Method for Identifying a Disease and Treatment Method

Abstract

Provided are methods for assessing abnormal glycosphingolipid processing and for determining the severity and / or progression of related diseases. Also provided are methods for assessing the possible therapeutic response of a subject to treatment with an agent that can modulate glycosphingolipid processing, as well as therapeutic methods that benefit from evaluating a patient according to the present disclosure. The methods of the present disclosure utilize an agent that can impose a load on the glycosphingolipid pathway in a cell (e.g., conduritol-β-epoxide), and subsequently monitor the recovery of this cell (e.g., monitor the recovery of glycosphingolipid flux in this cell).

Description

Technical Field

[0001] The present disclosure relates to a diagnostic method for evaluating abnormal glycosphingolipid processing, and a method for determining the severity and / or progression of related diseases such as Parkinson's disease. Also provided are methods for evaluating the possible therapeutic response of a subject to treatment with an agent capable of modulating glycosphingolipid processing, and related treatments.

Background Art

[0002] Glycosphingolipids (GSLs) are complex lipids involved in diverse cellular functions including plasma and vesicular membrane integrity, signal transduction, and cell - cell communication. The synthesis and breakdown of GSLs are essential for normal cell function and survival. Impairment of GSL breakdown is associated with many lysosomal storage diseases and neurodegenerative disorders.

[0003] The final reaction in the breakdown of certain GSLs is mediated by glucocerebrosidase (GC - ase, also called acid β - glucosidase), a hydrolase involved in the lysosomal breakdown of glucosylceramide (GL1). Lysosomal GC - ase is encoded by the gene GBA. Homozygous and compound heterozygous mutations in GBA cause Gaucher disease (GD), a lysosomal storage disease (LSD) characterized by inefficient clearance of GL1 and subsequent clinical symptoms. These patients exhibit altered glycosphingolipid levels that can be normalized by an increase in glucocerebrosidase activity and / or a decrease in GL1 synthesis, as demonstrated by the beneficial effects of enzyme replacement therapy or substrate reduction therapy, respectively. GBA mutations, even in the heterozygous state, increase the risk of developing Parkinson's disease (PD) and related synucleinopathies. Currently, GBA mutations represent the most common genetic risk factor for the development of these diseases. Interestingly, there is evidence for a decrease in GC - ase activity in the CNS of sporadic PD patients without known GBA mutations, suggesting another unknown mechanism associating decreased hydrolytic activity with the disease.

[0004] LSD is often associated with the accumulation of lysosomal substrates due to severe loss of function of one or more enzymes. For example, in GD, the loss of GCase activity results in the accumulation of GL1 and its deacylated derivative, glucosylsphingosine (lyso-GL1), while treatment with agents that can reduce the levels of these substrates (e.g., miglustat) can be used to treat the disease. Changes in the levels of these lysosomal substrates can function as important biomarkers for determining the effectiveness of therapeutic interventions. Unfortunately, such biomarkers have not been identified for PD. Despite the observation of reduced GCase activity in PD patients, little substrate accumulation is seen in accessible biological fluids from patients (as evaluated by the Parkinson’s Progression Markers Initiative), and little substrate accumulation is seen even in postmortem brain tissue samples. This is probably because patients are suffering from a loss of up to about 50% of GCase function and, in the absence of additional factors, this is sufficient to process the substrate in most cells and tissues. In these patients, accumulation may be limited to specific cell types or for a short period of time. These issues have made it difficult to evaluate, using conventional plasma biomarkers, which patients are likely to benefit from therapeutic interventions. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] The mechanisms for identifying individuals with altered GSL metabolism despite having a normal GBA genotype are very limited. A decrease in GC-ase enzyme activity in the blood, followed by confirmation of GBA mutations, can readily identify patients with GD. Identifying heterozygous patients poses various challenges. Genetic analysis can routinely identify established GBA mutations that reduce GC-ase activity, but may not be able to inform the relevance of novel mutations. Further limitations are shown by the biochemical quantification of GC-ase activity, as patients with heterozygous GBA mutations exhibit large variations in activity that can largely overlap with non-GBA mutation carriers. Additionally, the presence of PD patients without GBA mutations but with reduced GC-ase activity suggests that alternative detection methods are warranted. Similarly, a potentially harmful increase in GSL synthesis is another mechanism that cannot be revealed by any of the current methods. In summary, there are numerous mechanisms that can lead to changes in GSL flux (increased production and / or decreased degradation), and there is no appropriate method for identifying these abnormalities. Therefore, new methods are needed for the diagnosis and monitoring of conditions related to abnormal glycosphingolipid processing.

Means for Solving the Problems

[0006] Based on the data presented herein, the present application relates to alternative methods for assessing defects in the GSL pathway (e.g., partial reduction in the activity of associated enzymes that would not be captured using conventional sphingolipid biomarkers), thereby overcoming the deficiencies in conventional methods in the diagnosis and monitoring of disease states. The methods described herein present an acute load to the glycosphingolipid (GSL) pathway in a patient's cells and then measure the response of the GSL pathway to this load (e.g., changes in GSL flux). By using the way the GSL pathway recovers after the load, the type and severity of the patient's condition can be indicated. In a general sense, GSL pathway loading involves intervening in this pathway (e.g., upregulating or downregulating one or more enzymes involved in the GSL pathway), whereby differences between individuals become apparent when monitoring the recovery from the load. An example of GSL pathway loading described herein is the acute limitation of GC-ase activity that attempts to stress this sphingolipid catabolic pathway to the maximum extent possible. Conduritol-β-epoxide (CBE) is an irreversible covalent binder and inhibitor of glucocerebrosidase that can be used to acutely limit GC-ase activity. When cells or animals are treated with this enzyme, the existing pool of GC-ase will be permanently inactivated. Therefore, further degradation of the rapidly accumulating substrate is only possible via newly translated GC-ase enzymes. This requires that new enzyme molecules be efficiently synthesized, folded, and transported to the lysosome. By measuring the temporal changes and extent of GSL accumulation and recovery after CBE clearance or removal, it is possible to evaluate the overall efficiency of enzyme synthesis, transport, and catalytic activity. This method reveals significant GSL flux abnormalities that are not apparent by other methods and provides a more general assessment of the GSL pathway that can be used to investigate abnormal GSL processing, as well as disease states, progression, and treatment efficacy. Other suitable loading conditions will be apparent to those skilled in the art within the context of the present disclosure.

[0007] Accordingly, a first aspect provides a method of evaluating a subject for abnormal glycosphingolipid processing, the method comprising treating cells of the subject to impose a load on the glycosphingolipid pathway in the cells and monitoring the recovery of at least one glycosphingolipid after the load. In embodiments, imposing the load comprises contacting the cells with an agent that inhibits an enzyme involved in the glycosphingolipid pathway. In embodiments, the enzyme is glucocerebrosidase. In embodiments, the agent is selective for one enzyme involved in the glycosphingolipid pathway.

[0008] The agent can be an irreversible inhibitor of the enzyme; for example, the inhibitor can covalently bind to the enzyme. In embodiments, the agent has the formula (I):

Chemical formula

[0009] The step of treating the cells of the subject to impose a load on the glycosphingolipid pathway in the cells can include contacting the cells with conduritol-β-epoxide (CBE).

[0010] In an embodiment, this method is an in vitro method, where the cells are present in a sample obtained from a subject, and it is an in vitro method. In a particular embodiment, this sample is a tissue sample (e.g., a tissue sample from the subject's brain, liver, kidney, skin, or spleen), or this sample is a cell-containing blood sample (e.g., whole blood).

[0011] In an embodiment, this cell is a fibroblast, a peripheral blood mononuclear cell (PMBC), or an induced pluripotent stem cell (iPSC) derived from the somatic cells of the subject. In a particular embodiment, this cell is a peripheral blood mononuclear cell obtained (e.g., purified) from a blood sample collected from the subject.

[0012] In an embodiment, this cell expresses the neuroepithelial stem cell protein (nestin).

[0013] In an embodiment, monitoring the recovery of at least one glycosphingolipid after loading involves measuring the level of at least one glycosphingolipid in or produced by the cells multiple times after loading, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In some embodiments, monitoring the recovery of at least one glycosphingolipid after loading involves measuring the level of at least one glycosphingolipid in or produced by the cells until essentially reaching the pre-treatment (baseline) level, e.g., until reaching a level within about 10% or within about 5% of the pre-treatment level.

[0014] In embodiments, at least one glycosphingolipid is or comprises a lipid selected from glucosylceramide (GL1), ceramide, and glucosylsphingosine (lyso-GL1). In certain embodiments, at least one glycosphingolipid is or comprises total GL1, total ceramide, or total lyso-GL1. In embodiments, at least one glycosphingolipid is or comprises a lipid comprising a monounsaturated fatty acid moiety selected from, for example, C16:1, C18:1, C20:1, C22:1, or C24:1. In embodiments, at least one glycosphingolipid is or comprises a lipid comprising a fatty acid moiety selected from C24:0, C16:1 n-7, C18:1 n-7, C22:1 n-9, C24:1 n-9, and C18:2 cis. In some embodiments, monitoring the recovery of at least one glycosphingolipid after loading does not include monitoring the recovery of a lipid comprising a C18:0 fatty acid moiety and / or a lipid comprising a C18:1 fatty acid moiety.

[0015] This method may further comprise comparing the recovery of at least one glycosphingolipid after loading with the recovery of the same glycosphingolipid after the same loading to corresponding cells from a healthy individual. In embodiments, the comparing step comprises comparing one or more of the following parameters (derivable from a concentration vs. time plot): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) the time required to reach C max after loading (T max ); (iii) the area under the curve (AUC); and (iv) the time required for the level to return to the baseline, for example, the time required for the level to return within about 10% or within about 5% of the baseline level. In such embodiments, if the difference in the parameters is greater than about 10%, for example, greater than about 20%, greater than about 30%, greater than about 50%, greater than about 75%, greater than about 100%, greater than about 150%, or greater than about 200%, the subject may be evaluated as having abnormal glycosphingolipid processing.

[0016] A second aspect provides a method for determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject, the method comprising evaluating the subject with respect to abnormal glycosphingolipid processing according to the first aspect, and determining the severity of the disease based on how different the recovery is compared to the recovery of cells from a healthy individual similarly challenged. In embodiments, the disease is a lysosomal storage disease, such as Gaucher disease. In other embodiments, the disease is a synucleinopathy such as Parkinson's disease (PD), such as idiopathic PD.

[0017] In some embodiments, the mutation status of the GBA gene in the subject is unknown or the subject is evaluated as having only one or zero known mutant (e.g., non-functional or hypo-functional) GBA alleles. In some embodiments, the subject has not been previously diagnosed with a disease associated with abnormal glycosphingolipid processing, such as a lysosomal storage disease such as Gaucher disease or Parkinson's disease.

[0018] In other embodiments, the subject has been previously diagnosed with a disease associated with abnormal glycosphingolipid processing and the method determines the progression of the disease.

[0019] A third aspect is an in vitro method for diagnosing or monitoring the progression of a synucleinopathy such as Parkinson's disease (PD) in a subject, such as idiopathic PD, comprising: - obtaining a sample from the subject, optionally culturing the cells, the sample comprising fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from the somatic cells of the subject; - contacting the sample with a CBE; - measuring the levels of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cells over a plurality of times to obtain a response curve; and - comparing this reaction curve with a comparative reaction curve, which is a standard reaction curve created by subjecting the same sample obtained from (a) a healthy individual or (b) a patient with a confirmed diagnosis of PD (for diagnosing the subject) to a load, or the comparative curve is a reaction curve from a sample previously obtained from the same subject (for monitoring the progression of the disease in this subject), and comparing them provides a method comprising.

[0020] A fourth aspect is a method for evaluating a possible therapeutic response of a subject to treatment with an inhibitor of glucosylceramide synthase (GCS) or an activator of glucocerebrosidase, the method comprising contacting cells of the subject with a CBE to impose a load on the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the load, and if the recovery is slow or incomplete compared to the recovery of cells from a healthy individual similarly loaded, evaluating the subject as a candidate for treatment. In embodiments, the GCS inhibitor is miglustat or a pharmaceutically acceptable salt thereof. The cells can be fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from somatic cells of the subject. In certain embodiments, the cells are peripheral blood mononuclear cells obtained (e.g., purified) from a blood sample taken from the subject.

[0021] Related aspects provide the use of conduritol-β-epoxide in a method for diagnosing or determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject. In embodiments, the disease is a lysosomal storage disease (e.g., Gaucher's disease), or a synucleinopathy such as Parkinson's disease (e.g., idiopathic PD).

[0022] A fifth aspect is a method for evaluating a therapeutic response of a subject to treatment with an inhibitor of glucosylceramide synthase (GCS) or an activator of glucocerebrosidase (GCase), (a) At an initial time point, obtaining a first cell-containing sample from the subject; (b) Contacting the cells of the first cell-containing sample with a CBE to impose a load on the glycosphingolipid pathway in the cells and monitoring the recovery of at least one glycosphingolipid after the load; (c) At a second time point after treatment of the subject with an inhibitor of GCS or an activator of GC-ase, obtaining a second cell-containing sample from the subject; and (d) Contacting the cells of the second cell-containing sample with a CBE to impose a load on the glycosphingolipid pathway in the cells and monitoring the recovery of at least one glycosphingolipid after the load comprising, wherein if the recovery of the cells from the second cell-containing sample is faster or more complete compared to the recovery of the cells from the first cell-containing sample, the treatment is evaluated as positive, A method is provided. In an embodiment, the treatment of the subject is treatment with bengulstat, an inhibitor of GCS, or a pharmaceutically acceptable salt thereof.

[0023] In an embodiment, the cells of the first cell-containing sample and / or the cells of the second cell-containing sample are independently selected from fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from the somatic cells of the subject.

[0024] In some embodiments, steps (a) and (b) are performed in essentially the same manner as steps (c) and (d) of the method, the first cell-containing sample and the second cell-containing sample comprise essentially the same cell type, and / or at least one glycosphingolipid monitored in step (b) is the same as at least one glycosphingolipid monitored in step (d).

[0025] The sixth aspect is a method for treating a lysosomal storage disorder or synucleinopathy in a subject in need thereof, comprising administering to the subject an agent capable of treating the lysosomal storage disorder or synucleinopathy, the method comprising monitoring a treatment response according to the fifth aspect, and changing the dosage of the agent according to the result of the monitoring. In an embodiment, if the treatment is evaluated as positive, the dosage of the agent is kept the same, and if the treatment is evaluated as not positive, the dosage of the agent is increased, or if the treatment is evaluated as positive, the dosage of the agent is decreased, and the monitoring step is continued until the treatment is no longer evaluated as positive, and at this point of evaluation, the dosage is increased until the treatment is again evaluated as positive.

[0026] The seventh aspect is a method for treating a lysosomal storage disorder or synucleinopathy in a subject in need thereof, comprising administering an effective amount of an agent capable of treating the lysosomal storage disorder or the synucleinopathy, wherein the subject has been evaluated as having abnormal glycosphingolipid processing according to the method of the first aspect or an embodiment thereof, or the disease state or severity has been evaluated according to the method of the second or third aspect.

[0027] The eighth aspect is a method for treating or preventing the onset or progression of a lysosomal storage disorder or synucleinopathy in a subject who has been evaluated as having a risk of developing a lysosomal storage disorder or synucleinopathy according to the method of the second or third aspect, (a) initiating a series of therapeutic treatments in the subject; and optionally, (b) evaluating or repeating the evaluation of the risk of developing a lysosomal storage disorder according to the method of the second or third aspect, and optionally adjusting the therapeutic treatment based on the new evaluation is provided.

[0028] Further features and advantages of the compounds, compositions, and methods disclosed herein will be apparent from the following detailed description.

Brief Description of the Drawings

[0029]

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DETAILED DESCRIPTION OF THE INVENTION

[0030] Hereinafter, specific embodiments of the present disclosure will be described with reference to preparations and schemes, but it should be understood that such embodiments are merely illustrative and represent only a small part of many possible specific embodiments that can represent the application of the principles of the present disclosure. Various changes and modifications will be apparent to those skilled in the art in view of the benefits of the present disclosure and are considered to be within the spirit and scope of the present disclosure further defined in the appended claims.

[0031] Definitions 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 disclosure, exemplary methods, devices, and materials are described herein. All technical and patent publications mentioned herein are hereby incorporated by reference in their entirety. Nothing in this specification shall be construed as an admission that the present disclosure has a right to antedate such disclosure by virtue of prior disclosure.

[0032] The practice of the present disclosure, unless otherwise indicated, uses conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, for example: Michael R. Green and Joseph Sambrook, Molecular Cloning (4 th ed., Cold Spring Harbor Laboratory Press 2012); the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0033] All numerical designations (e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges) are approximate values that vary, where appropriate, in increments of, for example, 0.1 or 1.0, either (+) or (-). Although not necessarily explicitly stated, it should be understood that there is a term "about" used before all numerical designations to indicate the conventional level of variability. For example, a numerical designation that is "about" a given value can vary by ±10% of that value, or this variation can be ±5%, ±2%, or ±1% of this value. Also, although not necessarily explicitly stated, it should be understood that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0034] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells (including mixtures thereof). Unless otherwise specified or clear from the context, as used herein, the term "or" is understood to be inclusive. The term "comprising" is used herein to mean "including but not limited to" and is used synonymously with this phrase.

[0035] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of" is meant to exclude other elements that are essential for the stated purpose when used to define compositions and methods. Thus, a composition consisting essentially of the elements defined herein will not exclude minor impurities from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, etc. "Consisting of" is meant to exclude the exclusion of other components in excess of trace elements, as well as substantial method steps for administering the disclosed compositions, or process steps for manufacturing the composition or achieving the intended result. Embodiments defined by each of these transition terms are within the scope of the present disclosure. The use of the term "comprising" herein is intended to encompass both "consisting essentially of" and "consisting of".

[0036] "Subject", "individual", or "patient" are used interchangeably herein and refer to vertebrates such as mammals. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, livestock, sport animals, pets, horses, primates, and humans. In embodiments, mammals include horses, dogs, and cats. In embodiments, the mammal is a human.

[0037] As used herein, the term "healthy individual" typically refers to an individual who does not suffer from synucleinopathy and / or does not have any GBA mutations. A healthy individual may lack mutations in any gene encoding an enzyme involved in the glycosphingolipid pathway, for example, may lack mutations in genes encoding: ceramide synthase, glucosylceramide synthase, galactosylceramide synthase, lactosylceramide synthase, sphingomyelin synthase, ceramidase, glucocerebrosidase, saposin, galactosylceramide β-galactosidase, acid sphingomyelinase, arylsulfatase A, α-galactosidase A, β-hexosaminidase (e.g., Hex A or Hex B), sialidase, GM1-β-galactosidase, GM2 ganglioside activator protein, glucosyltransferase, and galactosyltransferase.

[0038] As used herein, "administering" is defined as a means of providing to a subject a drug or a composition containing the drug by a method that causes the drug to be present within the body of the subject. Such administration can be by any route, including, but not limited to, oral administration, transdermal (e.g., vaginal, rectal, oral mucosa) administration, administration by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, into the CNS), or administration by inhalation (e.g., oral or nasal). The pharmaceutical is, of course, administered in a form suitable for each route of administration.

[0039] "Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e., not causing the clinical symptoms of the disease to develop in a patient who may be susceptible to the disease but has not yet experienced or manifested the symptoms of the disease; (2) inhibiting the disease, i.e., stopping or reducing the development of the disease or its clinical symptoms; and / or (3) alleviating the disease, i.e., causing regression of the disease or its clinical symptoms.

[0040] The term "suffering from", when related to the term "treatment", refers to a patient or individual who has been diagnosed with a disease or is susceptible to a disease. A patient may also be said to be "at risk of suffering from" a disease, for example, due to a family history of the disease in their family line or the presence of a genetic mutation related to the disease. A patient at risk of a disease has not yet developed all or part of the characteristic pathological condition of this disease.

[0041] "Effective amount" or "therapeutically effective amount" means an amount sufficient to produce a beneficial or desired result. The effective amount can be administered in one or more doses, applications, or dosages. Such delivery depends on many variables, including the period during which individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, etc. However, the specific dosage level of a therapeutic agent for any particular subject depends on various factors (such as the activity of the specific compound utilized, the age, weight, general health status, sex, and diet of the subject, the time of administration, the rate of excretion, the combination of drugs, as well as the severity of the particular disorder being treated, and the dosage form). The treatment dosage can generally be titrated to optimize safety and effectiveness. Typically, the dose - effect relationship from in vitro tests and / or in vivo tests can first provide useful guidance regarding the appropriate dosage for patient administration. Generally, it would be desirable to administer an amount of the compound effective to achieve a serum level commensurate with the concentration found to be effective in vitro. These considerations, as well as effective formulations and administration procedures, are known in the art and are described in standard texts. Consistent with this definition, as used herein, the term "therapeutically effective amount" means an amount sufficient to treat (e.g., improve) one or more symptoms associated with a neurodegenerative condition. For example, oral administration may require a total daily dose of 0.1 mg to 1000 mg. The total daily dose can be administered as a single dose or divided doses, and the total daily dose can, at the discretion of the physician, fall outside the typical ranges described herein.

[0042] As used herein, the term "pharmaceutically acceptable excipient" encompasses any of the standard pharmaceutical excipients including carriers such as phosphate buffered saline, water, and emulsions (e.g., oil / water emulsion or water / oil emulsion), and various types of wetting agents. The pharmaceutical composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Remington’s Pharmaceutical Sciences (20th ed., Mack Publishing Co. 2000).

[0043] As used herein, the term "pharmaceutically acceptable salt" means a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt of a compound of the present disclosure that can be administered without producing any substantial undesirable biological effects or adverse interactions with any other constituent of the pharmaceutical composition that may be included.

[0044] The addition salts can be readily prepared using conventional techniques, for example, by treating a base compound with a predetermined amount of a selected mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as methanol or ethanol. Positively charged compounds (e.g., compounds containing quaternary ammonium) can also form salts with the anionic constituents of various inorganic and / or organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts are those that can form non-toxic acid addition salts, for example, those that can form salts containing pharmaceutically acceptable anions, for example, chlorides, bromides, iodides, nitrates, sulfates or bisulfates, phosphates or acid phosphates, acetates, lactates, citrates or acid citrates, tartrates or bitartrates, succinates, malates, maleates, fumarates, gluconates, saccharates, benzoates, methanesulfonates, and pamoates [i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts. The bases that can be used to prepare pharmaceutically acceptable base addition salts are those that can form non-toxic base addition salts, for example, those that can form salts containing pharmaceutically acceptable cations, for example, alkali metal cations (e.g., potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium, or other water-soluble amine addition salts (e.g., N-methylglucamine (meglumine)), lower alkanolammonium, and other such bases of organic amines that can form salts.

[0045] Any reference in this specification to a compound shall be construed as including a reference to its pharmaceutically acceptable salts, even if not explicitly stated. Thus, a reference to "benglutide" includes pharmaceutically acceptable salts of benglutide (e.g., benglutide malate). Similarly, a reference to "eliglustat" includes pharmaceutically acceptable salts of eliglustat (e.g., eliglustat hemicitrate). Benglutide is (S)-1-azabicyclo[2.2.2]octan-3-yl N-[2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl]carbamate. Eliglustat is N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]octanamide.

[0046] The recitation of embodiments with respect to a variable or aspect in this specification includes that embodiment as a single embodiment or in combination with any other embodiment or portion thereof.

[0047] The following abbreviations are used in this specification. AD Alzheimer's disease AUC Area Under the Curve (pharmacokinetics) CBE Conzilitol-β-epoxide Cmax Maximum Concentration (pharmacokinetics) CNS Central Nervous System DLB Dementia with Lewy bodies DNA Deoxyribonucleic acid EC 50 Half maximal effective concentration ERT Enzyme replacement therapy FD Fabry disease GBA Gene encoding GC-ase GC-ase Glucosylceramidase (also called acid β-glucosidase) GCS Glucosylceramide synthase GD Gaucher disease GL1 Glucosylceramide GL2 lactosylceramide GL3 globotriaosylceramide GM1 monosialotetrahexosylganglioside GM2 monosialotrihexosylganglioside GM3 monosialodihexosylganglioside GSL glycosphingolipid Hex β-hexosaminidase IC 50 Half-maximal inhibitory concentration iPSC induced pluripotent stem cell KD knockdown lyso-GL1 glucosylsphingosine lyso-GL2 lactosylsphingosine lyso-GL3 globotriaosylsphingosine LSD lysosomal storage disorder MPS mucopolysaccharidosis NCL neuronal ceroid lipofuscinosis OE overexpression PBMC peripheral blood mononuclear cell PCR polymerase chain reaction PC phosphatidylcholine PD Parkinson's disease RNA ribonucleic acid SAP saposin SD standard deviation SEM standard error of the mean SRT substrate reduction therapy Tmax Time at maximum concentration (pharmacokinetics) WT wild type

[0048] Method for imposing a load on GSL processing in cells As will be described in more detail in the examples below, the present disclosure is directed to a novel assay that can assess lipid flux in cells over a short period of time, for example, through perturbation of the sphingolipid pathway at the GC argenode (GCase node). The assay exemplified herein uses conduritol-β epoxide (CBE), an irreversible covalent binder and inhibitor of glucocerebrosidase, which, when administered intraperitoneally, for example, inhibits GC ase and enables measurement of subsequent lipid accumulation and recovery or flux of lipids (e.g., GL1 and / or lyso-GL1). The following three genotype conditions: homozygous knockout (Gba - / - ), heterozygous knockout (Gba - / + ), and wild type (Gba + / + ) using a GC ase mouse model to observe patterns of lipid flux correlated with CBE dose and genotype. It has also been observed that wild-type mice with different genetic backgrounds exhibit variable lipid fluxes depending on their genotype. These lipid flux patterns were reproduced in vitro using a human SH-SY5Y neuroblastoma cell line that was genetically modified to overexpress GBA or genetically modified to have reduced GC ase activity due to GBA knockdown. Lipid levels responsive to CBE treatment were shown to be inversely correlated with GBA levels.

[0049] New evidence suggests that patients who are carriers of idiopathic non-GBA mutations may exhibit reduced GCase activity and / or increased glycolipid accumulation in the central nervous system (CNS). This highlights the potential to expand the target population for substrate reduction therapy based on interrelated cellular and biochemical mechanisms (using drugs that modulate the GSL pathway, such as miglustat). From the results presented herein, it is suggested that elucidating GSL trafficking disorders by "loading" assays (e.g., using CBE, etc.) may expand the use of therapies targeting this pathway to a broader patient population. Data generated in vivo in mice and in vitro in human cell lines can be extrapolated to the clinical setting, and this disclosure describes how patient cells such as peripheral blood mononuclear cells (PBMCs) and / or fibroblasts can be obtained, cultured, and processed to reveal their susceptibility to GSL pathway perturbation. This, in turn, may help expand the patient population that would benefit from miglustat and related therapies.

[0050] Broadly speaking, this disclosure provides a method of imposing a load on the GSL pathway in cells from a subject and monitoring the response of the pathway to this load (e.g., recovery of the pathway). This can be used to assess a subject for abnormal glycosphingolipid processing (or to determine the severity of a disease associated with abnormal glycosphingolipid processing), or to diagnose, monitor, or assess a potential or actual therapeutic response of a subject to treatment with a drug that modulates the GSL pathway.

[0051] Thus, in a first aspect, this disclosure provides a method of assessing a subject for abnormal glycosphingolipid processing, the method comprising treating cells of the subject to impose a load on the glycosphingolipid pathway in the cells and monitoring the recovery of at least one glycosphingolipid after the load.

[0052] Typically, the load presented to the cell involves contacting the cell with an agent that modulates (e.g., inhibits) an enzyme involved in the glycosphingolipid pathway. Examples of enzymes that can be targeted include: (a) lipid synthases, such as ceramide synthase, glucosylceramide synthase, galactosylceramide synthase, lactosylceramide synthase, and sphingomyelin synthase; (b) lipid hydrolases, such as ceramidase, glucocerebrosidase, saposin (e.g., Sap A, B, C, or D), galactosylceramide β-galactosidase, acid sphingomyelinase, arylsulfatase A, α-galactosidase A, β-hexosaminidase (e.g., Hex A or Hex B), sialidase, GM1-β-galactosidase, and GM2 ganglioside-activating protein; and (c) glycosyltransferases, such as glucosyltransferase, and galactosyltransferase. In embodiments, the agent activates a lipid synthase enzyme. In other embodiments, the agent activates a glycosyltransferase enzyme. In other embodiments, the agent inhibits a lipid hydrolase. In one embodiment, the enzyme is glucocerebrosidase. In one embodiment, the enzyme is glucocerebrosidase and the agent is an inhibitor of glucocerebrosidase.

[0053] In embodiments, the agent is selective for one enzyme involved in the glycosphingolipid pathway. For example, the agent has at least 10-fold higher activity (e.g., as measured by IC 50 value or EC 50 value) for one enzyme compared to other enzymes involved in the glycosphingolipid pathway. In embodiments, the agent has 15, 20, 25, 30, 50, 75, 100, 150, 200, 500, or 1000-fold higher activity for one enzyme compared to other enzymes involved in the glycosphingolipid pathway.

[0054] ​In an embodiment, this agent is an irreversible regulator of an enzyme involved in the glycosphingolipid pathway. For example, this agent can covalently bind to this enzyme and block, for example, the active site of this enzyme, or block the interaction between this enzyme and another biological molecule necessary for its activity. Alternatively, this agent can target the enzyme for inactivation or degradation. In an embodiment, this agent is an irreversible inhibitor of an enzyme involved in the glycosphingolipid pathway. In one embodiment, this agent is an irreversible inhibitor that covalently binds to this enzyme.

[0055] Agents that strongly bind (e.g., covalently bind) to enzymes involved in the glycosphingolipid pathway are known to those skilled in the art (see, for example, Canals et al., Br J Pharmacol. (2011) 163(4):694 - 712 for details of agents targeting sphingomyelinase and ceramidase). Such agents are often structural analogs of the natural substrate of the enzyme, for example, mimicking the transition state of the natural reaction intermediate and / or being structural analogs containing reactive chemical groups that can form covalent bonds with the enzyme. Exemplary covalent regulators include the small - molecule inhibitors of glucocerebrosidase described by Kuo et al. (The FEBS Journal (2019) 286:584 - 600). This compound is a transition - state analog containing an epoxide group that captures a carboxylic acid on the enzyme. Among the inhibitors described by Kuo et al., is conduritol - β - epoxide (CBE), which is highly selective for glucocerebrosidase over other glycosidases (with an IC 50 value of about 0.3 μM for GC - ase compared to values in the range of 200 - 2000 μM for non - lysosomal glucosylceramidase, β - glucuronidase, and α - glucosidase).

[0056] Therefore, in an embodiment, this agent has the formula (I):

Chemical formula

[0057] In one embodiment, the agent is a CBE. Thus, from this aspect, the present disclosure provides a method of evaluating a subject with respect to abnormal glycosphingolipid processing, the method comprising contacting cells of the subject with a CBE to impose a load on the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after said load.

[0058] In practice, this method is typically performed on a sample obtained from the subject. Thus, in an embodiment, the method is an in vitro method, wherein the cells are present in a sample obtained from the subject. The method may include obtaining a sample from the subject for direct analysis. Alternatively, the method may be performed on a sample previously obtained from the subject, for example, on a sample that has been stored and optionally reconstituted (e.g., in the case of a dried blood sample, or a biopsy sample frozen using liquid nitrogen). The sample may be processed to enrich or concentrate (or may be enriched or concentrated) the cells to be loaded. Alternatively, or in addition, the sample may be processed to remove unwanted sample material (e.g., tissue such as connective tissue) (or may be removed).

[0059] The methods of the present disclosure can utilize tissue samples (e.g., samples of tissue from a subject's brain, liver, kidney, skin, or spleen). The methods of the present disclosure can also utilize samples of cell-containing biological fluids (e.g., whole blood) or cell-containing fractions thereof. Methods for obtaining such samples are known to those of skill in the art. Thus, in embodiments, the sample is a tissue sample (e.g., from a subject's brain, liver, kidney, skin, or spleen), or the sample is a cell-containing blood sample (e.g., whole blood). In certain embodiments, the tissue sample is a sample of brain, liver, kidney, skin, or spleen. In other certain embodiments, the blood sample is whole blood. Whole blood and cell-containing fractions thereof are particularly convenient for use in accordance with the methods.

[0060] In embodiments, the method treats cells of a particular type (e.g., lineage). Thus, the methods of the present disclosure can utilize a sample that contains the cell type, or a purified, passaged, and / or expanded cell population that can consist essentially of the cell type. The use of partially or fully purified cells and cell populations can improve the sensitivity of the method. Examples of cell types that can be used in accordance with the present disclosure include fibroblasts, peripheral blood mononuclear cells (PMBC), and induced pluripotent stem cells (iPSC). Methods for obtaining, purifying, passaging, and / or expanding such cells are known to those of skill in the art. It will be understood that the methods described herein as being performed on "cells" include methods in which a plurality of such cells are used and optionally other cells or cell types may also be present.

[0061] Therefore, in one embodiment, the cell is a fibroblast, and in related embodiments, the sample comprises (or consists essentially of) fibroblasts. Fibroblasts can be obtained, for example, from the subject's skin tissue (e.g., from a skin punch biopsy) using conventional techniques. In another embodiment, the cell is an iPSC, and in related embodiments, the sample comprises (or consists essentially of) iPSCs. iPSCs are generated from the patient's somatic cells by reprogramming fibroblasts that can be obtained, for example, as described herein. Other sources of somatic cells for the generation of iPSCs include keratinocytes (which can be obtained, for example, from hair follicles), peripheral blood cells, and renal epithelial cells (which can be obtained, for example, from urine). In another embodiment, the cell is a (e.g., purified) PBMC obtained from a blood sample collected from the subject; in related embodiments, the sample comprises (or consists essentially of) a (e.g., purified) PBMC obtained from a blood sample collected from the subject. In an embodiment, the cell expresses the neuroepithelial stem cell protein (nestin).

[0062] The method includes monitoring the recovery of the GSL pathway after loading. Based on the disclosure herein, there are various ways in which this monitoring can be achieved, and the most appropriate way may vary in different situations, for example, when different drugs are used to load different parts of the GSL pathway and / or when different samples or cell types are utilized. Those skilled in the art will understand that this may vary. The following discussion focuses on the situation exemplified below (i.e., monitoring GSL recovery after treatment of cells with CBE for specifically loading the GC ase node). If a load is applied to other parts of the GSL pathway, this assessment may be modified (as necessary) based on the present disclosure.

[0063] According to a first aspect of the present disclosure, monitoring the recovery of the GSL pathway against a load is achieved by monitoring the recovery of at least one glycosphingolipid. In embodiments where the load comprises contacting a cell with an agent that modulates (e.g., inhibits) an enzyme involved in the glycosphingolipid pathway, the at least one glycosphingolipid to be monitored comprises one or more of the following: (i) a substrate of the enzyme; (ii) an upstream precursor of the substrate of the enzyme; (iii) a product of the enzyme; and (iv) a downstream metabolite of the product of the enzyme.

[0064] Suitable glycosphingolipids that can be used to monitor recovery include GL1, lyso-GL1, GL2, lyso-GL2, GL3, lyso-GL3, galactosylceramide, GM1, GM2, and GM3, and, for example, ceramide and sphingosine. In embodiments, the at least one glycosphingolipid is (or comprises) a lipid selected from glucosylceramide (GL1), ceramide, and glucosylsphingosine (lyso-GL1). In embodiments, the at least one glycosphingolipid is (or comprises) total GL1, total ceramide, or total lyso-GL1. These embodiments are particularly useful when the agent is an inhibitor of GC-ase. In other embodiments, the at least one glycosphingolipid is (or comprises) total GL3, total ceramide, or total lyso-GL3.

[0065] In embodiments, the at least one glycosphingolipid is (or comprises) a lipid (e.g., GL1) that contains a fatty acid moiety having a low baseline abundance (i.e., a low abundance prior to any load being presented to the GSL pathway). Such a fatty acid can be found at a level of less than 25% of the total fatty acids on the lipid in question (on a molar basis), for example, less than 20%, less than 15%, less than 10%, less than 8%, less than 5%, less than 2%, or less than 1% of the total fatty acids on the lipid in question.

[0066] In an embodiment, at least one glycosphingolipid is (or comprises) a lipid containing a monounsaturated fatty acid moiety (e.g., GL1). For example, this fatty acid moiety can be selected from C16:1, C18:1, C20:1, C22:1, C23:1, C24:1, or C26:1. In an embodiment, this fatty acid moiety is selected from C16:1, C18:1, C20:1, C22:1, and C24:1. For example, this fatty acid moiety can be selected from C16:1, C18:1, and C20:1, or can be selected from C16:1 and C18:1. In one embodiment, at least one glycosphingolipid is (or comprises) a lipid containing a C16:1 fatty acid moiety. In other embodiments, at least one glycosphingolipid is (or comprises) a lipid containing a fatty acid moiety selected from C24:0, C16:1 n-7, C18:1 n-7, C22:1 n-9, C24:1 n-9, and C18:2 cis. In an embodiment, monitoring the recovery of at least one glycosphingolipid after loading does not include monitoring the recovery of a lipid containing a C18:0 fatty acid moiety (e.g., GL1) and / or a lipid containing a C18:1 fatty acid moiety.

[0067] In an embodiment, the method utilizes a single measurement to monitor GSL recovery, for example, measuring the level of at least one GSL at a single set point after loading. In other embodiments, this monitoring includes monitoring GSL recovery over a period of time, for example, measuring the level of at least one GSL at multiple time points after loading. For example, monitoring the recovery of at least one glycosphingolipid after loading may include measuring the level of at least one glycosphingolipid in or produced by the cell multiple times after said loading, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In an embodiment, monitoring the recovery of at least one glycosphingolipid after loading includes measuring the level of at least one glycosphingolipid in or produced by the cell at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment, monitoring the recovery of at least one glycosphingolipid after loading includes measuring the level of at least one glycosphingolipid in or produced by the cell until essentially reaching the pre-treatment (baseline) level, for example, until reaching a level within about 10% or within about 5% of the pre-treatment level. This provides information regarding the recovery of the GSL pathway in terms of both the time required to return to the baseline GSL level and the profile of GSL recovery (e.g., the shape of the curve when concentration is plotted as a function of time).

[0068] As an alternative to (or in addition to) monitoring the recovery of the GSL pathway in a cell as compared to the baseline level from the cell, it is useful to compare the recovery of the GSL pathway in the subject's cells with the recovery in the corresponding cells of a healthy individual. Such a comparison can be useful for identifying abnormal GSL processing, particularly when the abnormality is not extremely severe. Thus, in embodiments, the method further comprises comparing the recovery of at least one glycosphingolipid after loading with the recovery of the same glycosphingolipid after the same loading in corresponding cells from a healthy individual. In embodiments, the comparing step comprises comparing one or more of the following parameters (which can be derived, for example, from a concentration vs. time plot): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) the time (T max ) required to reach C max after loading; (iii) the area under the curve (AUC); and (iv) the time required for the level to return to the baseline, e.g., the time required for the level to return within about 10% or about 5% of the baseline level. Using these metrics, if the difference in the parameters is, for example, greater than about 10%, e.g., greater than about 20%, greater than about 30%, greater than about 50%, greater than about 75%, greater than about 100%, greater than about 150%, or greater than about 200%, the subject can be evaluated as having abnormal glycosphingolipid processing. In embodiments (e.g., when the sample is or comprises PBMCs obtained from the subject), the recovery of the glycosphingolipid is monitored over a period of up to about 72 hours after loading, e.g., over a period of up to about 64 hours, about 56 hours, about 48 hours, about 40 hours, about 32 hours, or about 24 hours after loading.

[0069] The methods of the present disclosure can also be used to evaluate disease states in an individual. Thus, in a second aspect, the present disclosure provides a method for determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject. This method comprises evaluating the subject for abnormal glycosphingolipid processing according to the aspects above, and determining the severity of the disease based on how different the recovery is compared to the recovery of cells from a healthy individual that was similarly challenged. Embodiments of the first aspect (e.g., regarding the agent used, sample type, and / or cell type, lipid being monitored, etc.) can be applied to this second aspect.

[0070] Diseases associated with abnormal glycosphingolipid processing include lysosomal storage diseases. Therefore, this aspect can be particularly useful for evaluating a subject known to have (or thought to have or predicted to have) a lysosomal storage disease. Such diseases typically involve changes in GSL levels, often as a result of deficiencies in one or more enzymes involved in GSL processing. As demonstrated in the examples below, a mouse model of Gaucher disease (GD) shows a significant difference in lipid flux after CBE treatment compared to wild-type mice.Thus, in embodiments, the method can be used to determine the severity of lysosomal storage disorders in a subject, such as Fabry disease, Krabbe disease, Gaucher disease (e.g., types 1, 2, and 3), Niemann-Pick disease (e.g., types A, B, and C), metachromatic leukodystrophy, Farber disease, Krabbe disease, galactosialidosis, Schindler disease, GM1 gangliosidosis, GM2 gangliosidosis (e.g., AB variant, Sandhoff disease, and Tay-Sachs disease), lysosomal acid lipase deficiency, Wolman disease, cholesterol ester storage disease, multiple sulfatase deficiency, Pompe disease, Danon disease, Salla disease, α-mannosidosis, β-mannosidosis, aspartylglucosaminuria, fucosidosis, MPS I (e.g., Hurler syndrome, Scheie syndrome, and Hurler-Scheie syndrome), MPS II (e.g., Hunter syndrome), MPS III (e.g., Sanfilippo syndromes A, B, C, and D), MPS IV (e.g., Morquio syndromes A and B), MPS VI (e.g., Maroteaux-Lamy syndrome), MPS VII (e.g., Sly syndrome), MPS IX (e.g., hyaluronidase deficiency), mucolipidosis (e.g., sialidosis, cell disease, pseudo-Hurler polydystrophy / phosphotransferase deficiency, and mucolipin 1 deficiency), and neuronal ceroid lipofuscinosis (e.g., Santavuori-Haltia disease / infantile NCL, Jansky-Bielschowsky disease / late infantile NCL, Batten-Spielmeyer-Vogt disease / juvenile NCL, Kufs disease / adult NCL, Finnish variant / type 5, late infantile variant / type 6, type 7, northern epilepsy / Turkish late infantile / type 8, German / Serbian late infantile / type 9, and congenital cathepsin D deficiency). In one embodiment, the disease is GD (e.g., type 1, 2, or 3 GD). In another embodiment, the disease is FD.

[0071] Diseases associated with abnormal glycosphingolipid processing include synucleinopathies. Thus, synucleinopathies are also included as other conditions that can be usefully investigated using the methods of the present disclosure. This is a neurodegenerative disease characterized by abnormal accumulation of aggregates of α-synuclein protein in neurons, nerve fibers, or glial cells. However, there is new evidence that synucleinopathies may be associated with abnormal GSL processing. Thus, in embodiments, the methods can be used to determine the severity of synucleinopathy in a subject, for example, the severity of a disease selected from Parkinson's disease (PD), such as idiopathic PD, or dementia with Lewy bodies (DLB). In embodiments, the disease is PD, for example, idiopathic PD.

[0072] The methods can be used to assess the disease state in an individual having a known or unknown genotype of a gene that represents a risk factor for developing a condition associated with abnormal GSL processing (e.g., lysosomal storage disease, or synucleinopathy). In embodiments, the mutation status of the GBA gene in the subject is unknown, and in other embodiments, the subject is evaluated as having only 1 or 0 known mutant (e.g., non-functional or hypofunctional) GBA alleles (see, e.g., Hruska et al., Hum. Mutat. (2008) 29(5):567-583). Similarly, the methods can be used to assess the disease state in an individual, regardless of whether the individual has been previously evaluated for a disease associated with abnormal glycosphingolipid processing. Thus, in embodiments, the subject has not been previously diagnosed with a disease associated with abnormal glycosphingolipid processing (e.g., a lysosomal storage disease such as Gaucher disease, or a synucleinopathy such as Parkinson's disease). In embodiments, the subject has not been previously diagnosed with Gaucher disease. In embodiments, the subject has not been previously diagnosed with Parkinson's disease. In other embodiments, the subject has been previously diagnosed with a disease associated with abnormal glycosphingolipid processing, and the methods determine the progression of the disease.

[0073] In a third aspect, the present disclosure is an in vitro method for diagnosing or monitoring the progression of synucleinopathy (e.g., as defined herein) in a subject, comprising: - obtaining a sample from the subject, optionally culturing the cells, the sample comprising fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from somatic cells of the subject; - contacting the sample with a CBE; - measuring the levels of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cells over a plurality of times to obtain a reaction curve; and - comparing the reaction curve with a comparison reaction curve, the comparison curve being a standard reaction curve created by loading the same sample obtained from (a) a healthy individual or (b) a patient with a confirmed diagnosis of PD (for diagnosing the subject), or the comparison curve being a reaction curve from a sample previously obtained from the same subject (for monitoring the progression of the disease in the subject). A method is provided that includes. Embodiments of the first and second aspects (e.g., regarding sample type, and / or cell type, lipid being monitored, disease state, etc.) are understood to be applicable to this third aspect.

[0074] In an embodiment, the sample is contacted with a CBE at a final concentration of from about 0.01 μM to about 100 μM. For example, the final concentration of the CBE can be from about 0.1 μM to about 10 μM, from about 0.3 μM to 8 μM, from about 0.5 μM to 5 μM, or from about 0.8 μM to 3 μM, for example, about 1 μM or about 2 μM. In an embodiment, an excipient such as sodium taurocholate is added to the sample to increase the dynamic range by, for example, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, for example, up to about 10-fold.

[0075] As with disease assessment, diagnosis, and monitoring, the method can also be usefully employed in the evaluation of the therapeutic response to treatment both before any treatment is initiated (e.g., predictive assessment) and during a treatment regimen (e.g., monitoring of response). Such methods typically evaluate the therapeutic response to treatment with a modulator of an enzyme involved in the glycosphingolipid pathway, imposing a load on the GSL pathway at or near the node represented by this enzyme. Glycosphingolipids closely related to this node (e.g., the substrate or product of the enzyme) are typically those monitored to assess recovery. Thus, for example, when a drug that is an inhibitor of glucosylceramide synthase (GCS) or an activator of glucocerebrosidase (GCase) is used, this load can be glucocerebrosidase (e.g., using CBE), and one or more of glucosylceramide (GL1), ceramide, and glucosylsphingosine (lyso-GL1) can be monitored.

[0076] Accordingly, in a fourth aspect, the present disclosure provides a method for evaluating a possible therapeutic response of a subject to treatment with an inhibitor of glucosylceramide synthase (GCS) or an activator of glucocerebrosidase (GCase), the method comprising contacting cells of the subject with CBE to impose a load on the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the load, and evaluating the subject as a candidate for treatment if the recovery is slow or incomplete compared to the recovery of cells from a healthy individual similarly loaded. It will be understood that embodiments of the foregoing aspects (e.g., with respect to sample type, and / or cell type, lipids monitored, etc.) can be applied to this fourth aspect.

[0077] In an embodiment, the method assesses a possible therapeutic response of a subject to treatment with a GCS inhibitor (e.g., selected from eliglustat and miglustat, including pharmaceutically acceptable salts thereof). In an embodiment, the GCS inhibitor is miglustat or a pharmaceutically acceptable salt thereof. In other embodiments, the method assesses a possible therapeutic response of a subject to treatment with a GCase activator selected from, for example, saposin C, ambroxol, N-(4-ethynylphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide, isofagomine, NN-DNJ, NCGC758, NCGC607, S-181, structurally targeted allosteric modulators (e.g., GT-02287 and GT-02329), and LTI-291.

[0078] In a fifth aspect, the present disclosure is a method of assessing a therapeutic response of a subject to treatment with an inhibitor of glucosylceramide synthase (GCS) or an activator of glucocerebrosidase (GCase), comprising: (a) obtaining a first cell-containing sample from the subject at a first time point; (b) contacting the cells of the first cell-containing sample with CBE to load the glycolipid pathway in the cells and monitoring the recovery of at least one glycolipid after the load; (c) obtaining a second cell-containing sample from the subject at a second time point after treatment of the subject with an inhibitor of GCS or an activator of GCase; and (d) contacting the cells of the second cell-containing sample with CBE to load the glycolipid pathway in the cells and monitoring the recovery of at least one glycolipid after the load wherein if the recovery of the cells from the second cell-containing sample is faster or more complete than the recovery of the cells from the first cell-containing sample, the treatment is evaluated as positive (e.g., successful). A method is provided. (For example, regarding sample type, and / or cell type, lipids to be monitored, etc.) It will be understood that embodiments of the foregoing aspects may be applied to this fifth aspect.

[0079] In an embodiment, steps (a) and (b) are performed in essentially the same manner as steps (c) and (d) of this method. In this way, the difference in GSL recovery in cells from the first sample and the second sample may best reflect the effectiveness of the treatment. Therefore, typically, the first cell-containing sample and the second cell-containing sample contain essentially the same cell type. For example, they are derived from the same type of tissue that has been handled and processed in essentially the same manner. Similarly, typically, at least one glycosphingolipid being monitored in step (b) may be the same as at least one glycosphingolipid being monitored in step (d).

[0080] The previous aspect also promotes the optimization of treatment methods in a subject, for example, by providing a method for treating a disease associated with abnormal glycosphingolipid processing in a subject, comprising administering to the subject an agent capable of treating the disease, monitoring the treatment response to the agent, and appropriately changing the dosage of the agent.

[0081] Therefore, in a sixth aspect, the present disclosure provides a method for treating a disease associated with abnormal glycosphingolipid processing in a subject in need thereof (for example, a lysosomal storage disease or synucleinopathy as defined herein), comprising administering to the subject an agent capable of treating the lysosomal storage disease or synucleinopathy, the method comprising monitoring the treatment response according to the fifth aspect (above), and changing the dosage of the agent according to the result of this monitoring. (For example, regarding sample type, and / or cell type, lipids to be monitored, etc.) It will be understood that embodiments of the foregoing aspects may be applied to this sixth aspect.

[0082] In an embodiment, when the treatment is evaluated as positive, the dosage of this drug is kept the same, and when the treatment is evaluated as not positive, the dosage of this drug is increased. In other embodiments, when the treatment is evaluated as positive, the dosage of this drug is decreased, and the monitoring step is continued until the treatment is no longer evaluated as positive, and at this point of evaluation, the dosage is increased until the treatment is evaluated as positive again. In an embodiment, the monitoring step is carried out at regular intervals to evaluate the continuous effectiveness of the treatment and / or to enable adjustment of the dosage so that the treatment can be evaluated as positive. The monitoring step can be carried out, for example, at intervals of 1, 2, 3, 4, 5, 6, 12, 18, 24 months, or more months. In an embodiment, the monitoring step is carried out more frequently (e.g., every 1, 2, 3, or 4 weeks) at the start of the treatment, and then less frequently (e.g., every 2, 3, 4, 6, or 12 months) when a stable treatment regimen has been established.

[0083] In an embodiment, the monitoring step is carried out in parallel with other known methods for monitoring the progression of a disease associated with abnormal glycosphingolipid processing (e.g., lysosomal storage disease). For example, the measuring step can be carried out in parallel with the measurement of the subject's platelet count, hemoglobin concentration, spleen volume, and / or liver volume. These measurements can be useful, for example, in determining whether the subject is experiencing more severe symptoms of lysosomal storage disease.

[0084] In a seventh aspect, the present disclosure provides a method of treating a lysosomal storage disease or synucleinopathy in a subject in need thereof, comprising administering an effective amount of a drug capable of treating a lysosomal storage disease or synucleinopathy (e.g., a drug described herein), wherein the patient has been evaluated as having abnormal glycosphingolipid processing according to the method described herein or the disease state or severity has been evaluated according to the method described herein.

[0085] In an eighth aspect, the present disclosure provides a method of treating or preventing the onset or progression of lysosomal storage disease or synucleinopathy in a subject who is evaluated as being at risk of developing lysosomal storage disease or synucleinopathy according to the methods described herein (e.g., the methods of the second or third aspect), the method comprising: (a) initiating a series of therapeutic treatments (e.g., a therapeutically effective amount of an agent capable of treating lysosomal storage disease or synucleinopathy, such as an agent described herein) in the subject; and optionally, (b) evaluating or repeating the evaluation of the risk of developing lysosomal storage disease or synucleinopathy according to the methods described herein and optionally adjusting the therapeutic treatment based on the new evaluation.

[0086] In embodiments, the method comprises administering one or more (e.g., two, three, four, or more) agents capable of treating or preventing the disease or disorder. For example, the method may comprise administering a lysosomal enzyme (ERT) such as imiglucerase, and / or a small molecule (SRT) such as, for example, benglutide or eliglustat. Alternatively, or in addition, the method may comprise administering one or more agents that reduce the symptoms of the disease or disorder, such as, for example, levodopa or a prodrug thereof (optionally in combination with carbidopa or a prodrug thereof), ambroxol, amantadine, a dopamine agonist, an MAO inhibitor, a COMT inhibitor, and an anticholinergic agent.

[0087] In a ninth aspect, the present disclosure provides the use of conduritol-β-epoxide (CBE) in a method of diagnosing or determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject. It will be understood that embodiments of the foregoing aspects (e.g., of the disease) may be applied to this ninth aspect.

[0088] Although generally described herein, the following non-limiting examples are provided to further illustrate the present disclosure.

Example

[0089] Example 1: In Vivo Proof-of-Concept Study in Mice This example aimed to test the hypothesis that transient inhibition of GCase and subsequent accumulation of GCase substrates may function as a sensitive assay to reveal defects in lipid flux. Initial proof-of-concept studies were performed in mice and transformed human cell lines.

[0090] Dose Response of GSL Pathway Loading in WT Mice In wild-type (WT) mice (Charles River Laboratories or Jackson Labs), the dose response to CBE was performed. Concentrations of CBE from 0.3 mg / kg to 100 mg / kg were investigated. GCase activity remaining 24 hours after CBE administration was measured using a 4-methylumbelliferone (4MU) hydrolysis assay. Briefly, cell lysates from various sample types were diluted in 0.1 M sodium acetate buffer (pH 4.5) containing 10 mM synthetic substrate 4-methylumbelliferyl-β-D-glucopyranoside and incubated at 37 °C for 1 hour. The reaction was terminated by adding 0.5 volume of 1 M glycine buffer (pH 12.5). The fluorescence of this reaction was measured using a Spectramax fluorometer (Ex365 / Em445; Molecular Devices, Sunnyvale, CA, USA). A standard curve was generated using Cerezyme® (imiglucerase).

[0091] Figure 1 shows the results of CBE administration in WT mice. Enzyme activity decreased in a dose-dependent manner, but beyond 30 mg / kg, further inhibition of GCase was either barely measurable or not measurable at all. From these data alone, it was difficult to determine whether this was due to enzyme saturation or to the sensitivity of the GCase activity assay. Measurement of downstream substrates allowed discrimination between these two cases. Lyso-GL1 levels in cortex, liver, and plasma all increased and rose as the CBE dose exceeded 30 mg / kg (Figure 2), suggesting that this substrate can act as a reporter of GCase activity over a wider range of concentrations.

[0092] Time-course of lipid flux after GSL pathway loading in Gba+ / - mice We investigated whether substrate accumulation could enable discrimination between mice with different copy numbers of the mutation in GBA1. We investigated the following three mouse models: Gba D409V / D409V 、Gba D409V / + 、and Gba + / + (which are homozygous for the deletion of the functional D409V mutation in GBA1, heterozygous for this mutation, or wild-type (WT) with respect to GBA1, respectively). The GCase substrate glucosylceramide (GL1), in addition to glucosylsphingosine (lyso-GL1), was investigated. Mice were administered a single dose of CBE and sacrificed at 3, 6, 24, 48, or 72 hours later. Substrate accumulation and dissipation were measured over time.

[0093] Between 24 and 48 hours, substrate accumulation reached a peak, and the accumulation of Lyso-GL1 in the cortex, liver, and plasma showed a higher sensitivity to the genetic background (Figure 3). After the peak, as the newly synthesized enzyme degraded these substrates, the substrate levels began to decline. A secondary pathway may also have contributed to the observed lipid reduction. As predicted, the peak lipid concentration was inversely correlated with the genotype. In addition, the recovery rate was correlated with the genotype, with WT mice recovering or approaching normal levels the fastest, followed by heterozygous mutant mice, and then homozygous mutant mice.

[0094] Time-course of lipid flux after GSL pathway loading in wild-type mice Although it was demonstrated that substrate accumulation and dissipation are sensitive measures of the amount of GC-ase activity, the next question to be tested was how substrate levels changed over time in wild-type mice after loading.

[0095] Three different wild-type mouse strains (C57BL / 6J, BALB / cJ, and FVB - Charles River Laboratories or Jackson Labs) were evaluated for GSL accumulation and recovery after CBE loading. These strains are all "normal" in the sense that they do not have any specific mutations but are genetically different from each other as individual people are. Importantly, the selected mice do not have mutations in GBA1. The time-course of CBE loading was performed in these three different strains, and the accumulation of lyso-GL1 and GL-1 was measured at 24, 48, and 72 hours after administration.

[0096] The results of the experiment are shown in FIGS. 4 and 5. There were no significant differences among the strains in GSL levels in the cortex (FIG. 4A). However, in the liver and plasma, C57BL / 6 mice accumulated much more lyso-GL1 than the BALB / cJ and FVB strains (FIGS. 4B and 4C). These results were similarly reflected in the total GL1 in the liver. Plasma GL1 levels showed a slightly different trend, with the highest levels observed in FVB mice. These results indicate that CBE loading may be able to distinguish between individuals who may have defects in the sphingolipid metabolic pathway even without GBA1 mutations. Analysis of the different isoforms of GL1 in the cortex showed a variable distribution of chain lengths among the strains (FIG. 5), and C57BL / 6 mice had higher levels of the C22, C23, C24, and C24:1 isoforms.

[0097] Example 2: In Vitro Studies in Human Cell Lines The in vivo experiment described above transiently inhibits GC-ase to increase the accumulation of sphingolipids, thereby providing a proof of concept for amplifying the signal from underlying genetic differences. However, such a method is not practical for use in patients because tissue samples must be extracted to perform the measurements. To bridge this feasibility gap, the levels of GBA1 transcript expression were investigated in various human cell lines.

[0098] First, the response of human SH-SY5Y neuroblastoma cells to CBE was studied. Cells were used without further modification (wild-type) or modified to reduce or increase Gba activity (knockdown and overexpression, respectively). This modification was effected by stable transfection with a plasmid expressing siRNA targeting GBA (KD) or a plasmid constitutively overexpressing GBA (OE). Human SH-SY5Y neuroblastoma cells (Sigma) were incubated with 100 μM CBE (Toronto Research Chemicals) for 24 hours. Enzyme activity and lipid levels were measured at -24 hours (before CBE incubation) and at 0, 24, and 48 hours after CBE removal. Figure 6 shows the effect of different incubation periods of wild-type cells with CBE on enzyme activity, from which it was concluded that an incubation period of 24 hours was suitable to obtain a good response. Figure 7 shows the response of three different cell types to various concentrations of CBE. A significant increase in enzyme activity was observed between knockdown and wild-type cells and between wild-type and overexpressing cells. Interestingly, it was found that the dynamic range of the assay also increased significantly when the cells were incubated with sodium taurocholate (Figure 8).

[0099] Next, the lipid response of cells to CBE incubation was investigated (Figure 9). No significant difference in total GL1 levels was observed in wild-type cells after treatment (Figure 9A). Some differences in lyso-GL1 levels after CBE treatment were observable among the three cell types (Figure 9B), but no measurable accumulation of lyso-GL1 was present when untreated. Under typical cell culture conditions, it was found impossible to measure significant differences in GL1 accumulation among cells expressing 40% of endogenous GBA1, the WT amount, or 300% of the WT amount. Furthermore, none of these cell lines showed measurable accumulation of lyso-GL1 under normal conditions. The results of genetic differences among cells could only be revealed by transient inhibition of GCase by CBE. Upon CBE treatment, cells accumulated GL1 and lyso-GL1 at levels inversely proportional to the level of GBA1 expression. This in vitro model suggests that the unique defects in the patient's sphingolipid metabolic pathway can be measured using cells collected from the patient and treated with CBE during culture.

[0100] The accumulation of various GL1 species after CBE treatment was also investigated. Generally, monounsaturated GL1 species (i.e., isoforms having one double bond in the fatty acid portion of the molecule) accumulated more compared to saturated species, similar to the shorter-chain-length isoforms (Figure 10). Considering the baseline abundance of GL1 species (Figure 11), it was observed that low-abundance isoforms were generally the ones that accumulated in response to CBE treatment.

[0101] Example 3: CBE Loading in Human PBMCs The effect of CBE loading in human peripheral blood mononuclear cells (PBMCs) was investigated.

[0102] PBMCs were obtained from a blood sample (Stem Cell Technologies, catalog number 70025, lot number 2105417005, donor ID CE0006419) using standard procedures. 25,000 cells were placed into individual wells of a multi-well plate and allowed to acclimate for 24 hours before being treated with 100 μM CBE for 24 hours. The culture medium was removed and replaced with fresh medium (without CBE), and lipids were extracted from these cells at time intervals of 0, 2, 4, 8, 16, and 24 hours. GL1 levels and lyso-GL1 levels were measured, and the levels of phosphatidylcholine (PC) were also measured. The PC levels were found to be little affected by the CBE treatment, and thus were used to normalize the levels of GL1 and lyso-GL1 (e.g., to minimize the effects of varying numbers of cells in each well and / or the effects of cell growth over time).

[0103] GL1 levels in PBMCs increased approximately 6-fold (from approximately 2 ng / ml to approximately 12 ng / ml) after CBE treatment, and lyso-GL1 levels increased from near the assay's lower limit of detection to approximately 0.08 ng / ml. The lyso-GL1 levels were near the lower limit of detection of the assay, and thus, greater error occurred. When normalized to total PC levels (100 - 600 ng / ml during the course of the experiment), the GL1 and lyso-GL1 levels were as shown in Figure 12. The GL1 levels (Figure 12A) and lyso-GL1 levels (Figure 12B) were normalized at 16 hours after treatment.

[0104] Analysis of the GL1 isoform was performed to check the stability of cell cultures. Cells were obtained and cultured as described above without CBE treatment and monitored over 72 hours. The lipid levels of the GL1 isoform are shown in Figure 13. Although the various isoforms show various absolute levels, there is a pattern for all species tested, namely, the levels rise almost linearly from 0 to 24 to 48 hours and then decline at 72 hours. The increase in levels up to 48 hours is due to cell growth, and the decline at 72 hours is hypothesized to be due to heterogeneous cell population growth and / or adhesion kinetics. From these results, it is suggested that the time-course analysis of GSL levels in PBMCs using this protocol typically should not proceed beyond 48 hours after loading.

[0105] In summary, PBMCs are a suitable cell type for monitoring GSL flux after loading, and the time-course can be established from cells obtained from approximately 1 ml of whole blood. Normalization of GSL levels to PC concentration can reduce systematic error. PC is not essential for the GSL pathway, and the concentration of PC is not affected by CBE treatment. GL1 appears to report better CBE loading and recovery compared to lyso-GL1. In healthy individuals, GL1 levels rise robustly after loading and return to baseline at 16 hours.

[0106] Although the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to illustrate and not to limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0107] In addition, when a feature or aspect is described in terms of a Markush group, it will be recognized by those skilled in the art that thereby such feature or aspect is also described in terms of any individual member or subgroup of members of the Markush group.

[0108] All publications, patent applications, patents, and other references mentioned in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, this specification, including definitions, will control.

Claims

1. An in vitro method for evaluating a subject with respect to abnormal glycosphingolipid processing, comprising treating the subject cells to load the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the load.

2. The loading comprises contacting the cells with a drug that inhibits an enzyme involved in the glycosphingolipid pathway (e.g., glucocerebrosidase), optionally, The aforementioned drug is i) Selective for one enzyme involved in the glycosphingolipid pathway, ii) An irreversible inhibitor of the enzyme, for example, the inhibitor covalently binds to the enzyme and / or iii) Equation (I): 【Chemistry 1】 A compound of or a pharmaceutically acceptable salt thereof, wherein the formula is X is -O- or -N(R 2 ) - and; R 1 These are -OH, -C(O)OH, and -CH 2 Selected from OH; R 2 If present, C is optionally substituted with one or more groups independently selected from -H, -OH, and halogens. 1 ~ 6 - An in vitro method according to claim 1, selected from alkyl groups.

3. The in vitro method according to claim 1, wherein the step of processing the target cells to load the glycosphingolipid pathway in the cells comprises contacting the cells with conzlitol-β-epoxide (CBE).

4. i) The cells are present in a sample obtained from the subject, for example, the sample is a tissue sample (e.g., a tissue sample derived from the brain, liver, kidney, skin, or spleen of the subject), or the sample is a cell-containing blood sample (e.g., whole blood), and / or ii) The cells are fibroblasts, peripheral blood mononuclear cells (PMBCs), or induced pluripotent stem cells (iPSCs) derived from somatic cells of the subject, for example, the cells are peripheral blood mononuclear cells obtained (e.g., purified) from a blood sample taken from the subject, and / or iii) The in vitro method according to claim 1, wherein the cells express neuroepithelial stem cell protein (nestin).

5. Monitoring the recovery of at least one glycosphingolipid after the aforementioned loading is necessary. i) After the load, multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more times, and / or ii) Essentially, until the baseline level is reached, for example, until the level is within approximately 10% or 5% of the baseline level, The in vitro method according to claim 1, comprising measuring the level of at least one glycosphingolipid in or produced by the cells.

6. The aforementioned at least one glycosphingolipid is i) Lipids selected from glucosylceramide (GL1), ceramide, and glucosylsphingosine (lyso-GL1), for example, the at least one glycosphingolipid being total GL1, total ceramide, or total lyso-GL1, and / or ii) The in vitro method according to claim 1, wherein the lipid is (a) a monounsaturated fatty acid moiety selected from, for example, C16:1, C18:1, C20:1, C22:1, or C24:1, or (b) a fatty acid moiety selected from C24:0, C16:1 n-7, C18:1 n-7, C22:1 n-9, C24:1 n-9, and C18:2 cis, or comprises

7. The in vitro method according to claim 1, wherein monitoring the recovery of at least one glycosphingolipid after the load does not involve monitoring the recovery of lipids containing a C18:0 fatty acid moiety and / or lipids containing a C18:1 fatty acid moiety.

8. The method further includes a step of comparing the recovery of the at least one glycosphingolipid after the aforementioned load with the recovery of the same glycosphingolipid in corresponding cells derived from a healthy individual after the same load, Optionally, the comparing step includes comparing one or more of the following parameters (which can be derived from a concentration vs. time plot): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) the time required to reach C max after loading (T max ); (iii) the area under the curve (AUC); and (iv) the time required for the level to return to the baseline, e.g., the time required for the level to return within about 10% or about 5% of the baseline level. For example, if the difference in the parameters is more than approximately 10%, and for example more than approximately 20%, more than approximately 30%, more than approximately 50%, more than approximately 75%, more than approximately 100%, more than approximately 150%, or more than approximately 200%, the subject is evaluated as having abnormal glycosphingolipid processing, claim The in vitro method described in 1.

9. An in vitro method for determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject, comprising: evaluating the subject with respect to abnormal glycosphingolipid processing according to any one of claims 1 to 8; and determining the severity of the disease based on how much the recovery differs from the recovery of cells from a similarly stressed healthy individual.

10. The aforementioned disease is a lysosomal storage disorder, for example, Gaucher disease, or The aforementioned disease is a synuclein disease such as Parkinson's disease (PD), for example, idiopathic PD, and / or The mutation status of the GBA gene in the subject is unknown, or the subject is assessed to have only one or zero known variant (e.g., non-functional or reduced-function) GBA alleles, and / or The subjects mentioned above have not been previously diagnosed with a disease related to abnormal glycosphingolipid processing, for example, they have not been previously diagnosed with lysosomal storage disorders such as Gaucher disease or Parkinson's disease, or The method according to claim 9, wherein the subject has been previously diagnosed with a disease related to abnormal glycosphingolipid processing, and the method determines the progression of the disease.

11. An in vitro method for diagnosing or monitoring the progression of synuclein diseases such as Parkinson's disease (PD), for example, idiopathic PD, in a subject, - A sample obtained from the subject, including fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from the subject's somatic cells, is cultured as an option, and then brought into contact with CBE; - Obtain a reaction curve by measuring the levels of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cells multiple times; and - The comparison is between the aforementioned response curve and a comparative response curve, wherein the comparative curve is either a standard response curve created by applying a load to the same sample obtained from (a) a healthy individual or (b) a patient with a confirmed diagnosis of PD (to diagnose the subject), or the comparative curve is a response curve obtained in advance from the same subject (to monitor the progression of the disease in the subject). In vitro methods including those mentioned above.

12. An in vitro method for evaluating a possible therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor (the GCS inhibitor being benglustat or a pharmaceutically acceptable salt thereof) or a glucocerebrosidase activator, comprising contacting the cells of the subject with CBE to load the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the load, wherein if the recovery is slower or incomplete compared to the recovery of cells from a similarly loaded healthy organism, the subject is evaluated as a candidate for treatment. Selectively, the cells are fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from the somatic cells of the subject. The method is in vitro, wherein the cells are peripheral blood mononuclear cells obtained (e.g., purified) from a blood sample taken from the subject.

13. An in vitro method using conzlitol-β-epoxide for diagnosing or determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject, An in vitro method in which, optionally, the disease is lysosomal accumulation (e.g., Gaucher disease) or a synuclein disease such as Parkinson's disease (e.g., idiopathic PD).

14. An in vitro method for evaluating the therapeutic response of a subject to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GCase) activator (for example, the treatment is with the GCS inhibitor benglustat or a pharmaceutically acceptable salt thereof), (a) At the initial stage, bring the cells of the first cell-containing sample obtained from the subject into contact with CBE to load the glycosphingolipid pathway in the cells and monitor the recovery of at least one glycosphingolipid after the load; and (b) At a second time point following treatment of the subject with the GCS inhibitor or GCase activator, the cells of a second cell-containing sample obtained from the subject are brought into contact with CBE to load the glycosphingolipid pathway in the cells, and the recovery of at least one glycosphingolipid after the load is monitored. Includes, If the recovery of the cells derived from the second cell-containing sample is faster or more complete than the recovery of the cells derived from the first cell-containing sample, the treatment is evaluated as positive. An in vitro method in which, optionally, the cells in the first cell-containing sample and / or the cells in the second cell-containing sample are independently selected from fibroblasts, peripheral blood mononuclear cells, or induced pluripotent stem cells derived from the somatic cells of the subject.

15. The in vitro method according to claim 14, wherein the first cell-containing sample and the second cell-containing sample comprise essentially the same cell type, and / or the at least one glycosphingolipid monitored in step (a) is the same as the at least one glycosphingolipid monitored in step (b).

16. A pharmaceutical composition for use in the treatment of a lysosomal storage disorder or synuclein disease of a required subject, comprising an agent capable of treating the lysosomal storage disorder or synuclein disease, wherein the use comprises monitoring the therapeutic response in accordance with claim 14 and changing the dose of the agent in accordance with the results of the monitoring. A pharmaceutical composition comprising: optionally, if the treatment is evaluated as positive, maintaining the same dose of the drug; if the treatment is evaluated as not positive, increasing the dose of the drug or decreasing the dose of the drug if the treatment is evaluated as positive; continuing the monitoring step until the treatment is no longer evaluated as not positive; and at the time of evaluation, increasing the dose until the treatment is again evaluated as positive.

17. A pharmaceutical composition for use in the treatment of a lysosomal storage disorder or synuclein disease in a subject of interest, comprising an agent capable of treating the lysosomal storage disorder or synuclein disease, wherein the subject is evaluated as having abnormal glycosphingolipid processing according to the method of any one of claims 1 to 8, or the disease state or severity is evaluated according to the method of claim 9 or 11.

18. A pharmaceutical composition for use in treating or preventing the onset or progression of a disease related to abnormal glycosphingolipid processing, wherein the method is: (a) the step of initiating a series of therapeutic procedures on the subject; and optionally, (b) Evaluating the subject with respect to abnormal glycosphingolipid processing and determining the severity of the disease with respect to abnormal glycosphingolipid processing in the subject according to the method of claim 9; (c) A step of adjusting the therapeutic measures based on the severity of the disease. A pharmaceutical composition containing the above.

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