g., D596V) Amino acid position 600 (e.g., F600L) Amino acid position 602 (e.g., R602x) Amino acid position 646 (e.g., F646V) Amino acid position 656 (e.g., C656Y, C656F) Amino acid position 657 (e.g., L657V) <![CDATA[amino acid position 667 (e.g., G667C¹, G667S)]]> Amino acid position 676 (e.g., Y676S)

¹ Russo et al., Acquired resistance to the TRK inhibitor Entrectinib in colorectal cancer, Cancer Discovery, Jan 2016; 6(1):36-44, 2016.

Table 18. Exemplary TrkB resistance mutations

Amino acid position 545 (e.g., G545R) Amino acid position 570 (e.g., A570V) Amino acid position 596 (e.g., Q596E, Q596P) Amino acid position 601 (e.g., V601G) Amino acid position 617 (e.g., F617L, F617C, F617I) Amino acid position 623 (e.g., G623S, G623R) Amino acid position 624 (e.g., D624V) Amino acid position 628 (e.g., F628x) Amino acid position 630 (e.g., R630K) Amino acid position 672 (e.g., F672x) Amino acid position 682 (e.g., C682Y, C682F) Amino acid position 683 (e.g., L683V) Amino acid position 693 (e.g., G693S) Amino acid position 702 (e.g., Y702x)

Table 19. Exemplary TrkC resistance mutations

¹ Drilon et al., What hides behind the MASC: clinical response and acquired resistance to entrectinib after ETV6-NTRK3 identification in a mammary analogue secretory carcinoma (MASC), Ann Oncol., May 2016; 27(5):920-6. doi:10.1093/annonc/mdw042.Epub2016 Feb 15.

In some embodiments, this document provides a method for treating a patient diagnosed with Trk-related cancer, comprising administering to the patient a therapeutically effective amount of the liquid formulation provided herein. For example, Trk-related cancers may be selected from the group consisting of: non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, acute myeloid leukemia, colorectal cancer, large cell neuroendocrine carcinoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, gastric cancer, Spitz carcinoma, papillary thyroid carcinoma, colon cancer, acute myeloid leukemia, gastrointestinal stromal tumor (GIST) (e.g., wild-type GIST detection of KIT/PDGFR/BRAF/SDH), sarcoma, pediatric glioma, intrahepatic cholangiocarcinoma, pilocytic astrocytoma, low-grade glioma, lung adenocarcinoma, salivary gland carcinoma, secretory breast cancer, fibrosarcoma, nephroma, and breast cancer.

In some implementations, TRK-related cancers are selected from the group consisting of: non-limiting examples of TRK-related cancers include: Spitz melanoma, Spitz tumors (e.g., metastatic Spitz tumors), non-small cell lung cancer (NSCLC), thyroid cancer (e.g., papillary thyroid carcinoma (PTC)), acute myeloid leukemia (AML), sarcoma (e.g., undifferentiated sarcoma or adult soft tissue sarcoma), pediatric glioma, colorectal cancer (CRC), glioblastoma multiforme (GBM), large cell neuroendocrine carcinoma (LCNEC), thyroid cancer, intrahepatic cholangiocarcinoma (ICC), pilocytic astrocytoma, low-grade glioma, head and neck squamous cell carcinoma, adenocarcinoma (e.g., lung adenocarcinoma), salivary gland carcinoma, Secretory breast cancer, breast cancer, acute myeloid leukemia, fibrosarcoma, renal cell carcinoma, melanoma, bronchogenic carcinoma, B-cell carcinoma, bronchial carcinoma, oral or pharyngeal cancer, hematologic malignancies, cervical cancer, gastric cancer, kidney cancer, liver cancer, multiple myeloma, ovarian cancer, pancreatic cancer, salivary gland cancer, small bowel or appendix cancer, testicular cancer, bladder cancer, uterine or endometrial cancer, inflammatory myofibroblastoma, gastrointestinal stromal tumor, non-Hodgkin's lymphoma, neuroblastoma, small cell lung cancer, squamous cell carcinoma, esophageal or gastric cancer, skin cancer, tumors (e.g., melanocytic tumors), Spitz nevus, astrocytoma, medulloblastoma, glioma, large cell neuroendocrine tumor, breast-like secretory carcinoma, non-papillary cell carcinoma, bone cancer, and rectal cancer.

In some implementations, fibrosarcoma is infantile fibrosarcoma.

In some implementations, the Trk-related cancer is LMNAa-NTRK1 fusion soft tissue sarcoma or EVT6-NTRK3 fusion papillary thyroid carcinoma.

In some embodiments, the liquid formulations described herein can be used to treat Trk-related cancers in pediatric patients. For example, the liquid formulations provided herein can be used to treat infantile sarcoma, neuroblastoma, congenital mesodermal nephroma, low-grade glioma, and pontine glioma.

In some embodiments, the liquid formulations provided herein can be used in combination with one or more other therapeutic agents or therapies that act through the same or different mechanisms of action to treat Trk-related cancers.

In some embodiments, other therapeutic agents are selected from the group consisting of receptor tyrosine kinase targeted therapeutic agents, including cabozantinib, crizotinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, pertuzumab, regorafenib, sunitinib, and trastuzumab.

In some embodiments, other therapeutic agents are selected from signal transduction pathway inhibitors, including, for example, Ras-Raf-MEK-ERK pathway inhibitors (e.g., sorafenib, trametinib, or vemurafenib), PI3K-Akt-mTOR-S6K pathway inhibitors (e.g., everolimus, rapamycin, perifoxine, or tesiromolimus), and modulators of apoptosis pathways (e.g., oxacral).

In some embodiments, other therapeutic agents are selected from the group consisting of: cytotoxic chemotherapy, including, for example, arsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel, pemetrexed, temozolomide, and vincristine.

In some implementations, other therapeutic agents are selected from the group of angiogenesis-targeted therapies, including, for example, aflibercept and bevacizumab.

In some implementations, other therapeutic agents are selected from the group of immune-targeting agents, including, for example, interleukin, ipilimumab, lanolizumab, nivolumab, and sipuleucel-T.

In some embodiments, other therapeutic agents are selected from agents that are active against the downstream Trk pathway, including, for example, NGF-targeting biological agents, such as NGF antibodies and panTrk inhibitors.

In some implementations, other therapeutic agents or therapies are radiation therapy, including, for example, radioactive iodine therapy, external beam radiation therapy, and radium-223 therapy.

In some implementations, other therapeutic agents include any of the therapies or therapeutic agents listed above, which are the standard of care in cancer where the cancer has an dysregulation of the NTRK gene, Trk protein, or their expression or activity or level.

Methods for detecting dysregulation of the NTRK gene, Trk protein, or their expression or activity or level include, for example, detecting NTRK gene translocations, such as using fluorescence in situ hybridization (FISH) (e.g., described in international applications PCT/US2013/061211 and PCT/US2013/057495, which are incorporated herein by reference).

In some embodiments, this document provides a method of treating a patient with cancer (e.g., Trk-related cancer), comprising administering to the patient a combination of the liquid formulation provided herein and at least one other therapy or therapeutic agent. In some embodiments, the at least one other therapy or therapeutic agent is selected from radiotherapy (e.g., radioactive iodine therapy, external beam radiotherapy, or radium-223 therapy), cytotoxic chemotherapy (e.g., arsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel, pemetrexed, temozolomide, or vincristine), and tyrosine kinase-targeted therapy (e.g., afatinib, cabozantinib, cetuximab, crizotinib, dabrafenib, erlotinib, etc.). Lotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, panitumab, pertuzumab, regorafenib, sunitinib, or trastuzumab), apoptosis regulators and signal transduction inhibitors (e.g., everolimus, piperazine, rapamycin, sorafenib, tesiromolimus, trametinib, or vemurafenib), immunotargeted therapies (e.g., adeleukin, interferon alpha-2b, ipilimumab, lanolizumab, nivolumab, prednisone, or sipuleucel-T), and angiogenesis-targeted therapies (e.g., aflibercept or bevacizumab), wherein the amount of the liquid formulation provided herein, in combination with other therapies or therapeutic agents, is effective in treating the cancers described herein.

In some embodiments, the other therapeutic agents are different TRK inhibitors. In some embodiments, the receptor tyrosine kinase targeted therapy is a multi-kinase inhibitor exhibiting TRK inhibitory activity (e.g., a TRK-targeted therapy inhibitor). In some embodiments, the TRK-targeted therapy inhibitor is selective for TRK kinases. Exemplary TRK kinase inhibitors, as measured in the assays described herein, may exhibit inhibitory activity ( _IC50 ) against TRK kinases of less than about 1000 nM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. In some embodiments, as measured in the assays, TRK kinase inhibitors may exhibit inhibitory activity ( _IC50 ) against TRK kinases of less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM. For example, the TRK inhibitor assay may be any assay provided in U.S. Patent No. 8,933,084 (e.g., Example A or B).

Non-limiting examples of receptor tyrosine kinase (e.g., Trk) targeted therapies include afatinib, cabozantinib, cetuximab, crizotinib, dabrafenib, entrectinib, erlotinib, gefitinib, imatinib, lapatinib, letatinib, nilotinib, pazopanib, panitumumab, pertuzumab, sunitinib, trastuzumab, 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidine-3-yl)-3-(4-methyl- 3-(2-methylpyrimidin-5-yl))-l-phenyl-1H-pyrazol-5-yl)urea, AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, DS-6051, GNF-5837, GTx-186, GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101, TPX-0005 and TSR-011. Other Trk-targeted therapies include U.S. Patent Nos. 8,450,322; 8,513,263; 8,933,084; 8,791,123; 8,946,226; 8,450,322; 8,299,057 and 8,912,194; U.S. Publication Nos. 2016/0137654; 2015/0166564; 2015/005 1222; 2015/0283132 and 2015/0306086; International Publication Nos. WO2010/033941; WO2010/048314; WO2016/077841; WO2011/146336; WO2011/006074; WO2010/033941; WO2012/158413; WO2 014078454; WO2014078417; WO2014078408; WO2014078378; WO2014078372; WO2014 078331; WO2014078328; WO2014078325; WO2014078323; WO2014078322; WO20151757 The entire contents of those described in WO2009/013126; WO2013/174876; WO2015/124697; WO2010/058006; WO2015/017533; WO2015/112806; WO2013/183578 and WO2013/074518 are incorporated herein by reference.

Other examples of Trk inhibitors can be found in U.S. Patent No. 8,637,516, International Publication No. WO2012/034091, U.S. Patent No. 9,102,671, International Publication No. WO2012/116217, U.S. Publication No. 2010/0297115, International Publication No. WO2009/053442, U.S. Patent No. 8,642,035, International Publication No. WO2009092049, U.S. Patent No. 8,691,221, and International Publication No. WO2006131952, the entire contents of which are incorporated herein by reference. Exemplary Trk inhibitors include GNF-4256 described in CancerChemother.Pharmacol.75(1):131-141,2015; and GNF-5837 (N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrolo-2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N'-[2-fluoro-5-(trifluoromethyl)phenyl]-urea) described in ACSMed.Chem.Lett.3(2):140-145,2012, the entire contents of which are incorporated herein by reference.

Other examples of Trk inhibitors include those disclosed in U.S. Publication No. 2010/0152219, U.S. Patent No. 8,114,989, and International Publication No. WO2006/123113, the entire contents of which are incorporated herein by reference. Exemplary Trk inhibitors include AZ623 described in Cancer 117(6):1321-1391, 2011; AZD6918 described in Cancer Biol. Ther. 16(3):477-483, 2015; AZ64 described in Cancer Chemother. Pharmacol. 70:477-486, 2012; AZ-23 ((S)-5-chloro-N-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine) described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451, the entire contents of which are incorporated herein by reference.

Trk inhibitors may include those described in U.S. Patent Nos. 7,615,383; 7,384,632; 6,153,189; 6,027,927; 6,025,166; 5,910,574; 5,877,016 and 5,844,092, the entire contents of which are incorporated herein by reference.

Other examples of Trk inhibitors include CEP-751 described in Int. J. Cancer 72:672-679, 1997; CT327 described in Acta Derm. Venereol. 95:542-548, 2015; compounds described in International Publication No. WO2012/034095; compounds described in U.S. Patent Nos. 8,673,347 and WO2007/022999; compounds described in U.S. Patent No. 8,338,417; compounds described in International Publication No. WO2016/027754; and U.S. Patent No. 9,242. The compounds described in 977; the compounds described in US Patent Publication No. 2016/0000783; sunitinib (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide) described in PLoS One 9:e95628,2014; the compounds described in International Publication No. WO2011/133637; the compounds described in US Patent 8,637,256; and the compounds described in Expert. Opin. Ther. Pat. 24(7):731-744,2014. Compounds; compounds described in Expert Opin. Ther. Pat. 19(3):305-319, 2009; (R)-2-phenylpyrrolidinyl-substituted imidazopyridazines, such as GNF-8625, (R)-1-(6-(6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-[2,4'-bipyridin]-2'-yl)piperidin-4-ol; GT described in PLoS One 8(12):e83380, 2013 x-186 and others; K252a((9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindole[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazinoct-1-one) described in Mol. Cell Biochem. 339(1-2):201-213, 2010; 4-aminopyrazolylpyrimidine described in J. Med. Chem. 51(15):4672-4684, 2008, for example AZ-23 (((S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine)); PHA-739358 (darusete) described in Mol. Cancer Ther. 6:3158, 2007; (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propionitrile) described in J. Neurochem. 72:919-924, 1999; IJAE 115:117, 2010 The following are described: GW441756 ((3Z)-3-[(1-methylindol-3-yl)methylene]-1H-pyrrolo[3,2-b]pyridin-2-one); milkiclib (PHA-848125AC) described in J. Carcinog. 12:22, 2013; AG-879 ((2E)-3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2-propenylthioamide); altiratinib (N-(4-((2-(cyclopropanecarbamoyl)pyridin-4-yl)oxy)-2,5-difluorophenyl)-N-(4-fluorophenyl)cyclopropane -1,1-Dicarboxamide); Cabozantinib (N-(4-((6,7-dimethoxyquinoline-4-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); Letatinib ((5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanedibenzo[b,H]cyclooctano[jkl]cyclopentano[e]-as-indene-13(6H)-one); Dolatinib (4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl) Quinoline-2(1H)-one mono-2-hydroxypropionate hydrate); sitravatinib (N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); ONO-5390556; regorafenib (4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridin-2-carboxamide hydrate); and VSR-902A; the entire contents of which are incorporated herein by reference.

The ability of a Trk inhibitor to act as a TrkA, TrkB, and/or TrkC inhibitor can be tested using the assays described in Examples A and B of U.S. Patent No. 8,513,263, which is incorporated herein by reference.

In some implementations, signal transduction pathway inhibitors include Ras-Raf-MEK-ERK pathway inhibitors (e.g., binimetinib, selumetinib, encorafinib, sorafenib, trametinib, and vemurafenib), PI3K-Akt-mTOR-S6K pathway inhibitors (e.g., everolimus, rapamycin, perifosib, sirolimus), and other kinase inhibitors such as basipatinib, brigatinib, capmatinib, danusertib, ibrutinib, milkiclib, quercetin, regorafenib, ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110, INCB40093, INCB50465. INCB52793, INCB54828, MGCD265, NMS-088, NMS-1286937, PF 477736 ((R)-amino-N-[5,6-dihydro-2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-1H-pyrrolo[4,3,2-ef][2,3]benzodiazepine-8-yl]-cyclohexaneacetamide), PLX3397, PLX7486, PLX8394, PLX9486, PRN1008, PRN1371, RXDX103, RXDX106, RXDX108 and TG101209 (N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide).

Non-limiting examples of checkpoint inhibitors include ipilimumab, tramemumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736, MSB0010718C, BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, and pembrolizumab.

In some embodiments, the cytotoxic chemotherapeutic agent is selected from arsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel, pemetrexed, temozolomide, and vincristine.

Non-limiting examples of angiogenesis-targeted therapies include aflibercept and bevacizumab.

In some implementations, the immune targeting agent is selected from adenoleukin, interferon alpha-2b, ipilimumab, lanolizumab, nivolumab, prednisone, and sipuleucel-T.

Non-limiting examples of radiotherapy include radioactive iodine therapy, external beam radiotherapy, and radium-223 therapy.

Other kinase inhibitors include, for example, U.S. Patent Nos. 7,514,446; 7,863,289; 8,026,247; 8,501,756; 8,552,002; 8,815,901; 8,912,204; 9,260,437; 9,273,051; U.S. Publication No. US2015/0018336; International Publication Nos. WO2007/002325; WO2007/002433; WO2008/080001; WO2008/079906; WO2008/079903; WO2008/079909; WO2008/080015; WO2009/007 748; WO2009/012283; WO2009/143018; WO2009/143024; WO2009/014637; 2009/152083; WO2010/11 1527; WO2012/109075; WO2014/194127; WO2015/112806; WO2007/110344; WO2009/071480; WO200 9/118411; WO2010/031816; WO2010/145998; WO2011/092120; WO2012/101032; WO2012/139930; W The entire contents of those described in WO2012/143248; WO2012/152763; WO2013/014039; WO2013/102059; WO2013/050448; WO2013/050446; WO2014/019908; WO2014/072220; WO2014/184069 and WO2016/075224 are incorporated herein by reference.

Other examples of kinase inhibitors include, for example, WO2016/081450; WO2016/022569; WO2016/011141; WO2016/011144; WO2016/011147; WO2015/191667; WO2012/101029; WO2012/113774; WO2015/191666; WO2015/1612 The entire contents of those described in WO2015/161274; WO2015/108992; WO2015/061572; WO2015/058129; WO2015/057873; WO2015/017528; WO2015/017533; WO2014/160521 and WO2014/011900 are incorporated herein by reference.

Other therapeutic agents include RET inhibitors, such as those described in U.S. Patent Nos. 8,299,057; 8,399,442; 8,937,071; 9,006,256 and 9,035,063; U.S. Publication Nos. 2014/0121239; 2011/0053934; 2011/0301157; 2010/0324065; 2009/0227556; 2009/0130229; 2009/0099167; 2005/0209195; and international publications. Those described in publication numbers WO2014/184069; WO2014/072220; WO2012/053606; WO2009/017838; WO2008/031551; WO2007/136103; WO2007/087245; WO2007/057399; WO2005/051366 and WO 2005/044835 and those described in J.Med.Chem.2012, 55(10), 4872-4876.

These other therapeutic agents may be used as part of the same or different dosage forms, along with one or more of the liquid formulations provided herein, via the same or different routes of administration, and in the same or different administration regimens, in accordance with standard pharmaceutical practices known to those skilled in the art.

This document also provides (i) a pharmaceutical combination for treating cancer (e.g., Trk-related cancer) in patients in need, comprising: (a) a liquid formulation provided herein, (b) other therapeutic agents, and (c) optionally at least one other additive, for simultaneous, separate, or sequential use in treating the cancerous disease, wherein the amounts of the liquid formulation provided herein and the other therapeutic agents together are effective in treating said cancer; (ii) a pharmaceutical composition comprising such a combination; (iii) the use of such a combination in the preparation of a pharmaceutical for treating cancer (e.g., Trk-related cancer); (iv) a commercial package or product comprising such a combination as a combined formulation for simultaneous, separate, or sequential use; and a method of treating cancer (e.g., Trk-related cancer) in patients in need.

Methods for treating subjects identified or diagnosed with Trk-related cancer (e.g., any Trk-related cancer described herein or known in the art) are also provided (e.g., subjects identified or diagnosed with Trk-related cancer by using a regulatory-approved (e.g., FDA-approved) kit or by dysregulation of the NTRK gene, Trk protein, or their expression or activity or level in a biopsy sample of the subject), comprising administering to the subject a therapeutically effective amount of the liquid formulation provided herein. The use of the liquid formulation provided herein in treating Trk-related cancer in subjects identified or diagnosed with Trk-related cancer (e.g., any Trk-related cancer described herein or known in the art) is also provided. Also provided is the use of the liquid formulation provided herein in the preparation of a medicament for treating subjects identified or diagnosed with Trk-related cancer (e.g., any Trk-related cancer described herein or known in the art) (e.g., subjects identified or diagnosed with Trk-related cancer by using a regulatory-approved (e.g., FDA-approved) kit or by identifying dysregulation of the NTRK gene, Trk protein, or their expression or activity or level in a biopsy sample of the subject).

Methods for treating subjects (e.g., subjects suspected of having Trk-related cancer, subjects presenting with one or more symptoms of Trk-related cancer, or subjects at high risk of developing Trk-related cancer) are also provided, comprising assaying samples obtained from the subject (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using regulatory-approved kits, such as those approved by the FDA) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, and administering (e.g., specific or selective administration) a therapeutically effective amount of the liquid formulation provided herein to the subject determined to have dysregulation of the NTRK gene, Trk protein, or their expression or activity or level. Other assays, non-limiting assays, that can be used with these methods are described herein. Other assays are also known in the art. The use of the liquid formulation provided herein in treating Trk-related cancer in a subject identified or diagnosed with Trk-related cancer through the following steps, said steps including assaying a sample obtained from the subject (e.g., in vitro assay) (e.g., assay using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using a regulatory-approved kit, such as an FDA-approved kit) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, wherein the presence of dysregulation of the NTRK gene, Trk protein, or their expression or activity or level indicates that the subject has Trk-related cancer. The use of the liquid formulation described herein in the manufacture of a medicament for treating a subject identified or diagnosed with Trk-related cancer through the following steps, said steps including assaying a sample obtained from the subject (e.g., in vitro assay) (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using a regulatory-approved kit, such as an FDA-approved kit) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, wherein the presence of dysregulation of the NTRK gene, Trk protein, or their expression or activity or level indicates that the subject has Trk-related cancer. Some embodiments of any of the methods or uses described herein also include recording in the subject's clinical record (e.g., a computer-readable medium) that a subject determined by assay to have dysregulation of the NTRK gene, Trk protein, or their expression or activity should be administered the liquid formulation described herein.

In some embodiments of any of the methods or uses described herein, the subject has been identified or diagnosed with cancer that has dysregulation of the NTRK gene, Trk protein, or their expression or activity, or levels (e.g., determined using an agency-approved, such as FDA-approved assay or kit). In some embodiments of any of the methods or uses described herein, the subject has a tumor that is positive for dysregulation of the NTRK gene, Trk protein, or their expression or activity, or levels (e.g., determined using an agency-approved assay or kit). In some embodiments of any of the methods or uses described herein, the subject may be a subject with a tumor that is positive for dysregulation of the NTRK gene, Trk protein, or their expression or activity, or levels (e.g., identified as positive using an agency-approved, such as FDA-approved assay or kit). In some embodiments of any of the methods or uses described herein, the subject may be a subject whose tumor has dysregulation of the NTRK gene, Trk protein, or their expression or activity, or levels (e.g., where the tumor is identified using an agency-approved, such as FDA-approved kit or assay). In some embodiments of any of the methods or uses described herein, the subject is suspected of having Trk-related cancer. In some embodiments of any of the methods or uses described herein, the subject has a clinical record indicating that the subject has a tumor with dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level (and optionally, the clinical record indicates that the subject should be treated with any of the compositions provided herein). In some embodiments of any of the methods or uses described herein, the subject is suspected of having Trk-related cancer. In some embodiments of any of the methods or uses described herein, the subject has a clinical record indicating that the subject has a tumor with dysregulation of the NTRK gene, Trk protein, or its expression or activity, or its level (and optionally, the clinical record indicates that the subject should be treated with any of the compositions provided herein).

Methods of treating subjects, comprising administering to a subject a therapeutically effective amount of the liquid formulation provided herein, the subject having a clinical record indicating dysregulation of the NTRK gene, Trk protein, or their expression or activity or level. Use of the liquid formulation provided herein in the manufacture of a medicament for treating Trk-related cancers in subjects having a clinical record indicating dysregulation of the NTRK gene, Trk protein, or their expression or activity or level. Use of the liquid formulation provided herein in the manufacture of a medicament for treating Trk-related cancers in subjects having a clinical record indicating dysregulation of the NTRK gene, Trk protein, or their expression or activity or level. Some implementations of these methods and uses may further include: steps of measuring samples obtained from the subject (e.g., in vitro assays) (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using regulatory-approved, such as FDA-approved kits) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or its expression or activity or level, and recording in the subject's clinical record (e.g., a computer-readable medium) information that the subject has been identified as having dysregulation of the NTRK gene, Trk protein, or its expression or activity or level.

Methods for selecting treatment for subjects (e.g., in vitro methods) are also provided, including the selection of treatment comprising administering a therapeutically effective amount of the liquid formulation provided herein to subjects identified or diagnosed with Trk-related cancer (e.g., any Trk-related cancer described herein or known in the art) using a regulatory-approved, e.g., dysregulation of the NTRK gene, Trk protein, or their expression or activity or level in a biopsy sample of the subject. Some embodiments may further include administering the selected treatment to subjects identified or diagnosed with Trk-related cancer. Some embodiments may further include a step of measuring samples obtained from the subject (e.g., in vitro assays) (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using a regulatory-approved, e.g., FDA-approved kit) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, and to identify or diagnose subjects identified as having dysregulation of the NTRK gene, Trk protein, or their expression or activity or level with Trk-related cancer.

Methods for selecting a treatment for a subject are also provided, the treatment comprising administering a therapeutically effective amount of the liquid formulation provided herein, wherein the method includes the steps of measuring a sample obtained from the subject (e.g., in vitro assays) (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using a regulatory-approved kit, such as an FDA-approved kit) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, and to identify or diagnose a subject with dysregulation of the NTRK gene, Trk protein, or their expression or activity or level as having Trk-related cancer, and selecting a therapeutic treatment comprising administering a therapeutically effective amount of the liquid formulation provided herein to the subject identified or diagnosed with Trk-related cancer. Some embodiments also include administering the selected treatment to the subject identified or diagnosed with Trk-related cancer.

Methods for selecting subjects for treatment, said treatment comprising administering a therapeutically effective amount of the liquid formulation provided herein, are also provided. These methods include selecting, identifying, or diagnosing subjects with Trk-related cancer, and selecting pediatric subjects for treatment comprising administering a therapeutically effective amount of the liquid formulation provided herein. In some embodiments, identifying or diagnosing a subject with Trk-related cancer may include steps of measuring samples obtained from the subject (e.g., in vitro assays) (e.g., assays using next-generation sequencing, immunohistochemistry, separation FISH, or dual fusion FISH analysis) (e.g., using a regulatory-approved kit, such as an FDA-approved kit) to determine whether the subject has dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, and identifying or diagnosing pediatric subjects identified as having dysregulation of the NTRK gene, Trk protein, or their expression or activity or level, or having Trk-related cancer. In some embodiments, treatment selection may be used as part of a clinical study that includes administering various treatments for Trk-related cancer.

In some embodiments of any of the methods or uses described herein, the assay used determines whether a subject has an dysregulation of the NTRK gene, Trk protein, or their expression or activity, or level, using a sample (e.g., a biological sample or biopsy sample, such as a paraffin-embedded biopsy sample) from a subject (e.g., a subject suspected of having Trk-related cancer, a subject with one or more symptoms of Trk-related cancer, and/or a subject at increased risk of having Trk-related cancer). This assay may include, for example, next-generation sequencing, immunohistochemistry, fluorescence microscopy, separation FISH analysis, Southern blotting, Western blotting, FACS analysis, Northern blotting, and PCR-based amplification (e.g., RT-PCR). As is well known in the art, the assay is typically performed, for example, with at least one labeled nucleic acid probe or at least one labeled antibody or its antigen-binding fragment. The assay may utilize other detection methods known in the art to detect dysregulation of the NTRK gene, Trk protein, or their expression or activity, or level (see, for example, references cited herein).

In some embodiments, the liquid formulations provided herein can be used to treat chronic and acute pain, including pain associated with cancer, surgery, and fractures. The liquid formulations provided herein can be used to treat a variety of types of pain, including inflammatory pain, neuropathic pain, and pain associated with cancer, surgery, and fractures. The liquid formulations provided herein can also be used to treat cancers, including neuroblastoma, ovarian cancer, pancreatic cancer, and colorectal cancer. The liquid formulations provided herein can also be used to treat inflammation and certain infectious diseases. Furthermore, the liquid formulations provided herein can be used to treat interstitial cystitis (IC), painful bladder syndrome (PBS), urinary incontinence, asthma, anorexia, atopic dermatitis, and psoriasis. The liquid formulations provided herein can also treat demyelination and myelination by blocking Sp35-TrkA interactions, thereby promoting myelination, neuronal survival, and oligodendrocyte differentiation. The liquid formulations provided herein can be used to treat a variety of types of pain, including inflammatory pain, neuropathic pain, surgical pain, and cancer-related pain. The liquid formulations provided herein can be used to treat bone-related diseases (e.g., diseases involving bone resorption). Examples of bone-related diseases include metastatic bone disease, treatment-induced bone loss, osteoporosis, rheumatoid arthritis, ankylosing spondylitis, Paget's disease, and periodontal disease. Osteoporosis can be attributed to (1) menopause in women, (2) aging in men or women, (3) poor bone growth during childhood and adolescence resulting in failure to reach peak bone mass, and/or (4) bone loss secondary to other diseases, eating disorders, medications, and/or medical treatments. Other osteolytic diseases that can be treated according to the methods provided herein are more localized. A specific example is metastatic tumor-induced osteolysis. In this case, bone cancer or bone metastases cause localized osteolysis, which causes pain, bone weakness, and fractures. This localized osteolysis also allows tumors to grow larger by creating more space for them in the bone and releasing growth factors from the bone matrix. Cancers known to cause tumor-induced osteolysis include hematologic malignancies (e.g., myeloma and lymphoma) and solid tumors (e.g., breast cancer, prostate cancer, lung cancer, kidney cancer, and thyroid cancer), all of which are considered for treatment in this disclosure. As used in this article, the term treatment includes both prevention and treatment of existing conditions.

Therefore, this document also provides methods for treating diseases or medical conditions in subjects in need, wherein the diseases or conditions can be treated with inhibitors of TrkA and/or TrkB (e.g., Trk-related cancers), comprising administering a liquid formulation provided by the present invention, in an amount effective in treating or preventing said disease. In one specific embodiment, this document provides a method for treating pain, cancer, inflammation, neurodegenerative diseases, or Trypanosoma cruzi infection in mammals, comprising administering a therapeutically effective amount of the liquid formulation provided herein to said mammals. In another embodiment, this document provides a method for treating osteolytic diseases in mammals, comprising administering a therapeutically effective amount of the liquid formulation provided herein to said subject in need.

The liquid formulations described herein can be used in combination with one or more other drugs that act through the same or different mechanisms of action. This combination therapy can be achieved by administering the individual components of the treatment simultaneously, sequentially, or separately. Examples include anti-inflammatory compounds, steroids (e.g., dexamethasone, cortisone, and fluticasone), analgesics such as NSAIDs (e.g., aspirin, ibuprofen, indomethacin, and ketoprofen), opioids (e.g., morphine), and chemotherapeutic agents.

In the field of medical oncology, it is common practice to treat each patient with cancer using a combination of different forms of therapy. In medical oncology, in addition to the compositions presented herein, other components of such a combination therapy can be, for example, surgery, radiation therapy, chemotherapy, signal transduction inhibitors, and/or monoclonal antibodies. Therefore, compounds of formula (I) can also be used as adjuvants in cancer treatment, i.e., they can be used in combination with one or more other therapies or therapeutic agents, such as chemotherapeutic agents that act through the same or different mechanisms of action.

Therefore, the liquid formulation provided herein can be administered in combination with one or more agents selected from mitosis inhibitors, alkylating agents, antimetabolites, antisense DNA or RNA, intercalating antibiotics, growth factor inhibitors, signal transduction inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modulators, anti-hormones, angiogenesis inhibitors, cell inhibitors, anti-androgens, targeted antibodies, HMG-CoA reductase inhibitors, and isoprenoid protein transferase inhibitors.

In some embodiments of any of the methods described herein, the liquid formulation provided herein is administered in combination with a therapeutically effective amount of at least one other therapeutic agent selected from one or more other therapies or treatments (e.g., chemotherapy).

Non-limiting examples of other therapeutic agents include: other receptor tyrosine kinase-targeting therapies (e.g., TRK kinase inhibitors), kinase-targeting therapies, signal transduction pathway inhibitors, checkpoint inhibitors, modulators of apoptosis pathways (e.g., oxacral); cytotoxic chemotherapy, angiogenesis-targeting therapies, immunotherapies, and radiation therapy.

Among the treatment methods described herein, the liquid formulations provided herein are particularly useful for treating subjects suffering from dysphagia (e.g., difficulty swallowing). For example, the liquid formulations provided herein can be used to treat cancer in subjects suffering from oropharyngeal dysphagia.

When the compounds disclosed herein have at least one chiral center, the compounds may exist as enantiomers accordingly. When the compounds have two chiral centers, the compounds may also exist as diastereomers. In other words, in addition to having the desired configuration specified by the term "(S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide" (hereinafter referred to as the (S,R) isomer), the compound of formula I may also exist in small amounts as the isomer (R)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate (hereinafter referred to as the (R,R) isomer), and/or may also exist in small amounts as (S)-N-(5-((S)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate (hereinafter referred to as the (R,R) isomer), and/or may also exist in small amounts as (S)-N-(5-((S)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide). Alkyl-1-carboxamide hydrogen sulfate (hereinafter referred to as the (S,S) isomer), and/or may be present in small amounts as the isomer (R)-N-(5-((S)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate (hereinafter referred to as the (R,S) isomer). It should be understood that all such isomers and mixtures thereof are included within the scope of the invention. Preferably, the compound is present as the (S,R) isomer in an excess of more than or equal to about 80%, more preferably in an excess of more than or equal to about 90%, even more preferably in an excess of more than or equal to about 95%, even more preferably in an excess of more than or equal to about 98%, and even more preferably in an excess of more than or equal to about 99%.

It should be understood that the crystalline form (I-HS) contains two asymmetric centers and can therefore be prepared and isolated in mixtures of isomers, such as racemic or diastereomeric mixtures, or in enantiomerically pure form. Stereoisomers are thus specified and defined when stereochemistry is indicated by solid wedges or dashed lines representing a particular configuration.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesirable toxicological effects. These pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound, in its free acid or free base form, with a suitable base or acid, respectively. In some embodiments, pharmaceutically acceptable salts are preferred over the corresponding free base or free acid because these salts impart greater stability or solubility to the molecule, thereby facilitating formulation. Basic compounds are generally capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids. Representative pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, hydrogen sulfate, aminosulfonate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylic acid, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, and chlorobenzoate. Methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannic acid salt, formate, stearate, ascorbate, palmitate, oleate, pyruvate, dihydroxynaphthyl salt, malonate, laurate, glutamate, glutamate, ethanolate, methanesulfonate (methanesulfonate), ethanesulfonate (ethanesulfonate), 2-hydroxyethanesulfonate, benzenesulfonate (benzenesulfonate), p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate), naphthalene-2-sulfonate, ethanedisulfonate and 2,5-dihydroxybenzoate.

Unless otherwise stated, the term "separate form" as used herein refers to a compound existing separately from any solid mixture, solvent system, or biological environment containing another compound. In some embodiments, the crystalline form (I-HS) exists in a separate form.

Unless otherwise stated, the term "substantially pure form" as used herein means that the molar percentage of impurities in the isolated compound or crystalline form is less than about 5 mol%, preferably less than about 2 mol%, more preferably less than about 0.5 mol%, and most preferably less than about 0.1 mol%. In some embodiments, the crystalline form (I-HS) is present in a substantially pure form.

As used herein, unless otherwise stated, when used to describe the crystalline form (I-HS), the term "substantially free of other amorphous, polymorphic, or crystalline forms" should mean that the molar percentage of other amorphous, polymorphic, or crystalline forms of the separated base in the crystalline form (I-HS) is less than about 5 mol%, preferably less than about 2 mol%, more preferably less than about 0.5 mol%, and most preferably less than about 0.1 mol%. In some embodiments, the crystalline form (I-HS) is present in a form substantially free of other amorphous, polymorphic, or crystalline forms.

The terms "polymorph" and "polymorphic form" refer to different crystal forms of a single compound. That is, polymorphs are different solids with the same molecular formula, but each polymorph can have different solid-state physical properties. Therefore, a single compound can produce multiple polymorphic forms, each with different and distinct solid-state physical properties, such as different solubility profiles, dissolution rates, melting temperatures, flowability, and/or different X-ray diffraction peaks. These differences in physical properties can affect drug parameters such as storage stability, compressibility and density (which can be important in formulation and product manufacturing) and dissolution rate (which can be a significant factor in bioavailability). Techniques for characterizing polymorphism include, but are not limited to, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), single-crystal X-ray diffraction (XRD), vibrational spectroscopy such as infrared (IR) and Raman spectroscopy, solid-state and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility measurement, dissolution measurement, elemental analysis, and Karl Fischer analysis.

The term "amorphous" refers to a solid in a non-crystalline state. Amorphous solids are a disordered arrangement of molecules and therefore do not have a distinguishable lattice or unit cell, and thus lack a definable long-range order. Solids in solid form can be determined by polarized light microscopy, X-ray powder diffraction ("XRPD"), differential scanning calorimetry ("DSC"), or other standard techniques known to those skilled in the art.

As used herein, unless otherwise stated, the term “treatment” and the like shall include the administration and care of a subject or patient (preferably a mammal, more preferably a human) to combat a disease, symptom, or disorder, and include the administration of the disclosed compound to relieve symptoms or complications, or to slow the progression of the disease, symptom, or disorder.

Unless otherwise stated, the term “prevention” as used herein shall include: (a) reducing the frequency of one or more symptoms; (b) mitigating the severity of one or more symptoms; (c) delaying or avoiding the development of other symptoms; and/or (d) delaying or avoiding the development of disease or condition.

As used herein, the term "Trk-related cancer" should be defined as including or having cancers associated with or having dysregulation of the NTRK gene, Trk protein, or their expression or activity or level (e.g., any type of NTRK gene, Trk protein, or their expression or activity or level as described herein). Non-limiting examples of Trk-related cancers are described herein.

As used herein, the term "pain" should be defined to include acute, chronic, inflammatory, and neuropathic pain, including diabetic neuropathy. Furthermore, pain can be centrally mediated, peripherally mediated, caused by structural tissue injury, soft tissue injury, or progressive disease. Any pain associated with centrally mediated, peripherally mediated, structural tissue injury, soft tissue injury, or progressive disease can be acute or chronic.

As used herein, unless otherwise stated, pain should include inflammatory pain, centrally mediated pain, peripherally mediated pain, visceral pain, structural pain, cancer pain, soft tissue injury-related pain, progressive disease-related pain, neuropathic pain, acute pain from acute injury, acute pain from trauma, acute pain from surgery, headache, toothache, back pain (preferably low back pain), chronic pain from neurological disorders, and chronic pain from post-stroke conditions.

Some embodiments include methods for treating pain, wherein the pain is acute pain. Some embodiments include methods for treating pain, wherein the pain is chronic pain. Some embodiments include methods for treating pain, wherein the pain is neuropathic pain, including diabetic neuropathy. Some embodiments include methods for treating pain, wherein the pain is inflammatory pain.

In some implementations, the pain is selected from osteoarthritis, rheumatoid arthritis, fibromyalgia, headache, toothache, burns, sunburn, animal bites (e.g., dog bites, cat bites, snake bites, spider bites, and insect bites), neurogenic bladder, benign prostatic hyperplasia, interstitial cystitis, rhinitis, contact dermatitis/allergy, pruritus, eczema, pharyngitis, mucositis, enteritis, cellulite, burning pain, sciatica, temporomandibular joint neuralgia, peripheral neuritis, polyneuritis, stump pain, phantom limb pain, postoperative intestinal obstruction, cholecystitis, post-mastectomy pain syndrome, oral neuropathic pain, Charcot pain, reflex sympathetic dystrophy, Guillain-Barré syndrome, willpower pain, and burning mouth syndrome. Postherpetic neuralgia, trigeminal neuralgia, peripheral neuropathy, bilateral peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, optic neuritis, postfebrile neuritis, migratory neuritis, segmental neuritis, Gombault neuritis, neuritis, cervical neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngeal neuralgia, migraine neuralgia, idiopathic neuralgia, intercostal neuralgia, mastalgia, metatarsalgia, nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder neuralgia, sphenopalatine neuralgia, supraorbital neuralgia, retinal neuralgia, inflammatory bowel disease, irritable bowel syndrome, childbirth, menstruation, cancer, low back pain, and a group consisting of low back pain and carpal tunnel syndrome pain.

Acute pain includes pain caused by acute injury, trauma, illness, or surgery (e.g., open-chest surgery, including open-chest or bypass surgery). Acute pain also includes, but is not limited to, headache, postoperative pain, kidney stone pain, gallbladder pain, obstetric pain, rheumatic pain, toothache, or pain caused by sports medicine injuries, carpal tunnel syndrome, burns, musculoskeletal sprains and strains, tendinitis, neck pain syndrome, indigestion, gastric ulcers, duodenal ulcers, dysmenorrhea, or endometriosis.

Chronic pain includes pain caused by inflammatory conditions, osteoarthritis, rheumatoid arthritis or sequelae of disease, acute injury or trauma. Chronic pain also includes, but is not limited to, headache, upper or lower back pain (selected from back pain caused by systemic, regional or primary spinal diseases (selected from radiculopathy)), bone pain (selected from bone pain due to osteoarthritis, osteoporosis, bone metastases or unknown cause), pelvic pain, spinal cord injury-related pain, cardiac chest pain, non-cardiac chest pain, central post-stroke pain, myofascial pain, cancer pain, AIDS pain, sickle cell pain, geriatric pain or pain caused by headache, migraine, trigeminal neuralgia, temporomandibular joint syndrome, fibromyalgia syndrome, osteoarthritis, rheumatoid arthritis, gout, fibrositis or thoracic outlet syndrome.

Neuropathic pain includes pain caused by chronic or debilitating conditions or disorders. Chronic or debilitating conditions or disorders that can cause neuropathic pain include, but are not limited to, painful diabetic peripheral neuropathy, postherpetic neuralgia, trigeminal neuralgia, post-stroke pain, multiple sclerosis-related pain, neuropathic pain such as idiopathic or post-traumatic neuropathy and mononeuritis, HIV-related neuropathic pain, cancer-related neuropathic pain, carpal tunnel-related neuropathic pain, spinal cord injury-related pain, complex regional pain syndrome, fibromyalgia-related neuropathic pain, lumbar and cervical pain, reflex sympathetic malnutrition, phantom limb syndrome, and other pain syndromes associated with chronic and debilitating diseases.

"Acute neurodegenerative disease or illness" includes, but is not limited to, various types of acute neurodegenerative diseases associated with neuronal death or damage, including cerebrovascular insufficiency, focal traumatic brain injury, diffuse brain injury, and spinal cord injury, i.e., cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, acute ischemic reperfusion, perinatal hypoxic-ischemic injury, cardiac arrest, and any type of intracranial hemorrhage (including but not limited to epidural, subdural, subarachnoid, and intracerebral), as well as intracranial and intervertebral lesions (including but not limited to contusions, penetrations, shearings, compressions, and tears) and shaken infant syndrome. In some embodiments, acute neurodegenerative disease is the result of stroke, acute ischemic injury, head injury, or spinal cord injury.

"Chronic neurodegenerative conditions or diseases" include, but are not limited to, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), chronic epilepsy associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxia, corticobasal degeneration, Guam ALS-Parkinson's-dementia syndrome, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, primary pathogenesis (including multiple system atrophy), primary progressive aphasia, striatum-nigrostriatum degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and olivocerebellar degeneration, Gilles de La Tourette Diseases including medullary and pseudobulbar palsy, spinal cord and spinal muscular atrophy (Kennedy's disease), multiple sclerosis, primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sach disease, Sandhoff disease, familial spastic disorders, Wohlfart-Kugelberg-Welander disease, spastic lower limb paralysis, progressive multifocal leukoencephalopathy, familial autonomic dysfunction (Riley-Day syndrome), and prions (including but not limited to Creutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease, Kuru, and fatal familial insomnia). In some embodiments, chronic neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, or cerebral palsy.

As used herein, the term "subject" refers to an animal, preferably a mammal, and most preferably a human, that has become the subject of treatment, observation, or experimentation. In some embodiments, the subject has experienced and/or exhibited at least one symptom of a disease or condition to be treated and/or prevented. In some embodiments, the patient is from birth to the first 28 days after birth, 29 days to less than 2 years of age, 2 years to less than 12 years of age, 12 years to 21 years of age (maximum but excluding the 22nd birthday), 22 years to 35 years of age, 35 years to 65 years of age, or older than 65 years of age. In some embodiments, the patient is a pediatric patient (i.e., a patient under 21 years of age at the time of diagnosis or treatment). The term "pediatric" can be further subdivided into various subgroups, including: neonates (from birth to the first 28 days after birth); infants (29 to less than two years of age); children (two to less than 12 years of age); and adolescents (12 to 21 years of age (maximum but excluding the 22nd birthday)). Berhman RE, Kliegman R, Arvin AM, Nelson WE. Nelson Textbook of Pediatrics, 15th Ed. Philadelphia: W.B. Saunders Company, 1996; Rudolph AM, et al. Rudolph’s Pediatrics, 21st Ed. New York: McGraw-Hill, 2002; and Avery MD, First LR. Pediatric Medicine, 2nd Ed. Baltimore: Williams &Wilkins; 1994. In some implementations, the patient is an elderly patient (e.g., a patient older than 65 years).

In some embodiments, the subject has been identified or diagnosed with cancer that is dysregulated in the NTRK gene, Trk protein, or their expression or activity, or their levels (e.g., determined using an agency-approved, such as FDA-approved assay or kit). In some embodiments, the subject has a tumor that is positive for dysregulation of the NTRK gene, Trk protein, or their expression or activity, or their levels (e.g., determined using an agency-approved assay or kit). The subject may be a subject with a tumor that is positive for dysregulation of the NTRK gene, Trk protein, or their expression or activity, or their levels (e.g., identified as positive using an agency-approved, such as FDA-approved assay or kit). The subject may be a subject whose tumor has dysregulation of the NTRK gene, Trk protein, or their expression or activity, or their levels (e.g., where the tumor is identified using an agency-approved, such as FDA-approved kit or assay). In some embodiments, the subject is suspected of having Trk-related cancer. In some embodiments, the subject has a clinical record indicating that the subject has a tumor that is dysregulated in the NTRK gene, Trk protein, or their expression or activity, or their levels (and optionally, the clinical record further indicates that the subject should be treated with any of the compositions provided herein).

The term “Trk” or “Trk protein” includes any Trk protein described herein (e.g., TrkA, TrkB, or TrkC protein).

The term “NTRK gene” includes any NTRK gene described herein (e.g., NTRK1, NTRK2, or NTRK3 gene).

The term “wild-type” or “wild-type” describes nucleic acids (e.g., NTRK gene or Trk mRNA) or proteins (e.g., Trk protein) found in the cells or tissues of subjects who do not have Trk-related cancer (and optionally do not have an increased risk of developing Trk-related cancer or disease and/or are not suspected of having Trk-related cancer or disease).

The term "regulatory agency" refers to the national agency that approves the medical use of pharmaceutical reagents in a country. For example, a non-restrictive example of a regulatory agency is the U.S. Food and Drug Administration (FDA).

The phrase “dysregulation of NTRK genes, Trk proteins, or their expression, activity, or levels” refers to gene mutations (e.g., NTRK gene translocations resulting in fusion protein expression, deletions in NTRK genes expressing Trk proteins including at least one amino acid deletion compared to wild-type Trk proteins, mutations in NTRK genes expressing Trk proteins with one or more point mutations, or alternative splicing of TrkmRNAs resulting in Trk proteins with at least one amino acid deletion compared to wild-type Trk proteins), or NTRK gene duplications resulting in overexpression of Trk proteins or autocrine activity produced by overexpression of NTRK genes in cells, leading to pathogenic increases in the activity of kinase domains of Trk proteins in cells (e.g., constitutively active kinase domains of Trk proteins). For example, dysregulation of NTRK genes, Trk proteins, or their expression, activity, or levels could be mutations in NTRK1, NTRK2, or NTRK3 genes encoding constitutive activity or proteins with increased activity compared to proteins encoded by NTRK1, NTRK2, or NTRK3 genes without mutations. For example, dysregulation of the NTRK gene, the Trk protein, or its expression, activity, or level can result from gene translocation, leading to the expression of a fusion protein containing a first part of TrkA, TrkB, or TrkC (containing a functional kinase domain) and a second part of a chaperone protein (i.e., not TrkA, TrkB, or TrkC). Genes encoding fusion proteins may include, for example, the following exons of the wild-type NTRK1 gene: exons 10-19, 12-19, 12-19, 13-19, 14-19, or 15-19. Genes encoding fusion proteins may include, for example, the following exons of the wild-type NTRK2 gene: exons 12-21, 13-21, 15-21, 16-21, or 17-21. Genes encoding fusion proteins may include, for example, the following exons of the wild-type NTRK3 gene: exons 17-22 or exons 16-22. Non-limiting examples of fusion proteins due to NTRK gene translocations are described in Tables 1, 3, and 4.

Dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level may include, for example, mutations in the NTRK1, NTRK2, or NTRK3 genes that result in TrkA, TrkB, or TrkC containing at least one (e.g., two, three, four, or five) point mutations (e.g., one or more point mutations listed in Table 6). Dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level may include, for example, mutations in the NTRK2 gene that result in a point mutation in the TrkB protein including V673M. Dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level may include, for example, mutations in the NTRK3 gene that result in a point mutation in the TrkC protein including H677Y.

Dysregulation of the NTRK gene, Trk protein, or its expression or activity or level can be a mutation in the NTRK1, NTRK2, or NTRK3 gene that results in the deletion of one or more consecutive amino acids in the TrkA, TrkB, or TrkC protein (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least thirty, at least thirty-four, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least ten ... 30, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, or at least 400 amino acids (except for the deletion of amino acids in the TrkA, TrkB, or TrkC kinase domains that would lead to inactivation of the kinase domain). In some embodiments, dysregulation of the NTRK gene, the Trk protein, or its expression or activity or level may include a deletion in the NTRK1 gene that results in the absence of an NGF binding site or a TrkA protein including exon 10 of an NGF binding site, the latter being associated with acute myeloid leukemia.

In some instances, dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level may include alternative splicing forms of TrkmRNA, such as the TrkAIII splice variant, or alternative splicing forms of TrkA mRNA, resulting in the production of a TrkA protein lacking the amino acid encoded by exon 10. In some instances, dysregulation of the NTRK gene, Trk protein, or its expression, activity, or level includes amplification of the NTRK gene (e.g., one, two, three, or four additional copies of the NTRK gene), which can lead to autocrine expression of the NTRK gene in the cell.

The term “Trk-related cancer or tumor” refers to cancer associated with dysregulation of the NTRK gene, Trk protein, or its expression or activity or level (e.g., cancer associated with at least one instance (e.g., two, three, four, or five instances) of dysregulation of the NTRK gene, Trk protein, or its expression or activity or level as described herein).

As used herein, the term “mammal” means warm-blooded animals that have or are at risk of developing the diseases described herein, including but not limited to guinea pigs, dogs, cats, rats, mice, hamsters, and primates, including humans.

As used herein, the term "therapeutic effective amount" refers to the amount of an active compound or agent that elicits a biological or pharmaceutical response in an tissue system, animal, or human being sought by a researcher, veterinarian, physician, or other clinician, including the reduction of symptoms of the disease or condition being treated. Specifically, when administered to a subject requiring such treatment, a therapeutic effective amount is sufficient to: (i) treat or prevent a particular disease, condition, or disorder that can be treated with TrkA and/or TrkB inhibitors; (ii) reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of crystalline form (I-HS) corresponding to such a therapeutic effective amount will vary depending on factors such as the disease condition and its severity, and the characteristics of the mammal requiring treatment (e.g., body weight), but can still be routinely determined by those skilled in the art.

As used herein, the term "composition" is intended to cover products containing specific amounts of specific ingredients, and any products produced directly or indirectly from combinations of specific amounts of specific ingredients.

To provide a more concise description, some of the quantitative expressions given herein are not limited by the term "about". It should be understood that, whether or not the term "about" is explicitly used, each quantity given herein refers to an actual given value, and also implies an approximation of such a given value that can be reasonably inferred based on ordinary techniques in the art, including approximations due to the experimental and/or measurement conditions of such a given value.

In some implementations, the term "about" is used herein to mean approximately, in a region, approximately, or about. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the stated value. Typically, the term "about" is used herein to modify values above and below the stated value by 10% of the difference.

The term "about" preceding the position of one or more peaks in an X-ray powder diffraction pattern indicates that all peaks in the preceding group are reported in angular position (2θ), with an allowed variation of ±0.3°. This ±0.3° variation is intended to be used when comparing two powder X-ray diffraction patterns. In practice, if a peak from one pattern is specified as a range of angular positions (2θ), i.e., the measured peak position is ±0.3°, and if these peak position ranges overlap, then the two peaks are considered to have the same angular position. For example, if a peak from one pattern is determined to have a position of 11.0°, then for comparative purposes, the allowed variation allows the peak to be specified as being in the range of 10.7°–11.3°.

The term “about” preceding DSC, TGA, TG, or DTA values is reported in °C, with an allowable variation of ±5 °C.

To provide a more concise description, some quantitative expressions in this document are specified as ranges from approximately quantity X to approximately quantity Y. It should be understood that a range is enumerated therein, which is not limited to the upper and lower limits stated therein, but includes the entire range from approximately quantity X to approximately quantity Y, or any range thereof.

In some embodiments, the liquid formulations provided herein contain about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, or about 500 mg per unit dose unit of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof. However, the dosage may vary depending on the patient's needs, the severity of the condition being treated, and the compound used. In some embodiments, the dosage is administered once daily (QD) or twice daily (BID).

The daily dose of the liquid formulation of compound (I) described herein, of a pharmaceutically acceptable salt or combination thereof, may vary in a wide range, or higher, from 1.0 to 10,000 mg/adult/day, or any range thereof. For oral administration, the composition is preferably provided in tablet form containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250, and 500 mg of the active ingredient for symptom-adjusting dosage for patients to be treated. An effective amount of the drug is typically provided at dose levels of about 0.1 mg/kg to about 1000 mg/kg body weight daily, or any range thereof. This range may be about 0.5 to about 500 mg/kg body weight daily, or any range thereof. This range may be about 1.0 to about 250 mg/kg body weight daily, or any range thereof. This range may be about 0.1 to about 100 mg/kg body weight daily, or any range thereof. In one instance, the range may be from about 0.1 to about 50.0 mg/kg body weight per day, or any amount or range thereof. In another instance, the range may be from about 0.1 to about 15.0 mg/kg body weight per day, or any range thereof. In yet another instance, the range may be from about 0.5 to about 7.5 mg/kg body weight per day, or any amount or range thereof thereof. The liquid formulations provided herein may be administered 1 to 4 times daily or in a single-day dose regimen.

The dosage will vary depending on the strength, method of administration, and progression of the disease condition. Furthermore, factors relevant to the specific patient being treated, including patient age, weight, diet, and time of administration, will necessitate dose adjustments. The optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the method of administration, formulation strength, administration route, and progression of the disease condition. Furthermore, factors relevant to the specific patient being treated, including patient age, weight, diet, and time of administration, will necessitate dose adjustments.

Those skilled in the art will recognize that in vivo and in vitro assays using appropriate, known and generally accepted cell and/or animal models can predict the ability of an investigational compound to treat or prevent a given condition.

Those skilled in the art will further recognize that human clinical trials can be conducted using methods well-known in the clinical and medical fields, including first-in-human dose range and efficacy trials in healthy patients and/or patients with a given condition. For example, appropriate doses for pediatric patients can be determined using known methods, including weight, age, and models such as pediatric modeling (CERTARA, Princeton, New Jersey), which can be used to establish pharmacokinetic methods for administration that take into account patient age, individual development of the clearance pathways of the compound of formula (I), its pharmaceutically acceptable salts or combinations thereof, and body surface area (BSA).

The liquid formulations described herein can be administered via a variety of routes, including oral administration, intranasal administration, and administration via enteral feeding or gastrostomy tubes.

The abbreviations found in the instruction manual have the following meanings:

ATP Adenosine triphosphate DI Deionized EtOH ethanol GC Gas chromatography MOPS 3-(N-morpholino)-propanesulfonic acid MTBE Methyl tert-butyl ethyl ether PDA Photodiode array RRT Relative retention time RT room temperature THF Tetrahydrofuran TMB 3,3',5,5'-Tetramethylbenzidine

The following examples are provided to illustrate the present invention and to aid in understanding it, and are not intended, nor should they be construed as limiting the invention as described in the following claims in any way.

In the examples described below, all temperatures are expressed in °C unless otherwise specified. Reagents were purchased from commercial suppliers such as Sigma-Aldrich Chemical Company, EMD, JT Baker, or Pharco-Aaper and were used without further purification unless otherwise specified. Tetrahydrofuran (THF), heptane, and other organic solvents were purchased from commercial suppliers such as Sigma-Aldrich Chemical Company, ACROS, Alfa-Aesar, Lancaster, TCI, or Maybridge and were used as is.

Those skilled in the art will recognize that, unless otherwise stated, reaction steps can be carried out under suitable conditions according to known methods to provide a desired product. Those skilled in the art will also recognize that the reaction steps disclosed herein can be carried out in a variety of solvents or solvent systems, and also in mixtures of suitable solvents or solvent systems. Those skilled in the art will recognize that in the specification and claims presented herein, where reagents or reagent classes/types (e.g., bases, solvents, etc.) are listed in more than one method step, each reagent is selected independently for each reaction step and can be different from each other. For example, where two steps of the method list organic or inorganic bases as reagents, the organic or inorganic base selected for the first step can be the same as or different from the organic or inorganic base in the second step.

The reactions listed below are typically carried out in “ACS grade” solvents under positive pressure of nitrogen (unless otherwise specified), and the reaction flasks are usually fitted with rubber septa for introducing substrates and reagents via syringes or feeding funnels.

Process monitoring and analysis were performed using acetonitrile and water/trifluoroacetic acid as mobile phases, employing two reversed-phase high-performance liquid chromatography (HPLC) systems. One system used an Agilent Zorbax Extend C18 column at 264 nm, while the other system (hereinafter, "TRK1PM1HPLC") comprised a Waters Xbridge Phenyl column at 268 nm. The former system was used unless otherwise specified. The silica from both systems was stirred with the compound in a flask, filtered through polypropylene cloth, and then analyzed.

The amorphous free base form of compound I : Approximately 1 g of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide was dissolved in a minimal amount of water and cooled to approximately -26°C, then dried in a freeze dryer for 24 hours. Approximately 20 mg of the amorphous material obtained from the freeze dryer was weighed in a vial, and 5 equal volumes of a suitable solvent system were added to it. The dissolution of the mixture was checked; if no significant dissolution was observed, the mixture was heated to approximately 40°C and checked again. This process was continued until dissolution was observed or until 100 volumes of solvent were added. The XRPD diagram of the amorphous material obtained from the freeze-drying experiment is shown in Figure 7.

The amorphous hydrogen sulfate of the compound of formula I was prepared as described in Example 14A of WO2010/048314 (see Example 3).

This document also provides a method for preparing the crystalline form (I-HS). In some embodiments, the method includes the steps shown in Scheme 1.

In some embodiments, this document provides a method for preparing the crystalline form (I-HS), which includes:

(a) Concentrated sulfuric acid was added to an EtOH solution of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide to form the hydrogen sulfate of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide;

(b) In step (a), heptane is added to the solution to form a slurry;

(c) Filter the slurry to separate (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate;

(d) Mix the (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate with a 5:95 w/w water/2-butanone solution;

(e) Heating the mixture from step (d) at about 65-70°C with stirring until the weight percentage of ethanol is about 0.5%, to form a slurry in the crystalline form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate; and

(f) The crystalline form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate was separated by filtration.

In some embodiments, the above method further includes: (b1) adding (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide to the solution of step (a) at room temperature, and stirring the solution until a slurry is formed.

In some embodiments, this document provides a method for preparing the crystalline form (I-HS), which includes:

(a) Reacting 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine with (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxysuccinate in the presence of a base to form (R)-5-(2-(2,5-difluorophenyl)pyrrolidine-1-yl)-3-nitropyrazolo[1,5-a]pyrimidine;

(b) Treat the (R)-5-(2-(2,5-difluorophenyl)pyrrolidone-1-yl)-3-nitropyrazolo[1,5-a]pyrimidine with Zn and hydrochloric acid to form (R)-5-(2-(2,5-difluorophenyl)pyrrolidone-1-yl)pyrazolo[1,5-a]pyrimidine-3-amine;

(c) Treat the (R)-5-(2-(2,5-difluorophenyl)pyrrolidone-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine with a base and phenyl chloroformate to form phenyl(R)-5-(2-(2,5-difluorophenyl)pyrrolidone-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamate tert-butyl ester;

(d) Reacting the phenyl(R)-5-(2-(2,5-difluorophenyl)pyrrolidone-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamate tert-butyl with (S)-pyrrolidone-3-ol to form (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidone-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidone-1-carboxamide;

(e) adding sulfuric acid to the (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide to form (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate; and

(f) Separation of the crystalline form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate.

In some embodiments of step (a) above, the base is an amine base, such as triethylamine.

In some embodiments of step (c) above, the base is an alkali metal base, such as an alkali metal carbonate, for example, potassium carbonate.

Formulation A

Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine

Step A—Preparation of sodium pyrazolo[1,5-a]pyrimidine-5-ol : A solution of 1H-pyrazol-5-amine and 1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1.05 equivalents) was placed in a round-bottom flask equipped with a mechanical stirrer, a steam boiler, a reflux condenser, a J-Kem temperature probe, and an _N2 adapter for positive _N2 pressure control. Under mechanical stirring, the solid was suspended in a nitrogen atmosphere by 4 volumes (4 mL/g) of anhydrous EtOH, followed by the addition of 2.1 equivalents of NaOEt (21% by weight EtOH solution), and then washed linearly with 1 volume (1 mL/g) of anhydrous EtOH. The slurry was heated to approximately 75°C and stirred under gentle reflux until less than 1.5 area % of 1H-pyrazole-5-amine was observed by TRK1PM1 HPLC. The reaction progress was tracked using 20 μL of the slurry diluted in 4 mL of deionized water and 5 μL of the sample injected at 220 nm.

One hour later, 2.5 volumes (2.5 mL/g) of heptane were added to the mixture, and the mixture was refluxed at 70 °C for 1 hour. The slurry was then cooled to room temperature overnight. The solid was collected by filtration through a benchtop funnel and polypropylene filter cloth. The reactor was rinsed and 4 volumes (4 mL/g) of heptane were added to the filter cake. The filter cake was pulled out, and the solid was transferred to a balanced drying tray and dried in a high vacuum oven at 45 °C until its weight was constant. A pale yellow solid, pyrazolo[1,5-a]pyrimidine-5-ol sodium salt, was given in 93–96% (corrected) and observed by HPLC to be greater than 99.5% area (1 mg/mL dilution in deionized water, TRK1PM1 at 220 nm).

Step B—Preparation of 3-nitropyrazolo[1,5-a]pyrimidine-5(4H)-one : Sodium pyrazolo[1,5-a]pyrimidine-5-ol, dissolved in 3.0 volume (3.0 mL/g) deionized water at 40–45 °C, was added to a balanced round-bottom flask. The solution was then concentrated in a water bath on a rotary evaporator under high vacuum at 65 °C until 2.4 x weight of the starting material (1.4 volume/1.4 mL/g deionized water content) was observed. Gas chromatography (GC) of residual EtOH (30 μL of solution dissolved in approximately 1 mL of MeOH) showed less than 100 ppm, followed by the observation of trace amounts of ethyl nitrate fumes upon addition of _HNO₃ . In some cases, an additional 1.5 volumes (1.5 mL/g) of DI water were added to the original solution, and then the solution was concentrated in a water bath on a rotary evaporator under high vacuum at 65 °C until 2.4 x the weight of the starting material (1.4 volumes/1.4 mL/g DI water content) was observed. Gas chromatography of the residual EtOH (30 μL of solution dissolved in about 1 mL of MeOH) showed << 100 ppm of residual EtOH, and no ethyl nitrate fumes were observed after the subsequent addition of _HNO3 .

A round-bottomed container equipped with a mechanical stirrer, steam boiler, reflux condenser, J-Kem temperature probe, and _N2 adapter for positive _N2 pressure control was filled with 3 vol% (3 mL/g, 10 equivalents) >90 wt% _HNO3 and cooled to approximately 10 °C under nitrogen atmosphere using an external ice-water cooling bath. Using a pressure-balanced feeding funnel, 1.75–1.95 volumes of a deionized aqueous solution of pyrazolo[1,5-a]pyrimidine- ₅ -ol sodium salt (1.16–1.4 mL deionized water/g pyrazolo[1,5-a]pyrimidine-5-ol sodium salt) was added to the HNO3 solution at a rate maintaining an internal temperature of 35–40 °C under cooling. Two azeotropes were observed, without any ethyl nitrate fumes. The azeotropic flask, delivery line (if applicable), and feeding funnel were rinsed with 2 × 0.1 volumes (2 × 0.1 mL/g) of deionized water added to the reaction mixture. Once the addition was complete, the temperature was gradually increased to approximately 45-50°C for about 3 hours. HPLC showed that the sodium pyrazolo[1,5-a]pyrimidin-5-ol salt was converted to 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one >99.5% of the area.

Step C—Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine : 3-nitropyrazolo[1,5-a]pyrimidine-5(4H)-one was added to a round-bottom flask equipped with a mechanical stirrer, heating mantle, reflux condenser, J-Kem temperature probe, and _N2 adapter for positive _N2 pressure control. Under mechanical stirring, the solid was suspended in 8 volumes (8 mL/g) of _CH3CN , and then 2,6-dimethylpyridine (lutitine) (1.05 equivalents) was added, followed by warming the slurry to approximately 50°C. Using a pressure-balanced feeding funnel, 0.33 equivalents of _POCl3 were added dropwise to the mixture. This addition produced a thick, beige trimer slurry, which was homogenized with stirring until a semi-mobile substance was observed. An additional 1.67 equivalents of _POCl3 was added to the mixture while stabilizing the temperature, and then the reaction mixture was warmed to mild reflux (78°C). Some swelling was observed in the warm mixture, which subsided as the thick slurry thinned.

The reaction mixture was refluxed until completely dissolved to a dark solution, and HPLC (20 μL diluted in ₅ mL CH3CN, TRK1PM1 HPLC, 5 μL injection, 268 nm) confirmed the absence of more trimers (RRT 0.92) and less than 0.5 area % of 3-nitropyrazolo[1,5-a]pyrimidine-5(4H)-one (RRT 0.79) by manually removing any interferences and early elution peaks associated with dimethylpyridine from the regio-integration. At a scale of 1.9 kg, 0 area % trimers, 0.25 area % of 3-nitropyrazolo[1,5-a]pyrimidine-5(4H)-one, and 99.5 area % of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine were observed at 268 nm using TRK1PM1 HPLC after 19 hours of gentle reflux.

Formulation B

Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxysuccinate

Step A—Preparation of (4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate tert-butyl ester : 2-bromo-1,4-difluorobenzene (1.5 equivalents) was dissolved in 4 volumes of THF (based on the weight of tert-butyl 2-oxopyrrolidine-1-carboxylate added) and cooled to approximately 5°C. Over 2 hours, a 2.0 M iPrMgCl solution in THF (1.4 equivalents) was added to the mixture while maintaining the reaction temperature below 25°C. The solution was cooled to approximately 5°C and stirred for 1 hour (GC analysis confirmed Grignard formation). Over approximately 30 minutes, a solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (1.0 equivalent) in 1 volume of THF was added while maintaining the reaction temperature below 25°C. The reaction was stirred at approximately 5°C for 90 minutes (2-oxopyrrolidine-1-carboxylate tert-butyl ester was confirmed to be less than 0.5 area % by HPLC). The reaction was quenched with 5 volumes of 2 M HCl aqueous solution while maintaining the reaction temperature below 45°C. The reactants were then transferred to a separatory funnel, 10 volumes of heptane were added, and the aqueous layer was removed. The organic layer was washed with 4 volumes of saturated NaCl aqueous solution, followed by the addition of 2 × 1 volumes of saturated NaCl aqueous solution. The organic layer solvent was converted to heptane (confirmed by GC as <1 wt% THF) at a distillation temperature of 35–55 °C, with a distillation pressure of 100–200 mm Hg and a minimum distillation volume of approximately 7 volumes for each 2 × 4 volumes of heptane added. The mixture was then diluted to 10 volumes with heptane while heating to approximately 55 °C to obtain a denser solid. The mixture was cooled to room temperature overnight. The slurry was cooled to below 5 °C and filtered through a polypropylene filter cloth. The wet filter cake was washed with 2 × 2 volumes of heptane. The solid was dried under vacuum at 55 °C until constant weight to give tert-butyl (4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate as a white solid with a theoretical yield of approximately 75% to 85%.

Step B—Preparation of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole : Tert-butyl (4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate was dissolved in 5 volumes of toluene containing 2.2 equivalents of 12M HCl. Mild exothermic reaction and gas escape were observed. The reaction was heated to 65°C for 12–24 hours and monitored by HPLC. After completion, the reaction was cooled to below 15°C using an ice/water bath. The pH was adjusted to approximately 14 with 3 equivalents of 2M NaOH aqueous solution (4.7 volumes). The reaction was stirred at room temperature for 1–2 hours. The mixture was transferred to a separatory funnel with toluene. The aqueous layer was removed, and the organic layer was washed with 3 volumes of saturated NaCl aqueous solution. The organic layer was concentrated into an oil and redissolved in 1.5 volumes of heptane. The obtained suspension was filtered through GF/F filter paper and concentrated into a pale yellow oily substance of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole, with a theoretical yield of 90% to 100%.

Step C—Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine : 0.2 mol% of chloro-1,5-cyclooctadiene iridium dimer and (R)-2-(2-(diphenylphosphino)phenyl)-4-isopropyl-4,5-dihydrooxazole (0.4 mol%) were suspended at room temperature in 5 volumes of MTBE (based on 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole). The mixture was stirred for 1 hour until most of the solid dissolved and the solution turned deep red. Catalyst formation was monitored using an HPLC/PDA detector. The reaction was cooled to below 5°C and 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole (1.0 equivalent) was added after rinsing with 0.5 volumes of MTBE. Diphenylsilane (1.5 equivalent) was added over approximately 20 minutes while maintaining the reaction temperature below 10°C. The reaction was stirred below 10°C for 30 minutes and then warmed to room temperature. The reaction was stirred overnight at room temperature. The reaction was confirmed to be complete by HPLC, and then cooled to below 5°C. The reaction was quenched with 5 volumes of 2M HCl aqueous solution, maintaining the temperature below 20°C. After 10 minutes, the ice/water bath was removed, and the reaction temperature was raised to room temperature while stirring for 2 hours. The mixture was transferred to a separatory funnel containing 3 volumes of MTBE. The aqueous layer was washed with 3.5 volumes of MTBE, and then 5 volumes of MTBE were added to the aqueous layer while adjusting the pH to approximately 14 by adding 0.75 volumes of 50% NaOH aqueous solution. The organic layer was washed with 5 volumes of saturated NaCl aqueous solution, then concentrated to an oil and diluted with 3 volumes of MTBE. The solution was filtered through a polypropylene filter cloth and washed with 1 volume of MTBE. The filtrate was concentrated to an oil of (R)-2-(2,5-difluorophenyl)-pyrrolidine, with a theoretical yield of 95%–100% and an ee of 75%–85%.

Preparation of step D—(R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxy-succinate : (R)-2-(2,5-difluorophenyl)-pyrrolidine (1.0 equivalent) was transferred to a round-bottom flask containing 15 volumes (corrected power) of EtOH (200 pf). D-malic acid (1.05 equivalent) was added, and the mixture was heated to 65°C. The solid dissolved completely at approximately 64°C. The solution was cooled to room temperature. At approximately 55°C, (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxy-succinate (approximately 50 mg, >97% ee) was added to the solution, and the mixture was stirred overnight at room temperature. The suspension was then filtered through a polypropylene filter cloth and washed with 2 × 1 volumes of EtOH (200 pf). The solid was dried under vacuum at 55 °C to give (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxy-succinate, with a theoretical yield of 75% to 90% and an ee of >96%.

Referring to Scheme 1, suitable bases include tertiary amine bases, such as triethylamine and _K₂CO₃ . Suitable solvents include ethanol, heptane, and tetrahydrofuran (THF). The reaction can be conveniently carried out at temperatures _ranging from 5°C to 50°C. The reaction progress is typically monitored using HPLC TRK1PM1.

Option 1

Compounds II (5-chloro-3-nitropyrazolo[1,5-a]pyrimidine) and III ((R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxysuccinate, 1.05 equivalents) were loaded into a round-bottom flask equipped with a mechanical stirrer, a J-Kem temperature probe, and an _N2 adapter for positive _N2 pressure control. A solution of 4:1 EtOH:THF (10 mL/g compound II) was added, followed by the addition of triethylamine (NEt ₃ , 3.50 equivalents) through a feeding funnel, with the temperature reaching approximately 40°C during the addition. Once the addition was complete, the reaction mixture was heated to 50°C and stirred for 0.5–3 hours to obtain compound IV.

Compound IV was added to a round-bottom flask equipped with a mechanical stirrer, a J-Kem temperature probe, and a _N2 inlet, followed by tetrahydrofuran (10 mL/g of compound IV). The solution was cooled to below 5°C in an ice bath, and Zn (9–10 equivalents) was added. Then, 6M HCl (9–10 equivalents) was added dropwise at this rate to maintain the temperature below 30°C (approximately 1.5 hours for a 1 kg scale). Once the exothermic reaction subsided, the reaction was warmed to room temperature and stirred for 30–60 minutes until compound IV was no longer detected by HPLC. At this point, an aqueous solution of potassium _{carbonate (K₂CO₃ ,} 2.0 equivalents) (5 mL/g of compound IV) was added in one go, followed by rapid dropwise addition of phenyl chloroformate (PhOCOCl, 1.2 equivalents). Gas escape ( _CO₂ ) was observed during both additions, and the temperature rose to approximately 30°C after the addition of phenyl chloroformate. The urethane formation was stirred at room temperature for 30–90 minutes. Then, HPLC analysis was performed immediately to ensure that the presence of amines was less than 1% by area and that the yield of compound VI in solution was high.

Add amine VII ((S)-pyrrolidine-3-ol, based on a theoretical yield of 1.1 equivalents for compound VI) and EtOH (10 mL/g compound VI) to the above solution. Add compound VII before or simultaneously with EtOH to avoid the formation of urethane impurities. Concentrate the above EtOH solution under reduced pressure using a batch concentrator to a minimum volume (4-5 mL/g) (HF level should be <5% by GC), and add back EtOH (10 mL/g compound VI) to give a total of 10 mL/g. The reaction is then heated at 50 °C for 9-19 hours or until HPLC shows compound VI to be less than 0.5 area%. The reaction is then cooled to room temperature, and sulfuric acid ( _H₂SO₄ , 1.0 equivalent to compound VI) is added through a feeding _funnel to give compound I-HS, typically exothermically at about 30 °C.

Example 1

Preparation of crystalline form (I-HS) (Method 1)

(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.500 g, 1.17 mmol) was dissolved in EtOH (2.5 mL) and cooled to approximately 5 °C. Concentrated sulfuric acid (0.0636 mL, 1.17 mmol) was added to the cooled solution and stirred for approximately 10 minutes while simultaneously warming to room temperature. Methyl tert-butyl ether (MTBE) (2 mL) was slowly added to the mixture, causing the product to gel. Then, EtOH (2.5 mL) was added to the mixture and heated to reflux temperature until all solids dissolved. After cooling to room temperature and stirring for approximately 1 hour, some solids formed. After cooling to approximately 5 °C, the solids were filtered and washed with MTBE. After filtration and air drying for about 15 minutes, (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate was separated as a solid.

Example 2

Preparation of crystalline form (I-HS) (Method 2)

Concentrated sulfuric acid (392 mL) was added to 3031 g of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide in 18322 mL of EtOH solution to form bisulfate. 2 g of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide bisulfate solution was added to the solution, and the mixture was stirred at room temperature for at least 2 hours to form a bisulfate slurry. Heptane (20888 g) was added, and the slurry was stirred at room temperature for at least 60 minutes. The slurry was filtered, and the filter cake was washed with a 1:1 heptane/EtOH solution. The solid was then vacuum dried at ambient temperature (oven temperature set at 15°C).

The dried hydrogen sulfate (6389 g, from 4 combined batches) was added to a 5:95 w/w water/2-butanone solution (total weight 41652 g). The mixture was heated at about 68 °C with stirring until the weight percentage of ethanol was about 0.5%, during which time a slurry was formed. The slurry was filtered, and the filter cake was washed with the 5:95 w/w water/2-butanone solution. The solid was then vacuum dried at ambient temperature (oven temperature set at 15 °C) to give the crystalline form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate.

Example 3

Preparation of amorphous AM (HS)

Sulfuric acid (0.1 M MeOH solution, 219.4 mL, 21.94 mmol) was slowly added to a MeOH solution of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (9.40 g, 21.94 mmol) in 220 mL of MeOH under rapid stirring at ambient temperature. After 30 minutes, the reaction was first concentrated to near dryness by rotary evaporator, and then concentrated under high vacuum for 48 hours to provide the amorphous form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide sulfate (11.37 g, 21.59 mmol, 98.43% yield). LCMS (apci m/z 429.1, M+H).

Example 4

Preparation of crystalline HCl salts of Formula I

A mixture of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.554 g, 1.29 mmol) in EtOH (6 mL, 200 standard) and MTBE (10 mL) was heated to 50 °C with stirring to obtain a solution. Then, concentrated hydrogen chloride (0.108 mL, 1.29 mmol) was added in a single batch. The reaction mixture was then cooled to ambient temperature first, and then further cooled to approximately 5 °C with stirring in an ice-water bath to induce crystallization. The suspension was stirred in an ice-water bath for 4 hours, then filtered under vacuum. The filter cake was washed with MTBE and dried under vacuum at 55°C to constant weight to give crystalline (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrochloride (0.534 g, 89% yield). LCMS (APCI m/z 429.2, M+H).

Preparation of crystalline HBr salts of Formula I

A mixture of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.505 g, 1.18 mmol) in EtOH (6 mL, 200 standard) and MTBE (10 mL) was heated to 50 °C with stirring to obtain a solution. Hydrogen bromide (33% aqueous solution) (0.213 mL, 1.18 mmol) was then added in a single batch. The reaction mixture was heated to reflux to obtain a mostly clear solution, and a small amount of oily residue was added to the glass wall of the reaction vessel. Upon cooling to ambient temperature, a precipitate formed, and the oily residue solidified. The mixture was reheated to 50 °C, then cooled to room temperature and stirred overnight. The suspension was vacuum filtered, the filter cake was washed with MTBE and vacuum dried at 55 °C to constant weight to give crystalline (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrobromide (0.51 g, 85% yield). LCMS (APCI m/z 429.3, M+H).

Preparation of crystalline methanesulfonate of Formula I

A mixture of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.532 g, 1.24 mmol) in EtOH (2.7 mL, 200 standard) and MTBE (5.3 mL) was heated to 50 °C with stirring to obtain a solution. Then, methanesulfonic acid (0.076 mL, 1.24 mmol) was added in a single batch. The reaction mixture was heated to reflux to obtain a mostly clear solution containing a small amount of particles. Upon cooling to ambient temperature, a precipitate and some oily residue appeared. Additional EtOH (0.5 mL, 200 standard) and methanesulfonic acid (0.010 mL) were added to obtain a solution. The reaction mixture was reheated to 50 °C, then cooled to room temperature and stirred for 1 hour. The suspension was vacuum filtered, the filter cake was washed with MTBE and vacuum dried at 55 °C to constant weight to give crystalline (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide methanesulfonate (0.51 g, 78% yield). LCMS (APCI m/z 429.4, M+H).

Preparation of crystalline camphor sulfonate of Formula I

A mixture of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.500 g, 1.17 mmol) and S-(+)-camphorsulfonic acid (0.271 g, 1.17 mmol) in EtOH (3 mL, 200 standard) and MTBE (5 mL) was heated to reflux with stirring to obtain a solution. Upon cooling to ambient temperature, a precipitate formed. The suspension was stirred overnight at room temperature, then vacuum filtered. The filter cake was washed with MTBE and vacuum dried at 55°C to constant weight to obtain crystalline (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide ((1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]hept-1-yl)methanesulfonate.

Example 5

Using (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidine-3- A liquid formulation of 1-(3-hydroxypyrrolidine-1-carboxamide) successfully treated infantile fibrosarcoma with NTRK3-ETV6 fusion.

Materials and methods

A multicenter, phase 1 dose-escalation pediatric study (ClinicalTrials.gov Identifier: NCT02637687) began in December 2015 to evaluate the safety and tolerability of compound I-HS (i.e., the bisulfate of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide) in patients with advanced solid or primary central nervous system tumors. Eligibility criteria included 1–21 years of age, regardless of the presence of known TRK alterations, and patients aged 1 month or older with known NTRK fusions and a diagnosis of infantile fibrosarcoma or congenital mesodermal nephroma. An oral liquid formulation of compound I-HS was developed for patients who could not swallow capsules. A pharmacokinetic approach to the dose was established using pediatric simulation modeling (CERTARA, Princeton, New Jersey), taking into account patient age, individual development, and body surface area (BSA) to eliminate the I-HS clearance pathway of the compound. The initial cohort was expected to select a pediatric dose equivalent to the exposure achieved in adult patients receiving a 100 mg BID dose, i.e., the recommended Phase 2 adult dose. Cycles were measured in 28-day increments with continuous dosing. Response assessments were scheduled every eight weeks using appropriate imaging modalities. Patients continued treatment until disease progression or evidence of intolerable toxicity.

A kit is provided comprising: a sealed graduated amber vial containing 7.6 g of compound I-HS; a sealed vial containing 51 g of W7 HP Pharma; a sealed vial containing 500 g of trisodium citrate dihydrate; a sealed vial containing 100 mL of sterile water; a sealed pint (approximately 473 mL) vial for ORA-; a funnel; a 28 mm push-in vial adapter; a box containing 56 units of 1-mL disposable syringes; a box containing 56 units of 5-mL disposable syringes; a pharmaceutical label indicating the concentration of compound I-HS (20 mg/mL); and combination instructions.

Prepare the liquid solution as shown in Figure 9. First, remove the seal (cap) from the bottle containing W7 HP Pharma. Next, using a funnel, add the contents of a 100 mL sterile water bottle to the bottle containing W7 HP Pharma. Then close the bottle with its cap and shake the bottle containing W7 HP Pharma and sterile water until all W7 HP dissolves. Allow 10 minutes for W7 HP Pharma to completely dissolve. Check the bottom and sides of the bottle to ensure all W7 HP Pharma is dissolved and there are no clumps at the bottom or adhering to the sides. Next, let the bottle stand without stirring for about 5 minutes to allow any bubbles generated by the dissolved W7 HP Pharma to dissipate. Then remove the seal (cap) from the graduated bottle containing compound I-HS. Using the same funnel as before, add the W7 HP Pharma solution to the graduated bottle containing compound I-HS. Cap the bottle and shake by hand until dissolved. Allow bubbles to reach the surface and produce a clear red solution. Using the same funnel as before, add the supplied volume to 300 mL. Cap the graduated bottle and gently invert it 10 times to mix with the compound I-HS/W7 HP solution, being careful not to introduce too many air bubbles into the formulation. Next, weigh 3.5 g of trisodium citrate dihydrate from the provided container and add it to the liquid formulation using the second funnel in the kit. Cap the bottle and invert it 10 times. Allow the air bubbles to rise to the top and check the contents of the bottle to ensure that all trisodium citrate dihydrate is completely dissolved; if not, invert the bottle 10 more times. Then, remove the cap from the graduated bottle and insert the provided 28 mm push-in bottle adapter (syringe adapter) into the bottle. Seal the bottle by firmly placing the cap on it. Then, administer the required amount of compound I-HS into the liquid formulation using a 1 mL or 5 mL syringe, according to the patient dosing regimen.

result

A healthy female was born with large blood vessels and a mass on the right side of her neck extending to her face, initially diagnosed and treated as a rapidly invasive congenital hemangioma. At 6 months of age, the mass grew rapidly, and surgical resection/debulking confirmed the diagnosis of IFS by fluorescence in situ hybridization (FISH) via ETV6 translocation. Within the first 7 days post-surgery, the tumor rapidly progressed, invading the oral cavity. Chemotherapy was initiated with vincristine, actinomycin-D, and cyclophosphamide, but the patient experienced disease progression during the first cycle. A new chemotherapy regimen consisting of ifosfamide and doxorubicin (ID) was started, along with debulking surgery and a tracheotomy for oropharyngeal obstruction. Two additional cycles of ID and four cycles of ifosfamide and etoposide had little effect on the tumor. The tumor progressed to involve the skull base, mastoid process, and cervical vascular system. Major surgical resection was performed in October 2015 by a multidisciplinary team of surgeons, but a clear surgical margin could not be achieved.

Five weeks post-surgical resection, MRI of the brain and neck revealed a hyperenhancing mass measuring 20 mm × 19 mm × 18 mm, involving the base of the skull in the middle cranial fossa, located only anterior and inferior to the structures of the inner ear (Figures 10A and 10B). Further chemotherapy was deemed futile due to the lack of response to all standard protocols. Repeat surgical resection was considered impossible. Treatment with radiation therapy was possible, but depending on the patient's age and the location of the disease, devastating long-term sequelae were anticipated.

At 16 months, the patient enrolled in a Phase 1 pediatric study of the oral selective TRK inhibitor compound I-HS. Parents noted increased engagement and playfulness throughout Cycle 1. At the end of Cycle 1 (day 28), MRI scans of the brain and neck showed a significant reduction in spacing and an increase in mass exceeding 90% of baseline (Figures 10C and 10D). At the end of Cycle 2, repeat scans confirmed the reduction in size and showed a sustained decrease in enhancement, confirming a partial response (Figures 10E and 10F). During the first two cycles, the patient experienced fever and PCR-confirmed influenza A (considered irrelevant) but no adverse events related to compound I-HS.

-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxy Liquid formulation of pyrrolidine-1-carboxamide

Liquid formulations of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide were prepared using the components listed in Table 16.

Table 16. Liquid formulations of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide.

(1) Including the API correction factor of 0.8137. Calculation: Free base molecular weight/salt molecular weight = 428.44/526.51. The density of the liquid formulation is 1.2 mg/mL.

(2) Label statement = 3518.8 g of API in salt form × 0.8137/171,648 g of total preparation * 1.2 g/mL density * 1000 mg/g.

(3) If necessary, an additional 5% of the total amount of sodium citrate may be added to the formulation to adjust the pH value.

References:

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2. Vaishnavi et al., Nature Med. 19: 1469-1472, 2013.

3. Greco et al., Mol. Cell. Endocrinol. 28:321, 2010.

4. Kim et al., PLoS ONE 9(3):e91940, 2014.

5. Vaishnavi et al., Nature Med. 19: 1469-1472, 2013.

6. Fernandez-Cuesta et al., “Cross-entity mutation analysis of lung neuroendocrine tumors sheds light into their molecular origin and identifies new therapeutic targets”, AACR Annual Meeting 2014, Abstract, April 2014.

7. Stransky et al., Nature Comm. 5:4846, 2014.

8. Ross et al., Oncologist 19:235-242, 2014.

9. Doebele et al., J. Clin. Oncol. 32: 5s, 2014.

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12.WO 2013/059740

13. Zheng et al., “Anchored multiplex PCR for targeted next-generation sequencing”, Nature Med., published online on November 10, 2014.

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16. Martin-Zanca et al., Nature 319:743, 1986.

17. Meyer et al., Leukemia 21: 2171-2180, 2007.

18. Reuther et al., Mol. Cell. Biol. 20: 8655-8666, 2000.

19. Marchetti et al., Human Mutation 29(5): 609-616, 2008.

20.Tacconelli et al., Cancer Cell 6:347, 2004.

21. Walch et al., Clin. Exp. Metastasis 17: 307-314, 1999.

22. Papatsoris et al., Expert Opin. Invest. Drugs 16(3): 303-309, 2007.

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25. Truzzi et al., J. Invest. Dermatol. 128(8): 2031, 2008.

26. Kolokythas et al., J. Oral Maxillofacial Surgery 68(6): 1290-1295, 2010.

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sequence list

<110> Loxo Oncology, Inc.

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Leu Pro Glu Ile Ser Val Ser His Val Asn Leu Thr Val Arg Glu Gly

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Val Asp Trp Ile Val Thr Gly Leu Gln Ser Ile Asn Thr His Gln Thr

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Val Thr Ser Glu Asp Asn Gly Phe Thr Leu Thr Cys Ile Ala Glu Asn

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His Asn Gly Gln Pro Leu Arg Glu Ser Lys Ile Ile His Val Glu Tyr

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Pro Glu Ser Thr Asp Asn Phe Ile Leu Phe Asp Glu Val Ser Pro Thr

405 410 415

Pro Pro Ile Thr Val Thr His Lys Pro Glu Glu Asp Thr Phe Gly Val

420 425 430

Ser Ile Ala Val Gly Leu Ala Ala Phe Ala Cys Val Leu Leu Val Val

435 440 445

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530 535 540

Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu

545 550 555 560

Ser Pro Thr Lys Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Asp

565 570 575

Pro Thr Leu Ala Ala Arg Lys Asp Phe Gln Arg Glu Ala Glu Leu Leu

580 585 590

Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Gly

595 600 605

Asp Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp

610 615 620

Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Met Ile Leu Val

625 630 635 640

Asp Gly Gln Pro Arg Gln Ala Lys Gly Glu Leu Gly Leu Ser Gln Met

645 650 655

Leu His Ile Ala Ser Gln Ile Ala Ser Gly Met Val Tyr Leu Ala Ser

660 665 670

Gln His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly

675 680 685

Ala Asn Leu Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val

690 695 700

Tyr Ser Thr Asp Tyr Tyr Arg Leu Phe Asn Pro Ser Gly Asn Asp Phe

705 710 715 720

Cys Ile Trp Cys Glu Val Gly Gly His Thr Met Leu Pro Ile Arg Trp

725 730 735

Met Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp

740 745 750

Val Trp Ser Phe Gly Val Ile Leu Trp Glu Ile Phe Thr Tyr Gly Lys

755 760 765

Gln Pro Trp Phe Gln Leu Ser Asn Thr Glu Val Ile Glu Cys Ile Thr

770 775 780

Gln Gly Arg Val Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr

785 790 795 800

Asp Val Met Leu Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Asn

805 810 815

Ile Lys Glu Ile Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr Pro

820 825 830

Ile Tyr Leu Asp Ile Leu Gly

835

Claims (11)

1. A liquid formulation comprising: (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide of formula (I), pharmaceutically acceptable salts thereof, or combinations thereof: 13% to 17% by weight of hydroxypropyl-β-cyclodextrin; 0.7% to 1.5% by weight of sodium citrate dihydrate; A sweetener comprising sucrose, glycerol, sorbitol, and flavoring agents, wherein the sweetener is buffered with citric acid and sodium phosphate, wherein the sweetener is preserved with methylparaben and potassium sorbate, and wherein the sweetener is present in an amount of 45% to 55% by weight; and Bitterness masking agent; in: The pH of the formulation is 2.5 to 5.5; and The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof in the liquid preparation is from 20 mg/mL to 30 mg/mL. 2. A liquid formulation comprising: (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide of formula (I), pharmaceutically acceptable salts thereof, or combinations thereof: 13% to 17% by weight of hydroxypropyl-β-cyclodextrin; 0.7% to 1.5% by weight of sodium citrate dihydrate; Including sweeteners such as sucralose; and A flavoring agent present in an amount of 0.01% by weight to 0.1% by weight; in: The pH of the formulation is 2.5 to 5.5; and The concentration of the compound of formula (I), its pharmaceutically acceptable salt, or a combination thereof in the liquid preparation is from 20 mg/mL to 30 mg/mL. 3. Use of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidine-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, a pharmaceutically acceptable salt thereof, or a combination thereof, for the preparation of a liquid formulation of claim 1 or 2 for the treatment of Trk kinase-mediated cancers in patients of need. 4. The use of claim 3, wherein the cancer is associated with one or more of the overexpression, activation, amplification, and mutation of Trk kinase. 5. The use of claim 3, wherein the cancer is identified as having an imbalance in the expression or level of the NTRK gene, Trk protein, or the NTRK gene. 6. The use of claim 5, wherein the dysregulation of the NTRK gene, Trk protein, or their expression or level is a chromosomal translocation leading to the translation of the Trk fusion protein. 7. The use of claim 6, wherein the Trk fusion protein is selected from TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C20ORF112-TrkA, DNER-TrkA, ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA6-TrkA, PEAR1-T rkA, MRPL24-TrkA, MDM4-TrkA, LRRC71-TrkA, GRIPAP1-TrkA, EPS15-TrkA, DYNC2H1-TrkA, CEL-TrkA, EPHB2-TrkA, TGF-TrkA, NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, S QSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB, DAB2IP-TrkB, ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC, and TEL-TrkC. 8. The use of claim 5, wherein the dysregulation of the NTRK gene, Trk protein, or their expression or level is one or more point mutations in the NTRK gene. 9. The use of claim 8, wherein one or more point mutations in the NTRK gene result in the translation of a TrkA protein having one or more of the following amino acid substitutions: R33W, A336E, A337T, R324Q, R324W, V420M, R444Q, R444W, G517R, G517V, K538A, R649W, R649L, R682S, V683G, R702C, and C1879T. 10. The use of claim 8, wherein one or more point mutations in the NTRK gene result in the translation of a TrkB protein having one or more of the following amino acid substitutions: A13T, E142K, R136H, V619M, F663L, G639R, G709C, G709S, and G709S. 11. The use of claim 8, wherein one or more point mutations in the NTRK gene result in the translation of a TrkC protein having one or more of the following amino acid substitutions: V603M, F617L, G623R, G696C, G696A, or G696S.