Escalating dose enzyme replacement therapy for acid sphingomyelinase deficiency

Abstract

To provide a favorable enzyme replacement therapy regimen using acid sphingomyelinase (ASM) for treatment of human subjects having acid sphingomyelinase deficiency (ASMD), and specifically, non-neurological manifestations of a Niemann-Pick disease (NPD), and in certain embodiments, NPD type B.SOLUTION: Initially, a low non-toxic dose is administered and then the dose is escalated in subsequent administrations. The highest dose of ASM tolerated by the patient can then be used as a maintenance dose. Alternatively, a therapeutically effective dose less than the highest dose tolerated is used as the maintenance dose.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] This application claims priority to U.S. Provisional Application No. 61 / 238,113, filed Aug. 28, 2009, which is incorporated by reference in its entirety.

[0002] 1. Introduction The present invention relates to escalating dose enzyme replacement therapy using acid sphingomyelinase (ASM) for the treatment of human subjects with acid sphingomyelinase deficiency (ASMD), specifically non-neurological manifestations of Niemann-Pick disease (NPD), in certain embodiments NPD type B.

[0003] 2. Background technology Acid sphingomyelinase EC 3.1.4.12 (ASM) is a lysosomal phosphodiesterase enzyme that hydrolyzes sphingomyelin, a phospholipid storage material found in the brain, liver, lungs, spleen, and lymph nodes, into ceramide and phosphorylcholine. Deficiency of ASM activity results in the inability of the body to break down sphingomyelin, causing a lysosomal storage disease called Niemann-Pick disease.

[0004] Niemann-Pick disease is a genetic autosomal recessive lipid storage disorder characterized by the excessive accumulation of sphingomyelin in the lysosomes of cells such as macrophages and neurons, which impairs normal cellular function. Niemann-Pick disease type A is a rapidly progressive neurodegenerative disease of infants that typically leads to death by 2–3 years of age. Niemann-Pick disease type B results in liver and spleen enlargement and respiratory distress, and typically leads to death by early adulthood. These two types of Niemann-Pick disease are both associated with ASM deficiency and are collectively referred to herein as Niemann-Pick disease, or ASM deficiency syndrome (ASMD). Other types of Niemann-Pick disease, such as type C, do not involve mutations in the ASM gene and are not directly attributable to the function of ASM. The nature of the biochemical and molecular disorders underlying the remarkable clinical diversity of subtypes A and B remains unclear. Patients with both subtypes have residual ASM activity (approximately 1–10% of normal), but biochemical analysis cannot reliably distinguish between the two phenotypes. Furthermore, the clinical course of NPD type B is highly variable and it is currently not possible to demonstrate a correlation between disease severity and residual ASM activity.

[0005] NPD occurs more frequently in people of Ashkenazi Jewish descent than in the general population. The incidence of Niemann-Pick disease type A in Ashkenazi Jews is approximately 1 / 40,000, with a gene frequency (q) of approximately 1 / 200, and a heterozygote carrier frequency (2 pq) of 1 / 100 (Goodman, 1979, "Genetic Disorders Among The Jewish People", John Hopkins Univ. Press, Baltimore, pp. 96-100). The incidence of heterozygote carriers of NPD type B in the Ashkenazi Jewish population is less frequent (Goodman, supra). The combined heterozygote carrier frequency for NPD types A and B has been estimated to be approximately 1 / 70 in people of Ashkenazi Jewish descent. Although enzymatic diagnosis of patients with either NPD type A or B can be made with certainty (Spence and Callahan, supra), enzymatic detection of obligate heterozygotes has proven problematic, especially using peripheral leukocytes as the enzyme source. Perhaps the occurrence of neutral sphingomyelinase in some sources and / or the presence of residual ASM activity resulting from mutant alleles 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 unequivocal results for heterozygotes. Epidemiological studies performed in individual countries have estimated the combined incidence of Niemann-Pick disease types A and B in several countries worldwide to be between 1 / 167,000 and 1 / 250,000 newborns (Miekle et al., 1999 JAMA 281 (3):249-254; Poorthuis et al., 1999 Hum Genet 105:151-156; Pinto et al., 2004 Euro. J. Hum. Gene. 12:87-92). The proportion of heterozygous carriers is thought to be between 1 / 200 and 1 / 250.

[0006] Enzyme replacement therapy has also been used for other lysosomal storage diseases. It seeks to replace the activity of a deficient enzyme with an exogenously supplied enzyme. In the case of enzyme replacement therapy for Niemann-Pick disease, the goal is to enable the affected individual to process sphingomyelin and prevent its accumulation in the lysosomes. To be effective, such therapy requires the continuous administration of a sufficient amount of replacement enzyme to initially break down the accumulated sphingomyelin, as well as replacement enzyme to prevent further accumulation of sphingomyelin.

[0007] 3. Overview of the Invention The present invention relates to dose-escalating enzyme replacement therapy for the treatment of human subjects with ASMD, particularly those with non-neurological manifestations of NPD, in particular the NPDB form. More specifically, ASM enzyme is administered to such patients at low, non-toxic doses initially, and titrated up with subsequent doses. The highest dose of ASM tolerated by the patient can then be used as a maintenance dose. Alternatively, a therapeutically effective dose lower than the highest tolerated dose can be used as a maintenance dose.

[0008] The present invention is based, in part, on the discovery that doses of ASM required to clear accumulated sphingomyelin substrate in human subjects (i.e., ASMD or Niemann-Pick disease patients) result in adverse side effects (including clinical signs of toxicity), which is particularly surprising in NPD type B patients, a less severe form of ASMD, who, although deficient, have at least some enzyme activity.

[0009] More specifically, treatment of NPD requires administration of high enough doses to achieve proper distribution of ASM enzyme in affected organs (e.g., liver, spleen, lungs, heart, kidneys and brain, among others). Studies in ASM knockout mouse models (ASKMO mice) have shown that the majority of administered recombinant human ASM (rhASM) is distributed to the liver and spleen, reducing substrate, but significantly less to the lungs, heart and brain (Miranda et al. FASEB 2000, 14:1988; He et al., 1999, Biochimica et Biophsyica Acta 1432: 251-264 see also Fig. 9B). Subsequent studies using higher doses of rhASM in the ASMKO mouse model reduced substrate, and no toxicity was observed at doses of 3.0 mg / kg or less. Indeed, clinical signs of toxicity were not observed until doses of 10 mg / kg or more were used."Dose Responsive Toxicological Findings Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice." C. Nickerson, J. Murray, A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept Pharm / Tox, Pathology, Genzyme Corp., Framingham, MA., American Society of Human Genetics 2005; and "Elevations of Pro-Inflammatory Cytokines and Decreases in Cardiovascular Hemodynamics Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice." J. Murray, A.M. D’ Angona, C. Nickerson, A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept. Pharmacology / Toxicology & Pathology, Genzyme Corp., Framingham, MA., Society of Toxicology 2006. See

[0010] Based on these ASKMO data, human subjects with non-neurogenic ASMD were treated with a modest maximum dose of 1.0 mg / kg rhASM, as described in Section 6 (below). Quite unexpectedly, toxicity in human subjects, including the development of clinically relevant adverse events, was observed at doses as low as 0.3 mg / kg. This result was particularly surprising because the ASM enzyme is not present in the knockout mouse model, which would have at least some enzymatic activity and thus reflect a more severe condition than human subjects with relatively mild disease.

[0011] Without intending to be bound by any theory, adverse side effects occurring with ASM treatment may be due to the breakdown of sphingomyelin substrate stored in ASMD patients and the release of the product ceramide, which induces proapoptotic and proinflammatory cytokine responses and hyperbilirubinemia. To address this issue, we have developed a regimen that allows the safe administration of high doses of ASM enzyme required to achieve proper distribution in diseased organs. According to this regimen, a very low dose of ASM is used for initial treatment to slowly break down the stored substrate, with fewer associated side effects. Since the substrate is depleted in the subject (the stored substrate is "depleted"), the dose can be safely titrated up.

[0012] According to this protocol, a non-toxic, low dose of ASM enzyme is initially administered to a patient with NPD disease, and the dose is gradually titrated up. As the dose of ASM enzyme is titrated up, the patient's total bilirubin concentration, acute phase reactant production, inflammatory mediator production, and related adverse events can be monitored. The administration of low doses of ASM and the titrated doses facilitate the weight loss of accumulated sphingomyelin. Once the patient is weighted down, a higher dose can be safely administered to the patient to ensure proper distribution of ASM enzyme to target organs (e.g., liver, spleen, lungs, heart, kidneys, brain, bone marrow, skeleton, joints, etc.). In certain embodiments, the highest dose tolerated by the patient can be used as the maintenance dose. In some embodiments, the maintenance dose can be increased or decreased over time depending on the patient's condition.

[0013] In certain embodiments, to ensure a stable response before increasing the dose to the next level, the patient's treatment is monitored by measuring plasma sphingomyelin concentration, plasma ceramide concentration, production of "acute phase reactants" and inflammatory mediators that are measures of inflammatory response, bilirubin concentration (total, direct, indirect), and / or other biochemical markers. These markers include, but are not limited to, C-reactive protein (CRP) or high-sensitivity CRP (hs-CRP), cytokines (e.g., IL-8, IL-6), calcitonin, and ferritin. In specific embodiments, the patient may be monitored for one or more associated adverse events, including, but not limited to, systemic symptoms (e.g., fever, nausea, vomiting, pain, myalgia) and jaundice.

[0014] A dose of less than 1 mg / kg is preferred for initiating treatment. The initial dose is gradually increased until a therapeutic dose is reached. Such dose escalation can be used to determine the highest tolerated dose. For example, once the patient's stored sphingomyelinase substrate has been reduced, the dose can be further increased until toxicity is observed. The maintenance dose can be adjusted accordingly and can be continually and periodically re-adjusted depending on the patient's condition.

[0015] In a specific embodiment, a method of treating a human subject with acid sphingomyelinase deficiency comprises: (a) a regimen to reduce accumulated sphingomyelin substrate in the human subject comprising (i) administering to the human subject a non-toxic, low initial dose of ASM; (ii) administering successively increasing doses of ASM and monitoring the subject after each successive administration for one or more adverse side effects as indicated by elevated bilirubin or related adverse events; and (b) a maintenance regimen comprising administering to the subject a maintenance dose up to the highest dose tolerated by the subject.

[0016] In another specific embodiment, a method of treating a human subject with acid sphingomyelinase deficiency comprises administering rhASM in an escalating dose regimen at successive doses of 0.1 mg / kg, 0.3 mg / kg, 0.6 mg / kg, and 1.0 mg / kg, where each dose of rhASM is administered at least twice, where each dose is administered every two weeks, and where the patient is monitored for toxic side effects before increasing the dose to the next level.

[0017] In another specific embodiment, described herein is an acid sphingomyelinase (ASM) for treating acid sphingomyelinase deficiency in a human subject, the ASM being formulated to be administered in a regimen to reduce stored sphingomyelin substrate comprising: (i) administering a non-toxic, low initial dose of ASM; (ii) administering successively increasing doses of ASM and monitoring the subject after each successive administration for one or more adverse side effects as indicated by elevated bilirubin or related adverse events; and (b) a maintenance regimen comprising administering to the subject a maintenance dose up to the highest dose tolerated by the subject.

[0018] In another specific embodiment, described herein is a recombinant human ASM for treating acid sphingomyelinase deficiency in a human subject, formulated for sequential administration in an escalating dose regimen of 0.1 mg / kg, 0.3 mg / kg, 0.6 mg / kg, and 1.0 mg / kg, wherein each dose is administered at least twice, each dose is administered every two weeks, and the subject is monitored for toxic side effects before increasing the dose to the next level.

[0019] Terminology As used herein, the terms "about" and "approximately" are used interchangeably in the context of any value to refer to a range around any value, where the resulting value is substantially the same as the explicitly recited value. In specific embodiments, "about" means within 10%, 15%, 25% of any value or range.

[0020] As used herein, the term "elderly" refers to humans aged 65 and older.

[0021] As used herein, the term "adult" refers to humans aged 18 years or older.

[0022] As used herein, the term "child" refers to a human between the ages of 1 and 18.

[0023] As used herein, the term "infant" refers to a human from newborn to 1 year of age.

[0024] As used herein, the term "infant" refers to a human between the ages of 1 and 3.

[0025] As used herein, the term "adverse event" refers to "any untoward medical manifestation in a patient or clinical trial subject administered a medicinal product," as defined in the Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology, version 3.1.1. A "related adverse event" is an adverse event that has a causal relationship to treatment.

[0026] As used herein, "maintenance dose" and similar terms refer to a dose administered to ASMD patients to maintain a desired therapeutic effect.In a specific embodiment, the maintenance dose is determined by (i) a reduction in spleen volume, as assessed by techniques known in the art, such as MRI; (ii) a reduction in liver sphingomyelin levels, as assessed by techniques known in the art, such as biochemical and / or histomorphometric analysis of liver samples; (iii) an increase in exercise capacity, as assessed by techniques known in the art, such as maximal workload by cycle ergometer exercise testing, including percent predicted maximal workload, peak oxygen consumption and carbon dioxide production; (iv) a reduction in lung capacity, as assessed by techniques known in the art, such as pulmonary diffusion capacity (DLco), percent predicted forced vital capacity (FVC), as measured by spirometry techniques, forced expiratory volume in 1 second (FEV1), as measured by spirometry techniques, and total lung capacity, as measured by American Thoracic Society, 1991, Am. Rev. Respir. Dis.144: 1202-1218, (v) a reduction in bronchoalveolar lavage (BAL) sphingomyelin, (vi) a reduction in liver volume as assessed by techniques known in the art, e.g., MRI, (vii) an improvement in lung appearance as assessed by techniques known in the art, e.g., high resolution computed tomography (CT) scan or chest x-ray, (viii) a reduction in liver, skin, plasma and dried blood spot (DBS) sphingomyelin concentrations as measured, e.g., by tandem mass spectrometry, (ix) a reduction or amelioration in the severity of ASMD and / or symptoms associated therewith, (x) a reduction in the duration of symptoms associated with ASMD, (xi) prevention of recurrence of symptoms associated with ASMD, (xii) a reduction in hospitalization of the subject, (vi) a reduction in the duration of hospitalization. (xiii) increased subject survival, (xiv) decreased mortality, (xv) decreased hospitalization rates, (xvi) decreased number of symptoms associated with ASMD, (xvii) increased symptom-free survival in ASMD patients, (xviii) improved neurological function (e.g., psychomotor function, social responsiveness, etc.), (xix) improved lung clearance as measured, for example, by BAL cell count and profile, (xx) decreased serum levels of chitotriosidase, (xxi) decreased serum levels of chemokine (cc) motif ligand 18 (CCL18), (xxii) improved lipid profile (e.g., HDL, LDL, cholesterol, triglycerides, and total cholesterol:HDL ratio), and (xxiii) improved quality of life as measured, for example, by a questionnaire. In certain embodiments, the maintenance dose is the highest or maximum dose tolerated by the patient.

[0027] In some embodiments, the maintenance dose is a dose between 0.5 mg / kg to 1.5 mg / kg, 0.75 mg / kg to 1.25 mg / kg, 1 mg / kg to 2.5 mg / kg, 1 mg / kg to 2.75 mg / kg, 1.5 mg / kg to 2.5 mg / kg, 1.5 mg / kg to 2.75 mg / kg, 2 mg / kg to 2.5 mg / kg, 2 mg / kg to 2.75 mg / kg, 2.5 mg / kg to 2.75 mg / kg, 2.5 mg / kg to 3 mg / kg, 3 mg / kg to 4 mg / kg, 3 mg / kg to 5 mg / kg, 4 mg / kg to 5 mg / kg, 2 mg / kg to 5 mg / kg, or 5 mg / kg to 10 mg / kg of ASM. In certain embodiments, the maintenance dose is between 5 mg / kg to 15 mg / kg, 10 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 20 mg / kg, 20 mg / kg to 30 mg / kg, 25 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg, 30 mg / kg to 45 mg / kg, or 40 mg / kg to 50 mg / kg of ASM. In some embodiments, the maintenance dose is 0.75 mg / kg, 0.80 mg / kg, 0.85 mg / kg, 0.90 mg / kg, 0.95 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.75 mg / kg, or 2 mg / kg of ASM. In certain embodiments, the maintenance dose is 2.5 mg / kg, 2.75 mg / kg, 3 mg / kg, 3.25 mg / kg, 3.5 mg / kg, 3.75 mg / kg, 4 mg / kg, 4.25 mg / kg, 4.5 mg / kg, 4.75 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, or 10 mg / kg of ASM. In some embodiments, the maintenance dose is 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg of ASM.In some embodiments, when the maximum dose is 10 mg / kg of ASM, the maintenance dose is at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, or at least 8 mg / kg of ASM. In certain embodiments, when the maximum dose is 50 mg / kg, the maintenance dose is at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, or at least 35 mg / kg of ASM.

[0028] As used herein, a "non-toxic dose" and similar terms refer to a dose that does not result in (i) any moderate or severe related adverse events, defined as clinical symptoms that interfere with normal daily function and require additional monitoring, intervention, or treatment, or abnormal laboratory values ​​or treatment findings of clinical concern that require further monitoring, treatment, or intervention (e.g., as described in the Clinical Data Interchange Standards Consortium Study Data Tabulation 2010). (ii) 1.5 mg / dL, 1.75 mg / dL, 2.0 mg / dL, 2.1 mg / dL, 2.2 mg / dL, 2.3 mg / dL, 2.4 mg / dL, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.75 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 3.1 mg / dL, 3.2 ... / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, or 4 mg / dL, or in the range of 2.1 mg / dL to 2.5 mg / dL, 2.1 mg / dL to 3.0 mg / dL, or 2.1 mg / dL to 4 mg / dL; (iii) a total bilirubin level of 8.2 μg / mL, 8.3 μg / mL, 8.5 μg / mL, 8.6 μg / mL, 8.7 μg / mL, 8.8 μg / mL, 8.9 μg / mL, 8.1 μg / mL, 8.2 μg / mL, 8.3 μg / mL, 8.4 μg / mL, 8.5 μg / mL, 8.6 μg / mL, 8.7 μg / mL, 8.8 μg / mL, 8.9 ...9 μg / mL, 8.1 μg / mL, 8.2 μg / mL, 8.3 μg / mL, 8.3 μg / mL, 8.3 μg / mL, 8.3 μg / 4μg / mL, 8.5μg / mL, 8.75μg / mL, 9μg / mL, 9.5μg / mL, 10μg / mL, 11μg / mL, 12μg / mL, 13μg / mL, 14μg / mL, 15μg / mL, 16μg / mL, 17μg / mL, 18μg / mL, 19μg / mL, 20μg / m L, 25μg / mL, 30μg / mL, 35μg / mL, 40μg / mL, 45μg / mL, 50μg / mL, 55μg / mL, 60μg / mL, 65μg / mL, 70μg / mL, 75μg / mL, or greater than 80μg / mL, or 8.2μg / mL~10μg / mL, 8.or (iv) a plasma ceramide concentration in the range of 5 μg / mL to 10 μg / mdL, 9 μg / mL to 12 μg / mL, 10 μg / mL to 12 μg / mL, 10 μg / mL to 15 μg / mL, 10 μg / mL to 20 μg / mL, 15 μg / mL to 20 μg / mL, or 20 μg / mL to 30 μg / mL. A "non-toxic dose" of ASM may vary, for example, depending on the stability of the enzyme used, the activity of the enzyme used, and / or the route of administration of the enzyme. For example, the dose of a modified ASM enzyme with increased activity may be lower than the dose of unmodified ASM. One of skill in the art can adjust the dose of the enzyme administered based on the stability of the enzyme, the activity of the enzyme, and / or the route of administration of the enzyme.

[0029] An acute phase response is an early response (generally, for example, within 12-72 hours) indicative of an inflammatory response following administration of ASM. The acute phase response can be assessed by changes in concentrations of acute phase reactants (e.g., CRP / hs-CRP, ferritin, fibrinogen, iron, or transferrin), changes in percent neutrophils, changes in prothrombin time, or changes in partial thromboplastin time. In specific embodiments, an increase in CRP / hs-CRP concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's CRP / hs-CRP concentration prior to ASM administration can serve as a measure of the acute phase response. In another specific embodiment, a plasma CRP / hs-CRP concentration that is greater than the normal plasma CRP / hs-CRP concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma CRP / hs-CRP concentration of greater than about 8.1 mg / L, 8.2 mg / L, 8.3 mg / L, 8.4 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L, 10 mg / L, 11 mg / L, or 12 mg / L, or in the range of 8.5 mg / L to 10 mg / L, or 8.5 mg / dL to 12 mg / L, or 10 mg / L to 12 mg / L, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0030] In a specific embodiment, an increase in ferritin 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's ferritin level before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma ferritin concentration greater than normal plasma ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, the ASM concentration is about 300ng / mL, 325ng / mL, 350ng / mL, 375ng / mL, 400ng / mL, 425ng / mL, 450ng / mL, 475ng / mL, 500ng / mL, 525ng / mL, 550ng / mL, 575ng / mL, 600ng / mL, 625ng / mL, 650ng / mL, 675ng / mL, 700ng / mL, 725ng / mL, 750ng / mL, 775ng / mL, 800ng / mL, Plasma ferritin concentrations greater than 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, 1050ng / mL, 1100ng / mL, 1150ng / mL, or 1200ng / mL or in the ranges of 600ng / mL to 800ng / mL, 650ng / mL to 850ng / mL, 600ng / mL to 1000ng / mL, 600ng / mL to 1200ng / mL, 800ng / mL to 1000ng / mL, 900ng / mL to 1000ng / mL, or 1000ng / mL to 1200ng / mL can be used as a measure of the acute phase response.

[0031] In a specific embodiment, an increase in plasma or serum IL-8 concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's IL-8 concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum IL-8 concentration that exceeds normal plasma IL-8 concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma IL-8 concentration of greater than about 24 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, or 900 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0032] In a specific embodiment, an increase in plasma or serum IL-6 concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's IL-6 concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum IL-6 concentration that is greater than normal plasma or serum IL-6 concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, plasma IL-6 concentrations of greater than about 4.4 pg / mL, 6 pg / mL, 8 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, or 30 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0033] In a specific embodiment, an increase in plasma or serum calcitonin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's calcitonin concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum calcitonin concentration that exceeds the normal plasma calcitonin concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma calcitonin concentration of greater than about 9.4 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, or 250 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0034] In a specific embodiment, an increase in fibrinogen 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's fibrinogen concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma fibrinogen concentration that exceeds normal plasma fibrinogen concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma fibrinogen concentration greater than about 350 mg / L, 375 mg / L, 400 mg / L, 425 mg / L, or 450 mg / L, or in the range of 350 mg / dL to 400 mg / dL, 350 mg / dL to 450 mg / dL, or 400 mg / dL to 450 mg / dL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0035] In one embodiment, an increase in the percent neutrophils of total white blood cells 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's percent neutrophils of total white blood cells prior to administration of ASM can be used as a measure of the acute phase response. In another embodiment, an increase in the percent neutrophils of total white blood cells over the normal percent neutrophils of total white blood cell concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, an increase in percent normal neutrophils in total white blood cell concentration of 70%, 75%, 80%, 85%, 90%, 95% or greater 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0036] As used herein, a "non-toxic low dose" and similar terms, in the context of an initial dose or dose administered to a subject, refers to an initial dose or dose of ASM administered to a subject to treat ASMD that is a non-toxic dosage. In certain embodiments, a non-toxic low dose is between 0.001 mg / kg and 0.01 mg / kg, between 0.001 mg / kg and 0.01 mg / kg, between 0.001 mg / kg and 0.05 mg / kg, between 0.001 mg / kg and 0.1 mg / kg, between 0.001 mg / kg and 0.5 mg / kg, between 0.05 mg / kg and 0.275 mg / kg, between 0.075 mg / kg and 0.275 mg / kg, between 0.05 mg / kg and 0.2 mg / kg, between 0.07 ... Doses of 0.1mg / kg to 0.275mg / kg, 0.1mg / kg to 0.25mg / kg, 0.1mg / kg to 1mg / kg, 0.5mg / kg to 1mg / kg, 0.75mg / kg to 1mg / kg, 0.1mg / kg to 2mg / kg, 0.5mg / kg to 2mg / kg, 0.75mg / kg to 2mg / kg, 1mg / kg to 2mg / kg or 1.25mg / kg to 2mg / kg, 1.5mg / kg to 2mg / kg or 1.75mg / kg to 2mg / kg. In some specific embodiments, the non-toxic low dose is a dose of 0.001 mg / kg, 0.005 mg / kg, 0.0075 mg / kg, 0.01 mg / kg, 0.0125 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.075 mg / kg, 0.1 mg / kg, 0.125 mg / kg, 0.15 mg / kg, 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg of ASM.

[0037] The terms "subject" and "patient" are used interchangeably herein to refer to a human. In a specific embodiment, the human has been diagnosed with ASMD.

[0038] As used herein, the term "therapeutically effective" in the context of administering a dose of ASM refers to an amount of ASM that provides a benefit or therapeutic effect. In specific embodiments, the term "therapeutically effective" in the context of administering a dose of ASM refers to an amount of ASM sufficient to achieve at least one, two, three, four or more of the following effects: (i) a reduction in spleen volume as assessed by techniques well known in the art, e.g., MRI; (ii) a reduction in liver sphingomyelin levels as assessed by techniques well known in the art, e.g., biochemical and / or histomorphometric analysis of liver samples; (iii) an increase in exercise capacity as assessed by techniques well known in the art, e.g., maximal workload by cycle ergometer exercise testing, including percent predicted maximal workload, peak oxygen consumption and carbon dioxide production; (iv) an increase in lung capacity as assessed by techniques well known in the art, e.g., pulmonary diffusion capacity (DLco), percent predicted forced vital capacity (FVC) as measured by, e.g., spirometry techniques, forced expiratory volume in one second (FEV1), as measured by, e.g., spirometry techniques, and total lung capacity, as measured by, e.g., American Thoracic Society, 1991, Am. Rev. Respir. Dis.144: 1202-1218, (v) a reduction in bronchoalveolar lavage (BAL) sphingomyelin, (vi) a reduction in liver volume as assessed by techniques known in the art, e.g., MRI, (vii) an improvement in lung appearance as assessed by techniques known in the art, e.g., high resolution CT scan or chest x-ray, (viii) a reduction in liver, skin, plasma and dried blood spot (DBS) sphingomyelin concentrations as measured, e.g., by tandem mass spectrometry, (ix) a reduction or amelioration in the severity of ASMD and / or symptoms associated therewith, (x) a reduction in the duration of symptoms associated with ASMD, (xi) prevention of recurrence of symptoms associated with ASMD, (xii) a reduction in hospitalization of the subject. (vi) reduced length of hospital stay, (xiii) increased survival of subjects, (xiv) reduced mortality, (xv) reduced hospitalization rates, (xvi) reduced number of symptoms associated with ASMD, (xvii) increased symptom-free survival in ASMD patients, (xviii) improved neurological function (e.g., psychomotor function, social responsiveness, etc.), (xix) improved lung clearance as measured, for example, by BAL cell count and profile, (xx) reduced serum levels of chitotriosidase, (xxi) reduced serum levels of chemokine (cc) motif ligand 18 (CCL18), (xxii) improved lipid profile (e.g., HDL, LDL, cholesterol, triglycerides, and total cholesterol:HDL ratio), and (xxiii) improved quality of life (QOL) as measured, for example, by questionnaires.

[0039] As used herein, the term "therapy" refers to any protocol, method, composition, formulation, and / or agent that can be used to treat, manage, or ameliorate ASMD or a condition or symptom related thereto. In certain embodiments, the term "therapy" refers to biological, adjunctive, and / or other therapies useful in treating, managing, preventing, or ameliorating ASMD or a condition or symptom related thereto. In embodiments, the term "therapy" refers to one or more of the following therapies: (i) enhancing the delivery of ASM to the lesion site; (ii) enhancing the activity of ASM; and (iii) enhancing the stability of ASM. In certain embodiments, the term "therapy" refers to a therapy other than ASM. In specific embodiments, "additional therapy" refers to a therapy other than ASM.

[0040] As used herein, "toxic effect" and similar terms refer to a related adverse event of moderate or severe severity following administration of a dose of ASM, defined as (i) clinical symptoms that interfere with normal daily functioning and require additional monitoring, intervention, or treatment, or abnormal laboratory values ​​or treatment findings of clinical concern that require further monitoring, treatment, or intervention.See, e.g., the Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology Version 3.1.1., (ii) 1.5 mg / dL, 1.75 mg / dL, 2.0 mg / dL, 2.1 mg / dL, 2.2 mg / dL, 2.3 mg / dL, 2.4 mg / dL, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.75 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 3.10 mg / dL, 3.25 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 3.10 mg / dL, 3.11 mg / dL, 3.12 mg / dL, 3.13 mg / dL, 3.14 mg / dL, 3.15 mg / dL, 3.16 mg / dL, 3.17 mg / dL, 3.18 mg / dL, 3.19 mg / dL, 3.26 mg / dL, 3.27 mg / dL, 3.28 mg / dL, 3.29 mg / dL, 3.30 mg / dL, 3.31 mg / dL, 3.32 mg / dL, 3.33 mg / dL, total bilirubin levels greater than 9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, or 4 mg / dL, or in the ranges of 2.1 mg / dL to 2.5 mg / dL, 2.1 mg / dL to 3.0 mg / dL, or 2.1 mg / dL to 4 mg / dL, (iii) 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, or 24 hours after administration of a dose of ASM; , 8.2 μg / mL, 8.3 μg / mL, 8.4 μg / mL, 8.5 μg / mL, 8.75 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, at 36, 48, or 72 hours. 14μg / mL, 15μg / mL, 16μg / mL, 17μg / mL, 18μg / mL, 19μg / mL, 20μg / mL, 25μg / mL, 30μg / mL, 35μg / mL, 40μg / mL, 45μg / mL, 50μg / mL, 55μg / mL, 60 or (iv) one, two, three, or all of the following: a plasma ceramide concentration greater than 65 μg / mL, 70 μg / mL, 75 μg / mL, or 80 μg / mL, or in the range of 8.2 μg / mL to 10 μg / mL, 8.5 μg / mL to 10 μg / mL, 9 μg / mL to 12 μg / mL, 10 μg / mL to 15 μg / mL, 10 μg / mL to 20 μg / mL, 15 μg / mL to 20 μg / mL, or 20 μg / mL to 30 μg / mL; or

[0041] The acute phase response can be assessed by changes in the concentration of acute phase reactants (e.g., C-reactive protein, ferritin, albumin, IL-8, IL-6, calcitonin, fibrinogen, iron, or transferrin), changes in the percentage of neutrophils, changes in prothrombin time, or changes in partial thromboplastin time. In a specific embodiment, an increase in CRP / hs-CRP concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's CRP / hs-CRP concentration before ASM administration can be used as a measure of the acute phase response. In another specific embodiment, a plasma CRP / hs-CRP concentration that exceeds normal plasma CRP / hs-CRP concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma CRP / hs-CRP concentration of greater than about 8.1 mg / L, 8.2 mg / L, 8.3 mg / L, 8.4 mg / L, 8.5 mg / L, 8.6 mg / L, 8.7 mg / L, 8.8 mg / L, 8.9 mg / L, 9 mg / L, 9.5 mg / L, 10 mg / L, 11 mg / L, or 12 mg / L, or in the range of 8.5 mg / L to 10 mg / L, or 8.5 mg / L to 12 mg / L, or 10 mg / L to 12 mg / L, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0042] In a specific embodiment, an increase in ferritin 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's ferritin level before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma ferritin concentration greater than normal plasma ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, the ASM concentration is about 600ng / mL, 625ng / mL, 650ng / mL, 675ng / mL, 700ng / mL, 725ng / mL, 750ng / mL, 775ng / mL, 800ng / mL, 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, 1050ng / mL, 1100ng / mL, or ... Plasma ferritin concentrations greater than 100ng / mL, 1150ng / mL, or 1200ng / mL or in the ranges of 600ng / mL to 800ng / mL, 650ng / mL to 850ng / mL, 600ng / mL to 1000ng / mL, 600ng / mL to 1200ng / mL, 800ng / mL to 1000ng / mL, 900ng / mL to 1000ng / mL, or 1000ng / mL to 1200ng / mL can be used as a measure of the acute phase response.

[0043] In a specific embodiment, an increase in fibrinogen 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's fibrinogen concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma fibrinogen concentration that exceeds normal plasma fibrinogen concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma fibrinogen concentration greater than about 350 mg / L, 375 mg / L, 400 mg / L, 425 mg / L, or 450 mg / L, or in the range of 350 mg / dL to 400 mg / dL, 350 mg / dL to 450 mg / dL, or 400 mg / dL to 450 mg / dL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0044] In specific embodiments, a decrease in albumin concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's albumin concentration before administration of ASM can be used as a measure of the acute phase response. In certain embodiments, a decrease in albumin concentration from the normal range of 3.5-5.0 g / dL to concentrations of 0.2, 0.4, 0.6, 1, 1.5, or 2.0 g / dL 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0045] In specific embodiments, a decrease in ferritin concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's ferritin concentration before ASM administration can be used as a measure of the acute phase response. In certain embodiments, a decrease in ferritin concentration from the normal range of 60-170 mcg / dL, such as concentrations of 20, 40, 60, 80, 100, 120, 140, or 160 mcg / dL 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0046] In specific embodiments, a decrease in transferrin concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's transferrin concentration before administration of ASM can be used as a measure of the acute phase response. In certain embodiments, a decrease in ferritin concentration from the normal range of 202-336 mg / dL to concentrations of 20, 40, 60, 80, 100, 120, 140, 160, 180 mg / dL 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0047] In one embodiment, an increase in the percent neutrophils of total white blood cells 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's percent neutrophils of total white blood cells prior to administration of ASM can be used as a measure of the acute phase response. In another embodiment, an increase in the percent neutrophils of total white blood cells over the normal percent neutrophils of total white blood cell concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, an increase in percent normal neutrophils in total white blood cell concentration of 70%, 75%, 80%, 85%, 90%, 95% or greater 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0048] 4. Brief description of the drawings Figure 1 is a diagrammatic representation of the protocol design.

[0049] FIG. 2 is a table showing the demographic and baseline characteristics of patients enrolled in the protocol described below.

[0050] 3A, 3B, and 3C are graphs showing plasma levels of ceramide (FIG. 3A), plasma levels of sphingomyelin (FIG. 3B), and dried blood spot levels of sphingomyelin (FIG. 3C) over time in different patients receiving different doses of rhASM. The right axis of the graphs shows patient number and dose of rhASM.

[0051] 4 is a graph showing total bilirubin levels measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and dose of rhASM.

[0052] Figures 5A-5G are graphs showing the levels of CRP / hs-CRP (Figure 5A), percent neutrophils (Figure 5B), fibrinogen (Figure 5C), ferritin (Figure 5D), IL-8 (Figure 5E), IL-6 (Figure 5F), and calcitonin (Figure 5G) measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graphs shows the patient number and the dose of rhASM administered.

[0053] FIG. 6 is a table of adverse events that occurred during treatment in four patients, each of whom received a different rhASM dose, and which were considered to be (probably, probably, or definitely) related to treatment.

[0054] FIG. 7 is a diagram showing the initial treatment period, consisting of the dose escalation and dose maintenance phases, of the Phase 2 rhASM repeat dose protocol for patients with ASMD.

[0055] 5. Detailed Description of the Invention The present invention relates to a dose-escalating enzyme replacement therapy for the treatment of human subjects with ASMD, particularly those with non-neurological manifestations of NPD, in particular the NPDB form. More specifically, the enzyme ASM is administered to such patients at low, non-toxic doses initially, and titrated up with subsequent doses. The highest dose of ASM tolerated by the patient can be used as a maintenance dose. Alternatively, a therapeutically effective dose lower than the highest tolerated dose can be used as a maintenance dose.

[0056] The treatment of NPD requires a sufficient dose to achieve proper distribution of ASM enzyme in affected organs (e.g., spleen, lung, heart, kidney and brain). After intravenous administration of recombinant human ASM to ASMKO mice, most of the ASM activity is distributed to the liver, and only a small amount of ASM enzyme activity is detected in other affected organs, such as the spleen, heart, kidney and lung (see, e.g., Figure 9B in He et al., 1999, Biochimia et Biophsyica Acta 1432: 251-264). Therefore, a very high dose is required to ensure distribution and delivery of the administered enzyme to the lungs, heart and kidneys of patients suffering from ASMD or Niemann-Pick disease.

[0057] Studies in the ASM knockout mouse model (ASKMO mice) showed that the majority of administered rhASM was distributed to the liver and spleen, reducing substrate, with significantly less distribution to the lungs, heart, and brain (Miranda et al. FASEB 2000, 14:1988). Subsequent studies using higher doses of rhASM in the ASMKO mouse model reduced substrate, and no toxicity was observed at doses below 3.0 mg / kg. In fact, clinical signs of toxicity were not observed until doses of 10 mg / kg or higher were used."Dose Responsive Toxicological Findings Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice" C. Nickerson, J. Murray, A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept Pharm / Tox, Pathology, Genzyme Corp., Framingham, MA., American Society of Human Genetics 2005; and "Elevations of Pro-Inflammatory Cytokines and Decreases in Cardiovascular Hemodynamics Following Intravenous Administration of Recombinant Human Acid Sphingomyelinase (rhASM) to Acid Sphingomyelinase Knock-out (ASMKO) Mice" J. Murray, A.M. D’Angona, C. Nickerson, A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept. Pharmacology / Toxicology & Pathology, Genzyme Corp., Framingham, MA., Society of Toxicology 2006. See

[0058] Based on these ASKMO data, human subjects with non-neurogenic ASMD were treated at a modest maximum dose of 1.0 mg / kg, as described in Section 6 (below). Quite unexpectedly, toxicity in human subjects, including the development of clinically symptomatic adverse events, was observed at doses as low as 0.3 mg / kg. This result was particularly surprising because the ASM enzyme is not present in the knockout mouse model, which would have at least some enzymatic activity and thus reflect a more severe condition than human subjects with relatively mild disease.

[0059] Without wishing to be bound by any theory, it is possible that administration of high doses of ASM to NPD patients may result in hydrolysis of large amounts of sphingomyelin resulting in high concentrations of ceramide, which may lead to the adverse side effects observed in these NPD patients. The ASM enzyme hydrolyzes sphingomyelin, a major component of the cell membrane (see, e.g., Milaus et al., 2010 FEBS Letters 584: 1887-1894), into ceramide and phosphocholine. Ceramide is known to play a role in cell death and is known to be a pro-apoptotic agent (see, e.g., Smith and Schuchman, 2008, FASEB 22: 3419-3431).

[0060] Furthermore, unlike other lysosomal enzymes associated with characterized lysosomal storage diseases, ASM hydrolyzes sphingomyelin at the neutral pH found in plasma and the acidic pH found in lysosomes (see, e.g., Schissel et al., 1998. J. Biol. Chem. 273: 2738-2746). The ability of the ASM enzyme to function in plasma results in hydrolysis of sphingomyelin found in lipoproteins and cell membranes, increasing the amount of the degradation product ceramide, which may result in the adverse side effects observed in NPD patients receiving high doses of ASM enzyme.

[0061] To solve the problem of achieving proper distribution of ASM enzyme to affected organs while avoiding or minimizing the toxicity associated with high doses of enzyme, the inventors have developed the regimen described herein, in which a non-toxic low dose of ASM enzyme is initially administered to NPD patients and the dose is titrated up over time. As the dose of ASM enzyme is titrated up, the patient can be monitored for total / direct / indirect bilirubin levels, production of acute phase reactants, inflammatory mediators, and associated adverse events. The administration of low doses of ASM and titration of the enzyme promotes debulking of accumulated sphingomyelin. Once the patient is debulked, higher doses can be safely administered to the patient to ensure proper distribution of ASM enzyme to target organs (e.g., liver, spleen, lungs, heart, kidneys, brain, bone marrow, skeleton, joints, etc.). In certain embodiments, the highest dose tolerated by the patient can be used as the maintenance dose. Alternatively, a therapeutically effective dose lower than the highest dose tolerated can be used as the maintenance dose. In some embodiments, the maintenance dose can be increased or decreased depending on the patient's condition.

[0062] In certain embodiments, to ensure a stable response before increasing the dose to the next level, the patient's treatment can be monitored by measuring plasma sphingomyelin concentration, plasma ceramide concentration, production of "acute phase reactants" and inflammatory mediators that are measures of inflammatory response, bilirubin concentration (total, direct, or indirect), and / or other biochemical markers. These markers include, but are not limited to, CRP / hs-CRP, cytokines (e.g., 1L-8, 1L-6), calcitonin, ferritin. In specific embodiments, the patient may be monitored for one or more associated adverse events, including, but not limited to, systemic symptoms (e.g., fever, nausea, vomiting, pain, myalgia, and jaundice).

[0063] 5.1. Dose Escalation Protocol A method of treating ASMD is described, which includes administering one or more initial low, non-toxic doses of ASM to the patient to reduce the amount of sphingomyelin accumulated in the subject. After a period of time, the dose of ASM can be gradually increased until a maximum dose that is therapeutically effective and tolerated by the patient is reached. Once this dose is identified, it can be used as a maintenance dose to treat the subject. The maintenance dose can be administered weekly, biweekly, or monthly to the subject to treat ASMD. In some embodiments, the subject receiving the maintenance dose is monitored every three months, every six months, or every year for one, two, three, or all of the following: (i) related adverse events, (ii) total / direct / indirect bilirubin concentrations, (iii) plasma ceramide concentrations, or (iv) acute phase responses. If the subject experiences an associated adverse event of moderate intensity (e.g., an associated moderate adverse event), a total bilirubin concentration that exceeds the total bilirubin value of a human without ASMD (e.g., a healthy individual), a plasma ceramide concentration that exceeds the plasma ceramide concentration of a human without ASMD (e.g., a healthy individual), or an acute phase reaction, the subject's dosage can be evaluated by a physician or other medical professional to determine whether the dose needs to be adjusted.

[0064] In one embodiment, a method of treating a human subject with acid sphingomyelinase deficiency comprises (a) a regimen for reducing the accumulated sphingomyelin substrate in the human subject, comprising (i) administering to the human subject a non-toxic, low initial dose of ASM, (ii) administering successively increasing doses of ASM and monitoring the subject after each successive dose for one or more adverse side effects as indicated by elevated bilirubin or related adverse events, and (b) a maintenance regimen comprising administering a maintenance dose to the subject up to the highest dose tolerated by the subject. In certain embodiments, the initial dose of ASM ranges from 0.1 mg / kg to 0.5 mg / kg or 0.1 mg / kg to 1 mg / kg. In some embodiments, the successively increasing doses are administered 1 week, 2 weeks, 3 weeks, or 4 weeks after the previous dose. In certain embodiments, the successively escalating dose is about 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 higher than the previous dose. In some embodiments, the successively escalating dose is 0.1-0.5 mg / kg, 0.1 mg / kg-1 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 higher than the previous dose. In certain embodiments, the highest dose tolerated by the subject is 1 mg / kg-2.5 mg / kg. In some embodiments, the peak dose is administered to the human subject as a maintenance dose.

[0065] In certain embodiments, a method of treating a human subject with ASMD includes (a) administering reduced ASM to reduce the amount of sphingomyelin accumulated in the human subject, where reduced ASM administration includes (i) administering a non-toxic low dose of ASM to the human subject, and (ii) administering successively increasing doses of ASM to the human if the human subject does not exhibit one or more side effects as indicated by elevated total bilirubin concentrations, elevated plasma ceramide concentrations, production of acute phase reactants, production of inflammatory mediators, or adverse events (e.g., Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology Version 3.1.1), and (b) maintenance ASM administration, where maintenance ASM administration includes repeatedly administering a maintenance dose of ASM to the human subject. In some embodiments, the patient is monitored for one or more adverse side effects or total bilirubin for a period of time (e.g., 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, one week, or until the next dose) after administration of a dose of ASM. In certain embodiments, the maintenance dose administered may be adjusted over the course of the patient's treatment. In some embodiments, the maintenance dose administered to a subject is the maximum dose tolerated by the subject.

[0066] In a specific embodiment, the method of treating ASMD comprises administering to a subject in need thereof a non-toxic initial low dose of ASM (e.g., a dose of 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, 0.1 mg / kg-0.5 mg / kg, or 0.5-1 mg / kg of ASM) followed after a period of time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks) by administration of the ASM using techniques known in the art (e.g., He et al., 2003, Analytical Biochemistry 314: The method includes administering successively increasing amounts of ASM to the subject until the level of ASM activity in one or more diseased organs, as measured in a subject with ASMD (e.g., using the techniques described in US Pat. No. 4,993,313, or 6,100,120), is at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 80%, 85%, 90%, 95% or more of the ASM activity in the corresponding organ in subjects without ASMD (e.g., healthy subjects or a population of 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, 175 or more subjects). In another embodiment, a method for treating ASMD comprises administering a 0.1 mg / kg dose of ASM to a subject in need thereof, followed after a period of time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks) by immunohistochemistry using techniques known in the art (e.g., He et al., 2003, Analytical Biochemistry 314: the method comprises administering successively increasing amounts of ASM to the subject until the level of ASM activity in one or more of the following diseased organs, as measured by a method using a human veterinary medicine (e.g., the techniques described in US Pat. No. 4,993,116-120), is 5%-10%, 5%-15%, 5%-20%, 10%-15%, 10%-20%, 15%-20%, 15%-25%, 25%-50%, 50%-75%, or 75%-95% of the normal activity of ASM in the corresponding organ in subjects without ASMD (e.g., healthy subjects or a population of 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, 175 or more subjects).In certain embodiments, the dose is gradually increased if the total bilirubin concentration is below 2.0 mg / dL or 2.1 mg / dL and the subject does not experience moderate or severe related adverse events. In certain embodiments, ASM activity in normal healthy subjects is estimated to be about 20-40 units / mg of protein in the brain, heart, kidney and liver based on ASM activity in similar organs of healthy mice using the assay described in Horinouchi et al., 1995, Nature Genetics 10: 288-293, which is incorporated herein by reference in its entirety. In certain embodiments, ASM activity in normal healthy subjects is estimated to be 15-25 units / mg of protein in the lung and 10-15 units / mg of protein in the spleen based on ASM activity in similar organs of healthy mice using the assay described in Horinouchi et al., 1995, Nature Genetics 10: 288-293. In certain embodiments, once ASM activity reaches normal or a certain percentage of normal in one or more affected organs, the subject can be administered a maintenance dose that is equal to or less than the highest dose tolerated by the subject. Over time, the maintenance dose can be adjusted depending on the subject's health status. Depending on the subject's condition, the maintenance dose can be increased or decreased.

[0067] In one embodiment, a method of treating ASMD includes (a) administering a non-toxic initial low dose of ASM to a human in need thereof; and (b) after administration of the dose of ASM, the human experiences (i) a severe associated adverse event, e.g., as defined by the Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology version 3.1.1., or (ii) a pulmonary embolism (PE) of 1.5 mg / dL, 1.75 mg / dL, 2.0 mg / dL, 2.1 mg / dL, 2.2 mg / dL, 2.3 mg / dL, 2.4 mg / dL, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 4.0 mg / dL, 4.1 mg / dL, 4.2 mg / dL, 4.3 mg / dL, 4.4 mg / dL, 4.5 mg / dL, 4.5 mg / dL, 4.6 mg / dL, 4.7 mg / dL, 4.8 mg / dL, 4.9 mg / dL, 5.0 mg / dL, 5.0 mg / dL, 5.1 mg / dL, 5.2 mg / dL, 5.3 mg / dL, 5.4 mg / dL, 5.5 mg / dL, 5.6 mg / dL, 5.7 mg / dL, 5.8 mg / dL, 5.9 mg / dL, 5.8 mg / >2.6mg / dL, 2.7mg / dL, 2.75mg / dL, 2.8mg / dL, 2.9mg / dL, 3.0mg / dL, 3.1mg / dL, 3.2mg / dL, 3.3mg / dL, 3.4mg / dL, 3.5mg / dL, 3.6mg / dL, 3.7mg / dL, 3.8mg / dL, 3.9mg / dL or 4mg / dL or >2.1mg / dL-2.5mg / dL, 2.1mg / dL-3.0mg / dL mg / dL, or total bilirubin levels in the range of 2.1 mg / dL to 4 mg / dL at 6, 8, 10, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM, respectively; and (iii) levels of 8.2 μg / mL, 8.3 μg / mL, 8.4 μg / mL, 8.5 μg / mL, 8.75 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 51 μg / mL, 52 μg / mL, 53 μg / mL, 54 μg / mL, 3μg / mL, 14μg / mL, 15μg / mL, 16μg / mL, 17μg / mL, 18μg / mL, 19μg / mL, 20μg / mL, 25μg / mL, 30μg / mL, 35μg / mL, 40μg / mL , 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, or greater than 80 μg / mL, or 8.2 μg / mL to 10 μg / mL, 8.and (iv) administering successively increasing doses of ASM to a human in need thereof, unless the human develops one or more of the following: a plasma ceramide concentration in the range of 5 μg / mL to 10 μg / mL, 9 μg / mL to 12 μg / mL, 10 μg / mL to 12 μg / mL, 10 μg / mL to 15 μg / mL, 10 μg / mL to 20 μg / mL, 15 μg / mL to 20 μg / mL, or 20 μg / mL to 30 μg / mL; or (iv) a side effect, including an acute phase reaction. According to this embodiment, successively increasing doses of ASM can be administered to a human in need thereof, unless the human develops one or more of the following: a plasma ceramide concentration in the range of 5 μg / mL to 10 μg / mL, 9 μg / mL to 12 μg / mL, 10 μg / mL to 12 μg / mL, 10 μg / mL to 15 μg / mL, 10 μg / mL to 20 μg / mL, 15 μg / mL to 20 μg / mL, or 20 μg / mL to 30 μg / mL. If the human develops one or more of the following, depending on the severity of the symptoms, the same dose that caused the development of the following items (i) to (iv) can be repeated or reduced to the previous dose.

[0068] In another embodiment, a method of treating ASMD includes: (a) administering an initial dose of ASM of 0.1 mg / kg to a human in need thereof; (b) monitoring, after administration of the dose of ASM, one or more of: (i) total bilirubin concentration; (ii) occurrence of associated adverse events; (iii) acute phase response; or (iv) plasma ceramide concentration in the human; (c) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of items (i)-(iv); and (d) repeating steps (b) and (c) following administration of the dose of ASM determined in the previous step (c). In another embodiment, a method of treating ASMD includes: (a) administering to a human in need thereof an initial dose of ASM of 0.1 mg / kg twice, 2-4 weeks apart; (b) monitoring, after administration of each dose of ASM, one or more of: (i) total bilirubin concentration; (ii) occurrence of associated adverse events; (iii) acute phase response; or (iv) plasma ceramide concentration in the human; (c) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of items (i)-(iv); and (d) repeating steps (b) and (c) following administration of the dose of ASM determined in the previous step (c). According to these embodiments, higher doses of ASM can be administered to a human one or more times at 2-4 week intervals if the initial dose of 0.1 mg / kg or the adjusted dose determined in step (c) results in: (i) a total bilirubin concentration of 2.0 mg / dL or less prior to the due date for administering another dose of ASM; (ii) no associated adverse events or only mild associated adverse events; (iii) plasma ceramides within the normal range 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 48 ​​hours, or 72 hours after administration of the last dose of ASM; or (iv) no acute phase response or a statistically insignificant acute phase response.However, the dose may be maintained or reduced if the initial dose of 0.1 mg / kg or the adjusted dose determined in step (c) results in any of the following: (i) a total bilirubin concentration of 2.1 mg / dL or greater before the deadline for administering another dose of ASM; (ii) an associated adverse event; (iii) plasma ceramides higher than the normal range 6, 8, 12, 16, 18, 24, 48, or 72 hours after administration of the last dose of ASM; or (iv) a statistically significant acute phase response.

[0069] In another embodiment, a method of treating ASMD includes: (A) administering to a human being in need thereof two initial doses of 0.1 mg / kg, two weeks apart; (B) following administration of each initial dose of ASM, monitoring (i) total bilirubin concentration, (ii) either the occurrence of an associated adverse event, or (iii) both (i) and (ii); (C) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of items (i)-(iii); and (D) repeating steps (B) and (C) following administration of the dose of ASM determined in the previous step (C), wherein (a) the total bilirubin concentration is 0.1 mg / kg, and (b) the bilirubin concentration is 0.1 mg / kg. If the bilirubin concentration is below 2.0 mg / dL or the human experiences mild related adverse events, the dose administered to the human is increased; (b) if the total bilirubin concentration is between 2.1 mg / dL and 3.1 mg / dL or the human experiences moderate related adverse events, the human continues to receive 1 to 4 doses at 2 to 4 week intervals at the current dose, and if after the last administered dose the total bilirubin concentration is still above 2.0 mg / dL, this dose is maintained; (c) if the total bilirubin concentration is above 3 mg / dL or the human experiences severe related adverse events, the dose administered to the human is decreased or no longer administered ASM.

[0070] In another embodiment, a method of treating ASMD comprises: (A) administering to a human being in need thereof two initial doses of 0.3 mg / kg, two weeks apart; (B) following administration of each dose of ASM, monitoring (i) the total bilirubin concentration; (ii) either the occurrence of an associated adverse event; or (iii) both (i) and (ii); (C) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of items (i)-(iii); and (D) repeating steps (B) and (C) following administration of the dose of ASM determined in the previous step (C), wherein (a) the total bilirubin concentration is increased or decreased; (b) if the total bilirubin concentration is between 2.1 mg / dL and 3.1 mg / dL or if the human experiences mild related adverse events, the dose administered to the human is increased; (b) if the total bilirubin concentration is between 2.1 mg / dL and 3.1 mg / dL or if the human experiences moderate related adverse events, the human continues to receive 1 to 4 doses at 2 to 4 week intervals at the current dose, and if after the last administered dose the total bilirubin concentration is still above 2.0 mg / dL, the dose is maintained; and (c) if the total bilirubin concentration is above 3 mg / dL or the human experiences severe related adverse events, the dose administered to the human is decreased or no longer administered ASM.

[0071] In a specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof a 0.1 mg / kg dose of ASM, followed two weeks later by administering a 0.3 mg / kg dose of ASM every two weeks. In another specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof a 0.1 mg / kg dose of ASM, followed two weeks after administering the 0.1 mg / kg dose of ASM, followed by a 0.3 mg / kg dose of ASM two weeks after administering the 0.1 mg / kg dose of ASM, followed by a 0.6 mg / kg dose of ASM two weeks after administering the 0.3 mg / kg dose of ASM. In another specific embodiment, the method of treating ASMD comprises administering to a subject in need thereof a 0.1 mg / kg dose of ASM, a 0.3 mg / kg dose of ASM two weeks after administration of the 0.1 mg / kg dose of ASM, a 0.6 mg / kg dose of ASM two weeks after administration of the 0.3 mg / kg dose of ASM, and a 1 mg / kg dose of ASM two weeks after administration of the 0.6 mg / kg dose of ASM. In certain embodiments, each dose may be repeated at least twice, preferably 2-4 times, before increasing the dose to the next level. According to these embodiments, the dose is increased only if the total bilirubin level is below 2.0 mg / dL and / or the subject experiences mild related adverse events. The dose is not increased if the total bilirubin level is between 2.1-3 mg / dL and / or the subject experiences moderate related adverse events. If the total bilirubin level exceeds 3.0 mg / dL of total bilirubin and / or the subject experiences severe related adverse events, reduce the dose to the previously tolerated dose.

[0072] In a specific embodiment, the patient is treated for ASMD according to the protocols described in Sections 8 or 9 et seq. (below), or similar protocols.

[0073] In specific embodiments, a method of treating ASMD comprises administering a dose of 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, or between 0.1 mg / kg and 0.5 mg / kg, or between 0.1 mg / kg and 1 mg / kg of ASM to a subject in need thereof, and gradually increasing the dose of ASM administered to the subject if, after a period of time (e.g., 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks), the total bilirubin concentration is less than or equal to 2.1 mg / dL and the subject does not experience moderate or severe related adverse events. In some embodiments, the dose of ASM is gradually increased until a maximum or highest dose that is therapeutically effective and tolerated by the subject is achieved. In certain embodiments, such highest or maximum tolerated dose is administered until accumulated sphingomyelin in the affected organ is reduced, after which the subject is administered a therapeutically effective maintenance dose that is lower than the maximum tolerated dose, hi some embodiments, the maintenance dose is tapered over time as the patient's condition improves.

[0074] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing one or more severe related adverse events or related adverse events (e.g., as defined in the Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology version 3.1.1). In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without increasing the total bilirubin concentration compared to the patient's total bilirubin concentration prior to administration of ASM, where this increase persists for more than 2 days, 3 days, 5 days, 1 week, 2 weeks, or 3 weeks. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing the total bilirubin concentration to be higher than normal total bilirubin concentration for more than 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours, 5 days, 1 week, 2 weeks, or 3 weeks after administration of ASM. A normal total bilirubin concentration in a person without ASMD (e.g., a healthy individual) is less than about 1.2 mg / dL. In certain embodiments, the highest therapeutically effective and tolerable dose is a dose that maintains a total bilirubin concentration of about 1.5 mg / dL, 1.75 mg / dL, 2.0 mg / dL, 2.1 mg / dL, 2.2 mg / dL, 2.3 mg / dL, 2.4 mg / dL, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 ...9 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3. The highest dose effective in treating ASMD without exceeding 0.4 mg / d, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.75 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL or 4 mg / dL or falling within the ranges of 2.1 mg / dL to 2.5 mg / dL, 2.1 mg / dL to 3.0 mg / dL, or 2.1 mg / dL to 4 mg / dL.

[0075] In another embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without increasing plasma ceramide concentrations above normal plasma ceramide concentrations 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of ASM, which in humans without ASMD (e.g., healthy humans) is about 1.5-8 μg / mL. In certain embodiments, the highest tolerated dose is one that results in a plasma ceramide concentration of about 8.2 μg / mL, 8.3 μg / mL, 8.4 μg / mL, 8.5 μg / mL, 8.75 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 51 μg / mL, 52 μg / mL, 53 μg / mL, 54 μg / mL, 55 μg / mL, 56 μg / mL, 57 μg / mL, 58 μg / mL, 59 μg / mL, 60 μg / mL, 61 μg / mL, 62 μg / mL, 63 μg / mL, 64 μg / mL, 65 μg / mL, 66 μg / mL, 67 μg / mL, 68 μg / mL, 69 μg / mL, 70 μg / mL, 71 μg / mL, 72 μg / mL, 75 μg / mL, 76 μg / mL, 77 10μg / mL, 35μg / mL, 40μg / mL, 45μg / mL, 50μg / mL, 55μg / mL, 60μg / mL, 65μg / mL, 70μg / mL, 75μg / mL, or 80μg / mL, or within the ranges of 8.2μg / mL to 10μg / mL, 8.5μg / mL to 10μg / mL, 9μg / mL to 12μg / mL, 10μg / mL to 15μg / mL, 10μg / mL to 20μg / mL, 15μg / mL to 20μg / mL, or 20μg / mL to 30μg / mL.

[0076] In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing an acute phase reaction. The acute phase reaction can be evaluated by changes in the concentration of acute phase reactants, changes in prothrombin time, changes in partial thromboplastin time, or changes in neutrophil percentage. For example, the acute phase reaction can be evaluated by an increase in one or more of the following factors after ASM administration to a subject compared to those before ASM administration to the subject or to those of a person without ASMD (e.g., a healthy person): neutrophil percentage, prothrombin time (PT), partial thromboplastin time (PTT), total bilirubin concentration, C-reactive protein (CRP / hs-CRP) concentration, serum amyloid A (SAA), serum amyloid P component, angiotensin-converting enzyme (ACE), ferritin concentration, IL-6 concentration, IL-8 concentration, calcitonin concentration, albumin concentration, or fibrinogen concentration. The acute phase response can also be assessed by a decrease in iron or albumin concentration after administration of ASM to a subject compared to the iron or albumin concentration in the subject before administration of ASM, or compared to the iron or albumin concentration in a human without ASMD (e.g., a healthy individual).

[0077] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing an increase in CRP / hs-CRP concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM compared to the patient's CRP / hs-CRP concentration before ASM administration. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing a plasma CRP / hs-CRP concentration to exceed normal plasma CRP / hs-CRP concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM. Normal plasma CRP / hs-CRP concentration in humans without ASMD (e.g., healthy individuals) is approximately less than 8 mg / dL. In certain embodiments, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing plasma CRP / hs-CRP concentrations to exceed about 8.1 mg / L, 8.2 mg / L, 8.3 mg / L, 8.4 mg / L, 8.5 mg / L, 8.6 mg / L, 8.7 mg / L, 8.8 mg / L, 8.9 mg / L, 9 mg / L, 9.5 mg / L, 10 mg / L, 11 mg / L, or 12 mg / L, or to fall within the range of 8.5 mg / L to 10 mg / L, or 8.5 mg / dL to 12 mg / L, or 10 mg / L to 12 mg / L, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM.

[0078] In a specific embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without causing an increase in ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's ferritin concentration before ASM administration. In another specific embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without causing a plasma ferritin concentration exceeding the normal plasma ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM. Normal plasma ferritin concentration in humans without ASMD (e.g., healthy individuals) is 10-30 ng / mL. In certain embodiments, the highest therapeutically effective and tolerated dose is one that results in a plasma ferritin concentration of about 300ng / mL, 325ng / mL, 350ng / mL, 375ng / mL, 400ng / mL, 425ng / mL, 450ng / mL, 475ng / mL, 500ng / mL, 525ng / mL, 550ng / mL, 575ng / mL, 600ng / mL, 625ng / mL, 650ng / mL, 675ng / mL, 700ng / mL, 725ng / mL, 750ng / mL, 775ng / mL, The highest dose effective in treating ASMD without exceeding 800ng / mL, 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, 1050ng / mL, 1100ng / mL, 1150ng / mL, or 1200ng / mL, or falling within the ranges of 600ng / mL to 800ng / mL, 650ng / mL to 850ng / mL, 600ng / mL to 1000ng / mL, 600ng / mL to 1200ng / mL, 800ng / mL to 1000ng / mL, 900ng / mL to 1000ng / mL, or 1000ng / mL to 1200ng / mL, can be used as a measure of the acute phase response.

[0079] In a specific embodiment, an increase in plasma or serum IL-8 concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's IL-8 concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum IL-8 concentration that exceeds normal plasma IL-8 concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma IL-8 concentration of greater than about 24 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, or 900 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0080] In a specific embodiment, an increase in plasma or serum IL-6 concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's IL-6 concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum IL-6 concentration that exceeds normal plasma IL-6 concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, plasma IL-6 concentrations of greater than about 4.4 pg / mL, 6 pg / mL, 8 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, or 30 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0081] In a specific embodiment, an increase in plasma or serum calcitonin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's calcitonin concentration before administration of ASM can be used as a measure of the acute phase response. In another specific embodiment, a plasma or serum calcitonin concentration that exceeds the normal plasma calcitonin concentration 6, 8, 12, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response. In certain embodiments, a plasma calcitonin concentration of greater than about 9.4 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, or 250 pg / mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM can be used as a measure of the acute phase response.

[0082] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in fibrinogen concentration 6, 8, 12, 18, 24, 36, 48 or 72 hours after administration of a dose of ASM compared to the patient's fibrinogen concentration before administration of ASM. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing a plasma fibrinogen concentration to exceed normal plasma fibrinogen concentration 6, 8, 12, 16, 18, 24, 36, 48 or 72 hours after administration of a dose of ASM. Normal plasma fibrinogen concentration in humans without ASMD (e.g., healthy individuals) is between 150 mg / dL and 300 mg / dL. In certain embodiments, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing a plasma fibrinogen concentration to exceed about 350 mg / dL, 375 mg / dL, 400 mg / dL, 425 mg / dL, or 450 mg / dL, or to fall within the range of 350 mg / dL to 400 mg / dL, 350 mg / dL to 450 mg / dL, or 400 mg / dL to 450 mg / dL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM.

[0083] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in the neutrophil percentage of total white blood cells 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's neutrophil percentage of total white blood cells prior to administration of ASM. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing the neutrophil percentage of total white blood cells to exceed the normal neutrophil percentage of total white blood cell concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM. The normal percentage of neutrophils of total white blood cells in humans without ASMD (e.g., healthy individuals) is between 45% and 60%. In certain embodiments, the highest therapeutically effective and tolerable dose is the highest dose that is effective in treating ASMD without causing a 70%, 75%, 80%, 85%, 90%, 95% or greater increase in the neutrophil percentage of total white blood cells 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours or 72 hours after administration of a dose of ASM.

[0084] In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in antibodies against the administered ASM. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing a hypersensitivity reaction. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing cytokine release syndrome.

[0085] In certain embodiments, the highest dose that is therapeutically effective and tolerated by the subject is 1 mg / kg to 2.5 mg / kg, 2 mg / kg to 3 mg / kg, 3 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, 15 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, 20 mg / kg to 30 mg / kg, or 25 mg / kg to 50 mg / kg. In some embodiments, the highest dose that is therapeutically effective and tolerated by the subject is 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg. In certain embodiments, the maximum dose that is therapeutically effective and tolerated by the subject is 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg.

[0086] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing one or more severe related adverse events or related adverse events (e.g., as defined in the Clinical Data Interchange Standards Consortium Study Data Tabulation Model Standard Terminology version 3.1.1). In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without increasing the total bilirubin concentration compared to the patient's total bilirubin concentration prior to administration of ASM, where this increase persists for more than 2 days, 3 days, 5 days, 1 week, 2 weeks, or 3 weeks. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing the total bilirubin concentration to be higher than normal total bilirubin concentration for more than 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours, 5 days, 1 week, 2 weeks, or 3 weeks after administration of ASM. A normal total bilirubin concentration in a person without ASMD (e.g., a healthy individual) is less than about 1.2 mg / dL. In certain embodiments, the highest therapeutically effective and tolerable dose is a dose that maintains a total bilirubin concentration of about 1.5 mg / dL, 1.75 mg / dL, 2.0 mg / dL, 2.1 mg / dL, 2.2 mg / dL, 2.3 mg / dL, 2.4 mg / dL, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 ...9 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3. The highest dose effective in treating ASMD without exceeding 0.4 mg / d, 2.5 mg / dL, 2.6 mg / dL, 2.7 mg / dL, 2.75 mg / dL, 2.8 mg / dL, 2.9 mg / dL, 3.0 mg / dL, 3.1 mg / dL, 3.2 mg / dL, 3.3 mg / dL, 3.4 mg / dL, 3.5 mg / dL, 3.6 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL or 4 mg / dL or falling within the ranges of 2.1 mg / dL to 2.5 mg / dL, 2.1 mg / dL to 3.0 mg / dL, or 2.1 mg / dL to 4 mg / dL.

[0087] In another embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without increasing plasma ceramide concentrations above normal plasma ceramide concentrations 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of ASM, which is about 1.5-8 μg / dL in humans without ASMD (e.g., healthy individuals). In certain embodiments, the highest tolerated dose is one that results in a plasma ceramide concentration of about 8.2 μg / mL, 8.3 μg / mL, 8.4 μg / mL, 8.5 μg / mL, 8.75 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 51 μg / mL, 52 μg / mL, 53 μg / mL, 54 μg / mL, 55 μg / mL, 56 μg / mL, 57 μg / mL, 58 μg / mL, 59 μg / mL, 60 μg / mL, 61 μg / mL, 62 μg / mL, 63 μg / mL, 64 μg / mL, 65 μg / mL, 66 μg / mL, 67 μg / mL, 68 μg / mL, 69 μg / mL, 70 μg / mL, 71 μg / mL, 72 μg / mL, 75 μg / mL, 76 μg / mL, 77 10μg / mL, 35μg / mL, 40μg / mL, 45μg / mL, 50μg / mL, 55μg / mL, 60μg / mL, 65μg / mL, 70μg / mL, 75μg / mL, or 80μg / mL, or within the ranges of 8.2μg / mL to 10μg / mL, 8.5μg / mL to 10μg / mL, 9μg / mL to 12μg / mL, 10μg / mL to 15μg / mL, 10μg / mL to 20μg / mL, 15μg / mL to 20μg / mL, or 20μg / mL to 30μg / mL.

[0088] In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing an acute phase reaction. The acute phase reaction can be evaluated by changes in the concentration of acute phase reactants, changes in prothrombin time, changes in partial thromboplastin time, or changes in neutrophil percentage. For example, the acute phase reaction can be evaluated by an increase in one or more of the following factors after ASM administration to a subject, compared to those before ASM administration to the subject or to those of a person without ASMD (e.g., a healthy individual): neutrophil percentage, prothrombin time (PT), partial thromboplastin time (PTT), total bilirubin concentration, C-reactive protein (CRP / hs-CRP) concentration, serum amyloid A (SAA), serum amyloid P component, angiotensin-converting enzyme (ACE), ferritin concentration, albumin concentration, or fibrinogen concentration. The acute phase response can also be assessed by a decrease in iron or albumin concentration after administration of ASM to a subject compared to the iron or albumin concentration in the subject before administration of ASM, or compared to the iron or albumin concentration in a human without ASMD (e.g., a healthy individual).

[0089] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing an increase in CRP / hs-CRP concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM compared to the patient's CRP / hs-CRP concentration before administration of ASM. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing a plasma CRP / hs-CRP concentration to exceed normal plasma CRP / hs-CRP concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM. Normal plasma CRP / hs-CRP concentration in humans without ASMD (e.g., healthy individuals) is approximately less than 8 mg / L. In certain embodiments, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing a plasma CRP / hs-CRP concentration to exceed about 8.1 mg / L, 8.2 mg / L, 8.3 mg / L, 8.4 mg / L, 8.5 mg / L, 8.6 mg / L, 8.7 mg / L, 8.8 mg / L, 8.9 mg / L, 9 mg / L, 9.5 mg / L, 10 mg / L, 11 mg / L, or 12 mg / L or to fall within the range of 8.5 mg / L to 10 mg / L, or 8.5 mg / L to 12 mg / L, or 10 mg / L to 12 mg / L, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM.

[0090] In a specific embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without causing an increase in ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's ferritin concentration before ASM administration. In another specific embodiment, the highest therapeutically effective and tolerable dose is the highest dose effective in treating ASMD without causing a plasma ferritin concentration exceeding the normal plasma ferritin concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM. Normal plasma ferritin concentration in humans without ASMD (e.g., healthy individuals) is 10-30 ng / mL. In certain embodiments, the highest therapeutically effective and tolerated dose is one that results in a plasma ferritin concentration of about 600ng / mL, 625ng / mL, 650ng / mL, 675ng / mL, 700ng / mL, 725ng / mL, 750ng / mL, 775ng / mL, 800ng / mL, 850ng / mL, or 900ng / mL at 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM. ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, 1050ng / mL, 1100ng / mL, 1150ng / mL, or 1200ng / mL, or within the ranges of 600ng / mL to 800ng / mL, 650ng / mL to 850ng / mL, 600ng / mL to 1000ng / mL, 600ng / mL to 1200ng / mL, 800ng / mL to 1000ng / mL, 900ng / mL to 1000 ng / mL, or 1000ng / mL to 1200ng / mL.

[0091] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in fibrinogen concentration 6, 8, 12, 18, 24, 36, 48 or 72 hours after administration of a dose of ASM compared to the patient's fibrinogen concentration before administration of ASM. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing a plasma fibrinogen concentration to exceed normal plasma fibrinogen concentration 6, 8, 12, 16, 18, 24, 36, 48 or 72 hours after administration of a dose of ASM. Normal plasma fibrinogen concentration in humans without ASMD (e.g., healthy individuals) is between 150 mg / dL and 300 mg / dL. In certain embodiments, the highest therapeutically effective and tolerated dose is the highest dose that is effective in treating ASMD without causing a plasma fibrinogen concentration to exceed about 350 mg / dL, 375 mg / dL, 400 mg / dL, 425 mg / dL, or 450 mg / dL, or to fall within the range of 350 mg / dL to 400 mg / dL, 350 mg / dL to 450 mg / dL, or 400 mg / dL to 450 mg / dL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration of a dose of ASM.

[0092] In a specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in the neutrophil percentage of total white blood cells 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM compared to the patient's neutrophil percentage of total white blood cells prior to administration of ASM. In another specific embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing the neutrophil percentage of total white blood cells to exceed the normal neutrophil percentage of total white blood cell concentration 6, 8, 12, 16, 18, 24, 36, 48, or 72 hours after administration of a dose of ASM. The normal percentage of neutrophils of total white blood cells in humans without ASMD (e.g., healthy individuals) is between 45% and 60%. In certain embodiments, the highest therapeutically effective and tolerable dose is the highest dose that is effective in treating ASMD without causing a 70%, 75%, 80%, 85%, 90%, 95% or greater increase in the neutrophil percentage of total white blood cells 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours or 72 hours after administration of a dose of ASM.

[0093] In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing an increase in antibodies against the administered ASM. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing a hypersensitivity reaction. In another embodiment, the highest therapeutically effective and tolerated dose is the highest dose effective in treating ASMD without causing cytokine release syndrome.

[0094] In certain embodiments, the highest dose that is therapeutically effective and tolerated by the subject is 1 mg / kg to 2.5 mg / kg, 2 mg / kg to 3 mg / kg, 3 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, 15 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, 20 mg / kg to 30 mg / kg, or 25 mg / kg to 50 mg / kg. In some embodiments, the highest dose that is therapeutically effective and tolerated by the subject is 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg. In certain embodiments, the maximum dose that is therapeutically effective and tolerated by the subject is 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg.

[0095] In certain embodiments, the ASM doses described herein are administered to a subject every 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In some embodiments, the ASM doses described herein are administered to a subject every 3-5 days, 3-7 days, 5-7 days, 5-10 days, 5-14 days, 7-14 days, 2-4 weeks, or 3-5 weeks. In certain embodiments, the frequency of administration of the ASM doses varies as the dose is adjusted. For example, a 0.3 mg / kg dose may be administered every 1 or 2 weeks, and a 1 mg / kg dose may be administered every 2 or 3 weeks.

[0096] In some embodiments, a dose of ASM (e.g., a maintenance dose) is administered to a subject for 12 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 52 weeks, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, or until the patient experiences a related adverse event, a total bilirubin level greater than the bilirubin level of a human without ASMD (e.g., a healthy individual), a plasma ceramide level greater than the plasma ceramide level of a human without ASMD (e.g., a healthy individual), or an acute phase reaction.

[0097] In certain embodiments, a non-toxic initial low dose of ASM is administered to a subject every 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In some embodiments, an ASM dose as described herein is administered to a subject every 3-5 days, 3-7 days, 5-7 days, 5-10 days, 5-14 days, 7-14 days, 2-4 weeks, or 3-5 weeks. In certain embodiments, a non-toxic initial low dose of ASM is administered for 4 weeks, 6 weeks, 8 weeks, 12 weeks, 14 weeks, or more.

[0098] In certain embodiments, the ASM dose escalation is about 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 higher than the previous dose. In some embodiments, the ASM dose escalation is 0.1-0.5 mg / kg, 0.1 mg / kg-1 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 higher than the previous dose.

[0099] The ASM dose described herein can be administered by any route that is beneficial for achieving a therapeutic effect. Specific routes of administration of the ASM dose include, but are not limited to, intravenous, intracerebroventricular, intradermal, transdermal, subcutaneous, intramuscular, intranasal, inhalation, intrapulmonary, topical, transmucosal, intracranial, intrathecal, epidural, and intrasynovial. In one embodiment, the ASM dose is administered systemically (e.g., parenterally) to a subject in need thereof. In another embodiment, the ASM dose is administered locally to a subject in need thereof.

[0100] ASM enzyme doses effective in treating somatic (non-central nervous) manifestations of ASMD cannot effectively cross the brain barrier. Thus, in specific embodiments, ASM is administered intracerebroventricularly to patients or intrathecally to the brain of NPD patients. For methods of intracerebroventricular delivery of lysosomal storage enzymes to the brain, see, for example, US Patent Application Publication Nos. 2009 / 0130079 and 2009 / 0123451, both of which are incorporated herein by reference in their entirety. In certain embodiments, ASM is administered intracerebroventricularly to patients. For methods of intracerebroventricular injection of ASM, see, for example, Dodge et al., 2009, Experimental Neurology 215: 349-357, which is incorporated herein by reference in its entirety. In some embodiments, ASM is administered to patients by indirect intracerebroventricular injection. See, for example, Yang et al., 2007, Experimental Neurology 207: 258-266. In some embodiments, modified forms of ASM that target a brain enzyme are administered to patients to treat ASMD, as described in Section 5.2 (below).

[0101] Only a small percentage of the ASM enzyme administered to a patient reaches the lungs. Thus, in a specific embodiment, ASM is administered to the patient's lungs. In certain embodiments, ASM is administered to the patient intranasally or by inhalation. For information regarding intranasal administration of ASM, see Ziegler et al., 2009, Molecular Genetics and Metabolism 97: 35-42, which is incorporated by reference in its entirety. In some embodiments, modified forms of ASM that target pulmonary enzymes are administered to a patient to treat ASMD, as described in Section 5.2 (below). In certain embodiments, ASM is administered to a patient using a nebulizer. Delivery of ASM to the lungs can be achieved by intrapulmonary instillation through a bronchoscope, a metered dose inhaler, or a nebulizer.

[0102] In certain embodiments, the ASM enzyme is administered systemically to the patient, but also locally to specific organs, such as the brain and lungs. In some embodiments, local administration of the ASM enzyme complements systemic administration of the enzyme. In specific embodiments, the ASM enzyme is administered locally, for example, to the brain or lungs, after systemic administration (e.g., intravenous administration) reduces the patient's accumulated sphingomyelin.

[0103] In certain embodiments, the patient's genotype is examined prior to administration of ASM. In some embodiments, the glycosylation pattern of ASM expressed by the patient is determined prior to administration of ASM. Administration of ASM similar / matching to that endogenously expressed by the patient may reduce potential immunogenicity.

[0104] In certain embodiments, the activity of endogenously expressed ASM is determined prior to administration of ASM. The activity of endogenously expressed ASM can be measured in DBS and cultured fibroblasts using techniques well known to those of skill in the art.

[0105] In a specific embodiment, the method of treating ASMD provided herein reduces spleen volume as assessed by techniques known in the art, for example, MRI. In another specific embodiment, the method of treating ASMD provided herein reduces liver sphingomyelin levels as assessed by techniques known in the art, for example, liver biochemical and / or histomorphometric analysis. In another specific embodiment, the method of treating ASMD provided herein increases exercise capacity as assessed by techniques known in the art, for example, maximal workload by cycle ergometer exercise testing, including percent predicted maximal workload, peak oxygen consumption, and carbon dioxide production. In another specific embodiment, the method of treating ASMD provided herein improves pulmonary function as assessed by techniques known in the art, for example, DLco, FVC, FEV, and / or TLC. In another specific embodiment, the method of treating ASMD provided herein reduces bronchoalveolar lavage (BAL) sphingomyelin. In another specific embodiment, the method of treating ASMD provided herein reduces liver volume as assessed by techniques known in the art, such as MRI. In another specific embodiment, the method of treating ASMD provided herein improves lung appearance as assessed by techniques known in the art, such as high resolution CT scan or chest X-ray. In another specific embodiment, the method of treating ASMD provided herein improves lung clearance. In another specific embodiment, the method of treating ASMD provided herein reduces sphingomyelin concentrations in liver, skin, plasma and DBS. In another specific embodiment, the method of treating ASMD provided herein reduces plasma chitotriosidase levels. In another specific embodiment, the method of treating ASMD provided herein reduces serum CCL18 levels. In another specific embodiment, the method of treating ASMD provided herein improves the lipid profile of the patient (e.g., lowering cholesterol).In another specific embodiment, the method of treating ASMD provided herein reduces or ameliorates the severity of ASMD and / or symptoms associated therewith. In another specific embodiment, the method of treating ASMD provided herein reduces the duration of symptoms associated with ASMD. In another specific embodiment, the method of treating ASMD provided herein prevents the recurrence of symptoms associated with ASMD. In another specific embodiment, the method of treating ASMD provided herein reduces hospitalization of the subject. In another specific embodiment, the method of treating ASMD provided herein reduces the duration of hospitalization. In another specific embodiment, the method of treating ASMD provided herein increases the survival of the subject. In another specific embodiment, the method of treating ASMD provided herein reduces the mortality rate of the subject. In another specific embodiment, the method of treating ASMD provided herein reduces the hospitalization rate of the subject. In another specific embodiment, the method of treating ASMD provided herein reduces the number of symptoms associated with ASMD. In another specific embodiment, the method of treating ASMD provided herein increases the symptom-free survival of the patient. In another specific embodiment, the methods of treating ASMD provided herein improve neurological function (eg, psychomotor function, social responsiveness, etc.) in a subject.

[0106] In another specific embodiment, the methods of treating ASMD provided herein improve the patient's quality of life. In certain specific embodiments, the methods of treating ASMD provided herein include the use of a pulmonary circulation monitoring instrument (PMI) for assessing dyspnea and fatigue, such as, for example, the Brief Fatigue Inventory (BFI; Mendoza et al., 1999, Cancer 85(5): 1186-1196), the Brief Pain Inventory-Short Form (BPI-SF; Cleeland et al., 1994, Ann Acad Med Singapore 23(2): 129-138), the ASM-Health Assessment Questionnaire (which is a composite of the BFI, BPI-SF, and Short Form 36 Health Survey), the Chronic Respiratory Disease Questionnaire Self-Administered Standardized (CRQ-SAS) for assessing dyspnea and fatigue, and the Chronic Respiratory Disease Questionnaire Self-Administered Standardized (CRQ-SAS; Schunemann et al., 2005, Eur. Respir. J. 25: 1186-1196). 31-40) improve patients' quality of life, as assessed by computer-adaptive tests to assess the Activity Measure for Post-Acute Care (AM-PAC), and functional mobility (e.g., mobility, self-care, applied cognition).

[0107] Acid sphingomyelinase enzyme ASM refers to any form of acid sphingomyelinase enzyme that retains the ability to hydrolyze sphingomyelin to ceramide and phosphorylcholine, as assessed by techniques known to those of skill in the art, such as those described in U.S. Patent Nos. 4,039,388, 4,082,781, 5,686,240, 7,563,591, and International Publication Nos. WO 2007 / 078806 and WO 2006 / 058385, which are incorporated herein by reference in their entireties. In a specific embodiment, acid sphingomyelinase enzyme has the ability to hydrolyze sphingomyelin to ceramide and phosphorylcholine in a fluorescence-based high performance liquid chromatography assay as described in He et al., 2003, Analytical Biochemistry 314: 116-120. In specific embodiments, the acid sphingomyelinase has at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30%-50%, 40%-50%, 50%-75%, 50%-90%, 75%-80%, 75%-90%, 75%-95%, or 85%-95% of the activity of ASM-1 (SEQ ID NO: 1, infra) as measured, e.g., in an assay described in He et al., 2003, Analytical Biochemistry 314: 116-120.

[0108] In a specific embodiment, the ASM is human ASM. There are various isoforms of human ASM that arise from alternative splicing. One human ASM isoform, human ASM isoform 1 (sometimes referred to as ASM-1), has the amino acid sequence found in UniProtKB / Swiss-Prot Accession No. P17405-1. Another human ASM isoform, human ASM isoform 2 (sometimes referred to as ASM-2), has the amino acid sequence found in UniProtKB / Swiss-Prot. Accession No. P17405-2. A third human ASM isoform, human ASM isoform 3 (sometimes referred to as ASM-3), has the amino acid sequence found in UniProtKB / Swiss-Prot. Accession No. P17405-3. The amino acid sequence of the most abundant isoform, ASM-1, is shown below.

[0109] [ka]

[0110] ASM-2 differs from ASM-1 in that amino acid residues 363-374 of ASM-1 (i.e., IGGFYALSPYPG (SEQ ID NO:2)) have been replaced with amino acids YLSSVETQEGKR (SEQ ID NO:3) and amino acid residues 375-418 of ASM-1 are absent in ASM-2. ASM-3 differs from ASM-1 in that amino acid residues 363-418 of ASM-1 are absent in ASM-3. ASM-2 and ASM-3 are included as ASMs that may be administered to a subject so long as they have enzymatic activity, e.g., as measured by the assay described in He et al., 2003, Analytical Biochemistry 314: 116-120. In specific embodiments, ASM-2 and ASM-3 may be included as ASMs that can be administered to a subject, so long as they have at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30%-50%, 40%-50%, 50%-75%, 50%-90%, 75%-80%, 75%-90%, 75%-95%, or 85%-95% of the activity of ASM-1 as measured, for example, in an assay described in He et al., 2003, Analytical Biochemistry 314: 116-120.

[0111] In specific embodiments, human ASM has the amino acid sequence of human acid sphingomyelinase disclosed in SEQ ID NO:1 (above), the amino acid sequence of human acid sphingomyelinase disclosed in U.S. Pat. No. 6,541,218 (e.g., SEQ ID NO:2 in U.S. Pat. No. 6,541,218), or the amino acid sequence disclosed in Figure 3 of Schuchman et al., 1991, J. Biol. Chem. 266: 8531-8539, each of which is incorporated herein by reference in its entirety.

[0112] In a specific embodiment, human ASM is the processed mature form. In another embodiment, human ASM is the immature unprocessed form. With respect to ASM-1, the immature form is 629 amino acids in length and contains a signal peptide found at amino acid residues 1-46. The mature form of ASM-1 lacks the signal peptide and consists of amino acid residue 47 through amino acid residue 629. Human ASM-1 contains a saposin B-type domain from amino acid residues 85-169. With respect to ASM-1, the following amino acid residues are glycosylated (specifically, N-linked glycosylated): 86, 175, 335, 395, and 520.

[0113] In addition to the isoforms of human ASM, there are various naturally occurring variants of human ASM. For example, naturally occurring variants of the human ASM gene with different numbers of hexanucleotide repeat units have been identified within the gene region encoding the putative signal peptide of ASM. For example, see Wan and Schuchman, 1995, Biochimica et Biophysica Acta 1270: 207-210, which describes the identification of five alleles corresponding to 9, 7, 6, 5 and 4 hexanucleotide repeats (incorporated herein in their entirety by reference). In addition, naturally occurring variants of human ASM with single amino acid changes have been identified. For information regarding single polymorphisms, see, for example, Schuchman et al., 1991, J. of Biol. Chem 266: 8531-8539 and Schuchman et al., 1991, Nucleic Acids Research 19(11): 3160 (incorporated herein in their entirety by reference). Any naturally occurring variants of human ASM are included as ASMs that can be administered to a subject, so long as they have enzymatic activity, as measured, for example, by the assay described in He et al., 2003, Analytical Biochemistry 314:116-120. In specific embodiments, any naturally occurring variant of human ASM may be included as an ASM that can be administered to a subject, so long as it has at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30%-50%, 40%-50%, 50%-75%, 50%-90%, 75%-80%, 75%-90%, 75%-95%, or 85%-95% of the activity of ASM-1 as measured, for example, in an assay described in He et al., 2003, Analytical Biochemistry 314: 116-120.

[0114] Many single nucleotide polymorphisms (SNPs) for the gene encoding human ASM have been identified (see, e.g., the NCBI website for examples of SNPs: ncbi.nlm.nih.gov / projects / SNP / ). To the extent that any SNPs in the gene encoding ASM have enzymatic activity, as measured, for example, by the assay described in He et al., 2003, Analytical Biochemistry 314: 116-120, they are included as ASMs that can be administered to a subject. In specific embodiments, any SNP in the gene encoding ASM may be included as an ASM that can be administered to a subject, so long as it has at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30%-50%, 40%-50%, 50%-75%, 50%-90%, 75%-80%, 75%-90%, 75%-95%, or 85%-95% of the activity of ASM-1 as measured, for example, in an assay as described in He et al., 2003, Analytical Biochemistry 314: 116-120. In certain embodiments, the ASM is a variant of human ASM. In specific embodiments, the variant of human ASM is one disclosed in U.S. Pat. No. 7,527,956, which is incorporated herein by reference in its entirety. In another embodiment, the variant of human ASM is one disclosed in International Publication No. WO 2008 / 136451, which is incorporated herein by reference in its entirety. In some specific embodiments, the ASM is a variant of human ASM that has one or more of the following properties: (i) increased targeting to disease sites (e.g., lung and / or brain) compared to unmodified human ASM, (ii) increased stability compared to unmodified human ASM, and (iii) increased activity compared to unmodified human ASM. Techniques well known in the art can be used to measure the stability, activity and targeting of ASM.In a specific embodiment, the technique described in Gamacho et al., 2008, J. Pharmacol. Exp. Ther. 325: 400-408 (incorporated herein in its entirety by reference) is used to evaluate the targeting of ASM. In another specific embodiment, the technique described in He et al., 1999, Biochimica et Biophysica Acta 1432: 251-264 or Dhami et al., 2001, Lab. Invest. 81: 987-999 (incorporated herein in its entirety by reference) is used to evaluate the stability of ASM. In another specific embodiment, the technique described in He et al., 2003, Analytical Biochemistry 314: 116-120 (incorporated herein in its entirety by reference) is used to evaluate the activity of ASM.

[0115] In one embodiment, the ASM is a modified form of human ASM that has increased enzymatic activity compared to unmodified human ASM, such as ASM-1. For example, see Qiu et al., 2003, J. Biol. Chem. 278(35): 32744-32752 and U.S. Patent No. 7,527,956 for mutant forms of recombinant human ASM that have higher activity than wild-type recombinant human ASM (e.g., ASM-1). In some embodiments, the addition of zinc cations causes the ASM to have higher enzymatic activity compared to unmodified human ASM (e.g., ASM-1). For a discussion of the role of zinc in ASM activity, see, for example, Schissel et al., 1998, J. Biol. Chem 273: 18250-18259, which is incorporated by reference in its entirety. In another embodiment, the ASM is a modified form of human ASM that has a greater affinity for its native receptor (e.g., mannose 6-phosphate or high mannose) compared to unmodified human ASM (e.g., ASM-1, etc.). In a specific embodiment, ASM is conjugated to an oligosaccharide to increase targeting of the enzyme to its native receptor, as described in U.S. Patent No. 7,001,994 and International Patent Application Publication Nos. WO2010 / 075010 and 2010 / 0173385, which are incorporated herein by reference in their entireties. In another embodiment, the ASM is a modified form of human ASM that binds to an alternative receptor that is different from ASM's native receptor (e.g., intercellular adhesion molecule (ICAM)-1, which may increase targeting to organs such as the lung). In one embodiment, the ASM is a modified form of human ASM that has a greater stability than unmodified human ASM (e.g., ASM-1, etc.).

[0116] In some embodiments, ASM is directly or indirectly conjugated or condensed to a targeting moiety such as insulin-like growth factor (IGF)-I, IGF-II, leptin, granulocyte colony-stimulating factor (G-CSF), or a humanized antibody that binds to the human insulin receptor (HIR).For a discussion of the use of IGF-I as a targeting moiety for lysosomal storage enzymes, see, for example, US Patent Application Publication No. 2009 / 0029467 and US Patent No. 7,560,424 (incorporated herein in its entirety by reference), and for a discussion of the use of IGF-II as a targeting moiety for lysosomal storage enzymes, see, for example, US Patent No. 7,396,811 (incorporated herein in its entirety by reference).The targeting moieties IGF-I and IGF-II can enhance the targeting of the ASM enzyme to lysosomes. For a discussion of the use of hormones such as G-CSF and leptin as targeting moieties, see, for example, International Patent Application Publication No. WO 2007 / 091250, and US Patent Application Publication No. 2010 / 0183577, which are incorporated herein by reference in their entirety. For a discussion of humanized monoclonal antibodies that bind to the human insulin receptor (HIR) conjugated to pharmaceutical agents such as enzymes, see US Patent Application Publication No. 2004 / 0101904, which is incorporated herein by reference in its entirety. Antibodies that bind to targeting moieties G-CSF, leptin, and HIR can facilitate targeting of the ASM enzyme to the brain.

[0117] In certain embodiments, the ASM is a modified form of ASM (e.g., a modified form of human ASM) that has reduced activity compared to unmodified ASM (e.g., unmodified human ASM such as ASM-1). In some embodiments, the ASM is a modified form of ASM (e.g., a modified form of human ASM) that has increased targeting to disease sites and reduced enzymatic activity compared to unmodified ASM (e.g., unmodified human ASM such as ASM-1).

[0118] In certain embodiments, ASM is a highly phosphorylated form of ASM. For a discussion of techniques for generating highly phosphorylated forms of lysosomal enzymes, see U.S. Patent Application Publication No. 2002 / 0150981, which is incorporated herein by reference.

[0119] ASM enzymes can be produced by any method known in the art, including, but not limited to, recombinant DNA techniques, cDNA cloning (see, e.g., Schuchman et al., 1991, J. Biol. Chem 266: 8531-8539 and U.S. Pat. No. 5,773,278 for cDNA cloning of human AMS, which are incorporated herein by reference in their entireties), gene cloning, gene activation (see, e.g., U.S. Pat. No. 5,641,670 for gene activation techniques, which are incorporated herein by reference in their entireties), or selected cell lines that produce high levels of ASM enzymes (e.g., mammalian, yeast, prokaryotic, insect cells (e.g., Sf9, Sf21, Trichoplusia ni, Spodoptera frugiperda, and Bombyx mori)] and plant cells). ASM and methods for ASM production are described, for example, in U.S. Patent Nos. 5,773,278, 6,541,218, 5,686,240, and 7,527,956, all of which are incorporated herein by reference in their entirety. In a specific embodiment, ASM is recombinantly produced ASM (e.g., recombinantly produced human ASM). Cellular expression systems that have the cellular machinery and elements for proper processing (i.e., signal cleavage, glycosylation, phosphorylation, and protein sorting) are preferred. For example, mammalian cellular expression systems are preferred for the expression of properly folded and processed biologically active enzymes, and such expression products should exhibit correct tissue targeting and no adverse immunological reaction when administered to humans.

[0120] In certain embodiments, ASM (e.g., human ASM) is produced by overexpression of ASM cDNA in mammalian cells. In a specific embodiment, human ASM is produced by overexpression of human ASM cDNA in Chinese Hamster Ovary (CHO) cells. For methods of overexpressing human ASM cDNA in CHO cells, see He et al., 1999, Biochimia et Biophsyica Acta 1432: 251-264 and U.S. Patent No. 6,541,218, both of which are incorporated herein by reference in their entireties. In certain embodiments, human ASM is produced by overexpression of human ASM cDNA in CHO cells that co-express α-2,6-sialyltransferase. See, for example, U.S. Patent No. 5,047,335, which describes CHO cells engineered to express α-2,6-sialyltransferase, which is incorporated herein by reference in its entirety.

[0121] In another specific embodiment, the mammalian cell is a human cell. Examples of human cells that can be used to recombinantly express ASM (e.g., human ASM) include Crucell Per.C6 cells, HT 1080 cells, HeLa cells, HEK 293 cells, 293T cells, WI38 cells, HuT292 cells, LF 1043 (HEL) cells, MRC-5 cells, TMK-1 cells, BT483 cells, Hs578T cells, HTB2 cells, HTB3 cells, HTB4 cells, BT 20 cells, T47D cells, CRL7O3O cells, HsS78Bst cells, 721 cells, A2780 cells, A172 cells, A253 cells, COR-L23 / R23 cells, COV-434 cells, DU145 cells, DuCaP cells, EM2 cells, Saos-2 cells, U373 cells, WM39 cells, L132 cells, A-5489 cells, G-293 cells, G-401 cells, CAKI-1 cells, RD cells, and YAR cells. Other exemplary human cells that can be used to express ASM include human cells listed in the Immuno Polymorphism Database of the European Molecular Biology Laboratory and the National Institutes of Health databases, and those commercially available, e.g., from the American Type Culture Collection (Manassas, VA), and Lifeline Cell Technology (Walkersville, MD). When using human cell lines for production, rhASM can be produced using cDNA, gDNA for hASM, or by gene activation techniques such as those described in U.S. Patent No. 5,641,670, which is incorporated herein by reference in its entirety.

[0122] Examples of other mammalian cells that can be used to produce ASM (e.g., human ASM) include, but are not limited to, Vero cells, VERY cells, BHK cells, COS cells, MDCK cells, or 3T3 cells. In certain embodiments, myeloma cells are used to produce ASM (e.g., human ASM). Non-limiting examples of myeloma cells include NS0 cells, 45.6 TG1.7 cells, AF-2 clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells, NCI-H929 cells, NP cells, NS0 / 1 cells, P3 NS1 Ag4 cells, P3 / NS1 / 1-Ag4-1 cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1 cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells, Y3.Ag.1.2.3 cells, and YO cells.

[0123] In some embodiments, plant cell culture systems are used for expression of ASM. For plant cells and methods for the production of proteins using plant cell culture systems, see, e.g., U.S. Pat. Nos. 5,929,304, 7,504,560, 6,770,799, 6,551,820, 6,136,320, 6,034,298, 5,914,935, 5,612,487, and 5,484,719, U.S. Patent Application Publication Nos. 2009 / 0208477, 2009 / 0082548, 2009 / 0053762, 2008 / 0038232, 2007 / 0275014, and 2006 / 0204487, and Shoji et al., 2008, Vaccine, 26(23):2930-2934, and D'Aoust et al., 2008, J. Plant Biotechnology, 6(9):930-940, which are incorporated by reference in their entireties. In particular, U.S. Patent Applications 2009 / 0208477, 2008 / 0038232, and 2006 / 0204487 describe the expression and production of enzymatically active high mannose lysosomal enzymes using transformed plant roots, specifically carrot cells. In a specific embodiment, the carrot cells are genetically engineered to express ASM. In certain embodiments, algae (e.g., the green alga Chlamydomonas reinhardtii) may be genetically engineered to express ASM (see, e.g., Rasala et al., 2010, Plant Biotechnology Journal, published online March 7, 2010, which is incorporated by reference in its entirety).

[0124] Tissue and cellular uptake of ASM is mediated by both high mannose residues (e.g., in macrophages) and mannose-6-phosphate (e.g., in the liver). Thus, production of ASM with altered glycan content and / or phosphorylation may be desirable to improve drug distribution. For example, ASM is produced by cells with a mutation (e.g., knockout) in at least one Golgi-processed mannosidase. In one embodiment, the mutation reduces gene expression, reduces protein or activity levels, or alters the distribution or other post-translational modifications (e.g., processing of the carbohydrate chain) of the mannosidase. In a specific embodiment, the mutation reduces the level of Golgi-processed mannosidase activity. The mutation can occur in class 1 processed mannosidase, class 2 processed mannosidase, class 1 processed mannosidase, and class 2 processed mannosidase. Class 1 processed mannosidases include Golgi mannosidase IA, Golgi mannosidase IB, and Golgi mannosidase IC. Class 2 processing mannosidases include Golgi mannosidase II. See, for example, International Patent Application Publication No. WO 02 / 15927 and U.S. Patent No. 7,138,262, which are incorporated by reference in their entireties, for methods of producing high mannose proteins.

[0125] In certain embodiments, cells expressing ASM are cultured in the presence of mannosidase inhibitors, such as antibodies, kifunensine, swainsonine, mannostatin, 6-deoxy-1,4-dideoxy-1,4-imino-D-mannitol (6-deoxy-DIM), or 6-deoxy-6-fluoro-1,4-dideoxy-1,4-imino-D-mannitol (6-deoxy-6-fluoro-DIM). Cultivation of ASM in the presence of such inhibitors can result in the production of high mannose ASM. In some embodiments, cells expressing ASM can contain a nucleic acid sequence, such as an antisense molecule or a ribozyme, that can bind to or inactivate a cellular mannosidase nucleic acid sequence (e.g., mRNA) and inhibit expression of the protein. See, for example, International Patent Application Publication No. WO 02 / 15927 and US Pat. No. 7,138,262, which are incorporated by reference in their entireties, for methods of producing high mannose proteins.

[0126] In certain embodiments, the carbohydrate chains of recombinantly expressed ASM enzymes are remodeled by sequence treatment with various enzymes, such as neuraminidase, galactosidase, and β-N-acetylglucosaminidase. See, e.g., U.S. Patent No. 5,549,892, incorporated herein by reference in its entirety, for methods of remodeling carbohydrate chains of lysosomal enzymes.

[0127] Mannose-6-phosphate (M6P)-mediated ASM uptake can be enhanced by modifying ASM to generate hyperphosphorylated mannose residues and M6P. For example, ASM can be modified by recombinantly introducing additional mannose-6-phosphate into ASM to enhance cellular uptake. See, for example, Matsuoka et al., 2010 Mole. Ther.18:1519-1526, which is incorporated herein by reference in its entirety. In other embodiments, ASM can be conjugated to phosphorylated oligosaccharide derivatives that contain mannose-6-phosphate (M6P). See, for example, U.S. Patent No. 7,001,994, U.S. Patent Application Publication No. US2010 / 0173385, and International Publication No. WO2010 / 075010. In another approach, yeast culture systems can be used to express recombinant ASM that contains additional hyperphosphorylated mannose-6-phosphate residues. See, e.g., Akeboshi et al., 2009 Glycobiology 19(9):1002-1009, incorporated herein by reference in its entirety.

[0128] Once ASM is produced, it can be isolated or purified by any method known in the art for protein isolation or purification, such as, for example, chromatography (e.g., ion exchange, high performance liquid chromatography, affinity, particularly affinity for ASM, with protein A, and sizing column chromatography), centrifugation, absorption differential solubility, or any other standard technique for protein isolation or purification. If the ASM enzyme is secreted by cultured cells, ASM can be readily recovered from the medium. For methods of purifying ASM, see, for example, He et al., 1999, Biochimia et Biophsyica Acta 1432: 251-264.

[0129] The ASM enzyme can be formulated for any route of administration (e.g., injection, subcutaneous, intramuscular, intrathecal, intracerebroventricular, intranasal, inhalation, or intradermal). The ASM enzyme can be provided in lyophilized form and reconstituted, for example, with sterile saline (e.g., 0.9% sodium chloride) or sterile water prior to use. Alternatively, the ASM enzyme can be provided in an aqueous solution. In certain embodiments, the ASM is administered to the subject in a formulation that includes zinc. In some embodiments, the ASM enzyme is infused into the patient using, for example, a syringe pump or an infusion bag with a pump.

[0130] In certain embodiments, ASM is administered to a subject in a carrier such as liposome or polycationic carrier. For carriers that can be used for administering ASM, see, for example, U.S. Patent No. 5,716,614, which is incorporated herein by reference in its entirety. In some embodiments, ASM is administered to a subject in an ICAM-1 targeted nanocarrier. For ASM delivery using ICAM-1 targeted nanocarriers, see, for example, Muro et al., 2006, Mol. Ther. 13(1): 135-141, which is incorporated herein by reference in its entirety.

[0131] Patient population In a specific embodiment, the subject treated for ASMD according to the methods provided herein is a human who has or has been diagnosed with one or more mutations in the gene encoding acid sphingomyelinase. In a particular embodiment, the subject treated for ASMD according to the methods provided herein is a human who has or has been diagnosed with Niemann-Pick disease (NPD). In one embodiment, the subject treated for ASMD according to the methods provided herein is a human who has or has been diagnosed with NPD type A. In another specific embodiment, the subject treated for ASMD according to the methods provided herein is a human who has or has been diagnosed with NPD type B.

[0132] In certain embodiments, the subject treated for ASMD according to the methods provided herein has one or more mutations in the SMPD1 gene. In some embodiments, the mutation is a missense mutation. In other embodiments, the mutation is a deletion resulting in the deletion of one or more amino acid residues. In specific embodiments, the subject treated for ASMD according to the methods provided herein has one or more mutations.

[0133] [Table 1] TIFF0007605949000003.tif218169 TIFF0007605949000004.tif218169 TIFF0007605949000005.tif103169

[0134] For mutations in the acid sphingomyelinase gene (designated SMPD1), see, e.g., Simonaro et al., 2002, Am. J. Hum. Genet. 71: 1413-1419, incorporated herein by reference in its entirety.

[0135] In certain embodiments, a subject treated for ASMD in accordance with the methods provided herein endogenously expresses ASM at 2-5%, 5-10%, 5-15%, 5-20%, 5%-30%, or 20%-30% of the activity of normal human ASM (e.g., ASM-1). In some embodiments, a subject treated for ASMD in accordance with the methods provided herein endogenously expresses ASM at less than 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. Patent Nos. 4,039,388, 4,082,781, 5,686,240, 7,563,591, and International Publication Nos. WO 2007 / 078806 and WO 2006 / 058385, which are incorporated by reference in their entireties. In a specific embodiment, a fluorescence-based high performance liquid chromatography assay is used to measure ASM activity as described in He et al., 2003, Analytical Biochemistry 314: 116-120, which is incorporated by reference herein.

[0136] In certain embodiments, the subject treated for ASMD according to the methods provided herein shows one or more symptoms of NPD.Symptoms of NPD include, but are not limited to, abdominal distension, hepatomegaly, splenomegaly, hepatosplenomegaly, neutropenia, pulmonary disease, lymphadenopathy, histochemical presence of NPD-specific foam cells, anemia (e.g., microcytic anemia), thrombocytopenia, repeated vomiting, chronic constipation, growth retardation (e.g., linear growth and weight loss), delayed puberty, repeated bruising, repeated bleeding, atherogenic lipid profile (high cholesterol, triglycerides, LDL, and low HDL), pain (headache, back, limbs, abdomen), fatigue, early satiety, low endurance, osteopenia, neurological signs, and dyspnea (e.g., interstitial lung disease, shortness of breath).Neurological signs of NPD include cherry red spots, hypotension, muscle weakness, psychomotor retardation, spasticity, social reluctance, irritability, and convulsions.

[0137] In certain embodiments, the subject treated for ASMD according to the methods provided herein is a human subject that exhibits two or more clinical features consistent with non-neurogenic NPD.In a specific embodiment, the subject treated for [ASMD] according to the methods described herein is a human subject that exhibits two or more clinical features consistent with non-neurogenic NPD (thrombocytopenia, anemia, neutropenia, hepatomegaly, splenomegaly, and pulmonary disease).In other embodiments, the subject treated for ASMD according to the methods provided herein is a human subject that exhibits two or more clinical features consistent with neurogenic NPD.

[0138] In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human having a spleen volume that is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, or 12-fold larger than the spleen volume of a healthy individual, as assessed by techniques well known in the art, e.g., magnetic resonance imaging (MRI). In specific embodiments, a subject treated for ASMD according to the methods provided herein is a human having a spleen volume that is 8-fold larger than the spleen volume of a healthy individual, as assessed by techniques well known in the art, e.g., MRI. In some embodiments, a subject treated for ASMD according to the methods provided herein is a human having a spleen volume that is 8-12-fold, 9-12-fold, 10-12-fold, or 12-14-fold larger than the spleen volume of a healthy individual, as assessed by techniques well known in the art, e.g., MRI. In a specific embodiment, a subject treated for ASMD in accordance with the methods provided herein is a human with a spleen volume greater than or equal to 8 times normal (MN: multiplicative to normal) (i.e., 1.6% of body weight) as assessed by techniques well known in the art, such as, for example, MRI.

[0139] In certain embodiments, subjects treated for ASMD according to the methods provided herein have a lung diffusion capacity (DL) of less than that of a healthy individual. CO ) DL between 20%-80%, 25%-80%, 30%-80%, 40%-80%, 50%-80%, or 60%-80% of predicted value COIn certain embodiments, the subject treated for ASMD according to the methods provided herein is a human having a predicted DL of a healthy individual. CO DL between 20%-90%, 25%-90%, 30%-90%, 40%-90%, 50%-90%, 60%-90%, or 70%-90% CO DL is a human being who has CO DL measures the diffusion rate per minute of a diffusion-limited gas (e.g., carbon monoxide) across the alveolar-capillary membrane. CO DL can be calculated by comparing the amount of carbon monoxide exhaled after inhaling a known amount of carbon monoxide. CO To assess the .alpha.-amino acid sequence, techniques known in the art can be used.

[0140] In certain embodiments, the subject treated for ASMD according to the methods provided herein is a human infant. In some embodiments, the subject treated for ASMD according to the methods provided herein is a human aged 2-3 months, 2-6 months, 3-6 months, 4-6 months, 5-8 months, or 6-9 months. In other embodiments, the subject treated for ASMD according to the methods provided herein is a human child. In some embodiments, the subject treated for ASMD according to the methods provided herein is a human aged 1-3 years, 2-3 years, 3-5 years, 4-5 years, 5-7 years, or 6-9 years.

[0141] In certain embodiments, the subject treated for ASMD according to the methods provided herein is an adult. In other embodiments, the subject treated for ASMD according to the methods provided herein is an elderly person. In some embodiments, the subject treated for ASMD according to the methods provided herein is a human aged 10-18 years, 10-20 years, 12-20 years, 15-20 years, 20-25 years, 21-25 years, 21-30 years, 25-30 years, 30-35 years, 35-40 years, 40-45 years, 45-50 years, 50-55 years, 55-60 years, 60-65 years, 65-70 years, 70-75 years, 75-80 years, 80-85 years, 85-90 years, 90-95 years, or 95-100 years.

[0142] In a specific embodiment, the subject treated for ASMD according to the methods provided herein is a human female. In another embodiment, the subject treated for ASMD according to the methods provided herein is a human male. In a specific embodiment, the subject treated for ASMD according to the methods provided herein is a human female who is not pregnant or breastfeeding. In another embodiment, the subject treated for ASMD according to the methods provided herein is a human female who is pregnant or will become pregnant or breastfeeding.

[0143] In certain embodiments, the subject treated for ASMD according to the methods provided herein is a human who has not undergone a major organ transplant (e.g., liver or bone marrow transplant). In other embodiments, the subject treated for ASMD according to the methods provided herein is a human who has undergone a major organ transplant (e.g., liver or bone marrow transplant). In some embodiments, the subject treated for ASMD according to the methods provided herein is a human who has not undergone a total or partial splenectomy. In other embodiments, the subject treated for ASMD according to the methods provided herein is a human who has undergone a total or partial splenectomy.

[0144] In certain embodiments, subjects treated for ASMD according to the methods provided herein are humans who do not have or have not been diagnosed with one of the following: active hepatitis B, active hepatitis C, human immunodeficiency virus (HIV) infection, cirrhosis of the liver, or significant cardiac disease (e.g., moderate or severe pulmonary hypertension or valvular insufficiency, or left ventricular ejection fraction of less than 50%, less than 40%, less than 30%, or less than 20% by echocardiography). In other embodiments, subjects treated for ASMD according to the methods provided herein are humans who have or have been diagnosed with one or more of the following: active hepatitis B, active hepatitis C, human immunodeficiency virus (HIV) infection, cirrhosis of the liver, or significant cardiac disease (e.g., moderate or severe pulmonary hypertension or valvular insufficiency, or left ventricular ejection fraction of less than 50%, less than 40%, less than 30%, or less than 20% by echocardiography).

[0145] In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human who does not have any of the following: an International Normalized Ratio (INR) greater than 1.25, 1.5, 1.75, or 2; an INR greater than 60×10 3 In other embodiments, the subject to be treated for ASMD in accordance with the methods provided herein is a human with one or more of the following: an international normalized ratio (INR) of greater than 1.25, 1.5, 1.75, or 2; 3 platelet count less than 1 / μL, alanine aminotransferase (ALT) greater than 250 IU / L, aspartate aminotransferase greater than 250 IU / L, or total bilirubin greater than 1.5 mg / dL, 1.75 mg / dL, or 2 mg / dL.

[0146] In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human not taking any of the following medications: chlorpromazine, imipramine, or desipramine. In other embodiments, a subject treated for ASMD according to the methods provided herein is a human taking one or more of the following medications: chlorpromazine, imipramine, or desipramine.

[0147] In certain embodiments, subjects treated for ASMD according to the methods provided herein are humans who are not taking herbal supplements or medications that may cause or prolong bleeding (e.g., anticoagulants, ibuprofen, aspirin, garlic supplements, ginkgo biloba, and ginseng) or that are potentially hepatotoxic (e.g., 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors, erythromycin, valproic acid, antidepressants, kava, and echinacea). In other embodiments, subjects treated for ASMD according to the methods provided herein are humans who are taking one or more of the following herbal supplements or medications: anticoagulants, ibuprofen, aspirin, garlic supplements, ginkgo biloba, ginseng, 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors, erythromycin, valproic acid, antidepressants, kava, and echinacea.

[0148] In specific embodiments, the subject treated for ASMD in accordance with the methods provided herein is a human who meets one, two or more, or all of the criteria for a subject in the Examples in Sections 6, 8, 9 and following.

[0149] 5.3.Treatment monitoring According to the methods provided herein, multiple parameters (e.g., factors or markers) can be monitored before, during, and / or after administration of a dose of ASM. In certain embodiments, a physical examination is performed before administration of ASM as needed or recommended during the course of treatment with ASM. The physical examination can include evaluation of the following: general appearance, skin, head, ears, eyes, nose, and throat (HEENT), lymph nodes, heart, lungs, abdomen, limbs / joints, neurological, mental status, and reflexes. In certain embodiments, vital signs, continuous heart rate, respiratory rate, temperature, and oxygen saturation can be assessed before, during, and / or after administration of a dose of ASM. Heart rate can be monitored continuously by telemetry, for example, starting 6 hours prior to administration of a dose of ASM and up to 72 hours after administration of a dose of ASM.

[0150] In certain embodiments, blood white blood cell percentage, blood urea nitrogen (BUN), bicarbonate, creatinine, glucose, uric acid, calcium, phosphate, albumin, total protein, sodium, potassium, chloride, lactate dehydrogenase, creatinine kinase, MB fraction of creatinine kinase, and urinalysis (including urine color, appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, hemoglobin, and microscopy, if necessary) may be performed at designated times before, during, and / or after administration of the ASM dose. In certain embodiments, liver tests, coagulation studies, and / or fasting lipid profiles may be performed before, during, and / or after administration of the ASM dose. Liver function tests may include evaluation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (AP), gamma-glutamyltransferase (GGT), total bilirubin, and direct bilirubin concentrations. Coagulation studies may include evaluation of prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), D-dimer concentration, and fibrinogen concentration. Fasting lipid profile evaluation may include evaluation of total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), and triglycerides.

[0151] In certain embodiments, a skin biopsy is performed before, during, and / or after administration of a dose of ASM. In some embodiments, a liver biopsy is performed before, during, and / or after administration of a dose of ASM. Sphingomyelin levels in skin and liver biopsies can be assessed by metamorpha histological analysis.

[0152] In certain embodiments, pulmonary function tests are performed before, during, or / and after administration of a dose of ASM. Calibration of pulmonary function testing equipment and test administration protocols can be standardized according to American Thoracic Society (ATS) guidelines (ATS, 1991, Am Rev Respir Dis 144: 1202-1218). In certain embodiments, percent predicted forced vital capacity (FVC) is assessed before, during, and after administration of a dose of ASM. FVC is the total volume of air exhaled during a forced breath. FVC can be measured using standard spirometry techniques.

[0153] In certain embodiments, forced expiratory volume in 1 second (FEV1) can be performed before, during, and after administration of a dose of ASM. FEV1 is the amount of air exhaled in the first second of the FVC. FEV1 can be measured using standard spirometry techniques.

[0154] In certain embodiments, total lung capacity (TLC) can be assessed before, during, and after administration of a dose of ASM. TLC is the total volume of air in the lungs after a maximal inspiratory effort. TLC can be measured using whole-body plethysmography.

[0155] In certain embodiments, DLco can be assessed before, during, and after administration of a dose of ASM. DLco measures the diffusion-limited diffusion rate per minute of gas (carbon monoxide, CO) across the alveolar-capillary membrane. DLco can be calculated by comparing the amount of exhaled CO after inhaling a known amount of carbon monoxide (CO). Helium, which does not diffuse across the alveolar-capillary membrane, can be included as a tracer in the inspired CO to control for air trapping.

[0156] In certain embodiments, chest x-rays (posterior, anterior and lateral) can be obtained before, during and / or after administration of a dose of ASM. In certain embodiments, an abdominal MRI is obtained before, during and / or after administration of a dose of ASM.

[0157] In certain embodiments, blood samples are taken before, during and / or after administration of a dose of ASM to assess biomarkers, bilirubin concentration, neutrophil percentage of total white blood cells. In specific embodiments, the concentration of one or more of the following biomarkers is assessed using techniques well known in the art: CRP / hs-CRP concentration, sphingomyelin concentration, iron concentration, ferritin concentration, calcitonin concentration, albumin concentration, SAA, serum amyloid P component, ACE, CCL18, chitotriosidase, transferrin, fibrinogen concentration, and plasma sphingomyelin concentration, plasma ceramide concentration. In specific embodiments, the concentration of one or more of the following cytokines is assessed using techniques well known in the art: IL-6 and IL-8.

[0158] In certain embodiments, the concentration of antibodies to ASM (e.g., anti-recombinant human ASM IgG and / or anti-recombinant human ASM IgE) is assessed before, during, and / or after administration of a dose of ASM. In some embodiments, the concentration of one or more complement factors is assessed before, during, and / or after administration of a dose of ASM. In certain embodiments, the concentration of serum tryptase is assessed before, during, and / or after administration of a dose of ASM. In certain embodiments, skin tests are assessed to determine IgE-mediated responses to ASM before, during, and / or after administration of a dose of ASM.

[0159] In certain embodiments, the pharmacokinetic profile of ASM is measured before, during, and / or after administration of a dose of ASM. In some embodiments, BAL cell counts are measured before, during, and / or after administration of a dose of ASM. See Section 8 et al. (below) for other parameters that may be assessed before, during, and / or after administration of a dose of ASM.

[0160] In certain embodiments, the factors or markers described herein are evaluated 1 week, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes before administration of a dose of ASM. In some embodiments, the factors or markers described herein are evaluated during administration of a dose of ASM. For example, the factors or markers described herein are evaluated during administration of a dose of ASM. In certain embodiments, the factors or markers described herein are evaluated 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more after administration of a dose of ASM. In certain embodiments, the factors or markers described herein are evaluated hours or weeks after a particular dose of ASM. For example, the factors or markers described herein are evaluated every 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after a particular number of doses of ASM. In certain embodiments, the factors or markers are evaluated every 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some examples, the factors or markers are evaluated every 1-2 months, 1-4 months, 2-4 months, 2-4 months, 3-4 months, 2-5 months, 3-5 months, 4-5 months, 2-6 months, 3-6 months, 4-6 months, or 5-6 months. In certain embodiments, the factors or markers are evaluated every 6-8 months, 6-12 months, 8-12 months, 9-12 months, or 10-12 months. In some embodiments, none of the factors or markers described herein are assessed before, during and / or after administration of a dose of ASM.

[0161] In certain embodiments, the results of the evaluation of one or more factors or markers indicate that the dosage of ASM should be adjusted.

[0162] 5.4. Biological Samples According to the methods described herein, biological samples undergo one or more pretreatment steps prior to detection and / or measurement of cell populations, factors or markers (e.g., biomarkers). In certain embodiments, biological fluids are pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps. In other embodiments, tissue samples are pretreated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization, or homogenization followed by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps. In some embodiments, the sample is pretreated by removing specific types of cells from the sample or by removing debris from the sample prior to determining the number or amount of specific cell types in the sample according to the methods described herein.

[0163] The biological sample may be a tissue sample, biological fluid, secretion, or any other sample from a human subject. In some embodiments, the biological sample is a blood sample or a bone marrow sample. In certain embodiments, the biological sample is a tissue sample (e.g., a liver, skin, or lung biopsy). In some embodiments, the biological sample is a biological fluid, such as urine. In certain embodiments, the biological sample is a sputum or nasal secretion sample. In some embodiments, the biological sample is a mouth swab.

[0164] Techniques well known in the art can be used to assess the presence, absence, number, amount or percentage of a particular type of cell present in a biological sample. For example, cells can be sorted using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorter (FACS) is a known method of sorting particles, including cells, based on the fluorescent properties of the particles. See, for example, Kamarch, 1987, Methods Enzymol 151:150-165. Laser excitation of fluorescent moieties on individual particles creates a small charge, allowing electromagnetic separation of positively and negatively charged particles from a mixture. Antibodies or ligands used to detect antigenic determinants on the cell surface of specific cells are labeled with a fluorescent dye, such as FITC or phycoerythrin. Cells are incubated with the fluorescently labeled antibody or ligand for a time sufficient for the labeled antibody or ligand to bind to the cells. The cells are processed through a cell sorter to separate the cells of interest from other cells. FACS sorted particles can be placed directly into individual wells of a microtiter plate to facilitate separation.

[0165] Cells can also be separated using magnetic beads. For example, they can be sorted using magnetic activated cell sorting (MACS) technology, a method for separating particles based on their ability to bind to magnetic beads (0.5-100 μm in diameter). A variety of useful modifications can be performed on magnetic microspheres, including the covalent attachment of antibodies that specifically recognize cell solid-phase surface molecules or haptens. A magnetic field is then applied to physically manipulate the selected beads. In a specific embodiment, antibodies against blood cell surface markers are bound to the magnetic beads. The beads are then mixed with a blood cell culture to allow binding. The cells are then passed through a magnetic field to select cells with the blood cell surface marker of interest. In this way, these cells can be isolated.

[0166] In some embodiments, the surface of a culture dish is coated with an antibody and used to select cells in a method called panning. A separate dish can be coated with an antibody specific for a particular cell. The cells are first added to a dish coated with an antibody specific for the blood cells of interest. After thorough rinsing, the cells that remain attached to the culture dish are cells that express the cell marker of interest. Examples of cell surface antigenic determinants or markers include, but are not limited to, CD2 on T lymphocytes and natural killer cells, CD3 on T lymphocytes, CD11a on white blood cells, CD28 on T lymphocytes, CD19 on B lymphocytes, CD20 on B lymphocytes, CD21 on B lymphocytes, CD22 on B lymphocytes, CD23 on B lymphocytes, CD29 on white blood cells, CD14 on monocytes, CD41 on platelets, CD61 on platelets, CD66 on granulocytes, CD67 on granulocytes, and CD68 on monocytes and macrophages.

[0167] The presence, absence, concentration or amount of markers (including biomarkers) or factors can be evaluated using techniques well known in the art. The presence, absence, concentration or amount of markers (including biomarkers) or factors can be measured at the protein level and / or RNA level using techniques well known to those skilled in the art. At the protein level, immunoassays such as ELISA and immunoprecipitation and Western blot can be used to measure the presence, absence, concentration or amount of markers (including biomarkers) or factors. In addition, FACS can be used to measure the presence, absence, concentration or amount of markers (including biomarkers) or factors. At the RNA level, RT-PCR and Northern blot can be used to measure the presence, absence, concentration or amount of markers (including biomarkers) or factors.

[0168] Combination therapy In some embodiments, the method of treating ASMD involves administering ASM in combination with one or more additional therapeutic agents.As used herein, the term "combined" in the context of administering ASM refers to administering a dose of ASM before, simultaneously with, or after administration of one or more additional therapeutic agents (e.g., drugs) used to treat ASMD.The use of the term "combined" does not limit the order in which ASM and one or more additional therapeutic agents are administered to a subject. In specific embodiments, the interval between the dose of ASM and the administration of the one or more additional therapeutic agents can be about 1-5 minutes, 1-30 minutes, 30-60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, or any time period in between. In certain embodiments, the doses of ASM and the one or more additional therapeutic agents are administered less than one day, one week, two weeks, two weeks, three weeks, one month, two months, three months, six months, one year, two years, or five years apart.

[0169] In some embodiments, the combination therapy provided herein involves administering a dose of ASM every 2-4 weeks, and administering one or more additional therapeutic agents once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 2 months (e.g., about 8 weeks), once every 3 months (e.g., about 12 weeks), or once every 4 months (e.g., about 16 weeks). In certain embodiments, the ASM and one or more additional therapeutic agents are administered to the subject in a cyclical manner. Cycling therapy involves administering ASM for a period of time, followed by administration of one or more additional therapeutic agents for a period of time, and repeating this sequential administration. In certain embodiments, cycling therapy may include a drug holiday during which ASM or additional therapeutic agent is not administered for a period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years). In one embodiment, the number of administration cycles is 1 to 12 cycles, 2 to 10 cycles, or 2 to 8 cycles.

[0170] In some embodiments, the methods of treating ASMD provided herein include administering ASM as a single agent for a period of time prior to administering ASM in combination with an additional therapeutic agent. In certain embodiments, the methods of treating ASMD provided herein include administering only the additional therapeutic agent for a period of time prior to administering ASM in combination with the additional therapeutic agent.

[0171] In some embodiments, administration of ASM and one or more additional therapeutic agents in accordance with the methods described herein has an additive effect compared to administration of ASM or one or more of the additional therapeutic agents alone, hi some embodiments, administration of ASM and one or more additional therapeutic agents in accordance with the methods described herein has a synergistic effect compared to administration of ASM or one or more of the additional therapeutic agents alone.

[0172] As used herein, the term "synergistic" refers to the effect of administering a dose of ASM in combination with one or more additional therapeutic agents (e.g., drugs) where the combination is more effective than the additive effect of any two or more single therapeutic agents (e.g., drugs). In specific embodiments, the synergistic effect of the combination therapy allows for the use of lower dosages (e.g., suboptimal dosages) of ASM or additional therapeutic agents and / or allows for less frequent administration of ASM or additional therapeutic agents to a subject. In certain embodiments, the ability to utilize lower dosages of ASM or additional therapeutic agents and / or allow for less frequent administration of ASM or additional therapeutic agents reduces the toxicity associated with administration of ASM or additional therapeutic agents to a subject without decreasing the efficacy of ASM or additional therapeutic agents in treating ASMD. In some embodiments, the synergistic effect results in improved efficacy of ASM and additional therapeutic agents in treating ASMD. In some embodiments, the synergistic effect of the combination of ASM and one or more additional therapeutic agents avoids or reduces adverse or undesirable side effects associated with the use of any single therapeutic agent.

[0173] In specific embodiments, one or more additional therapeutic agents are administered to a subject in combination with ASM to reduce or ameliorate one or more of the following that may be associated with administration of a particular dose of ASM: (i) associated adverse events; (ii) total bilirubin levels that exceed the bilirubin levels of humans without ASMD (e.g., healthy subjects); (iii) plasma ceramide levels that exceed the plasma ceramide levels of humans without ASMD (e.g., healthy subjects); or (iv) acute phase reactions. In specific embodiments, one or more additional therapeutic agents are administered to a subject in combination with ASM to improve lung function while minimizing one or more of the following that may be associated with administration of a particular dose of ASM: (i) associated adverse events; (ii) total bilirubin levels that exceed the bilirubin levels of humans without ASMD (e.g., healthy subjects); (iii) plasma ceramide levels that exceed the plasma ceramide levels of humans without ASMD (e.g., healthy subjects); or (iv) acute phase reactions.

[0174] In certain embodiments, ASM is administered in combination with one or more additional therapeutic agents to a subject to control or reduce symptoms associated with ASMD. In some embodiments, the one or more additional therapeutic agents administered in combination with ASM to a subject are analgesics. Specific examples of therapeutic agents that can be administered in combination with ASM include, but are not limited to, N-acetyl-L-cysteine ​​(NAC), S-adenosyl-L-methionine (SAM), interleukin (IL)-6 antibody, IL-6 receptor antibody, dexamethasone, L-Nil (selective inhibitor of inducible NOS), L-NAME (selective inhibitor of NOS), basic fibroblast growth factor (b-FGF), imipramine (sphingomyelinase inhibitor), D609 (sphingomyelinase inhibitor), and N-oleoylethanolamine (NOE, ceramide inhibitor).

[0175] In certain embodiments, chaperones, such as small molecule chaperones, are administered in combination with ASM.For example, for the drugs (e.g., small molecules) that can be administered to subjects in combination with ASM, see U.S. Patent No. 7,750,050 and International Publication Nos. WO 2004 / 045574 and WO 2010 / 015816, which are incorporated herein by reference.In some embodiments, chaperones (e.g., small molecule chaperones) perform one, two or all of the following actions: increase the targeting of ASM to the lesion site, stabilize the activity of ASM, and enhance the activity of ASM.

[0176] In some embodiments, a glucocorticosteroid, such as dexamethasone, is administered in combination with the ASM. For agents (e.g., glucocorticosteroids) that may be administered in combination with ASM, see, e.g., U.S. Patent No. 7,658,916, which is incorporated herein by reference in its entirety.

[0177] In some embodiments, a substrate reducer is administered to a subject in combination with ASM. In a specific embodiment, a molecule (e.g., a small molecule) that reduces the amount of sphingomyelin, decreases the rate of sphingomyelin synthesis, or both, is administered to a subject in combination with ASM. For sphingomyelin synthase inhibitors, such as tricyclodecan-9-yl-xanthogenate and sphingomyelin synthase siRNA, see, for example, Li et al. 2007, Biochim Biophys Act 1771(9):1186-1194 (incorporated herein by reference in its entirety).

[0178] In some embodiments, a therapeutic agent that reduces the immunogenic potential of ASM is administered to the subject in combination with ASM, hi certain embodiments, Benadryl is administered to the subject in combination with ASM.

[0179] The combination of ASM and one or more additional therapeutic agents can be administered to a subject in the same pharmaceutical composition. Alternatively, the ASM and one or more additional therapeutic agents can be administered to a subject simultaneously in separate pharmaceutical compositions. The ASM and one or more additional therapeutic agents can be administered to a subject sequentially in separate pharmaceutical compositions. The ASM and one or more additional therapeutic agents can also be administered to a subject by the same route of administration or by different routes of administration.

[0180] 5.6.Medicines In one aspect, a final packaged and labeled pharmaceutical product is described herein. In one embodiment, the pharmaceutical product comprises a unit dosage form of ASM in a suitable vessel or container (e.g., a glass vial or other sealed container). In some embodiments, the unit dosage form is a lyophilized formulation of ASM, and under these circumstances, the pharmaceutical product may include a second container of sterile saline or sterile water for reconstituting the lyophilized formulation of ASM. In other embodiments, the unit dosage form is a water-soluble formulation of ASM that does not require reconstitution prior to administration to a subject. In specific embodiments, the unit dosage form of ASM comprises an amount of ASM sufficient to administer a non-toxic, low dose of the enzyme to a subject. In certain embodiments, the unit dosage form is suitable for a selected route of administration of the ASM to a subject. In specific embodiments, the unit dosage form is suitable for intravenous delivery to a subject.

[0181] In one embodiment, the pharmaceutical product comprises a unit dosage form of the ASM in a suitable vessel or container (e.g., a glass vial or other sealed container) and an infusion or syringe pump for administration of the ASM to a subject.

[0182] As with other pharmaceutical products, packaging materials and containers are designed to ensure product stability during storage and delivery. In addition, the pharmaceutical product includes instructions for use or other informational materials to advise physicians, technicians, or patients on the appropriate method of treating ASMD. That is, the pharmaceutical product includes instruction means that indicate or suggest a dosing regimen, including but not limited to actual doses (e.g., dose escalation protocols), monitoring procedures, and other monitoring information. In certain embodiments, the pharmaceutical product includes a method for determining the genotype of the patient (e.g., PCR primers for the SMPD1 gene).

[0183] 6. Example 1: Clinical Trial of Recombinant Human Acid Sphingomyelinase (rhASM) Enzyme Replacement Therapy in Adults with ASM Deficiency 6.1. Introduction ASMD is an autosomal recessive lysosomal storage disorder that occurs when sphingomyelin is not broken down normally into ceramide and phosphorylcholine. As a result, sphingomyelin accumulates intracellularly, primarily in the reticuloendothelial system, leading to hepatosplenomegaly, anemia, thrombocytopenia, and interstitial lung disease. Growth retardation and an atherogenic lipid profile are also common findings. Patients with little or no residual ASM activity are most severely affected, exhibiting childhood onset, growth failure, neurodegeneration, and death by age 3 (Niemann-Pick disease type A, NPD A). Patients with higher residual ASM activity have variable ages of onset, heterogeneous findings and physical symptoms, little or no neurological impairment, and generally survive into adulthood (NPD B). Currently, there is no treatment for patients with ASMD.

[0184] Enzyme replacement therapy (ERT) has been used successfully to treat several lysosomal storage disorders, including Gaucher disease, mucopolysaccharidoses I, II, and VI, Fabry disease, and Pompe disease. Recombinant human lysosomal enzymes are administered intravenously and internalized by receptor-mediated endocytosis for subsequent targeting to lysosomes. Proof of principle for the treatment of ASMD was demonstrated by the laboratory of Dr. Edward Schuchman (Mount Sinai Medical Center), where intravenous injection of recombinant human ASM (rhASM) effectively reduced sphingomyelin levels in the liver and spleen, and to a lesser extent in the lungs, in an ASM knockout mouse (ASMKO) model (Miranda, et al., FASEB 2000;14:1988). However, rhASM cannot cross the blood-brain barrier, and therefore did not reduce sphingomyelin levels in the brain. ASMKO mice have no residual ASM activity or protein and rapidly develop severe neurological disease, making these animals the best model of NPD type A (see Buccinna et al., 2009, J. Neurochem. 109:105-115).

[0185] Further studies confirmed that biweekly administration of rhASM reduced sphingomyelin levels in ASMKO mice in a dose-dependent manner (0.3-3 mg / kg). The no observed adverse effect levels (NOAELs) for single and repeated dosing were determined to be 0.3 mg / kg and 3 mg / kg, respectively, in ASMKO mice. Subsequent attempts to deplete pulmonary sphingomyelin levels with higher doses of rhASM caused unexpected toxicity. At doses of 10 mg / kg and above, only ASMKO mice, but not normal animals, experienced hepatitis, adrenal hemorrhage, cardiovascular shock and death accompanied by large elevations in cytokine levels, suggesting cytokine release syndrome. The toxicity and cytokine elevations seen with high doses of rhASM could be ameliorated or prevented by pretreating ASMKO mice several times with lower doses of rhASM, suggesting that the rate and amount of sphingomyelin degradation plays an important role.

[0186] In ASMKO mice, a model of NPD type A, no toxicity was observed following single doses of rhASM at or below 0.3 mg / kg and repeated doses of rhASM at or below 3.0 mg / kg, and no severe toxicity was observed until single doses of rhASM at or above 10 mg / kg were administered. Thus, a conservative starting dose of 0.03 mg / kg of rhASM was selected for single dosing in human subjects to ensure a 10-fold safety margin relative to the single dose NOAEL (0.3 mg / kg) observed in ASMKO mice. A maximum dose of 1.0 mg / kg of rhASM was selected for single dosing in human subjects to ensure a 10-fold safety margin relative to the dose at which severe toxicity was observed in ASMKO mice (10 mg / kg). Upon completion of the protocol specified below, toxicity in human subjects was unexpectedly observed at doses as low as 0.3 mg / kg of rhASM.

[0187] Materials and Methods 6.2.1. Design of human protocols This protocol was a single-center, single-dose, dose-escalation phase 1 study. The primary objective of the study was to evaluate the safety and pharmacokinetics of a single dose of rhASM in adults with non-neuropathic ASMD (Niemann-Pick B). Single doses of 0.03, 0.1, 0.3, 0.6, and 1.0 mg / kg of rhASM were infused sequentially in dose cohorts. The initial study design called for a minimum of 15 patients (five cohorts with three patients each). Due to difficulties with patient enrollment, the protocol was modified to enroll three patients each in the first two cohorts and two patients each in the last three cohorts. An independent data monitoring committee monitored the study, and all protocol procedures were approved by the IRB. Figure 1 shows the patient flow of the protocol.

[0188] 6.2.2.Patient To be eligible for the protocol, patients had to be 18-65 years of age, have deficiency of ASM enzyme activity, spleen volume ≥2x normal, AST and ALT ≤250 IU / L, bilirubin ≤3.6 mg / dL, INR ≤1.5, DLco >30% of predicted, and platelets ≤60,000 / mL. Patients were excluded if they had cirrhosis (by liver biopsy), significant cardiac disease, had undergone total splenectomy, or were taking medications or herbal supplements that were potentially hepatotoxic, promoted bleeding, or inhibited rhASM.

[0189] A total of 13 patients were enrolled, with 11 patients infused with rhASM. rhASM was produced by overexpression of ASM cDNA in Chinese hamster ovary cells. The mean age of infused patients was 30.8 years, all were white (non-Hispanic / non-Latino), and the mean spleen volume was 10.8 times normal. One patient had undergone partial splenectomy, and the remaining patient's spleens were intact at the time of protocol enrollment. Figure 2 shows the demographics and baseline characteristics of patients on this protocol.

[0190] Once screening was completed and eligibility was confirmed, patients were admitted to the cardiac intensive care unit (CCU) overnight for baseline telemetry and were infused with rhASM the following morning. Patients were monitored by telemetry for 72 hours after infusion (24 hours in the CCU and 48 hours in the general clinical research center). Patients had an overnight visit on day 14 and an outpatient visit on day 28.

[0191] The following describes the medical examinations performed during the protocol.

[0192] Physical Examination Days 0, 1, 2, 14 and 28 Chemistry, hematology, and urinalysis - pre-infusion, then 24, 48, 72 hours, 14 and 28 days Liver function tests - before infusion, then every 12 hours for up to 72 hours, on days 14 and 28. Aldosterone, cortisol, delta-4-androstenedione - before infusion, then every 12 hours for up to 72 hours ACTH stimulation test - screening, day 14 Telemetry - continuous ECG up to 72 hours, echocardiogram - pre-infusion, end of infusion, and 1, 2, 6, 12 and 4 hours post-infusion, day 14 Cardiac biomarkers (BNP, cardiac troponin I, CPK-MB) - pre-infusion and then 2, 6, 12 and 24 hours Plasma sphingomyelin and ceramide levels - before infusion, then 24, 48, and 72 hours, and on days 14 and 28 Pulmonary function test and CXR-screening, day 14 Liver and spleen volumes by MRI – Screening, day 14 Liver and skin biopsy – Screening, Day 14 Biomarkers – Pre-dose, Days 14 and 28 Anti-rhASM IgG test - pre-infusion, day 28 Pharmacokinetics - within 30 minutes prior to infusion, 15 minutes after infusion start, at the end of infusion, and at 15, 30, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours, and 72 hours after infusion.

[0193] Cytokines (IL-1a, IL-1b, IL-6, G-CSF, GM-CSF, MIP-1a, TNF-a) - pharmacokinetic time points (above) and day 14 6.3.Results As shown in Figures 3A and 3B, plasma ceramide levels and plasma sphingomyelin levels were determined at several time points, respectively. Plasma ceramide levels showed a dose-dependent increase up to 6 hours and peaked at 18–72 hours. Plasma sphingomyelin levels were normal at baseline and did not show a consistent trend over time. In patient no. 12112, who received the highest dose (1 mg / kg), plasma sphingomyelin levels increased after dosing and peaked at 72 hours.

[0194] Figure 4 shows total bilirubin levels determined over several time points in the protocol. Total bilirubin showed a dose-dependent rise through 24 hours, peaking at 48–60 hours. Patient #12112, who received the highest dose (1 mg / kg), had a maximum total bilirubin of 4.7 mg / dL. Proportional increases were seen in direct and indirect bilirubin. No increases were seen in ALT, AST, or alkaline phosphatase. Mild increases in GGT were seen through 72 hours in two patients who received the highest dose (1 mg / kg, patient #12112) and the lowest dose (0.03 mg / kg, patient #10503) (not shown). Hemoglobin and hematocrit levels remained stable throughout the protocol, indicating that hemolysis was not responsible for the increase in bilirubin levels.

[0195] Figures 5A-5G show the levels of acute phase response (inflammatory) markers, CRP / hs-CRP, neutrophil%, fibrinogen, ferritin, IL-8, IL-6, and calcitonin, respectively, determined at several time points for several patients. CRP / hs-CRP showed a transient dose-dependent increase up to 24 hours, peaking at 48-72 hours, and returning to normal by day 14. Other acute phase reactants also showed increases (neutrophil%, fibrinogen, ferritin, PT and PTT) and decreases (iron, albumin). Certain inflammatory biomarkers (e.g., cytokines, IL-6 and IL-8) and calcitonin also showed significant dose-dependent increases, peaking at 48 hours post-infusion. Among these inflammatory mediators, the greatest changes occurred in, in descending order, IL-8 (peaked at 33.8 times the upper limit of normal), calcitonin (peaked at 33.4 times the upper limit of normal), CRP (peaked at 9.8 times the upper limit of normal), and ferritin (peaked at 3.9 times the upper limit of normal). No trends were seen in platelet counts or fibrin split products (not shown). Elevations in laboratory acute-phase reactants correlated in some patients with systemic clinical symptoms of fever, nausea, vomiting, headache, pain, and myalgia.

[0196] Figure 6 is a table of adverse events that occurred during treatment for four patients, each of whom received a different dose of rhASM, and were considered to be (probably, probably, or definitely) related to treatment. As shown in the table, patient number 11509, who received rhASM 0.3 mg / kg, experienced moderate to severe adverse events beginning on day 2.

[0197] Regarding the associated adverse events reported in the protocol, there were no significant cardiovascular changes by telemetry, ECG, echocardiogram, or biomarkers (BNP, cardiac troponin I, CPK-MB) or adrenal hormone insufficiency. One patient (no. 12112, 1.0 mg / kg cohort) had an increase in cortisol levels in the morning 24 hours after dosing, which was considered to possibly represent a normal physiological stress response to some ongoing moderate associated adverse events. In addition, 4 of 6 patients receiving rhASM at 0.3 mg / kg or higher experienced a total of 19 clinical and laboratory-related adverse events that were assessed as drug-related. The intensity of the associated adverse events ranged from mild to severe, but most were moderate and did not require intervention. These included fever (n=2), pain [muscle pain, abdominal, leg, and lower back pain] (n=2), nausea (n=2), scleral icterus and urinary urobilinogen (n=1), fatigue (n=1), vomiting (n=1), lymphocytic infiltration / hepatocyte degeneration on liver biopsy (n=1), acute phase reaction (n=4), increased bilirubin (n=2), and increased fibrin D dimer (n=1). Onset of clinical symptoms began 12 hours after infusion and resolved by 72 hours, except for lower back pain in one patient, which began at 72 hours. On day 14, liver biopsy from one patient (rhASM 0.6 mg / kg, patient no. 12313) showed two new foci of lymphocytic infiltration, one small (0.1 mm in diameter) and the other moderate (0.5 mm in diameter), associated with hepatocyte degeneration. The protocol was discontinued when the first patient in cohort 5 (rhASM 1 mg / kg, patient no. 12112) experienced dose-limiting toxicity of hyperbilirubinemia (peak 4.7 mg / dL).

[0198] The results showed an unexpected dose-dependent delayed onset of clinical and laboratory adverse events at doses of rhASM 0.3 mg / kg and above in humans with ASMD. Furthermore, two important safety laboratory findings were described in light of the results of this protocol. Regarding antibilirubinemia, a dose-proportional increase in direct and indirect bilirubin was observed, but no signs of markers consistent with liver damage (AST, ALT, AP) or hemolysis (hemoglobin and hematocrit) were noted. Two patients had mildly elevated GGT at 48-72 hours, and one patient had two new foci of lymphocytic infiltration, one of which was associated with hepatocellular degeneration.

[0199] Another finding was the acute phase response, i.e. inflammation. Results showed increases in CRP / hs-CRP, neutrophil%, ferritin, IL-6, IL-8, calcitonin, fibrinogen, PY, PTT, and decreases in iron and albumin in response to increasing doses of rhASM. Systemic clinical symptoms of fever, nausea, vomiting, pain, and myalgia are likely related to the acute phase response. Results showed no signs of cytokine release syndrome associated with cardiovascular changes, and no signs of renal hormone disorders.

[0200] 6.4. Conclusion The above protocol represents the first attempt at enzyme replacement therapy in adult patients with ASMD. At bioactive doses in humans, rhASM did not produce cytokine release syndrome associated with cardiovascular changes or adrenal hormone insufficiency. The main safety findings were dose-dependent hyperbilirubinemia and acute phase reactions. Both adverse events are probably related to the breakdown of sphingomyelin to ceramide and phosphorylcholine, although the exact molecular mechanisms are not fully understood. Several safety biomarkers were identified, including bilirubin, ceramide, CRP / hs-CRP, 1L-8, 1L-6, calcitonin, and ferritin. Furthermore, based on hyperbilirubinemia findings, the maximum starting dose of rhASM that was tolerated was 0.6 mg / kg.

[0201] In addition, given that the NOAEL ("no observed adverse effect level") in ASMKO mice was 0.3 mg / kg, it was surprising that the onset of adverse events accompanied by clinical symptoms in patients was observed at a dose as low as 0.3 mg / kg. Notably, clinical symptoms of toxicity in ASMKO mice were not observed until doses of 10 mg / kg or higher were used.

[0202] 7. Example 2: Toxicity Protocol of Repeated Dose Intravenous Injection Followed by Weight Loss Phase in Acid Sphingomyelin Knockout Mice This example describes an investigation of the potential toxicity of repeated intravenous administration of recombinant human acid sphingomyelinase (rhASM) in acid sphingomyelinase knockout (ASMKO) mice, followed by a weight loss phase. Having established that toxic side effects begin to be observed in ASMKO mice with an initial dose of 10 mg / kg rhASM, the following study was designed to determine whether administration of increasing doses of recombinant human acid sphingomyelinase (rhASM) to ASMKO mice would sufficiently reduce accumulated sphingomyelin such that higher doses of rhASM could be administered to lesion target sites such as the lungs and brain of ASMKO mice with minimal or no observable toxicity.

[0203] JK ASMKO mice were administered 3 mg / kg rhASM on study days (SD) 1, 3, 5, and 7 (weakening phase). Beginning on SD day 9, mice received rhASM therapeutic doses of 3, 10, or 30 mg / kg every other week for 13 weeks (7 doses) (treatment phase).

[0204] rhASM was administered intravenously to male and female ASMKO mice via a bolus injection into the lateral tail vein. Groups 1–3 received 3 mg / kg rhASM during the weight loss phase (SD days 1, 3, 5, and 7). Groups 1, 2, and 3 received 3, 10, and 30 mg / kg rhASM, respectively, during the treatment phase (SD days 9, 23, 37, 51, 65, 79, and 93). One subset of mice from each group (the first two mice per sex in group 1, and the first four mice per sex in groups 2–3) was bled pretest as well as 5 min and 4 h after treatment phase doses 1, 4, and 7 for analysis of toxicokinetics (TK) (5 min) and ceramide levels (4 h). Another subset of mice in each group (the two last mice per sex in group 1, and the four last mice per sex in groups 2-3) were bled pre-study as well as 4 and 24 hours after treatment doses 1, 4, and 7 for analysis of rodent multi-analyte profiles (4 hours) and acute phase protein / liver function test levels (24 hours). See study design in Table 2 (below).

[0205] Because of the possibility of anaphylactic reactions beginning with the first treatment dose and each dose thereafter, after test article administration each mouse was closely observed for signs of anaphylaxis (e.g., restlessness, chewing, face rubbing, urticaria, edema, lethargy, and sparse fur). Mice were administered diphenhydramine (DPH) beginning before the first treatment dose and before each subsequent dose.

[0206] DPH at a concentration of 5 mg / mL was prepared by diluting the 50 mg / mL commercial stock solution with sterile 0.9% saline. DPH was administered intraperitoneally (IP) at a dose of 20 mg / kg (4 mL / kg) based on recent body weight, 10–20 min prior to administration of rhASM to avoid potential anaphylactic reactions. If the test animal exhibited a hypersensitivity reaction following test article administration despite DPH pretreatment, a second dose of 10 mg / kg (2 mL / kg) of DPH was administered IP.

[0207] All mice were euthanized by CO2 asphyxiation on SD day 100. After euthanasia, blood was collected by cardiac puncture for clinical pathology and immunogenicity analysis. After blood collection, necropsy was performed on all mice. All tissues were preserved in 10% neutral buffered formalin (NBF) for subsequent histopathological analysis. Portions of liver, spleen, kidney, and lung were preserved in 2% glutaraldehyde / 2% paraformaldehyde for analysis of sphingomyelin loading. Portions of liver, spleen, kidney, and lung were also harvested and stored at or below -70°C for possible future analysis.

[0208] After necropsy, all remaining tissues, including the carcass, were placed in 10% NBF.

[0209] [Table 2]

[0210] In-life findings: Day 1 of the study is considered SD 1 (day 1 of dosing). Animals were weighed weekly during the course of the study starting on SD -1 and then every Monday thereafter. Clinical observations were performed once daily Monday through Friday. All abnormal and normal findings were recorded. Post-dose clinical observations / scoring were performed immediately before, 10-20 minutes, and 50-70 minutes after administration of each dose. All abnormal and normal findings were recorded. Any adverse events were consulted with the attending veterinarian and / or study director and appropriate action was taken based on their advice.

[0211] Euthanasia: If an adverse event occurred affecting the health and welfare of the animals, the animals were euthanized by CO2 asphyxiation and the thoracic, abdominal and cranial cavities were dissected and placed in 10% neutral buffered formalin (NBF) for possible future analysis. If an animal died, the whole carcass was preserved in 10% NBF for possible future analysis. At the end of the study, all surviving animals were euthanized by CO2 asphyxiation.

[0212] Sample Collection: Dose Analysis: Approximately five hundred microliters (500 μL) of test article was collected from all dose levels within 1-10 minutes of each daily dose prescription and stored on dry ice until transferred to a freezer set to maintain a temperature of -70°C or below. Dose analysis samples were transferred on SD days 1, 3, 5, 7, 9, 23, 37, 51, 65, 79, and 93 and stored at -70°C or below until analysis. Dose analysis samples were measured by A280 assay.

[0213] Blood Collection: Mice were anesthetized with a mixture of isoflurane and oxygen to collect blood for analysis of TK, rodent multi-analyte profile, and acute phase protein / liver function test levels. Blood collections were performed on SD days -1, 9, 51, and 93 (pre-study and 1st, 4th, and 7th treatment doses, respectively) according to the blood collection tables (see Tables 4 and 5, below) and the text below. rhASM Toxicokinetics: In the first two mice per sex in group 1, and the first four mice per sex in groups 2-3, blood samples were collected pre-study (SD -1) and 5 min after the first, fourth, and seventh treatment doses for analysis of peak rhASM levels. Blood for all animals was collected from the retro-orbital venous plexus of unconscious mice. Approximately 60 μL of whole blood was collected in hematocrit tubes and allowed to clot at room temperature for at least 1 min. Serum was prepared from these samples by centrifugation at 10,000 revolutions per minute (RPM) for 5 min. After centrifugation, serum was collected and stored on dry ice until transferred to a freezer set to maintain a temperature of -70°C or less. Once transferred, all samples were stored at -70°C or less until analysis. TK samples were transferred on SD days 1, 9, 51, and 93. TK samples were measured by enzyme-linked immunosorbent assay (ELISA). Analysis of Ceramide Levels: In the first two mice per sex in group 1, and the first four mice per sex in groups 2-3, blood samples were collected pre-study (SD -1) and 4 hours after the first, fourth, and seventh treatment phase doses for ceramide level analysis. Blood for these animals was collected from the retro-orbital venous plexus of unconscious mice. Approximately 240 μL of whole blood was collected in potassium EDTA tubes and placed on a Nutator mixer for up to 30 minutes to prevent clot formation. Plasma was prepared from these samples by centrifugation at 10,000 RPM for 5 minutes. After centrifugation, the plasma was collected and stored on dry ice until it was transferred to a freezer set to maintain a temperature below -70°C. Once transferred, all samples were stored below -70°C until analysis by the mass spectrometry group. Ceramide samples were transferred by SD day 94. Ceramide samples were measured by mass spectrometry. Rodent Multianalyte Profile (MAP) Analysis: For the last two mice per sex in group 1, and the last four mice per sex in groups 2-3, blood samples were collected pre-study (SD -1) and 4 hours after the first, fourth, and seventh treatment doses for rodent multianalyte profile analysis. Blood for these animals was collected from the retro-orbital venous plexus of unconscious mice. Approximately 150 μL of whole blood was collected into serum separator tubes and allowed to clot for at least 30 minutes. Serum was prepared from these samples by centrifugation at 10,000 RPM for 5 minutes. After centrifugation, serum was collected and stored on dry ice until transferred to a freezer set to maintain a temperature of -70°C or lower. Once transferred, all samples were stored at -70°C or lower until shipped by SD 94 for rodent multianalyte profile analysis. Samples were shipped on dry ice to Rules Based Medicine. The Rodent Multianalyte Profile Table (Table 3, see below) lists the analytes that were measured.

[0214] [Table 3]

[0215] Analysis of Acute Phase Protein / Liver Function Test Levels: In the last two mice per sex in group 1, and the last four mice per sex in groups 2-3, blood samples were collected pre-study (SD -1) and 24 hours after the first, fourth, and seventh treatment doses for analysis of acute phase protein (serum amyloid A and serum amyloid P) and liver function (bilirubin and alanine aminotransferase (ALT)) levels. Blood for these animals was collected from the retro-orbital venous plexus of unconscious mice. Approximately 150 μL of whole blood was collected into serum separator tubes and allowed to clot for at least 30 minutes. Serum was prepared from these samples by centrifugation at 10,000 RPM for 5 minutes. After centrifugation, serum was collected and stored on dry ice until it was transferred to a freezer set to maintain a temperature of -70°C or lower. Once transferred, all samples were stored at -70°C or lower until they were shipped by SD day 94 for acute phase protein / liver function test analysis. Samples were shipped to Analytics on dry ice.

[0216] [Table 4]

[0217] [Table 5]

[0218] autopsy Terminal blood collection for clinical pathology and immunogenicity analysis: After euthanasia, cardiac puncture was performed on all animals for whole blood collection (approximately 500 μL or more) from all main study animals for the purpose of clinical pathology and immunogenicity analysis. Approximately 150 μL of whole blood was placed into a potassium EDTA tube and gently inverted for hematology parameter analysis. After gentle inversion, all samples were stored at room temperature with shaking on a Nutator mixer until analysis, when they were stored at 2-10 °C. The remaining blood (collected by cardiac puncture following collection of 150 μL for hematology analysis) was placed into a serum separator tube, allowed to clot at room temperature for at least 30 minutes, and centrifuged at 10,000 RPM for 5 minutes to collect serum. Approximately 30 μL of serum was placed into an Eppendorf tube for immunogenicity analysis, and the remaining serum was placed into an Eppendorf tube for clinical chemistry analysis. Immunogenicity samples were stored on dry ice until storage in a freezer set to maintain temperature at or below -70 °C. Immunogenicity samples were measured by ELISA. Clinical chemistry samples were placed on dry ice until all samples were stored at -20°C or below until analysis. The Hematology and Clinical Chemistry Specimen Table (Table 6, see below) lists the specimens that were measured.

[0219] [Table 6]

[0220] The results of hematology and clinical chemistry analyses of all animals were interpreted by a qualified pathologist.

[0221] Tissue Collection: After euthanasia on SD day 100, all animals were necropsied for tissue collection. Necropsy included examination of the external features of the carcass, external body orifices, abdominal and thoracic cavities, organs and tissues. Gross findings were recorded. The tissue collection table (Table 7, see below) lists the tissues that were collected in 10% NBF. Gross lesions were also collected and preserved in 10% NBF. The remaining carcasses were preserved in 10% NBF.

[0222] [Table 7]

[0223] Organ Weights: At necropsy, the organs in bold above were weighed. Paired organs were weighed together. Weights were recorded on an organ weight form. Percentage of organ weight to body and organ to brain ratios were calculated based on body weights taken before necropsy. Histopathology: Samples from all animals and gross lesions of test animals were transferred to histopathology. The above archived tissues from each animal were embedded in paraffin. All tissues were sectioned, stained with hematoxylin and eosin, and examined microscopically. All sections were examined microscopically by a qualified pathologist for evaluation of histological changes.

[0224] Liver, spleen, kidney, and lung samples from all animals were collected and fixed in 2% glutaraldehyde / 2% paraformaldehyde for analysis of sphingomyelin loading. These tissues were postfixed in potassium dichromate / osmium tetroxide and embedded in Epon. One-micron sections were stained with tannic acid and toluidine blue for light microscopy. Sphingomyelin loading in each sample was quantified by computer morphometry using MetaMorph software by a qualified pathologist.

[0225] Liver, spleen, kidney, and lung samples from all animals were collected, frozen in liquid nitrogen, and stored frozen at or below −70°C for possible future analysis.

[0226] Sample Analysis: Dose analysis was determined by A280 analysis and toxicokinetics, and immunogenicity samples were analyzed by ELISA.

[0227] Ceramide analysis was determined by mass spectrometry.

[0228] Hematology and clinical pathology samples were measured using a Sysmex XTV 2000 IV Hematology Analyzer and a Radnox Daytona Clinical Chemistry Analyzer, respectively.

[0229] Tissue samples were processed and histopathological analyses were interpreted by qualified pathologists according to SOPs. Results of clinical pathological analyses were interpreted by qualified pathologists according to SOPs.

[0230] 8. Example 3: Escalating Doses of Recombinant Human Acid Sphingomyelinase (rhASM) Enzyme Replacement Therapy 8.1 Introduction The following protocol will be used for intrapatient dose escalation as an option to achieve higher repeat doses of rhASM. The safety, efficacy, and pharmacokinetics (PK) of rhASM infusions at 0.3, 0.6, and 1.0 mg / kg administered every 2 weeks (q2w) for 40 weeks will be evaluated, as well as the long-term safety and efficacy of rhASM infusions.

[0231] Materials and Methods 8.2.1. Protocol Design Twelve NP-B patients of either sex were treated. Patients were initially administered at least one dose of 0.1 mg / kg rhASM. Patients who tolerated the 0.1 mg / kg infusion were then administered escalating doses of 0.3 mg / kg, 0.6 mg / kg, and 1 mg / kg every 2 weeks as tolerated. Patients who tolerated 1.0 mg / kg were then stratified by spleen volume (<12 or >12 times normal) and randomized to receive one of two target doses of 1.0 mg / kg or 3.0 mg / kg rhASM, with patients randomized to the 3.0 mg / kg arm being titrated to 2.0 mg / kg and then 3.0 mg / kg as tolerated. Patients who did not tolerate the 0.1 mg / kg dose of rhASM were replaced (replacing / substitution). If the patient is unable to tolerate the dose escalation, the maximum tolerated dose will be maintained for a 26-week maintenance dose period.

[0232] All patients will be dose-titrated from 0.1 mg / kg to the target dose. Dose adjustments during the titration period will occur every 2 weeks (if tolerated) until the target dose is reached. Following the initial infusion (rhASM 0.1 mg / kg) and subsequent doses, patient outcomes (i.e., occurrence / severity of adverse events (AEs), and serum bilirubin, hsCRP, other acute phase response proteins, ceramides) will be reassessed. The next dose administered during the dose-titration period will be determined according to the following criteria: 1. Total bilirubin ≤ 2.0 mg / dL or mild AE → Titrate to next dose (if applicable) 2. Total bilirubin level 2.1-3.0 mg / dL or moderate AEs → no dose escalation, repeat current dose 3. Total bilirubin > 3.0 mg / dL or severe AE → Reduce dose to previously administered / tolerated dose. Serious adverse events (SAEs) judged to be related to rhASM, failure to reduce bilirubin levels to <2.0 mg / dL prior to the next scheduled dose, or AEs that raise concerns regarding the safety of rhASM at the administered dose may be considered dose-limiting toxicities (DLTs). If a patient experiences a DLT, further dosing for that patient should be temporarily discontinued. Patients may be rechallenged to receive the dose that caused the DLT and, if tolerated, treatment may proceed as originally planned.

[0233] If a patient is unable to tolerate two doses of 0.1 mg / kg (i.e., initial infusion and one rechallenge), they will not be treated with the escalating dose, will be discontinued and replaced in the study. Patients who tolerate the 0.3 mg / kg dose will receive an initial infusion of rhASM (0.1 mg / kg) followed two weeks later by 0.3 mg / kg rhASM (assuming the dose escalation criteria are met after the 0.1 mg / kg dose). Subsequent rhASM doses will be administered every 2 weeks for up to 18 weeks during the dose escalation period. If the patient is successfully escalated to 0.3 mg / kg and subsequently meets criteria number 2 or number 3 (above), the patient may be rechallenged twice. If the rechallenge is not successful (i.e., the target dose cannot be reached), the patient will continue on the 0.1 mg / kg rhASM dose for the remainder of the 40-week treatment period.

[0234] Patients treated with the 0.6 mg / kg dose regimen will receive an initial infusion of rhASM (0.1 mg / kg), followed by one 0.3 mg / kg rhASM, then one 0.6 mg / kg rhASM every 2 weeks for the remainder of the 40 week treatment period (assuming the previous 0.1 mg / kg and 0.3 mg / kg rhASM infusions were well tolerated). Patients treated with the 1.0 mg / kg dose regimen will receive an initial infusion of rhASM (0.1 mg / kg), followed by one 0.3 mg / kg rhASM, then one 0.6 mg / kg rhASM, then one 1.0 mg / kg rhASM every 2 weeks for the remainder of the 40 week treatment period (assuming the previous 0.1 mg / kg, 0.3 mg / kg, and 0.6 mg / kg rhASM infusions were well tolerated). If the patient is successfully titrated from 0.3 mg / kg to 0.6 mg / kg or from 0.6 mg / kg to 1.0 mg / kg and then meets criteria #2 or #3 (above), the patient may be re-challenged twice. If re-challenging is not successful (i.e., the target dose cannot be reached), the patient will continue on the lower tolerated dose for the remainder of the 40-week treatment period.

[0235] After the 40 week treatment period, patients may continue treatment at a maintenance dose level.

[0236] 8.2.2.Patient Each patient must meet the following eligibility criteria to be treated according to these regimens: 1. Confirmed ASM deficiency consistent with Niemann-Pick (NP) disease 2. Diffusing capacity of the lungs (DLco) is greater than 20% and less than 80% of the predicted normal value 3. Spleen volume is more than 8 times the normal volume (more than 1.6% of body weight) 4. Female patients of childbearing potential must have a negative serum pregnancy test for β-hCG and agree to use a medically acceptable method of contraception for the duration of the protocol.

[0237] 8.2.3. Treatment Patients receive a single dose of 0.1 mg / kg rhASM IV over approximately 35 minutes. Dosing guidelines are summarized in Table 8 below. Patients who tolerate an initial infusion of 0.1 mg / kg rhASM are titrated to the target therapeutic dose (rhASM 0.3, 0.6 or 1.0 mg / kg). All patients are titrated from 0.1 mg / kg to the target dose. Dose adjustments during the titration period are made every 2 weeks until the target dose is reached.

[0238] [Table 8] TIFF0007605949000013.tif64169

[0239] The degree of splenomegaly at baseline for each patient can be recorded as a factor of normal (times above normal) as an index of disease severity. Normal spleen volume is equal to 0.2% of body weight.

[0240] At each visit, patients should be assessed for new or ongoing adverse events (AEs).

[0241] Clinical endpoints can be measured as the percent change from baseline to week 26 of the maintenance dose period. The primary efficacy endpoint is a reduction in spleen volume as measured by MRI. Secondary efficacy endpoints include a reduction in liver sphingomyelin levels, an increase in exercise capacity as measured by % predicted maximum workload on cycle ergometer testing, improved lung function as % predicted DLco, and increased lung clearance as measured by bronchoalveolar lavage (BAL) cell counts and profiles, and sphingomyelin, ceramide, cytokine, and chitotriosidase levels. Tertiary efficacy endpoints included reduction in liver volume as measured by MRI, improvement in lung function as measured by predicted FVC, FEV1, TLC%, improvement in lung appearance on high resolution CT scan and chest x-ray, improvement in lipid profile as measured by HDL, LDL, total cholesterol, triglyceride levels and the ratio of HDL cholesterol to total cholesterol, improvement in platelet count, reduction in hemoglobin, skin, plasma and sphingomyelin levels in DBS, as well as improvement in other biomarkers such as CCL18, ACE.

[0242] 9. Example 4: Escalating Doses of Recombinant Human Acid Sphingomyelinase (rhASM) Enzyme Replacement Therapy Introduction The protocol described below utilizes a repeat-dose, dose comparison to evaluate the safety, efficacy, and pharmacokinetics of recombinant human acid sphingomyelinase (rhASM) in adult patients with ASMD. Protocol objectives include: (i) evaluating the safety of rhASM dose escalation, (ii) evaluating the safety, efficacy, and pharmacokinetics of the maximum tolerated or randomized dose of rhASM administered intravenously every 2 weeks for 26 weeks, and (iii) evaluating the long-term safety and efficacy of rhASM infusions administered intravenously every 2 weeks from week 26 through completion of the protocol.

[0243] Materials and Methods 9.2.1.Patient Each patient must meet the following eligibility criteria to be treated according to these regimens: 1. Confirmed acid sphingomyelinase (ASM) deficiency consistent with Niemann-Pick (NPD) disease. 2. Two or more characteristic clinical features, including thrombocytopenia, anemia, neutropenia, hepatomegaly, splenomegaly, and pulmonary disease consistent with non-neurogenic NPD 3. Pulmonary Diffusing Capacity for Carbon Monoxide (DL CO ) >20% and <80% of predicted normal 4. Spleen volume ≥8x normal (MN) (i.e., ≥1.6% of body weight). Partial splenectomy performed ≥1 year prior to screening / baseline is permitted if residual spleen volume is ≥8x normal. 5. Female patients of childbearing potential must have a negative serum pregnancy test for beta-human chorionic gonadotropin (β-HGC) and agree to use a medically acceptable method of contraception for the duration of the protocol.

[0244] 9.2.2. Protocol Design Patients who tolerate rhASM at 0.1 mg / kg may enroll in the protocol. Each patient's participation in the protocol may consist of three periods. 1. Screening / Baseline (days ‐60 to ‐1). 2. Primary treatment period (approximately 32-46 weeks). Dose escalation (DE) phase (primary DE - approximately 6 to 16 weeks). -Once rhASM has been safely titrated from 0.1 to 0.3, 0.6, and 1.0 mg / kg, patients will be stratified and randomized by spleen volume (<12 vs. ≥12 times normal) to continue at 1.0 mg / kg for the remainder of the protocol or continue dose escalation to 2.0 mg / kg and 3.0 mg / kg followed by rhASM at 3.0 mg / kg or maximum tolerated dose (secondary DE-4 weeks). - Non-randomized patients will remain at the maximum tolerated dose (<1.0 mg / kg rhASM). Dose maintenance (DM) phase (26 weeks at maximum tolerated dose or randomized dose). 3. Long-term treatment duration (at least 182 weeks at maximum tolerated dose or randomized dose).

[0245] Screening / baseline assessments can be completed at least 24 hours prior to the initial infusion of rhASM. Enrollment in the protocol is contingent on patients being able to tolerate two doses of rhASM at 0.1 mg / kg. Initially, all eligible patients will receive a single dose of rhASM 0.1 mg / kg on dose escalation (DE) day 1. Patients who cannot tolerate this dose will be re-challenged 2 weeks later. Patients who cannot tolerate two doses of rhASM 0.1 mg / kg (i.e., the initial dose and the re-challenge dose) will be discontinued from the study and replaced to ensure that approximately 12 patients (those who tolerate rhASM at 0.1 mg / kg) are enrolled.

[0246] The dose of rhASM can be titrated from 0.1 to 0.3 to 0.6 to 1.0 mg / kg every 2 weeks during the primary DE phase. Patients can safely titrate their rhASM dose from 0.1 to 1.0 mg / kg every 2 weeks during the primary DE phase (shown in FIG. 7).

[0247] During the primary and secondary DE phases, the next dose of rhASM to be administered can be determined according to the following dose escalation criteria. 1. If total bilirubin (highest value prior to the next scheduled dose of rhASM, including pre-infusion blood draw) is <2.0 mg / dL or there are no or mild related adverse events (AEs) → titrate to next dose. 2. If total bilirubin is >2.0 mg / dL but <3.0 mg / dL or there are mild related AEs → repeat same dose. If total bilirubin remains above 2.0 mg / dL immediately prior to the next dose, temporarily discontinue further administration to the patient (potential dose-limiting toxicity [DLT]). 3. If total bilirubin is 3.0 mg / dL or higher or if there are severe related AEs, reduce the dose. If total bilirubin remains above 2.0 mg / dL immediately prior to the next dose → temporarily discontinue further dosing of the patient (possible DLT). 4. For the primary DE phase only, patients may be re-dosed as many times as needed (at specific dose levels) before being randomized or continuing at maximum tolerated dose until the end of the 16-week primary DE phase (re-dosing is not permitted in the secondary DE phase). 5. Enrolled patients (i.e., those who can tolerate 0.1 mg / kg of rhASM) cannot be replaced if they are unable to escalate to the 1.0 mg / kg dose and are unable to be randomized or if they are unable to tolerate the target randomized dose (i.e., 1.0 mg / kg or 3.0 mg / kg of rhASM). These patients may continue on the protocol at the maximum tolerated dose.

[0248] If an infusion is missed, the patient may receive the same dose as the previous infusion. If it exceeds the visit window ±5 days, the infusion cannot be given and the patient should receive the next infusion at the scheduled time for that infusion. For the purposes of this protocol, results may be considered indicative of potential dose-limiting toxicity (DLT) of rhASM at any dose if any of the following criteria are met. A serious adverse event (SAE) judged by an investigator to be related to rhASM, or Total bilirubin remains above 2.0 mg / dL immediately prior to the next dose, or · An adverse event (AE) that, in the physician's opinion, raises concern regarding the safety of rhASM at the administered dose.

[0249] The severity of the related adverse event is assessed by a physician. Mild related adverse events are usually temporary and may require only minimal treatment or therapeutic intervention. Moderate related adverse events are usually alleviated with additional specific therapeutic intervention. The event interferes with daily routine activities and causes discomfort, but does not pose a significant or permanent risk of harm to the subject. Serious adverse events may interrupt daily routine activities or significantly affect clinical status or require intensive therapeutic intervention.

[0250] After dose escalation, all patients who safely tolerate 1.0 mg / kg rhASM by week 16 can be stratified by spleen volume (<12 vs ≥12 times normal) and randomized 1:1 to one of the following: 1.0 mg / kg rhASM (or maximum tolerated dose) every 2 weeks for 26 weeks, or Continue rhASM dose escalation (DE) to 2.0 mg / kg and 3.0 mg / kg, then rhASM 3.0 mg / kg (or maximum tolerated dose) every 2 weeks for 26 weeks.

[0251] During the secondary DE phase, patients randomized to the 3.0 mg / kg dose group must titrate their rhASM dose to 3.0 mg / kg over 4 weeks before initiating the 26-week DM phase at 3.0 mg / kg (or maximum tolerated dose, see Figure 7).

[0252] The primary DE phase ranges in duration from approximately 6 to 16 weeks for each patient. This depends on the individual patient's tolerability issues, if any, that they experience during dose escalation of rhASM. During the primary DE phase, each patient may have their rhASM dose titrated from 0.1 mg / kg to 1.0 mg / kg (shown in Figure 7) every 2 weeks.

[0253] The primary DE phase may be completed when the patient receives and tolerates a dose of 1.0 mg / kg of rhASM or reaches week 16, whichever occurs first. If criteria that would lead to dose rechallenge or dose reduction are not met, the patient may receive a single infusion of each dose (i.e., a total of four rhASM infusions) and enter the 6-week primary DE phase. However, if criteria that would lead to dose rechallenge or dose reduction are met, the length of the primary DE phase may exceed 6 weeks, but not exceed 16 weeks.

[0254] Dose escalation was performed, and patients who safely tolerated 1.0 mg / kg by week 16 could be assigned to one of two target dose arms (i.e., rhASM 1.0 mg / kg or 3.0 mg / kg).

[0255] Patients randomized to 3.0 mg / kg rhASM may enter a secondary DE phase consisting of two infusions (see Figure 7). In this phase, the first infusion (i.e., 2.0 mg / kg) is administered 2 weeks after the first tolerated dose of 1.0 mg / kg, and the second infusion (i.e., 3.0 mg / kg or maximum tolerated dose) is administered 4 weeks after the first tolerated dose of 1.0 mg / kg. Thus, the secondary DE phase may be completed 4 weeks after the first tolerated dose of 1.0 mg / kg. Rechallenges are not permitted. If a patient does not tolerate the first dose of 2.0 mg / kg, the second dose of rhASM may be reduced to the maximum tolerated dose (i.e., 1.0 mg / kg) or temporarily discontinued if there is potential for dose-limiting toxicity (DLT).

[0256] Patients may continue to receive either the maximum tolerated or randomized dose of rhASM IV every 2 weeks during the 26-week DM phase of the first-line treatment period. This may include both randomized and non-randomized patients.

[0257] As shown in Figure 7, patients in the 1.0 mg / kg dose group can begin the DM phase with a first dose of 1.0 mg / kg after randomization, while patients in the 3.0 mg / kg dose group can begin DM with a third dose of rhASM after randomization. Subsequent rhASM doses can be administered IV every 2 weeks for the 26 weeks of the DM phase to all randomized patients.

[0258] Non-randomized patients may begin the DM phase with their first dose of rhASM 2 weeks after the 16-week DE phase. These patients may continue on the maximum tolerated dose of rhASM (<1.0 mg / kg) every 2 weeks for a total of 26 weeks.

[0259] If a randomized patient experiences a moderate / severe related AE (i.e., infusion-related reaction (IAR)) during the infusion in the DM phase, the patient may discontinue and restart (i.e., interrupt) the rhASM infusion or reduce the infusion rate. If no potential DLT occurs, the patient may continue on this dose of rhASM for the remainder of the DM phase.

[0260] After completion of all protocol assessments at Week 26, all patients (including non-randomized patients) will enter the long-term treatment period and may continue rhASM treatment (at the rhASM dose administered during the DM phase) IV every 2 weeks for at least 182 weeks [3.5 years] or until the end of the protocol. The long-term treatment period includes a protocol completion (or patient withdrawal / discontinuation) visit and a safety follow-up phone call (30-37 days after the last dose of rhASM). Individual patient doses of rhASM may be adjusted in the long-term treatment period based on the results of the primary treatment period analysis. Any necessary dose escalation can be performed as previously described.

[0261] 9.3.Treatment Patients will receive intravenous infusions of 0.1, 0.3, 0.6, and 1.0 mg / kg rhASM every 2 weeks during the primary DE phase. Patients randomized to 3.0 mg / kg rhASM will receive 2.0 mg / kg before receiving 3.0 mg / kg (or maximum tolerated dose) and 2 weeks later during the secondary DE phase. During the 26-week DM phase, patients will receive 1.0 mg / kg or 3.0 mg / kg (or maximum tolerated dose) every 2 weeks following the DE phase. During the long-term treatment period, the rhASM dose administered during the DM phase will be administered IV every 2 weeks.

[0262] Patients will receive each intravenous infusion of rhASM over approximately 35-135 minutes, depending on dose (see Table 9 below). Protocol treatment (rhASM) will be infused using a standard programmable infusion pump and a 0.22 micron low protein binding in-line filter. To calculate the dose in mg / kg, use actual body weight for patients with a body mass index (BMI) ≤ 30 (weight in kg) / (height in m), and for patients with a BMI > 30, use the patient's height to calculate the weight corresponding to a BMI of 30.

[0263] [Table 9] TIFF0007605949000015.tif223169 TIFF0007605949000016.tif135169

[0264] Clinical endpoints can be measured as the percent change from baseline in spleen volume times normal (measured by magnetic resonance imaging) after 26 weeks of treatment with the maximum tolerated or randomized dose of rhASM. Secondary efficacy endpoints include liver volume (measured by MRI), lung imaging (by high-resolution computed tomography and chest x-ray), pulmonary function tests (including percent predicted pulmonary diffusing capacity for carbon monoxide, percent predicted forced vital capacity, forced expiratory volume in 1 second, and total lung capacity), exercise capacity by cycle ergometer testing (including percent predicted maximum workload, peak oxygen consumption, and carbon dioxide production), change in Physician's Global Assessment, efficacy biomarkers such as serum chitotriosidase, CCL18, and ACE, and hematology parameters such as platelet count and hemoglobin levels. Efficacy assessments can also include fasting lipids, coagulation tests, disease-related biomarkers, medical outcome measures, and patient photographs.

[0265] Safety assessments may be performed before, during and / or after administration of a dose. Safety assessments that may be performed include: physical examination with neurological assessment, vital signs (e.g., systolic and diastolic blood pressure, temperature, heart rate, respiratory rate and oxygen saturation), ECG, CHO with Doppler, blood chemistry (e.g., sodium, potassium, calcium, chloride, blood urine nitrogen, creatinine, uric acid, ALT, aspartate aminotransferase, total bilirubin, lactate dehydrogenase, alkaline phosphate, total protein, albumin, glucose, cholesterol, etc.), hematology (e.g., complete blood count), urinalysis, biomarker analysis for biomarkers associated with cancer, hormones, cytokines (e.g., IL-8 and IL-6), cardiovascular risk, acute phase reactants and other cellular processes, serum anti-rhASM IgG concentrations, serum anti-rhASM IgE concentrations, serum tryptase activity, and skin tests for hypersensitivity reactions. Additionally, pharmacokinetic and pharmacodynamic assessments can be performed before, during and / or after administration. For example, sphingomyelin levels can be assessed in liver and skin by biopsy, and in plasma and DBS by tandem mass spectrometry.

[0266] All references cited in this specification are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

[0267] The present invention is not intended to be limited in scope by the specific embodiments described herein. Of course, various modifications of the present invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. [Brief description of the drawings]

[0268] [Figure 1] Figure 1 is a diagrammatic representation of the protocol design. [Diagram 2]FIG. 2 is a table showing the demographic and baseline characteristics of patients enrolled in the protocol described below. [Figure 3A] 3A is a graph showing plasma levels of ceramide over time in different patients receiving different doses of rhASM. The right axis of the graph shows patient number and dose of rhASM. [Figure 3B] 3B is a graph showing plasma levels of sphingomyelin over time in different patients receiving different doses of rhASM, with the right axis of the graph showing patient number and dose of rhASM. [Figure 3C] 3C is a graph showing sphingomyelin levels in dried blood spots over time for different patients receiving different doses of rhASM, with the right axis of the graph showing patient number and rhASM dose. [Figure 4] 4 is a graph showing total bilirubin levels measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and dose of rhASM. [Figure 5A] 5A is a graph showing the levels of CRP / hs-CRP measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and dose of rhASM. [Figure 5B] Figure 5B is a graph showing the percentage of neutrophils measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and dose of rhASM. [Figure 5C] Figure 5C is a graph showing fibrinogen measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and rhASM dose. [Figure 5D] Figure 5D is a graph showing ferritin measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and rhASM dose. [Figure 5E]Figure 5E is a graph showing IL-8 measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and rhASM dose. [Figure 5F] Figure 5F is a graph showing IL-6 measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and rhASM dose. [Figure 5G] Figure 5G is a graph showing calcitonin measured over time in different patients receiving different doses of rhASM during the protocol. The right axis of the graph shows patient number and dose of rhASM. [Figure 6] FIG. 6 is a table of adverse events that occurred during treatment for four patients, each of whom received a different dose of rhASM. [Figure 7] FIG. 7 is a diagram showing the initial treatment period, consisting of the dose escalation and dose maintenance phases, of the Phase 2 rhASM repeat dose protocol for patients with ASMD.

Claims

1. A composition for treating a human subject having acid sphingomyelinase deficiency (ASMD), comprising: The composition comprises recombinant human acid sphingomyelinase (rhASM); The composition is administered in an escalating dose regimen comprising administering to the human subject the following doses of rhASM: (a) 0.1 mg / kg, (b) 0.3 mg / kg, and (c) 0.6 mg / kg, Each dose of rhASM was administered 2 weeks after the previous dose; Each dose is administered at least once before escalating the dose to the next level; The composition, wherein the concentration of alanine aminotransferase (ALT) and the concentration of aspartate aminotransferase (AST) in a blood sample from the human subject are assessed within 72 hours prior to administering a dose of rhASM to the human subject.

2. The composition described in claim 1, wherein the regimen further comprises (d) a dose of 1 mg / kg.

3. The composition described in claim 2, wherein the regimen further comprises (e) a dose of 2 mg / kg.

4. The composition described in claim 3, wherein the regimen further comprises (f) a dose of 3 mg / kg.

5. The composition described in any one of claims 1 to 4, wherein the ASMD is Niemann-Pick disease (NPD) type B.

6. A composition described in any one of claims 1 to 5, wherein, in the case of a subject with a BMI > 30, the subject's weight is calculated using the subject's height.

7. The composition described in any one of claims 1 to 6, wherein the human subject is an adult human.

8. The composition of claim 1, wherein the dose is administered intravenously.

9. The composition described in any one of claims 1 to 8, wherein the regimen further comprises a maintenance dose regimen comprising administering to the subject a maintenance dose that is less than or equal to a maximum dose tolerated by the subject.

10. The composition described in claim 9, wherein the maintenance dose is administered to the subject every 1 to 4 weeks.

11. The composition described in claim 9 or 10, wherein the maintenance dose is 3 mg / kg.

12. A composition for treating a human subject having acid sphingomyelinase deficiency (ASMD), comprising: The composition comprises recombinant human acid sphingomyelinase (rhASM); (a) administering to the human subject a dose of 0.001 mg / kg to 0.05 mg / kg of rhASM; and (b) the composition is subsequently administered in an escalating dose regimen comprising administering to the human subject doses of rhASM of (i) 0.1 mg / kg, (ii) 0.3 mg / kg, and (iii) 0.6 mg / kg; Each dose of rhASM was administered 2 weeks after the previous dose; Each dose is administered at least once before escalating the dose to the next level; The composition, wherein the concentration of alanine aminotransferase (ALT) and the concentration of aspartate aminotransferase (AST) in a blood sample from the human subject are assessed within 72 hours prior to administering a dose of rhASM to the human subject.

13. The composition of claim 12, wherein the regimen further comprises (iv) a dose of 1 mg / kg.

14. The composition described in claim 13, wherein the regimen further comprises (v) a dose of 2 mg / kg.

15. The composition of claim 13, wherein the regimen further comprises (vi) a dose of 3 mg / kg.

16. The composition of any one of claims 12 to 15, wherein the dose is administered intravenously.

17. The composition described in any one of claims 12 to 16, wherein the ASMD is Niemann-Pick disease (NPD) type B.

18. The composition of claim 12, wherein, for a subject with a BMI > 30, the subject's weight is calculated using the subject's height.

19. The composition of any one of claims 12 to 18, wherein the human subject is a human child.

20. The composition of any one of claims 12 to 19, wherein the regimen further comprises a maintenance dose regimen comprising administering to the subject a maintenance dose that is less than or equal to a maximum dose tolerated by the subject.

21. The composition described in claim 20, wherein the maintenance dose is administered to the subject every 1 to 4 weeks.

22. The composition described in claim 20 or 21, wherein the maintenance dose is 3 mg / kg.

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