Methods for Treating and Monitoring Parkinson's Disease

Abstract

The present disclosure relates to methods for treating Parkinson's disease in a subject with the compounds provided herein, pharmaceutical compositions comprising said compounds, and methods for monitoring a subject's response to treatment.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 182,207, filed April 30, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to methods for treating and / or monitoring Parkinson's disease. [Background technology]

[0003] A combination of genetic and biochemical evidence has implicated certain kinase functions in the pathogenesis of neurodegenerative disorders (Christensen, KV (2017) Progress in Medicinal Chemistry 56:37-80; Fuji, RNet al (2015) Sci. Transl. Med. 7(273):ra15; Taymans, JMet al (2016) Curr. Neuropharm. 14(3):214-225). Parkinson's disease is a neurodegenerative disorder that affects the nervous system and exhibits both motor and non-motor symptoms. Although the exact cause of Parkinson's disease is unknown, a combination of genetic and environmental factors is thought to contribute to the pathogenesis of the disease. Genes implicated in Parkinson's disease include Park8, which encodes the complex signaling protein leucine-rich repeat kinase 2 (LRRK2), a key therapeutic target in Parkinson's disease (PD). Mutations in Park8 have been found in both familial and non-familial (sporadic) forms of Parkinson's disease, and elevated kinase activity of LRRK2 has been implicated in the pathogenesis of Parkinson's disease. Mutations in the LRRK2 gene are the most frequent genetic cause of familial Parkinson's disease and are the main driver of lysosomal dysfunction that contributes to the pathogenesis of Parkinson's disease and the formation of neurodegeneration (Chai C, et al. Curr Genomics. 2013; 14: 464-471; Healy DG, et al. Lancet Neurol. 2008; 7: 583-590; Henry AG, et al. Human Mol. Gen. 2015; 24: 6013-6028; Cookson MR, et al. Nat. Rev. Neurosci. 2016; 11: 791-797). LRRK2 regulates lysosomal biogenesis and function, which is impaired in Parkinson's disease and can be reversed by LRRK2 inhibition, potentially positively modifying disease progression in patients with inherited LRRK2 mutations and also in patients with sporadic Parkinson's disease.

[0004] A combination of genetic and biochemical evidence supports the model that LRRK2 kinase function is causally implicated in the pathogenesis of sporadic and familial forms of PD, and therefore LRRK2 kinase inhibitors may be useful for treatment (Christensen, KV (2017) Progress in Medicinal Chemistry 56:37-80). Inhibition of LRRK2 kinase activity is under investigation as a treatment for Parkinson's disease (Fuji, et al., 2015; Taymans, JMet al (2016) Current Neuropharmacology 14(3):214-225).

[0005] LRRK2 kinase inhibitors have been investigated to treat Alzheimer's disease, Parkinson's disease, ALS, and other neurodegenerative diseases (Estrada, AA et al (2015) Jour. Med. Chem. 58(17):6733-6746; Estrada, AA et al (2013) Jour. Med. Chem. 57:921-936; Chen, H. et al (2012) Jour. Med. Chem. 55:5536-5545; Estrada, AA et al (2015) Jour. Med. Chem. 58:6733-6746; Chan, BK et al (2013) ACS Med. Chem. Lett. 4:85-90; US 8354420; US 8569281; US ​​8791130; US 8796296; US WO 2011 / 151360; WO 2012 / 062783; WO 2013 / 079493).

[0006] It is known that administration of various LRRK2 kinase inhibitors induces changes in lysosomal morphology and tissue levels of lysosomal-associated lipids. Accordingly, administration of the LRRK2 inhibitors GNE-7915 and GNE-0877 in monkeys resulted in a decrease in urinary di-22:6-BMP (Fuji RN, et al (2015) Sci.Transl.Med.7(273):273ra215; Baptista MA, et al Baptista et al., (2020) Sci.Transl.Med.12(540).

[0007] Di-22:6-BMP is a phospholipid that is normally localized to the inner membrane of lysosomes and late endosomes and is responsible for lysosomal degradation. Enlarged and increased numbers of lysosomes containing stacked whorls of membrane and lipids were also observed in the proximal tubules of the kidneys of LRRK2 knockout mice (Herzig MC, et al. (2011) Hum. Mol. Genet. 20(21):4209-4223), suggesting phospholipid membrane accumulation in lysosomes. Drug-induced phospholipidosis (PLD) is an acquired lysosomal storage disorder characterized by excessive accumulation of phospholipids and drugs in lysosomes in various tissues, such as the kidney, heart, and lung (Shayman JA, et al (2013) Biochim. Biophys. Acta. 1831(3):602-611; Atashrazm, F. (2016) Clinical Pharmacology: Advances and Applications 8:177-189). Summary of the Invention

[0008] There is a need for methods for treating Parkinson's disease and / or monitoring the progress of the treatment.

[0009] The following brief summary is not intended to be inclusive of all features and aspects of the present invention, nor is it intended to imply that the present invention must include all features and aspects discussed in this summary.

[0010] The present disclosure relates to a method for treating Parkinson's disease, comprising administering to a subject in need thereof about 70 to about 800 mg / day of Compound I, N2-(3-(2-(2H-1,2,3-triazol-2-yl)propan-2-yl)-1-cyclopropyl-1H-pyrazol-5-yl)-N4-ethyl-5-(trifluoromethyl)pyrimidine-2,4-diamine: [ka] or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0011] In another embodiment, a method for treating Parkinson's disease comprises administering to a subject in need thereof about 70 to about 800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0012] In one aspect, the disclosure provides a method for treating Parkinson's disease with about 70 to about 225 mg / day of Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0013] In another aspect, the disclosure relates to a method of treating Parkinson's disease with about 70 to about 80 mg / day of Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0014] In other embodiments, about 70 mg, about 75 mg, about 80 mg, about 105 mg, about 130 mg, about 150 mg, about 225 mg, about 250 mg, about 300 mg or about 400 mg is administered to the subject.

[0015] In one embodiment, Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof, is administered orally.

[0016] In one embodiment, Compound I, or a pharma- ceutically acceptable salt or deuterated analogue thereof, is administered once daily.

[0017] In another embodiment, Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof, is administered twice daily.

[0018] In other aspects, the methods provided herein are for treating a human. In yet other aspects, the methods are for treating familial Parkinson's disease. In yet other aspects, the methods are for treating sporadic Parkinson's disease.

[0019] In yet another aspect, the method results in a reduction of phosphorylated S935 LRRK2 (pS935) in whole blood of the subject.

[0020] In yet another aspect, the method results in a decrease in phosphorylated ras-related protein Rab10 (pRab10) in peripheral blood mononuclear cells (PBMCs) of the subject.

[0021] In yet other embodiments, the methods result in a reduction of lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in the subject's urine.

[0022] In another aspect, there is provided a method for reducing phosphorylated S935 LRRK2 (pS935) in whole blood in a subject suffering from Parkinson's disease, the method comprising administering to a subject in need thereof about 70-800 mg / day of Compound I, or a pharma- ceutical acceptable salt or deuterated analog thereof.

[0023] In one embodiment, pS935 is reduced by at least 41-97%.

[0024] In yet another aspect, there is provided a method for reducing phosphorylated ras-related protein Rab10 (pRab10) in peripheral blood mononuclear cells (PBMCs) of a subject suffering from Parkinson's disease, the method comprising administering to a subject in need thereof about 70-800 mg / day of Compound I, or a pharma- ceutical acceptable salt or deuterated analog thereof.

[0025] In one embodiment, pRab10 is reduced by at least 44-97%.

[0026] In another aspect, there is provided a method for reducing urinary lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in a subject suffering from Parkinson's disease, comprising administering to a subject in need thereof about 70-800 mg / day of Compound I, or a pharma- ceutical acceptable salt or deuterated analog thereof.

[0027] In one embodiment, BMP(22:6 / 22:6) or BMP(22:6 / 22:6) / creatinine is reduced by 22-86% or by at least 40%.

[0028] In another aspect, there is provided a use of an LRRK2 inhibitor for treating Parkinson's disease, wherein the inhibitor is administered to a subject in need thereof at about 70-800 mg / day, and is Compound I, or a pharma- ceutical acceptable salt or deuterated analog thereof.

[0029] In one aspect, there is provided a use of an LRRK2 inhibitor in the manufacture of a medicament for treating Parkinson's disease, wherein the inhibitor is administered to a subject in need thereof at about 70-800 mg / day, and is Compound I, or a pharma- ceutical acceptable salt or deuterated analogue thereof.

[0030] In another embodiment, a method is provided for evaluating a treatment by detecting a decrease in phosphorylated S935 LRRK2 (pS935), phosphorylated ras-related protein Rab10 (pRab10), or lysosomal lipid 2:6-bis[monoacylglycerol]phosphate (BMP) in a patient sample.

[0031] In one aspect, a method is provided for monitoring a subject's response to a treatment method provided herein, the method comprising: (a) measuring the amount of one or more pS935, pRab10 and / or BMP species in a sample from a subject treated with about 70-800 mg / day of Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof; (b) comparing the difference in amount between the one or more pS935, pRab10 and / or BMP species measured in (a) and one or more reference values; and (c) determining from the comparison whether the compound, pharmaceutical composition, or dosing regimen thereof has improved the levels of one or more pS935, pRab10 and / or BMP species for treating Parkinson's disease.

[0032] In another embodiment, the method further comprises altering the dosage or frequency of administration of Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof, or the course of treatment administered to the patient.

[0033] In yet another aspect, the present invention provides a pharmaceutical composition comprising 70 to 800 mg of Compound I, [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0034] In another aspect, the present invention relates to a pharmaceutical composition comprising about 70 to 225 mg of Compound I.

[0035] In yet another aspect, the present invention relates to a pharmaceutical composition of Compound I suitable for administration of about 225 mg per day or up to 800 mg per day.

[0036] In yet another aspect, the present invention relates to a pharmaceutical composition comprising about 70 mg, about 75 mg, about 80 mg, about 105 mg, about 130 mg, about 150 mg, about 225 mg, about 250 mg, about 300 mg, or about 400 mg of Compound I.

[0037] In another aspect, the present invention relates to pharmaceutical compositions of Compound I suitable for oral administration.

[0038] In another aspect, the present invention relates to pharmaceutical compositions of Compound I suitable for administration once, twice or three times daily.

[0039] Features and advantages of the present invention will become apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views, and in which the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0040] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0041] [Figure 1] The proposed mechanism of action of LRRK2 is shown, comparing Parkinson's disease cells with LRRK2 inhibitor-treated cells. aSyn = α-synuclein; GBA = β-glucocerebrosidase; LRRK2 = leucine-rich repeat kinase 2; Rabs = Rab GTPase. [Diagram 2]The Phase 1 study design is shown. This double-blind, placebo-controlled Phase 1 study included a single ascending dose (SAD) and a 10-, 14-, and 28-day multiple ascending dose (MAD) portion in healthy volunteers. BID = twice daily; PBO = placebo; QD = once daily. [Diagram 3] The Phase 1b study design is shown. The study was a double-blind, placebo-controlled, parallel-design Phase 1b study with once-daily dosing for 28 days in patients with Parkinson's disease. [Figure 4A] Target engagement in a Phase 1 study is shown. BL = baseline; IQR = interquartile range; MAD = multiple dose escalation. Percent reduction in whole blood pS935 is shown (from baseline to day 10). Abbreviations: IQR = interquartile range; pS935 LRRK2 = leucine-rich repeat kinase 2 serine 935 phosphorylation; QD = once daily; BID - twice daily. [Figure 4B] Target engagement in a Phase 1 study is shown. BL = baseline; IQR = interquartile range; MAD = multiple dose escalation. Percent reduction in whole blood pS935 is shown (from baseline to day 14). Abbreviations: IQR = interquartile range; pS935 LRRK2 = leucine-rich repeat kinase 2 serine 935 phosphorylation; QD = once daily; BID - twice daily. [Figure 5A] Pathway engagement in a Phase 1 study. Percent reduction of pRab10 from PBMCs (baseline to day 10) is shown. [Figure 5B] Pathway engagement in a Phase 1 study. Percent reduction of pRab10 from PBMCs is shown (from baseline to day 14). [Figure 6A] 1 shows target and pathway engagement in a Phase 1b study. Percent reduction in whole blood pS935 is shown (from baseline to day 28). [Figure 6B] 1 shows target and pathway engagement in a Phase 1b study. Percent reduction of pRab10 from PBMCs (baseline to day 28). [Figure 7A]Figure 1 shows lysosomal engagement in a Phase 1 / 1b study with Compound I. Figure 1 shows the percent reduction in BMP (22:6 / 22:6) in Phase I healthy volunteers (Parts B, D, and E MAD cohorts) (from baseline to Day 10 [Part B], Day 28 [Part D], and Day 14 [Part E]). BMP concentrations were normalized to creatinine concentrations (ng / mg). [Figure 7B] Figure 1 shows lysosomal engagement in a Phase 1 / 1b study with Compound I. Figure 1 shows the percent reduction in urinary BMP (22:6 / 22:6) / creatinine in Phase 1b patients with Parkinson's disease (from baseline to Day 10 [Part B], to Day 28 [Part D], and to Day 14 [Part E]). BMP concentrations were normalized to creatinine concentrations (ng / mg). [Figure 8] Demographic and clinical characteristics of patients with Parkinson's disease in a Phase 1b study. H&Y, Hoehn and Yahr; MDS-UDPRS III, International Movement Disorder Society-Uniform Parkinson's Disease Rating Scale; MAO-B, monoamine oxidase; PD, Parkinson's disease; QD, once daily. [Figure 9] Treatment-emergent adverse events in the MAD cohort from the Phase 1 study in healthy volunteers are shown. *Procedure-related included (in order of frequency): procedural pain, procedural headache, postprocedural complications, puncture site pain, puncture site pruritus, puncture site pain, catheter site pain, postprocedural discomfort, device dermatitis, and catheter site erythema. Separate analyses of 1 or more TEAEs in 2 or more subjects per treatment arm included the following additional TEAEs not listed above: earache (n=2; 105 mg QD 10 day cohort); nasopharyngitis (n=2; 225 mg QD 28 day cohort); asymptomatic COVID-19 (n=2; 400 mg BID 14 day cohort); somnolence (n=2; 250 mg BID 14 day cohort). Two subjects also experienced syncopal dizziness associated with the lumbar puncture (one each in the 150 and 225 mg QD 28-day cohorts). [Figure 10]Treatment-emergent adverse events in a Phase 1b study in patients with Parkinson's disease are shown. GERD, gastroesophageal reflux disease; TEAEs, treatment-emergent adverse events. aProcedure-related included (in order of frequency): procedural pain, postprocedural bruising, postprocedural hematoma, and procedural headache; bHypotension and orthostatic hypotension, which occurred in the same two patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. In general, the nomenclature used in connection with and in the art of cell and molecular biology and chemistry is well known and commonly used in the art. Certain experimental techniques that are not specifically defined are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For the sake of clarity, the following terms are defined below.

[0043] The words "comprise", "comprising", "include", "including" and "includes", when used in the present specification and claims, are intended to specify the presence of stated features, integers, components, or steps, but do not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0044] The terms "treat" and "treatment" refer to both therapeutic treatments and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) undesired physiological changes or disorders, such as the proliferation, development or spread of lysosomal dysfunction disorders. For the purposes of this invention, advantageous or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, remission or alleviation of disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a condition or disorder, as well as those prone to have a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0045] The term "about" indicates that a value includes the variation of error inherent in the method used to determine the value or the variation that exists in experiments. The term "about" can refer to a variation of + / - 10%.

[0046] The term "amount" refers to the level or concentration of a molecule, compound, or agent (e.g., a pS935, pRab10, or BMP molecule). This term includes absolute amounts or concentrations as well as relative amounts or concentrations. In some embodiments, a reference standard (e.g., an internal pS935, pRab10, or BMP standard) is used for calibration to determine the absolute amount or concentration of a molecule, compound, or agent present (e.g., in a sample) and / or to normalize to a control to determine the relative amount or concentration of a molecule, compound, or agent present.

[0047] The phrase "therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats a particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. Efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or by determining the response rate (RR).

[0048] The term "detection" includes any means of detection, including direct and indirect detection.

[0049] "Changes" or "modulations" in the status of biomarkers, including LRRK2 mutations or BMP amounts, when occurring in vitro or in vivo, are detected by analysis of the biological sample using one or more methods commonly used in establishing pharmacodynamics, including: (1) sequencing genomic DNA or reverse transcription PCR products of the biological sample, thereby detecting one or more mutations; (2) assessing gene expression levels by quantitating message levels or assessing copy number; and (3) analyzing proteins by immunohistochemistry, immunocytochemistry, ELISA, or mass spectrometry, thereby detecting protein degradation, stabilization, or post-translational modifications, such as phosphorylation or ubiquitination.

[0050] The term "subject" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep. In some embodiments, the subject is a human.

[0051] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when said event or circumstance does not occur.

[0052] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic product.

[0053] Any compound or structure shown herein is intended to represent the unlabeled form of the compound as well as isotopically labeled forms. Isotopically labeled compounds have the structure depicted herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include, respectively: 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I and other isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine. Various isotopically labeled compounds of the present disclosure, e.g., 3 H, 13 C and 14 Those incorporating a radioactive isotope such as C. Such isotopically labeled compounds may be useful in detection or imaging techniques such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) including metabolic studies, reaction kinetic studies, drug or substrate tissue distribution assays, or in radiation treatment of patients.

[0054] The present disclosure also includes "deuterated analogs" of the compounds described herein in which one to n hydrogens attached to a carbon atom have been replaced by deuterium, where n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and are therefore useful for increasing the half-life of any compound when administered to a mammal, particularly a human. See, for example, Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol.Sci.5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example, by using starting materials in which one or more hydrogens have been replaced by deuterium.

[0055] Deuterium-labeled or substituted therapeutic compounds of the present disclosure may have improved DMPK (drug metabolism and pharmacokinetic) properties related to distribution, metabolism and excretion (ADME). Substitution with heavy isotopes such as deuterium may confer certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life, reduced dosage requirements and / or improved therapeutic index. 18 F, 3 H, 11 C labeled compounds can be useful for PET or SPECT or other imaging studies.The isotopically labeled compounds of the present disclosure can generally be prepared by carrying out the procedures disclosed in the following schemes or in the Examples and Preparations by using readily available isotopically labeled reagents instead of nonisotopically labeled reagents.It is understood that deuterium in this context is considered as a substituent in the compounds described herein.

[0056] The concentration of such heavy isotopes, specifically deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present disclosure, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", the position is understood to have hydrogen at its natural abundance isotopic composition. Thus, in the compounds of the present disclosure, any atom specifically designated as deuterium (D) is intended to represent deuterium.

[0057] In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0058] Pharmaceutically acceptable salts of the compounds described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms and other substances that are useful in the preparation of pharmaceutical compositions that are suitable for veterinary or human pharmaceutical use.

[0059] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, perphosphate, isonicotinate, lactate, salicylate, percitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthalene)-sulfonate. Other salts include acid salts such as the coformers listed above. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharma- ceutically acceptable salt may have more than one charged atom in its structure. Instances in which multiple charged atoms are part of a pharma-ceutically acceptable salt may have multiple counter ions. Thus, a pharma-ceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0060] The desired pharma- ceutically acceptable salts can be prepared by any suitable method available in the art.For example, treatment of the free base with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, methanesulfonic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acids such as glucuronic acid or galacturonic acid, alpha hydroxy acids such as citric acid or tartaric acid, amino acids such as aspartic acid or glutamic acid, aromatic acids such as benzoic acid or cinnamic acid, sulfonic acids such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.Acids that are generally considered suitable for the formation of pharma- ceutically useful or acceptable salts from basic pharmaceutical compounds are described, for example, in Stahl PH, Wermuth CG, editors.Handbook of Pharmaceutical Salts;Properties, Selection and Use, 2 nd Revision(International Union of Pure and Applied Chemistry).2012, New York:Wiley-VCH;S.Berge et al,Journal of Pharmaceutical Sciences(1977)66(1)1 19;P.Gould,International J.of Pharmaceutics(1986)33 201 217;Anderson et al,The Practice of Medicinal Chemistry(1996),Academic Press,New York;Remington's Pharmaceutical Sciences,18 th ed., (1995) by Mack Publishing Co., Easton PA; and The Orange Book (Food & Drug Administration, Washington, DC, web site), the disclosures of which are incorporated herein by reference.

[0061] The phrase "pharmacologically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or with the mammal being treated therewith.

[0062] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable additive" or "additive" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0063] Targets and pathway biomarkers of LRRK2 activity Lysosomal dysfunction is a central pathophysiology of Parkinson's disease (PD) in patients with and without known genetic drivers of PD. Elevated LRRK2 kinase activity impairs lysosomal function and drives familial PD. LRRK2 inhibition can restore normal lysosomal function and reduce toxicity in (PD) models. Inhibition of LRRK2 may be a therapeutically advantageous approach for many forms of PD, including idiopathic PD. LRRK2 disease-causing mutations increase kinase activity.

[0064] The level of LRRK2-dependent lysosomal function can be determined by measuring the presence (e.g., in a sample, cell, tissue, and / or subject) of phosphorylated LRRK2 (pS935), phosphorylated ras-related protein Rab10 (pRab10), or bis(monoacylglycero)phosphate (BMP).

[0065] BMP BMP is represented by the following formula: [ka] The lysosomes are negatively charged glycerophospholipids having a pH of 10-20 (eg, at the pH normally present in the lysosome).

[0066] The BMP molecule contains two fatty acid side chains. R and R' in the above formula represent independently selected saturated or unsaturated aliphatic chains, each typically containing 14, 16, 18, 20, or 22 carbon atoms. When the fatty acid side chain is unsaturated, it may contain 1, 2, 3, 4, 5, 6, or more carbon-carbon double bonds. In addition, the BMP molecule may contain one or two alkyl ether substituents, in which the carbonyl oxygen of one or both fatty acid side chains is replaced with two hydrogen atoms.

[0067] The nomenclature used herein to describe specific BMP species refers to species having two fatty acid side chains, where the structure of the fatty acid side chain is shown in parentheses in the BMP format (e.g., BMP(18:1_18:1)). The numerals follow the standard fatty acid notation format of "number of fatty acid carbon atoms:number of double bonds". The "e-" prefix is ​​used to indicate the presence of an alkyl ether substituent in which the carbonyl oxygen of the fatty acid side chain is replaced with two hydrogen atoms. For example, the "e" in "BMP(16:0e_18:0)" indicates that the side chain with 16 carbon atoms is an alkyl ether substituent.

[0068] BMPs are unusual in that they have a sn-1;sn-1' structural arrangement (i.e., based on phosphate-linked glycerol carbons) that is not observed in other glycerophospholipids. The synthesis of BMPs involves several acylation and diacylation steps, and a transacylase activity that reorients the glycerol backbone to result in the unusual structural arrangement. The sn-1;sn-1' arrangement is thought to contribute to BMPs' resistance to cleavage by many phospholipases and their stability in late endosomes and lysosomes. BMPs are found in small amounts in many different cell types, but BMP content is significantly higher in macrophages and also in lysosomes of the liver and other tissue types.

[0069] Consistent with their function as digestive organelles, lysosomes contain large amounts of hydrolases at acidic pH (i.e., a pH of about 4.6 to about 5). Various cellular components and foreign antigens are captured by receptors on the cell surface for uptake and delivery to the lysosome. Within the cell, receptors such as the mannose-6-phosphate receptor bind to hydrolases and direct them down the biosynthetic pathway to the lysosome. The captured molecules pass through an intermediate heterogeneous set of organelles known as endosomes, which act as sorting stations that recycle hydrolases and other materials before they are directed to the lysosome. There, hydrolases are activated and undesirable materials are digested. In particular, the inner membranes of mature or "late" endosomes and lysosomes contain large amounts of BMPs.

[0070] Being negatively charged at lysosomal pH, BMPs can dock with luminal acid hydrolases that are positively charged at acidic pH and require a water-lipid interface for activation. By so binding, BMPs can stimulate several lysosomal lipid-degrading enzymes, including acid sphingomyelinase, acid ceramidase, acid phospholipase A2, and acid lipase, which has the ability to hydrolyze triacylglycerol and cholesterol esters.

[0071] Endosome membranes are extensions of lysosomal membranes, which function to sort and recycle materials back to the plasma membrane and endoplasmic reticulum. Thus, low-density lipoproteins (LDL) internalized in the liver reach late endosomes, where the component cholesterol esters are hydrolyzed by acidic cholesterol ester hydrolase. The distinctive network of BMP-rich membranes contained within late endosomes is a key element of cholesterol homeostasis, regulating cholesterol trafficking by acting as a collection and redistribution point for free cholesterol. For example, when lysosomal membranes are incubated with anti-BMP antibodies, significant amounts of cholesterol accumulate.

[0072] In some embodiments of the method of the present disclosure, the abundance of a single BMP species is measured. In some embodiments, the abundance of two or more BMP species is measured. In some embodiments, the abundance of at least 2, 3, 4, 5 or more BMP species is measured. When the abundance of two or more BMP species is measured, any combination of different BMP species can be used.

[0073] In some cases, one or more BMP species may be differentially expressed (e.g., at higher or lower abundance) in one type of sample when compared to each other, such as, for example, a cell-based sample (e.g., cultured cells) versus a tissue-based or blood sample. Thus, in some embodiments, the selection of one or more BMP species (i.e., for measuring abundance) depends on the type of sample. In some embodiments, for example, when the sample (e.g., the test sample and / or the reference sample) is a bone marrow-derived macrophage (BMDM), the one or more BMP species comprises BMP (18:1_18:1). In other embodiments, for example, when the sample comprises tissue (e.g., brain tissue, liver tissue) or plasma, urine, or CSF, the one or more BMP species comprises BMP (22:6_22:6);

[0074] In some embodiments, an internal BMP standard (e.g., BMP (14:0_14:0)) is used to measure the abundance of one or more BMP species in a sample and / or to determine a reference value (e.g., to measure the abundance of one or more BMP species in a reference sample). For example, a known amount of an internal BMP standard can be added to a sample (e.g., a test sample and / or a reference sample) to serve as a calibration point so that the amount of one or more BMP species present in the sample can be determined. In some embodiments, a reagent (e.g., methanol) used in the extraction or isolation of BMP from a sample is "spiked" with an internal BMP standard. Typically, the internal BMP standard will not naturally occur in a subject.

[0075] Typically, the abundance of each of one or more BMP species in test sample is compared with one or more reference values ​​(e.g., corresponding reference values).In some embodiments, BMP values ​​are measured before treatment and at one or more time points after treatment.Abundance values ​​taken at later time points can be compared with pre-treatment values ​​and also with control values, such as those of healthy or diseased controls, to determine how the subject responds to treatment.The one or more reference values ​​may be from different cells, tissues, or fluids that correspond to the cells, tissues, or fluids of test sample.

[0076] In some embodiments, reference value is the abundance of one or more BMP species measured in reference sample.Reference value can be the value of measured abundance (e.g., the value of abundance measured in reference sample), or can be derived from or extrapolated from the value of measured abundance.In some embodiments, for example, when reference value is obtained from a large number of samples or a population of subjects, reference value is a range of values.Furthermore, reference value can be expressed as a single value (e.g., the value of measured abundance, the mean value, or the median value) or a range of values, with or without standard deviation or standard error.

[0077] In some embodiments, both the first test sample and the second test sample are obtained from a subject (e.g., a target subject) after the subject has been treated. That is, the first test sample is obtained from the subject during treatment at an earlier time than the second test sample. In some embodiments, the first test sample is obtained before the subject is treated for Parkinson's disease with an LRRK2 inhibitor, and the second test sample is obtained after the subject is treated for the disorder with an LRRK2 inhibitor (i.e., a post-treatment test sample). In some embodiments, more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pre-treatment and / or post-treatment test sample is obtained from the subject. Furthermore, the number of pre-treatment and post-treatment test samples obtained does not have to be the same.

[0078] Di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) is an LRRK2-dependent indicator of lysosomal function and dysfunction (Fuji et al. 2015; Liu, N. et al, (2014) Toxicol. Appl. Pharmacol. 279:467-476; US 8313949) and has the structure: [ka] and has been named 1-(((1-(((4E,7E,10E,13E,16E,19E)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-2-hydroxyethoxy)(11-oxidanyl)phosphoryl)oxy)-3-hydroxypropan-2-yl(4E,7E,10E,13E,16E,19E)-docosa-4,7,10,13,16,19-hexaenoate. This family of glycerophospholipids is susceptible to rapid acyl migration, resulting in phosphate exchange and racemization of the stereocenter.

[0079] pRab10 Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are found in both familial and non-familial (sporadic) forms of Parkinson's disease (PD). Several different mutations have been identified as pathogenic mutations, including mutations 11122V, Nl437H, Rl441C / G / H, RI 728H, Rl628P, Yl699C, G2019S, 12020T, T2031S, and G2385R, and other mutations in LRRK2 have been associated with susceptibility to PD. At least some of the known pathogenic mutations in LRRK2 have been found to affect its kinase activity, and thus LRRK2 inhibitors have been proposed as treatments for PD.

[0080] Several proteins have been identified as possible physiological substrates of LRRK2, including Rab10, a member of the Rab GTPase family. Phosphorylation of Rab proteins has been detected in human cells overexpressing LRRK2 and Rab10. Furthermore, increased phosphorylation of Rab10 has been detected in various PD-linked LRRK2 mutants compared to wild-type LRRK2. Enhanced phosphorylation of Rab10 in the presence of LRRK2 variants suggests the presence of increased LRRK2 kinase activity of pathogenic variants in vivo. Thus, in some embodiments, phosphorylation of Rab10 represents a useful clinical marker for identifying patients with pathogenic mutations in LRRK2, such as 11122V, Nl437H, Rl441C / G / H, RI 728H, Rl628P, Yl699C, G2019S, I2020T, T2031S or G2385R mutations, and in other embodiments, Rl441C, Rl441G, Yl699C, G2019S, or I2020T mutations.

[0081] Monoclonal antibodies have been generated that specifically bind to phosphorylated Rab10 protein endogenously expressed in human biological samples, such as human peripheral blood mononuclear cells. See PCT / US2018 / 037809, filed June 15, 2018, published as WO 2018 / 232278 on December 20, 2018, which is incorporated herein by reference in its entirety for all purposes. In contrast, known polyclonal antibodies against phosphorylated Rab10 or phosphorylated Rab8a do not show a significant decrease in detectable phosphorylated Rab10 in response to treatment with LRRK2 inhibitors. It has also been found that the levels of phosphorylated Rab10 and phosphorylated Rab8a proteins are dose-dependently decreased in response to treatment with LRRK2 inhibitors, as measured using anti-phosphorylated Rab10 monoclonal antibodies.

[0082] pS935 The G2019S mutation, described above, is in the activation loop of LRRK2 and is the most common genetic cause of PD. G2019S causes increased LRRK2 kinase activity, resulting in toxicity. A marker for LRRK2 activity is phosphorylation of serine 935 (pS935). pS935 is decreased in response to all known LRRK2 kinase inhibitors and is therefore a useful biomarker for it.

[0083] BMP detection techniques: In some embodiments, mass spectrometry (MS) is used to detect and / or measure the abundance of one or more BMP species by the methods of the present disclosure. Mass spectrometry is an established technique in which compounds are ionized and the resulting ions are selected by their mass-to-charge ratio (abbreviated as m / Q, m / q, m / Z, or m / z). A sample (e.g., containing BMP molecules), which may exist in gas, liquid, or solid form, is ionized, and then the resulting ions are accelerated in an electric and / or magnetic field so that they are separated by their mass-to-charge ratio. The ions eventually impact an ion detector to generate a mass spectrogram. Sometimes the mass-to-charge ratio of the detected ions, together with their relative abundance, can be used to identify the parent compound by correlating known masses (e.g., of whole or intact molecules) to the masses of the detected ions and / or by recognizing patterns detected in the mass spectrogram.

[0084] In some embodiments, high performance liquid chromatography (HPLC) is used in combination with mass spectrometry. HPLC provides advanced separation by forcing analytes in a mobile phase under pressure through a stationary phase, typically a tightly packed column. In the established technique of LC / MS, HPLC serves as the separation front end and mass spectrometry serves as the characterization back end.

[0085] pRab10 and pS935 detection As discussed above for BMPs, pRab10 and pS935 can also be detected using MS. However, in one embodiment of the present invention, pRab10 and pS935 are detected using antibodies specific for these molecules, as described in the Examples below. These antibodies can be used for detection in immunoassays. One such commercially available assay is sold by Meso Scale Diagnostics, LLC. (MSD) (Rockville, Maryland).

[0086] Methods for Treating Parkinson's Disease Methods for treating diseases or conditions mediated at least in part by LRRK2 are generally described in U.S. Pat. No. 10,590,114, and compounds for use in such methods are described in U.S. Pat. No. 9,932,325, both of which are incorporated by reference herein in their entireties for all purposes.

[0087] A method for treating Parkinson's disease, comprising administering to a subject in need thereof about 70 to 800 mg / day of [ka] or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0088] A daily dosage may be described as a dose or the total amount of Compound I or a pharma- ceutically acceptable salt or deuterated analogue thereof administered per day. The daily dosage of Compound I or a pharma- ceutically acceptable salt or deuterated analogue thereof may be about 70-800 mg, about 70-225 mg / day, or about 70-80 mg / day.

[0089] In certain embodiments, the dose may be 70, 75, 80, 105, 130, 150, 225, 250, 300 or 400 mg. In some embodiments, the compound or its pharma- ceutically acceptable salt or deuterated analogue may be administered once a day (QD). In other embodiments, administration is twice a day (BID).

[0090] In some embodiments, a pharmaceutical composition comprising about 75 mg of Compound I in tablet form is provided.

[0091] In some embodiments, a subject in need thereof is administered two tablets, each containing about 75 mg of Compound I. In some embodiments, a subject in need thereof is administered two tablets, each containing about 75 mg of Compound I, once daily, for a total dose of about 150 mg / day.

[0092] In some embodiments, a subject in need thereof is administered three tablets, each containing about 75 mg of Compound I. In some embodiments, a subject in need thereof is administered three tablets, each containing about 75 mg of Compound I, once daily, for a total dose of about 225 mg / day.

[0093] In other embodiments, the compound of the present disclosure can be administered in combination with an additional agent that has activity for treating Parkinson's disease.For example, in some embodiments, the compound is administered in combination with one or more additional therapeutic agents that are useful for treating Parkinson's disease.In some embodiments, the additional therapeutic agent is L-dopa (e.g., Sinemet®), dopaminergic agonist (e.g., Ropinerol or Pramipexole), catechol-O-methyltransferase (COMT) inhibitor (e.g., Entacapone), L-monoamine oxidase (MAO) inhibitor (e.g., Selegiline or Rasagiline) or agent that increases dopamine release (e.g., Zonisamide).

[0094] Methods for Treating Parkinson's Disease with LRRK2 Inhibitors In one embodiment, a method for treating Parkinson's disease is provided comprising administering to a subject in need thereof about 75-225 mg of Compound I: [ka] once daily.

[0095] In another embodiment, a method for treating Parkinson's disease is provided comprising administering to a subject in need thereof about 75-225 mg of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0096] Methods for reducing phosphorylated S935 LRRK2 (PS935) in whole blood in subjects suffering from Parkinson's disease In one embodiment, a method for reducing phosphorylated S935 LRRK2 (PS935) in whole blood in a subject suffering from Parkinson's disease comprises administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0097] In another embodiment, a method for reducing phosphorylated S935 LRRK2 (PS935) in whole blood in a subject suffering from Parkinson's disease comprises administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0098] Methods for reducing phosphorylated ras-related protein RAB10 (PRAB10) in peripheral blood mononuclear cells (PBMCs) of subjects suffering from Parkinson's disease In one embodiment, a method for decreasing phosphorylated ras-related protein RAB10 (PRAB10) in peripheral blood mononuclear cells (PBMCs) of a subject suffering from Parkinson's disease is provided comprising administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0099] In another embodiment, a method for decreasing phosphorylated ras-related protein RAB10 (PRAB10) in peripheral blood mononuclear cells (PBMCs) of a subject suffering from Parkinson's disease is provided comprising administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0100] Methods for reducing urinary lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in subjects suffering from Parkinson's disease In one embodiment, a method for reducing urinary lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in a subject suffering from Parkinson's disease is provided comprising administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0101] In another embodiment, a method for reducing urinary lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in a subject suffering from Parkinson's disease is provided comprising administering to a subject in need thereof about 70-800 mg / day of Compound I: [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof, and a pharma- ceutically acceptable carrier.

[0102] Use of LRRK2 inhibitors to treat Parkinson's disease 2. Use of an LRRK2 inhibitor for treating Parkinson's disease, comprising administering the inhibitor to a subject in need thereof at about 70-800 mg / day, [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof.

[0103] Use of LRRK2 inhibitors in the manufacture of a medicament for treating Parkinson's disease 2. Use of an LRRK2 inhibitor in the manufacture of a medicament for treating Parkinson's disease, comprising administering the inhibitor to a subject in need thereof at about 70-800 mg / day, [ka] or a pharma- ceutically acceptable salt or deuterated analogue thereof.

[0104] Methods for monitoring response to LRRK2 inhibitor compounds 1. A method for monitoring a subject's response to a treatment method provided herein, comprising: (a) measuring the amount of one or more pS935, pRab10 and / or BMP species in a test sample from a subject having Parkinson's disease, wherein the test sample or the subject is being treated with about 70-800 mg / day of Compound I, or a pharma- ceutical acceptable salt or deuterated analog thereof; (b) comparing the difference in amount between the one or more BMP species measured in (a) and one or more reference values; and (c) determining from the comparison whether the LRRK2 inhibitor compound or pharmaceutical composition thereof, or a dosing regimen thereof, improved the level of one or more BMP species for treating Parkinson's disease. The method includes:

[0105] In one embodiment, the method further comprises: (d) maintaining or adjusting the amount or frequency of administration of Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof, to a test sample or a subject; and (e) administering the compound or a pharma- ceutically acceptable salt or deuterated analogue thereof to a test sample or to a subject. Includes.

[0106] In one exemplary embodiment, the one or more BMP species comprises BMP(22:6_22:6).

[0107] In an exemplary embodiment, the LRRK2 inhibitor is Compound I, or a pharma- ceutically acceptable salt or deuterated analog thereof.

[0108] In an exemplary embodiment, the one or more BMP species includes BMP(22:6_22:6).

[0109] In an exemplary embodiment of the above method, the reference value is determined in a reference sample obtained from a reference subject or a population of reference subjects.

[0110] In an exemplary embodiment of the above method, the reference subject or population of reference subjects are healthy controls.

[0111] In an exemplary embodiment of the above method, the reference subject or population of reference subjects does not have a lysosomal dysfunction disorder or reduced levels of pS935, pRab10 or BMP.

[0112] In an exemplary embodiment of the above method, subjects having or at risk of having Parkinson's disease have elevated levels of pS935, pRab10 or BMP species in bone marrow-derived macrophages compared to healthy controls or controls not related to Parkinson's disease.

[0113] In an exemplary embodiment of the above method, a subject having or at risk of having Parkinson's disease has reduced levels of pS935, pRab10 or BMP species in the liver, brain, cerebrospinal fluid, plasma, or urine compared to healthy controls or controls not related to Parkinson's disease.

[0114] In exemplary embodiments of the above methods, the amount of pS935, pRab10 or BMP species in a test sample from a subject having or at risk of having Parkinson's disease has a difference of at least about 1.2-fold, 1.5-fold, or 2-fold compared to a reference value from a control, such as a healthy control or a control not related to Parkinson's disease.

[0115] In an exemplary embodiment of the above method, the amount of pS935, pRab10 or BMP species in a test sample from a subject having or at risk of having Parkinson's disease is between about 1.2-fold and about 4-fold different compared to a reference value from a control, such as a healthy control or a control not associated with a lysosomal dysfunction disorder.

[0116] In an exemplary embodiment of the above method, the reference value is the value of pS935, pRab10 or a BMP species before treatment.

[0117] In exemplary embodiments of the above methods, the reduction in the level of pS935, pRab10 or BMP species is an improvement over the level of pS935, pRab10 or BMP species before treatment, compared to a reference value of a control, such as a healthy control or a control not associated with a lysosomal dysfunction disorder.

[0118] In exemplary embodiments of the above methods, the reduction in the levels of pS935, pRab10 or BMP species has a difference of 5% to 90%, preferably about 50 to 70%, more than about 50%, or more than about 70%, compared to a control.

[0119] In exemplary embodiments of the above methods, the test or reference sample or the one or more reference values ​​comprises or relates to a cell, a tissue, whole blood, plasma, serum, cerebrospinal fluid, interstitial fluid, sputum, urine, lymph, or a combination thereof.

[0120] In exemplary embodiments of the above methods, the cell is a peripheral blood mononuclear cell (PBMC), a bone marrow derived macrophage (BMDM), a retinal pigment epithelial (RPE) cell, a blood cell, a red blood cell, a white blood cell, a neuronal cell, a microglial cell, a brain cell, a cerebral cortex cell, a spinal cord cell, a bone marrow cell, a liver cell, a kidney cell, a splenocyte, a lung cell, an eye cell, a chorionic villus cell, a muscle cell, a skin cell, a fibroblast cell, a cardiac cell, a lymph node cell, or a combination thereof.

[0121] In an exemplary embodiment of the above method, the cells are cultured cells.

[0122] In exemplary embodiments of the above methods, the tissue comprises brain tissue, cerebral cortex tissue, spinal cord tissue, liver tissue, kidney tissue, muscle tissue, heart tissue, eye tissue, retinal tissue, lymph node, bone marrow, skin tissue, vascular tissue, lung tissue, spleen tissue, valve tissue, or a combination thereof.

[0123] In an exemplary embodiment of the above method, the test sample comprises endosomes, lysosomes, extracellular vesicles, exosomes, microvesicles, or a combination thereof.

[0124] In exemplary embodiments of the above methods, the one or more pS935, pRab10 or BMP species comprises two or more pS935, pRab10 or BMP species.

[0125] In an exemplary embodiment of the above method, the test sample comprises plasma, urine, cerebrospinal fluid (CSF), and / or brain or liver tissue, and the one or more BMP species comprises BMP(22:6_22:6).

[0126] In an exemplary embodiment of the above method, the test sample comprises CSF or urine and the one or more BMP species comprises BMP(22:6_22:6).

[0127] In exemplary embodiments of the above methods, the abundance of one or more pS935, pRab10 or BMP species is measured using liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), enzyme-linked immunosorbent assay (ELISA), or a combination thereof.

[0128] In exemplary embodiments of the above methods, an internal pS935, pRab10 or BMP standard is used when determining the amount of one or more pS935, pRab10 or BMP species.

[0129] In an exemplary embodiment of the above method, the internal pS935, pRab10 or BMP standard comprises a pS935, pRab10 or BMP species that is not naturally occurring in the subject and / or reference subject or population of reference subjects.

[0130] In an exemplary embodiment of the above method, the internal BMP standard comprises BMP(14:0_14:0).

[0131] In an exemplary embodiment of the above method, the lysosomal dysfunction disorder is a disorder associated with BMP expression, processing, glycosylation, cellular uptake, transport, and / or function.

[0132] In an exemplary embodiment of the above method, the subject has one or more mutations in the LRRK2 expressed gene.

[0133] In an exemplary embodiment of the above method, the disorder is associated with reduced levels of BMP in a tissue.

[0134] In an exemplary embodiment of the above method, the disorder is associated with elevated levels of BMP in urine.

[0135] In an exemplary embodiment of the above method, the disorder is associated with elevated levels of BMP in urine.

[0136] In exemplary embodiments of the above methods, the subject and / or reference subject is a human, a non-human primate, a rodent, a dog, or a pig.

[0137] Pharmaceutical Compositions and Modes of Administration Provided herein is a pharmaceutical composition comprising Compound I or its pharma- ceutically acceptable salt or deuterated analogue and one or more pharma- ceutical acceptable vehicles selected from carriers, adjuvants, and additives.Suitable pharma- ceutical acceptable vehicles can include, for example, inert solid diluents and bulking agents, diluents including sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizing agents, and adjuvants.Such compositions are prepared by methods well known in the pharmaceutical field.See, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & CT Rhodes, Eds.).

[0138] Oral administration may be, for example, administration by capsule or tablet. In preparing pharmaceutical compositions, active ingredients are usually diluted with additives and / or enclosed in carriers, which may be in the form of capsules, sachets, paper or other containers. When additives serve as diluents, they may be in the form of solid, semi-solid, or liquid substances that act as vehicles, carriers or mediums for active ingredients. Some examples of suitable additives include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulation may additionally include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propylhydroxy-benzoates; sweeteners; and flavoring agents. EXAMPLES

[0139] The compositions and processes of the present invention will be better understood in connection with the following examples, which are intended as illustrations only and do not limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications can be made without departing from the spirit of the invention and the scope of the appended claims, including, but not limited to, with respect to the processes, formulations and / or methods of the invention.

[0140] Overview of the disease PD is the second most common neurodegenerative disease, affecting approximately 1%-2% of individuals over the age of 65 (de Rijk MD,et al., J Neurol Neurosurg Psychiatry.1997;62(1):10-5; Blin P et al.,Eur J Neurol.2015;22(3):464-71), and prevalence is predicted to increase substantially as the world's population ages (Dorsey ER et al,Neurology.2007;68(5):384-6). The estimated prevalence of PD in Europe and North America ranges from 66 to 12,500 per 100,000 (von Campenhausen et al., Eur Neuropsychopharmacol. 2005;15(4):473-90) and 572 per 100,000 (Marras et al., NPJ Parkinsons Dis. 2018;4:21), respectively. The incidence of PD increases with age and is rare before the age of 50 (de Lau and Breteler, Lancet Neurol. 2006;5(6):525-35; Twelves et al., Mov Disord. 2003;18(1):19-31). Severe disability or death can be expected in 35% of patients within 5 years of onset, 65% within 10 years of onset, and 80% within 15 years of onset (Poewe, J Neurol. 2006;253 Suppl 7:VII2-6; Schrag and Bank, Mov Disord. 2006;21(11):1839-43; Mov Disord. 2010;25 Suppl 1:S131-5).

[0141] Currently approved treatments for PD improve motor symptoms but do not address the underlying cause of the disease. Over time, these symptomatic treatments lose effectiveness and are associated with an increase in the frequency and severity of adverse effects, such as movement disorders and hallucinations. In addition, non-motor symptoms including depression, anxiety, sleep disorders, cognitive impairment, and dementia are common disabling features of PD but are inadequately addressed by current treatments (Aarsland et al., Arch Neurol. 1996;53(6):538-42; Truong et al., J Neurol Sci. 2008;266(1-2):216-28; Lyons and Pahwa, Am J Manag Care. 2011;17 Suppl 12:S308-14; Khoo et al., Neurology. 2013;80(3):276-81; Seppi et al., Mov Disord. 2019;34(2):180-98; FDA 2016). Thus, PD patients inevitably experience increasing disability over the years to decades they live with the disease (Hely et al., Mov Disord. 2005;20(2):190-9). Thus, there is a significant need for effective disease-modifying treatments to prevent progressive motor and non-motor disorders that are not addressed by current treatments.

[0142] LRRK2 mutations are a proven cause of PD and are responsible for approximately 4%-5% of familial PD cases (Healy et al., Lancet Neurol. 2008;7(7):583-90; Chai C,et al., Curr Genomics 2013;14(8):486-501). Familial LRRK2 mutations are transmitted in an autosomal dominant pattern of inheritance with incomplete penetrance (Marder et al., Neurology 2015;85(1):89-95). In addition, variants in the LRRK2 gene are a genetic risk factor and are responsible for 1%-2% of "sporadic" PD cases (Healy, 2008; Chai et al, Curr Genomics 2013;14(8):486-501; Hernandez et al., J Neurochem 2016;139(Suppl 1):50-74; Cookson, Biochem Soc Trans. 2016;44(6):1603-10).

[0143] Rationale for Treating with Investigational Drugs in Disease Compound 1 is a selective, orally bioavailable, CNS-penetrant, reversible inhibitor of LRRK2 for treating patients with PD. Inhibition of the genetically validated target, LRRK2 kinase, improves lysosomal function in LRRK2-PD and potentially in iPD. Compound 1 may intervene in key disease pathways in PD to prevent or inhibit the accumulation of motor and non-motor disorders that define PD progression.

[0144] LRRK2 encodes a multidomain protein that contains a guanosine triphosphatase (GTPase) domain, a kinase domain, and several potential protein-protein interaction domains. The majority of identified pathogenic mutations in LRRK2 are located within its catalytic domain, including the most common mutation associated with LRRK2-PD, G2019S. These mutations increase LRRK2 kinase activity through a direct mechanism within the kinase domain or through an indirect mechanism (West et al., Human Mol Gen. 2007;16(2):223-32; Sheng et al., Sci Transl Med. 2012;4(164):164ra161). The G2019S point mutation increases LRRK2 activity by approximately two-fold, and protective LRRK2 variants are associated with modest decreases in LRRK2 kinase activity, suggesting that moderate changes in LRRK2 kinase activity contribute to lifetime risk of PD (Khan et al., Brain. 2005;128(Pt 12):2786-96; Jaleel et al., Biochem J. 2007;405(2):307-17; West et al., Human Mol Gen. 2007;16(2):223-32; Sheng et al., Sci Transl Med. 2012;4(164):164ra161; Steger et al., eLife. 2016;5:e12813; Ross et al., Lancet Neurol. 2011;10(10):898-908).

[0145] Although the exact pathogenic mechanism remains unknown, LRRK2 is thought to play a role in intracellular trafficking within the endo-lysosomal system (Henry et al. 2015; Cookson et al., 2015). The direct effect of kinase-activating LRRK2 mutations can increase phosphorylation of Rab GTPases, key regulators of intracellular trafficking (Steger et al., 2016;). Rab phosphorylation is thought to promote the accumulation of inactive Rabs within the lysosomal membrane, thus disrupting vesicle trafficking. Alterations in both lysosomal and cellular functions are associated with LRRK2 mutations. Cellular data show that inhibition of G2019S mutant LRRK2 activity in cells reverses lysosomal abnormalities (Khan et al., 2005; West et al., 2007; Sheng et al., 2012; Steger et al., 2016; Schapansky et al., Neurobiol Dis. 2018; 111:26-35; Hockey et al., J Cell Sci. 2015; 128(2):232-8; Henry et al., 2015; Wallings et al., Hum Mol Genet. 2019a; 28(16):2696-710; Rivero-Rios et al., J Biol Chem. 2019; 294(13):4738-58).

[0146] Current evidence supports LRRK2 inhibition to correct disease-associated lysosomal dysfunction, independent of LRRK2 mutation status. LRRK2 activity, as measured by pS1292 LRRK2 and Rab10 threonine 73 phosphorylation (pT73 Rab10), is elevated in the substantia nigra of brains collected postmortem from patients with iPD, suggesting that LRRK2 overactivity may drive PD pathogenesis in non-LRRK2 carrier populations (Di Maio et al., Sci Transl Med. 2018;10(451):eaar5429). Lysosomal dysfunction may be a central mechanism for intracellular protein accumulation, leading to the accumulation of α-synuclein and the formation of Lewy bodies, which are cardinal pathological features of iPD (Dehay et al., Mov Disord. 2013;28(6):725-32; Tofaris, Mov Disord. 2012;27(11):1364-9). A role for LRRK2 in α-synuclein accumulation and the resulting pathology has been suggested by in vitro and in vivo studies. Primary neuronal cultures expressing G2019S-LRRK2 develop α-synuclein inclusions that can be reduced by LRRK2 inhibitor treatment. In vivo, infection of the transgenic G2019S-LRRK2 rat model of PD with a virus overexpressing α-synuclein can induce dopaminergic neuronal degeneration, which can be attenuated by LRRK2 inhibitor treatment (Daher et al., J Biol Chem. 2015;290(32):19433-44; Volpicelli-Daley et al. J Neurosci. 2016;36(28):7415-27). This is further supported by data showing significant protection from pathology when reducing LRRK2 protein levels by 50% in a mouse model of PD overexpressing pathogenic α-synuclein (Zhao et al. Mol Ther Nucleic Acids. 2017;8:508-19).These data strongly support the concept that hyperactivity of LRRK2 has an effect on lysosomal function and may contribute to neurodegeneration in iPD, and that kinase inhibitors have the potential to restore lysosomal function and improve patient outcomes in the setting of iPD.

[0147] In addition, LRRK2 inhibition may correct disease-associated lysosomal dysfunction associated with other genetic variants leading to PD. LRRK2 kinase inhibition may correct signaling defects, including increased phosphorylated Rab10, associated with PD-linked mutations in the lysosomal trafficking molecules VPS35 and Rab29 (Purlyte et al., EMBO J. 2018;37(1):1-18; Mir et al., Biochem J. 2018;475(11):1861-83). Furthermore, patients with homozygous loss-of-function mutations in the gene encoding glucosylceramidase beta (GBA) develop the lysosomal storage disorder Gaucher disease, while subjects with heterozygous mutations in GBA are at high risk for PD. In fibroblasts derived from Gaucher disease patients, there is a near-complete loss of lysosomal protein turnover activity that can be partially corrected by LRRK2 inhibition. Thus, inhibiting increased LRRK2 kinase activity may alleviate LRRK2-mediated pathology including lysosomal dysfunction, as well as lysosomal dysfunction independent of LRRK2 overactivity, supporting the therapeutic potential of Compound I in a broad population of patients with PD (Di Maio et al., 2018, Ysselstein et al., Nat Commun. 2019;10(1):55702019, Sanyal et al., Front Neurosci. 2020;14:442).

[0148] In summary, LRRK2 activity is linked to central mechanisms of PD (iPD and LRRK2-PD) pathology through its role in lysosomal function, and LRRK2 kinase inhibitors, such as compound I, represent a new class of therapeutic agents with the potential to address the underlying biology of PD in patients with and without LRRK2 mutations.

[0149] Example 1: LRRK2 Kinase Inhibition in Compound I Phase 1 Study In the Phase 1 study in healthy volunteers, participant demographics were as follows:

[0150] 184 HVs (145 active, 39 placebo) were treated with single or repeated once-daily (QD) or twice-daily (BID) doses for up to 28 days in the following study parts: Part A (SAD; young HV; n = 48): 100% male and median age 25 years (range 18–50 years); Part B (10-day MAD; young HV; n = 80): 99% male and median age 26.5 years (range 18–50 years); Part C (SAD; elderly HV; n = 8): 50% male and median age 69 years (range 67–74 years); Part D (28-day MAD; young HV; n=17): 100% male and median age 29 years (range 18-39 years); and Part E (14-day MAD; young HV; n=31): 100% male and median age 30 years (range 18-50 years).

[0151] Example 2: Kinase activity in Parkinson's disease and LRRK2 risk variants Current evidence suggests that reduction of LRRK2 kinase activity, whether associated with familial LRRK2 pathogenic mutations or not, is a viable therapeutic strategy for treating patients with PD. The majority of identified pathogenic mutations in LRRK2 increase LRRK2 kinase activity through direct mechanisms within the kinase domain or through indirect mechanisms (West et al., 2007; Sheng et al., 2012). LRRK2 kinase activity, as measured by pS1292 LRRK2 and pT73 Rab10, is elevated in the substantia nigra of brains collected postmortem from patients with iPD, suggesting that LRRK2 kinase overactivity in the brain may drive PD pathogenesis in non-LRRK2 mutation carrier populations (Di Maio et al., 2018). In addition, current evidence suggests that LRRK2 inhibition may correct disease-associated lysosomal dysfunction, such as reduced GBA activity, independent of LRRK2 mutation status (Ysselstein et al., 2019). Thus, LRRK2 kinase inhibitors, such as Compound I, represent a new class of therapeutic agents with the potential to address the underlying biology of PD in patients with and without LRRK2 mutations.

[0152] The most common disease-causing variant in LRRK2 (G2019S) increases LRRK2 kinase activity by approximately two-fold; therefore, normalization can be predicted with a 50% reduction in LRRK2 kinase activity.

[0153] pS935 LRRK2, a pharmacodynamic marker of LRRK2 kinase inhibition pS935 LRRK2 in whole blood is the primary pharmacodynamic marker used to quantify LRRK2 inhibition in the Compound I clinical study. pS935 LRRK2 has been demonstrated to be sensitive to pharmacological inhibition of LRRK2 kinase (Fell et al., J Pharmacol Exp Ther. 2015; 355(3): 397-409; Fuji et al., 2015; Henderson et al., J Med Chem. 2015; 58(1): 419-32), and a decrease in pS935 LRRK2 is measurable in whole blood in human subjects following treatment with LRRK2 inhibitors. Furthermore, animal studies have demonstrated that the exposure-response of pS935 LRRK2 in the periphery closely parallels that in the CNS (e.g., on average, a 50% reduction in pS935 LRRK2 in the periphery corresponds to an approximately 50% reduction in the central nervous system), demonstrating that peripheral LRRK2 inhibition likely mirrors brain inhibition in human subjects.

[0154] Because the primary pathological findings of PD are in the brain, direct quantification of LRRK2 inhibition in the CNS would be preferable to quantify peripheral LRRK2 inhibition as a pharmacodynamic measure. Researchers in this field are therefore developing assays to quantify LRRK2 phosphorylation in the CSF, matrices that would reflect LRRK2 inhibition in the brain. As these assays progress, they will be implemented in clinical studies to quantify LRRK2 inhibition in the CNS and measure the relationship between peripheral and central LRRK2 inhibition in human subjects.

[0155] Based on the 2-fold increase in kinase activity associated with the LRRK2 G2019S point mutation, a reduction in whole blood pS935 LRRK2 of 50% or greater at trough is the minimum pharmacodynamic target in >50% of study subjects. Clinical studies to date have demonstrated that exposures resulting in a mean reduction in pS935 LRRK2 of 85%-90% are safe and generally well tolerated in healthy subjects and subjects with PD. Specifically, there were no apparent changes in potential LRRK2 on-target kidney or lung function over exposures of up to 28 days.

[0156] Lysosomal biomarkers regulated by BMP and LRRK2 activity The lysosomal lipid BMP 22:6 / 22:6 (referred to throughout the document as "BMP") measured in urine is a mechanistic marker of lysosomal pathway modifications downstream of LRRK2. BMP is a lysosomal phospholipid found exclusively on intraluminal vesicles of late endosomes and lysosomes (Bissig and Gruenberg, Cold Spring Harb Perspect Biol. 2013; 5(10): a016816). Individuals with genetic or drug-induced lysosomal dysfunction, including those with the G2019S LRRK2 mutation, have elevated levels of urinary BMP (Lecommandeur et al., J Lipid Res. 2017; 58(7): 1306-14; Alcalay et al., Mov Disord. 2013; 28(14): 1966-71). LRRK2 inhibition has been shown to reduce urinary BMP in both animal models and humans (Fuji et al. 2015; Alcalay et al., 2020; Figures 4A, 6A, 7A, 7B).

[0157] Lysosomal dysfunction is a common hallmark of PD, and it is hypothesized that therapeutic approaches aimed at improving PD-linked defects in lysosomal homeostasis, including LRRK2 inhibition, may have a meaningful impact on disease progression (Wallings et al., Trends Neurosci. 2019b; 42(12):899-912). A reduction in urinary BMP may, but does not necessarily, indicate defective lysosomal pathway regulation in patients with PD. The primary pathological findings in PD are in the CNS; therefore, efficacy of therapeutics likely requires modulation of the lysosomal pathway in the CNS. While there is evidence that urinary BMP is a measure of lysosomal function, it is a peripheral marker, and therefore additional biomarkers of LRRK2 pathway activity and lysosomal function in the CNS will be further investigated for the relationship between urinary BMP and central lysosomal function.

[0158] Example 3: Clinical Safety Safety data from the initial Phase 1 in human study in healthy subjects and a Phase 1b study in subjects with PD are summarized below.

[0159] Phase 1, safety, tolerability, pharmacokinetic, and pharmacodynamic study in healthy subjects This was a Phase 1, randomized, placebo-controlled, double-blind FIH study designed to determine in healthy subjects the safety, tolerability, PK, and pharmacodynamics of Compound I. Open-label safety data from the repeat-dose cohorts (Parts B, D, and E) are summarized below.

[0160] Safety and tolerability in healthy subjects after repeated dosing in Part B Part B of the study consisted of sequential multiple ascending dose (MAD) cohorts. Healthy subjects were enrolled in eight cohorts (Cohorts B1–B8; n=10 / cohort) and received Compound I 15, 30, 45, 70, 105, 150, 225, or 300 mg or placebo (4:1 ratio) QD for 10 days.

[0161] In Part B, Compound I was generally well tolerated in healthy subjects at multiple doses up to 300 mg QD for 10 days. No deaths, other SAEs (serious adverse events), AESIs (adverse events of special interest), or treatment-emergent adverse events (TEAEs) leading to discontinuation of study drug were reported. A total of 56 (86.2%) Compound I-treated subjects and 13 (86.7%) placebo-treated subjects experienced one or more TEAEs (Figure 9). The most common TEAEs among Compound I-treated subjects were headache (29 [45%] subjects); treatment-related (32 [49%] subjects); fatigue (6 [9.2%] subjects); and nausea (6 [9.2%] subjects). Of the Compound I-treated subjects, 55 (84.6%) experienced one or more mild TEAEs, 9 (13.8%) subjects experienced one or more moderate TEAEs, and no subjects experienced severe TEAEs.

[0162] No clinically significant changes were observed in safety laboratory, vital signs, ECG, neurological examination, pulmonary function test (PFT), or Columbia-Suicide Severity Rating Scale (C-SSRS) results.

[0163] Effect of treatment on pS935 and pRab10 Procedure for pS935 in whole blood and pRab10 in PBMCs: Sample preparation Whole blood preparation for the pS935 assay: Frozen human whole blood samples were thawed and lysed directly in 96-well plates (100 μl whole blood sample with 100 μl lysis buffer). Prior to the MSD assay, samples were spun (2,500×g for 20 min at 4° C.).

[0164] PBMC preparation for pRab10 assay: Blood was collected into CPT-sodium heparin tubes (BD BDAM362780) and PBMCs were isolated according to the manufacturer's protocol. PBMCs were pelleted by centrifugation at maximum speed and then resuspended in PBMC lysis buffer (1× cell lysis buffer [CST catalog #9803] containing PhosSTOP phosphatase inhibitors [Roche 04906837001], complete protease inhibitors [Roche 04693159001], and benzonase [Sigma E8263]). Lysates were kept on ice for 20 minutes and subsequently centrifuged at maximum speed for 20 minutes at 4°C. Supernatants were aliquoted and stored at -80°C for later immunoassay analysis.

[0165] MSD assay The capture antibody was biotinylated using EZ-Link™ NHS-LC-LC-Biotin (Thermo Fisher, #21343) and the detection antibody was conjugated using Sulfo-TAG NHS-ester (MSD, R31AA-1). 96-well (or 384-well) MSD GOLD Small Spot Streptavidin plates (MSD L45SSA-1) were coated with 25 μl (or 15 μl for 384-well plates) of capture antibody diluted in Diluent100 (MSD, R50AA-2) for 1 hour at room temperature with shaking at 700 rpm (1000 rpm for 384-well). After TBST washing (3×), 25 μl of sample was added to each well (10 μl for 384-well) and incubated overnight at 4° C. with shaking at 700 rpm. After TBST washing (3x), 25 μl (15 μl for 384-well) of detection antibody was added to each well together with rabbit (Rockland Antibodies D610-1000) and mouse gamma globin fraction (D609-0100) diluted in TBST containing 25% MSD blocker A (MSD R93AA-1). After 1 h incubation at room temperature and 700 rpm followed by TBST washing (3x), 150 μl of MSD read buffer (MSD R92TC, diluted 1:1 with water) is added (35 μl for 384-well) and the plate is read on an MSD Sector S 600. [Table 1]

[0166] FIG. 4 shows that at trough (pre-dose) and steady state (day 10 for cohort B, day 28 for cohort D, and day 14 for cohort E), Compound I treatment resulted in a robust reduction in pS935 of 80% or more at the highest dose and a reduction of 50% or more at the lowest clinically relevant dose compared to baseline.

[0167] Figure 5 shows that at trough (pre-dose), compound I reduced phosphorylation of Rab10 (pRab10), a direct substrate of LRRK2 kinase, in peripheral mononuclear cells (PBMCs) by more than 70% at the maximum steady-state dose in HV (day 10 in cohort B, day 28 in cohort D, and day 14 in cohort E).

[0168] Safety and tolerability in healthy subjects after repeated dosing in Part D Part D consisted of a repeat-dose cohort of healthy subjects (Cohort D1) receiving either Compound I 225 mg (n=13) or placebo (n=4) QD for 28 days.

[0169] In Part D, Compound I was generally well tolerated in healthy subjects at a dose of 225 mg QD for 28 days. No deaths, other SAEs, or AESIs were reported. One subject in the placebo group experienced a TEAE after the seventh dose, leading to discontinuation of study drug (transaminases elevated; judged by the investigator to be moderate in severity and unrelated to study drug). One subject withdrew consent for personal reasons after the eighth dose and was replaced. A total of 10 (77%) subjects in the Compound I 225 mg QD group and 2 (50%) subjects in the placebo group experienced one or more TEAEs (Figure 9). The most common TEAEs among Compound I-treated subjects were headache (3 [23%] subjects) and procedure-related (3 [23%] subjects). Of the Compound I-treated subjects, 10 (77%) experienced one or more mild TEAEs, and no subjects experienced moderate or severe TEAEs.

[0170] No other clinically significant changes were observed in safety laboratory, vital signs, ECG, neurological examination, PFT, or C-SSRS results.

[0171] Safety in healthy subjects after repeated dosing in Part E Part E consists of consecutive MAD cohorts. Healthy subjects in cohort E1 received Compound I 150 mg or placebo BID (4:1 ratio) for 14 days (n=9), and subjects in cohort E2 received Compound I 250 mg or placebo BID (4:1 ratio) for 14 days (n=11). Subjects in ongoing cohort E3 are receiving Compound I 400 mg or placebo BID (4:1 ratio) for 14 days. Open-label safety data in cohorts E1 and E2 are summarized below.

[0172] Based on open-label safety data from cohorts E1 and E2, Compound I was generally well tolerated in healthy subjects at 150 and 250 mg BID over 14 days. No SAEs or AESIs were reported. Two study-drug related discontinuations occurred in Part E: one subject had moderate nausea, headache, impaired concentration, and diarrhea (250 mg BID); a second subject had severe headache and fatigue with moderate nausea (400 mg). One subject (250 mg BID) discontinued the study early; the subject withdrew consent on day 3 as a result of moderate TEAEs of nausea, headache, impaired attention, and diarrhea, all of which resolved on the day of occurrence after treatment with acetaminophen and were deemed related to study drug by the investigator.

[0173] All 25 (100.0%) Compound I-treated subjects and 6 (100.0%) placebo-treated subjects experienced one or more TEAEs in the study. The most common TEAE among Compound I-treated subjects was headache (21 [84%] subjects vs. 4 [67%] placebo subjects). The majority of reported TEAEs were mild or moderate in severity. One subject (250 mg BID) experienced a severe TEAE (procedural headache; determined by the investigator to be unrelated to study drug). Moderate TEAEs were reported in two subjects receiving Compound I 250 mg BID: headache (n=1) and headache, nausea, distractibility, and diarrhea (n=1). Severe TEAEs were reported in two subjects: procedural headache (n=1; 250 mg BID); headache and fatigue (n=1; 40 mg BID).

[0174] No clinically significant changes were observed in vital signs, ECG, telemetry, safety laboratories (including liver and renal function tests), PFTs, neurological examination, or C-SSRS results.

[0175] Example 4: Phase 1b, safety, tolerability, pharmacokinetic, and pharmacodynamic study in subjects with Parkinson's disease Participant demographics A total of 36 patients with PD (26 active, 10 placebo) were treated with up to 300 mg QD for 28 days (Figure 8).

[0176] This was a Phase 1b, randomized, placebo-controlled, double-blind study designed to determine the safety, tolerability, PK, and pharmacodynamics of Compound I in subjects with PD. The study has been completed and clinical trial reporting is ongoing. In part 1, subjects received Compound I 80 mg or placebo QD (1:1 ratio) for 28 days (n=8). In part 2, subjects received Compound I 80 or 130 mg or placebo QD (1:2:1 ratio) for 28 days (n=17). In part 3, subjects received Compound I 300 mg or placebo QD (4:1 ratio) for 28 days (n=11).

[0177] Based on open-label safety data, Compound I was generally well tolerated in subjects with PD at 80, 130, or 300 mg QD over 28 days (summarized in Figure 10). No SAEs or AESIs were reported. Two (5.6%) subjects discontinued the study due to a hypotensive TEAE after the first dose. In one of these subjects (130 mg QD), the event was asymptomatic and was determined by the investigator to be severe and not related to study drug. This subject had been taking tamsulosin until 1 day prior to the event and had a documented history of autonomic dysregulation. In the second subject (300 mg QD), the event was mildly symptomatic on standing and was determined by the investigator to be mild and related to study drug. Two additional subjects experienced TEAEs of mild orthostatic hypotension (80 mg QD) and mild hypotension and orthostatic hypotension (300 mg QD), which resolved while on study drug.

[0178] Figure 6 shows that Compound I treatment resulted in robust reductions in pS935 in whole blood at trough (pre-dose) and steady state (day 28) across all dose levels studied. Compound I reduced pRab10 in PBMCs in patients with PD at trough (pre-dose) and steady state across all doses.

[0179] A total of 23 (88.5%) Compound I-treated subjects and 5 (50%) placebo-treated subjects experienced one or more TEAEs during the study (Figure 10). The most common TEAE among Compound I-treated subjects was headache (11 [42%] subjects vs. 2 [20%] subjects on placebo). The majority of TEAEs were mild or moderate in severity. Two subjects experienced severe TEAEs: one subject had hypotension (130 mg QD; determined to be unrelated to study drug and due to a pre-existing orthostatic effect) and one subject had a headache (300 mg QD; determined to be related to the lumbar puncture procedure and unrelated to study drug). Moderate TEAEs were experienced by five (19%) Compound I-treated subjects and one (10.0%) placebo-treated subject: parkinsonism (diminished parkinsonian symptoms) (80 mg QD; n=1), fungal skin infection (130 mg QD; n=1), neck stiffness (130 mg QD; n=1), headache (130 and 300 mg QD; n=2), myalgia (300 mg QD; n=1), and constipation (placebo QD; n=1).

[0180] No other clinically significant individual changes or notable trends from baseline were observed in safety laboratory (including liver and renal function tests), ECG, neurological assessment, vital signs, PFT, or C-SSRS results.

[0181] Dose selection for further studies will be based on population PK / pharmacodynamic relationships of Compound I plasma concentrations and validated target engagement biomarkers at exposures demonstrated to be safe and well tolerated.

[0182] conclusion Safety: Compound I was generally well tolerated over a wide range of doses for up to 28 days in patients with HV and PD. The most common TEAE in Compound I-treated participants was headache. There were no clinically meaningful changes in pulmonary or renal function.

[0183] Pharmacokinetics: Compound I demonstrated high CSF penetration based on CSF / unbound plasma ratios. Treatment with Compound I achieved robust target and pathway engagement at doses that were generally well tolerated in patients with HV and PD.

[0184] Pharmacodynamics: Robust target and pathway engagement was achieved in both patients with HV and PD with Compound I treatment. In addition, Compound I treatment resulted in a dose-dependent decrease in urinary lysosomal lipid BMP22:6, a marker of lysosomal function, in both patients with HV and PD.

[0185] To date, no dose-related significant safety concerns have been observed, and Compound I has been found to be safe and generally well tolerated in over 200 subjects at doses up to 400 mg twice daily (BID) for 14 days in healthy volunteers, or at doses of 225 mg for 28 days.

[0186] Example 5: Pharmaceutical Composition An immediate release tablet formulation containing 75 mg of Compound I mixed with the ingredients shown in the table below was prepared in an anhydrous granulation process. [Table 2]

[0187] The patent and scientific literature referenced herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

[0188] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, and the descriptions and examples should not be construed as limiting the scope of the invention. Accordingly, all suitable modifications and equivalents may be deemed to fall within the scope of the invention as defined by the following claims.

Claims

1. A pharmaceutical composition for treating a subject suffering from Parkinson's disease, comprising: Compound I: 【Chemistry 1】 or a pharma- ceutically acceptable salt or deuterated analog thereof, The compound is administered to the subject at about 70-800 mg / day. The pharmaceutical composition.

2. 10. The method of claim 1, wherein about 70-225 mg, or about 70-80 mg, of the compound is administered to the subject.

3. 10. The pharmaceutical composition of claim 1, wherein about 70 mg, about 75 mg, about 80 mg, about 105 mg, about 130 mg, about 150 mg, about 225 mg, about 250 mg, about 300 mg, or about 400 mg of the compound is administered to the subject.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound is administered orally.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein the compound is administered once daily or twice daily.

6. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutical composition (i) results in a reduction in phosphorylated S935 LRRK2 (pS935) in the whole blood of the subject, (ii) results in a reduction in phosphorylated ras-related protein Rab10 (pRab10) in peripheral blood mononuclear cells (PBMCs) of the subject, or (iii) results in a reduction in lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in the urine of the subject.

7. A pharmaceutical composition for treating a subject suffering from Parkinson's disease, comprising: Compound I: 【Chemistry 2】 Including, about 75-225 mg of the compound is administered to the subject once daily; The pharmaceutical composition.

8. 8. The pharmaceutical composition of claim 7, wherein about 75 mg, about 150 mg, or about 225 mg of the compound is administered to the subject.

9. A pharmaceutical composition for (i) reducing phosphorylated S935 LRRK2 (pS935) in whole blood of a subject suffering from Parkinson's disease, (ii) reducing phosphorylated ras-related protein Rab10 (pRab10) in peripheral blood mononuclear cells (PBMCs) of a subject suffering from Parkinson's disease, or (iii) reducing lysosomal lipid 22:6-bis[monoacylglycerol]phosphate (BMP) in the urine of a subject suffering from Parkinson's disease, comprising: Compound I: 【Chemistry 3】 or a pharma- ceutically acceptable salt or deuterated analog thereof, About 70-800 mg / day of the compound is administered to the subject; The pharmaceutical composition.

10. The pharmaceutical composition of claim 9, which (i) reduces pS935 by 41 to 97%, (ii) reduces pRab10 by 44 to 97%, or (iii) reduces BMP(22:6 / 22:6) / or BMP(22:6 / 22:6) / creatinine by 22 to 86%.

11. The pharmaceutical composition according to any one of claims 1 to 3 and 7 to 10, wherein the subject is a human.

12. The pharmaceutical composition according to any one of claims 1 to 3 and 7 to 10, wherein the Parkinson's disease is familial or sporadic.

13. 70 to 800 mg of Compound I, 【Chemistry 4】 or a pharma- ceutically acceptable salt or deuterated analogue thereof and a pharma- ceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, comprising about 70 to 225 mg of Compound I.

15. 14. The pharmaceutical composition of claim 13, suitable for administration of about 800 mg or about 225 mg per day.

16. 14. The pharmaceutical composition of claim 13, comprising about 70 mg, about 75 mg, about 80 mg, about 105 mg, about 130 mg, about 150 mg, about 225 mg, about 250 mg, about 300 mg, or about 400 mg of Compound I.

17. 14. The pharmaceutical composition of claim 13, suitable for oral administration.

18. 14. The pharmaceutical composition of claim 13, suitable for administration once daily, twice daily, or three times daily.

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