Markers for acid sphingomyelinase dysfunction and their use

Abstract

To provide a method of screening, diagnosing, monitoring and / or treating acid sphingomyelinase (ASM) disorders such as Niemann-Pick disease.SOLUTION: Provided is a method to determine effectiveness of doses of recombinant human acid sphingomyelinase (rhASM) or modified rhASM in treating acid sphingomvelinase deficiency (ASMD) in a human subject. In the method, a level of lyso-sphingomyelin (lyso-SPM) in a biological sample collected from the subject three days or more after the doses of rhASM or modified rhASM are administered to the subject, is inspected, and a reduction in the level of lyso-SPM in comparison with a reference level, indicates the effectiveness of the doses of rhASM or modified rhASM.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61 / 832,302, filed Jun. 7, 2013, which is hereby incorporated 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 Art

[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. Lack of ASM activity can prevent the body from breaking down SPM. In patients with ASM disorders, SPM accumulates mainly in macrophages, but also in hepatocytes and other cell types, causing marked hepatosplenomegaly, thrombocytopenia, interstitial lung disease and coronary artery disease. SPM does not increase significantly in plasma, whole blood or urine, and its use as a non-invasive biomarker is limited.

[0004] Currently, the diagnosis of ASM disorder requires invasive testing and / or time-consuming examinations, such as evaluation of suspicious clinical symptoms, liver or lung biopsy, testing of ASM activity in blood samples (if false-negative and positive cases are reported), and / or genetic testing (e.g., SMPD1 gene mutation analysis). Treatment of ASM disorder may include the administration of replacement enzymes. High-dose enzyme replacement therapy can lead to the production of toxic or harmful metabolites. Therefore, there is a need to develop improved methods for screening, diagnosing, and / or monitoring the course of treatment of ASM disorder. Disclosed herein is a method for non-invasively screening, diagnosing, monitoring treatment, and / or adjusting the dosage of therapeutic agents for treating ASM disorder, the method comprising measuring the level of lyso-SPM (sphingosylphosphorylcholine or lyso-sphingomyelin) in a biological sample. High levels of lyso-SPM can be used to screen or diagnose ASM disorder. High levels of lyso-SPM 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, thereby allowing for calibration of enzyme therapy to reduce SPM accumulation while avoiding the adverse side effects of the treatment. High levels of lyso-SPM can also be used to monitor the long-term effectiveness of the course of treatment for ASM impairment (for example, if lyso-SPM levels do not decrease throughout the course of treatment, this may indicate an ineffective treatment).

[0005] Niemann-Pick disease (NPD) is a genetic autosomal recessive lipid storage disorder characterized by the 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 leading to death by 2-3 years of age. Niemann-Pick disease type B ("NPD-B") results in hepatomegaly and spleen enlargement and respiratory distress, and generally 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 (ASMD). Each of these forms of Niemann-Pick disease is collectively referred to herein as Niemann-Pick disease (NPD).

[0006] NPD is more prevalent in individuals of Ashkenazi Jewish descent than in the general population. The incidence of NPD-A among Ashkenazi Jews is approximately 1 / 40,000, with a gene frequency (q) of approximately 1 / 200 and a heterozygous carrier frequency (2pq) of 1 / 100 (Non-Patent Literature 1). The incidence of heterozygous carriers of NPD-B among Ashkenazi Jewish populations is even lower (ibid.). The heterozygous carrier frequency for NPD types A and B combined is estimated to be approximately 1 / 70 in individuals of Ashkenazi Jewish lineage (ibid.). Epidemiological studies conducted in various countries have estimated the incidence of NPD types A and B combined in several countries worldwide to be between 1 / 167,000 and 1 / 250,000 in newborns (Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4). The proportion of heterozygous carriers 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 the excessive accumulation of sphingomyelin in lysosomes. Furthermore, the clinical course of NPD-B is highly variable, and it is currently impossible to determine a correlation between disease severity and 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. Moreover, enzymatic detection of obligate heterozygotes has proven problematic, particularly when using peripheral leukocytes as the enzyme source. One possible reason is that the presence of residual ASM activity resulting from the development of neutral sphingomyelinase and / or mutated alleles in some sources may contribute to the inability to reliably distinguish carriers for either disease subtype. Clear results for heterozygotes have not been obtained even when using cultured dermal fibroblasts that do not express neutral sphingomyelinase. 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) is used to treat various lysosomal storage diseases. In particular, ERT for Tay-Sachs, Pompe, and Niemann-Pick diseases is being examined; see Patent Documents 1 and 2, which are incorporated herein by reference. ERT attempts to replenish deficient and / or lacking enzymes with externally supplied enzymes. In the case of Niemann-Pick disease ERT, the goal is to enable the affected individual to process sphingomyelin and prevent its accumulation in lysosomes. To be effective, such therapy requires not only a sufficiently large amount of replacement enzyme to first break down the accumulated sphingomyelin, but also continuous administration of replacement enzyme to prevent further accumulation of sphingomyelin. However, the metabolism of accumulated sphingomyelin can lead to the production of toxic or harmful metabolites. Therefore, careful adjustment of ERT is necessary to effectively reduce accumulated sphingomyelin in patients without producing high levels of metabolites that may cause harmful side effects.

[0009] As previously stated, SPM does not significantly increase in plasma, whole blood, or urine, limiting its use as a non-invasive biomarker for screening, diagnosing, or monitoring ASM dysfunction. As disclosed herein, lyso-SPM (sphingosylphosphorylcholine or lyso-sphingomyelin), a deacylated form of SPM, significantly increases in tissues, including peripheral tissues, of patients with and / or being treated for ASM dysfunction, making it a promising marker for screening, diagnosing, and / or monitoring ASM dysfunction. This dichotomy, in which changes in the levels of both acylated and deacylated sphingoglycolipids in plasma are detectable, stands in contrast to many other lysosomal storage disorders. Given that changes in SPM levels are not detectable in the plasma of patients with or being treated for ASM dysfunction, it might have been assumed that lyso-SPM would also be unsuitable for screening, diagnosing, or monitoring. However, As disclosed in the details, lyso-SPM has been found to be detected at different levels in biological samples from various tissues, including peripheral tissues such as plasma.

[0010] Lyso-sphingolipids (lyso-SLs), including lyso-SPM, are deacylated forms of sphingolipids; several have been shown to be elevated in some lysosomal storage disorders. (Non-Patent Literature 5). The mechanisms by which lyso-SPM and other lyso-SLs are produced are not well understood. The absence of a simultaneous elevation of sphingosine suggests that deacylation of the corresponding sphingolipid is a possible pathway of production. However, to date, only sphingomyelin deacylase has been identified from the stratum corneum of atopic dermatitis subjects. (Non-Patent Literature 6). Expression of this deacylase appears to be limited to selected cell types under certain physiological conditions. Purified ASM from the placenta, brain, and urine has been shown not to hydrolyze lyso-SPM. (Non-Patent Literature 7; Non-Patent Literature 8; Non-Patent Literature 9). The lack of understanding of the biosynthetic pathway of lyso-SPM further highlights the difficulty in predicting lyso-SPM expression levels 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 literature 10; Non-patent literature 11. There are no reports on the levels of lyso-SPM in organs other than the spleen and liver of NPD-B patients, and the brain of NPD-A subjects with little or no ASM activity and who develop severe neuropathic diseases. [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) [Overview of the project] [Problems that the invention aims to solve]

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

[0015] The methods disclosed herein also include methods for treating human subjects having acid sphingomyelinase (ASM) disorder. In some embodiments, the method involves administering to a subject a first dose of a therapeutic agent having a first concentration for treating the ASM disorder; and, if it is determined that the subject has lyso-SPM levels below a reference level 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. It includes. The method includes identifying patients with ASM disorders by measuring lyso-SPM in a biological sample from peripheral tissue and detecting changes in lyso-SPM levels. The method also includes detecting the levels of one or more toxicity markers, including lyso-SPM, that change as a result of treating a patient with a therapeutic agent, such as an agent that reduces the SPM level in the patient's tissue, to monitor or adjust the treatment of patients with ASM disorders. The method enables non-invasive evaluation of such patients.

[0016] The methods disclosed herein can also be used, in some embodiments, to screen for, diagnose, monitor the progress of treatment of, and / or adjust treatment of ASM disorders such as NPD. For example, the method includes adjusting the dosage of a therapeutic agent administered to a patient to treat an ASM disorder by measuring a toxicity marker (e.g., lyso-SPM) to manage the level of toxic metabolites resulting from the treatment. The methods disclosed herein can also be used, in some embodiments, to monitor the long-term effectiveness of the course of treatment of an ASM disorder (e.g., if the level of lyso-SPM does not decrease over the course of treatment, this may indicate an ineffective treatment). The methods disclosed herein can also be used, in some embodiments, to screen subjects (e.g., pre-symptomatic patients) for an increase in lyso-SPM as an early sign of an ASM disorder. Subjects identified as having an increase in lyso-SPM in the screening are then subjected to additional evaluations (e.g., ASM blood tests, genetic tests, etc.) to diagnose / confirm the diagnosis of an ASM disorder, while subjects in whom no increase in lyso-SPM is seen are not subjected to additional evaluations. Such screening can potentially reduce the cost of testing.

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

Brief Description of the Drawings

[0018] [Figure 1] Figure 1A is a plot showing the ratio of the SPM concentration in dried blood spots (DBS) from NPD-A and NPD-B patients to the average concentration value in DBS from normal control samples. Figure 1B is a plot showing the ratio of the lyso-SPM concentration in DBS from NPD-A and NPD-B patients to the average concentration value in DBS from normal control samples. [Figure 2] Histogram showing the fold increase in the concentration (vertical axis) of lyso-SPM in DBS from ASM knockout mice at specified time points (1, 2, 4, 6, 24, 48, and 72 hours after administration) compared to the concentration 5 minutes after a single administration of 0, 3, or 20 mg / kg of rhASM. [Figure 3] Concentration (ng / ml) of lyso-SPM in DBS collected from wild-type (C57BL / 6) or ASM knockout (ASMKO) mice after administration of a single dose (10 mg / kg) or a reduced-dose regimen (3 mg / kg) of rhASM followed by administration of a 20 mg / kg dose of rhASM 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 dosing group were euthanized after 24 hours. [Figure 4]This histogram shows the concentration of lyso-SPM (ng / ml) in DBS from human Niemann-Pick patients collected before rhASM administration and 24, 48, and 72 hours after rhASM administration over a 26-week period. The x-axis shows 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). In week 26, samples were collected only before administration and 24 and 48 hours after administration. [Modes for carrying out the invention]

[0019] Herein, references are made in detail to several exemplary embodiments relating to the present disclosure, some examples of these embodiments are illustrated in the accompanying drawings. Where possible, the same reference numerals are used throughout the drawings to refer to the same or similar parts.

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

[0021] Section headings used herein are for structural purposes only and should not be interpreted as limiting the subject matter described. All documents, or parts thereof, cited herein, including but not limited to patents, patent applications, articles, books, and scholarly papers, are explicitly incorporated herein by reference in their entirety for any purpose.

[0022] Disclosed herein are methods for screening, diagnosing, monitoring the progression of treatment, and / or adjusting the dosage of therapeutic agents for treating ASM disorders, such as NPD. “ASM disorder” may encompass any disorder associated with reduced or impaired expression of acid sphingomyelinase. ASM disorder may also encompass any other disorder associated with the accumulation of sphingomyelin in tissues.

[0023] In some embodiments, the method includes administering to a subject a first dose of a therapeutic agent having a first concentration for treating ASM disorder; and, if it is determined that the subject has a lyso-SPM level below a reference level (e.g., a level in a sample from a control subject without 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 biological samples from human subjects. The measured levels of lyso-SPM can also be used to screen for, diagnose, monitor the progress of treatment for ASM disorders, and / or adjust the dosage of therapeutic agents for treatment. The method is based on the finding that lyso-SPM is significantly elevated in biological samples from peripheral tissues of patients with ASM disorders such as NPD, enabling non-invasive assessment of such patients, including screening and diagnosing ASM disorders, as well as monitoring / calibrating / controlling treatments for ASM disorders. The method is also based on the finding that accumulated ASM may be broken down during treatment, potentially leading to elevated 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 (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) designed to suppress SPM levels at doses that prevent excessive metabolite production. The measurement of the increase in lyso-SPM concentration is It can be used to detect the production of such metabolites and to calibrate treatments to avoid the production of excessively high levels (e.g., toxic levels) of metabolites. In some embodiments, the level of lyso-SPM in patients being treated for ASM disorders determines whether to increase, decrease, repeat, delay, or discontinue the dose.

[0025] The detection of 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 may include taking a biological sample from the subject, measuring the level of lyso-sphingomyelin (lyso-SPM) in the sample, comparing the measured level of lyso-SPM in the sample to a reference level, and detecting an adverse side effect if the level of lyso-SPM in the sample is elevated. In some embodiments, if the level of lyso-SPM is elevated 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 in the subject is elevated by a predetermined amount over time throughout the course of treatment (i.e., above a 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 from the treatment. In some embodiments, the treatment is enzyme replacement therapy (ERT), and the dose of ERT is controlled by taking one or more biological samples from the patient, examining each sample for an increase in lyso-SPM, and setting the dose of ERT to a level that does not result in high lyso-SPM levels exceeding a predetermined threshold. Controlling the dose of the therapeutic agent may include increasing, decreasing or maintaining the concentration of the therapeutic agent, and / or discontinuing the treatment. In some embodiments, one or more biological samples are taken after administering a certain dose of the therapeutic agent and / or immediately before 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 a dried blood spot) is higher than a reference level of approximately 100–700 ng / ml (e.g., higher than approximately 100, 200, 250, 300, 400, 500, 600 or 700 ng / ml or any intermediate concentration), or if the level of lyso-SPM in a biological sample is elevated by at least approximately 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 more (or any intermediate value) above the reference level. For example, such elevation is at least approximately 3 times. The reference level may be, for example, the level in a sample from a control subject without ASM impairment, or the baseline level measured in a patient before treatment with a certain dose of the therapeutic agent.

[0026] Furthermore, disclosed herein are methods for treating subjects having ASM disorders (e.g., NPD). In various embodiments, the method includes administering a therapeutic agent to treat the ASM disorder in increasing concentrations in a series of doses (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) and monitoring the subject for high lyso-SPM levels in a biological sample after each dose (e.g., 1 minute, 5 minutes, 10 minutes, 30 minutes or 45 minutes after administration, or 1, 2, 3, 4, 5, 10, 12, 15 or 20 hours after administration, or 1 day, 2 days, 5 days, 1 week, 2 weeks, 3 weeks or 4 weeks, or at any intermediate point) or immediately before the next dose. Monitoring of the subject may include taking 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 ASM impairment, or the level in an ASM patient before treatment), and detecting high levels of lyso-SPM in the sample compared 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 ASM impairment. In some embodiments, the reference level is set after the administration of an initial low dose of ERT, or before the administration of any ERT, for the subject. This refers to the level of lyso-SPM measured in a biological sample taken from an elephant. In some embodiments, the elevation of lyso-SPM levels in a biological sample (e.g., a serum sample, plasma sample, or blood sample such as a dried blood spot) is, for example, above a reference level of about 100-700 ng / ml. In some embodiments, the elevation of lyso-SPM levels 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 more (or any intermediate value) above the reference level. For example, the level is at least about 3 times higher. In some embodiments, if an elevation of lyso-SPM levels is detected after the 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 subjects. In various embodiments, the method includes taking a biological sample from a subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample to a reference level, and detecting / diagnosing an ASM disorder if the level of lyso-SPM in the sample is elevated compared to a reference sample. In some embodiments, the reference sample is a sample from a control subject that does not have an ASM disorder. In some embodiments, the lyso-SPM level in a biological sample (e.g., a blood sample such as a plasma sample, serum sample, or dried blood spot) is defined as being 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 intermediate value) 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, ASM impairment can be screened and / or diagnosed if the concentration is higher than the reference level of at least approximately 200–2000 ng / ml (e.g., above approximately 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 intermediate concentration), such as at least approximately 700 ng / ml and / or at least approximately 900 ng / ml. In some embodiments, biological samples from normal subjects may contain lyso-SPM levels in the range of approximately 25–200 ng / ml (e.g., approximately 25, 50, 75, 100, 125, 150, 175 or 200 ng / ml, or any intermediate concentration).

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

[0029] The methods disclosed herein relate to the measurement of lyso-SPM from various biological samples, including samples from peripheral tissues. Any method can be used to collect, prepare, and quantify the level of lyso-SPM from the biological sample. The level of lyso-SPM in the biological sample can be quantified using a mass spectrometer such as LC / MS / MS, or a spectrometer such as an electromagnetic frequency analyzer such as UV-VIS, IR, or NMR. In some embodiments, the level of lyso-SPM is Methods for quantification may include collecting a biological sample (e.g., by arterial or venous puncture, tissue biopsy, buccal oral 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 the level 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 the spleen, lungs, heart, liver, kidneys, and / or brain tissue, can also be used. Samples from combinations of two or more tissues (e.g., two, three, four, five, 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 before detection and / or measurement of lyso-SPM in the sample. In some embodiments, samples are pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps. In other embodiments, samples are pretreated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization and / or homogenization, followed by centrifugation, filtration, precipitation, dialysis and / or chromatography. In some embodiments, samples are pretreated before evaluation of lyso-SPM by removing a certain type of cell from the sample or by removing necrotic tissue fragments from the sample.

[0032] In various embodiments, a 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 levels of lyso-SPM and / or other markers in a sample can be evaluated quantitatively or semi-quantitatively. In some embodiments, a device for quantifying the levels 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) that react with the detection agent to emit a detectable signal, such as its intensity, brightness, color, etc., can be applied (e.g., by comparison with signals from one or more reference samples). For example, immunoassays such as ELISA, immunoprecipitation, and Western blotting, as well as further methods for quantifying lyso-SPM and / or other markers in biological samples, such as fluorescent cell sorting (FACS), fluorescence resonance energy transfer (FRET), RT-PCR, and / or Northern blotting, can also be used.

[0033] Management of ASM disorders In various embodiments, lyso-SPM levels can be measured as part of the treatment of ASM disorders. For example, ASM disorders are Niemann-Pick disease (NPD), e.g., NPD-A, NPD-B, or NPD-C. Treatment of ASM disorders may include the administration of one or more therapeutic agents that reduce the level of SPM in the patient's tissues (e.g., ERT, chaperone therapy, and / or substrate reduction therapy). For example, this is disclosed in U.S. Patent No. 2011 / 0052559, which is incorporated herein by reference in its entirety. Intra-patient dose-escalation enzyme replacement therapy (ERT) may also be used, such as the ASM dose-escalation protocol (see paragraphs

[0063] to

[0075] , for example, for describing the dose-escalation protocol).

[0034] In various embodiments, methods are disclosed for monitoring subjects for adverse side effects (e.g., production of toxic or harmful levels of metabolites) during dose-escalation therapy for ASM impairment by monitoring markers such as lyso-SPM. The methods may include taking 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, and detecting an adverse side effect if the level of lyso-SPM in the sample is elevated compared to a reference sample. One or more samples may be taken and evaluated after administration of a certain dose of the therapeutic agent or before administration of the next dose. For example, a sample may be taken and evaluated 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes after administration of a certain therapeutic dose, or 1, 2, 3, 5, 10, 12, 15, or 20 hours later, or 1, 2, 3, 4, or 5 days later, or 1, 2, 3, or 4 weeks later, or at any intermediate point, or before administration of the next dose. In some embodiments, the reference sample is a sample from a control subject without ASM impairment. In some embodiments, the reference sample is an initial biological sample from the subject before administration of a high-concentration dose of the therapeutic agent (or before administration of any therapeutic agent). In some embodiments, if the level of lyso-SPM increases by a predetermined amount or exceeds 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-concentration) dose of the therapeutic agent (e.g., a high-concentration ERT), this can be used as an indication of an adverse side effect of 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 no adverse side effect has occurred.

[0035] In various embodiments, treatments for ASM impairment are provided. These treatments may include the administration of one or more therapeutic agents that reduce the level of SPM in the patient's tissues (e.g., ERT, chaperone therapy, and / or substrate reduction therapy). In some embodiments, the treatment may include ERT (e.g., ASM replacement therapy). The treatment may include monitoring the subject for elevations in lyso-SPM levels between treatments and adjusting the concentration of the therapeutic agent (e.g., the concentration of the ERT dose) to reduce lyso-SPM levels below a predetermined threshold level. In some embodiments, monitoring may include evaluating the sample for elevations in lyso-SPM levels after each dose of the therapeutic agent or before each subsequent dose of the therapeutic agent. In some embodiments, monitoring after each dose is optional and may be 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 may include administering ERT (e.g., ASM replacement therapy) in increasing concentrations in a series of doses, taking a biological sample from the patient after a certain number of doses (e.g., after each dose or before each subsequent dose), detecting the level of lyso-SPM in the sample (e.g., using LC / MS / MS), and monitoring the subject for high lyso-SPM levels after each dose or before the next dose. For example, a biological sample may be taken and monitored 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes after a certain dose of ERT has been administered, or 1, 2, 3, 5, 10, 12, 15, or 20 hours later, or 1, 2, 3, 4, or 5 days later, or 1, 2, 3, or 4 weeks later, or at any intermediate point, or before the next dose. Monitoring a subject involves taking 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, and comparing the level of lyso-SPM in the sample to a reference level. If the ERT level increases by a predetermined amount compared to the sample, the dose of ERT may be adjusted. In some embodiments, the reference sample is a sample from a control subject that does not have an 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 lyso-SPM level increases by a predetermined amount in the subject sample and / or is above a reference threshold, this can be used as an indication that the ERT dose should be reduced, delayed, or terminated to avoid the production of toxic or harmful levels of metabolites. In some embodiments, if the lyso-SPM level is below the threshold, a subsequent dose of the same or higher concentration may be administered.

[0037] In various embodiments, dose-escalation therapy is provided, which involves administering ERT to a target while increasing the dose over time in order to reduce accumulated SPM without producing toxic or harmful levels of metabolites resulting from the rapid hydrolysis of accumulated SPM. In some embodiments, since high lyso-SPM may indicate the production of toxic or harmful levels of metabolites, biological samples from the patient are monitored for increases in lyso-SPM levels during dose-escalation therapy. In some embodiments, if levels of lyso-SPM exceeding a threshold concentration are detected, or if a significant increase in lyso-SPM levels is detected compared to the level in the previous sample, the dose increase of ERT is delayed or not administered. ERT can be administered via any route suitable for achieving the therapeutic effect, including intravenous, intradermal, subcutaneous, intraperitoneal, intrapulmonary, topical, intranasal, intracranial, or intramuscular.

[0038] In some embodiments, the ERT may include administration of an acid sphingomyelinase (ASM), such as recombinant human ASM (rhASM), or the ERT may include administration of a modified ASM (e.g., modified rhASM). The modified ASM may include any modifications to the enzyme that do 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% or any intermediate percentage of the enzymatic activity of the unmodified ASM). The hydrolytic ability of modified ASM can be evaluated by the art, including in U.S. Patents 4,039,388, 4,082,781, 5,686,240, and 7,563,591, which are incorporated herein by reference in their entirety, and in International Application Numbers WO2007 / 078806 and WO2006 / 058385.

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

[0108] to

[0117] and

[0124] to

[0127] , which discusses human ASM isoforms and their enzyme conjugates and their use in ERT).

[0040] In some embodiments, enzyme replacement therapy is administered to the subject in escalating doses after a non-toxic initial low dose. The maximum dose of enzyme tolerable by the subject without producing toxic or harmful levels of metabolites (detectable, for example, by monitoring levels of toxicity markers such as lyso-SPM) can then be used as a maintenance dose. Alternatively, a therapeutically effective dose below the maximum tolerable dose can be used as a maintenance dose. The therapeutically effective dose may include any dose sufficient to reduce the concentration of accumulated sphingomyelin in the ASM subject by at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% (or any intermediate percentage) after one or more doses. can.

[0041] Treatment of ASM disorders such as NPD requires high doses of therapeutic agents (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) sufficient to achieve proper distribution of the drug in affected organs (e.g., spleen, lungs, liver, heart, kidneys, and brain). Studies have shown that after intravenous administration of recombinant human ASM to ASM knockout mice, most ASM activity is distributed to the liver, with only small amounts of ASM enzyme activity detected in other organs such as the spleen, heart, kidneys, and lungs. See, for example, He et al., Biochimia et Biophsyica Acta 1432: pp. 251-264 (1999). Therefore, administration of high concentrations of therapeutic agents (e.g., high concentrations of replacement enzymes) is necessary to ensure proper distribution and delivery in subjects with ASM disorders such as NPD, particularly in the lungs, liver, heart, and kidneys.

[0042] Studies in ASM knockout mice have also shown that sufficiently high doses of enzyme replacement therapy may lead to the production of toxic or, conversely, 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). While not bound by theory, high doses of ASM administered to NPD subjects may lead to the hydrolysis of large amounts of accumulated sphingomyelin to 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 cause the toxic side effects observed in ASM knockout mice and NPD subjects.

[0043] Therefore, calibration and adjustment of therapies (e.g., ERT, chaperone therapy, and / or substrate reduction therapy) are required to provide a therapeutic concentration sufficient 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 by managing the dose escalation protocol by evaluating the levels of toxicity markers such as lyso-SPM in patient samples after a certain number of doses or after each dose, and by adjusting the dosing regimen described herein as necessary. Managing the therapeutic dose may include increasing, decreasing or maintaining the concentration of the therapeutic, and / or discontinuing the 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 a subject in order to reduce the amount of sphingomyelin accumulated in the subject. Subsequently, the dose of the therapeutic agent can be systematically increased in higher concentrations until the subject tolerates it and reaches the highest dose at which it is therapeutically effective. In some embodiments, the therapeutic agent is a replacement enzyme (e.g., rhASM) and is administered such that the enzyme activity in one or more affected organs (e.g., organs showing elevated lysosomal levels of SPM in patients with ASM impairment) 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 intermediate percentage) of the activity level in the corresponding organ of a subject without ASM impairment (e.g., a healthy individual).

[0045] In some embodiments, a method for treating ASM impairment includes (a) (i) administering an initial low dose to the target of rhASM or modified rhASM; and (ii) administering subsequent higher doses to the target of rhASM or modified rhASM (dose escalation), thereby accumulating the target. (b) administering an enzyme replacement regimen to reduce the sphingomyelin substrate; (c) monitoring the subject for one or more additional markers relating to elevated lyso-SPM levels and / or adverse side effects after a certain number of doses in steps (a)(i) and (a)(ii) or after each dose (e.g., using LC / MS / MS to quantify lyso-SPM concentration); (d) repeating, reducing and / or terminating the dose escalation protocol after elevated lyso-SPM levels are detected and / or after one or more additional adverse side effects are detected. In some embodiments, this further includes administering the patient a maintenance dose at or below the highest dose the subject has tolerated, and, if applicable, monitoring for elevated lyso-SPM levels during administration of the maintenance regimen. In some embodiments, the initial dose range of rhASM or modified rhASM can be approximately 0.03 mg / kg to approximately 1.0 mg / kg, or approximately 0.1 mg / kg to approximately 0.5 mg / kg (the dose concentration is measured as mg of enzyme per kg of body weight). In some embodiments, each subsequent dose with an increased enzyme concentration is administered approximately 1, 2, 3, 4, 5, 6, or 7 days after the previous dose, or 1, 2, 3, 4, or 5 weeks later. In some embodiments, the subsequent doses of the increased enzyme concentration can be between approximately 0.1 and 5 mg / kg (e.g., approximately 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 intermediate concentration).

[0046] In some embodiments, a dose of replacement enzyme at a given concentration is administered at least twice (e.g., at least 2, 3, 4, or 5 times) before the next 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 intermediate value) higher than the previous dose. In some embodiments, subsequent higher doses are approximately 0.03–0.1 mg / kg, 0.1 mg / kg–0.5 mg / kg, 0.5 mg / kg–1 mg / kg, 0.5 mg / kg–2 mg / kg, 1 mg / kg–2 mg / kg, 2 mg / kg–4 mg / kg, or 2 mg / kg–5 mg / kg (or any intermediate value) higher than the previous dose. In some embodiments, the highest tolerable dose in which the subject is able to tolerate the drug without producing toxic or harmful metabolites is approximately 1.0 mg / kg–3.0 mg / kg. In some embodiments, the highest tolerable dose is then administered to the human subject as a maintenance dose. In some embodiments, the maintenance dose is administered approximately every 1–8 weeks (e.g., every 1, 2, 3, 4, 5, 6, 7 or 8 weeks, or at any intermediate interval).

[0047] Once the maximum tolerable dose (e.g., a dose that does not produce toxic or, conversely, toxic levels of metabolites) is determined, this maximum tolerable dose can be used as the maintenance dose for treating the aforementioned subjects. The maintenance dose may be administered daily, once weekly, twice weekly, once monthly, twice monthly, or once every three months (or at any intermediate interval). Monitoring for elevated lyso-SPM levels can be done during the administration of the maintenance regime, for example, 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 at any intermediate interval. If high lyso-SPM levels (e.g., levels exceeding the reference level of approximately 100–700 ng / ml, or levels at least approximately 1.1–10 times higher than the reference level) are detected, the maintenance dose may be reduced or discontinued.

[0048] During treatment (e.g., during ERT), several other parameters can be measured in combination with lyso-SPM as part of the treatment to monitor the subject and / or to determine the maximum dose that the subject tolerates. For example, the subject can be measured using SPM. Further monitoring can be performed by measuring plasma ceramide levels and / or bilirubin concentrations. Subjects may also be monitored for the production of “acute phase reactants” and inflammatory mediators, which are measures of the inflammatory response, as well as / or other biochemical markers. These other biochemical markers include, but are not limited to, CRP / hs-CRP, cytokines (e.g., 1L-8, II-6), calcitonin, and ferritin. In some embodiments, monitoring one or more of the parameters listed above can be performed before increasing the dose to high concentrations to ensure a stable response to the treatment. In some embodiments, subjects may be monitored for one or more associated adverse events, including systemic symptoms (e.g., fever, nausea, vomiting, pain, muscle aches, and jaundice). Combinations of markers can also be monitored (e.g., lyso-SPM levels can be monitored in combination with bilirubin and / or ceramide levels). Appropriate threshold levels that can be monitored in conjunction with lyso-SPM are disclosed, for example, in U.S. Patent Application No. 2011 / 0052559, the entirety of which is incorporated herein by reference (see, for example, paragraphs

[0067] to

[0086] ).

[0049] In some embodiments, subjects receiving a dose escalation protocol (e.g., an ERT dose escalation protocol) are monitored for toxic or adverse side effects after each therapeutic dose (e.g., approximately 1 minute, 5 minutes, 10 minutes, 30 minutes, or 45 minutes after administration, or 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours after administration, or 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks or longer, or any intermediate period) or before administration of a high concentration of the therapeutic agent (e.g., by monitoring the level of one or more toxicity markers such as lyso-SPM). In some embodiments, subjects are monitored after each maintenance dose (e.g., approximately 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours after administration, or 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks or longer, or any intermediate period) or before administration of a subsequent maintenance dose of the therapeutic agent. In some embodiments, subjects receive a maintenance dose for one, two, three years or longer and are regularly monitored for toxic or adverse side effects. Monitoring may include monitoring related adverse events in addition to monitoring the toxicity markers described above. If a subject experiences an adverse event or if one or more of the subject's markers being monitored indicate an adverse side effect (e.g., elevated levels of lyso-SPM are detected), the administration of the maintenance dose may be discontinued or adjusted (e.g., a low concentration of ERT may be administered) to reduce or minimize the undesirable side effect.

[0050] In various embodiments, subjects can be monitored for toxic and / or conversely harmful doses of ERT by measuring lyso-SPM levels in biological samples taken after administration of a dose of ERT (e.g., after administration of rhASM or modified rhASM). In some embodiments, the method may include taking a biological sample from a subject, measuring the level of lyso-SPM in the sample, comparing the level of lyso-SPM in the sample to a reference level, and detecting adverse side effects if the level of lyso-SPM in the sample is elevated compared to a reference sample or elevated by a specific amount compared to a reference sample. In some embodiments, a next 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 the previous dose. Examples of appropriate threshold levels are described herein. In some embodiments, the level of lyso-SPM is also monitored during the administration of maintenance doses. In some embodiments, if the lyso-SPM level exceeds a predetermined threshold during maintenance dose administration, the maintenance dose is discontinued, or a lower dose is administered that does not result in a lyso-SPM level exceeding the predetermined threshold.

[0051] In some embodiments, adverse side effects of ERT can be detected if 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 if 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 concentration). In some embodiments, adverse side effects can be detected if 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 times or more (or any intermediate value) higher than the reference level. For example, the elevation may be at least about 3 times.

[0052] In some embodiments, the methods for treating ASM impairment provided herein reduce spleen volume as assessed by techniques well known in the art, such as MRI. In some embodiments, the methods reduce liver sphingomyelin levels as assessed by techniques well known in the art, such as biochemical analysis and / or histomorphometric analysis of liver samples. In some embodiments, the methods increase exercise capacity as assessed by techniques well known in the art, such as maximum workload by cycle ergometry, including percentage predicted maximum workload, peak oxygen consumption, and / or carbon dioxide production. In some embodiments, the methods improve lung function and / or lung clearance as assessed by techniques well known in the art, such as DLco, FVC, FEV, and / or TLC. In some embodiments, the methods reduce bronchoalveolar lavage (BAL) sphingomyelin. In some embodiments, the methods improve the appearance of the lungs as assessed by techniques well known in the art, such as high-resolution CT scans and / or chest X-rays.

[0053] In various embodiments, the methods for treating ASM disorder 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 patient's lipid profile (e.g., lower cholesterol). In some embodiments, the methods improve one or more neurological functions of the subject (e.g., psychomotor function, social reactivity, etc.). In some embodiments, the methods reduce or improve the severity and / or duration of one or more symptoms associated with ASM disorder and / or the disorder. In some embodiments, the methods prevent recurrence of symptoms associated with ASM disorder. In some embodiments, the methods increase the survival rate of the subject after treatment.

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

[0055] In some embodiments, a method for screening and / or diagnosing ASM impairment in a subject includes taking 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, and detecting / diagnosing ASM impairment if the level of lyso-SPM in the sample is elevated compared to the reference level. In some embodiments, the reference level is the level of ASM impairment. This is the level of lyso-SPM measured in a sample from a control subject that does not have ASM. In some embodiments, ASM impairment 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, ASM impairment is at least about 200 to 2000 ng / ml (e.g., about 200, 200 ng / ml), such as the level of lyso-SPM in a biological sample (e.g., blood sample) being higher than about 200 ng / ml, higher than about 300 ng / ml, higher than about 400 ng / ml, higher than about 500 ng / ml, higher than about 525 ng / ml, higher than about 575 ng / ml, and / or higher than about 700 ng / ml. ASM can be detected and / or diagnosed if it is higher than a reference level (higher 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 intermediate concentration). In some embodiments, ASM disorder can be detected and / or diagnosed if the level of lyso-SPM in a biological sample (e.g., a blood sample) is about 1 to 10 times higher than the reference level.

[0056] Biological samples from various tissues can be used with 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 can also be used, such as spleen, lung, liver, heart, kidney, and / or brain tissue. Samples from combinations of two or more tissues (e.g., two, three, four, five, or more tissues) can be used. In some embodiments, ASM disorders (e.g., NPD) can be detected / diagnosed by measuring lyso-SPM levels in biological samples taken from peripheral tissues. In some embodiments, peripheral tissues 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 may further include administering a therapeutic agent (e.g., enzyme replacement therapy) to a subject if an ASM disorder is detected / diagnosed. In some embodiments, enzyme replacement therapy includes administering rhASM or modified rhASM to a subject.

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

[0059] In some embodiments, the kit may further include a device for measuring lyso-SPM levels and / or other markers in a biological sample. For example, the kit may be used to detect lyso-SPM or other markers in a biological sample. The present invention may include antibodies and / or detection agents. In some embodiments, the detection agent is contained in or placed on the tissue collection device (e.g., an antibody or chemical indicator impregnated into the test specimen), while in other embodiments, the detection agent is provided separately from the collection device.

[0060] In some embodiments, the kit may further include a device for quantifying or semi-quantifying the level of lyso-SPM and / or other markers in the sample. For example, a reagent (e.g., a colorimetric analyte) can be provided that reacts with a detection agent to emit 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 comparison with signals from one or more reference samples). In some embodiments, the device may include a device for separating lyso-SPM from other components of the sample, for example, using liquid chromatography and / or mass spectrometry. In some embodiments, the device for quantifying the level of lyso-SPM and / or other markers in the sample may be a mass spectrometer such as LC / MS / MS, or a spectrometer such as an electromagnetic frequency analyzer such as UV-VIS, IR, or NMR.

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

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

[0063] Target group In various embodiments, the subjects used herein are humans who have been screened for ASM disorder. In various embodiments, the subjects used herein are humans who have or have been diagnosed with ASM disorder resulting in excessive accumulation of lysosomal SPM in one or more affected organs, and who are diagnosed with ASM disorder according to the methods provided herein. In some embodiments, the subjects have one or more mutations in the gene encoding acid sphingomyelinase, e.g., deletions, frameshifts, missense mutations and / or nonsense mutations. In certain embodiments, the subjects have NPD. In one embodiment, the subjects have 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 to be treated for ASM impairment according to the method provided herein is referred to in its entirety in U.S. Patent Application No. 2011 / 005255, which is incorporated herein by reference. The patient has one or more of the mutations shown in Table 1 of Issue 9. For mutations in the acid sphingomyelinase gene (specifically SMPD1), see Simonaro et al., Am. J. See also Hum. Genet. 71: pp. 1413-1419 (2002).

[0065] In some embodiments, subjects undergoing screening, diagnosis, or treatment for ASM disorder according to the methods provided herein endogenously express ASM at approximately 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, subjects endogenously express ASM at 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of the activity of normal human ASM, e.g., ASM-1. For techniques that can be used to measure the activity of ASM, see, for example, U.S. Patents 4,039,388, 4,082,781, 5,686,240, and 7,563,591, as well as International Publication Numbers WO2007 / 078806 and WO2006 / 058385 (these are incorporated herein by reference in their entirety); also see the fluorescence-based high-performance liquid chromatography assay described in He et al., Analytical Biochemistry 314: pp. 116-120 (2003).

[0066] In various embodiments, subjects undergoing screening, diagnosis, or treatment for ASM disorder according to the methods provided herein may exhibit one or more symptoms of NPD. Symptoms of NPD include, but are not limited to, abdominal distension, hepatomegaly, splenomegaly, hepatosplenomegaly, neutropenia, lung disease, lymphadenopathy, the presence of histochemically characteristic NPD foam cells, anemia (e.g., microcytic anemia), thrombocytopenia, recurrent vomiting, chronic constipation, growth retardation (e.g., linear growth and weight loss), delayed puberty, recurrent bruising, recurrent bleeding, atherosclerotic lipid profile (high cholesterol, triglycerides, LDL and / or low HDL), pain (headache, back, limbs, abdomen), fatigue, early satiety, poor endurance, osteopenia, neurological signs, and dyspnea (e.g., interstitial lung disease and / or shortness of breath). Neurological signs of NPD include cherry-red spots, hypotension, muscle weakness, psychomotor delay, spasticity, social unresponsiveness, irritability, and / or seizures.

[0067] In some embodiments, the subjects receiving screening, diagnosis, or treatment for ASM disorder according to the methods provided herein are human infants. In other embodiments, the subjects are human children. In some embodiments, the subjects are human adults (18 years of age or older). In some embodiments, the subjects are human females. In other embodiments, the subjects are human males. In some embodiments, the subjects are human females who are not pregnant or lactating.

[0068] Examples The following embodiments serve to illustrate the present disclosure and are not intended to limit it. [Examples]

[0069] material and method Whole blood samples used in this study were obtained from 20 subjects previously diagnosed with NPD-B and 20 healthy adults after written informed consent (ProMedDx, (Purchased from LLC, Norton, MA). Venous blood was collected using EDTA-containing Vacutainer® blood collection tubes (Becton, Dickinson and Company, Franklin Lakes, NJ), transported overnight in frozen packs, and stored at 4°C. Within 48 hours of collection, the blood was mixed by inverting the blood collection tubes multiple times, and 75 μl of blood per spot was placed on Whatman903® sample collection paper and dried 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 activator C16-d4; PAF C16-D4, Cayman Chemical Company, Ann Arbor, Michigan) was used as an internal standard. Dried blood spots (DBS) from a 3.2 mm perforator were extracted into 200 μL of methanol / acetonitrile / water (80 / 15 / 5) containing 0.8 ng of internal standard, vortexed for 30 minutes, and sonicated for 10 minutes. The eluent was removed at a concentration of 16,200 g by centrifugation for 5 minutes, and then 30 μL was injected into an API Qtrap 4000 LC / MS / MS system (AB Sciex, Toronto, Canada) linked to an Agilent 1100 high-pressure liquid chromatography (HPLC) system (Agilent, Palo Alto, CA). HPLC was performed using a normal-phase silica column with a methanol / acetonitrile / water mixture as the mobile phase in a homogeneous concentration manner. Mass spectrometry (MS) was performed using multiple reaction monitoring (MRM) with the following transitions: m / z lyso-SPM at 465.4 > 184.1 and PAF C16-D4 at 528.5 > 184.1. 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. Twelve isoforms of SPM were monitored and summarized. [Examples]

[0071] Diagnosis of NPD While SPM levels are known to increase more than tenfold in the liver and spleen of NPD-B subjects, SPM levels in the plasma of NPD-B subjects do not increase to a noticeable degree and overlap with those of normal controls. As shown in Figure 1A, SPM levels 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 increase in SPM in DBS of NPD subjects may be related to already high levels and rapid turnover of SPM in circulation.

[0072] Unlike SPM, lyso-SPM levels were clearly elevated in DBS from NPD-A and NPD-B subjects. As shown in Figure 1B, lyso-SPM levels were elevated in NPD-A and NPD-B patients, as indicated by the ratio of lyso-SPM concentration in dried blood spots (DBS) of NPD-A and NPD-B patients to the mean concentration value in DBS of normal control samples. Lyso-SPM levels were not correlated with the amount of residual ASM activity or the age of the subject at collection. In other words, lyso-SPM increased in peripheral tissue (DBS) of NPD subjects to a level distinguishable from normal controls. [Examples]

[0073] The effect of dose escalation in mice Previous studies in ASM knockout mice (Niemann-Pick model mice) showed that no clinical symptoms of toxicity were observed until a single dose of 10 mg / kg or higher was 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 Cyt 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, a single intravenous dose of rhASM was administered to ASM knockout mice at one of three different concentrations: 0 mg / kg, 3 mg / kg (non-toxic dose, no clinically apparent side effects were observed), or 20 mg / kg (toxic dose). Three male and three female mice were assigned to each dose group (18 animals in total), and blood samples were collected at the time points shown in Table 1.

[0075] [Table 1]

[0076] We quantified lyso-SPM levels in DBS samples and 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 multiplier of increase in lyso-SPM concentration in DBS samples at each time point from administration, compared to the concentration 5 minutes after administration. Surprisingly, the multiplier of increase in plasma lyso-SPM concentration in response to toxic doses (20 mg / kg) of rhASM was significantly higher than the multiplier of increase in plasma lyso-SPM concentration in response to non-toxic doses (3 mg / kg) of rhASM. This result suggests that lyso-SPM is a useful marker for measuring the toxic effects of therapeutic agents that reduce accumulated SPM levels in patients with ASM impairment. [Examples]

[0077] Comparison of single-dose and dose-reducing regimens Dried blood spots were collected from wild-type (C57BL / 6) or ASM knockout (ASMKO) mice after administration of a single dose or reduced dose regimen of rhASM. Five ASMKO mice were administered a single dose of 10 mg / kg of rhASM. Five C57BL / 6 mice and five ASMKO mice were treated with a reduced dose regimen of 3 mg / kg of 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 dose group were euthanized 24 hours later.

[0078] All 70 blood spot samples were prepared using a lipid multiplex extraction procedure and analyzed by LC / MS / MS. Briefly, a single DBS spot was perforated from each sample card and placed in individual Eppendorf tubes. Then, 200 microliters of an 80:15:5 solution (MeOH:ACN:H2O) was added to each tube, followed by vortexing for 30 minutes, sonication for 10 minutes, and centrifugation to precipitate into arbitrary microparticles. The concentration of lyso-SPM in each sample was determined using a lyso-SPM calibration curve (without internal standard).

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

[0080] Human trials Blood samples were collected from Niemann-Pick patients being treated with rhASM. A representative example is shown in Figure 4. Samples were collected before rhASM administration and 24, 48, and 72 hours after administration of specified doses (0.1, 0.3, 0.6, 1, 2, or 3 mg / kg) over a 26-week period (on day 1, and weeks 2, 4, 6, 8, 10, 12, 14, and 26). In week 26, samples were collected only before administration and 24 and 48 hours after administration. Lyso-SPM levels were measured in ng / ml. The data show an overall decreasing trend in lyso-SPM levels after repeated administration of high concentrations, although there were increases and decreases in lyso-SPM levels between pre-administration and 72 hours after each administration. Samples taken 24 hours after early administration may not have shown a sharp increase in post-administration lyso-SPM concentration up to that point. The administered dose may also have been below the concentration required to observe a significant surge in lyso-SPM levels after administration.

[0081] The embodiments described above are illustrative and not limiting. Other embodiments of the disclosed devices and methods will be apparent to those skilled in the art from the detailed description and practice of the devices and methods disclosed herein.

Claims

1. A method for determining the efficacy of recombinant human acid sphingomyelinase (rhASM) or modified rhASM doses in the treatment of acid sphingomyelinase deficiency (ASMD) in human subjects, wherein the method is The method involves examining the level of lyso-sphingomyelin (lyso-SPM) in a biological sample taken from a subject three days or more after administration of rhASM or a modified rhASM dose, wherein a decrease in the level of lyso-SPM compared to a reference level indicates the effectiveness of the rhASM or modified rhASM dose.

2. The method according to claim 1, comprising the step of determining efficacy by examining the level of lyso-sphingomyelin (lyso-SPM) in a biological sample taken after repeated administration of rhASM or modified rhASM to the subject.

3. The method according to claim 2, wherein the method comprises determining the level of lyso-SPM in a biological sample immediately before the repeated administration of rhASM or modified rhASM.

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

5. The method according to any one of claims 1 to 4, wherein the biological sample is whole blood, dried blood spot, plasma, or serum.

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

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

8. The method according to any one of claims 1 to 7, wherein the rhASM or modified rhASM dose, or the repeated dose of rhASM or modified rhASM, is a maintenance dose at a dose concentration less than or equal to the highest dose concentration administered to the subject.

9. The method according to any one of claims 2 to 8, wherein repeated administration of rhASM or modified rhASM is performed two weeks after the previous dose.

10. The method according to any one of claims 2 to 9, wherein one or more dose concentrations of rhASM or modified rhASM are administered at least twice.

11. The method according to any one of claims 1 to 10, wherein the dose is 0.03 mg / kg to 3 mg / kg.

12. The method according to any one of claims 1 to 11, wherein the first dose is 0.1 mg / kg.

13. The method according to any one of claims 1 to 12, wherein the maintenance dose is 1, 2, or 3 mg / kg.

14. The method according to any one of claims 1 to 13, wherein the administration is intravenous.

15. A method for determining the efficacy or toxicity of recombinant human acid sphingomyelinase (rhASM) or modified rhASM doses in the treatment of Niemann-Pick disease type A or B in human subjects, wherein the rhASM or modified rhASM is: (a) A first dose of rhASM or modified rhASM at 0.03 or 0.1 mg / kg, and subsequent doses in which the dose concentration increases until it reaches 3 mg / kg; (b) One or more maintenance doses of 3 mg / kg or less It is formulated to be delivered intravenously. Each dose is delivered two weeks after the previous dose. The aforementioned method, (c) From the subjects, measure the level of lyso-sphingomyelin (lyso-SPM) in the collected biological sample within (i) 24 hours, (ii) 48 hours, or (iii) 72 hours or more after dose administration. Including, here, An increase in the level of lyso-SPM measured in (i) or (ii) compared to the reference level indicates toxicity of rhASM or modified rhASM dose, and The method wherein a decrease in the level of lyso-SPM measured at (iii) compared to a reference level indicates the effectiveness of the rhASM or modified rhASM dose.

16. The method according to claim 15, wherein the maintenance dose is 1, 2, or 3 mg / kg.

17. The method according to claim 15 or 16, wherein determining efficacy or toxicity involves measuring the level of lyso-sphingomyelin (lyso-SPM) in a biological sample taken from a subject, wherein, prior to treatment, the subject has elevated levels of lyso-SPM compared to a healthy control.

18. The method according to any one of claims 15 to 17, wherein the biological sample is a dried blood spot.

19. The biological sample is tested before the subsequent dose, as described in any one of claims 15 to 18. The method.

20. The method according to any one of claims 1 to 19, wherein the subject is an adult patient.

21. The method according to any one of claims 1 to 19, wherein the subject is a pediatric patient.

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