Markers for acid sphingomyelinase disorders and uses thereof

Abstract

Methods for screening, diagnosing, monitoring and / or treating acid sphingomyelinase (ASM) disorders, such as Niemann-Pick disease, are provided. [Solution] A method for treating a human subject with an acid sphingomyelinase (ASM) disorder, comprising: (a) administering to the subject a first dose of a therapeutic agent for treating the ASM disorder, the therapeutic agent having a first concentration; and (b) if the subject is determined to have a level of lyso-sphingomyelin (lyso-SPM) below the reference level after administration of the first dose, administering to the subject a second dose of a therapeutic agent having a second concentration equal to or greater than the first concentration.

Description

[Technical Field]

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application No. 61 / 832,302, filed June 7, 2013, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods for screening, diagnosing, monitoring and / or treating acid sphingomyelinase (ASM) disorders, such as Niemann-Pick disease. [Background technology]

[0003] Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase enzyme that hydrolyzes sphingomyelin (SPM), a phospholipid storage substance found in the brain, liver, lung, spleen, and lymph nodes, into ceramide and phosphorylcholine. Deficiency of ASM activity can result in the body's inability to degrade SPM. In patients with ASM disorders, SPM accumulates primarily in macrophages, but also in hepatocytes and other cell types, leading to marked hepatosplenomegaly, thrombocytopenia, interstitial lung disease, and coronary artery disease. SPM is not significantly elevated in plasma, whole blood, or urine, limiting its use as a noninvasive biomarker.

[0004] Currently, diagnosing ASM disorders requires invasive and / or time-consuming testing, such as evaluation of suspicious clinical symptoms, liver or lung biopsy, testing for ASM activity in blood samples (when false-negative and positive cases are reported), and / or genetic testing (e.g., SMPD1 gene mutation analysis). Treatment of ASM disorders may involve administration of replacement enzymes. High-dose enzyme replacement therapy can result in the production of toxic or harmful metabolites. Therefore, there is a need for improved methods for screening, diagnosing, and / or monitoring the course of treatment for ASM disorders. Disclosed herein are methods for noninvasively screening, diagnosing, monitoring therapy, and / or adjusting the dosage of a therapeutic agent for treating ASM disorders, which methods include measuring the level of lyso-SPM (sphingosylphosphorylcholine or lyso-sphingomyelin) in a biological sample. High levels of lyso-SPM can be used to screen for or diagnose ASM disorders. High levels of lyso-SPMs can also be used as a signal or indicator of the production of one or more toxic metabolites associated with excessively high doses of enzyme replacement therapy, allowing for the calibration of enzyme therapy to reduce SPM accumulation while avoiding the adverse side effects of the therapy. High levels of lyso-SPMs can also be used to monitor the long-term effectiveness of a course of treatment for ASM disorders (e.g., if lyso-SPM levels do not decrease over the course of treatment, this may indicate ineffective treatment).

[0005] Niemann-Pick disease (NPD) is a genetic, autosomal recessive lipid storage disorder characterized by excessive accumulation of SPM in lysosomes of cells such as macrophages and neurons, which impairs normal cellular function. Niemann-Pick disease type A ("NPD-A") is a rapidly progressive neurodegenerative disease of infants, typically resulting in death by age 2–3 years. Niemann-Pick disease type B ("NPD-B") results in liver and spleen enlargement and respiratory distress, and typically leads to death by early adulthood. Other types of Niemann-Pick disease, such as type C ("NPD-C"), are also associated with the accumulation of SPM and / or lyso-SPM. These are also referred to herein as ASM disorders (ASMDs). These forms of Niemann-Pick disease are collectively referred to herein as Niemann-Pick disease (NPD).

[0006] NPD occurs more frequently in individuals of Ashkenazi Jewish ancestry than in the general population The incidence of NPD-A in Ashkenazi Jews is approximately 1 / 40,000, with a gene frequency (q) of approximately 1 / 200 and a heterozygote carrier frequency (2pq) of 1 / 100 (Non-Patent Document 1). The incidence of heterozygote carriers of NPD-B in the Ashkenazi Jewish population is lower (ibid.). The combined heterozygote carrier frequency for NPD types A and B has been estimated to be approximately 1 / 70 in individuals of Ashkenazi Jewish descent (ibid.). Epidemiological studies conducted in various countries have estimated the combined incidence of NPD types A and B in newborns across several countries worldwide to be between 1 / 167,000 and 1 / 250,000 (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4). The heterozygote carrier rate is thought to be between 1 / 200 and 1 / 250 in individuals.

[0007] Patients with either NPD-A or NPD-B have residual ASM activity (approximately 1–10% of normal), which is insufficient to prevent excessive accumulation of sphingomyelin in lysosomes. Furthermore, the clinical course of NPD-B is highly variable, and it is currently impossible to correlate the severity of the disease with residual ASM activity. Enzymatic diagnosis of patients with either NPD-A or NPD-B can be performed using blood samples, but this diagnosis is often preceded by invasive procedures such as liver or lung biopsy. Furthermore, enzymatic detection of obligate heterozygotes has proven problematic, especially when peripheral leukocytes are used as the enzyme source. The occurrence of neutral sphingomyelinase in some sources and / or the presence of residual ASM activity resulting from mutant alleles may contribute to the inability to reliably identify carriers for either disease subtype. The use of cultured skin fibroblasts, which do not express neutral sphingomyelinase, has not yielded clear heterozygous results. Therefore, there is a need for alternative methods to accurately detect, screen, diagnose and treat ASM disorders such as NPD.

[0008] Enzyme replacement therapy (ERT) has been used to treat various lysosomal storage diseases. See U.S. Patent Nos. 5,629,999 and 5,729,999, which are incorporated herein in their entireties and discuss ERT in Tay-Sachs, Pompe, and Niemann-Pick diseases, among others. ERT attempts to replace deficient and / or defective enzymes with exogenously supplied enzymes. In the case of Niemann-Pick disease, the goal is to enable the affected individual to process sphingomyelin and prevent its accumulation in lysosomes. To be effective, such therapy requires a sufficient amount of replacement enzyme to first degrade the accumulated sphingomyelin, as well as continuous administration of replacement enzyme to prevent further sphingomyelin accumulation. However, metabolism of accumulated sphingomyelin can result in the production of toxic or harmful metabolites. Therefore, careful titration of ERT is necessary to effectively reduce accumulated sphingomyelin in patients without producing high levels of metabolites that could lead to harmful side effects.

[0009] As mentioned above, SPMs are not significantly elevated in plasma, whole blood, or urine, limiting their use as noninvasive biomarkers for screening, diagnosing, or monitoring treatment for ASM disorders. As disclosed herein, the deacylated form of SPM, lyso-SPM (sphingosylphosphorylcholine or lyso-sphingomyelin), is significantly elevated in tissues, including peripheral tissues, of patients with ASM disorders and / or undergoing treatment for ASM disorders, making it a promising marker for screening, diagnosing, and / or monitoring treatment for ASM disorders. This dichotomy, in which changes in the levels of both acylated and deacylated glycosphingolipids in plasma are detectable, is in contrast to many other lysosomal storage disorders. Given that changes in SPM levels cannot be detected in the plasma of patients with ASM disorders or undergoing treatment for ASM disorders, it might have been assumed that lyso-SPMs would also be unsuitable for screening, diagnosing, or monitoring treatment. However, the present invention As disclosed herein, lyso-SPM has been found to be detectable at different levels in biological samples from various tissues, including peripheral tissues such as plasma.

[0010] Lyso-sphingolipids (lyso-SLs), including lyso-SPMs, are deacylated forms of sphingolipids; several species have been shown to be elevated in several lysosomal storage disorders (NPL 5). The mechanism by which lyso-SPMs and other lyso-SLs are produced is not fully understood. The lack of a concomitant increase in sphingosine suggests that deacylation of the corresponding sphingolipid is a likely route of production. However, the only sphingomyelin deacylase identified to date is derived from the stratum corneum of subjects with atopic dermatitis (NPL 6). Expression of this deacylase appears to be restricted to selected cell types under some physiological conditions. Purified ASM from placenta, brain, and urine has been shown not to hydrolyze lyso-SPMs (NPL 7; NPL 8; NPL 9). The limited understanding of the biosynthetic pathway of lyso-SPMs further emphasizes the difficulty of predicting a priori the expression levels of lyso-SPMs in patients with ASM disorders.

[0011] Lyso-SPM has a short half-life in the blood in vitro because it is rapidly metabolized to sphingosine-1-phosphate via autotaxin, an extracellular enzyme with lysophospholipase D activity. (Non-Patent Document 10; Non-Patent Document 11) Except for the spleen and liver of NPD-B patients and the brain of NPD-A subjects who have little or no ASM activity and develop severe neuropathic disease, there are no reports on the levels of lyso-SPM in other organs. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 7,001,994 [License 2] U.S. Patent Application No. 2011 / 0052559 [Non-licensed literature]

[0013] [Non-licensed Document 1] Goodman, 1979 "Genetic Disorders Among The Jewish People", John Hopkins Univ. Press, Baltimore, pp. 96~100 [Non-licensed Document 2] Miekle, 1999 JAMA 281(3): pages 249~254 [Non-licensed Document 3] Poorthuis, 1999 Hum. Genet. 105:151~156 pages [Non-licensed Document 4] Pinto, Euro. J. Hum. Gene. 2004, pp. 87-92. [Non-licensed Document 5] Galbiati, "Combined hematopoietic and lentiviral gene-transfer therapies in newborn Twitcher mice reveal contemporaneous neurodegeneration and demyelination in Krabbe disease", J. Neurosci. Res. 87: 1748~1759 pages (2009) [Non-licensed Document 6] Murata, "Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?", J.Invest.Dermatol.106:1242~1249 pages (1996) [Non-licensed Document 7] Pentchev et al., "The isolation and characterization of sphingomyelinase from human placental tissue", Biochim.Biophys.Acta.488:312-321 (1977) [Non-patent document 8] Yamanaka and Suzuki, "Acid sphingomyelinase of human brain: purification to homogeneity", J.Neurochem.38:1753-1764 (1982) [Non-Patent Document 9] Quintern et al., “Acid sphingomyelinase from human urine: purification and characterization,” Biochim. Biophys. Acta. 922:323–336 (1987) [Non-Patent Document 10] Tokumura et al., “Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase,” J. Biol. Chem. 277:39436–39442 (2002) [Non-Patent Document 11] Clair et al., “Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate,” Cancer Res. 63:5446–5453 (2003) Summary of the Invention [Problem to be solved by the invention]

[0014] As disclosed herein, lyso-SPMs are detected at altered concentrations in biological samples (e.g., samples taken from peripheral tissues) from patients with ASM disorders and / or undergoing treatment. Changes in levels may reflect the transient effects of treatment (e.g., lyso-SPM levels may serve as a marker for the production of acutely toxic metabolites in response to ERT). Changes in levels may also be used diagnostically (e.g., changes in lyso-SPM levels may serve as a diagnostic or screening marker for identifying symptomatic or presymptomatic subjects suffering from ASM disorders). Changes in levels may also be used to monitor the long-term effectiveness of a course of treatment for ASM disorders (e.g., if lyso-SPM levels do not decrease over the course of treatment, this may indicate ineffective treatment). Thus, disclosed herein are novel methods for screening for, diagnosing, monitoring the progress of treatment, and / or adjusting the dosage of therapeutic agents for the treatment of ASM disorders, such as NPD, using novel biomarkers, including lyso-SPMs (sphingosylphosphorylcholine or lyso-sphingomyelin). Monitoring the course of treatment can include detecting a decrease in the level of one or more markers of toxicity (e.g., lyso-SPMs) over the course of treatment, indicating an effective treatment regimen, or detecting no change in the level of the marker over time, indicating an ineffective regimen. [Means for solving the problem]

[0015] The methods disclosed herein also include methods of treating a human subject with an acid sphingomyelinase (ASM) disorder. In some embodiments, the method comprises administering to the subject a first dose of a therapeutic agent for treating the ASM disorder, the therapeutic agent having a first concentration; and, if the subject is determined to have a level of lyso-SPM below the reference level after administration of the first dose, administering to the subject a second dose of the therapeutic agent having a second concentration equal to or greater than the first concentration. The method includes identifying a patient with an ASM disorder by measuring lyso-SPM in a biological sample from a peripheral tissue and detecting changes in lyso-SPM levels. The method also includes monitoring or adjusting treatment of a patient with an ASM disorder by detecting changes in the levels of one or more toxicity markers, including lyso-SPM, as a result of treating the patient with a therapeutic agent, e.g., an agent that reduces SPM levels in the patient's tissue. The method allows for noninvasive evaluation of such patients.

[0016] The methods disclosed herein, in some embodiments, can also be used to screen for, diagnose, monitor treatment progress, and / or adjust treatment for ASM disorders, such as NPD. For example, the methods include adjusting the dose of a therapeutic agent administered to a patient to treat an ASM disorder by measuring toxicity markers (e.g., lyso-SPMs) to manage levels of toxic metabolites resulting from the treatment. The methods disclosed herein, in some embodiments, can also be used to monitor the long-term effectiveness of a course of treatment for an ASM disorder (e.g., if lyso-SPM levels do not decrease over the course of treatment, this may indicate ineffective treatment). The methods disclosed herein, in some embodiments, can also be used to screen subjects (e.g., pre-symptomatic patients) for elevated lyso-SPMs as an early sign of an ASM disorder. Subjects identified in the screening as having elevated lyso-SPMs are then subjected to additional evaluation (e.g., ASM blood testing, genetic testing, etc.) to diagnose / confirm the diagnosis of an ASM disorder, while subjects without elevated lyso-SPMs are not subjected to additional evaluation. Such screening could potentially reduce testing costs.

[0017] The methods disclosed herein can include measuring lyso-SPM in a biological sample from a human subject and administering a therapeutic agent (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) to treat the ASM disorder at a concentration optimized based on the measured level of lyso-SPM. In various embodiments, the biological sample is a peripheral sample. In some embodiments, the biological sample can be a sample of plasma, whole blood (e.g., a dried blood spot), serum, and / or urine. Measuring lyso-SPM levels using a peripheral sample can avoid the need for invasive procedures such as liver biopsy. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1A is a plot showing the ratio of SPM concentrations in dried blood spots (DBS) from NPD-A and NPD-B patients to the mean concentration values ​​in DBS from normal control samples, and Figure 1B is a plot showing the ratio of lyso-SPM concentrations in DBS from NPD-A and NPD-B patients to the mean concentration values ​​in DBS from normal control samples. [Figure 2] Histogram showing the fold increase in lyso-SPM concentration (vertical axis) in DBS from ASM knockout mice at the indicated time points (1, 2, 4, 6, 24, 48, and 72 hours after administration) compared to the concentration 5 minutes after a single dose of 0, 3, or 20 mg / kg rhASM. [Figure 3] The concentration of lyso-SPM (ng / ml) in DBS samples from wild-type (C57BL / 6) or ASM knockout (ASMKO) mice after administration of a single dose of rhASM (10 mg / kg) or a reduced-dose regimen (3 mg / kg) followed by a 20 mg / kg dose of rhASM is shown. Blood samples were collected at the following time points: 5 minutes, 4 hours, 6 hours, 24 hours, and 72 hours after administration. Animals in the 10 mg / kg group were euthanized after 24 hours. [Figure 4]This histogram shows the concentration (ng / ml) of lyso-SPM in DBS samples from a human Niemann-Pick patient taken before administration of rhASM and 24, 48, and 72 hours after administration over a 26-week period. The dose (0.1, 0.3, 0.6, 1, 2, or 3 mg / kg) and administration day (day 1, weeks 2, 4, 6, 8, 10, 12, 14, and 26) are indicated on the horizontal axis. At week 26, samples were collected only before administration and 24 and 48 hours after administration. DETAILED DESCRIPTION OF THE INVENTION

[0019] Reference will now be made in detail to several exemplary embodiments according to the present disclosure, some examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0020] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Any range described herein will be understood to include all values ​​between the endpoints.

[0021] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and journal articles, are expressly incorporated herein by reference in their entirety for any purpose.

[0022] Disclosed herein are methods for screening, diagnosing, monitoring the progress of treatment, and / or adjusting the dosage of a therapeutic agent for treating an ASM disorder, such as NPD. "ASM disorder" can include any disorder associated with decreased expression or impaired function of acid sphingomyelinase. ASM disorder can also include any other disorder associated with the accumulation of sphingomyelin in tissues.

[0023] In some embodiments, the method includes administering to the subject a first dose of a therapeutic agent for treating an ASM disorder, the first dose having a first concentration; and if the subject is determined to have a level of lyso-SPM below a reference level (e.g., a level in a sample from a control subject not having an ASM disorder, or a baseline level measured in the patient before treatment) after administration of the first dose, administering to the subject a second dose of the therapeutic agent having a second concentration greater than or equal to the first concentration.

[0024] In some embodiments, the method involves collecting and measuring lyso-sphingomyelin (lyso-SPM) in a biological sample from a human subject. The measured lyso-SPM levels can also be used to screen for, diagnose, monitor treatment progress, and / or adjust the dosage of a therapeutic agent for treating an ASM disorder. The method is based on the discovery that lyso-SPM is significantly elevated in biological samples from peripheral tissues of patients with an ASM disorder, such as NPD, allowing for noninvasive evaluation of such patients, including screening and diagnosing the disorder and monitoring / calibrating / managing treatments for the disorder. The method is also based on the discovery that degradation of accumulated ASM during treatment can increase the levels of markers (e.g., lyso-SPM) that signal the production of toxic or harmful metabolites, and that harmful levels of these metabolites can be avoided by administering therapeutic agents designed to suppress SPM levels at doses that prevent excessive metabolite production (e.g., ERT, chaperone therapy, and / or substrate reduction therapy). The measurement of the increase in lyso-SPM concentration These assays can be used to detect the production of such metabolites and can be used to calibrate treatments to avoid the production of excessively high (e.g., toxic) levels of the metabolites. In some embodiments, the level of lyso-SPM in a patient undergoing treatment for an ASM disorder determines whether to increase, decrease, repeat, delay, or discontinue the dose.

[0025] Detecting high lyso-SPM levels in subjects with ASM disorders (e.g., NPD patients) can be used as part of a method for monitoring adverse side effects during treatment. For example, the method can include obtaining a biological sample from the subject, measuring the level of lyso-sphingomyelin (lyso-SPM) in the sample, comparing the measured lyso-SPM level in the sample with a reference level, and detecting adverse side effects if the level of lyso-SPM in the sample increases. In some embodiments, if the level of lyso-SPM increases by a predetermined amount compared to a reference sample (e.g., a reference level in a sample from a control subject without ASM disorders) or if the level of lyso-SPM increases by a predetermined amount in a subject over time during the course of treatment (i.e., an increase above the baseline level measured in the patient before treatment, also referred to herein as the reference level), the measured level of lyso-SPM can be used as an indicator of adverse side effects caused by treatment. In some embodiments, the treatment is enzyme replacement therapy (ERT), and the dosage of ERT is managed by obtaining one or more biological samples from the patient, testing each sample for elevated lyso-SPMs, and titrating the ERT dose to a level that does not result in elevated lyso-SPM levels above a predetermined threshold. Managing the dosage of the therapeutic agent can include increasing, decreasing, or maintaining the concentration of the therapeutic agent and / or discontinuing treatment. In some embodiments, one or more biological samples are obtained after administering a dose of the therapeutic agent and / or immediately prior to administering a subsequent dose of the therapeutic agent.In some embodiments, adverse side effects may be detected if the level of lyso-SPM in a biological sample (e.g., a blood sample such as plasma, serum, or dried blood spot) is higher than a reference level of about 100-700 ng / ml (e.g., higher than about 100, 200, 250, 300, 400, 500, 600, or 700 ng / ml, or any intermediate level), or if the level of lyso-SPM in the biological sample is elevated by at least about 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or more (or any intermediate value) above the reference level. For example, the increase is at least about 3-fold. The reference level can be, for example, the level in a sample from a control subject without ASM disorder, or the baseline level measured in the patient prior to treatment with a dose of a therapeutic agent.

[0026] Also disclosed herein are methods of treating a subject with an ASM disorder (e.g., NPD). In various embodiments, the methods include administering a therapeutic agent for treating the ASM disorder in successive doses at increasing concentrations (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) and monitoring the subject for elevated lyso-SPM levels in a biological sample after each dose (e.g., 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes, or 1, 2, 3, 4, 5, 10, 12, 15, or 20 hours, or 1 day, 2 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, or any time in between) or immediately prior to the next dose. Monitoring the subject can include obtaining a biological sample from the subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample to a reference level (e.g., the level in a sample from a donor without an ASM disorder or the level in an ASM patient before treatment), and detecting an elevated level of lyso-SPM in the sample relative to the reference level. In some embodiments, the reference level is the level of lyso-SPM measured in a biological sample from a control subject without an ASM disorder. In some embodiments, the reference level is the level of lyso-SPM measured in a control subject after administration of an initial low dose of ERT or before administration of any ERT. The level of lyso-SPM measured in a biological sample taken from an elephant. In some embodiments, the elevated level of lyso-SPM in a biological sample (e.g., a blood sample such as a serum sample, plasma sample, or dried blood spot) is above a reference level, e.g., about 100-700 ng / ml. In some embodiments, the elevated level of lyso-SPM in a biological sample (e.g., a blood sample) is at least about 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times (or any value in between) higher than the reference level. For example, the level is at least about 3 times higher. In some embodiments, if elevated levels of lyso-SPM are detected after administration of a previous dose, a high-concentration dose of ERT is not administered.

[0027] In addition to treatments and monitoring of treatment methods, disclosed herein are methods for screening and / or diagnosing ASM disorders (e.g., NPD) in a subject. In various embodiments, the method includes collecting a biological sample from a subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample with a reference level, and detecting / diagnosing an ASM disorder if the level of lyso-SPM in the sample is elevated compared to the reference sample. In some embodiments, the reference sample is a sample from a control subject who does not have an ASM disorder. In some embodiments, the level of lyso-SPM in a biological sample (e.g., a blood sample such as a plasma sample, serum sample, or dried blood spot) is at least about 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times or more (or any value in between) higher than the reference level, or at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, at least about 525 ng / ml, at least about 575 ng / ml, If the level is higher than a reference level of at least about 200-2000 ng / ml, such as at least about 700 ng / ml and / or at least about 900 ng / ml (e.g., greater than about 200, 250, 300, 350, 400, 450, 500, 525, 550, 575, 600, 625, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ng / ml, or any level in between), an ASM disorder can be screened for and / or diagnosed. In some embodiments, a biological sample from a normal subject may contain lyso-SPM levels in the range of about 25-200 ng / ml (e.g., about 25, 50, 75, 100, 125, 150, 175, or 200 ng / ml, or any concentration in between).

[0028] Measurement of lyso-SPM For a discussion of the chemical structure of lyso-SPM, see Ito et al., J. Biol. Chem. 270:24370-4 (1995); see also Caymen Chemical Co. Item Number 10007947, as provided in the Caymen Chemical Co. catalog: https: / / www.caymanchem.com / app / template / Product.vm / catalog / 10007947.

[0029] The methods disclosed herein involve measuring lyso-SPM from various biological samples, including samples from peripheral tissues. Any method for collecting, preparing, and quantifying the levels of lyso-SPM from a biological sample can be used. The levels of lyso-SPM in a biological sample can be quantified using a spectrometer, such as a mass analyzer, such as LC / MS / MS, or an electromagnetic frequency analyzer, such as UV-VIS, IR, or NMR. In some embodiments, the levels of lyso-SPM can be measured using a spectrometer, such as a mass analyzer, such as LC / MS / MS, or an electromagnetic frequency analyzer, such as UV-VIS, IR, or NMR. Methods of quantification include obtaining a biological sample (e.g., by arterial or venipuncture, tissue biopsy, buccal swab, urine sample, etc.), detecting and / or separating lyso-SPM from other components in the sample (e.g., using antibodies, indicator chemicals, mass spectrometers such as LC / MS / MS, or electromagnetic frequency analyzers such as UV-VIS, IR, or NMR), and comparing the level of lyso-SPM to that in a reference sample.

[0030] Lyso-SPM levels can be measured in biological samples from various tissues using the methods described herein. For example, biological samples from peripheral tissues such as plasma, whole blood (e.g., dried blood spots), serum, skin, and / or urine can be collected for use in detecting high lyso-SPM levels. Biological samples from other tissues, such as spleen, lung, heart, liver, kidney, and / or brain tissue, can also be used. Samples from a combination of two or more tissues (e.g., 2, 3, 4, 5, or more tissues) can be used. In some embodiments, the use of biological samples from peripheral tissues can avoid the need for invasive procedures such as liver biopsy.

[0031] In some embodiments, biological samples are subjected to one or more pretreatment steps prior to detection and / or measurement of lyso-SPMs in the sample. In some embodiments, the sample is pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps. In other embodiments, the sample is pretreated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization, and / or homogenization followed by centrifugation, filtration, precipitation, dialysis, and / or chromatography. In some embodiments, the sample is pretreated by removing certain cell types from the sample or removing debris from the sample prior to assessment of lyso-SPMs.

[0032] In various embodiments, the biological sample is evaluated using a device for quantifying or semi-quantifying the level of one or more markers in the sample. For example, the level of lyso-SPM and / or other markers in the sample can be assessed quantitatively or semi-quantitatively. In some embodiments, a device for quantifying the level of lyso-SPM and / or other markers in a biological sample, such as tandem liquid chromatography-mass spectrometry (e.g., LC / MS / MS), can be used. In some embodiments, one or more antibodies or other detection agents that bind to lyso-SPM and / or other markers in the biological sample can be used. One or more agents (e.g., colorimetric agents) can be applied that react with the detection agent to produce a detectable signal whose intensity, brightness, color, etc. can be used to quantitatively or semi-quantitatively determine the level of lyso-SPM and / or other markers in the sample (e.g., by comparing the signal with that from one or more reference samples). Additional methods for quantifying lyso-SPM and / or other markers in biological samples can also be used, such as immunoassays such as ELISA, immunoprecipitation and Western blot, as well as fluorescence activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), RT-PCR and / or Northern blot.

[0033] Managing ASM failure actions In various embodiments, lyso-SPM levels can be measured as part of a treatment for an ASM disorder. For example, the ASM disorder is Niemann-Pick disease (NPD), e.g., NPD-A, NPD-B, or NPD-C. Treatment for an ASM disorder can include administering one or more therapeutic agents (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) that reduce the level of SPM in the patient's tissues. For example, the methods disclosed in U.S. Patent Application Publication No. 2011 / 0052559, incorporated herein by reference in its entirety, can be used to treat the ASM disorder. Intra-patient dose-escalating enzyme replacement therapy (ERT), such as the ASM dose-escalation method described in

[0063] to

[0075] , which describes the dose-escalation protocol, may also be used.

[0034] In various embodiments, methods are disclosed for monitoring a subject for adverse side effects (e.g., production of toxic or harmful levels of metabolites) during dose-escalation therapy for an ASM disorder by monitoring markers such as lyso-SPM. The method can include obtaining a biological sample from the subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample with a reference level, and detecting an adverse side effect if the level of lyso-SPM in the sample is elevated compared to the reference sample. One or more samples can be obtained and evaluated after administration of a dose of a therapeutic agent or before administration of the next dose. For example, samples can be obtained and evaluated 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes after administration of a therapeutic dose, or 1, 2, 3, 5, 10, 12, 15, or 20 hours, or 1, 2, 3, 4, or 5 days, or 1, 2, 3, or 4 weeks, or at any intermediate time point, or before administration of the next dose. In some embodiments, the reference sample is a sample from a control subject without an ASM disorder. In some embodiments, the reference sample is an initial biological sample from the subject prior to administration of a high dose of the therapeutic agent (or prior to administration of any therapeutic agent). In some embodiments, if the level of lyso-SPM increases by a predetermined amount or above a predetermined threshold compared to the level in the reference sample after administration of the initial dose of the therapeutic agent or after administration of an increased (i.e., high) dose of the therapeutic agent (e.g., a high concentration of ERT), this can be used as an indication of adverse side effects from the treatment. In some embodiments, if the level of lyso-SPM does not increase or does not increase above a threshold level, this can be used as an indication that adverse side effects are not occurring.

[0035] In various embodiments, methods of treating ASM disorders are provided. The treatments can include administering one or more therapeutic agents that reduce the level of SPM in a patient's tissues (e.g., ERT, chaperone therapy, and / or substrate reduction therapy). In some embodiments, the treatments can include ERT (e.g., ASM replacement therapy). The treatments can include monitoring the subject for elevated levels of lyso-SPM during the treatment and adjusting the concentration of the therapeutic agent (e.g., the concentration of an ERT dose) to reduce lyso-SPM levels below a predetermined threshold level. In some embodiments, the monitoring includes evaluating a sample for elevated levels of lyso-SPM after administration of each dose of the therapeutic agent or before administration of each subsequent dose of the therapeutic agent. In some embodiments, monitoring after each dose is optional and is performed periodically after a certain number of doses of the therapeutic agent or before a certain number of subsequent doses of the therapeutic agent.

[0036] In some embodiments, the treatment method can include administering successive doses of ERT (e.g., ASM replacement therapy) at increasing concentrations, collecting biological samples from the patient after a set number of doses (e.g., after each dose or before each subsequent dose), detecting the level of lyso-SPM in the samples (e.g., using LC / MS / MS), and monitoring the subject for elevated lyso-SPM levels after each dose or before the next dose. For example, biological samples can be collected and monitored 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes, or 1, 2, 3, 5, 10, 12, 15, or 20 hours, or 1, 2, 3, 4, or 5 days, or 1, 2, 3, or 4 weeks, or at any intermediate time point, or before the next dose. Monitoring the subject includes obtaining a biological sample from the subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample with a reference level, and determining whether the level of lyso-SPM in the sample is a reference level. The method can include adjusting the dose of ERT if the level of lyso-SPM increases by a predetermined amount compared to the reference sample. In some embodiments, the reference sample is a sample from a control subject without ASM disorder. In some embodiments, the reference sample is an initial biological sample from the subject before administration of a high-concentration dose of ERT (or before administration of any ERT). In some embodiments, if the level of lyso-SPM increases by a predetermined amount in the subject sample and / or is higher than the reference threshold, this can be used as an indication that the ERT dosage should be reduced, delayed, or terminated to avoid production of toxic or harmful levels of metabolites. In some embodiments, if the level of lyso-SPM is below the threshold, a subsequent dose of equal or higher concentration can be administered.

[0037] In various embodiments, a dose-escalation therapy is provided, which involves administering increasing doses of ERT to a subject over time to reduce accumulated SPMs without producing toxic or harmful levels of metabolites due to rapid hydrolysis of the accumulated SPMs. In some embodiments, during dose-escalation therapy, biological samples from the patient are monitored for increasing levels of lyso-SPMs, as high lyso-SPMs may indicate the production of toxic or harmful levels of metabolites. In some embodiments, if a level of lyso-SPM above a threshold concentration is detected or if a significant increase in lyso-SPM levels compared to levels in a previous sample is detected, the ERT dose increase is delayed or not administered. ERT can be administered by any route suitable to achieve a therapeutic effect, including intravenously, intradermally, subcutaneously, intraperitoneally, intrapulmonary, topically, intranasally, intracranially, or intramuscularly.

[0038] In some embodiments, ERT can include administration of acid sphingomyelinase (ASM), such as recombinant human ASM (rhASM), or ERT can include administration of modified ASM (e.g., modified rhASM). Modified ASM can include any modification to the enzyme that does not significantly alter the enzyme's ability to hydrolyze lysosomal sphingomyelin to ceramide and phosphorylcholine (e.g., the modified ASM exhibits at least about 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.5, or 99.9% of the enzymatic activity of unmodified ASM, or any percentage in between). The hydrolytic ability of the modified ASM can be assessed by techniques well known to those skilled in the art, such as those described in U.S. Pat. Nos. 4,039,388, 4,082,781, 5,686,240, and 7,563,591, and International Application Nos. WO2007 / 078806 and WO2006 / 058385, which are incorporated herein by reference in their entireties.

[0039] In some embodiments, ERT can include administration of recombinant human ASM (rhASM) or modified rhASM. There are various ASM isoforms known in the art, all of which can be used in the methods disclosed herein. See, for example, U.S. Application No. 2011 / 0052559, which is incorporated herein by reference in its entirety (see, e.g., paragraphs

[0108] -

[0117] and

[0124] -

[0127] for a discussion of human ASM isoforms and their enzyme conjugates and their use in ERT).

[0040] In some embodiments, enzyme replacement therapy is administered to a subject at a non-toxic initial low dose followed by subsequent incremental doses. The maximum dose of enzyme that the subject can tolerate without producing toxic or harmful levels of metabolites (e.g., as detected by monitoring the level of a toxicity marker, such as lyso-SPM) can then be used as a maintenance dose. Alternatively, a therapeutically effective dose less than the maximum tolerated dose can be used as a maintenance dose. A therapeutically effective dose can include any dose sufficient to reduce the concentration of accumulated sphingomyelin in an ASM subject by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% (or any percentage in between) after one or more administrations. can.

[0041] Treatment of ASM disorders such as NPD requires a high dose of a therapeutic agent (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) sufficient to achieve proper distribution of the therapeutic agent in affected organs (e.g., spleen, lungs, liver, heart, kidneys, and brain). It has been shown that after intravenous administration of recombinant human ASM to ASM knockout mice, most of the ASM activity is distributed to the liver, with only slight ASM enzyme activity being detected in other organs, such as the spleen, heart, kidneys, and lungs. See, for example, He et al., Biochimia et Biophysica Acta 1432:251-264 (1999). Therefore, to ensure proper distribution and delivery in, for example, the lungs, liver, heart, and kidneys, subjects with ASM disorders such as NPD require the administration of high concentrations of a therapeutic agent (e.g., high concentrations of replacement enzymes).

[0042] Studies in ASM knockout mice have also shown that enzyme replacement therapy at sufficiently high doses can result in the production of toxic or otherwise harmful metabolites of sphingomyelin. See, for example, C. Nickerson et al., American Society of Human Genetics (2005); and J. Murray et al., Society of Toxicology (2006). Without being bound by theory, administration of high doses of ASM to NPD subjects may result in the hydrolysis of large amounts of accumulated sphingomyelin into ceramide and phosphorylcholine. Ceramide is known to play a role in cell death and is known to be a potential pro-apoptotic agent. See, for example, Smith and Schuchman, FASEB 22:3419-3431 (2008). Therefore, ceramide may be responsible for the toxic side effects observed in ASM knockout mice and NPD subjects.

[0043] Therefore, titration and adjustment of therapies (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) is required to provide a sufficient concentration of the therapeutic agent to reduce and prevent future accumulation of lysosomal sphingomyelin throughout the affected organ, while also avoiding the production of excessive concentrations of toxic metabolites. In some embodiments, this adjustment is made through managing a dose-escalation protocol by assessing the levels of toxicity markers, such as lyso-SPM, in patient samples after a set number of doses or after every dose, and adjusting the dosing regimen described herein as needed. Managing the dose of the therapeutic agent can include increasing, decreasing, or maintaining the concentration of the therapeutic agent and / or discontinuing treatment.

[0044] In various embodiments, the dose-escalation method of treatment involves administering one or more initial low doses of a therapeutic agent (e.g., a replacement enzyme) to the subject to reduce the amount of sphingomyelin accumulated in the subject. Subsequently, doses of the therapeutic agent can be administered at systematically higher concentrations until the subject reaches a maximum therapeutically effective dose that is tolerated. In some embodiments, the therapeutic agent is a replacement enzyme (e.g., rhASM) and is administered so that the enzyme activity in one or more affected organs (e.g., organs exhibiting elevated lysosomal levels of SPM in patients with ASM disorders) is at least about 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 80%, 85%, 90%, 95% (or any percentage in between) of the activity level in the corresponding organ in a subject without ASM disorders (e.g., a healthy individual).

[0045] In some embodiments, the method of treating an ASM disorder comprises (a) administering to a subject an accumulation of rhASM or modified rhASM comprising: (i) administering to a subject an initial low dose of rhASM or modified rhASM; and (ii) administering to a subject subsequent higher doses of rhASM or modified rhASM (dose escalation). (b) monitoring the subject for one or more additional markers of elevated lyso-SPM levels and / or adverse side effects after a set number of doses or after each dose in steps (a)(i) and (a)(ii) (e.g., using LC / MS / MS to quantify lyso-SPM concentrations); (c) repeating, decreasing, and / or terminating the dose escalation protocol after elevated lyso-SPM levels are detected and / or one or more additional adverse side effects are detected. In some embodiments, the method further comprises administering to the patient a maintenance dose at or below the highest dose tolerated by the subject, and optionally monitoring for elevated lyso-SPM levels during administration of the maintenance regimen. In some embodiments, the initial dose of rhASM or modified rhASM can range from about 0.03 mg / kg to about 1.0 mg / kg, or from about 0.1 mg / kg to about 0.5 mg / kg (dosage concentrations are measured as mg enzyme per kg body weight). In some embodiments, each subsequent dose of increasing enzyme concentration is administered about 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, 4, or 5 weeks after the previous dose. In some embodiments, the subsequent dose of increasing enzyme concentration can be between about 0.1 and 5 mg / kg (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg / kg, or any concentration in between).

[0046] In some embodiments, at least two doses (e.g., at least 2, 3, 4, or 5) of a given concentration of replacement enzyme are administered before a subsequent higher concentration dose is administered, in some embodiments, the subsequent higher dose is about 0.03 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg (or any value in between) higher than the previous dose. In some embodiments, subsequent higher doses are about 0.03 to about 0.1 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to 2 mg / kg, about 2 mg / kg to about 4 mg / kg, or about 2 mg / kg to 5 mg / kg (or any value in between) higher than the previous dose. In some embodiments, the highest dose tolerated by a subject without producing toxic or harmful metabolites is about 1.0 mg / kg to about 3.0 mg / kg. In some embodiments, the highest tolerated dose is then administered to a human subject as a maintenance dose. In some embodiments, a maintenance dose is administered about every 1 to 8 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or any interval in between).

[0047] Once a maximum tolerated dose (e.g., a dose that does not produce toxic or adversely toxic levels of metabolites) is identified, the maximum tolerated dose can be used as a maintenance dose for treating the subject. The maintenance dose can be administered daily, weekly, twice weekly, monthly, twice monthly, or quarterly (or at any intermediate interval). Monitoring for elevated lyso-SPM levels can be performed during administration of the maintenance regimen, e.g., 1, 2, 3, 5, 10, 12, 15, or 20 hours after administration of the maintenance dose, or 1, 2, 3, 4, or 5 days, or 1, 2, 3, or 4 weeks, or any intermediate period. If elevated lyso-SPM levels (e.g., levels above a reference level of about 100-700 ng / ml, or levels at least about 1.1-10 times higher than the reference level) are detected, the maintenance dose can be reduced or discontinued.

[0048] During therapy (e.g., during ERT), several other parameters can be measured in combination with lyso-SPM to monitor the subject and / or as part of the therapy to determine the maximum dose that the subject will tolerate. For example, a subject can be administered SPM The subject may be further monitored by measuring blood glucose levels, plasma ceramide levels, and / or bilirubin concentrations. The subject may also be monitored for the production of "acute phase reactants" and inflammatory mediators, which are measures of the inflammatory response, and / or other biochemical markers. These other biochemical markers include, but are not limited to, CRP / hs-CRP, cytokines (e.g., 1L-8, 1L-6), calcitonin, and ferritin. In some embodiments, one or more of the above-listed parameters may be monitored before increasing the dose to a higher concentration to ensure a stable response to therapy. In some embodiments, the subject may be monitored for one or more associated adverse events, including systemic symptoms (e.g., fever, nausea, vomiting, pain, myalgia, and jaundice). Combinations of markers may also be monitored (e.g., lyso-SPM levels may be monitored in combination with bilirubin and / or ceramide levels). Suitable threshold levels that can be monitored in combination with lyso-SPM are disclosed, for example, in U.S. Application No. 2011 / 0052559 (see, e.g., paragraphs

[0067] to

[0086] ), the entire contents of which are incorporated herein by reference.

[0049] In some embodiments, subjects undergoing a dose escalation protocol (e.g., an ERT dose escalation protocol) are monitored for toxic or adverse side effects (e.g., by monitoring the levels of one or more toxicity markers, such as lyso-SPM) after each therapeutic administration (e.g., about 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes, or 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, or 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks or more, or any intermediate period) or before administering a higher concentration of the therapeutic agent. In some embodiments, subjects are monitored after each administration of a maintenance dose (about 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, or 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks or more, or any intermediate period) or before administering a subsequent maintenance dose of the therapeutic agent. In some embodiments, the subject receives maintenance dose for 1, 2, 3 or more years, and is regularly monitored for toxic or harmful side effects.Monitoring may include monitoring related adverse events in addition to monitoring the above-mentioned toxicity markers.If the subject experiences adverse events or one or more of the markers of the subject being monitored show harmful side effects (for example, if an increase in the level of lyso-SPM is detected), the administration of maintenance dose can be terminated or adjusted (for example, a low concentration of ERT can be administered) to reduce or minimize undesirable side effects.

[0050] In various embodiments, a subject can be monitored for toxic and / or adverse doses of ERT by measuring lyso-SPM levels from a biological sample collected after administration of a dose of ERT (e.g., after administration of rhASM or modified rhASM). In some embodiments, a method can include collecting a biological sample from a subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample with a reference level, and detecting adverse side effects if the level of lyso-SPM in the sample is elevated compared to the reference sample or elevated by a specified amount compared to the reference sample. In some embodiments, a subsequent dose of ERT at a higher concentration is administered only if the level of lyso-SPM in the biological sample does not exceed a specified threshold after administration of the previous dose. Examples of suitable threshold levels are described herein. In some embodiments, the level of lyso-SPM is also monitored during administration of a maintenance dose. In some embodiments, if the level of lyso-SPM exceeds a predetermined threshold during administration of a maintenance dose, the maintenance administration is discontinued or a lower dose is administered that does not result in the level of lyso-SPM exceeding the predetermined threshold.

[0051] In some embodiments, adverse side effects of ERT can be detected when the level of lyso-SPM in a biological sample is higher than a predetermined reference level. In some embodiments, adverse side effects can be detected when the level of lyso-SPM in a biological sample (e.g., a blood sample) is higher than a reference level of about 100-700 ng / ml (e.g., higher than about 100, 200, 250, 300, 400, 500, 600, or 700 ng / ml, or any intermediate level). In some embodiments, adverse side effects can be detected when the level of lyso-SPM in a biological sample (e.g., a blood sample) is at least about 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or more (or any intermediate value) higher than the reference level. For example, the increase can be at least about 3-fold.

[0052] In some embodiments, the methods of treating ASM disorders provided herein reduce spleen volume as assessed by techniques known in the art, such as MRI. In some embodiments, the methods reduce liver sphingomyelin levels as assessed by techniques known in the art, such as biochemical and / or histomorphometric analysis of liver samples. In some embodiments, the methods increase exercise capacity as assessed by techniques known in the art, such as maximal workload by cycle ergometry, including percent predicted maximum workload, peak oxygen consumption, and / or carbon dioxide production. In some embodiments, the methods improve pulmonary function and / or lung clearance as assessed by techniques known in the art, such as DLCO, FVC, FEV1, and / or TLC. In some embodiments, the methods reduce bronchoalveolar lavage (BAL) sphingomyelin. In some embodiments, the methods improve lung appearance as assessed by techniques known in the art, such as high-resolution CT scan and / or chest x-ray.

[0053] In various embodiments, the methods of treating ASM disorders provided herein reduce sphingomyelin concentrations in the liver, skin, and / or plasma, and / or reduce plasma chitotriosidase, CCL18 levels, lyso-SPM, ceramide, and / or bilirubin. In some embodiments, the methods improve the lipid profile of the patient (e.g., lower cholesterol). In some embodiments, the methods improve one or more neurological functions in a subject (e.g., psychomotor function, social responsiveness, etc.). In some embodiments, the methods reduce or ameliorate the severity and / or duration of an ASM disorder and / or one or more symptoms associated with the disorder. In some embodiments, the methods prevent the recurrence of symptoms associated with an ASM disorder. In some embodiments, the methods increase the survival rate of a subject after treatment.

[0054] Screening and / or diagnosis of ASM disorders In various embodiments, disclosed herein are methods for screening for and / or diagnosing an ASM disorder. In some embodiments, the ASM disorder is Niemann-Pick disease (NPD). In some embodiments, the ASM disorder is NPD type A, type B, and / or type C. In some embodiments, an ASM disorder (e.g., NPD) can be screened for and / or diagnosed by measuring lyso-SPM levels in a biological sample obtained from a subject.

[0055] In some embodiments, a method for screening for and / or diagnosing an ASM disorder in a subject comprises obtaining a biological sample from the subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample with a reference level, and detecting / diagnosing an ASM disorder if the level of lyso-SPM in the sample is elevated compared to the reference level. In some embodiments, the reference level is an ASM disorder. The level of lyso-SPM measured in a sample from a control subject who does not have ASM. In some embodiments, an ASM disorder can be detected / diagnosed when the level of lyso-SPM in a biological sample from a subject is higher than a predetermined reference level. In some embodiments, an ASM disorder is detected / diagnosed when the level of lyso-SPM in a biological sample (e.g., a blood sample) is at least about 200-2000 ng / ml (e.g., about 200, 2000 ng / ml), such as greater than about 200 ng / ml, greater than about 300 ng / ml, greater than about 400 ng / ml, greater than about 500 ng / ml, greater than about 525 ng / ml, greater than about 575 ng / ml, and / or greater than about 700 ng / ml. In some embodiments, an ASM disorder can be detected and / or diagnosed when the level of lyso-SPM in a biological sample (e.g., a blood sample) is about 1-10 times higher than the reference level (e.g., greater than 50, 300, 350, 400, 450, 500, 525, 550, 575, 600, 625, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ng / ml, or any concentration in between).

[0056] Biological samples from various tissues can be used in the screening and diagnostic methods described herein. For example, biological samples from peripheral tissues such as plasma, whole blood (e.g., dried blood spots), serum, skin, and / or urine can be used to monitor for elevated lyso-SPM levels. Biological samples from other tissues, such as spleen, lung, liver, heart, kidney, and / or brain tissue, can also be used. Samples from a combination of two or more tissues (e.g., 2, 3, 4, 5, or more tissues) can be used. In some embodiments, ASM disorders (e.g., NPD) can be detected / diagnosed by measuring lyso-SPM levels in a biological sample taken from a peripheral tissue. In some embodiments, the peripheral tissue can be plasma, whole blood (e.g., dried blood spots), serum, and / or urine. The use of peripheral samples can avoid the need for invasive procedures such as liver biopsy.

[0057] In various embodiments, the screening and / or diagnostic methods disclosed herein can further include administering a therapeutic agent (e.g., enzyme replacement therapy) to the subject if an ASM disorder is detected / diagnosed. In some embodiments, the enzyme replacement therapy includes administering rhASM or modified rhASM to the subject.

[0058] kit In various embodiments, kits are disclosed herein that include a device for collecting a biological sample containing lyso-SPMs and / or other markers for ASM disorders and instructions for using the kit to measure the level of lyso-SPMs and / or other markers in the biological sample. In some embodiments, the device for collecting the biological sample can include a test tube, syringe, and / or other container for storing a liquid sample, and / or a test strip, urine dipstick, etc. Any other device for collecting a biological sample known in the art can also be used. In some embodiments, the biological sample is a sample from a peripheral tissue, such as plasma, whole blood (e.g., dried blood spot), serum, skin, and / or urine. Biological samples can also be collected from other tissues, such as spleen, lung, heart, liver, kidney, and / or brain tissue, and the kit includes a device for collecting samples from the listed tissues. Samples from a combination of two or more tissues can be used.

[0059] In some embodiments, the kit can further include a device for measuring the level of lyso-SPM and / or other markers in a biological sample. For example, the kit can include a device for use in detecting lyso-SPM or other markers in a biological sample. In some embodiments, the detection agent is contained within or located on the tissue collection device (e.g., an antibody or chemical indicator impregnated in a test strip), while in other embodiments, the detection agent is provided separately from the collection device.

[0060] In some embodiments, the kit can further include a device for quantifying or semi-quantifying the level of lyso-SPM and / or other markers in a sample. For example, an agent (e.g., a colorimetric agent) can be provided that reacts with the detection agent to produce a detectable signal whose intensity, brightness, color, etc. can be used to quantitatively or semi-quantitatively determine the level of lyso-SPM and / or other markers in a sample (e.g., by comparing it to the signal from one or more reference samples). In some embodiments, the device can include a device for separating lyso-SPM from other components of the sample, e.g., using liquid chromatography and / or mass spectrometry. In some embodiments, the device for quantifying the level of lyso-SPM and / or other markers in a sample is a spectrometer, such as a mass analyzer such as LC / MS / MS, or an electromagnetic frequency analyzer such as UV-VIS, IR, or NMR.

[0061] In some embodiments, the kit can further include instructions for comparing the level of lyso-SPM and / or other markers in the sample to a reference level, and detecting the presence of toxic levels of one or more metabolites and / or adverse side effects during treatment of an ASM disorder if the level of lyso-SPM and / or other toxicity markers in the sample is elevated compared to one or more reference levels. In some embodiments, the kit can further include instructions for comparing the level of lyso-SPM in the sample to levels in a reference sample, and screening for and / or diagnosing an ASM disorder if the level of lyso-SPM in the sample is elevated compared to levels in the reference sample.

[0062] In some embodiments, the kit can be used as part of a therapy for and / or to diagnose an ASM disorder. In some embodiments, the ASM disorder is NPD-A, NPD-B, or NPD-C.

[0063] Target population In various embodiments, the subject used herein refers to a person who is being screened for ASM disorder. In various embodiments, the subject used herein refers to a person who is diagnosed or treated for ASM disorder according to the methods provided herein, and has or has been diagnosed as having an ASM disorder that causes excessive accumulation of lysosomal SPM in one or more affected organs. In some embodiments, the subject has one or more mutations, such as deletions, frameshifts, missense mutations and / or nonsense mutations, in the gene encoding acid sphingomyelinase. In certain embodiments, the subject has NPD. In one embodiment, the subject has NPD-A, NPD-B or NPD-C.

[0064] In some embodiments, the subject has one or more mutations in the SMPD1 gene. In some embodiments, the mutation is ΔR608 (deletion of arginine 608). In some embodiments, the mutation is a missense mutation. In some embodiments, the missense mutation is L302P, H421Y, or R496L. In other embodiments, the mutation is a deletion resulting in the deletion of one, two, three, or more amino acid residues. In specific embodiments, the subject being treated for ASM disorder according to the methods provided herein is a patient with a mutation in the SMPD1 gene described in U.S. Application No. 2011 / 005255, which is incorporated herein by reference in its entirety. 9, Table 1. For mutations in the acid sphingomyelinase gene (designated SMPD1), see Simonaro et al., Am. J. See also Hum. Genet. 71:1413-1419 (2002).

[0065] In some embodiments, a subject being screened for or diagnosed or treated for an ASM disorder according to the methods provided herein endogenously expresses ASM at about 2-5%, 5-10%, 5-15%, 5-20%, 5-30%, 20-30%, or 5-35% of the activity of normal (e.g., unmutated) human ASM, e.g., ASM-1. In some embodiments, the subject endogenously expresses ASM at less than 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the activity of normal human ASM, e.g., ASM-1. For techniques that can be used to measure ASM activity, see, e.g., U.S. Pat. Nos. 4,039,388, 4,082,781, 5,686,240, and 7,563,591, and International Publication Nos. WO2007 / 078806 and WO2006 / 058385, which are incorporated by reference in their entireties; see also the fluorescence-based high-performance liquid chromatography assay described in He et al., Analytical Biochemistry 314:116-120 (2003).

[0066] In various embodiments, a subject being screened for, diagnosed with, or treated for an ASM disorder according to the methods provided herein may exhibit one or more symptoms of NPD, including, but not limited to, abdominal distension, hepatomegaly, splenomegaly, hepatosplenomegaly, neutropenia, pulmonary disease, lymphadenopathy, histochemical presence of characteristic NPD foam cells, anemia (e.g., microcytic anemia), thrombocytopenia, recurrent vomiting, chronic constipation, failure to thrive (e.g., decreased linear growth and weight), delayed puberty, recurrent bruising, recurrent bleeding, atherogenic lipid profile (high cholesterol, triglycerides, LDL and / or low HDL), pain (headache, back, limbs, abdomen), fatigue, early satiety, low endurance, osteopenia, neurological signs, and dyspnea (e.g., interstitial lung disease and / or shortness of breath). Neurological signs of NPD include cherry-red spotting, hypotension, muscle weakness, psychomotor retardation, spasticity, social reluctance, irritability, and / or convulsions.

[0067] In some embodiments, the subject being screened or diagnosed or treated for ASM disorder according to the methods provided herein is a human infant. In another embodiment, the subject is a human child. In some embodiments, the subject is a human adult (18 years of age or older). In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In some embodiments, the subject is a human female who is not pregnant or lactating.

[0068] Example The following examples serve to illustrate, but not limit, the present disclosure. [Example]

[0069] material and method Whole blood used in this study was obtained from 20 subjects previously diagnosed with NPD-B and 20 healthy adults after written informed consent (ProMedDx, (Purchased from Becton, Dickinson and Company, Norton, MA). Venous blood was collected into EDTA-containing Vacutainer® collection tubes (Becton, Dickinson and Company, Franklin Lakes, NJ), shipped overnight in freezer packs, and stored at 4°C. Within 48 hours of collection, the blood was mixed by inverting the tubes multiple times, and 75 μl of blood per spot was placed on Whatman 903® specimen collection paper and allowed to dry at room temperature for at least 4 hours.

[0070] To quantify lyso-SPM, 1-O-hexadecyl-(7,7,8,8-d4)-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor C16-d4; PAF C16-D4, Cayman Chemical Company, Ann Arbor, Michigan) was used as an internal standard. Dried blood spots (DBS) from 3.2 mm punches were extracted into 200 μL of methanol / acetonitrile / water (80 / 15 / 5) containing 0.8 ng of the internal standard, vortexed for 30 min, and sonicated for 10 min. The eluent was removed by centrifugation at 16,200 g for 5 minutes, after which 30 μL was injected into an API Qtrap 4000 LC / MS / MS (AB Sciex, Toronto, Canada) system interfaced with an Agilent 1100 high-pressure liquid chromatography (HPLC) system (Agilent, Palo Alto, CA). HPLC was performed isocratically using a normal-phase silica column with a mixture of methanol / acetonitrile / water as the mobile phase. Mass spectrometry (MS) was performed in multiple reaction monitoring (MRM) mode with the following transitions: m / z 465.4 > 184.1 for lyso-SPM and 528.5 > 184.1 for PAF C16-D4. C12-SPM (Avanti Polar Lipids, Alabaster, Alabama) was used as an internal standard to quantify SPM. The extraction and LC / MS / MS procedures were similar to those used for lyso-SPM, except that the eluent was diluted 320-fold before injection, and 12 isoforms of SPM were monitored and summarized. [Example]

[0071] Diagnosing NPD Although SPM levels are known to be elevated over 10-fold in the liver and spleen of NPD-B subjects, SPM levels in the plasma of NPD-B subjects are not appreciably elevated and overlap with SPM levels in normal controls. As shown in Figure 1A, the levels of SPM in NPD-A and NPD-B patients were not significantly elevated, as indicated by the ratio of SPM concentrations in dried blood spots (DBS) of NPD-A and NPD-B patients to the mean concentration values ​​in DBS of normal control samples. SPM is a major component of cell membranes and lipoproteins, and the slight elevation of SPM in DBS of NPD subjects may be related to the already high levels and rapid turnover of SPM in the circulation.

[0072] Unlike SPM, lyso-SPM levels were clearly elevated in DBS samples from NPD-A and NPD-B subjects. As shown in Figure 1B, the levels of lyso-SPM were elevated in NPD-A and NPD-B patients, as indicated by the ratio of lyso-SPM concentrations in dried blood spots (DBS) from NPD-A and NPD-B patients to the mean concentration values ​​in DBS samples from normal control samples. Lyso-SPM levels did not correlate with the amount of residual ASM activity or the age of the subjects at the time of sampling. Thus, lyso-SPM was elevated in peripheral tissues (DBS) from NPD subjects at levels discernible from those of normal controls. [Example]

[0073] Effect of dose escalation in mice Previous studies in ASM knockout mice (Niemann-Pick model mice) showed that clinical signs of toxicity were not observed until single doses of 10 mg / kg or higher were used. Nickerson et al., "Dose-Responsive Toxicological Findings Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice," American Society of Human Genetics, 2005; and Murray et al., "Elevations of Pro-Inflammatory Cytokines," okines and Decreases in Cardiovascular Hemodynamics Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice,” Society of Toxicology 2006.

[0074] In this study, ASM knockout mice were administered a single intravenous dose of rhASM at one of three different concentrations: 0 mg / kg, 3 mg / kg (a non-toxic dose; no clinically apparent side effects were observed), or 20 mg / kg (a toxic dose). Three male and three female mice were assigned to each treatment group (18 animals in total), and blood samples were collected at the time points shown in Table 1.

[0075] [Table 1]

[0076] Quantification of lyso-SPM levels in DBS samples showed that lyso-SPM levels increased with increasing rhASM dose concentration, peaking approximately 6 hours after administration in the 3 mg / kg and 20 mg / kg treatment groups. See Figure 2, a histogram showing the fold increase in lyso-SPM concentrations in DBS at each sampled time point relative to the 5 min post-dose concentration. Surprisingly, the fold increase in plasma lyso-SPM concentrations in response to a toxic dose (20 mg / kg) of rhASM was significantly higher than the fold increase in plasma lyso-SPM concentrations in response to a non-toxic dose (3 mg / kg) of rhASM. This result suggests that lyso-SPM may be useful as a marker for measuring the toxic effects of therapeutic agents that reduce accumulated SPM levels in patients with ASM disorders. [Example]

[0077] Comparison of single-dose and reduced-dose regimens Dried blood spots were collected from wild-type (C57BL / 6) or ASM knockout (ASMKO) mice after administration of a single dose or a reduced dose regimen of rhASM. Five ASMKO mice received a single dose of 10 mg / kg rhASM. Five C57BL / 6 and five ASMKO mice were treated with a reduced dose regimen of 3 mg / kg rhASM followed by a dose of 20 mg / kg. Blood samples were collected at the following time points: 5 minutes, 4 hours, 6 hours, 24 hours, and 72 hours after administration. Animals in the 10 mg / kg group were euthanized 24 hours later.

[0078] A total of 70 blood spot samples were prepared using a lipid multiplex extraction procedure and analyzed by LC / MS / MS. Briefly, a single DBS spot was punched from each sample card and placed in an individual Eppendorf tube. 200 microliters of an 80:15:5 solution (MeOH:ACN:HO) was then added to each tube, followed by vortexing for 30 minutes, sonication for 10 minutes, and centrifugation to spin down any particulates. The concentration of lyso-SPM in each sample was determined using a lyso-SPM calibration curve (no internal standard).

[0079] The data in Figure 3 show that lyso-SPM levels were elevated only in the ASMKO group treated with a single (high) dose of 10 mg / kg rhASM, in contrast to the ASMKO group subjected to the dose-reduction regimen. At such a high dose, at least 50% of the mice would die between 24 and 72 hours, whereas all mice in the dose-reduction regimen would survive. These results suggest that lyso-SPM may be useful as a marker for measuring the toxic effects of high-dose therapeutic agents. [Example]

[0080] Human trials Blood samples were collected from Niemann-Pick patients undergoing treatment with rhASM. A representative example is shown in Figure 4. Over a 26-week period (day 1, weeks 2, 4, 6, 8, 10, 12, 14, and 26), samples were collected pre-dose and 24, 48, and 72 hours after administration of the indicated doses (0.1, 0.3, 0.6, 1, 2, or 3 mg / kg) of rhASM. At week 26, samples were collected only pre-dose and 24 and 48 hours after administration. Lyso-SPM levels were measured in ng / ml. The data show a general trend toward decreased lyso-SPM levels after repeated administration at higher concentrations, although there were some increases and decreases in lyso-SPM levels between pre-dose and 72 hours after each dose. Early post-dose samples were collected 24 hours after administration, and it is possible that the rapid increase in post-dose lyso-SPM levels was not observed until that time point. The dose administered may also have been below the concentration required to observe a significant spike in lyso-SPM levels after administration.

[0081] The foregoing examples are illustrative of the present disclosure and are not intended to be limiting in any way. Other embodiments of the disclosed devices and methods will be apparent to those skilled in the art from the specification and practice of the devices and methods disclosed herein.

Claims

1. A method for calibrating the dosage of a therapeutic agent for the treatment of acid sphingomyelinase deficiency (ASMD) in human subjects, wherein the therapeutic agent comprises recombinant human ASM (rhASM) or modified rhASM, and the method comprises measuring the level of lyso-sphingomyelin (lyso-SPM) in a biological sample taken from a subject to which a first dose of the therapeutic agent has been administered. If the lyso-SPM level is below the reference level, the second dose of the therapeutic agent administered to the subject is increased or maintained relative to the first dose. The method wherein, if the level of lyso-SPM exceeds a reference level, a second dose of the therapeutic agent administered to the subject is reduced relative to the first dose.

2. The method according to claim 1, wherein the biological sample is a whole blood sample, a dried blood spot, a plasma sample, or a serum sample.

3. The method according to any one of claims 1 to 3, wherein the reference level is the baseline lyso-SPM level of the subject before treatment with the therapeutic agent.

4. The method according to any one of claims 1 to 3, wherein the ASMD is Niemann-Pick disease type B.

5. The method according to any one of claims 1 to 3, wherein the ASMD is Niemann-Pick disease type A / B.

6. The method according to any one of claims 1 to 5, wherein the first and second doses are 0.03 mg / kg to 3 mg / kg.

7. The method according to claim 6, wherein the first and second doses are adjacent doses selected from 0.03, 0.1, 0.3, 0.6, 1, 2, and 3 mg / kg.

8. The method according to any one of claims 1 to 7, wherein the therapeutic agent is administered intravenously to the subject. 。

9. The method according to any one of claims 1 to 7, wherein the first and second doses are administered at intervals of two weeks.

10. The method according to any one of claims 1 to 7, wherein the subject has been determined to have elevated blood concentrations of lyso-SPM compared to a healthy control before treatment.

11. The method according to any one of claims 1 to 8, wherein the subject is an adult patient with ASMD.

12. The method according to any one of claims 1 to 8, wherein the subject is a pediatric patient with ASMD.

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