Methods for identifying and treating disease

Abstract

Methods for assessing abnormal glycosphingolipid processing, as well as methods for determining the severity and / or progression of a disease associated therewith, are provided. Also provided are methods for assessing the possible therapeutic response of a subject to treatment with an agent capable of modulating processing of glycosphingolipids, as well as methods of treatment that benefit from assessing a patient according to the present disclosure. The methods of the present disclosure employ an agent capable of stimulating the glycosphingolipid pathway in a cell (e.g., cyclohexentetrol-beta epoxide), and then monitor the recovery of the cell, e.g., monitor the recovery of glycosphingolipid flux in the cell.

Description

[0001] The present disclosure relates to diagnostic methods for evaluating abnormal glycosphingolipid processing, and to methods for determining the severity and / or progression of diseases associated therewith, such as Parkinson's disease. Methods for evaluating a subject's potential therapeutic response to treatment with an agent that can modulate glycosphingolipid processing, and related treatments are also provided. Summary of the invention

[0002] Glycosphingolipids (GSLs) are complex lipids that are responsible for a variety of cellular functions, including the integrity of plasma and vesicle membranes, signal transduction, and intercellular communication. GSL synthesis and degradation are essential for normal cell function and survival. Impaired GSL degradation is associated with many lysosomal storage disorders and neurodegenerative diseases.

[0003] The final reaction of the breakdown of certain GSLs is mediated by glucocerebrosidase (GCase, also known as acid β-glucosidase), a hydrolase responsible for the lysosomal degradation of glucosylceramide (GL1). Lysosomal GCase is encoded by the gene GBA. Homozygous and compound heterozygous mutations in GBA cause Gaucher disease (GD), a lysosomal storage disorder (LSD) characterized by inefficient clearance of GL1 and subsequent clinical manifestations. These patients present altered glycosphingolipid levels that can be normalized by increasing glucocerebrosidase activity and / or decreasing 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 that GC enzyme activity is reduced in the CNS of sporadic PD patients without known GBA mutations, suggesting the existence of additional unknown mechanisms linking reduced hydrolytic activity to disease.

[0004] Due to the severe loss of function of one or more enzymes, LSD is usually manifested as the accumulation of lysosomal substrates. For example, in GD, the loss of GC enzyme activity leads to the accumulation of GL1 and its deacylated derivative glucosylsphingosine (lyso-GL1), and treatment with drugs that can reduce the levels of these substrates (e.g., venglustat) can be used to treat the disease. Changes in the levels of these lysosomal substrates can be used as important biomarkers to determine the efficacy of therapeutic interventions. Unfortunately, such biomarkers for PD have not yet been found. Although the GC enzyme activity of PD patients is observed to be reduced, there is almost no obvious substrate accumulation in the biological fluids that can be obtained from patients (as evaluated by the Parkinson's Progression Markers Initiative), or even in brain tissue samples collected after death. This may be because patients lose up to about 50% of the GC enzyme function, which is sufficient to process its substrates in most cells and tissues in the absence of other factors. In these patients, accumulation may be limited to specific cell types, or limited to a short period of time. These challenges make it difficult to use conventional plasma biomarkers to assess which patients may benefit from therapeutic interventions.

[0005] Mechanisms for identifying individuals with a normal GBA genotype but altered GSL metabolism are very limited. GD patients can be easily identified by reduced GC enzyme activity in the blood and then confirmation of GBA mutations. The identification of heterozygous patients presents different challenges. Although genetic analysis can routinely identify GBA mutations that have been identified to reduce GC enzyme activity, it may not be able to tell the relevance of new mutations. Biochemical quantification of GC enzyme activity exhibits additional limitations because patients with heterozygous GBA mutations show a large variability in activity, which may overlap greatly with non-GBA mutation carriers. In addition, the existence of PD patients without GBA mutations with reduced GC enzyme activity suggests that alternative detection methods are necessary. Similarly, a potentially harmful increase in GSL synthesis is another mechanism that cannot be revealed by any current method. In summary, there are multiple mechanisms that can lead to changes in GSL flux (increased production and / or reduced degradation), but there is no appropriate method to identify these abnormalities. Therefore, new methods are needed to diagnose and monitor conditions associated with abnormal glycosphingolipid processing.

[0006] Based on the data presented herein, the present application relates to an alternative method for assessing GSL pathway defects, such as partial reductions in the activity of related enzymes, which cannot be captured using traditional sphingolipid biomarkers, thereby overcoming the deficiencies of conventional methods in diagnosing and monitoring disease conditions. The method described herein presents an acute stimulation (challenge) of the glycosphingolipid (GSL) pathway in the cells of a patient, and then measures the response of the GSL pathway to the stimulation, such as changes in GSL flux. The way in which the GSL pathway recovers after stimulation can be used to indicate the type and severity of the patient's condition. In a general sense, GSL pathway stimulation involves intervening in the pathway (e.g., upregulating or downregulating one or more enzymes involved in the GSL pathway), which reveals differences between individuals when monitoring recovery from the stimulation. An example of GSL pathway stimulation described herein is acute restriction of GC enzyme activity to maximize the stress on this sphingolipid degradation pathway as much as possible. Cyclohexene tetraol-β epoxide (CBE) is an irreversible covalent binder and inhibitor of glucocerebrosidase, which can be used to strongly restrict GC enzyme activity. When cells or animals are treated with this enzyme, the pre-existing GC enzyme pool is permanently inactivated. Therefore, only newly translated GC enzymes can further degrade rapidly accumulated substrates. This requires new enzyme molecules to be effectively synthesized, folded and transported to lysosomes. By measuring the time course and extent of GSL accumulation and recovery after CBE clearance or removal, the overall efficiency of enzyme synthesis, transport and catalytic activity can be evaluated. This method reveals important GSL flux abnormalities that were not otherwise obvious, and it provides a more general assessment of GSL pathways that can be used to study abnormal GSL processing as well as disease states, progression and therapeutic efficacy. In the context of the present disclosure, other suitable stimulation conditions will be clear to those skilled in the art.

[0007] Thus, a first aspect provides a method for assessing abnormal glycosphingolipid processing in a subject, the method comprising treating a cell of the subject to stimulate a glycosphingolipid pathway in the cell, and monitoring the recovery of at least one glycosphingolipid after the stimulation. In embodiments, the stimulation comprises contacting the cell 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 an enzyme involved in the glycosphingolipid pathway.

[0008] The agent may be an irreversible inhibitor of the enzyme, for example, the inhibitor may be covalently bound to the enzyme. In an embodiment, the agent is a compound of formula (I):

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein:

[0011] X is -O- or -N(R 2 )-;

[0012] R 1 Selected from -OH, -C(O)OH and -CH 2 OH; and

[0013] R 2 , when present, is selected from -H and C optionally substituted with one or more groups independently selected from -OH and halogen 1-6 alkyl.

[0014] The step of treating cells of the subject to stimulate a glycosphingolipid pathway in the cells may comprise contacting the cells with cyclohexenetetraol-β epoxide (CBE).

[0015] In embodiments, the method is an in vitro method, wherein the cell is present in a sample obtained from the subject. In specific embodiments, the sample is a tissue sample (e.g., from the brain, liver, kidney, skin or spleen of the subject), or wherein the sample is a blood sample (e.g., whole blood) containing cells.

[0016] In embodiments, the cell is a fibroblast, a peripheral blood mononuclear cell (PMBC) or an induced pluripotent stem cell (iPSC) derived from the somatic cell of the experimenter. In specific embodiments, the cell is a peripheral blood mononuclear cell obtained (e.g., purified) from a blood sample taken from the experimenter.

[0017] In embodiments, the cells express neuroepithelial stem cell protein (nestin).

[0018] In embodiments, monitoring the recovery of at least one glycosphingolipid after the stimulation comprises measuring the level of at least one glycosphingolipid in or produced by the cell multiple times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times after the stimulation. In some embodiments, monitoring the recovery of at least one glycosphingolipid after the stimulation comprises measuring the level of at least one glycosphingolipid in or produced by the cell until it substantially reaches the pre-treatment (baseline) level, e.g., a level within about 10% or about 5% of the pre-treatment level.

[0019] In an embodiment, the at least one glycosphingolipid is or comprises a lipid selected from the following: glucosylceramide (GL1), ceramide and glucosylsphingosine (lyso-GL1). In a specific embodiment, the at least one glycosphingolipid is or comprises total GL1, total ceramide or total lyso-GL1. In an embodiment, the at least one glycosphingolipid is or comprises a lipid containing a monounsaturated fatty acid portion, and the monounsaturated fatty acid portion is, for example, selected from C16:1, C18:1, C20:1, C22:1 or C24:1. In an embodiment, the at least one glycosphingolipid is or comprises a lipid containing a fatty acid portion, and the fatty acid portion is selected from C24:0, C16:1n-7, C18:1n-7, C22:1n-9, C24:1n-9 and C18:2 cis. In some embodiments, monitoring the recovery of at least one glycosphingolipid after the stimulation does not include monitoring the recovery of lipids containing C18:0 fatty acid portions and / or lipids containing C18:1 fatty acid portions.

[0020] The method may further comprise the step of comparing the recovery of the at least one glycosphingolipid after the stimulation with the recovery of one or more of the same glycosphingolipids in corresponding cells from a healthy individual after the same stimulation. In an embodiment, the comparison step involves comparing one or more of the following parameters (which may be derived from a plot of concentration versus time): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) after stimulation, the max The time required (T max ); (iii) area under the curve (AUC); and (iv) time required for the level to return to baseline, e.g., to within about 10% or within about 5% of the baseline level. In such embodiments, if the difference in one or more of the parameters is greater than about 10%, e.g., greater than about 20%, 30%, 50%, 75%, 100%, 150%, or 200%, the subject is assessed as having abnormal glycosphingolipid processing.

[0021] A second aspect provides a method for determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject, the method comprising assessing the subject's abnormal glycosphingolipid processing according to the first aspect, and determining the severity of the disease based on the extent to which the recovery differs from the recovery of cells stimulated in the same manner from healthy individuals. In embodiments, the disease is a lysosomal storage disease, such as Gaucher disease. In other embodiments, the disease is a synuclein disease such as Parkinson's disease (PD), such as idiopathic PD.

[0022] In some embodiments, the mutation status of the GBA gene in the subject is unknown, or the subject has been assessed as having only one or zero known mutant (e.g., non-functional or reduced-function) 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 has not been diagnosed with Parkinson's disease.

[0023] In other embodiments, the subject has been previously diagnosed with a disease associated with aberrant glycosphingolipid processing, and the method determines progression of the disease.

[0024] A third aspect provides an in vitro method for diagnosing a synucleinopathy such as Parkinson's disease (PD), e.g., idiopathic PD, or monitoring the progression thereof in a subject, the method comprising:

[0025] - obtaining from the subject a sample comprising fibroblasts, peripheral blood mononuclear cells or induced pluripotent stem cells derived from somatic cells of the subject, and optionally culturing the cells;

[0026] - contacting the sample with CBE;

[0027] - measuring the level of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cell multiple times to obtain a response curve; and

[0028] - comparing the response curve with a comparison response curve, wherein the comparison curve is a standard response curve generated by stimulating the same sample obtained from (a) a healthy individual or (b) a patient diagnosed with PD (to diagnose the subject), or the comparison curve is a response curve from a sample previously obtained from the same subject (to monitor the progression of the subject's disease).

[0029] A fourth aspect provides a method for evaluating a subject's possible therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase activator, the method comprising contacting the subject's cells with a CBE to stimulate the glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation, wherein if the recovery is slower or less complete than the recovery of cells stimulated in the same manner from a healthy individual, the subject is evaluated as a candidate for treatment. In an embodiment, the GCS inhibitor is vinlusstat or a pharmaceutically acceptable salt thereof. The cell may be a fibroblast derived from a somatic cell of the subject, a peripheral blood mononuclear cell, or an induced pluripotent stem cell. In a specific embodiment, the cell is a peripheral blood mononuclear cell obtained (e.g., purified) from a blood sample taken from the subject.

[0030] Related aspects provide for the use of cyclohexene tetraol-β epoxide in a method for diagnosing a disease associated with abnormal glycosphingolipid processing in a subject or determining its severity. In embodiments, the disease is a lysosomal storage disease (e.g., Gaucher disease) or a synucleinopathy such as Parkinson's disease (e.g., idiopathic PD).

[0031] A fifth aspect provides a method for evaluating a subject's therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GC enzyme) activator, the method comprising:

[0032] (a) obtaining a first cell-containing sample from the subject at a first time point;

[0033] (b) contacting cells of the first cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation;

[0034] (c) obtaining a second cell-containing sample from the subject at a second time point after treating the subject with a GCS inhibitor or a GC enzyme activator; and

[0035] (d) contacting cells of said second cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in said cells, and monitoring the recovery of at least one glycosphingolipid after said stimulation,

[0036] Wherein the treatment is assessed as positive if the recovery in the cells from the second cell-containing sample is faster or more complete than the recovery in the cells from the first cell-containing sample. In an embodiment, the subject's treatment is with the GCS inhibitor venlostat or a pharmaceutically acceptable salt thereof.

[0037] In embodiments, 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 somatic cells of the subject.

[0038] In some embodiments: steps (a) and (b) are performed in substantially the same manner as steps (c) and (d) of the method; the first cell-containing sample and the second cell-containing sample comprise substantially the same cell types; and / or the at least one glycosphingolipid monitored in step (b) is the same as the at least one glycosphingolipid monitored in step (d).

[0039] A sixth aspect provides a method of treating a lysosomal storage disease or a synucleinopathy in a subject in need thereof, the method comprising administering to the subject an agent capable of treating the lysosomal storage disease or the synucleinopathy, wherein the method involves monitoring the treatment response according to the fifth aspect, and adjusting the dose of the agent according to the results of the monitoring. In embodiments, if the treatment is assessed as positive, the dose of the agent remains the same, and if the treatment is assessed as not positive, the dose of the agent is increased; or if the treatment is assessed as positive, the dose of the agent is reduced, and the monitoring step is continued until the treatment is no longer assessed as positive, at which time the dose is increased until the treatment is again assessed as positive.

[0040] The seventh aspect provides a method for treating a lysosomal storage disease or a synucleinopathy in a subject in need thereof, the method comprising the step of administering an effective amount of an agent capable of treating the lysosomal storage disease or the synucleinopathy, wherein the subject has been assessed as having abnormal glycosphingolipid processing according to the method of the first aspect or an embodiment thereof, or has had his or her disease state or severity assessed according to the method of the second aspect or the third aspect.

[0041] The eighth aspect provides a method for treating or preventing the development or progression of a lysosomal storage disease or synucleinopathy in a subject assessed as being at risk of developing a lysosomal storage disease or synucleinopathy according to the method of the second aspect or the third aspect, the method comprising the following steps:

[0042] (a) starting the subject on a course of therapeutic treatment; and optionally

[0043] (b) assessing the risk of developing a lysosomal storage disease according to the method of the second aspect or the third aspect or repeating said assessment and optionally adjusting said therapeutic treatment based on the new assessment.

[0044] Additional features and advantages of the compounds, compositions, and methods disclosed herein will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shown is a CBE dose titration study in WT mice that revealed that after 24 h, the cortex ( Figure 1 A) and liver ( Figure 1 B) Dose-dependent reduction in GC enzyme activity in both. N = 4 / group (8 for untreated and 100 mg / kg plasma). Columns sharing the same letter are not significantly different from each other. Error bars show SEM.

[0046] Figure 2Shown is a CBE dose titration study in WT mice that revealed a dose-dependent increase in lipid accumulation after 24 hours. Figure 2 A) Liver Figure 2 B) and plasma ( Figure 2 C) Lyso-GL1 was detected in 1% 4% leukemia cells. N = 4 / group (8 for untreated and 100 mg / kg plasma). Results for lyso-GL1 are μg / g wet tissue. Columns sharing the same letter are not significantly different from each other. Error bars show SEM.

[0047] Figure 3 Shows Gba D409V / D409V , Gba D409V / + and Gba + / + CBE time course studies in mice revealed peak GSL accumulation at 24-48 hours with a time-dependent recovery lasting up to 72 hours with genotype-phenotype correlation. Following a single intraperitoneal CBE injection, homozygous mice showed increased GSL accumulation compared to heterozygous and WT mice in all regions evaluated, namely cortex ( Figure 3 A) Liver Figure 3 B) and plasma ( Figure 3 C) shows higher GL1 and lyso-GL1 peak accumulation. For each figure, the top line (with squares) is homozygous D409V mice, the middle line (with upward triangles) is heterozygous mice, and the bottom line (downward triangles) is wild-type mice. N=10 / group. Data are shown as mean ± SEM.

[0048] Figure 4 Shown is a CBE time course study in different WT mouse strains revealing differences in lipid flux. Figure 4 A) Liver Figure 4 B) and plasma ( Figure 4 C) measured GSL levels. All mice showed peak glycosphingolipid levels (lyso-GL1, left; total GL1, right) at 24 hours. However, C57BL / 6 mice showed increased sensitivity to GC enzyme inhibition, as shown in liver and plasma lyso-GL1 levels and liver GL1 levels. Plasma GL1 levels reveal different trends, wherein compared with BALB / cJ mice, FVB and C57BL / 6 mice show increased total GL1 levels. The difference in total GL1 levels was not observed in the cortex. For each figure, the light gray line with circle shows the result of BALB / cJ mice, the black line with square represents C57BL / 6 mice, and the dark gray line with triangle represents FVB mice. Data are shown as mean ± SD.

[0049] Figure 5Shown is a CBE time course study in different WT mouse strains measuring the levels of different GL1 isoforms in the cortex. Each graph reports the amount of GL1 isoform measured: Figure 5 A shows the C22 subtype; Figure 5 B shows the C23 subtype; Figure 5 C shows the C24 subtype; and Figure 5 D shows the C24:1 monounsaturated isoform. After evaluation, the distribution of chain lengths between strains was variable, with C57BL / 6 mice having higher levels of C22 ( Figure 5 A) and C23( Figure 5 B) C24( Figure 5 C) and monounsaturated C24:1( Figure 5 D) Subtype. For each figure, the light grey line with circles shows the results for BALB / cJ mice, the black line with squares represents C57BL / 6 mice, and the dark grey line with triangles represents FVB mice. Data are shown as mean ± SD.

[0050] Figure 6 Shown are the responses of human cells to different durations of CBE incubation, namely: 2 hours (black diamonds, top); 24 hours (squares, middle); and 48 hours (grey diamonds, bottom). Data are shown as mean ± SD.

[0051] Figure 7 Shown are CBE responses of wild-type human cells (black line with diamonds - 100% relative activity to untreated) compared to GBA knockdown cells (black line with squares - 40% decrease in activity) and GBA overexpressing cells (grey line with diamonds - 300% increase in activity). Data are shown as mean ± SD.

[0052] Figure 8 The effect of including sodium taurocholate in the incubation mixture is shown. The response of wild-type cells (dark gray line with diamonds, second from the bottom) increased approximately 10-fold when incubated with sodium taurocholate (black line with squares, top). For GBA knockdown cells (light gray line with diamonds, bottom), a similar increase in response was observed when incubated with sodium taurocholate (light gray with squares, second from the top). Data are shown as mean ± SD.

[0053] Fig. 9 Shown is the response of human SHSY-5Y neuroblastoma cells to treatment with 100 μM CBE for 24 hours. Total GL1 levels in wild-type cells ( Fig. 9A) No significant differences were observed when treated with (white bars, right) or without (black bars, left) CBE. Minor differences in lyso-GL1 levels were observed between knockdown (KD), wild-type (WT), and overexpression (OE) cells ( Fig. 9 B). In each case, lyso-GL1 levels were very low without treatment (left bar, black). Error bars show SD and data are expressed in relative (arbitrary) units "AU" normalized to cell number.

[0054] Fig.10 The relative accumulation of different GL1 isoforms in human cells after treatment with CBE is shown. GL1 species are arranged by fatty acid chain length (C14 to C26), with saturated species on the left and monounsaturated species on the right. Error bars show SD.

[0055] Fig.11 Baseline abundance of GL1 isoforms in human cells is shown. Fig.10 Likewise, GL1 species are arranged by fatty acid chain length (C14 to C26) with saturated species on the left and monounsaturated species on the right. Error bars show SD, and data are expressed in relative (arbitrary) units "AU" normalized to cell number.

[0056] Fig.12 GSL levels in human PBMCs after CBE stimulation for up to 24 hours (grey circles) are shown compared to untreated controls (black squares). Lipid levels were normalized to PC concentration in the samples. Fig.12 A) and lyso-GL1( Fig.12 B) levels increased significantly after stimulation and returned to baseline by 16 hours. Data are shown as mean ± SEM.

[0057] Fig.13 The accumulation of GL1 subtypes in untreated cells over 72 hours is shown. Types are represented by their fatty acid carbon chain lengths and saturation (unless otherwise specified, e.g., for C24:1, otherwise fully saturated). For each subtype, the columns are respectively baseline (black), 24 hours (grey), 48 hours (dots), and 72 hours (stripes) from left to right. Data are shown as mean ± SEM. DETAILED DESCRIPTION

[0058] Although specific embodiments of the present disclosure will now be described with reference to preparations and schemes, it should be understood that such embodiments are by way of example only and are only a few of the 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 given the benefit of this disclosure, and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0059] definition

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any method and material similar to or equivalent to those described herein can be used in the implementation and testing of the present disclosure, exemplary methods, devices and materials are now described. All technologies and patents cited herein are incorporated herein by reference in their entirety. Any content of this article should not be construed as admitting that the present disclosure is not entitled to such disclosure content earlier than the prior public text.

[0061] Unless otherwise indicated, the present disclosure will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Michael R. Green and Joseph Sambrook, Molecular Cloning (4th ed., Cold Spring Harbor Laboratory Press 2012); Book series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; Book series Methods in Enzymology (Academic Press, Inc., NY); 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, 5th ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acids Hybridization; Anderson (1999) Nucleic AcidHybridization; Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor) Laboratory); Makrides editor (2003) GeneTransfer and Expression in Mammalian Cells;Mayer and Walker (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: ALaboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (editor) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press). ;

[0062] All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc. (including ranges) are approximate and vary (+) or (-) in increments such as 0.1 or 1.0 where appropriate. It should be understood that, although not always explicitly stated, all numerical expressions are preceded by the term "about", which is used to indicate a normal level of variability. For example, a numerical designation of "about" a given value may vary by ±10% of the value; alternatively, the variation may be ±5%, ±2%, or ±1% of the value. It should also be understood that, although not always explicitly stated, the agents described herein are exemplary only and equivalents thereof are known in the art.

[0063] Unless the context clearly indicates otherwise, as used in the specification and claims, the singular forms "a", "an", and "the" include plural referents. For example, the term "cell" includes a plurality of cells, including mixtures thereof. Unless otherwise specified or obvious from the context, as used herein, the term "or" should be understood to be inclusive. The term "including" is used herein to mean and is used interchangeably with the phrase "including but not limited to".

[0064] As used herein, the term "comprising" or "comprises" is intended to indicate that compositions and methods include the listed elements, but do not exclude other elements. When used to define compositions and methods, "essentially consisting of" shall mean excluding other elements that have any significance for the purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc. "Consisting of" shall mean excluding other ingredients more than trace elements and substantial method steps for applying the compositions of the present disclosure, or process steps for producing the compositions or achieving the desired results. Embodiments defined by each of these transitional terms are within the scope of the present disclosure. The use of the term "comprising" herein is intended to cover both "essentially consisting of" and "consisting of".

[0065] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, rats, rabbits, monkeys, cattle, sheep, pigs, dogs, cats, livestock, sports animals, pets, horses, primates, and humans. In embodiments, mammals include horses, dogs, and cats. In embodiments, the mammal is a human.

[0066] As used herein, the term "healthy individual" generally refers to an individual who does not suffer from synucleinopathy and / or does not have any GBA mutation. A healthy individual may lack a mutation in any gene encoding an enzyme involved in the glycosphingolipid pathway, such as a mutation in a gene encoding the following enzymes: 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 activating protein, glucosyltransferase, and galactosyltransferase.

[0067] "Administration" is defined herein as a means of providing a medicament or a composition comprising the medicament to a subject in a manner that allows the medicament to enter the subject's body. Such administration can be by any route, including but not limited to oral, transdermal (e.g., vaginal, rectal, oral mucosa), by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, into the CNS), or by inhalation (e.g., oral or nasal). Pharmaceutical preparations are of course administered in a form suitable for each route of administration.

[0068] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e., preventing the clinical symptoms of the disease from occurring in a patient who may be susceptible to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and / or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

[0069] The term "suffering from" as it relates to the term "treating" refers to a patient or individual who has been diagnosed with or is susceptible to the disease. A patient may also be said to be "at risk" for the disease because of, for example, a history of the disease in their family lineage or because of the presence of a genetic mutation associated with the disease. A patient at risk for the disease has not yet developed all or part of the characteristic lesions of the disease.

[0070] "Effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a beneficial or desired result. The effective amount can be administered in one or more administrations, applications or dosings. Such delivery depends on many variables, including the time period of use of a single dosage unit, the bioavailability of the therapeutic agent, the route of administration, etc. However, it should be understood that the specific dosage level of the therapeutic agent for any particular subject depends on a variety of factors, including, for example, the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the excretion rate, the drug combination, and the severity and form of administration of the specific disorder being treated. The therapeutic dose can usually be titrated to optimize safety and efficacy. Typically, the dose-effect relationship from in vitro and / or in vivo testing can initially provide useful guidance on the appropriate dose for patient administration. Generally, people will wish to administer an amount of compound that effectively achieves a serum level commensurate with the concentration found to be effective in vitro. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to treat (e.g., improve) one or more symptoms associated with a neurodegenerative disorder. For example, oral administration may require a total daily dose of 0.1 mg to 1000 mg. The total daily dose may be administered in a single or divided dose and may exceed the typical range given herein at the discretion of the physician.

[0071] As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical excipient, including carriers such as phosphate buffered saline solution, water, and emulsions (such as oil-in-water or water-in-oil emulsions) and various types of wetting agents. Pharmaceutical compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences (20th edition, Mack Publishing Co. 2000).

[0072] 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 disclosed herein, which may be administered without producing any one or more substantial undesirable biological effects, or without producing any one or more deleterious interactions with any other components that may be contained in the pharmaceutical composition.

[0073] Addition salts can be easily prepared using conventional techniques, for example, by treating the basic compound with a selected inorganic acid or organic acid of a specified amount in an aqueous solvent medium or in a suitable organic solvent (e.g., such as methanol or ethanol). Positively charged compounds (e.g., containing quaternary ammonium) can also form salts with the anionic components of various inorganic acids and / or organic acids. The acid that can be used to prepare pharmaceutically acceptable acid addition salts is those that can form nontoxic acid addition salts, and the nontoxic acid addition salts for example contain the salt of a pharmacologically acceptable anion, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, sugar acid salt, benzoate, mesylate and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)] salt. Bases that can be used to prepare pharmaceutically acceptable base addition salts are those that can form non-toxic base addition salts, such as salts containing pharmacologically acceptable cations such as alkali metal cations (e.g., potassium ion and sodium ion), alkaline earth metal cations (e.g., calcium ion and magnesium ion), ammonium ion, or other water-soluble amine addition salts (e.g., N-methylglucamine (meglumine), lower alkanolammonium); and other such bases of organic amines.

[0074] Although not expressly stated, any reference to a compound herein shall be deemed to include a reference to a pharmaceutically acceptable salt thereof. Thus, reference to "venlustat" includes pharmaceutically acceptable salts of venlustat, such as venlustat malate. Similarly, reference to "eliglustat" includes pharmaceutically acceptable salts of venlustat, such as eliglustat hemi-tartrate. Venlustat is (S)-1-azabicyclo[2.2.2]oct-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-benzodioxane-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]octanamide.

[0075] The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0076] This article uses the following abbreviations:

[0077] AD Alzheimer's disease

[0078] AUC Area under the curve (pharmacokinetics)

[0079] CBE Cyclohexene tetraol-β-epoxide

[0080] Cmax Maximum concentration (pharmacokinetics)

[0081] CNS Central Nervous System

[0082] DLB Dementia with Lewy Bodies

[0083] DNA deoxyribonucleic acid

[0084] EC 50 Half maximum effective concentration

[0085] ERT enzyme replacement therapy

[0086] FD Fabry disease

[0087] GBA Gene encoding GC enzyme

[0088] GC enzyme glucocerebrosidase (also called acid beta-glucosidase)

[0089] GCS glucosylceramide synthase

[0090] GD Gaucher disease

[0091] GL1 Glucosylceramide

[0092] GL2 Lactose Ceramide

[0093] GL3 Globulin triacylceramide

[0094] GM1 Monosialotetrahexosylganglioside

[0095] GM2 Monosialotriahexosylganglioside

[0096] GM3 monosialodihexosylganglioside

[0097] GSL Glycosphingolipids

[0098] Hex β-hexosaminidase

[0099] IC 50 Half maximum inhibitory concentration

[0100] iPSC Induced Pluripotent Stem Cell

[0101] KD Knockdown

[0102] lyso-GL1 glucosphingosine

[0103] lyso-GL2 lactose sphingosine

[0104] lyso-GL3 spherical triacylsphingosine

[0105] LSD Lysosomal Storage Disorders

[0106] MPS Mucopolysaccharidosis

[0107] NCL Neuronal ceroid lipofuscinosis

[0108] OE overexpression

[0109] PBMC peripheral blood mononuclear cells

[0110] PCR polymerase chain reaction

[0111] PC Phosphatidylcholine

[0112] PD Parkinson's disease

[0113] RNA Ribonucleic acid

[0114] SAP

[0115] SD Standard deviation

[0116] SEM standard error of the mean

[0117] SRT Substrate Reduction Therapy

[0118] Tmax Time to maximum concentration (pharmacokinetics)

[0119] WT Wild type

[0120] Methods for stimulating GSL processing in cells

[0121] As explained in more detail in the following examples, the present disclosure relates to a novel assay that can evaluate lipid flux in cells over a short period of time via perturbations of the sphingolipid pathway, for example, at a GC enzyme node. The assay exemplified herein uses cyclohexene tetraol-β epoxide (CBE), an irreversible covalent conjugate and inhibitor of glucocerebrosidase, which inhibits the GC enzyme when, for example, administered intraperitoneally, allows measurement of subsequent lipid accumulation and recovery or flux of lipids (e.g., GL1 and / or lyso-GL1). A GC enzyme mouse model was used under the following three genotype conditions: homogeneous knockout (Gba - / -), heterogeneous knockout (Gba - / + ) and wild type (Gba + / + ), lipid flux patterns associated with CBE dose and genotype have been observed. It has also been observed that wild-type mice of different genetic backgrounds exhibit variable lipid fluxes dependent on their genotype. These lipid flux patterns were recapitulated in vitro using human SHSY-5Y neuroblastoma cell lines that have been genetically modified to overexpress GBA or have reduced GC enzyme activity due to GBA knockdown. Lipid levels in response to CBE treatment were shown to be negatively correlated with GBA levels.

[0122] Emerging evidence suggests that idiopathic, non-GBA mutation carrier patients may exhibit reduced GC enzyme activity and / or increased glycosphingolipid accumulation in the central nervous system (CNS). This highlights the potential to expand the target population for substrate reduction therapies (using drugs that modulate the GSL pathway, such as vinlukast) based on the relevant cellular and biochemical mechanisms. The results presented herein suggest that revealing impaired GSL flux through "stimulation" assays (e.g., using CBEs, etc.) has the potential to expand the use of therapies targeting this pathway to a wider patient population. Data generated in vivo in mice and in vitro in human cell lines can be extrapolated to clinical settings, and the present disclosure describes how to collect, culture, and process patient cells, such as peripheral blood mononuclear cells (PBMCs) and / or fibroblasts, to reveal susceptibility to perturbations of the GSL pathway. This in turn helps to expand the patient population that will benefit from vinlukast and related therapies.

[0123] Broadly speaking, the disclosure provides methods for stimulating a GSL pathway in cells from a subject and monitoring the response of the pathway to the stimulation (e.g., its recovery). This can be used to assess a subject's abnormal glycosphingolipid processing (or determine the severity of a disease associated therewith), or diagnose a disease associated therewith or monitor the progression of the disease, or assess a subject's potential or actual therapeutic response to treatment with a drug that can modulate a GSL pathway.

[0124] Thus, in a first aspect, the present disclosure provides a method of assessing aberrant glycosphingolipid processing in a subject, the method comprising treating cells of the subject to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation.

[0125] Typically, the stimulation provided to the cell involves contacting the cell with an agent that regulates (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, sapogenin (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 glucosyltransferases and galactosyltransferases. In embodiments, the agent activates a lipid synthase. In other embodiments, the agent activates a glycosyltransferase. 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 a glucocerebrosidase inhibitor.

[0126] In an embodiment, the agent is selective for one enzyme involved in the glycosphingolipid pathway. For example, the agent is at least 10 times more active against the one enzyme than against other enzymes involved in the glycosphingolipid pathway (e.g., as determined by IC 50 or EC 50 In embodiments, the agent is 15, 20, 25, 30, 50, 75, 100, 150, 200, 500, or 1000 times more active against the one enzyme than against other enzymes involved in the glycosphingolipid pathway.

[0127] In an embodiment, the agent is an irreversible modulator of an enzyme involved in a glycosphingolipid pathway. For example, the agent can be covalently bound to the enzyme, for example to block the active site of the enzyme or to block the interaction between the enzyme and another biomolecule required for its activity. Alternatively, the agent can target the enzyme for inactivation or degradation. In an embodiment, the agent is an irreversible inhibitor of an enzyme involved in a glycosphingolipid pathway. In one embodiment, the agent is an irreversible inhibitor covalently bound to the enzyme.

[0128] Agents that strongly bind (e.g., covalently bind) to enzymes involved in the glycosphingolipid pathway are known to those skilled in the art (for details on agents targeting sphingomyelinase and ceramidase, see, e.g., Canals et al., Br J Pharmacol. (2011) 163 (4): 694-712). Such agents are typically structural analogs of one or more natural substrates of the enzyme, such as transition states that mimic natural reaction intermediates and / or contain reactive chemical groups that can form covalent bonds with the enzyme. Exemplary covalent modulators include small molecule inhibitors of glucocerebrosidase described by Kuo et al. (The FEBS Journal (2019) 286: 584-600). These compounds are transition state analogs that contain epoxide groups that capture carboxylic acids on the enzyme. The inhibitors described by Kuo et al. include cyclohexene tetraol-β epoxide (CBE), which is highly selective for glucocerebrosidase over other glycosidases (IC 200 for GC enzyme). 50 The values ​​were approximately 0.3 μM, compared to values ​​in the range of 200-2000 μM for the non-lysosomal glucosylceramidase, β-glucuronidase, and α-glucosidase).

[0129] Thus, in an embodiment, the agent is a compound of formula (I):

[0130]

[0131] or a pharmaceutically acceptable salt thereof, wherein:

[0132] X is -O- or -N(R 2 )-;

[0133] R 1 Selected from -OH, -C(O)OH and -CH 2 OH; and

[0134] R 2 , when present, is selected from -H and C optionally substituted with one or more groups independently selected from -OH and halogen 1-6 alkyl.

[0135] In one embodiment, the agent is a CBE. Thus, in view of this aspect, the present disclosure provides a method of assessing abnormal glycosphingolipid processing in a subject, the method comprising contacting a cell of the subject with a CBE to stimulate a glycosphingolipid pathway in the cell, and monitoring the recovery of at least one glycosphingolipid after the stimulation.

[0136] In practice, the method will usually be carried out on a sample obtained from a subject. Therefore, in an embodiment, the method is an in vitro method, wherein the cell is present in a sample obtained from the subject. The method may include obtaining a sample from a subject for the step of direct analysis. Alternatively, the method may be carried out on a sample that was previously obtained from a subject and may, for example, have been stored and optionally reconstructed (e.g., in a dried blood sample or a biopsy sample frozen with liquid nitrogen). The sample may (or may have) be processed to enrich or concentrate cells to be stimulated. Alternatively or additionally, the sample may (or may have) be processed to remove unwanted sample material, such as tissue, such as connective tissue.

[0137] The method of the present disclosure can adopt tissue samples, such as tissue samples from the brain, liver, kidney, skin or spleen of the subject. The method of the present disclosure can also adopt samples of biological fluids containing cells (such as whole blood or its cell-containing fractions). The method for obtaining such samples is known to those skilled in the art. Therefore, in embodiments, the sample is a tissue sample (for example, from the brain, liver, kidney, skin or spleen of the subject), or the sample is a blood sample containing cells (for example, whole blood). In specific embodiments, the tissue sample is a brain, liver, kidney, skin or spleen sample. In other specific embodiments, the blood sample is whole blood. Whole blood and its cell-containing fractions are particularly convenient for use according to the method of the present invention.

[0138] In embodiments, the method processes cells of a particular type (e.g., pedigree). Therefore, the method of the present disclosure can adopt a sample comprising the cell type, or they can adopt a cell colony of purification, passage and / or amplification that can be substantially composed of the cell type. The sensitivity of the inventive method can be improved using partially or completely purified cells and cell colonies. The example of cell types that can be used according to the present disclosure includes fibroblasts, peripheral blood mononuclear cells (PMBCs) and induced pluripotent stem cells (iPSCs). Methods for obtaining, purifying, passage and / or amplifying such cells are known to those skilled in the art. It should be understood that the method described herein carried out on "cell" encompasses such a method, wherein a plurality of such cells are used, optionally wherein other cells or cell types may also be present.

[0139] Therefore, in one embodiment, the cell is a fibroblast; in a related embodiment, the sample comprises fibroblasts (or is substantially composed of fibroblasts). Fibroblasts can be obtained from, for example, the dermal tissue of a subject using conventional techniques, such as from a skin puncture biopsy. In another embodiment, the cell is an iPSC; in a related embodiment, the sample comprises iPSC (or is substantially composed of iPSC). iPSC is produced by somatic cells of the patient, for example, by reprogramming of fibroblasts that can be obtained as described herein. Other sources of somatic cells for producing iPSC 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 PBMC obtained (e.g., purified) from a blood sample taken from a subject; in a related embodiment, the sample comprises PBMC (or is substantially composed of PBMC), which is obtained (e.g., purified) from a blood sample taken from a subject. In an embodiment, the cell expresses neuroepithelial stem cell protein (nestin).

[0140] The methods of the present invention relate to monitoring the recovery of the GSL pathway after stimulation. Based on the disclosure herein, those skilled in the art will appreciate that there are different ways to achieve such monitoring, and that the most appropriate way (or ways) may be different in different situations, for example, when different agents are used to stimulate different parts of the GSL pathway and / or when different samples or cell types are used. The discussion below focuses on the case exemplified below, i.e., monitoring GSL recovery after treating cells with CBE to specifically stimulate the GC enzyme node. If other parts of the GSL pathway are stimulated, this assessment can be modified (if necessary) based on the disclosure.

[0141] According to a first aspect of the present disclosure, monitoring the recovery of the GSL pathway to stimulation is achieved by monitoring the recovery of at least one glycosphingolipid. In embodiments where the stimulation involves contacting the cell with an agent that modulates (e.g., inhibits) an enzyme involved in the glycosphingolipid pathway, the at least one glycosphingolipid monitored includes one or more of: (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.

[0142] Suitable glycosphingolipids that can be used to monitor recovery include GL1, lyso-GL1, GL2, lyso-GL2, GL3, lyso-GL3, galactocerebroside, GM1, GM2 and GM3, and for example ceramide and sphingosine. In an embodiment, the at least one glycosphingolipid is (or comprises) selected from the following lipids: glucosylceramide (GL1), ceramide and glucosylsphingosine (lyso-GL1). In an embodiment, 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 a GC enzyme inhibitor. In other embodiments, the at least one glycosphingolipid is (or comprises) total GL3, total ceramide or total lyso-GL3.

[0143] In an embodiment, the at least one glycosphingolipid is (or comprises) a lipid (e.g., GL1) containing a fatty acid moiety that has a low baseline abundance, i.e., low abundance, before any stimulus is provided to the GSL pathway. Such fatty acids may be found at a level less than 25% of the total fatty acids on the lipid in question (on a molar basis), such as a level 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.

[0144] In an embodiment, the at least one glycosphingolipid is (or comprises) a lipid containing a monounsaturated fatty acid moiety (e.g., GL1). For example, the 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, the fatty acid moiety is selected from C16:1, C18:1, C20:1, C22:1, and C24:1. For example, the fatty acid moiety can be selected from C16:1, C18:1, and C20:1, or selected from C16:1 and C18:1. In one embodiment, the at least one glycosphingolipid is (or comprises) a lipid containing a C16:1 fatty acid moiety. In other embodiments, the at least one glycosphingolipid is (or comprises) a lipid containing a fatty acid moiety selected from C24:0, C16:1n-7, C18:1n-7, C22:1n-9, C24:1n-9, and C18:2 cis. In embodiments, monitoring the recovery of at least one glycosphingolipid after stimulation does not include monitoring the recovery of lipids comprising a C18:0 fatty acid moiety (e.g., GL1) and / or a lipid comprising a C18:1 fatty acid moiety.

[0145] In embodiments, the methods of the present invention employ a single measurement to monitor GSL recovery, e.g., measuring the level of the at least one GSL at a single set time point after stimulation. In other embodiments, the monitoring involves monitoring GSL recovery over a period of time, e.g., measuring the level of the at least one GSL at more than one time point after stimulation. For example, monitoring the recovery of at least one glycosphingolipid after stimulation may include measuring the level of at least one glycosphingolipid produced in or by the cell multiple times after the stimulation, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In embodiments, monitoring the recovery of at least one glycosphingolipid after stimulation includes measuring the level of at least one glycosphingolipid produced in or by the cell at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In embodiments, monitoring the recovery of at least one glycosphingolipid after stimulation includes measuring the level of at least one glycosphingolipid produced in or by the cell until it substantially reaches the pre-treatment (baseline) level, e.g., a level within about 10% or about 5% of the pre-treatment level. This provides information about the recovery of the GSL pathway, including both the time required to recover to baseline GSL levels and the curve of GSL recovery (eg, the shape of the curve when plotting concentration versus time).

[0146] As an alternative (or supplement) to monitoring the recovery of a GSL pathway in a cell compared to the baseline level of 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 help identify abnormal GSL processing, particularly when the abnormality is not very severe. Therefore, in an embodiment, the method further comprises the step of comparing the recovery of at least one of the glycosphingolipids after the stimulation with the recovery of one or more of the same glycosphingolipids in the corresponding cells from a healthy individual after the same stimulation. In an embodiment, the comparison step involves comparing one or more of the following parameters (which can be derived, for example, from a graph of concentration versus time): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) after stimulation, the max The time required (T max); (iii) area under the curve (AUC); and (iv) the time required for the level to return to baseline, e.g., to within about 10% or about 5% of the baseline level. Using these metrics, if the difference in one or more of the parameters is, e.g., greater than about 10%, e.g., greater than about 20%, 30%, 50%, 75%, 100%, 150%, or 200%, the subject is assessed as having abnormal glycosphingolipid processing. In embodiments (e.g., where the sample is or comprises PBMC obtained from a subject), the recovery of one or more glycosphingolipids is monitored for a period of time of up to about 72 hours after stimulation, e.g., up to about 64 hours, about 56 hours, about 48 hours, about 40 hours, about 32 hours, or about 24 hours after stimulation.

[0147] The method of the present disclosure can also be used to assess the disease state of an individual. Therefore, 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. The method comprises assessing the abnormal glycosphingolipid processing of the subject according to the aforementioned aspects, and determining the severity of the disease based on the extent to which the recovery differs from the recovery of cells stimulated in the same manner from healthy individuals. The embodiments of the first aspect (e.g., regarding the medicament to be used, the sample type and / or cell type, the lipid to be monitored, etc.) can be applied to this second aspect.

[0148] Diseases associated with abnormal glycosphingolipid processing include lysosomal storage diseases. Therefore, this aspect may be particularly useful for evaluating subjects known to suffer from (or believed or predicted to suffer from) lysosomal storage diseases. Such diseases generally involve changes in GSL levels, which are generally due to the lack of one or more enzymes associated with GSL processing. As shown in the following examples, a mouse model of Gaucher disease (GD) showed significant differences in lipid flux after CBE treatment compared to wild-type mice. Thus, in embodiments, the methods can be used to determine the severity of a lysosomal storage disease in a subject, such as a disease selected from the group consisting of Fabry disease, Krabbe disease, Gaucher disease (e.g., type 1, type 2, and type 3), Niemann-Pick disease (e.g., type A, type B, and type C), metachromatic leukodystrophy, Farber disease, Krabbe disease, galactosialidase storage disease, 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, Sala disease, alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucosidosis, MPS MPS I (e.g., Hurler syndrome, Scheherazade syndrome, and Hurler-Schneider syndrome), MPS II (e.g., Hunter syndrome), MPS III (e.g., Sanfilippo syndrome types A, B, C, and D), MPS IV (e.g., Morquio syndrome types A and B), MPS VI (e.g., Maroteaux-Lamy syndrome), MPS VII (e.g., Slater syndrome), MPS IX (e.g., hyaluronidase deficiency), mucolipidosis (e.g., sialidosis, inclusion body cell disease, pseudo-Hurler polydystrophy / phosphotransferase deficiency, and mucolipidin 1 deficiency), and neuronal ceroid lipofuscinosis (e.g., Santavuori-Haltiadisease / infantile NCL, Jansky-Bielschowsky disease In one embodiment, the disease is GD (e.g., GD type 1, type 2, or type 3).In another embodiment, the disease is FD.

[0149] Diseases associated with abnormal glycosphingolipid processing also include synucleinopathies. Therefore, other conditions that can be usefully studied using the methods of the present disclosure include synucleinopathies. These are neurodegenerative diseases characterized by abnormal accumulation of alpha-synuclein aggregates in neurons, nerve fibers or glial cells. However, there is a large amount of new evidence that synucleinopathies may be associated with abnormal GSL processing. Therefore, in embodiments, the method can be used to determine the severity of a subject's synucleinopathies, such as diseases selected from Parkinson's disease (PD) such as idiopathic Parkinson's disease or Lewy body dementia (DLB). In embodiments, the disease is PD, such as idiopathic PD.

[0150] The methods of the present invention can be used to assess the disease state of an individual with a known or unknown genotype of a gene, which represents a risk factor for a condition associated with abnormal GSL processing (e.g., a lysosomal storage disease or a synuclein disease). In embodiments, the mutation status of the GBA gene in the subject is unknown; in other embodiments, the subject has been assessed as having only one or zero known mutations (e.g., non-functional or reduced-function) GBA alleles (see, e.g., Hruska et al., Hum. Mutat. (2008) 29 (5): 567-583). Similarly, the methods of the present invention can be used to assess the disease state of an individual, regardless of whether they have previously been assessed for a disease associated with abnormal glycosphingolipid processing. Thus, in embodiments, the subject has not previously been diagnosed with a disease associated with abnormal glycosphingolipid processing, e.g., a lysosomal storage disease such as Gaucher disease, or a synuclein disease such as Parkinson's disease). In embodiments, the subject has not previously been diagnosed with Gaucher disease. In embodiments, the subject has not previously been diagnosed with Parkinson's disease. In other embodiments, the subject has been previously diagnosed with a disease associated with aberrant glycosphingolipid processing, and the method determines progression of the disease.

[0151] In a third aspect, the present disclosure provides an in vitro method for diagnosing a synucleinopathy (e.g., as defined herein) or monitoring the progression thereof in a subject, the method comprising:

[0152] - obtaining from the subject a sample comprising fibroblasts, peripheral blood mononuclear cells or induced pluripotent stem cells derived from somatic cells of the subject, and optionally culturing the cells;

[0153] - contacting the sample with CBE;

[0154] - measuring the level of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cell multiple times to obtain a response curve; and

[0155] - comparing the response curve to a comparison response curve, wherein the comparison curve is a standard response curve generated by stimulating the same sample obtained from (a) a healthy individual or (b) a patient diagnosed with PD (to diagnose the subject), or the comparison curve is a response curve from a sample previously obtained from the same subject (to monitor the progression of the subject's disease). It should be understood that the embodiments of the first and second aspects (e.g., with respect to sample type and / or cell type, lipid to be monitored, disease state, etc.) apply to this third aspect.

[0156] In embodiments, the sample is contacted with CBE at a final concentration of between about 0.01 μM and about 100 μM. For example, the final concentration of CBE can be between about 0.1 μM and about 10 μM, between about 0.3 μM and 8 μM, between about 0.5 μM and 5 μM, or between about 0.8 μM and 3 μM, e.g., about 1 μM or about 2 μM. In embodiments, an excipient such as sodium taurocholate is added to the sample to increase the dynamic range, e.g., by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, e.g., up to about 10-fold.

[0157] In addition to disease assessment, diagnosis and monitoring, the methods of the present invention can also be usefully used to evaluate the therapeutic response to treatment before any treatment begins (e.g., predictive assessment), and during a treatment regimen (e.g., monitoring response). Such methods will generally evaluate the therapeutic response to treatment with a modulator of an enzyme involved in the glycosphingolipid pathway, and will stimulate the GSL pathway at or near a node represented by the enzyme. Glycosphingolipids (e.g., substrates or products of enzymes) that are closely associated with the node are typically those that are monitored to evaluate recovery. Thus, for example, when using an agent that is a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GC enzyme) activator, the stimulation can be glucocerebrosidase (e.g., using CBE), and one or more of glucosylceramide (GL1), ceramide, and glucosylsphingosine (lyso-GL1) can be monitored.

[0158] Thus, in a fourth aspect, the present disclosure provides a method for assessing a subject's potential therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GCase) activator, the method comprising contacting cells of the subject with a CBE to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation, wherein the subject is assessed as a candidate for treatment if the recovery is slower or less complete than the recovery of cells stimulated in the same manner from a healthy individual. It should be understood that the embodiments of the preceding aspects (e.g., regarding sample type and / or cell type, lipids to be monitored, etc.) may be applicable to this fourth aspect.

[0159] In an embodiment, the method evaluates the subject's possible therapeutic response to treatment with a GCS inhibitor (e.g., selected from ilurustat and vinlustat, including pharmaceutically acceptable salts thereof). In an embodiment, the GCS inhibitor is vinlustat or a pharmaceutically acceptable salt thereof. In other embodiments, the method evaluates the subject's possible therapeutic response to treatment with a GC enzyme activator, which is selected from, for example, sapocin 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.

[0160] In a fifth aspect, the present disclosure provides a method for evaluating a subject's therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GC enzyme) activator, the method comprising:

[0161] (a) obtaining a first cell-containing sample from the subject at a first time point;

[0162] (b) contacting cells of the first cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation;

[0163] (c) obtaining a second cell-containing sample from the subject at a second time point after treating the subject with a GCS inhibitor or a GC enzyme activator; and

[0164] (d) contacting cells of said second cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in said cells, and monitoring the recovery of at least one glycosphingolipid after said stimulation,

[0165] Wherein if the recovery in the cells from the second cell-containing sample is faster or more complete than the recovery in the cells from the first cell-containing sample, the treatment is evaluated as positive (e.g., successful). It should be understood that the embodiments of the preceding aspects (e.g., regarding sample type and / or cell type, lipids to be monitored, etc.) can be applied to this fifth aspect.

[0166] In embodiments, steps (a) and (b) will be performed in substantially the same manner as method steps (c) and (d). In this way, the difference between the recovery of GSLs in cells from the first sample and the second sample can best reflect the efficacy of the treatment. Thus, typically the first cell-containing sample and the second cell-containing sample will contain substantially the same cell type, for example, they will be derived from the same type of tissue treated and processed in substantially the same manner. Likewise, it is typically the case that the at least one glycosphingolipid monitored in step (b) is typically the same as the at least one glycosphingolipid monitored in step (d).

[0167] The foregoing aspects also facilitate optimization of treatment methods for a subject, for example by providing a method for treating a disease associated with abnormal glycosphingolipid processing in a subject, the method comprising administering to the subject an agent capable of treating the disease, monitoring the therapeutic response to the agent, and altering the dosage of the agent accordingly.

[0168] Thus, in a sixth aspect, the disclosure provides a method of treating a disease associated with abnormal glycosphingolipid processing (e.g., a lysosomal storage disease or a synucleinopathy as defined herein) in a subject in need thereof, the method comprising administering to the subject an agent capable of treating the lysosomal storage disease or the synucleinopathy, wherein the method involves monitoring a therapeutic response according to the fifth aspect (above), and adjusting the dose of the agent according to the results of the monitoring. It should be understood that the embodiments of the preceding aspects (e.g., regarding sample type and / or cell type, lipids to be monitored, etc.) may be applicable to this sixth aspect.

[0169] In embodiments, if treatment is evaluated as positive, the dosage of medicament remains unchanged, and if treatment is evaluated as inactive, the dosage of medicament is increased. In other embodiments, if treatment is evaluated as active, the dosage of medicament is reduced, and the monitoring step is continued until treatment is no longer evaluated as active, and the dosage is increased until treatment is again evaluated as active. In embodiments, the monitoring step is performed at regular intervals, so that treatment can be evaluated as active to assess the sustained efficacy of treatment and / or allow adjustment of dosage. The monitoring step can be performed at intervals of, for example, 1, 2, 3, 4, 5, 6, 12, 18, 24 months or more. In embodiments, the monitoring step is performed more frequently (for example, every 1, 2, 3 or 4 weeks) at the beginning of treatment, and once a stable treatment regimen is established, the monitoring step is performed less frequently (for example, every 2, 3, 4, 6 or 12 months).

[0170] In embodiments, the monitoring step is performed along with other known methods for monitoring the progression of a disease associated with abnormal glycosphingolipid processing (e.g., a lysosomal storage disease). For example, the measuring step can be performed along with measurements of the subject's platelet count, hemoglobin concentration, spleen volume, and / or liver volume. These measurements may be useful, for example, in determining whether a subject is experiencing more severe symptoms of a lysosomal storage disease.

[0171] In a seventh aspect, the present disclosure provides a method of treating a lysosomal storage disease or a synucleinopathy in a subject in need thereof, the method comprising the step of administering an effective amount of an agent capable of treating the lysosomal storage disease or the synucleinopathy (e.g., an agent as described herein), wherein the patient has been assessed as having abnormal glycosphingolipid processing according to the methods described herein, or has had his or her disease state or severity assessed according to the methods described herein.

[0172] In an eighth aspect, the present disclosure provides a method for treating or preventing the development or progression of a lysosomal storage disease or a synucleinopathy in a subject assessed as being at risk for developing a lysosomal storage disease or a synucleinopathy according to a method as described herein (e.g., the method of the second aspect or the third aspect), the method comprising the following steps: (a) starting the subject on a course of therapeutic treatment (e.g., a therapeutically effective amount of an agent capable of treating the lysosomal storage disease or the synucleinopathy, such as an agent described herein); and optionally (b) assessing the risk of developing a lysosomal storage disease or a synucleinopathy according to a method as described herein or repeating the assessment, and optionally adjusting the therapeutic treatment based on the new assessment.

[0173] In embodiments, the method includes administering one or more (e.g., two, three, four or more) agents that can treat or prevent the disease or disorder. For example, the method may include administering a lysosomal enzyme (ERT) such as imiglucerase, and / or a small molecule (SRT) such as vinlusstat or ilulosstat. Alternatively or additionally, the method may include administering one or more agents that alleviate the symptoms of the disease or disorder, such as selected from levodopa or its prodrug (optionally in combination with carbidopa or its prodrug), ambroxol, amantadine, dopamine agonists, MAO inhibitors, COMT inhibitors, and anticholinergic agents.

[0174] In a ninth aspect, the disclosure provides the use of cyclohexene tetraol-β epoxide (CBE) in a method for diagnosing a disease associated with abnormal glycosphingolipid processing in a subject or determining its severity. It should be understood that the embodiments of the preceding aspects (e.g., regarding diseases) can be applicable to this sixth aspect.

[0175] Having been generally described herein, the following non-limiting examples are provided to further illustrate the disclosure.

[0176] Example

[0177] Example 1: In vivo proof-of-concept study in mice

[0178] This example was designed to test the hypothesis that transient inhibition of GC enzyme and subsequent accumulation of GC enzyme substrate can serve as a sensitive assay for revealing lipid flux defects.Preliminary proof-of-concept studies were performed in mice and transformed human cell lines.

[0179] Dose response of GSL pathway stimulation in WT mice

[0180] Dose responses to CBE were performed in wild-type (WT) mice (Charles River Laboratories or Jackson Labs). Concentrations of CBE between 0.3 mg / kg and 100 mg / kg were studied. The 4-methylumbelliferyl (4MU) hydrolysis assay was used to measure the GC enzyme activity remaining 24 hours after CBE administration. Briefly, cell lysates from different sample types were diluted into 0.1 M sodium acetate buffer (pH 4.5) containing 10 mM synthetic substrate 4-methylumbelliferyl-β-D-pyranoglucoside and incubated at 37°C for 1 hour. The reaction was terminated by adding 0.5 volumes of 1 M glycine buffer (pH 12.5). The fluorescence of the reaction was measured using a Spectramax fluorometer (Ex365 / Em445; Molecular Devices, Sunnyvale, CA, USA). The 4MU hydrolysis assay was used. (imiglucerase) to generate a standard curve.

[0181] Figure 1 Results of CBE administration in WT mice are shown. Although there was a dose-dependent decrease in enzyme activity, little or no further inhibition of GC enzyme was measured above 30 mg / kg. Based on these data alone, it is difficult to determine whether this is due to saturation of the enzyme or due to the sensitivity of the GC enzyme activity assay. Measurements of downstream substrates allow for the distinction between these two scenarios. Lyso-GL1 levels in the cortex, liver, and plasma all increased with increasing CBE doses above 30 mg / kg ( Figure 2 ), which suggests that this substrate can serve as a reporter of GC enzyme activity over a wider concentration range.

[0182] Time course of lipid flux following GSL pathway stimulation in Gba+ / - mice

[0183] We investigated whether substrate accumulation could distinguish mice carrying different copy numbers of mutations in GBA1. Three mouse models were studied: GBA D409V / D409V , Gba D409V / + and Gba + / + , which were homozygous for the loss-of-function D409V mutation in GBA1, heterozygous for the mutation, or WT for GBA1. In addition to glucosylceramide (GL1), the GC enzyme substrate glucosylceramide (GL1) was also studied. Mice were administered a single dose of CBE and sacrificed 3, 6, 24, 48, or 72 hours later. Substrate accumulation and dissipation were measured over time.

[0184] Substrate accumulation peaked between 24 and 48 hours, with Lyso-GL1 accumulation in cortex, liver, and plasma showing greater sensitivity to genetic background ( Figure 3 ). After the peak, substrate levels begin to decline as newly synthesized enzymes degrade these substrates. Secondary pathways may also contribute to the observed lipid reduction. As predicted, peak lipid concentrations were negatively correlated with genotype. In addition, the rate of recovery was related to genotype: WT mice recovered to or near normal levels the fastest, followed by heterozygous mutant mice, and then homozygous mutant mice.

[0185] Time course of lipid flux following GSL pathway stimulation in wild-type mice

[0186] Having demonstrated that accumulation and dissipation of substrate is a sensitive measure of the amount of GC enzyme activity, the question next tested was how substrate levels change over time after stimulation in wild-type mice.

[0187] 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 stimulation. These strains are all "normal" in the sense that they do not have any specific mutations, but are genetically different from each other, just like individual humans. Importantly, the mice selected did not have a mutation in GBA1. A CBE stimulation time course was performed in these three different strains, and lyso-GL1 and GL-1 accumulation was measured at 24, 48, and 72 hours after administration.

[0188] The experimental results are shown in Figure 4 and Figure 5 There were no significant differences in GSL levels in the cortex between strains ( Figure 4 A). However, in liver and plasma, C57BL / 6 mice accumulated more lyso-GL1 ( Figure 4 B and Figure 4 C). These results were similar for total GL1 in the liver. Plasma GL1 levels showed a slightly different trend, with the highest levels observed in FVB mice. These results suggest that CBE stimulation can distinguish individuals with possible defects in sphingolipid metabolic pathways, even if they do not have GBA1 mutations. Analysis of the different isoforms of GL1 in the cortex showed variable chain length distribution between strains ( Figure 5 ), among which C57BL / 6 mice have higher levels of C22, C23, C24, and C24:1 isoforms.

[0189] Example 2: In vitro studies in human cell lines

[0190] The in vivo experiments described above provide proof of concept for transient inhibition of GC enzymes to increase accumulation of sphingolipids, thereby amplifying signals from underlying genetic differences. However, such approaches are impractical for use in patients because tissue samples must be extracted for measurement. To address this gap in feasibility, human cell lines with varying levels of GBA1 transcript expression were studied.

[0191] First, the response of human SHSY-5Y neuroblastoma cells to CBE was studied. Cells were used without other changes (wild type), or cells were modified to reduce or increase Gba activity (knockdown and overexpression, respectively). The modification was achieved by stable transfection with a plasmid (KD) expressing siRNA targeting GBA or a plasmid (OE) constitutively overexpressing GBA. Human SHSY-5Y 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 0, 24 and 48 hours after CBE removal. Figure 6 The effect of different durations of incubation of wild-type cells with CBE on enzyme activity is shown, from which it was concluded that a 24-hour incubation period was suitable to obtain a good reaction. Figure 7 The responses of three different cell types to different concentrations of CBE are shown. A significant increase in enzyme activity was observed between knockdown cells and wild-type cells, as well as between wild-type cells and overexpressing cells. Interestingly, when cells were also incubated with sodium taurocholate, the dynamic range of the assay was found to increase significantly ( Figure 8 ).

[0192] The lipid responses of cells to CBE incubation were then investigated ( Fig. 9 No significant differences in total GL1 levels were observed in wild-type cells after treatment ( Fig. 9 A). After CBE treatment, some differences in lyso-GL1 levels could be measured between the three cell types ( Fig. 9 B), in the absence of treatment, there was no measurable accumulation of lyso-GL1. Under typical cell culture conditions, it was found that no significant differences in GL1 accumulation could be measured between cells expressing 40% of endogenous GBA1, WT amounts, or 300% of WT amounts. Furthermore, under normal conditions, none of these cell lines had measurable lyso-GL1 accumulation. The consequences of genetic differences between cells could only be revealed by transient inhibition of the GC enzyme by CBE. Following CBE treatment, cells accumulated GL1 and lyso-GL1 at levels that were inversely proportional to GBA1 expression levels. This model in vitro system demonstrates that cells harvested from patients and treated with CBE in culture can be used to measure intrinsic defects in patients' sphingolipid metabolic pathways.

[0193] The accumulation of different GL1 species after CBE treatment was also investigated. In general, it was found that monounsaturated GL1 species (i.e., isoforms with one double bond in the fatty acid portion of the molecule) accumulated more than saturated species, and that isoforms with shorter chain lengths accumulated more than isoforms with longer chain lengths ( Fig.10 ). Considering the baseline abundance of GL1 species ( Fig.11), it was observed that the low-abundance isoforms were generally the ones that accumulated in response to CBE treatment.

[0194] Example 3: CBE stimulation in human PBMCs

[0195] The effects of CBE stimulation in human peripheral blood mononuclear cells (PBMCs) were investigated.

[0196] PBMCs are obtained from blood samples (Stem Cell Technologies, catalog number 70025, batch number 2105417005, donor number CE0006419) using standard procedures. 25,000 cells are added to a single well of a multiwell plate and allowed to adapt for 24 hours, and then they are treated with 100 μM CBE for 24 hours. The culture medium is removed and replaced with fresh culture medium (without CBE), and lipids are extracted from the cells at time intervals of 0, 2, 4, 8, 16 and 24 hours. GL1 and lyso-GL1 levels, as well as the level of phosphatidylcholine (PC) are measured. It is found that PC levels are largely unaffected by CBE treatment, and are therefore used to normalize the levels of GL1 and lyso-GL1 (e.g., to minimize the effects of different numbers of cells in each well, and / or the effects of cell proliferation during time).

[0197] After CBE treatment, GL1 levels in PBMCs increased approximately 6-fold (from approximately 2 ng / ml to approximately 12 ng / ml), and lyso-GL1 levels increased from barely detectable levels to approximately 0.08 ng / ml. Lyso-GL1 levels were near the lower limit of detection of the assay, so there was a large error. When normalized to total PC levels (100-600 ng / ml during the experiment), GL1 and lyso-GL1 levels were as follows Fig.12 As shown. At 16 hours after treatment, GL1 levels ( Fig.12 A) and lyso-GL1 levels ( Fig.12 B) has normalized.

[0198] The GL1 isoform was analyzed to check the stability of the cell culture. Cells were obtained as described above without CBE treatment and cultured and monitored for 72 hours. The lipid levels of the GL1 isoform are shown in Fig.13Although different subtypes exhibited different absolute levels, there was a pattern across all tested species where levels increased in a roughly linear manner from 0 to 24 to 48 hours and then declined at 72 hours. It is hypothesized that the increase in levels to 48 hours is due to cell proliferation, while the decrease at 72 hours may be due to heterogeneous cell population growth and / or adhesion dynamics. These results suggest that the time course for analyzing GSL levels in PBMCs using this protocol should generally not exceed 48 hours after stimulation.

[0199] In summary, PBMCs are a suitable cell type to monitor GSL flux after stimulation - a time course can be established from cells obtained from about 1 ml of whole blood. Normalization of GSL levels to PC concentration can reduce systematic errors. PC is not essential for the GSL pathway, and its concentration is not affected by CBE treatment. Compared with lyso-GL1, GL1 appears to report better results in CBE stimulation and recovery. In healthy individuals, GL1 levels increase robustly after stimulation, which return to baseline by 16 hours.

[0200] *****

[0201] It should be understood that although the present disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate rather than limit the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be clear to those skilled in the art to which the present disclosure belongs.

[0202] In addition, where features or aspects are described in terms of Markush groups, those skilled in the art will recognize that such features or aspects are also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0203] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Claims

1. A method for assessing abnormal glycosphingolipid processing in a subject, the method comprising treating cells of the subject to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation.

2. The method of claim 1, wherein the stimulation comprises contacting the cell with an agent that inhibits an enzyme involved in the glycosphingolipid pathway.

3. The method of claim 2, wherein the enzyme is glucocerebrosidase.

4. The method of claim 2 or 3, wherein the agent is selective for an enzyme involved in the glycosphingolipid pathway.

5. The method of any one of claims 2 to 4, wherein the agent is an irreversible inhibitor of the enzyme, for example, wherein the inhibitor is covalently bound to the enzyme.

6. The method according to any one of claims 2 to 5, wherein the agent is a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -N(R 2 )-; R 1 is selected from -OH, -C(O)OH and -CH2OH; and R 2 , when present, is selected from -H and C1 optionally substituted with one or more groups independently selected from -OH and halogen - 6 alkyl.

7. The method of claim 1, wherein the step of treating the subject's cells to stimulate a glycosphingolipid pathway in the cells comprises contacting the cells with cyclohexenetetraol-β-epoxide (CBE).

8. The method according to any one of claims 1 to 7, wherein the method is an in vitro method, wherein the cells are present in a sample obtained from the subject.

9. The method of claim 8, wherein the sample is a tissue sample (e.g., brain, liver, kidney, skin, or spleen from the subject), or wherein the sample is a blood sample (e.g., whole blood) containing cells.

10. The method according to any one of claims 1 to 9, wherein the cell is a fibroblast, a peripheral blood mononuclear cell (PMBC) or an induced pluripotent stem cell (iPSC) derived from a somatic cell of the subject.

11. The method of claim 10, wherein the cells are peripheral blood mononuclear cells obtained (eg, purified) from a blood sample taken from the subject.

12. The method according to any one of claims 1 to 11, wherein the cells express neuroepithelial stem cell protein (nestin).

13. The method of any one of claims 1 to 12, wherein monitoring the recovery of at least one glycosphingolipid after the stimulation comprises measuring the level of at least one glycosphingolipid in or produced by the cell multiple times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, after the stimulation.

14. The method of any one of claims 1 to 13, wherein monitoring the recovery of at least one glycosphingolipid after the stimulation comprises measuring the level of at least one glycosphingolipid in or produced by the cell until it substantially reaches a pre-treatment (baseline) level, such as a level within about 10% or within about 5% of the pre-treatment level.

15. The method according to any one of claims 1 to 14, wherein the at least one glycosphingolipid is or comprises a lipid selected from the group consisting of glucosylceramide (GL1), ceramide and glucosylsphingosine (lyso-GL1).

16. The method of claim 15, wherein the at least one glycosphingolipid is or comprises total GL1, total ceramide or total lyso-GL1.

17. The method according to any one of claims 1 to 16, wherein the at least one glycosphingolipid is or comprises a lipid containing a monounsaturated fatty acid moiety, for example selected from C16:1, C18:1, C20:1, C22:1 or C24:

1.

18. The method according to any one of claims 1 to 16, wherein the at least one glycosphingolipid is or comprises a lipid containing a fatty acid moiety selected from the group consisting of C24:0, C16:1n-7, C18:1n-7, C22:1n-9, C24:1n-9 and C18:2 cis.

19. The method of any one of claims 1 to 18, wherein monitoring the recovery of at least one glycosphingolipid after the stimulation does not include monitoring the recovery of lipids comprising C18:0 fatty acid moieties and / or lipids comprising C18:1 fatty acid moieties.

20. The method according to any one of claims 1 to 19, further comprising the step of comparing the recovery of the at least one glycosphingolipid after the stimulation with the recovery of one or more of the same glycosphingolipids after the same stimulation in corresponding cells from a healthy individual.

21. A method according to claim 20, wherein the comparing step involves comparing one or more of the following parameters (which can be derived from a plot of concentration versus time): (i) the maximum deviation of the concentration from the baseline (C max ); (ii) after stimulation, the max The time required (T max ); (iii) the area under the curve (AUC); and (iv) the time required for the level to return to baseline, e.g., to within about 10% or to within about 5% of the baseline level.

22. The method of claim 21, wherein the subject is assessed as having abnormal glycosphingolipid processing if the difference in one or more of the parameters is greater than about 10%, such as greater than about 20%, 30%, 50%, 75%, 100%, 150% or 200%.

23. A method for determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject, the method comprising assessing the subject's abnormal glycosphingolipid processing according to any one of claims 1 to 22, and determining the severity of the disease based on the extent to which the recovery differs from the recovery of cells stimulated in the same manner from healthy individuals.

24. The method of claim 23, wherein the disease is a lysosomal storage disease, such as Gaucher disease.

25. The method of claim 23, wherein the disease is a synucleinopathy such as Parkinson's disease (PD), e.g. idiopathic PD.

26. The method of any one of claims 23 to 25, wherein the mutation status of the GBA gene in the subject is unknown, or wherein the subject has been assessed as having only one or zero known mutated (e.g., non-functional or reduced-function) GBA alleles.

27. The method according to any one of claims 23 to 26, wherein the subject has not been previously diagnosed with a disease associated with abnormal glycosphingolipid processing, for example, has not been diagnosed with a lysosomal storage disease such as Gaucher disease, or has not been diagnosed with Parkinson's disease.

28. The method of any one of claims 23 to 26, wherein the subject has been previously diagnosed with a disease associated with abnormal glycosphingolipid processing and the method determines progression of the disease.

29. An in vitro method for diagnosing a synucleinopathy, such as Parkinson's disease (PD), e.g., idiopathic PD, or monitoring the progression thereof in a subject, the method comprising: - obtaining from the subject a sample comprising fibroblasts, peripheral blood mononuclear cells or induced pluripotent stem cells derived from somatic cells of the subject, and optionally culturing the cells; - contacting the sample with CBE; - measuring the level of glucosylceramide (GL1) and / or glucosylsphingosine (lyso-GL1) in or produced by the cell multiple times to obtain a response curve; and - comparing the response curve with a comparison response curve, wherein the comparison curve is a standard response curve generated by stimulating the same sample obtained from (a) a healthy individual or (b) a patient diagnosed with PD (to diagnose the subject), or the comparison curve is a response curve from a sample previously obtained from the same subject (to monitor the progression of the subject's disease).

30. A method for evaluating a subject's possible therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase activator, the method comprising contacting cells of the subject with a CBE to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid following the stimulation, wherein if the recovery is slower or less complete than the recovery of cells from a healthy individual stimulated in the same manner, the subject is evaluated as a candidate for treatment.

31. The method of claim 30, wherein the GCS inhibitor is venlukastat or a pharmaceutically acceptable salt thereof.

32. The method of claim 30 or 31, wherein the cell is a fibroblast, a peripheral blood mononuclear cell, or an induced pluripotent stem cell derived from a somatic cell of the subject.

33. The method of claim 32, wherein the cells are peripheral blood mononuclear cells obtained (eg, purified) from a blood sample taken from the subject.

34. Use of cyclohexenetetrol-β epoxide in a method for diagnosing or determining the severity of a disease associated with abnormal glycosphingolipid processing in a subject.

35. Use according to claim 34, wherein the disease is a lysosomal storage disease (eg Gaucher disease) or a synucleinopathy such as Parkinson's disease (eg idiopathic PD).

36. A method for evaluating a subject's therapeutic response to treatment with a glucosylceramide synthase (GCS) inhibitor or a glucocerebrosidase (GCase) activator, the method comprising: (a) obtaining a first cell-containing sample from the subject at a first time point; (b) contacting cells of the first cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in the cells, and monitoring the recovery of at least one glycosphingolipid after the stimulation; (c) obtaining a second cell-containing sample from the subject at a second time point after treating the subject with a GCS inhibitor or a GC enzyme activator; and (d) contacting cells of said second cell-containing sample with a CBE to stimulate a glycosphingolipid pathway in said cells, and monitoring the recovery of at least one glycosphingolipid after said stimulation, Wherein said treatment is assessed as positive if recovery in cells from said second cell-containing sample is faster or more complete than recovery in cells from said first cell-containing sample.

37. The method of claim 36, wherein the subject is treated with the GCS inhibitor venlostat or a pharmaceutically acceptable salt thereof.

38. The method of claim 36 or 37, wherein 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 somatic cells of the subject.

39. A method according to any one of claims 36 to 38, wherein: Steps (a) and (b) are performed in substantially the same manner as steps (c) and (d) of the method; the first cell-containing sample and the second cell-containing sample comprise substantially the same cell type; and / or the at least one glycosphingolipid monitored in step (b) is the same as the at least one glycosphingolipid monitored in step (d).

40. A method of treating a lysosomal storage disease or a synucleinopathy in a subject in need thereof, the method comprising administering to the subject an agent capable of treating the lysosomal storage disease or the synucleinopathy, wherein the method involves monitoring a treatment response according to any one of claims 36 to 39, and adjusting the dosage of the agent based on the results of the monitoring.

41. A method according to claim 40, wherein if the treatment is assessed as positive, the dosage of the agent remains the same, and if the treatment is assessed as not positive, the dosage of the agent is increased; or wherein if the treatment is assessed as positive, the dosage of the agent is reduced, and the monitoring step is continued until the treatment is no longer assessed as positive, at which time the dosage is increased until the treatment is again assessed as positive.

42. A method of treating a lysosomal storage disease or a synucleinopathy in a subject in need thereof, the method comprising the step of administering an effective amount of an agent capable of treating the lysosomal storage disease or the synucleinopathy, wherein the subject has been assessed as having abnormal glycosphingolipid processing according to the method of any one of claims 1 to 22, or has had their disease state or severity assessed according to the method of any one of claims 23 to 29.

43. A method of treating or preventing the development or progression of a lysosomal storage disease or synucleinopathy in a subject assessed as being at risk for developing a lysosomal storage disease or synucleinopathy according to any one of claims 23 to 29, the method comprising the steps of: (a) starting the subject on a course of therapeutic treatment; and optionally (b) assessing the risk of developing a lysosomal storage disease according to the method of any one of claims 23 to 29 or repeating said assessment, and optionally adjusting said therapeutic treatment based on the new assessment.

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