WO2018107040A1 - 1-(2-fluorophényl)-n-[1-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide, ses formes solides et leurs utilisations pharmaceutiques - Google Patents

1-(2-fluorophényl)-n-[1-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide, ses formes solides et leurs utilisations pharmaceutiques Download PDF

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WO2018107040A1
WO2018107040A1 PCT/US2017/065335 US2017065335W WO2018107040A1 WO 2018107040 A1 WO2018107040 A1 WO 2018107040A1 US 2017065335 W US2017065335 W US 2017065335W WO 2018107040 A1 WO2018107040 A1 WO 2018107040A1
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compound
vlcfa
lpc
levels
theta
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John J. Court
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Vertex Pharmaceuticals Incorporated
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/38—Nitrogen atoms
    • C07D231/40—Acylated on said nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • Adrenoleukodystrophy (also known as X-linked adrenoleukodystrophy or X- adrenoleukodystrophy (X-ALD)) patients suffer from debilitating, and often fatal, neurological effects and adrenal insufficiency often associated with one or more mutations in the ATP binding cassette
  • ABCD1 plays a critical role in very long chain fatty acid (VLCFA)
  • ALD ALD-degrading-degrading-degrading
  • ALD ALD worldwide. The overall incidence of ALD is estimated to be 1 in 17,000 newborns (males and females).
  • ALD cerebral ALD
  • adrenomyeloneuropathy adrenomyeloneuropathy
  • CALD CALD is the more extreme form, which presents with rapidly progressive inflammatory
  • CALD CALD
  • ALD will develop adrenomyeloneuropathy (AMN), a slowly progressive axonopathy with first symptoms appearing around 20 to 30 years of age.
  • AMN is characterized by chronic myelopathy with progressive spastic paraparesis, sensory ataxia, sphincter dysfunction and impotence, commonly associated with
  • VLCFA level may be sufficient to prevent cerebral ALD, delay onset, and/or reduce disease severity and progression.
  • ACOX1 Acyl-CoA oxidase
  • DBP D-Bifunctional protein
  • HSCT hematopoietic stem cell transplant
  • ABCD 1 protein also known as ALD protein
  • ALD protein can lead to transport defects of VLCFA into the peroxisome due to, for example, loss of protein expression or the protein being misfunctional or non-functional.
  • Deficiency of Acyl-CoA Binding Domain Containing 5 (ACBD5), Acyl-CoA oxidase (ACOX1), or D-Bifunctional protein can lead to defects in VLCFA degradation within the peroxisome due to, for example, loss of protein expression or the protein being misfunctional or non-functional.
  • the solid state compound described herein can reduce VLCFA levels (also referred to herein as VLCFA concentration) and can be useful for treating (including reducing symptoms of, preventing the onset of, or both) ALD and other diseases, disorders, or conditions associated with accumulation of VLCFA, associated with impaired peroxisomal function (e.g., impaired transport of VLCFA into the peroxisomes or impaired degradation/metabolism of VLCFA (e.g., impaired peroxisomal oxidation within peroxisomes)), or associated with a benefit from a treatment that lowers VLCFA levels.
  • the compound provided herein can enter the central nervous system (CNS) (e.g., brain, spinal cord, or both). Therefore, in some embodiments, Compound A can reduce VLCFA levels in the CNS. In some embodiments, Compound A provided herein can reversibly reduce VLCFA levels.
  • VLCFA levels are reduced when a cell or subject is treated with Compound A herein and, when treatment with Compound A has been stopped or discontinued, the VLCFA levels return back to about the VLCFA baseline levels prior to treatment.
  • the invention relates to l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3- yl]cyclopropanecarboxamide ("Compound A”) in solid form, as described herein.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising 1- (2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide in solid form and a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • the present invention provides a method for treating a disease, disorder or condition responsive to reduction of VLCFA levels in a patient comprising administering to the patient an effective amount of l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide in solid form, further described herein.
  • the subject can be a mammal.
  • the subject can be a human.
  • the subject has ALD.
  • the present invention provides a method of treating, preventing, or ameliorating one or more symptoms of a subject with ALD, its phenotypes, or other disease, disorder or condition responsive to reduction of VLCFA levels in a subject.
  • symptoms include, but are not limited to, decreased sensitivity to stimulus (e.g., in appendages and hands), seizures, coma, death, bladder misfunction, sphincter dysfunction, misfunction of gait, ability to walk, inability to see/hear, those associated with adrenal gland insufficiency (e.g., weakness/fatigue, nausea, abdominal pain, low blood pressure), or associated with peripheral neuropathy.
  • the present invention provides a method for reduction of VLCFA levels.
  • the reduction is reversible.
  • the reduction can be achieved in a cell (e.g., the cell used in an in vitro assay; cell in vitro; or cell ex vivo), the cell of a patient, by administering to the patient, or to the cell of the patient, or to a biological sample from the patient and comprising the cell, an effective amount of Compound A described herein.
  • the reduction can be achieved in a tissue, e.g., the tissue of a patient, by administering to the patient, or to the tissue of the patient, or to a biological sample from the patient and comprising the tissue, an effective amount of Compound A described herein.
  • the tissue can be brain tissue, adrenal gland tissue, muscle tissue, nerve (e.g., peripheral nerve) tissue, adipose tissue, testes tissue, eye tissue, or liver tissue.
  • the reduction can be achieved in a biological fluid, e.g., the biological fluid of a patient, by administering to the patient, or to the biological fluid of the patient, or to a sample from the patient and comprising the biological fluid, an effective amount of Compound A described herein.
  • the biological fluid can be cerebrospinal fluid (CSF), blood, or any fraction of blood, e.g., serum, or can be from the skin (e.g., skin oil).
  • FIG. 1 shows does response in adrenoleukodystrophy (ALD) patient fibroblasts (AMN 1, CALD 1, AMN 2) and healthy human fibroblasts (Healthy 1, Healthy 2) (FIG. 1A), ALD patient B- lymphocytes (CALD 1, Heterozygous (Het) Female 1, Heterozygous (Het) Female 2) (FIG. IB), and human microglia (FIG. 1C) with administration of Compound A.
  • ALD adrenoleukodystrophy
  • the LPC level is depicted as C26:0 LPC/C16:0 LPC level, indicating that the C26:0 LPC measurement was normalized (i.e., divided by) the C16:0 LPC measurement, for example, as shown in FIG. 1A, FIG. IB, and FIG. 1C, via mass spectroscopy.
  • AMN adrenomyeloneuropathy; AMN 1 are cells from one male patient and AMN 2 are cells from a different male patient; he CALD 1 cell line from which fibroblasts in FIG.
  • Het Female 1 are cells from one heterozygous female and Het Female 2 are cells from a different heterozygous female; healthy 1 and healthy 2 are control cell lines from two human fibroblast cell lines in which the humans do not have ABCDl mutations.
  • FIG. 2 shows reduction of a VLCFA level, specifically C26:0 LPC level in vivo in blood following administration of Compound A, from ABCDl knockout (KO) mice, wild-type (WT) rats, and cynomolgus monkeys, each as further described below.
  • ABCDl KO mice received no treatment, vehicle (2% D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS)), or 1, 8, or 16 mg/kg Compound A PO QD daily for 14 days (FIG. 2A).
  • TPGS D-a-Tocopherol polyethylene glycol 1000 succinate
  • WT and ABCDl KO mice received 0.5 to 64 mg/kg Compound A PO QD and LPC levels, depicted as C26:0 LPC/C16:0 LPC level, were examined after 28 days of dosing (FIG. 2B).
  • WT rats received 2% TPGS vehicle or 30, 100, or 300 mg/kg Compound A PO QD for 7 days and LPC levels, depicted as C26:0 LPC/C16:0 LPC level, were examined (FIG. 2C).
  • Male cynomolgus monkeys received 30 mg/kg Compound A PO QD for 7 days and LPC levels, depicted as C26:0
  • LPC/C16:0 LPC level were examined (FIG. 2D).
  • FIG. 3 shows reduction of VLCFA level, specifically C24:0 LPC level and C26:0 LPC level, in the brain following administration of Compound A in adult female ABCD1 KO mice.
  • Ten mg/kg Compound A in ABCD1 KO mice induced significant reduction in brain C24:0 LPC (FIG. 3E) and in brain C26:0 LPC level (about 40% reduction for C26:0 LPC level) (FIG.
  • FIG. 3F C16:0 LPC
  • FIG. 3B C18:0 LPC
  • FIG. 3C C20:0 LPC
  • FIG. 3D C22:0 LPC
  • Data shown for C18:0, C20:0, C22:0, C24:0, and C26:0 LPCs were normalized by the C16:0 LPC signal counts.
  • P values versus ABCD1 KO vehicle controls are indicated as follows: *P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001; error bars indicate standard deviation.
  • Mice received vehicle (2% TPGS), 1 mg/kg Compound A or 10 mg/kg Compound A PO QD for 3 months.
  • Ten mg/kg Compound A induced a significant reduction in brain C24:0 SC-VLCFA level and in brain C26:0 SC-VLCFA level (about a 65% reduction in brain C26:0 VLCFA level), each after 3 months of dosing (** PO.01, **** PO.0001, respectively) (FIG.
  • FIG. 4E and FIG. 4F respectively. Levels of other VLCFA are shown for comparison (FIG. 4A: C16:0 VLCFA; FIG. 4B: C18:0 VLCFA; FIG. 4C: C20:0 VLCFA; FIG. 4D: C22:0 VLCFA).
  • FIG. 5 shows the response latency (in seconds) of male ABCD1 KO mice that received prophylactic or therapeutic dosing of Compound A in response to an infrared source on each hind paw.
  • the dashed line indicates historical WT mouse responses
  • error bars indicate standard error of the mean
  • * corresponds to Tukey's post-hoc test between groups and indicates a significant difference from vehicle treated mice during that month.
  • FIG. 6 shows a thermal ellipsoid plot of two molecules of crystalline Compound A in lattice structure Form A. Disordered components have been omitted for figure clarity.
  • FIG. 7 shows a thermal ellipsoid plot of two molecules of crystalline Compound A in lattice structure Form B. Disordered components have been omitted for figure clarity.
  • FIG. 8 shows an X-ray powder diffractogram of crystalline Compound A, taken over a range of 3°-40° 2 theta with a step size of 0.014° and a dwell time of 0.25s per step.
  • FIG. 9 shows an X-ray powder diffractogram of crystalline Compound A, taken over a range of 3°-40° 2 theta with a step size of 0.013° and a dwell time of 10.2s per step.
  • FIG. 10 shows an X-ray powder diffractogram of amorphous Compound A, taken over a range of 4.9948°-40° 2 theta with a step size of 0.0131° and a dwell time of 18.87s per step.
  • the inv ound is the inv ound
  • the crystalline solid form of the free Compound A is characterized by an X-ray powder diffraction pattern comprising one or more peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the peak positions set forth in Table 3 or Table 4 below.
  • the X-ray powder diffraction pattern comprises at least one peak position, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the X-ray powder diffraction pattern comprises at least two peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97. In other embodiments, the X-ray powder diffraction pattern comprises at least three peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the X-ray powder diffraction pattern comprises at least four peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97. In other embodiments, the X-ray powder diffraction pattern comprises at least five peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the X-ray powder diffraction pattern comprises peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • at least one of the peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta) is selected from the group consisting of 7.67, 10.27, and 19.97.
  • at least two of the peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta) are selected from the group consisting of 7.67, 10.27, and 19.97.
  • the X-ray powder diffraction pattern further comprises at least two peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 1 1.28, 12.10, 13.10, 14.36, 15.68, 16.37, 17.66, and 18.70.
  • the X-ray powder diffraction pattern further comprises at least four peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), selected from the group consisting of 1 1.28, 12.10, 13.10, 14.36, 15.68, 16.37, 17.66, and 18.70.
  • the crystalline solid form of the free Compound A comprises Form A, wherein Form A is characterized by an X-ray powder diffraction pattern comprising peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), of 7.67, 10.27, 1 1.28, 13.10, 13.65, 15.37, 15.68, 16.37, 16.84, 17.66, 18.70, and 19.97.
  • the crystalline solid form of the free Compound A comprises Form B, wherein Form B is characterized by an X-ray powder diffraction pattern comprising peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta or ⁇ 0.1 degrees 2-theta), of 7.67, 10.27, 12.10, 13.65, 14.36, 15.37, 15.68, 16.84, 17.66, 18.70, and 19.97.
  • the crystalline solid form of the free Compound A is characterized by an X-ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 8.
  • the crystalline solid form of the free Compound A is characterized by an X-ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 9.
  • the free compound l-(2-fluorophenyl)-N-[l-(2-fluoro-4- pyridyl)pyrazol-3-yl]cyclopropanecarboxamide (Compound A) is in crystalline solid form characterized by an X-ray powder diffraction pattern comprising at least one peak position, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the free compound l-(2-fluorophenyl)-N-[l-(2-fluoro-4- pyridyl)pyrazol-3-yl]cyclopropanecarboxamide (Compound A) is in crystalline solid form characterized by an X-ray powder diffraction pattern comprising at least three peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the free compound l-(2-fluorophenyl)-N-[l-(2-fluoro-4- pyridyl)pyrazol-3-yl]cyclopropanecarboxamide (Compound A) is in solid form characterized by an X-ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 8.
  • a seventh embodiment relates to a pharmaceutical composition
  • a pharmaceutical composition comprising l-(2- fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide in solid form according any of embodiments one through six, and a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • An eighth embodiment relates to a method of treating a disease, disorder or condition in a subject comprising administering to the subject an effective amount of the free compound of any one of embodiments one through six or the pharmaceutical composition of embodiment seven.
  • a ninth embodiment relates to the method set forth in the eighth embodiment wherein the disease, disorder or condition is associated with (1) one or more mutations of ABCD l transporter protein, (2) impaired peroxisomal beta-oxidation, (3) mutations of at least one of Acyl-CoA oxidase, D- Bifunctional protein, or ACBD5, or (4) accumulation of very long chain fatty acid (VLCFA) levels.
  • VLCFA very long chain fatty acid
  • a tenth embodiment relates to a method of treating ALD comprising administering to a subject an effective amount of the free compound of any one of embodiments one through six or the pharmaceutical composition of embodiment seven.
  • An eleventh embodiment relates to a method of reduction of very long chain fatty acids (VLCFA) levels in a subject comprising administering to the subject an effective amount of a free compound of any one of embodiments one through six or a pharmaceutical composition of embodiment seven.
  • VLCFA very long chain fatty acids
  • free compound refers to the non-salt form of the compound having the structure indicated by the chemical name or structure.
  • solid form when referring to Compound A means that Compound A is in the solid state.
  • crystalline refers to a solid material whose constituent particles (e.g., molecules) are arranged spatially in a regular and repeating lattice.
  • amorphous refers to a non-crystalline solid material whose constituent particles (e.g., molecules) are not arranged in a regular and repeating lattice pattern.
  • the term "similar,” when referring to two or more X-ray powder diffraction patterns, means that the patterns would be understood by a person of ordinary skill in the art to represent the same crystalline form and that the patterns are the same, except for the types of variations that would be expected by a person of ordinary skill in the art to arise from experimental variations, such as instrumentation used, time of day, humidity, season, pressure, temperature, etc.
  • VLCFA very long chain fatty acids
  • fatty acid moieties having greater than or equal to 22 carbons in the carbon chain length (e.g., at least 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbons long) of the main fatty acid side chain and can be saturated (i.e., without double- bonds; also called straight-chain) or unsaturated (e.g., monounsaturated with 1 double bond or polyunsaturated with at least 2 double bonds).
  • VLCFA refers to fatty acid moieties having greater than or equal to 24 carbons in the carbon chain length (e.g., at least 24, 25, 26, 27, 28, 29, or 30 carbons long) of the main fatty acid side chain and are saturated. In some embodiments, VLCFA refers to fatty acid moieties having 26 carbons in the carbon chain of the main fatty acid side chain and are saturated.
  • VLCFA is a straight-chain VLCFA such as lignocerotic acid, which is a C24:0 straight-chain VLCFA, and cerotic acid, which is a C26:0 straight-chain VLCFA.
  • C##:# means that there are ##-number of carbons in the carbon chain-length and that there is # instances of double-bonds in the carbon chain.
  • C26:0 means that the carbon chain of the VLCFA has 26 carbons in the carbon chain-length and zero instances of double-bonds in the carbon chain.
  • VCLFA include straight-chain VLCFA (SC-VLCFA) and VLCFA incorporation products (i.e., fatty-acid moieties that are generated from SC-VLCFA by incorporating SC- VLCFA into their structure), such as, but not limited to, lysophosphatidylcholines (LPC), sphingomyelins (SM), acyl carnitines, cholesterol esters, and ceramides.
  • LPC VLCFA are generated from straight chain VLCFA (SC-VLCFA) and are used clinically for newborn screening (Vogel et al., Mol. Genet. Metab. (2015) 1 14(4):599-603).
  • Compound A, compositions thereof, and methods of using any of the foregoing, as described further herein, are useful for reduction of VLCFA levels in the CSF, blood, skin oil, brain, adrenal gland, nerve, adipose, muscle, liver, and/or other tissues.
  • the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are unsaturated.
  • the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are saturated (also called straight-chain).
  • the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are monounsaturated.
  • the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are polyunsaturated. In some embodiments, the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are SC-VLCFA. In some embodiments, the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are VLCFA
  • the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are LPC. In some embodiments, the methods described herein are useful for reduction of a VLCFA level, wherein the VLCFA has at least 24 carbons in the chain length, at least 26 carbons, at least 28 carbons, or at least 30 carbons in the chain length. In some embodiments, the methods described herein are useful for reduction of a VLCFA level, wherein the VLCFA has 26 carbons in the chain length. In some embodiments, the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are C24:0 SC-VLCFA or C26:0 SC-VLCFA.
  • the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are C24:0 LPC or C26:0 LPC.
  • the phrase "reduction of VLCFA levels" or “reduction of a VLCFA level” means reduction of at least one or more types of VLCFA (which include VLCFA incorporation products) and optionally can be further specified in context.
  • reduction of VLCFA levels means that the levels of VLCFA in the cell or patient, following treatment with one or more chemical entities described herein, are reduced compared to the baseline levels of VLCFA before treatment with Compound A described herein.
  • the reduction of VLCFA levels means that the levels of VLCFA for cells or patients, either directly or via a sample, are reduced by at least about 25%, or at least by about 30%, or at least by about 33%, or by about 30% to about 80% relative to the baseline untreated levels after the cell or patient are treated with Compound A as described herein.
  • phrases such as deficiency of a protein means that there are mutations that lead, for example, to a loss of protein expression or to a loss of protein function, or to a loss of protein trafficking to its place of function, or to two or all of these losses.
  • the invention relates to a method of preparing a crystalline solid form of the free Compound A, comprising contacting the free Compound A with a solvent and isolating the crystalline solid form.
  • the solvent is an organic solvent, a mixture of organic solvents, or a mixture of one or more organic solvents and water.
  • the solvent is an alcoholic solvent, such as methanol, ethanol, or isopropanol.
  • the solvent is a hydrocarbon solvent, such as hexane, heptane, or cyclohexane.
  • the solvent is an organic ester solvent, such as ethyl acetate or isopropyl acetate.
  • the solvent is a mixture of an alcoholic solvent (e.g., isopropanol) and water.
  • the solvent is selected from the group consisting of methylcyclohexane, diethyl ether, acetonitrile, tetrahydrofuran, 2- methyltetrahydrofuran, methyl t-butyl ether, 1,4-dioxane, methyl ethyl ketone, dichloromethane, 1,2- dichloroethane, dimethylsulfoxide, N,N-dimethylformamide, l-methyl-2-pyrrolidinone, chlorobenzene, pyridine, nitromethane, and toluene.
  • the term "contacting,” when referring to contacting the free Compound A with a solvent includes dissolving some or all of the compound in the solvent and suspending the compound in the solvent.
  • the term "isolating,” when referring to the crystalline solid form of the free Compound A, means separating the crystalline solid form from a solvent (e.g., by filtration or decantation).
  • "isolating” comprises stirring a mixture of the free Compound A and the solvent (e.g., a solution or suspension) for a period of time (e.g., up to 24 hours, up to 2 days, or up to 4 days) and filtering the mixture to obtain the crystalline solid form.
  • "isolating” comprises evaporating the solvent to afford the crystalline solid form (e.g., evaporating the solvent under reduced pressure in a rotary evaporator).
  • the present invention also provides forms of Compound A and compositions that are useful for reduction of VLCFA levels or for treating disorders related to impaired peroxisomal function (e.g., impaired transport of VLCFA into the peroxisomes or impaired VLCFA degradation/metabolism within the peroxisomes) or accumulation of very long -chain fatty acids (VLCFA).
  • impaired peroxisomal function e.g., impaired transport of VLCFA into the peroxisomes or impaired VLCFA degradation/metabolism within the peroxisomes
  • VLCFA very long -chain fatty acids
  • compositions that comprise any of the forms of Compound A as described herein, and additionally comprise a
  • the pharmaceutically acceptable carrier, adjuvant, or excipient includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • REMINGTON THE SCIENCE AND PRACTICE OF PHARMACY, 20 th Edition, A.R. Gennaro (ed.), Lippincott Williams & Wilkins: Baltimore, MD (2000) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof.
  • any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.
  • Some examples of materials which can serve as pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- poly oxypropylene -block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; ex
  • Compound A of the invention can be formulated into pharmaceutical compositions for administration to animals or humans.
  • these pharmaceutical compositions comprise an amount of Compound A described herein effective to treat or prevent the diseases or conditions described herein and a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • the exact amount of compound required for treatment will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular agent, its mode of administration, and the like.
  • the various forms of Compound A of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of
  • compositions optionally further comprise one or more additional therapeutic agents.
  • additional therapeutic agents optionally further comprise one or more additional therapeutic agents.
  • Adrenoleukodystrophy also known as X-linked adrenoleukodystrophy or X- adrenoleukodystrophy (X-ALD)
  • ALD Adrenoleukodystrophy
  • X-ALD X-linked adrenoleukodystrophy
  • ABCD1 ATP Binding Cassette protein D l
  • VLCFA elongation occurs via the successive addition of 2 carbon atom units by ELOVL family members (Jakobsson A., et al. Prog. Lipid Res. 2006; 45 :237-249).
  • ELOVL6 elongates shorter VLCFA;
  • ELOVL7 elongates mid-range VLCFA; and
  • ELOVL 1 is primarily responsible for the synthesis of C26:0 (T. Sassa, et al. J.
  • ALD is associated with impaired peroxisomal beta-oxidation and accumulation of very long-chain fatty acids (VLCFA) in tissues and body fluids (e.g., plasma, cerebrospinal fluid (CSF)). Mutations in the ABCD1 gene impair the degradation of VLCFA by preventing their transportation into peroxisomes where they are broken down by beta-oxidation. This disruption in the VLCFA degradation process results in the accumulation of VLCFA, for example, C24:0 and C26:0, in plasma and tissues. ALD patients accumulate C26:0 (and longer carbon chain lengths) VLCFA and their incorporation products, including
  • lysophosphatidylcholines LPC
  • sphingomyelins sphingomyelins
  • acylcamitines cholesterol esters and ceramides.
  • VLCFA VLCFA
  • ABCD1 KO mice exhibit a thickening of myelin that appears to disrupt peripheral axons and leads to AMN-like symptoms.
  • A. Pujol et al., Human Molecular Genetics 2002, 1 1 : 499-505 mutations in either Acyl-CoA oxidase or D- Bifunctional protein also lead to accumulation of VLCFA and fatal demyelinating disorders, supporting the hypothesis that increased VLCFA cause the underlying pathophysiology of ALD.
  • High levels of C26:0 have been correlated with pathogenic effects.
  • C26:0 decreases the response of adrenocortical cells to adrenocorticotropic hormone stimulation.
  • a pathogenic role for C26:0 is further supported by its disruptive effects on the structure, stability and function of cell membranes (J.K. Ho et al., J. Clin. Invest. 1995, 96: 1455-1463; R.A. Knazek et al., J. Clin. Invest. 1983, 72:245-248), and by its possible contribution to oxidative stress. (S. Fourcade et al., Hum. Mol. Genet. 2008, 17: 1762-1773; J.M. Powers et al., J. Neuropathol. Exp. 2005, 64: 1067-1079).
  • Compound A is useful for treating at least one of the following diseases: ALD and its phenotypes (e.g., CALD and AMN), ACOX deficiency, DBP deficiency, ACBD5 deficiency, or Zellweger spectrum disorders (ZSDs).
  • ALD and its phenotypes e.g., CALD and AMN
  • ACOX deficiency e.g., DBP deficiency
  • ACBD5 deficiency e.g., ACBD5 deficiency
  • ZSDs Zellweger spectrum disorders
  • VLCFA are synthesized by the fatty acid elongation cycle, and the rate-limiting step is enzymatically catalyzed by the elongation of very long -chain fatty acids (ELOVL).
  • ELOVL very long -chain fatty acids
  • ELOVLl is the primary enzyme responsible for the synthesis of C22:0 to C26:0 VLCFA that are accumulated in ALD patients. (Orfman).
  • compounds that inhibit ELOVLl may be useful in suppressing the synthesis of VLCFA and therefore useful in the treatment of disorders such as ALD.
  • certain compounds described herein, such as Compound A inhibit ELOVLl, which may cause the reduction in VLCFA levels observed herein.
  • the present invention provides forms of Compound A that reduce a VLCFA level and compositions comprising Compound A in solid form, as described above.
  • the present invention provides methods and uses for treating or preventing a disease, condition, or disorder responsive to reduction in VLCFA level, which employ administering Compound A of the invention, or a pharmaceutical composition of the invention comprising Compound A. Such methods and uses typically employ administering an effective amount of Compound A or pharmaceutical composition thereof to a patient or subject.
  • the reduction in VLCFA level is reversible.
  • disease disease
  • disorder condition
  • condition may be used interchangeably herein to refer to any deviation from or interruption of the normal structure or function of any body part, organ, or system that is manifested by a characteristic set of symptoms and signs.
  • Diseases, disorders and conditions of particular interest in the context of the present invention are those responsive to reduction of VLCFA level.
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “patient” refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), particularly a mammal including non-primates (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, or mouse) and primates (e.g., a monkey, chimpanzee or human), and more particularly a human.
  • non-primates e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, or mouse
  • primates e.g., a monkey, chimpanzee or human
  • the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In some embodiments, the subject is a human.
  • a farm animal e.g., a horse, cow, pig or sheep
  • a pet e.g., a dog, cat, guinea pig or rabbit.
  • the subject is a human.
  • an "effective amount” refers to an amount sufficient to elicit the desired biological response.
  • certain examples of the desired biological response is to treat or prevent a disease, condition or disorder responsive to reduction in VLCFA level, or to enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against a disease, condition or disorder responsive to reduction in VLCFA level.
  • the precise amount of compound administered to a subject will depend on the mode of administration, the type and severity of the disease, condition, or disorder and on the characteristics of the patient, such as general health, age, sex, body weight and tolerance to drugs. Persons skilled in the art will be able to determine appropriate dosages depending on these and other factors.
  • an "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the person skilled in the art according to the condition of the patient, the type of condition(s) being treated and the amount of a compound described herein being used.
  • chemical entities described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.
  • the chemical entities and compositions, according to the methods of the present invention may be administered using any amount and any route of administration effective for eliciting the desired biological response.
  • the terms “treat,” “treatment” and “treating” can refer to both therapeutic and prophylactic treatments.
  • therapeutic treatments include the reduction, amelioration, slowing or arrest of the progression, severity and/or duration of one or more conditions, diseases or disorders and/or of one or more symptoms (specifically, one or more discernible symptoms) thereof, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as Compound A or composition of the invention).
  • treatment refers to reduction or amelioration of the progression, severity and/or duration of one or more conditions, diseases or disorders, resulting from the administration of one or more therapies.
  • treatment refers to reduction or amelioration of the severity and/or duration of one or more conditions, diseases or disorders, resulting from the administration of one or more therapies. In some embodiments, treatment refers to reduction or amelioration of the progression, severity and/or duration of one or more symptoms (specifically, one or more discernible symptoms) of one or more conditions, diseases or disorders, resulting from the administration of one or more therapies. In some embodiments, treatment refers to reduction or amelioration of the severity and/or duration of one or more symptoms (specifically, one or more discernible symptoms) of one or more conditions, diseases or disorders, resulting from the administration of one or more therapies.
  • Prophylactic treatments include prevention or delay of the onset of one or more conditions, diseases or disorders and/or of one or more symptoms (specifically, one or more discernible symptoms) thereof, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as Compound A or composition of the invention).
  • treatment refers to prevention or delay of the onset of one or more conditions, diseases or disorders resulting from the administration of one or more therapies.
  • treatment refers to prevention or delay of the onset of one or more symptoms (specifically, one or more discernible symptoms) of one or more conditions, diseases or disorders resulting from the administration of one or more therapies.
  • the invention provides co-administering to a patient an additional therapeutic agent, wherein said additional therapeutic agent is appropriate for the disease, condition or disorder being treated; and said additional therapeutic agent is administered together with Compound A of the invention as a single dosage form, or separately from said compound as part of a multiple dosage form.
  • therapies e.g., prophylactic and/or therapeutic agents
  • the use of the terms does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a patient, nor does it require administration in any specific proximity in time, so long as in the judgment of a suitable physician the patient is understood to be receiving the one or more therapies at the same time. For example, receiving therapy A on days 1-5 of a 28-day schedule and therapy B on days 1, 8 and 15 of a 21-day schedule would be considered “in combination" or a "co-administration".
  • Co-administration also encompasses administration of the first and second amounts of the compounds of the co-administration in an essentially simultaneous manner, such as in a single pharmaceutical composition, for example, capsule or tablet having a fixed ratio of first and second amounts, or in multiple, separate capsules or tablets for each.
  • co-administration also encompasses use of each compound in a sequential manner in either order.
  • Therapies which may be used in combination with the chemical entities of the present invention include Lorenzo's Oil (4: 1 glycerol trioleate and glyceryl trierucate), allogenic hematopoietic stem cell transplant, autologous hematopoietic stem cell transplant, corticosteroid replacement therapy and CNS gene replacement therapy.
  • compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray or via inhalation, or the like, depending on the identity and/or severity of the disease being treated.
  • the chemical entities of the invention may be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 50 mg/kg, , of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), derivatized/modified beta-cyclodextrin, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, sodium lauryl sulfate, d-a-to
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • a compound of the present invention In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide.
  • the rate of compound release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or
  • microemulsions that are compatible with body tissues.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert,
  • excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents (or disintegrant) such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as tal
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active compounds can also be in microencapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • buffering agents include polymeric substances and waxes.
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention.
  • the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body.
  • Such dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavouring or colouring agents may also be added.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suppositories can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
  • the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
  • the pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • compositions should be formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of Compound A can be administered to a patient receiving these compositions.
  • Compound A will also depend upon the particular compound in the composition.
  • additional drugs which are normally administered to treat or prevent that condition, may be administered together with
  • Compound A or a pharmaceutical composition thereof.
  • Those additional agents may be administered separately, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together Compound A in a single composition.
  • compositions thereof are also useful in biological samples.
  • the invention relates to a reduction in VLCFA level in a biological sample, which method comprises contacting said biological sample with Compound A or a composition thereof comprising.
  • biological sample means an in vitro or an ex vivo sample, including cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof Enumerated Embodiments
  • the X-ray powder diffraction pattern comprises at least five peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), selected from the group consisting of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • the X-ray powder diffraction pattern comprises peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), of 7.67, 10.27, 13.65, 15.37, 16.84, and 19.97.
  • Form A is characterized by an X-ray powder diffraction pattern comprising peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), of 7.67, 10.27, 11.28, 13.10, 13.65, 15.37, 15.68, 16.37, 16.84, 17.66, 18.70, and 19.97.
  • Form B is characterized by an X-ray powder diffraction pattern comprising peak positions, in degrees 2-theta ( ⁇ 0.2 degrees 2-theta), of 7.67, 10.27, 12.10, 13.65, 14.36, 15.37, 15.68, 16.84, 17.66, 18.70, and 19.97.
  • a pharmaceutical composition comprising a chemical entity of any one of embodiments 1-186 and a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • a method of treating a disease, disorder or condition in a subject comprising administering to the subject an effective amount of the free compound of any one of embodiments 1- 19 or the pharmaceutical composition of embodiment 20.
  • VLCFA very long chain fatty acid
  • a method of reduction of very long chain fatty acids (VLCFA) levels in a subject comprising administering to the subject an effective amount of the free compound of any one of embodiments 1-19 or the pharmaceutical composition of embodiment 20.
  • VLCFA very long chain fatty acids
  • VLCFA very long chain fatty acids
  • a method of reduction of a very long chain fatty acids (VLCFA) level in a cell comprising administering to the cell an effective amount of the free compound of any one of embodiments 1- 19 or the pharmaceutical composition of embodiment 20.
  • VLCFA very long chain fatty acids
  • a method of reduction of a very long chain fatty acids (VLCFA) level in the brain of a subject comprising administering systemically to the subject an effective amount of a free compound that penetrates the blood-brain-barrier to provide reduction in the VLCFA level in the brain of the subject.
  • VLCFA very long chain fatty acids
  • VLCFA is VLCFA comprising at least 24 carbons.
  • administering systemically to the subject comprises administering via oral administration, intravenous injection, or subcutaneous injection to the subject.
  • administering systemically to the subject comprises administering via oral administration to the subject.
  • Example 1 Chemical synthesis of 1 -(2-fluorophenyl)-N-(l-(2 -fluoropyridin-4-yl)- 1H- pyrazol-3-yl)cyclopropane-l-carboxamide (Compound A)
  • Step 1 2-fluoro-4-(3-nitro-lH-pyrazol-l-yl)pyridine
  • the off-white solid was collected by vacuum filtration.
  • the solid was re-suspended in water (2 L) and filtered, and this step was repeated once further.
  • the product was dried under vacuum, then suspended in heptane (4L), stirred 3 h at room temperature, and filtered.
  • the solid was washed with two further portions of heptane (2 L each) and dried under vacuum to provide 2-fluoro-4-(3-nitro-lH-pyrazol- l-yl)pyridine (426.3 g of 92% purity, 87% yield).
  • Step 2 l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine
  • Step 1 2-fluoro-4-(3-nitro-lH-pyrazol-l-yl)pyridine
  • a reactor was charged with 3-nitro-lH-pyrazole (300 g, 2.67 mol, limiting reagent).
  • 2-fluoro-4-(3-nitro-lH-pyrazol- l-yl)pyridine was separated from 2,4-bis(3-nitro-lH-pyrazol- l-yl)pyridine (formed as a side product) by recrystallization.
  • a reactor was charged with crude 2-fluoro- 4-(3-nitro-lH-pyrazol- l-yl)pyridine (944.1 g), dichloromethane (8.5 L, 9 vol.), and methanol ( 19.8 L, 21 vol.), and the agitation was set to 150 rpm.
  • the slurry was stirred at 39 °C for about 4 h, and then the jacket temperature was ramped down to 20 °C, and stirring was continued for 30 minutes.
  • Step 2 l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine
  • the batch was cooled to 30 °C and filtered over a Celite pad to remove the catalyst.
  • the filter cake was washed with 2: 1 tetrahydrofuran methanol (1.76 L, 2 vol.), the tetrahydrofuran/methanol mother liquors were stripped to dry solid, and two chases of isopropyl alcohol (each 5 volumes) were performed to remove as much tetrahydrofuran as possible.
  • the solids were then taken up in 8 volumes of isopropyl alcohol (6.5 L) and heated to 80°C. Once temperature was reached, 4 volumes of water (3.2 L) were added over 1 hour to afford a clear, yellow solution.
  • the solution was cooled to 70°C and was seeded with crystals of l-(2-fluoropyridin-4-yl)-lH-pyrazol-3 -amine (0.05 wt%, 4 g). Crystals were allowed to grow as the batch was cooled from 70 °C to 60 °C over 1 hour, and then another 12 volumes of water (9.7 L) were added over two hours. Once the water addition was complete, the batch was cooled from 60 °C to 20 °C over 5 hours and was then filtered and washed with 2 volumes of 2: 1 water: isopropyl alcohol (2.4 mL). The solids were dried in an oven at 45 °C with a nitrogen sweep until a constant weight was obtained. l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine was obtained in 88% yield.
  • Example 1.2 Chemical synthesis of l-(2-fluorophenyl)-N-(l-(2-fluoropyridin-4-yl)-lH- pyrazol-3-yl)cyclopropane-l-carboxamide (Compound A)
  • Step 1 To a solution/suspension of l-(2-fluorophenyl)cyclopropane-l-carboxylic acid (266 g, 1.46 mol, 1.3 eq) in thionyl chloride (SOCh; 295 mL, 4.04 mol, 3.6 eq) at room temperature was added DMF (800 ⁇ , 10.33 mmol, 0.01 eq). The resultant solution was stirred 1 hour (h) at room temperature and 3 h at 30 °C. The solvent was removed in vacuo, and excess thionyl chloride and HCl were removed by azeotrope with toluene (100 mL).
  • Step 2 To a 0 °C suspension of l-(2-fluoropyridin-4-yl)-lH-pyrazol-3 -amine (200 g, 1.12 mol, 1.0 eq) and triethylamine (Et3N; 391 mL, 2.81 mol, 2.5 eq) in THF (1.6 L) was added l-(2- fluorophenyl)cyclopropanecarbonyl chloride (290 g, 1.46 mol, 1.3 eq) slowly over 1 h so as to maintain the reaction temperature below 8 °C. The reaction mixture was stirred a further for 1 h in the ice-bath then warmed to room temperature for approximately 16 h.
  • Et3N triethylamine
  • Example 1.2A Chemical synthesis of l-(2-fluorophenyl)-N-(l-(2-fluoropyridin-4-yl)- lH- pyrazol-3-yl)cyclopropane-l-carboxamide (Compound A) (alternate synthesis)
  • Step 1 A reactor was charged with l-(2-fluorophenyl)cyclopropane- l-carboxylic acid (1750.6 g, 9.72 mol, limiting reagent), and toluene (3.5 L, 2 vol) was added. Thionyl chloride (1417 mL, 19.43 mol, 2 eq) was added to reactor, and the reaction was heated to 35-40 °C. Upon completion of the reaction, toluene (7 L, 4 vol) was added to the reactor, and the reaction mixture was distilled to dryness to obtain l-(2-fluorophenyl)cyclopropanecarbonyl chloride in 98% yield as a yellow oil .
  • Step 2 A reactor was charged with l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine (1499.9 g, 8.42 mol, limiting reagent) and tetrahydrofuran ( 15 L, 10 vol). Triethylamine (2.35 L, 16.84 mol, 2 eq) was added at 13 °C. A solution of l-(2-fluorophenyl)cyclopropanecarbonyl chloride (1672.4 g, 8.42 mol, 1.0 eq) in tetrahydrofuran (3.0 L, 2 vol) was added to the reactor, while maintaining a temperature of 13 - 18 °C.
  • X-ray Powder Diffraction Crystalline Compound A, prepared according to Example 1.2 (above), was analyzed by X-ray powder diffraction (XRPD) analysis. XRPD measurements were performed using a Bruker D8 advance diffractometer at room temperature with copper radiation (1.54060 A). The X-ray generator was operating at a voltage of 40 kV and a current of 40 mA. The powder sample was placed in a silicon or PMM plastic holder. The data were recorded in a theta-theta scanning mode over the range of 3°-40° 2 theta with a step size of 0.014° and a dwell time of 0.25s per step. The measured XRPD pattern is shown in FIG. 8. The XRPD peak positions and D spacings are listed in Table 3. The form(s) corresponding to each peak were determined by comparison to simulated XRPD patterns generated from the single crystal X-ray diffraction analysis.
  • the data were recorded in a theta-theta scanning mode over the range of 3°-40° 2 theta with a step size of 0.013° and a dwell time of 10.2s per step.
  • the XRPD pattern of the sample retrieved at 2 days is shown in FIG. 9.
  • the XRPD and D spacings are listed in Table 4. XRPD analysis of the sample retrieved at 2 weeks yielded similar results.
  • HEK293 cells are treated with Compound A using the representative manual protocols described below.
  • the protocols below are also adapted to a semi-automated protocol using standard methods in the art.
  • HEK293 cells are maintained in FreeStyle F 17 media (Gibco # A13835) supplemented with PenStrep (1%, Gibco # 15070-063), Glutamax (2%, Gibco # 35050-061), and Pluronic (0.1%, Gibco # 24040-032) ("supplemented media"). Suspension cultures are grown in disposable Erlenmeyer flasks at about 120 rpm, 37 °C, 5% CO2, and 80% humidity. Cell densities are kept between about 0.5 and 3 million cells per mL, in about 50 - 200 mL per flask.
  • Treatment of cells with compounds provided herein The cells are treated with Compound A using either a total of 900uL cell media volume (high-volume assay) or a total of 200uL cell media volume (low -volume assay).
  • high-volume assay 450 of supplemented media plus 13C-acetate (1.0 mg/mL, Sigma Aldrich # 282014) are added to 0.5 of Compound A in DMSO in a polypropylene v-bottom plate (Costar #3363) in 1 of 3 dilution schemes.
  • the contents of each well are mixed and transferred to a sterile polypropylene deep-well v-bottom plate (Costar #3960).
  • 450 of cultured HEK293 cells in supplemented media at a density of 1.0 million cells/mL is added to each well.
  • 100 of supplemented media plus 13C-acetate ( 1.0 mg/mL, Sigma Aldrich # 282014) are added to either 0.1 ⁇ or 1.0 uL of Compound A in DMSO in a polypropylene v-bottom plate (Costar #3363) in 1 of 3 dilution schemes.
  • 100 ⁇ of cultured HEK293 cells in supplemented media at a density of 1.0 million cells/mL is added to each well.
  • the high volume and low volume plates are sealed with either AirPore Tape Sheets (Qiagen # 19571) or Duetz plate covers to control evaporation and placed into a shaking incubator at 225 rpm, 37 °C, 5% C02, and 80% humidity for 48 hours.
  • AirPore Tape Sheets Qiagen # 19571
  • Duetz plate covers to control evaporation and placed into a shaking incubator at 225 rpm, 37 °C, 5% C02, and 80% humidity for 48 hours.
  • the 3 dilution schemes used are as follows:
  • treated cells are harvested by centrifugation at 1690xg for 10 minutes.
  • 200 uL treated cells are transferred to a polypropylene v-bottom plate (Costar #3363) prior to centrifugation.
  • the incubation plate is centrifuged directly, without a transfer step. The supernatant is then discarded and the analytes are extracted using 1 of 2 different extraction schemes.
  • the cell pellet is visibly broken up by mixing the cell pellet up and down in 100 ⁇ of hexane/isopropanol (60:40) 20 times.
  • the resulting mixture is transferred to a 0.45 ⁇ Durapore membrane (Millipore #MSH VN4510) atop a polypropylene v-bottom plate (Costar #3363) and filtered by centrifugation at 1690xg for 5 minutes.
  • 120 ⁇ of n-butanol containing 10 nM C 13 :0 lysophosphatidylcholine is added to the filtrate as an injection control standard, then the entire volume is transferred to a new Durapore membrane / v-bottom plate.
  • the cell pellet is visibly broken up by mixing the cell pellet up and down in 180 ⁇ of methanol containing 10 nM C 13:0 lysophosphatidylcholine 20 times.
  • peak areas for the 13C-labeled C26:0 are normalized to the median signal of the lowest tested concentration (negative control).
  • IC50 values for a set of control compounds are found to be within acceptable variance regardless of the assay volume, extraction scheme, or dilution scheme utilized.
  • the average IC50 HEK293 IC50 of Compound A was 0.013 ⁇ .
  • Example 2.2 Reduction in C26:0 LPC concentration in human HEK and patient cells in vitro
  • Lysophosphatidylcholine (LPC) VLCFA were generated from straight chain VLCFA (SC- VLCFA) and were used clinically for newborn screening (Vogel et al., Mol. Genet. Metab. (2015) 114(4):599-603).
  • LPC VLCFA level measured as LPC synthesis
  • human HEK cells 2, patient derived cells, and 3) human microglia, which are disease relevant CNS cells.
  • Compound A's dose response relationships and IC50 values were measured in HEK cells, primary patient fibroblasts, immortalized patient lymphocytes, and a human microglial cell line.
  • 13C Labeled sodium acetate Sigma Aldrich # 282014
  • HEK293 cells HEK293 cell culture protocol and treatment with compound, such as Compound A, was described in example 2.1.
  • Human microglia Immortalized human microglia (Applied Biological Materials (ABM); catalog # T0251; Richmond BC, Canada) were grown and sub-cultured following the subculturing protocols from ABM except DMEM (high glucose, pyruvate; LifeTech Cat. No. 11995) was used instead of Prigrow III medium and standard tissue culture grade flasks and plates were used. Microglia cells were grown to about 80% confluence and the media was aspirated and washed once with DPBS. TryplE (or trypsin) was added and incubated for about 5 min until the cells detached. An equal volume of media was used to neutralize the detachment media and the cells were collected and counted. The cells were spun down at 1000 rpm for 5 min and brought back up in complete media and plated as required at the desired density the day before treatment.
  • DMEM high glucose, pyruvate; LifeTech Cat. No. 11995
  • Cell assays for microglia cells were run in 12 well tissue culture treated plates. Assays run in 12 well plates were done either in 900 or 1000 ul of media plus Compound A, which was added to 12 well plated by changing the media with media containing 1 mg/ml 13 C-Sodium acetate. Cells were treated with Compound A for about 2 days at a dose of 2uM, along with a 2-fold dilution scheme across 11 points to generate a 12 points IC50 curve. After about 2 days compound treatment, the cells were harvested.
  • the media (with compound treatment) was aspirated from the well. About 1-2 ml of DPBS was added to wash the cells. 100 ul of TryplE was added to the cells and allowed to incubate at room temperature or 37°C for 5 min. The cells were scraped and transferred to a polypropylene V-bottomed 96 well plate. Each well was then washed with another 100 ul of DPBS, scraped and transferred again to the same polypropylene V-bottomed 96 well plate. The polypropylene plate was then centrifuged at 3000 rpm for 10 minutes. The supernatant was then removed. The plate was sealed with a plate tape and put at -80°C for further VLCFA extraction and VLCFA quantitation on LC-MS, as described below.
  • B-Lymphocytes Immortalized primary patient lymphocytes cell lines (cell lines GM 13496, GM 13497, and GM04674) were obtained from the Coriell Cell Repository at the Coriell Institute for Medical Research. Lymphocytes were cultured and plated at a desired cell density, such as lxlO 5 cells/well. Media used was RPMI + 2 mM Glutamine or Glutamax + 15% FBS (not heat inactivated). Assays were completed similar to the protocol described for microglia cells except that round bottom 96 well plates were used and the assays were performed in 200 ul of complete media with 1 mg/ml 13C- sodium acetate.
  • Lymphocytes were treated with Compound A for about two days at the following doses: 2, 0.964, 0.464, 0.224, 0.108, 0.0519, 0.025, 0.0121, 0.0058, 0.0028, 0.00135, and 0.00065 ⁇ .
  • lymphocytes were harvested by spinning down at 3000 rpm for 10 min and removing the supernatant. The plate was sealed with a plate tape and put at -80°C for further VLCFA extraction and VLCFA quantitation on LC-MS, as described below.
  • Patient fibroblasts Primary patient fibroblasts were obtained from Coriell Institute for Medical Research. Fibroblasts were cultured by passing the cells at about 95% confluency (nearly 100%), aspirating the media, washing the plate with DPBS, adding TryplE (preferred) or trypsin to dislodge the cells and leave at 37 ° C for 5-10 min, collecting cells with at least as much volume as TryplE used to neutralize the trypsin, count the cells and calculating cell density. Fibroblasts were plated at a desired cell density, such as 1.9xl0 5 cell/well, in 12 well plates the day before dosing with Compound A.
  • a desired cell density such as 1.9xl0 5 cell/well
  • 13C-acetate 1.0 mg/mL, Sigma Aldrich # 282014
  • Compound A were diluted in media and simultaneously added to a 50% confluent fibroblast culture in 12 well plates, following removal of the growth media. The cells were incubated at 37°C, 5% CO2, and 80% humidity for 48 hours with
  • the cells were harvested similarly to the protocol described for microglia. The plate was sealed with a plate tape and put at -80°C for further VLCFA extraction and VLCFA quantitation on LC-MS, as described below.
  • VLCFA extraction and quantitation on LCMS Treated cells were transferred to a polypropylene v-bottom plate and then centrifuged at 1690xg for 10 minutes. The supernatant was discarded and the cell pellet was disrupted by trituration in 100 uL of hexane (60%) / isopropanol (40%). The resulting mixture was transferred to a 0.45um Durapore membrane (Millipore #MSH VN4510) atop a polypropylene v-bottom plate and filtered by centrifugation at 1690xg for 5 minutes.
  • Millipore #MSH VN4510 0.45um Durapore membrane
  • lysophosphatidylcholine indicated fatty acid elongation. Specifically, C16:0, C18:0, C20:0, C22:0, C24:0, and C26:0 LPC levels were measured via mass spectroscopy as described above and IC50 values indicated half maximal reduction in C26:0 LPC levels.
  • Results: C26:0 LPC levels normalized by C16:0 LPC are shown in FIG. 1A, FIG. IB, and FIG. 1C.
  • Compound A lowered LPC C26:0 levels in human HEK293, patient fibroblasts (CALD1, AMN1, AMN2), patient-derived lymphocytes (CALD, Het Female 1, Het Female 2), and human microglia (see FIG. 1A, FIG. IB, and FIG. 1C, and Table 5 below).
  • Compound A reduced C26:0 LPC synthesis in HEK cells, yielding an IC50 of 8 nM.
  • the potency of Compound A for ALD patient fibroblasts, lymphocytes, and microglia was similar to the potency for HEK cells.
  • ALD adrenoleukodystrophy
  • AMN adrenomyeloneuropathy
  • CALD cerebral obstructive pulmonary disease
  • IC50 values indicate half maximal reduction in C26:0 LPC. Each number indicates a separate measurement.
  • Example 2.3 Reduction of plasma C26:0 LPC in vivo in a mouse model, wild-type rats, and wild-type monkeys.
  • Bioanalysis of LPC in whole blood and brain tissue A LC-MS/MS method of analyzing Lysophosphatidylcholine (LPC) in whole blood (dried blood spot card, DBS) and brain tissue samples was developed for measuring the abundance of saturated C16, C18, C20, C22, C24 and C26 LPC in DBS and brain samples.
  • Whole blood was collected with Whatman DMPK-C DBS card at an approximate volume of 20 at each time point. Brain tissue was collected at the end point of the study. Samples were prepared and LC-MS/MS analysis was performed as described below.
  • Sample preparation for LPC bioanalysis For DBS bioanalysis, the DBS card was punched at 3 mm in diameter using a semi -automated DBS card puncher. To each punched spot 200 of pure methanol was added. The vial was vortexed at low speed for 20 minutes and centrifuged at 4000 rpm for 20 minutes. The clear supernatant was injected onto LC-MS/MS for analysis.
  • brain tissue bioanalysis brain tissue was collected in a tared homogenization tube pre-filled with metal bead and weighted. To each sample vial two parts weight of methanol was added. The sample was homogenized using Precellys-24 at 5000 rpm for 20 seconds with one cycle.
  • LC-MS/MS Analysis The supernatant obtained from each sample was injected into a LC- MS/MS system (Agilent Technologies, Santa Clara, CA and Applied Biosystems, Framingham, MA) for analysis. All six LPC components (C16:0, C18:0, C20:0, C22:0, C24:0 and C26:0) were injected into a LC- MS/MS system (Agilent Technologies, Santa Clara, CA and Applied Biosystems, Framingham, MA) for analysis. All six LPC components (C16:0, C18:0, C20:0, C22:0, C24:0 and C26:0) were
  • Ions of Ql were monitored at m/z of 496.6, 524.6, 552.6, 580.6, 608.6 and 636.6 for LPC 16:0, LPC 18:0, LPC 20:0, LPC 22:0, LPC 24:0 and LPC 26:0, respectively.
  • a common Q3 ion m/z of 184.2 was used for all LPC analyses.
  • C16:0LPC levels were expressed as a concentration. All other LPC levels were expressed relative to C16.
  • a one-way ANOVA with Dunnett's multiple comparisons test was performed to assess differences in LPC levels among the different groups. A value of ⁇ 0.05 was considered statistically significant. All statistical analyses were conducted using Prism Software version 7.01 (GraphPad, La Jolla, CA).
  • the vehicle used was 2% D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS) and Compound A doses were prepared in 2% TPGS.
  • ABCD1 KO mice showed 5 -fold higher blood C26:0 LPC levels than WT mice, consistent with the elevations seen in human ALD patients (Van debeek 2016).
  • Interperitoneal dosing, at 2 or 20 mg/kg (data not shown) or oral (PO) dosing at 1, 8, or 16 mg/kg (FIG. 2A) yielded similar results.
  • a dose response was observed between 1 and 8 mg/kg.
  • Plasma C26:0 LPC levels dropped over the first 8 days before plateauing at near WT baseline levels.
  • LPC/vehicle LPC levels C26:0 LPC levels were normalized to CI 6:0 LPC levels and vehicle controls
  • ABCD1 knockout mice without treatment, vehicle, 1, 8, or 16 mg/kg Compound A PO QD daily for 14 days. Error bars indicate standard deviation.
  • WT mice treated with Compound A also showed a reduction in VLCFA levels following Compound A treatment.
  • the maximal effect plateau in WT mice was reached between the 2 mg/kg and 16 mg/kg doses, and resulted in about a 65% reduction in C26:0 LPC levels to below baseline levels.
  • P value versus ABCD1 KO vehicle controls was 0.0001 at 0.5 mg/kg and higher doses (P ⁇ 0.0001); error bars indicated standard deviation.
  • the vehicle used was 2% TPGS and Compound A doses were prepared in 2% TPGS.
  • Dried Blood Spot (DSB) samples were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 hours post dose on Day 1 and Day 7, respectively. In addition, DSB samples were collected for all animals prior to dosing on study Days 3, 4 and 6. DBS cards were stored at 4°C until they could be analyzed for VLCFAs. DBS samples were prepared and analyzed using LC-MS/MS as described above.
  • Reversible reducing effect on LPC level The C26:0 LPC reducing effect of Compound A was found to be reversible.
  • vehicle 1 or 8 mg/kg of Compound A PO (orally) QD (once per day) for 14 days (i.e., day 7 through day 21)
  • treatments with Compound A and vehicle were discontinued and blood LPC levels were assessed for another 2 weeks.
  • the vehicle used was 2% TPGS and Compound A doses were prepared in 2% TPGS.
  • DBS cards were stored at 4°C until they could be analyzed for lysophosphatidyl cholines (LPCs).
  • DBS samples were prepared and analyzed using LC-MS/MS as described above. Since this study is longitudinal (multiple time points), a two-way ANOVA was performed to assess differences in LPC levels among the different groups. A value of P ⁇ 0.05 was considered statistically significant. All statistical analyses were conducted using Prism Software version 7.01. LPC levels returned to baseline levels in approximately 1 week after compound discontinuation, mirroring the kinetics observed following Compound A initiation (FIG. 2F).
  • Example 2.4 Reduction of C26:0 LPC and SC-VLCFA levels in wild-type and ABCD1 KO brains.
  • VLCFA LPC, SC-VLCFA, acyl-carnitines
  • LCMS liquid chromatography-mass spectrometry
  • VLCFA including straight chain very long chain fatty acids (SC-VLCFA), acyl carnitines, and lysophosphatidylcholines (LPC), in the brain were examined.
  • SC-VLCFA were expected to be rapidly incorporated into other forms and acyl carnitines were expected to be rapidly degraded, contributing to a short expected half-life for these forms.
  • LPC was expected to integrate into membranes, contributing to a longer expected half-life.
  • Brain C26:0 LPC levels in ABCD1 KO mouse were approximately 8 fold higher than in WT mice. There were no changes in LPC levels at either dose after 2 weeks of dosing (not shown).
  • Brain sample preparation (i) 3 volumes of MeOH was add to each sample; (ii) homogenized tissue samples with FastPrep (FP120) at 4.5 intensity for 25 seconds; and (iii) aliquoted tissue lysates.
  • FP120 FastPrep
  • VLCFA e.g., spingomyelin (SM) and LPC and derivatized VLCFA (FA-DMAE)
  • Phase A 50%MeOH/5mM AF; Phase B: 2-propanol
  • Phase A H2O/0.1%FA
  • Phase B ACN/0.1%FA
  • ABCD1 KO mice were used as a functional model of AMN. ABCD1 KO mice display a progressive loss of sensitivity to painful thermal stimulus similar to symptoms observed in AMN patients such as decreased sensitivity to touch. To determine the effect of Compound A on thermal sensitivity, Compound A was dosed PO QD either prophylactically or therapeutically to determine whether ABCD1 KO mice have different latency thresholds for the Plantar test (Hargreaves apparatus) response compared to wild-type (WT) mice. [00165] For the prophylactic study, mice were tested beginning at 10 months of age (before the loss of pain sensitivity) using doses of either 5 or 20 mg/kg.
  • mice were tested beginning at 18 months of age, after there was already a significant loss of pain sensitivity, using doses of either 32 or 64 mg/kg. Mice did not have a significant drop in body weight or any other noticeable adverse effect during Compound A treatment in either experiment.
  • the Plantar test (using a Hargreaves apparatus) was used and measured the latency to respond to a thermal stimulus using the following protocol. An individual mouse was placed into an individual compartment with a glass floor for about 10-15 minutes until they were settled. Each individual mouse was given three trials with an infrared source on each hind paw (alternated hind paws each time, and waited 5 minutes between each trial). The infrared source was placed under the glass floor and was positioned by the operator directly beneath the hind paw. A trial was commenced by depressing a key/button which turned on the infrared source and started a digital timer. When a response was observed (paw withdrawal), the key/button was released and the latency to respond was recorded (in seconds).
  • Compound A treated mice developed smaller deficits than vehicle treated mice. Dosing was initiated at 10 months of age, before the mice show deficits in thermal sensitivity.
  • mice dosed with Compound A exhibited lower latencies than vehicle treated mice, indicating a restoration or preservation of thermal pain sensitivity and slowing of disease progression.
  • Two-way ANOVA revealed a significant effect of time (p ⁇ 0.0001), treatment (p ⁇ 0.0001) and an interaction (p ⁇ 0.0001).
  • mice showed statistically significant improvements relative to their 18 month baseline scores.
  • Two-way ANOVA revealed a significant effect of time (p ⁇ 0.0001), treatment
  • Cryopreserved human hepatocytes (Lot Hue50c), monkey hepatocytes (cynomolgus; Lot Cy328), dog hepatocytes (beagle, Lot Db235), rat hepatocytes (Sprague Dawley; NNH), and mouse hepatocytes (CD-I; Lot Mc522) were obtained from ThermoFisher (Paisley, UK).
  • Compound A (1 ⁇ ) was incubated with hepatocytes from each species (0.5 million cells/mL, suspension) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4- (2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES, 9 mM) and fructose (2.2 mM) (pH

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Abstract

L'invention concerne un composé pyrazole à substitution en position 1 et 3 sous forme solide, des compositions pharmaceutiques de celui-ci, utile pour la réduction des niveaux d'acides gras à chaîne très longue et pour le traitement de diverses maladies, troubles et états pathologiques, tels que l'adrénoleucodystrophie (ALD).
PCT/US2017/065335 2016-12-09 2017-12-08 1-(2-fluorophényl)-n-[1-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopropanecarboxamide, ses formes solides et leurs utilisations pharmaceutiques WO2018107040A1 (fr)

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