ITS SOLID FORMS AND PHARMACEUTICAL USES THEREOF

BACKGROUND

[0001] Adrenoleukodystrophy (ALD) (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

transporter D 1 (ABCD1) gene. ABCD1 plays a critical role in very long chain fatty acid (VLCFA)

degradation and, as such, ALD patients typically have elevated VLCFA levels that are thought to be causative of the pathology in ALD. The prevalence of ALD is 1 in 20,000 to 50,000 individuals

worldwide. The overall incidence of ALD is estimated to be 1 in 17,000 newborns (males and females).

In males there are two predominant phenotypes: cerebral ALD (CALD) and adrenomyeloneuropathy

(AMN). CALD is the more extreme form, which presents with rapidly progressive inflammatory

demyelination of the brain, leading to rapid cognitive and neurological decline. If untreated, CALD

patients die within approximately 2 years of symptom onset. Over the course of their lifetime,

approximately 60% of males with ALD will develop CALD, most frequently between the ages of about 3 and about 12 (35 to 40%), with continued (albeit decreasing) risk during adulthood. Adult males with

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

primary adrenocortical and/or testicular insufficiency. Approximately 7,000 to 10,000 males in the US and EU combined will develop AMN. Women with ALD are also affected and not merely carriers: >80% of these individuals develop signs and symptoms of myelopathy by the age of 60 years. Approximately 12,000 to 15,000 women in the US and EU combined will eventually develop AMN. Female ABCD1 heterozygotes exhibit approximately half the plasma VLCFA elevation observed in males, never develop the cerebral form of the disease, and develop more modest, but debilitating, AMN-like symptoms later in life. Therefore, about a 50% to about a 75% reduction in VLCFA levels relative to a patient's baseline

VLCFA level may be sufficient to prevent cerebral ALD, delay onset, and/or reduce disease severity and progression.

[0002] Mutations in any of three separate genes in the VLCFA degradation pathway have been

associated with VLCFA accumulation and demyelinating diseases in humans. In addition to mutations in ABCD1, mutations in Acyl-CoA oxidase (ACOX1) or D-Bifunctional protein (DBP) also are associated with accumulation of VLCFA and demyelinating disorders, supporting the hypothesis that increased VLCFA cause the underlying pathophysiology of ALD.

[0003] There are few treatment options available for ALD patients and their families. One treatment for CALD is an allogenic hematopoietic stem cell transplant (HSCT), but this is effective only if the disease is identified early and a match can be found. Allogenic HSCT is a high-risk procedure, with significant mortality associated with the ablation procedure and graft versus host disease. HSCT is currently used for children affected with CALD; limited data is available regarding effectiveness in adults with CALD, and it has no effect on the subsequent development of AMN in adults. Another treatment for ALD, though not approved for such, has been Lorenzo's oil (LO). Research has suggested that LO has not been able to correct accumulation of VLCFA in brains of ALD patients (Rasmussen et al,

Neurochem. Res. ( 1994) 19(8): 1073-82; Poulos et al., Ann Neurol. (1994) 36(5):741-6). Accordingly, there is a need for the development of therapeutic agents useful in the treatment of ALD (for example, CALD, AMN, or both) or other disorders associated with deficiency in very long-chain fatty acids (VLCFA) degradation, associated with deficiency in VLCFA transport into the peroxisomes, associated with accumulation of very long-chain fatty acids (VLCFA), or associated with a benefit from a treatment that lowers VLCFA levels. Deficiency of ABCD 1 protein (also known as 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.

[0004] 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. In some embodiments, 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.

Reversibly reducing VLCFA means that the 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.

SUMMARY

[0005] In one aspect, 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.

[0006] In some aspects, the present invention provides a pharmaceutical composition comprising 1- (2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclo propanecarboxamide in solid form and a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0007] In some aspects, 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]cyc lopropanecarboxamide in solid form, further described herein. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some embodiments, the subject has ALD.

[0008] In some aspects, 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. Examples of 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.

[0009] In some aspects, the present invention provides a method for reduction of VLCFA levels. In some embodiments, the reduction is reversible. In some embodiments, 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. In some embodiments, 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. In certain embodiments, 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. In some embodiments, 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. In certain embodiments, 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).

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] 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. In FIG. 1A, FIG. IB, and FIG. 1C, the level of VLCFA, lysophosphatidylcholine (LPC), was measured in human fibroblast and lymphocyte cells (FIG. 1 A and IB, respectively) from both ALD and healthy patients and in human microglia cells, each grown with ¹³ C-acetate in the presence of increasing concentrations of Compound A for about 48 hr. 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. 1A were derived is different from the CALD 1 cell line from which B-lymphocytes in FIG. IB were derived; 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.

[0011] 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). 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). Compound A was dosed PO QD at 1 and 10 mg/kg to adult female ABCD 1 KO mice (n = 6), with groups analyzed at 3 months and, as shown, and LPC levels, depicted as C26:0 LPC/C16:0 LPC level, in the blood were maintained at near WT levels through 3 months dosing (FIG. 2E; P values versus ABCD1 KO vehicle controls (*** P<0.001, **** P<0.0001); error bars indicate standard deviation). Discontinuation of Compound A returns blood LPC levels, depicted as C26:0 LPC/C16:0 LPC level, to about baseline level in adult female ABCD1 KO mice (n=5) (FIG. 2F; error bars indicate standard deviation. For FIG. 2A to FIG. 2F, the vehicle used was 2% D-a- Tocopherol polyethylene glycol 1000 succinate (TPGS) and Compound A doses were prepared in 2% TPGS. As used herein, mpk means mg/kg.

[0012] 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. ABCD1 KO mice received vehicle (n=6), 1 mg/kg Compound A (n=6), or 10 mg/kg Compound A (n=6) PO QD for 3 months. WT mice also received vehicle for 3 months (n=6). 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. 3F), with 1 mg/kg Compound A showing about a 30% reduction in brain C26:0 LPC level, each after 3 months of dosing. Levels of other LPC are shown for comparison (FIG. 3A: 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.

[0013] Fig. 4 shows reduction of VLCFA level, specifically C24:0 SC-VLCFA level and C26:0 SC- VLCFA level, in the brain following administration of Compound A, in wild-type mice (n=6) and adult female ABCD1 KO mice (n=6) for 3 months. 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. 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).

[0014] 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. FIG. 5A shows the response latency from the prophylactic dosing of Compound A PO QD at 5mg/kg (data shown with squares), Compound A PO QD at 20 mg/kg (data shown with triangles), and 2% TPGS vehicle (data shown with circles) (n = 8-10 mice per group). FIG. 5B shows the response latency from the therapeutic dosing of Compound A PO QD at 32 mg/kg (data shown with squares), Compound A PO QD at 64 mg/kg (data shown with triangles), and 2% TPGS vehicle (data shown with circles) (n = 8-10 mice per group). In FIG. 5 A and FIG. 5B, the dashed line indicates historical WT mouse responses, error bars indicate standard error of the mean, and * corresponds to Tukey's post-hoc test between groups and indicates a significant difference from vehicle treated mice during that month.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

DETAILED DESCRIPTION

[0020] In one embodiment, the inv ound

l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl] cyclopropanecarboxamide (Compound A) in solid form.

[0021] In a second embodiment, the free compound

l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl] cyclopropanecarboxamide (Compound A) is in crystalline solid form.

[0022] In some embodiments, 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. In other embodiments, 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. In other embodiments, 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. In other embodiments, 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. In other embodiments, 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. In some of the foregoing embodiments, 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. In some of the foregoing embodiments, 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. In some of the foregoing embodiments, three of the peak positions, in degrees 2-theta (±0.2 degrees 2-theta or ±0.1 degrees 2-theta), are 7.67, 10.27, and 19.97. In some of the foregoing embodiments, 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. In some of the foregoing embodiments, 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.

[0023] In some embodiments, 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.

[0024] In some embodiments, 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.

[0025] In some embodiments, 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.

[0026] In some embodiments, 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.

[0027] In a third embodiment, 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.

[0028] In a fourth embodiment, 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.

[0029] In a fifth embodiment, 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.

[0030] In a sixth embodiment, the free compound

l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl] cyclopropanecarboxamide (Compound A) is in amorphous solid form.

[0031] A seventh embodiment relates to a pharmaceutical composition comprising l-(2- fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl]cyclopro panecarboxamide in solid form according any of embodiments one through six, and a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] As used herein, the term "free compound" refers to the non-salt form of the compound having the structure indicated by the chemical name or structure.

[0037] As used herein, the term "solid form," when referring to Compound A means that Compound A is in the solid state.

[0038] As used herein, the term "crystalline" refers to a solid material whose constituent particles (e.g., molecules) are arranged spatially in a regular and repeating lattice.

[0039] As used herein, the term "amorphous" refers to a non-crystalline solid material whose constituent particles (e.g., molecules) are not arranged in a regular and repeating lattice pattern.

[0040] As used herein, 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.

[0041] As used herein, the term "including" and other forms thereof such as "include", "includes", etc. are intended to be open-ended unless otherwise specified or clear from context. That is, "including" is to be understood as "including but not limited to" unless otherwise specified or clear from context. The phrase "such as" is similarly intended to be open-ended unless otherwise specified or clear from context.

[0042] As used herein, the term "very long chain fatty acids" (VLCFA) refers to 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). [0043] In some embodiments, 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.

[0044] A non-limiting example of 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. It is understood by one of ordinary skill in the art that 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. Thus, 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. In some embodiments, the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are unsaturated. In some embodiments, the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are saturated (also called straight-chain). In some embodiments, the methods described herein are useful for reduction of VLCFA levels wherein the VLCFA are monounsaturated. In some embodiments, 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

incorporation products. In some embodiments, 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. In some embodiments, the methods described herein are useful for reduction of VLCFA levels, wherein the VLFCA are C24:0 LPC or C26:0 LPC. As used herein, 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. In some embodiments, 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. In some embodiments, 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.

[0045] As used here, phrases such as deficiency of a protein (e.g., ABCD 1 protein, ACOX1, ACBD5, and DBP) 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.

Preparation of Crystalline Compound A

[0046] In another aspect, 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.

[0047] In some embodiments, the solvent is an organic solvent, a mixture of organic solvents, or a mixture of one or more organic solvents and water. In other embodiments, the solvent is an alcoholic solvent, such as methanol, ethanol, or isopropanol. In other embodiments, the solvent is a hydrocarbon solvent, such as hexane, heptane, or cyclohexane. In other embodiments, the solvent is an organic ester solvent, such as ethyl acetate or isopropyl acetate. In other embodiments, the solvent is a mixture of an alcoholic solvent (e.g., isopropanol) and water. In other embodiments, 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. [0048] As used herein, 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.

[0049] As used herein, 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). In some embodiments, "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. In other embodiments, "isolating" comprises evaporating the solvent to afford the crystalline solid form (e.g., evaporating the solvent under reduced pressure in a rotary evaporator).

Pharmaceutical Compositions

[0050] 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).

[0051] In some aspects the present invention provides pharmaceutically acceptable compositions that comprise any of the forms of Compound A as described herein, and additionally comprise a

pharmaceutically acceptable carrier, adjuvant or excipient.

[0052] The pharmaceutically acceptable carrier, adjuvant, or excipient, as used herein, 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. Except insofar as 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.

[0053] 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; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate, sodium stearyl fumarate, and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0054] Compound A of the invention can be formulated into pharmaceutical compositions for administration to animals or humans. In some embodiments, 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.

[0055] 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. The expression "dosage unit form" as used herein 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

administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

[0056] In some embodiments, these compositions optionally further comprise one or more additional therapeutic agents. Some embodiments provide a simultaneous, separate or sequential use of a combined preparation.

Pharmacology

[0057] Adrenoleukodystrophy (ALD), also known as X-linked adrenoleukodystrophy or X- adrenoleukodystrophy (X-ALD), is a metabolic disorder in which patients accumulate VLCFA due to the absence or misfolding of ALD protein, a peroxisomal endoplasmic reticulum membrane protein encoded by the ATP Binding Cassette protein D l (ABCD1) transporter gene. (Mosser, et al. Nature (1993), 361 : 726-730) This transporter ALD protein is required for the import of VLCFA into peroxisomes where they are degraded through beta-oxidation by proteins including Acyl-CoA oxidase (ACOX1) and D- Bifunctional protein. 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. Lipid Res. 55(3), (2014): 524-530). 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, acylcamitines, cholesterol esters and ceramides. These accumulating VLCFA are thought to be particularly detrimental to the central nervous system;

accumulation of C26:0 VLCFA are thought to be the pathological factor disrupting the fatty acid-rich myelin sheath, the adrenal glands and Leydig cells in testes; 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). Interestingly, 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. [0058] High levels of C26:0 have been correlated with pathogenic effects. (R. Orfman et al., EMBO Mol. Med. 2010, 2:90-97). For example, C26:0 decreases the response of adrenocortical cells to adrenocorticotropic hormone stimulation. (R.W. Whitcomb et al., J. Clin. Invest. 1988, 81 : 185-188). 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).

[0059] Mutations in other proteins of the VLCFA degradation pathway, Acyl-CoA oxidase, D- Bifunctional protein (DBP), Acyl-CoA binding domain containing protein 5 (ACBD5), also contribute to VLCFA accumulation and demyelinating diseases in humans.

[0060] In some embodiments, 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).

[0061] 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). Of the seven known ELOVL isozymes, ELOVLl is the primary enzyme responsible for the synthesis of C22:0 to C26:0 VLCFA that are accumulated in ALD patients. (Orfman). Accordingly, compounds that inhibit ELOVLl may be useful in suppressing the synthesis of VLCFA and therefore useful in the treatment of disorders such as ALD. Without being bound by theory, certain compounds described herein, such as Compound A, inhibit ELOVLl, which may cause the reduction in VLCFA levels observed herein.

Uses and Methods of Treatment

[0062] In some aspects, the present invention provides forms of Compound A that reduce a VLCFA level and compositions comprising Compound A in solid form, as described above. In some aspects, 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. In some embodiments, the reduction in VLCFA level is reversible.

[0063] The terms, "disease", "disorder", and "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.

[0064] As used herein, 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. In some embodiments, 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.

[0065] As used herein, an "effective amount" refers to an amount sufficient to elicit the desired biological response. In the present invention, 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. When co-administered with other agents, 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. For example, 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.

[0066] As used herein, the terms "treat," "treatment" and "treating" can refer to both therapeutic and prophylactic treatments. For example, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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.

[0067] In some embodiments, 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.

[0068] As used herein, the terms "in combination" or "co-administration" can be used

interchangeably to refer to the use of more than one therapy (e.g., one or more 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". [0069] 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. In addition, such co-administration also encompasses use of each compound in a sequential manner in either order.

[0070] 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.

Modees of Administration and Dosage Forms

[0071] The pharmaceutically acceptable 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. In certain embodiments, 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.

[0072] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, 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-tocopheryl polyethylene glycol succinate (TPGS; also called vitamin E-TPGS or tocophersolan), and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0073] 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0074] 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.

[0075] 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. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other 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.

[0076] 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.

[0077] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert,

pharmaceutically acceptable 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 talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0078] 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.

[0079] 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. In such solid dosage forms 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. In the case of capsules, tablets and pills, 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. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0080] 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. Additionally, 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.

[0081] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

[0082] 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, 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. 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. Other commonly used surfactants, such as d-a-tocopheryl polyethylene glycol succinate (TPGS; also called vitamin E-TPGS or tocophersolan), Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. [0083] The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavouring or colouring agents may also be added.

[0084] Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These 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. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0085] The pharmaceutical 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.

[0086] 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.

[0087] For topical applications, 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. Alternatively, 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.

[0088] For ophthalmic use, 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. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum. [0089] The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such 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.

[0090] The amount of Compound A that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, the 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.

[0091] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug

combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of Compound A will also depend upon the particular compound in the composition.

Administering with Another Agent

[0092] Depending upon the particular conditions to be treated or prevented, additional drugs, which are normally administered to treat or prevent that condition, may be administered together with

Compound A or a pharmaceutical composition thereof.

[0093] 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.

Biological Samples

[0094] Compound A and compositions thereof are also useful in biological samples. In some aspects, 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. The term "biological sample", as used herein, 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

[0095] In some embodiments, provided are:

1. A free compound

l-(2-fluorophenyl)-N-[l-(2-fluoro-4-pyridyl)pyrazol-3-yl] cyclopropanecarboxamide (Compound A) in solid form.

2. The free compound of embodiment 1 in crystalline solid form.

3. The free compound of embodiment 2, wherein the crystalline solid form is 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.

4. The free compound of embodiment 3, wherein the X-ray powder diffraction pattern comprises at least two 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.

5. The free compound of embodiment 4, wherein the X-ray powder diffraction pattern comprises 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.

6. The free compound of embodiment 5, wherein the X-ray powder diffraction pattern comprises at least four 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.

7. The free compound of embodiment 6, wherein 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. 8. The free compound of embodiment 7, wherein 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.

9. The free compound of any one of embodiments 3-5, wherein at least one of the peak positions, in degrees 2-theta (±0.2 degrees 2-theta), is selected from the group consisting of 7.67, 10.27, and 19.97.

10. The free compound of any one of embodiments 4-6, wherein at least two of the peak positions, in degrees 2-theta (±0.2 degrees 2-theta), are selected from the group consisting of 7.67, 10.27, and 19.97.

11. The free compound of any one of embodiments 5-7, wherein three of the peak positions, in degrees 2-theta (±0.2 degrees 2-theta), are 7.67, 10.27, and 19.97.

12. The free compound of any one of embodiments 3-11, wherein the X-ray powder diffraction pattern further comprises at least two peak positions, in degrees 2-theta (±0.2 degrees 2-theta), selected from the group consisting of 11.28, 12.10, 13.10, 14.36, 15.68, 16.37, 17.66, and 18.70.

13. The free compound of any one of embodiments 3-11, wherein the X-ray powder diffraction pattern further comprises at least four peak positions, in degrees 2-theta (±0.2 degrees 2-theta), selected from the group consisting of 11.28, 12.10, 13.10, 14.36, 15.68, 16.37, 17.66, and 18.70.

14. The free compound of embodiment 2, wherein the crystalline solid form 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), 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.

15. The free compound of embodiment 2, wherein the crystalline solid form 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), of 7.67, 10.27, 12.10, 13.65, 14.36, 15.37, 15.68, 16.84, 17.66, 18.70, and 19.97.

16. The free compound of embodiment 2, wherein the crystalline solid form is characterized by an X- ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 8. 17. The free compound of embodiment 2, wherein the crystalline solid form is characterized by an X- ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 9.

18. The free compound of embodiment 1 in amorphous solid form.

19. The free compound of embodiment 18, wherein the amorphous solid form is characterized by an X-ray powder diffraction pattern similar to the X-ray powder diffraction shown in FIG. 10.

20. A pharmaceutical composition comprising a chemical entity of any one of embodiments 1-186 and a pharmaceutically acceptable carrier, adjuvant, or excipient.

21. 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.

22. The method of embodiment 21, wherein the disease, disorder or condition is associated with one or more mutations of ABCD 1 transporter protein.

23. The method of embodiment 21, wherein the disease, disorder or condition is associated with impaired peroxisomal beta-oxidation.

24. The method of embodiment 21, wherein the disease, disorder or condition associated with mutations of at least one of Acyl-CoA oxidase, D-Bifunctional protein, or ACBD5.

25. The method of embodiment 21, wherein the disease, disorder or condition is associated with accumulation of very long chain fatty acid (VLCFA) levels.

26. The method of embodiment 25, wherein the VLCFA are 24 to 26 carbons long.

27. The method of embodiment 25, wherein the VLCFA are incorporation products. 28. A method of treating ALD comprising administering to a subject an effective amount of the free compound of any one of embodiments 1- 19 or the pharmaceutical composition of embodiment 20.

29. The method of embodiment 28, wherein ALD is the CALD phenotype.

30. The method of embodiment 28, wherein ALD is the AMN phenotype.

31. 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.

32. A method of reduction of very long chain fatty acids (VLCFA) levels in a biological sample of a subject comprising administering to the subject an effective amount of the free compound of any one of embodiments 1-19.

33. 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.

34. 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.

35. The method of embodiment 34, wherein the VLCFA is VLCFA comprising at least 24 carbons.

36. The method of embodiment 34, wherein the VLCFA is VLCFA having 26 carbons.

37. The method of any one of embodiments 34-36, wherein the free compound is the free compound of any one of embodiments 1-19. 38. The method of any one of embodiments 34-37, wherein administering systemically to the subject comprises administering via oral administration, intravenous injection, or subcutaneous injection to the subject.

39. The method of any one of embodiments 34-37, wherein administering systemically to the subject comprises administering via oral administration to the subject.

40. The method of any one of embodiments 34-39, where in the reduction in a VLCFA level in the brain of the subject is at least about 30% when measured as a reduction in LPC 26:0 following administration of the free compound to the subject.

41. The method of embodiment 40, where in the reduction in LPC 26:0 following administration of the free compound to the subject is measured from a sample of cerebrospinal fluid (CSF) from the subject.

[0096] EXAMPLES

[0097] Example 1. Chemical synthesis of 1 -(2-fluorophenyl)-N-(l-(2 -fluoropyridin-4-yl)- 1H- pyrazol-3-yl)cyclopropane-l-carboxamide (Compound A)

intermediate

[0099] Step 1 : 2-fluoro-4-(3-nitro-lH-pyrazol-l-yl)pyridine

[00100] To a 0 °C solution of 3-nitro- lH-pyrazole (250.0 g, 2.17 mol, 1.0 eq) in anhydrous DMF (2.5 L; 10.2 vol eq) under nitrogen was added NaH (95.42 g of 60 %w/w, 2.39 mol, 1.1 eq) in batches over 30 min while maintaining temperature below 8 °C. The mixture was stirred for 1 h then 2,4-difluoropyridine (300 mL, 3.29 mol, 1.5 eq) was added, and the reaction was warmed to room temperature and stirred for approximately 16 hours (h). The reaction mixture was diluted with water (12.5 L) and stirred vigorously for 1 h. 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). 1Η NMR (400 MHz, DMSO-d6) δ 9.01 (d, J = 2.8 Hz, 1H), 8.45 (d, J = 5.7 Hz, 1H), 7.95 (ddd, J = 5.7, 1.9, 1.2 Hz, 1H), 7.81 (t, J = 1.4 Hz, 1H), 7.46 (d, J = 2.8 Hz, 1H) ppm. ESI-MS m/z calc. 208.04, found 209.01 (M+l).

[00101] Step 2: l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine

[00102] A mixture of 2-fluoro-4-(3-nitropyrazol-l-yl)pyridine (200.0 g, 893.6 mol, 1.0 eq), 10% Pd/C (18.60 g of 10 %w/w, 17.48 mmol, 0.02 eq), ammonium formate (572.95 g, 8.814 mol, 10 eq), methanol (500 mL; 2.7 vol eq), and dioxane (1.0 L; 5.4 vol eq) was stirred at 50°C until starting materials were consumed, which was about 2.5 h. The reaction mixture was hot-filtered through Celite, and the filter cake was washed with dioxane (500 mL) and methanol (250 mL). The combined filtrate was concentrated to a white solid. The solid was suspended in water (3L), stirred overnight (about 16 h), and filtered. Water (1L) was added, mixture stirred, filtered, and dried on vac line for about 6 h. The product was dried at 55 °C under vacuum overnight to provide l-(2-fluoropyridin-4-yl)-lH-pyrazol-3 -amine (145.0 g, 89% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 2.8 Hz, 1H), 8.14 (d, J = 5.8 Hz, 1H), 7.56 (dt, J = 5.7, 1.7 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 5.91 (d, J = 2.8 Hz, 1H), 5.47 (s, 2H) ppm. ESI-MS ra/z calc. 178.07, found 178.98 (M+l). -(2-fluoropyridin-4-yl)-lH-pyrazol-3 -amine (alternate synthesis)

[00104] Step 1 : 2-fluoro-4-(3-nitro-lH-pyrazol-l-yl)pyridine

[00105] A reactor was charged with 3-nitro-lH-pyrazole (300 g, 2.67 mol, limiting reagent).

Anhydrous DMF (2.4 L, 8 vol.) was added, and stirring was begun. The solution was cooled to 13 °C, and K3PO4 ( 1.13 kg, 5.33 mol, 2 eq) was added. 2,4-difluoropyridine (613.9 g, 5.33 mol, 2 eq) was added to the reactor, and the reaction was stirred until complete. The reaction mixture was filtered, and the filtrate was transferred slowly into a reactor containing water (6 L, 20 vol.). The resulting slurry was stirred for lh. The slurry was then filtered, and the wet cake was washed with water and dried in a vacuum oven at 60 °C. Crude 2-fluoro-4-(3-nitro- lH-pyrazol-l-yl)pyridine was isolated in 89% yield as an off white solid.

[00106] 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. The reaction mixture was filtered, and the wet cake was rinsed with methanol (0.5 L, 0.6 vol.). The filtrate was concentrated, and the resulting slurry was filtered. The wet cake was rinsed with methanol and then dried in a vacuum oven at 50-55 °C with nitrogen bleed. 2-fluoro-4-(3-nitro- lH-pyrazol-l-yl)pyridine was isolated in 75% yield (708 g) as a white solid.

[00107] Step 2: l-(2-fluoropyridin-4-yl)-lH-pyrazol-3-amine

[00108] 2-fluoro-4-(3-nitro-lH-pyrazol-l-yl)pyridine (808 g, 3.88 mol, 1 eq), 3% platinum on carbon catalyst (66% wet) (37.9 g, 1.94 mol, 0.0005 eq), and 2: 1 tetrahydrofuran: methanol (13.6 L, 17 vol.) were loaded into a jacketed hydrogenator. The hydrogenator was purged with nitrogen and was then purged with hydrogen. The hydrogen was charged to a pressure of 3.0 bar, and the jacket temperature was ramped to 50 °C over 1 hour. Stirring was maintained between about 800 and 1,000 RPM. The batch was stirred until complete conversion was achieved (-10 hours). 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.

[00109] Example 1.2. Chemical synthesis of l-(2-fluorophenyl)-N-(l-(2-fluoropyridin-4-yl)-lH- pyrazol-3-yl)cyclopropane-l-carboxamide (Compound A)

[00110] 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 μί, 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). l-(2-fluorophenyl)cyclopropanecarbonyl chloride (290 g, 100%) was obtained as a clear yellow oil. 1H NMR (400 MHz, CDC13) δ 7.44 - 7.24 (m, 2H), 7.24 - 7.05 (m, 2H), 2.11 - 1.96 (m, 2H), 1.59 - 1.43 (m, 2H) ppm. ESI-MS ra/z calc. 198.02, found 199.63 (M+l) ⁺ .

[00111] 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. After water (200 mL) was added and stirred for about 20 minutes, the THF was removed in vacuo. The resultant mixture was partitioned between ethyl acetate (6.5 L) and aqueous 5% Na2C03 (3 L). The layers were separated, and the organic layer was washed with aqueous 5% Na2C03 (3 L), dried and concentrated. The crude residue was purified by silica gel chromatography (linear gradient of 0 - 100% ethyl acetate/heptane). Relevant fractions were combined and concentrated to provide the desired product, which was re-suspended in heptane (4L) and circulated on a rotary evaporator at atmospheric pressure for approximately 16 h. The product was collected by filtration, washed twice with heptane, and dried in vacuo to provide l-(2-fluorophenyl)-N-(l- (2-fluoropyridin-4-yl)-lH-pyrazol-3-yl)cyclopropane-l-carbox amide (300 g, 78% yield; white crystalline solid). 1H-NMR (400 MHz, DMSO- 6) δ 9.59 (s, 1H), 8.63 (d, J = 2.8 Hz, 1H), 8.25 (d, J = 5.7 Hz, 1H), 7.71 (dt, J = 5.7, 1.5 Hz, 1H), 7.55 - 7.44 (m, 2H), 7.44 - 7.33 (m, 1H), 7.28 - 7.13 (m, 2H), 6.88 (d, J = 2.8 Hz, 1H), 1.71 - 1.54 (m, 2H), 1.25 - 1.08 (m, 2H) ppm.

[00112] 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)

[00113] 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 .

[00114] 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. Upon reaction completion, methanol (0.75 L0.5 vol) was added, and the mixture was stirred for no less than 30 minutes. Water (6 L, 4 vol) was added to the reactor at 14 °C, and the mixture was allowed to warm up to ambient temperature. The reaction mixture was extracted with ethyl acetate (7.5 L, 5 vol), and the organic layer was washed with 1 Ν HQ (6.76 L, 4.5 vol), followed by water (6 L, 4 vol). The organic layer was concentrated, isopropyl alcohol (1 1.25 L, 7.5 vol.) was added, and the mixture was heated to 75 °C. Water (3.8 L, 2.5 vol) was added to the reactor over lh, while maintaining a temperature greater than 70 °C. Seed crystals of l-(2-fluorophenyl)-N-(l-(2-fluoropyridin-4-yl)-lH-pyrazol-3- yl)cyclopropane-l- carboxamide (28.7 g, 0.08 mol, 0.01 eq) were added at 55 °C, and the mixture was stirred for 30 minutes. Water (7.5 L, 5 vol) was added to the reactor at 50- 55 °C over 5 h, and then the jacket was ramped down to 20 °C over 5 hours. Stirring was continued at 20 °C for 30 minutes, and then the batch was filtered and washed with 1 : 1 isopropyl alcohokwater (3.8 L). The wet cake was transferred to drying trays and dried in a vacuum oven at 45 °C with nitrogen bleed. l-(2-fluorophenyl)-N-(l-(2-fluoropyridin-4-yl)-lH- pyrazol-3-yl)cyclopropane-l-carboxamide was obtained in 83.5% yield.

[00115] Crystallin e Forms of Compoun d A

[00116] Single-Crystal X-ray Analysis: Single crystals of Compound A were grown by dissolving 206 mg of Compound A powder in 1 mL of 1,2-dichloroethane and vapor diffusing the solution with pentane over 2 days. Several crystals from the crop were analyzed by single-crystal X-ray diffraction analysis. X-ray diffraction data were acquired at 298 K on a Bruker diffractometer equipped with Cu K _a radiation (λ = 1.5478) and a hybrid pixel area detector. The structure was solved and refined using SHELX program (Sheldrick, G.M., Acta Cryst, (2008) A64, 112-122).

[00117] It was observed that the unit cell characteristics of crystalline Compound A varied from crystal to crystal within the crop. The crystals were observed to have varying amounts of two lattice structures, which are identified herein as "Form A" and "Form B." The average unit cell characteristics of Forms A and B are reported in Table 2. As used herein, the term "average unit cell" refers to as the unit cell determined from reflections arising from Bragg diffraction only, ignoring any contribution by diffuse scatter or satellite reflections. Thermal ellipsoid plots of Compound A in Forms A and B are shown in FIGs. 6 and 7, respectively.

Table 2. Avera e Unit Cell Characteristics for Crystalline Compound A (Forms A and B)

[00118] 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.

[00119] Table 3. XRPD Signals for Crystalline Compound A (Forms A and B)

[00120] In a separate experiment, a mortar was charged with 100 mg of crystalline Compound A, and the powder was ground with a pestle. The powder was placed in a vial with a magnetic stir-bar and 2 mL of isopropyl alcohol and left to stir for two weeks. Samples were retrieved for XRPD analysis at 2 days and after 2 weeks. XRPD measurements were performed using a PANalytical Empyrean diffractometer at room temperature with copper radiation (1.54060 A). The X-ray generator was operating at a voltage of 40kV and a current of 35 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 0.013° 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.

[00121] Table 4. XRPD Signals for Crystalline Compound A (Forms A and B) after Stirring

22.74 3.91

23.19 3.84

23.54 3.78

25.84 3.45

26.42 3.37

26.67 3.34

27.03 3.30

28.93 3.09

29.33 3.05

Amorphous Form of Compound A

[00122] Crystalline Compound A (1.19 g) was dissolved in THF and Water (25 mL, 4: 1 v/v) and lyophilized for 18 hr. The resulting solid was analyzed by XRPD analysis. XRPD measurements were performed using a PANalytical Empyrean diffractometer at room temperature with copper radiation (1.54060 A). The X-ray generator was operating at a voltage of 45 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 4.9948°-40° 2 theta with a step size of 0.0131° and a dwell time of 18.87s per step. The XRPD pattern of the solid material is shown in FIG. 10 and lacks discernible peaks (except at diffraction angles below 12 degrees 2-theta), confirming that the solid was substantially amorphous.

[00123] Example 2. IC50 Assays; In Vitro and In Vivo Studies

[00124] Example 2.1. HEK293 VLCFA-LPC IC ₅₀ Determination.

[00125] 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.

[00126] Cell Culture Growth Conditions: 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.

[00127] 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). In the 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. In the low volume assay, 100 of supplemented media plus 13C-acetate ( 1.0 mg/mL, Sigma Aldrich # 282014) are added to either 0.1 μί or 1.0 uL of Compound A in DMSO in a polypropylene v-bottom plate (Costar #3363) in 1 of 3 dilution schemes. 100 μί 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. For both the high and low volume assays the 3 dilution schemes used are as follows:

a) Top dose of 5uM with a 2.5 fold dilution scheme across 9 points to generate a 10-point IC50 curve b) Top dose of 5uM with a 2.5 fold dilution scheme across 7 points to generate a 8-point IC50 curve c) Top dose of 0.2uM with a 2.5 fold dilution scheme across 7 points to generate a 8-point IC50 curve

[00128] Following incubation, treated cells are harvested by centrifugation at 1690xg for 10 minutes. In the high volume assay 200 uL treated cells are transferred to a polypropylene v-bottom plate (Costar #3363) prior to centrifugation. In the low volume assay 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. In the first scheme, the cell pellet is visibly broken up by mixing the cell pellet up and down in 100 μί of hexane/isopropanol (60:40) 20 times. The resulting mixture is transferred to a 0.45 μπι Durapore membrane (Millipore #MSH VN4510) atop a polypropylene v-bottom plate (Costar #3363) and filtered by centrifugation at 1690xg for 5 minutes. 120 μί 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. In the second scheme, the cell pellet is visibly broken up by mixing the cell pellet up and down in 180 μί of methanol containing 10 nM C 13:0 lysophosphatidylcholine 20 times. The resulting mixture is transferred to a 0.45 μπι Durapore membrane (Millipore #MSH VN4510) atop a polypropylene v-bottom plate and filtered by centrifugation at 1690xg for 5 minutes. In both schemes, the plates are then sealed with pierceable capmats (Micronic MP53017) and stored at -20 °C until analyzed. [00129] UHPLC /Mass Spectrometry Readout: The filtered organic extraction is analyzed with a 1290 Agilent Infinity Series UHPLC coupled to an ABI Sciex QTrap 6500 mass spectrometer.

Separation of the derivatized VLCFA, lysophosphatidylcholine, of varying chain lengths (e.g., C16:0, C18:0, C20:0, C22:0, C24:0, and C26:0) is achieved using an Ascentis Express HILIC column (2.7 micron, 5 cm x 2.1 mm, Sigma #53934-U). The UHPLC mobile phases consisted of 100% water with 20 mM ammonium formate (solvent A) and acetonitrile (90%) / water (10%) with 20 mM ammonium formate (solvent B). The peak area for the mass spectrometry transition monitoring 13C-labeled C26:0 lysophosphatidylcholine (638.500/104.100 m/z) is used to generate IC50 values by fitting the data to a four parameter dose response (Y=Bottom + (Top-Bottom)/ (1+10 ^Λ ((LogIC5o-X)*Hill Slope)). In dilution scheme a), peak areas for the 13C-labeled C26:0 are normalized to the median signal of the lowest tested concentration (negative control). In dilution schemes b) and c), peak areas for the 13C-labeled C26:0 are normalized between the average signal of 8 DMSO-treated wells (negative control) and the average signal of 8 established C26:0 LPC-lowering compound-treated wells (positive control). IC50 values are generated using either GraphPad Prism (La Jolla, CA) or GeneData Analyzer Software (Basel,

Switzerland). 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 μΜ.

[00130] Example 2.2. Reduction in C26:0 LPC concentration in human HEK and patient cells in vitro

[00131] 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). In vitro efficacy studies were performed by measuring LPC VLCFA level (measured as LPC synthesis) in various cell lines, specifically in 1) 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. To measure LPC VLCFA synthesis, the foregoing cells were grown in the presence of ¹³ C labeled acetate (13C Labeled sodium acetate; Sigma Aldrich # 282014) and

Compound A (prepared in DMSO) for about 48 hours. Primary patient fibroblasts and immortalized primary patient lymphocytes were acquired from the Coriell Cell Repository at the Coriell Institute for Medical Research. [00132] HEK293 cells: HEK293 cell culture protocol and treatment with compound, such as Compound A, was described in example 2.1.

[00133] 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.

[00134] 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.

[00135] Upon the completion of the compound treatment, 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.

[00136] 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 μΜ. At completion of the assay, 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.

[00137] 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. 13C-acetate (1.0 mg/mL, Sigma Aldrich # 282014) and 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

Compound A at the following doses: 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.001953125, and 0.000976563 μΜ. Upon the completion of the compound treatment, 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.

[00138] 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. 120 uL of n-butanol containing 10 nM C13:0 lysophosphatidylcholine was added to the filtrate, then the entire volume was transferred to a new Durapore membrane / v-bottom plate. The resulting mixture was filtered as before followed by centrifugation at 1690xg for 10 minutes. The plates were then sealed with pierceable capmats (Micronic MP53017) and stored at -20°C until further analyzed using UPHLC/Mass Spectrometry Readout, as described above in example 2.1, which measured the integration of ¹³ C into

lysophosphatidylcholine (LPC) 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.

[00139] 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). Specifically, 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.

[00140] Table 5. Compound A Potencies Across Cell Types

Note: ALD: adrenoleukodystrophy; AMN: adrenomyeloneuropathy; CALD: cerebral

adrenoleukodystrophy; Het: heterozygous; LPC: lysophosphatidylcholine; 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.

[00141] 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.

[00142] 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. For 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. A lOOmg aliquot of homogenate was used for analysis. To each sample vial 400 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.

[00143] 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

chromatographically separated using a Series 1290 binary pump and a Phenomenex (Torrance, CA) Kinetex C18 analytical column (2.1x100mm, 5μιη particle diameter) with a 10-min gradient. A 5% acetonitrile in water solution was used as the aqueous phase and a 40% acetonitrile/60% methanol solution in 1% 2 Mol ammonium acetate was used as the organic mobile phase for achieving the chromatographic analysis. LPCs were detected by an AB Sciex API-6500 triple quadrupole MS with electrospray ionization in the mode of multiple reaction monitoring. 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).

[00144] Dosing in ABCD1 knockout mice: To determine the effect of Compound A on blood VLCFA levels, Compound A was administered to ABCD1 knockout (KO) mice, a model that reproduces the C26:0 VLCFA accumulation observed in ALD patients. Specifically, Compound A was administered orally (PO) QD at 1, 8, or 16 mg/kg to ABCD1 KO mice (n = 5 per group). DBS were collected on day 0 (pre-dosing), and daily through 14 days of dosing. DBS cards were stored at 4°C in sealed ziplock bags with desiccant until they could be analyzed for LPC using the sample preparation and LC-MS/MS as described above. 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. FIG. 2A shows LPC/vehicle LPC levels (C26:0 LPC levels were normalized to CI 6:0 LPC levels and vehicle controls) for 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.

[00145] Daily oral dosing in ABCD1 knockout mice: To establish the dose response relationship, WT and ABCD1 KO mice were treated with Compound A at doses ranging from 0.5 to 64 mg/kg PO once daily (QD) for 28 days (FIG. 2B). The vehicle used is 2% D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS) and Compound A doses were prepared in 2% TPGS. Mice were dosed daily (QD) orally (PO) with Compound A for 28 days (n=5 mice per group). DBS were collected (n=2 per mouse per time point) and 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.

[00146] The lowest dose tested, 0.5 mg/kg, yielded a statistically significant reduction in C24:0 and C26:0 LPC levels compared to vehicle controls (50% reduction, one-way ANOVA with Dunnett's multiple comparisons test, p = 0.0001). The dose response in ABCD1 KO mice plateaued with a reduction of approximately 75% in C26:0 LPC levels between the 4 mg/kg and 8 mg/kg doses. Blood area under the concentration time-curves (AUCs) were 1951 (±289) ng.h/ml and 3487 (±657) ng.h/ml at the 4 mg/kg and 8 mg/kg doses, respectively. This maximal effect plateau arose at approximately WT baseline LPC levels. 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. In FIG. 2B, 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.

[00147] Reduction of plasma C26:0 LPC in vivo in rats and monkeys: Compound A was dosed PO (orally by oral gavage) QD at 30, 100, and 300 mg/kg in wild-type (WT) rats (n = 5) for 7 days (FIG. 2C). The lowest dose tested in rats, 30mg/kg, yielded about a 65% reduction in C26:0 LPC levels compared to vehicle controls. The 100 and 300mg/kg doses yielded about 75% and about 85% reductions, respectively compared to vehicle controls. C26:0 LPC levels in the blood were reduced to below WT baseline. The vehicle used was 5% TPGS and Compound A doses were prepared in 5% TPGS. Dried Blood Spot (DSB) samples were collected on day 7, at termination of the experiment. DBS cards were stored at 4°C until they could be analyzed for LPC. DBS samples were prepared and analyzed using LC-MS/MS as described above.

[00148] Compound A was dosed PO QD at 30 mg/kg in wild-type male cynomolgus monkeys (n = 5) for 7 days (FIG. 2D) and showed about a 50% reduction in blood C26:0 LPC after 7 days of dosing. 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.

[00149] In FIG. 2C and FIG. 2D, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with Dunnett's multiple comparisons test; error bars indicate standard deviation.

[00150] Long term dosing in ABCD1 knock-out mice: To examine whether continuous dosing maintained efficacy in blood, WT mice were dosed with vehicle (n=6) and female ABCD 1 KO mice (n=6 per group) were dosed for 3 months with vehicle or with Compound A at 1 or 10 mg/kg PO QD. The vehicle used was 2% TPGS and Compound A doses were prepared in 2% TPGS. DBS were collected on day 0 (pre-dose), day 1, and weekly through 12 weeks of dosing. DBS cards were stored at 4°C in sealed ziplock bags with desiccant until they could be analyzed for VLCFAs. DBS samples were prepared and analyzed using LC-MS/MS as described above. Blood C26:0 LPC levels, depicted as C26:0 LPC/C16:0 LPC level, were assessed (FIG. 2E). A dose response was observed; the 1 mg/kg dose induced approximately a 65% reduction in C26:0 LPC levels in vivo and the 10 mg/kg dose induced

approximately a 70% reduction in C26:0 LPC levels in vivo. C26:0 LPC/C16:0 LPC levels in the blood were maintained at near WT levels following 3 months of dosing. A one-way ANOVA with Dunnett's multiple comparisons test yielded P value of < 0.001 and 0.0001, respectively for the 1 and lOmg/kg groups. Error bars indicated standard deviation.

[00151] Reversible reducing effect on LPC level: The C26:0 LPC reducing effect of Compound A was found to be reversible. After treating WT mice with vehicle (n=5) and adult female ABCD1 KO mice (n=5 per group) with 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 were collected (n=2 per mouse per time point) on day 0, day 7 (before dosing with Compound A or vehicle), days 14 and 21 (while on Compound A treatment or on vehicle), as well as days 24, 28, 32 and 36 (after treatment with Compound A or vehicle were discontinued). 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).

[00152] Example 2.4. Reduction of C26:0 LPC and SC-VLCFA levels in wild-type and ABCD1 KO brains.

[00153] To examine the effect of Compound A on VLCFA levels in the CNS, female ABCD 1 KO mice were treated with vehicle, 1 or 10 mg/kg PO QD for 2 weeks (14 days; n=5 per group), 1 month (28 days; n=5 per group), 2 months (56 days; n=6 per group), or 3 months (84 days; n=6 per group). The brain samples used in this study were from the same mice used in the long term dosing study in ABCD1 knock-out mice and WT mice (see Example 2.3). Brain tissue samples were collected after 2, 4, 8, or 12 weeks of dosing with vehicle or Compound A. Brain samples were frozen at -70°C and were analyzed for VLCFA (LPC, SC-VLCFA, acyl-carnitines) via liquid chromatography-mass spectrometry (LCMS) as described below. The vehicle used was 2% TPGS and Compound A doses were prepared in 2% TPGS.

[00154] Levels of 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.

[00155] Compound A reduced C26:0 SC-VLCFA levels in the brains of ABCD 1 KO mice after 2 months of treatment (data not shown), and levels were significantly reduced after 3 months (FIG. 4F). In this experiment, C26:0 SC-VLCFA levels in ABCD1 KO mice were 10 fold higher than in WT mice (Poulos A., et al., Ann. Neurol. (1994) 36(5):741-6; Asheuer M, et al., Hum. Mol. Genet. (2005) 14(10): 1293-303). There were no changes in SC-VLCFA levels at either lmg/kg or 10 mg/kg dose after

2 weeks of dosing (not shown). The 1 mg/kg dose of Compound A reduced C26:0 SC-VLCFA levels by about 30% at 2 months (not shown) and about 50% at 3 months (FIG. 4F). The 10 mg/kg dose yielded a more rapid reduction followed by an apparent plateau, reducing C26:0 SC-VLCFA by about 55% by month 2 (not shown) and by about 65% by month 3 (FIG. 4F). Ten mg/kg of Compound A also induced a significant reduction in brain C24:0 SC-VLCFA level after 3 months of dosing (P<0.01) (FIG. 4E). In FIG. 4, P values versus ABCD1 KO vehicle controls are as follows: *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001; and error bars indicated standard deviation.

[00156] Compound A reduced C26:0 acyl carnitine levels in the brains of ABCD1 KO mice as well. After 2 months of treatment, C26:0 acyl carnitine levels showed about a 50% reduction at 1 mg/kg and about a 70% reduction at 10 mg/kg. Data for acyl carnitine levels are not shown.

[00157] LPC levels in the brains of ABCD 1 KO mice showed more modest changes in response to Compound A. FIG. 3F shows levels of normalized C26:0 LPC in brains of wild-type adult female mice (n=6) treated with vehicle and of adult female ABCD 1 KO mice treated with vehicle (n=6), treated with 1 mg/kg of Compound A PO QD for 3 months (n=6), and treated with 10 mg/kg Compound A PO QD for 3 months (n=6). 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). One mg/kg Compound A induced about a 30% reduction in brain C26:0 LPC at 2 months (not shown) that was maintained through month 3 (FIG. 3F). Ten mg/kg Compound A induced about a 40% reduction in brain C26:0 LPC at 2 months (not shown) and 3 months (FIG. 3F). Both one mg/kg and ten mg/kg of Compound A induced a reduction in brain C24:0 LPC levels (normalized by C16:0 LPC levels) (FIG. 3E). 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 indicated standard deviation.

[00158] These long term brain studies indicated that Compound A induced significant reductions in VLCFA levels in the brains of ABCD1 KO mice, a preclinical model of CLD. Specifically, there were significant reductions in brain C26:0 LPC (FIG. 3F) and SC-VLCFA (FIG. 4F) levels at both doses by

3 months of dosing. LPC levels exhibited more modest changes, while acyl carnitines and straight chain VLCFA levels showed robust changes after 8 weeks of dosing. [00159] 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.

[00160] LPC and acylcarnitine extraction with CHC13 '/MeOH liquid-liquid extraction: Added 1 mL MeOH, then added 1 mL CH3CI to the brain tissue lysates; incubated 30 minutes at room temperature; added 1 mL CHCI3 and 0.75 mL H2O; incubated 30 minutes; centrifuged max for 10 minutes; transferred lower layer to new vials; organic phase was dried using Turbo-Vac. The resulting residue was reconstituted with MeOH.

[00161] 3-step chemical derivatization ofSC-VLCFA using dimethylaminoethanol (VLCFA-DMAE) : (i) added oxalyl chloride (2 mol/1 oxalyl chloride in CH2C12, 200ul) to the dried mixture, incubated at 65°C for 5 minutes; (ii) added 60 uL dimethylaminoethanol, incubated at 25°C for 5 minutes and dried down; (iii) added 100 uL methyl iodide, incubated briefly and dried down. The resulting residue was reconstituted with ethanol (EtOH).

[00162] LCMSMS detection of VLCFA (e.g., spingomyelin (SM) and LPC and derivatized VLCFA (FA-DMAE)):

LPC Detection:

Column: Discovery C18, 2.1x20mm

Phase A: 50%MeOH/5mM AF; Phase B: 2-propanol

MS: 4000 Qtrap operated in ESI MRM positive mode

FA-DMAE Detection:

Column: Synergi Polar RP, 2x150mm

Phase A: H2O/0.1%FA; Phase B: ACN/0.1%FA

MS: 4000 Qtrap operated in ESI MRM positive mode

[00163] Example 2.5. Thermal Pain Sensitivity in ABCD1 KO Mice in Prophylactic and Therapeutic Dosing Models

[00164] 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. For the therapeutic study, 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).

[00166] Prophylactic treatment with Compound A at 5 or 20 mg/kg reduced the loss of thermal pain sensitivity in ABCD l KO mice (n = 8-10 mice per group) (FIG. 5A). 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. Ten-month-old ABCD l KO mice initially had response latencies around 4 seconds, similar to WT mice (indicated by the dashed horizontal line in FIG. 5A). Mice dosed with vehicle had a significant increase in response latencies over the 6 month period, consistent with a loss of thermal pain 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).

[00167] Therapeutic treatment with Compound A reversed the loss of thermal pain sensitivity in older ABCD l KO mice (n = 8-10 mice per group) (FIG. 5B). Dosing was initiated at 18 months of age, after the mice developed deficits in thermal sensitivity, which occurs around 15 months of age. Eighteen- month-old ABCD l KO mice have response latencies of approximately 6 seconds, which are significantly longer than WT mice (indicated by the dashed horizontal line in FIG. 5B). The Plantar test (using a Hargreaves apparatus) was used and measured the latency to respond to a thermal stimulus using the previously described protocol. Baseline measurements were performed before dosing was initiated and used to randomize mice into treatment groups. Mice dosed with vehicle had a gradual increase in response latencies over several months, consistent with further losses in thermal pain sensitivity as the mice age. Mice dosed with Compound A showed a statistically significant improvement in response latencies compared to vehicle treated mice, suggesting slowing or an arrest of disease progression.

Therapeutically treated 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

(p=0.0053) and an interaction (pO.0001).

[00168] Example 3. Metabolic Stability of Compound A

[00169] The metabolic intrinsic clearance (CLmt) of Compound A was determined in human, monkey, dog, rat, and mouse hepatocytes. 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). In separate experiments, Compound A (1 μΜ) 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

7.5). Samples were quenched with acetonitrile and analyzed by LC-MS/MS. The mean CLint for Compound A in human, monkey, dog, rat, and mouse hepatocytes after incubation for 4 hours was determined to be <2.5, <2.5, 7.2, 23.6 and 10.7 μί/πιίη/ιηίΐΐίοη cells. Based on these date, Compound A was low to moderately metabolized in hepatocytes in mouse, rat, dog, monkey, and human, and the rank order of stability at 1 μΜ was approximately human>monkey>dog>mouse >rat.