RU2833674C2 - Enzyme replacement therapy with dose escalation for treating acid sphingomyelinase deficiency - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and represents a method of treating acid sphingomyelinase (asmd) deficiency in a human subject, involving administering recombinant human acid sphingomyelinase (rhasm) to a human subject in a dose-increasing regimen.

EFFECT: invention provides higher clinical effectiveness in acid sphingomyelinase deficiency.

30 cl, 7 dwg, 9 tbl, 4 ex

Description

This application claims priority to U.S. Provisional Patent Application No. 61/238,113, filed August 28, 2009, which is incorporated herein by reference in its entirety.

1. Introduction

The present invention relates to dose escalating enzyme replacement therapy with acid sphingomyelinase (ASM) for the treatment of human subjects having acid sphingomyelinase deficiency (ASMD), and in particular the non-neurological manifestations of Niemann-Pick disease (NPD), and in certain embodiments NPD type B.

2. prerequisites

Acid sphingomyelinase, E.C. 3.1.4.12, (ASM) is a lysosomal phosphodiesterase enzyme that hydrolyzes sphingomyelin, a phospholipid storage substance found in the brain, liver, lungs, spleen, and lymph nodes, into ceramide and phosphorylcholine. Deficiencies in ASM activity result in the body's inability to break down sphingomyelin, causing the lysosomal storage disease called Niemann-Pick disease.

Niemann-Pick disease is an inherited, autosomal recessive lipid storage disorder characterized by 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 disorder of neonates and is usually fatal by age two to three years. Niemann-Pick disease type B results in enlarged liver and spleen and fatal respiratory failure, usually occurring in adolescence. These two forms of Niemann-Pick disease, both associated with ASM deficiencies, are collectively referred to herein as Niemann-Pick disease or ASM deficiency (ASMD). Other types of Niemann-Pick disease, such as type C, do not involve mutations in the ASM gene and do not directly contribute to ASM function. The nature of the biochemical and molecular defects that underlie the significant clinical heterogeneity between subtypes A and B remains unknown. Although patients with both subtypes have residual ASM activity (approximately 1-10% of normal), biochemical analysis cannot reliably distinguish between the two phenotypes. Moreover, the disease course of NPD type B is highly variable, and it is currently not possible to correlate disease severity with the level of residual ASM activity.

NPD occurs more frequently among individuals of Ashkenazi Jewish descent than in the general population. The incidence of type A disease among Ashkenazi Jews is estimated to be approximately 1 in 40,000, the gene frequency (q) approximately 1 in 200, and the heterozygous carrier frequency (2 pq) 1 in 100 (Goodman, 1979, in Genetic Disorders Among The Jewish People, John Hopkins Univ. Press, Baltimore, pp. 96-100). The heterozygous carrier frequency of type B NPD in the Ashkenazi Jewish population is less common (Goodman, previously). The combined heterozygous carrier frequency for types A and B NPD has been estimated to be approximately 1 in 70 among individuals of Ashkenazi Jewish descent. Although enzyme testing of patients with either type A or B NPD can be performed reliably (Spence and Callahan, previously), enzyme testing in obligate heterozygotes has proven problematic, particularly with the use of leukocytes as the enzyme source. It is likely that the occurrence of neutral sphingomyelinases in multiple sources and/or the presence of residual ASM activity resulting from the mutant allele have contributed to the failure to reliably discriminate carriers for either subtype of the disease. Even the use of cultured skin fibroblasts, which do not express neutral sphingomyelinase, has been inconclusive with heterozygotes. In epidemiologic studies conducted in individual countries, the combined incidence of Niemann-Pick disease A and B in some countries worldwide is estimated to range from 1 in 167,000 to 1 in 250,000 births (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 heterozygous carrier rate is thought to range from 1 in 200 to 1 in 250 individuals.

Enzyme replacement therapy has been used for other lysosomal storage diseases. Enzyme replacement therapy attempts to replace deficient enzyme activity with exogenously supplied enzyme. In the case of enzyme replacement therapy for Niemann-Pick disease, the goal would be to enable the affected individual to process sphingomyelin and avoid its accumulation within lysosomes. To be effective, such therapy would initially require substantially larger amounts of replacement enzyme to break down the accumulated sphingomyelin, as well as continuous administration of replacement enzyme to avoid further accumulation of sphingomyelin.

3. brief description

The present invention relates to a dose escalation enzyme replacement therapy for the treatment of human subjects having ASMD - in particular, subjects having non-neurological manifestations of NPD and, in particular embodiments, NPD type B. More particularly, the enzyme, ASM, is administered to such patients at an initial low, non-toxic dose, which is then increased in subsequent administrations. The highest dose of ASM that is tolerated by the patient can then be used as a maintenance dose. Alternatively, a therapeutically effective dose that is less than the highest tolerated dose can be used as a maintenance dose.

The present invention is based, in part, on the discovery that the doses of ASM that would be required to eliminate accumulated sphingomyelin substrate in human subjects, i.e., ASMD patients or Niemann-Pick patients, result in toxic side effects (including clinical signs of toxicity). This is particularly unexpected in patients with a less severe form of ASMD, NPD type B, who have a deficiency but at least some enzyme activity.

More specifically, treatment of NPD will require doses high enough to achieve sufficient distribution of the ASM enzyme to the diseased organs (e.g., liver, spleen, lung, heart, kidney, and brain, among others). Studies in the ASM knockout mouse model (ASKMO mouse) have shown that the majority of administered recombinant human ASM (rhASM) is distributed to the liver and spleen, where it reduces substrate, but to a much lesser extent to the lung, heart, and brain (Miranda et al. FASEB 2000, 14:1988; see also Fig. 9B He et al., 1999, Biochimica et Biophsyica Acta 1432: 251-264). In subsequent studies using higher doses of rhASM in the ASMKO mouse model, substrate was reduced and no toxicity was observed at doses ≤3.0 mg/kg; in fact, clinical signs of toxicity were not observed up to doses ≥10 mg/kg. See 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.

Based on these ASKMO data, we treated human subjects with non-neuronopathic ASMD with a conservative maximum dose of 1.0 mg/kg rhASM, as described in Section 6 below. Quite surprisingly, toxicity in human subjects, including the occurrence of clinically symptomatic adverse events, was observed at doses up to 0.3 mg/kg! This result was particularly surprising since the ASM enzyme is absent in the knockout mouse model, which should reflect a more severe condition than these human subjects who have at least some enzymatic activity and a relatively mild form of the disease.

Without intending to be bound by any theory, the toxic side effects that occur with ASM treatment may be the result of the breakdown of the stored sphingomyelin substrate in a patient with ASMD and the release of the product, ceramide, which is pro-apoptotic and induces a pro-inflammatory cytokine response and hyperbilirubinemia. To address this issue, we have developed a regimen that safely delivers the high doses of ASM enzyme needed to achieve sufficient distribution to diseased organs. In this regimen, initial treatment with very low doses of ASM is used to achieve a slow breakdown of stored substrate that is associated with fewer side effects. As the subject becomes depleted of substrate (as the reserve substrate “depletes” in mass), the dose can be safely increased.

According to this protocol, a patient with NPD is initially administered a low, non-toxic dose of the ASM enzyme and the dose is increased over time. As the dose of the ASM enzyme is increased, the patient can be monitored for total bilirubin concentration, production of acute phase reactants, production of inflammatory mediators, and related adverse events. Administration of a low dose of ASM and increasing the dose facilitates reduction of the accumulated sphingomyelin mass. As the patient's sphingomyelin mass is reduced, higher doses can be safely administered to the patient to ensure sufficient distribution of the ASM enzyme to target organs (e.g., liver, spleen, lungs, heart, kidney, brain, bone marrow, skeleton, joints, etc.). In certain embodiments, the maximum dose tolerated by the patient can be used as a maintenance dose. In some embodiments, based on the patient's condition, the maintenance dose can be increased or decreased over time.

In certain embodiments, the patient's treatment is monitored by measuring plasma sphingomyelin levels, plasma ceramide levels, production of "acute phase reactants" and inflammatory mediators, which are a measure of inflammatory responses, bilirubin concentrations (total, direct and indirect) and/or other biochemical markers to ensure a stable response before increasing the dose to the next level. 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 particular embodiments, the patient may be monitored for one or more associated adverse events, which may include, but are not limited to, systemic symptoms (e.g., fever, nausea, vomiting, pain, myalgia) and jaundice.

Doses of less than 1 mg/kg are preferred for initiating treatment. The initial dose is sequentially increased until a therapeutic dose is reached. This dose escalation can be used to determine the highest tolerated dose. For example, once a patient's accumulated sphingomyelinase substrate mass is reduced, the dose can be further increased as long as toxicity is not observed. The maintenance dose can be adjusted accordingly and can be continuously and periodically readjusted depending on the patient's condition.

In a specific embodiment, a method of treating a human subject having acid sphingomyelinase deficiency comprises: (a) a regimen for reducing the mass of accumulated sphingomyelin substrate in a human subject, comprising: (i) administering an initial low, non-toxic dose of ASM to the human subject; (ii) administering successively higher doses of ASM to the human subject and monitoring the subject for one or more adverse side effects after each subsequent dose, as determined by elevated bilirubin or a related adverse event; and (b) a maintenance regimen comprising administering a dose equal to or less than the highest dose tolerated by the subject as a maintenance dose for the subject.

In another specific embodiment, a method of treating a human subject having acid sphingomyelinase deficiency comprises administering rhASM in a dose escalation schedule with the following sequential doses: 0.1 mg/kg; 0.3 mg/kg; 0.6 mg/kg; and 1.0 mg/kg, wherein each dose of rhASM is administered at least twice, and each dose is administered at two-week intervals, and wherein the patient is monitored for toxic side effects before escalating the dose to the next level.

In another specific embodiment described herein, there is provided acid sphingomyelinase (ASM) for use in the treatment of acid sphingomyelinase deficiency in a human subject, formulated for administration: (a) in a regimen for reducing the mass of accumulated sphingomyelin substrate, comprising: (i) administering an initial low, non-toxic dose of acid sphingomyelinase (ASM); (ii) administering successively higher doses of ASM, and monitoring the subject for one or more undesirable side effects after each subsequent dose, as determined by increased bilirubin or a related adverse event; and (b) in a maintenance regimen, comprising administering a dose equal to or less than the highest dose tolerated by the subject, as a maintenance dose for the subject.

In another specific embodiment described herein, there is provided recombinant human ASM for use in the treatment of acid sphingomyelinase deficiency in a human subject, formulated for administration in a dose escalation regimen with the following sequential doses: 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, and each dose is administered at two-week intervals, and wherein the subject is monitored for toxic side effects before escalating the dose to the next level.

3.1. TERMINOLOGY

As used herein, the terms "approximately" and "approximately" are used interchangeably in the context of a given value to denote a range around the given value, where the resulting value is substantially the same as the exact value listed. In a particular embodiment, "approximately" means within 10%, 15%, 25% of the given value or range.

As used in this document, the term "elderly person" refers to a person 65 years of age or older.

As used in this document, the term "adult" refers to a person who is 18 years of age or older.

As used in this document, the term "human child" refers to a person between the ages of 1 and 18 years.

As used in this document, the term "human infant" refers to a human being from birth to 1 year of age.

As used in this document, the expression "human child of nursery age" refers to a human being who is between 1 year and 3 years of age.

As used in this document, the term "adverse event" refers to "any untoward medical occurrence in a patient or clinical trial subject who has been administered a pharmaceutical product" as defined in the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1. A "related adverse event" is an adverse event that has a causal relationship to treatment.

As used herein, the term "maintenance dose(s)" and the like refers to a dosage administered to patients with ASMD to maintain a desired therapeutic effect. In particular embodiments, the maintenance dose(s) maintains one, two, three, four or more of the following desired therapeutic effects: (i) a decrease in spleen volume, as assessed by techniques known in the art, such as MRI (magnetic resonance imaging); (ii) a decrease in liver sphingomyelin levels, as assessed by techniques known in the art, such as biochemical analysis 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 using bicycle ergometry, including relative estimated maximal workload, peak oxygen consumption and carbon dioxide production; (iv) an increase in pulmonary function as assessed by techniques known in the art, such as those described in American Thoracic Society, 1991, Am. Rev. Respir. Dis. 144: 1202-1218, such as diffusing capacity of the lungs (DL _CO ), forced vital capacity (FVC) as measured by, for example, spirometric techniques, forced expiratory volume in 1 second (FEV ₁ ) as measured by, for example, spirometric techniques, and total lung capacity; (v) a decrease in bronchoalveolar lavage (BAL) sphingomyelin; (vi) a decrease in liver volume as assessed by techniques known in the art, such as MRI; (vii) an improvement in lung appearance as assessed by techniques known in the art, such as high-resolution computed tomography (CT) imaging or chest radiography; (viii) reducing the concentration of sphingomyelin in the liver, skin, plasma and dried blood spot (DBS), as measured by, for example, tandem mass spectrometry; (ix) reducing or ameliorating the severity of ASMD and/or a symptom associated with it; (x) reducing the duration of a symptom associated with ASMD; (xi) preventing the recurrence of a symptom associated with ASMD; (xii) reducing the frequency of hospitalization of the subject; (vi) reducing the duration of hospitalization; (xiii) increasing the survival of the subject; (xiv) reducing mortality; (xv) reducing the frequency of hospitalization; (xvi) reducing the number of symptoms associated with ASMD; (xvii) increasing the symptom-free survival of patients with ASMD; (xviii) improving neurological function (e.g., psychomotor function, social responsibility, etc.); (xix) improving lung clearance, as measured by, for example, cell count and BAL profile; (xx) reducing serum chitotriosidase levels; (xxi) a decrease in serum chemokine (cc) motif ligand 18 (CCL18) levels; (xxii) an improvement in lipid profile (e.g., HDL [high-density lipoprotein, or HDL], LDL [low-density lipoprotein, or LDL], cholesterol, triglycerides, and total cholesterol:HDL ratio); and (xxiii) an improved quality of life, as assessed by, for example, a questionnaire. In certain embodiments, the maintenance dose is the highest or maximum dose tolerated by the patient.

In some embodiments, the maintenance dose is a dose of 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 ASM. In certain embodiments, the maintenance dose is a dose of 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 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, 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, at least 8 mg/kg of ASM, with the highest dose being 10 mg/kg of ASM. In certain embodiments, 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 ASM, with the highest dose being 50 mg/kg.

As used herein, the term "non-toxic dose(s)" or the like refers to a dosage administered to patients with ASMD that does not result in one, two, three, or all of the following: (i) a moderate or severe associated adverse event, as defined by a clinical symptom that interferes with normal daily functioning and requires additional monitoring, intervention, or treatment, or, an abnormal laboratory value or procedural outcome of a clinical task that requires further follow-up, treatment, or investigation. See, for example, the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1; (ii) the total bilirubin value is greater than 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, or 4 mg/dL, or is 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 that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours, 5 days, one week, two weeks, or three weeks after an ASM dose; (iii) plasma ceramide concentration is greater than 8.2 μg/dL, 8.3 μg/dL, 8.4 μg/dL, 8.5 μg/dL, 8.75 μg/dL, 9 μg/dL, 9.5 μg/dL, 10 μg/dL, 11 μg/dL, 12 μg/dL, 13 μg/dL, 14 μg/dL, 15 μg/dL, 16 μg/dL, 17 μg/dL, 18 μg/dL, 19 μg/dL, 20 μg/dL, 25 μg/dL, 30 μg/dL, 35 μg/dL, 40 μg/dL, 45 mcg/dL, 50 mcg/dL, 55 mcg/dL, 60 mcg/dL, 65 mcg/dL, 70 mcg/dL, 75 mcg/dL, or 80 mcg/dL, or in the range of 8.2 mcg/dL to 10 mcg/dL, 8.5 mcg/dL to 10 mcg/dL, 9 mcg/dL to 12 mcg/dL, 10 mcg/dL to 12 mcg/dL, 10 mcg/dL to 15 mcg/dL, 10 mcg/dL to 20 mcg/dL, 15 mcg/dL to 20 mcg/dL, or 20 mcg/dL to 30 mcg/dL at 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM; or (iv) an acute phase response. The "non-toxic dose" of ASM may vary depending on, for example, the stability of the enzyme administered, the activity of the enzyme administered, and/or the route of administration of the enzyme. For example, the dose of a modified ASM with increased activity may be lower than the dose of unmodified ASM. One skilled in the art will be able to adjust the dose of enzyme administered based on the stability of the enzyme, the activity of the enzyme, and/or the route of administration of the enzyme.

An acute phase response is an early response (typically, for example, within 12-72 hours) after administration of ASM that is characteristic of an inflammatory response. An acute phase response can be assessed by a change in the concentration of an acute phase agent (such as, for example, CRP/hs-CRP, ferritin, fibrinogen, iron, or transferrin), a change in the percentage of neutrophils, a change in prothrombin time, or a change in partial thromboplastin time. In a particular embodiment, an increase in the CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM, relative to the CRP/hs-CRP concentration in the patient before administration of ASM, can be used as a measurement 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 at 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 may 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 at 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 measurement of the acute phase response.

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

In a specific embodiment, an increase in the concentration of IL-8 in plasma or serum at 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, relative to the concentration of IL-8 in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of IL-8 in plasma or serum that is greater than a normal concentration of IL-8 in plasma at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement 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 at 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 measurement of the acute phase response.

In a specific embodiment, an increase in the concentration of IL-6 in plasma or serum at 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, relative to the concentration of IL-6 in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of IL-6 in plasma or serum that is greater than the normal concentration of IL-6 in plasma or serum at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement of the acute phase response. In certain embodiments, a plasma IL-6 concentration 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 at 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 measurement of the acute phase response.

In a specific embodiment, an increase in the concentration of calcitonin in plasma or serum at 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, relative to the concentration of calcitonin in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of calcitonin in plasma or serum that is greater than the normal concentration of calcitonin in plasma at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement 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 at 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 measurement of the acute phase response.

In a specific embodiment, an increase in fibrinogen concentration at 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, relative to the fibrinogen concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a plasma fibrinogen concentration that is greater than the normal plasma fibrinogen concentration at 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 measurement of the acute phase response. In certain embodiments, a plasma fibrinogen concentration of greater than about 350 mg/dL, 375 mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, 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 at 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 measurement of the acute phase response.

In one embodiment, an increase in the relative content of neutrophils to the total number of white blood cells at 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 relative content of neutrophils to the total number of white blood cells in the patient before administration of ASM can be used as a measurement of the acute phase response. In another embodiment, an increase in the relative content of neutrophils to the total number of white blood cells that is greater than the normal relative content of neutrophils to the total concentration of white blood cells at 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 measurement of the acute phase response. In certain embodiments, an increase in the relative proportion of neutrophils to total white blood cells that is 70%, 75%, 80%, 85%, 90%, 95%, or greater at 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 measurement of the acute phase response.

As used herein, the term "low, non-toxic dose(s)" or the like in the context of an initial dose or doses administered to a subject refers to a dosage that is the first dose or doses of ASM administered to a subject for the treatment of ASMD that is non-toxic. In certain embodiments, the low, non-toxic dose(s) is a dose of 0.001 mg/kg to 0.01 mg/kg, 0.001 mg/kg to 0.01 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.1 mg/kg, 0.001 mg/kg to 0.5 mg/kg, 0.05 mg/kg to 0.275 mg/kg, 0.075 mg/kg to 0.275 mg/kg, 0.05 mg/kg to 0.2 mg/kg, 0.075 mg/kg to 0.2 mg/kg, 0.1 mg/kg to 0.275 mg/kg, 0.1 mg/kg to 0.25 mg/kg, 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, 0.75 mg/kg to 1 mg/kg, 0.1 mg/kg to 2 mg/kg, 0.5 mg/kg to 2 mg/kg, 0.75 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg or 1.25 mg/kg to 2 mg/kg, 1.5 mg/kg to 2 mg/kg or 1.75 mg/kg to 2 mg/kg ASM. In some specific embodiments, the low, non-toxic 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.

The terms "subject" and "patient" are used interchangeably herein to refer to a human being. In a particular embodiment, the human being is or has been diagnosed as having ASMD.

As used herein, the term "therapeutically effective" in the context of administering a dose of ASM to a subject refers to an amount of ASM that results in a beneficial or therapeutic effect. In particular embodiments, the term "therapeutically effective" in the context of administering a dose of ASM to a subject refers to an amount of ASM that is sufficient to achieve at least one, two, three, four or more of the following effects: (i) a decrease in spleen volume, as assessed by techniques known in the art, such as MRI; (ii) a decrease in liver sphingomyelin levels, as assessed by techniques known in the art, such as biochemical analysis 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 using bicycle ergometry, including percent estimated maximal workload, peak oxygen consumption and carbon dioxide production; (iv) an increase in pulmonary function as assessed by techniques known in the art, such as those described in American Thoracic Society, 1991, Am. Rev. Respir. Dis. 144: 1202-1218, such as diffusing capacity of the lungs (DL _CO ), forced vital capacity (FVC) as measured by, for example, spirometric techniques, forced expiratory volume in 1 second (FEV ₁ ) as measured by, for example, spirometric techniques, and total lung capacity; (v) a decrease in bronchoalveolar lavage (BAL) sphingomyelin; (vi) a decrease in liver volume as assessed by techniques known in the art, such as MRI; (vii) an improvement in lung appearance as assessed by techniques known in the art, such as high-resolution CT imaging or chest radiography; (viii) reducing the concentration of sphingomyelin in skin, plasma and dried blood spot (DBS), as measured by, for example, tandem mass spectrometry; (ix) reducing or alleviating the severity of ASMD and/or a symptom associated with it; (x) reducing the duration of a symptom associated with ASMD; (xi) preventing the recurrence of a symptom associated with ASMD; (xii) reducing the frequency of hospitalization of the subject; (vi) reducing the duration of hospitalization; (xiii) increasing the survival of the subject; (xiv) reducing mortality; (xv) reducing the frequency of hospitalization; (xvi) reducing the number of symptoms associated with ASMD; (xvii) increasing the symptom-free survival of patients with ASMD; (xviii) improving neurological function (e.g., psychomotor function, social responsibility, etc.); (xix) improving lung clearance, as measured by, for example, cell count and BAL profile; (xx) reducing serum chitotriosidase levels; (xxi) reduction in serum CCL18 levels; (xxii) improvement in lipid profile (e.g. HDL, LDL, cholesterol, triglycerides and total cholesterol:HDL ratio); and (xxiii) improved quality of life as assessed by e.g. questionnaire.

As used herein, the terms "therapy" and "therapy" may refer to any protocol(s), method(s), composition(s), formulation(s), and/or agent(s) that can be used in the treatment, management, or amelioration of ASMD or a condition or symptom associated therewith. In certain embodiments, the terms "therapy" and "therapy" refer to biological therapy, adjunctive therapy, and/or other therapies useful in the treatment, management, prevention, or amelioration of ASMD or a condition or symptom associated therewith. In embodiments, the term "therapy" refers to a therapy that achieves one, two, or more of the following: (i) enhances delivery of ASM to disease sites, (ii) enhances ASM activity, and (iii) enhances ASM stability. In certain embodiments, the term "therapy" refers to a therapy other than ASM. In particular embodiments, "additional therapy" and "additional therapies" refer to a therapy other than ASM.

As used herein, the expression "toxic effect(s)" or the like refers to one, two, three, or all of the following following administration of an ASM dose(s): (i) a moderate or severe associated adverse event that is defined as a clinical symptom that interferes with normal daily functioning and requires additional monitoring, intervention, or treatment, or an abnormal laboratory value or procedural outcome of a clinical task that requires further monitoring, treatment, or investigation. See, for example, the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1; (ii) a total bilirubin value greater than 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, 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 that persists for more than 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours, 5 days, one week, two weeks, or three weeks after administration of a dose of ASM; (iii) plasma ceramide concentration is greater than 8.2 μg/dL, 8.3 μg/dL, 8.4 μg/dL, 8.5 μg/dL, 8.75 μg/dL, 9 μg/dL, 9.5 μg/dL, 10 μg/dL, 11 μg/dL, 12 μg/dL, 13 μg/dL, 14 μg/dL, 15 μg/dL, 16 μg/dL, 17 μg/dL, 18 μg/dL, 19 μg/dL, 20 μg/dL, 25 μg/dL, 30 μg/dL, 35 μg/dL, 40 μg/dL, 45 mcg/dL, 50 mcg/dL, 55 mcg/dL, 60 mcg/dL, 65 mcg/dL, 70 mcg/dL, 75 mcg/dL, or 80 mcg/dL, or in the range of 8.2 mcg/dL to 10 mcg/dL, 8.5 mcg/dL to 10 mcg/dL, 9 mcg/dL to 12 mcg/dL, 10 mcg/dL to 12 mcg/dL, 10 mcg/dL to 15 mcg/dL, 10 mcg/dL to 20 mcg/dL, 15 mcg/dL to 20 mcg/dL, or 20 mcg/dL to 30 mcg/dL at 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of an ASM dose; or (iv) an acute phase response.

The acute phase response can be assessed by a change in the concentration of an acute phase agent (such as, for example, C-reactive protein, ferritin, albumin, IL-8, IL-6, calcitonin, fibrinogen, iron, or transferrin), a change in the relative neutrophil count, a change in prothrombin time, or a change in partial thromboplastin time. In a particular embodiment, an increase in the CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM relative to the patient's CRP/hs-CRP concentration before administration of ASM can be used as a measurement 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 at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of an ASM dose can be used as a measure of the acute phase response. In certain embodiments, the plasma CRP/hs-CRP concentration is greater than about 8.1 mg/dL, 8.2 mg/dL, 8.3 mg/dL, 8.4 mg/dL, 8.5 mg/dL, 8.6 mg/dL, 8.7 mg/dL, 8.8 mg/dL, 8.8 mg/dL, 8.9 mg/dL, 9 mg/dL, 9.5 mg/dL, 10 mg/dL, 11 mg/dL, or 12 mg/dL, or in the range of 8.5 mg/dL to 10 mg/dL, or 8.5 mg/dL to 12 mg/dL, or 10 mg/dL to 12 mg/dL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours post-dose, ASM can be used as a measure of the acute phase response.

In a specific embodiment, an increase in ferritin concentration at 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, relative to the ferritin concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a plasma ferritin concentration that is greater than the normal plasma ferritin concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement of the acute phase response. In certain embodiments, the plasma ferritin concentration is greater than about 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL, 1050 ng/mL, 1100 ng/mL, 1150 ng/mL, or 1200 ng/mL, or in the range of 600 ng/mL to 800 ng/mL, 650 ng/mL to 850 ng/mL, 600 ng/mL up to 1000 ng/mL, 600 ng/mL to 1200 ng/mL, 800 ng/mL to 1000 ng/mL, 900 ng/mL to 1000 ng/mL, or 1000 ng/mL to 1200 ng/mL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after dosing ASM can be used as a measure of the acute phase response.

In a specific embodiment, an increase in fibrinogen concentration at 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, relative to the fibrinogen concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In another specific embodiment, a plasma fibrinogen concentration that is greater than the normal plasma fibrinogen concentration at 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 measurement of the acute phase response. In certain embodiments, a plasma fibrinogen concentration of greater than about 350 mg/dL, 375 mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, 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 at 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 measurement of the acute phase response.

In a particular embodiment, a decrease in albumin concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM, relative to the albumin concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In certain embodiments, a decrease in albumin concentration by the following amount 0.2, 0.4, 0.6, 1, 1.5, 2.0 g/dL from the normal range of 3.5-5.0 g/dL at 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 measurement of the acute phase response.

In a particular embodiment, a decrease in ferritin concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM, relative to the ferritin concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In certain embodiments, a decrease in ferritin concentration by the following amount 20, 40, 60, 80, 100, 120, 140, 160 mcg/dL from the normal range of 60-170 mcg/dL at 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 measurement of the acute phase response.

In a particular embodiment, a decrease in transferrin concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM, relative to the transferrin concentration in the patient before administration of ASM, can be used as a measurement of the acute phase response. In certain embodiments, a decrease in transferrin concentration by the following amount 20, 40, 60, 80, 100, 120, 140, 160, 180 mg/dL from the normal range of 202-336 mg/dL at 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 measurement of the acute phase response.

In one embodiment, an increase in the relative content of neutrophils to the total number of white blood cells at 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 relative content of neutrophils to the total number of white blood cells of patients before administration of ASM, can be used as a measurement of the acute phase response. In another embodiment, an increase in the relative content of neutrophils to the total number of white blood cells that is greater than the normal relative content of neutrophils to the total concentration of white blood cells at 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 measurement of the acute phase response. In certain embodiments, an increase in the relative proportion of neutrophils to total white blood cells that is 70%, 75%, 80%, 85%, 90%, 95%, or greater at 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 measurement of the acute phase response.

4. brief description of graphic materials

Fig.1 is a graphical representation of the protocol diagram.

Figure 2 is a table showing the demographics and baseline characteristics of patients included in the protocol described below.

Figures 3A, 3B, and 3C are graphs showing plasma ceramide levels (Figure 3A), plasma sphingomyelin levels (Figure 3B), and dried blood spot sphingomyelin levels (Figure 3C) over time in different patients treated with different doses of rhASM. The right axes of the graphs show the number of patients and the dosage of rhASM.

Figure 4 is a graph showing total bilirubin levels determined over time in different patients who were administered different doses of rhASM during the protocol. The right axis of the graph shows the number of patients and the dose of rhASM.

Figures 5A-5G are graphs showing the levels of CRP/hs-CRP (Figure 5A), percentage of neutrophils (Figure 5B), fibrinogen (Figure 5C), ferritin (Figure 5D), IL-8 (Figure 5E), IL-6 (Figure 5F), and calcitonin (Figure 5G) determined over time in different patients who were administered different doses of rhASM during the protocol. The right axes of the graphs show the number of patients and the dosage of rhASM.

Figure 6 is a table of treatment-emergent adverse events for four patients, each on a different dose of rhASM, where events are considered related (possibly, probably, or definitely) to treatment.

Figure 7 is a diagram showing the primary treatment period, which consists of dose escalation and dose maintenance phases, of the Phase 2 rhASM repeat dose protocol in patients with ASMD.

5. detailed description of the invention

The present invention relates to a dose escalation enzyme replacement therapy for the treatment of human subjects having ASMD - particularly subjects having non-neurological manifestations of NPD, and in certain embodiments, NPD type B. More particularly, the enzyme, ASM, is administered to such patients at an initial low, non-toxic dose, which is then increased 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 that is less than the highest tolerated dose can be used as a maintenance dose.

Treatment of NPD requires doses high enough to achieve sufficient distribution of the ASM enzyme to the diseased organs (e.g., spleen, lung, heart, kidney, and brain). Following intravenous administration of recombinant human ASM to ASMKO mice, the majority of ASM activity is distributed to the liver, with small amounts of ASM enzymatic activity detected in other diseased organs such as the spleen, heart, kidney, and lung (see, e.g., Fig. 9B He et al., 1999, Biochimia et Biophsyica Acta 1432: 251-264). Thus, very high doses would be necessary to ensure distribution and delivery of the administered enzyme to the lung, heart, and kidney of patients suffering from ASMD or Niemann-Pick disease.

Studies in the ASM knockout mouse model (ASKMO mouse) showed that the majority of administered rhASM was distributed to the liver and spleen, where it reduced substrate, but to a much lesser extent in the lung, heart, and brain (Miranda et al. FASEB 2000, 14:1988). In subsequent studies using higher doses of rhASM in the ASMKO mouse model, substrate was reduced and toxicity was not observed at doses ≤3.0 mg/kg; in fact, clinical signs of toxicity were not observed until doses ≥10 mg/kg were used. See 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.

Based on these ASKMO data, we treated human subjects with non-neuronopathic ASMD with a conservative maximum dose of 1.0 mg/kg as described in Section 6 below. Surprisingly, toxicity in human subjects, including the occurrence of clinical adverse events, was observed with doses as low as 0.3 mg/kg! This result was particularly surprising since the ASM enzyme is absent in the knockout mouse model, which should reflect a more severe condition than human subjects who have at least some enzyme activity and relatively mild disease.

Without being bound by theory, administration of high doses of ASM to patients with NPD may result in the hydrolysis of large amounts of sphingomyelin into large concentrations of ceramide, which may produce the toxic side effects observed in these patients with NPD. The ASM enzyme hydrolyzes sphingomyelin, which is a major component of the plasma membrane of cells (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).

Moreover, 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 the lysosome (see, e.g., Schissel et al., 1998. J. Biol. Chem. 273: 2738-2746). The ability of the ASM enzyme to function in plasma may result in the hydrolysis of sphingomyelin found in lipoproteins and the plasma membrane of cells, which may increase the amount of the breakdown product, ceramide, which may cause the toxic side effects observed in NPD patients administered high doses of the ASM enzyme.

To address the problem of achieving sufficient distribution of the ASM enzyme to diseased organs while avoiding or minimizing toxicity associated with high doses of the enzyme, the inventors have developed treatment regimens as described herein that initially administer a low, non-toxic dose of the ASM enzyme to a patient with NPD and increase the dose over time. As the dose of the ASM enzyme is increased, the patient can be monitored for total/direct/indirect bilirubin concentrations, production of acute phase reactants, production of inflammatory mediators, and related adverse events. Administration of a low dose of ASM and increase in enzyme facilitates reduction of accumulated sphingomyelin mass. Once the patient's accumulated sphingomyelin mass decreases, the patient can be safely administered higher doses of the ASM enzyme to ensure sufficient distribution of the ASM enzyme to target organs (e.g., liver, spleen, lungs, heart, kidney, brain, bone marrow, skeleton, joints, etc.). In certain embodiments, the maximum dose tolerated by the patient can be used as a maintenance dose. Alternatively, a therapeutically effective dose that is less than the highest tolerated dose can be used as a maintenance dose. In some embodiments, the maintenance dose can be increased or decreased based on the patient's condition.

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

5.1. DOSE ESCREATION PROTOCOL

Methods for treating ASMD are described comprising administering one or more initial, low, non-toxic doses of ASM to a subject to reduce the amount of sphingomyelin that has accumulated in the subject. After a certain period of time, the dose of ASM can be increased until the highest dosage tolerated by the subject that is therapeutically effective is reached. Once this dosage is determined, 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 3 months, every 6 months, or every year for one, two, three, or all of the following: (i) associated adverse events; (ii) total/direct/indirect bilirubin concentrations; (iii) plasma ceramide concentration; or (iv) acute phase response. If a subject experiences an associated adverse event of moderate intensity (e.g., an associated moderate adverse event), a total bilirubin concentration greater than the total bilirubin value for a person without ASMD (e.g., a healthy person), a plasma ceramide concentration greater than the plasma ceramide concentration for a person without ASMD (e.g., a healthy person), or an acute phase response, then the dose administered to the subject may be evaluated by a physician or other health care professional to determine whether the dose needs to be adjusted.

In one embodiment, a method of treating a human subject having acid sphingomyelinase deficiency comprises: (a) a regimen for reducing accumulated sphingomyelin substrate mass in a human subject, comprising: (i) administering an initial low, non-toxic dose of ASM to the human subject; (ii) administering successively higher doses of ASM to the human subject and monitoring the subject for one or more undesirable side effects after each subsequent dose as indicated by elevated bilirubin or a related adverse event; and (b) a maintenance regimen comprising administering a dose equal to or less than the highest tolerated dose by the subject as a maintenance dose to the subject. In certain embodiments, the initial dose ranges are from 0.1 mg/kg to 0.5 mg/kg or from 0.1 mg/kg to 1 mg/kg of ASM. In some embodiments, the successively higher doses are administered one, two, three, or four weeks after the previous dose. In certain embodiments, the sequentially higher 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 higher dose is 0.1 to 0.5 mg/kg, 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg, 2 mg/kg to 4 mg/kg, or 2 mg/kg to 5 mg/kg higher than the previous dose. In certain embodiments, the highest dose tolerated by the subject is 1 mg/kg to 2.5 mg/kg. In some embodiments, the highest dose is administered to the human subject as a maintenance dose.

In certain embodiments, a method of treating a human subject having ASMD comprises: (a) weight-loss administration of ASM to reduce the amount of sphingomyelin that has accumulated in the human subject, wherein the weight-loss administration of ASM comprises: (i) administering a low, non-toxic dose of ASM to the human subject; and (ii) administering successively higher doses of ASM to the human unless the human subject exhibits one or more undesirable side effects as indicated by an elevated total bilirubin concentration, an elevated plasma ceramide concentration, production of acute phase reactants, production of inflammatory mediators, or an adverse event (e.g., such as defined using the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1); and (b) maintenance administration of ASM, wherein the maintenance administration of ASM comprises repeat administration of a maintenance dose of ASM to the human subject. In some embodiments, the patient is monitored for a period of time after administration of the ASM dose (e.g., 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, every week, or until the next dose) for one or more undesirable side effects or total bilirubin. In certain embodiments, the maintenance dose that is administered can be adjusted during the course of treatment of the patient. In some embodiments, the maintenance dose administered to the subject is the highest dose tolerated by the subject.

In a specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof an initial low, non-toxic 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 to 0.5 mg/kg, or 0.5 to 1 mg/kg of ASM) and, after a certain period of time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks), sequentially increasing the dose of ASM administered to the subject until the level of ASM activity in one or more diseased organs 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 a subject(s) without ASMD (e.g., a healthy subject or a population of 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, 175 or more subjects), as measured by techniques known in the art, such as, for example, the technique described in He et al., 2003, Analytical Biochemistry 314: 116-120. In another embodiment, a method of treating ASMD comprises administering to a subject in need thereof a dose of 0.1 mg/kg ASM and, after a certain period of time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks), sequentially increasing the dose of ASM administered to the subject until the level of ASM activity in one or more of the following diseased organs is from 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, 10% to 20%, 15% to 20%, 15% to 25%, 25% to 50%, 50% to 75%, or 75% to 95% of the normal ASM activity in the corresponding organ in a subject(s) without ASMD (e.g., a healthy subject or a population of 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, 175 or more subjects), as measured by techniques known in the art, such as, for example, the technique described in He et al., 2003, Analytical Biochemistry 314: 116-120. In certain embodiments, the dose is sequentially increased if the total bilirubin concentration is less than or equal to 2.0 mg/dL or 2.1 mg/dL and the subject does not experience a moderate or severe related adverse event. In particular embodiments, ASM activity in normal healthy subjects is estimated to be about 20-40 units/mg protein for the brain, heart, kidney, and liver, based on ASM activity in the same organs in healthy mice using the assay described in Horinouchi et al., 1995, Nature Genetics 10: 288-293 (which is incorporated by reference herein in its entirety). In certain embodiments, ASM activity in normal healthy subjects is estimated to be about 15-25 units/mg protein for the lung and 10-15 units/mg protein for the spleen, based on ASM activity in the same organs in healthy mice using the assay described in Horinouchi et al., 1995, Nature Genetics 10: 288-293. In certain embodiments, once the ASM activity reaches normal or a certain percentage of normal in one or more diseased organs, the subject may be administered a dose equal to or less than the highest dose tolerated by the subject as a maintenance dose. Over time, the maintenance dose may be adjusted depending on the health of the subject. Depending on the conditions of the subject, the maintenance dose may be increased or decreased.

In one embodiment, a method of treating ASMD comprises: (a) administering to a human in need thereof an initial low, non-toxic dose of ASM; and (b) administering successively higher doses of ASM if the human does not exhibit one, two, three, or four of the following adverse events following administration of a dose of ASM: (i) a severe associated adverse event as defined using, for example, the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1; (ii) the total bilirubin value is greater than 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, or 4 mg/dL, or is 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 that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, one week, two weeks, or three weeks after an ASM dose; (iii) plasma ceramide concentration is greater than 8.2 μg/dL, 8.3 μg/dL, 8.4 μg/dL, 8.5 μg/dL, 8.75 μg/dL, 9 μg/dL, 9.5 μg/dL, 10 μg/dL, 11 μg/dL, 12 μg/dL, 13 μg/dL, 14 μg/dL, 15 μg/dL, 16 μg/dL, 17 μg/dL, 18 μg/dL, 19 μg/dL, 20 μg/dL, 25 μg/dL, 30 μg/dL, 35 μg/dL, 40 μg/dL, 45 mcg/dL, 50 mcg/dL, 55 mcg/dL, 60 mcg/dL, 65 mcg/dL, 70 mcg/dL, 75 mcg/dL, or 80 mcg/dL, or in the range of 8.2 mcg/dL to 10 mcg/dL, 8.5 mcg/dL to 10 mcg/dL, 9 mcg/dL to 12 mcg/dL, 10 mcg/dL to 12 mcg/dL, 10 mcg/dL to 15 mcg/dL, 10 mcg/dL to 20 mcg/dL, 15 mcg/dL to 20 mcg/dL, or 20 mcg/dL to 30 mcg/dL at 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM; or (iv) an acute phase response. According to this embodiment, successively higher doses of ASM may be administered to a human in need thereof until the human exhibits any one or more of (i)-(iv). In the event that the human exhibits any one or more of (i)-(iv), then depending on the severity of that exhibit, the same dose that resulted in the exhibiting of (i)-(iv) may be repeated or the dose may be reduced to the previous dose.

In another embodiment, a method of treating ASMD comprises: (a) administering to a human in need thereof an initial dose of 0.1 mg/kg ASM; (b) monitoring one or more of the following events in the human after administration of the ASM dose: (i) total bilirubin concentration, (ii) occurrence of an associated adverse event; (iii) acute phase response; or (iv) plasma ceramide concentration; (c) determining whether to adjust (e.g., increase or decrease) or maintain the ASM dose based on one or more of (i)-(iv); and (d) repeating steps (b) and (c) after administration of the ASM dose determined in previous step (c). In another embodiment, a method of treating ASMD comprises: (a) administering to a human in need thereof two initial doses of 0.1 mg/kg ASM 2-4 weeks apart; (b) monitoring one or more of the following events in the human after each ASM dose: (i) total bilirubin concentration, (ii) occurrence of an associated adverse event; (iii) an acute phase response; or (iv) a plasma ceramide concentration; (c) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of (i)-(iv); and (d) repeating steps (b) and (c) after administering the dose of ASM determined in the preceding step (c). According to these embodiments, the human may be administered a higher dose of ASM one or more times after 2 to 4 weeks 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 less than or equal to 2.0 mg/dL at the deadline for administering another dose of ASM; (ii) no associated adverse events or only mild associated adverse events; (iii) plasma ceramide within the normal range at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 48 hours, or 72 hours after the last dose of ASM; or (iv) no acute phase response, or an acute phase response that is not statistically significant. 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 the following: (i) a total bilirubin concentration of 2.1 mg/dL or greater up to the deadline for administration of another dose of ASM; (ii) an associated adverse event; (iii) plasma ceramide above the normal range at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 48 hours, or 72 hours after the last dose of ASM; or (iv) an acute phase response that is statistically significant.

In another embodiment, a method of treating ASMD comprises: (A) administering to a human in need thereof two initial doses of 0.1 mg/kg 2 weeks apart; (B) monitoring any of the following: (i) a total bilirubin concentration, (ii) the occurrence of an associated adverse event, (iii) both (i) and (ii) in the human after administration of each initial dose of ASM; (C) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of (i)-(iii), and (D) repeating steps (B) and (C) after administration of the dose of ASM determined in previous step (C), wherein (a) the dose administered to the human is increased if the total bilirubin concentration is less than or equal to 2.0 mg/dL or the human has a mild associated adverse event; (b) the person continues to receive the current dose 1-4 times every 2-4 weeks if the total bilirubin concentration is between 2.1 mg/dL and 3.1 mg/dL, or the person has a moderate associated adverse event, and this dose is maintained if the total bilirubin concentration remains greater than 2.0 mg/dL after the last dose administered; (c) the dose administered to the person is reduced or ASM is no longer administered if the total bilirubin concentration is greater than 3 mg/dL, or the person has a severe associated adverse event.

In another embodiment, a method of treating ASMD comprises: (A) administering to a human in need thereof two initial doses of 0.3 mg/kg 2 weeks apart; (B) monitoring any of the following: (i) a total bilirubin concentration, (ii) the occurrence of an associated adverse event, (iii) both (i) and (ii) in the human following administration of each dose of ASM; (C) determining whether to adjust (e.g., increase or decrease) or maintain the dose of ASM based on one or more of (i)-(iii), and (D) repeating steps (B) and (C) following administration of the dose of ASM determined in previous step (C), wherein (a) the dose administered to the human is increased if the total bilirubin concentration is less than or equal to 2.0 mg/dL or the human experiences a mild associated adverse event; (b) the person continues to receive the current dose 1-4 times every 2-4 weeks if the total bilirubin concentration is between 2.1 mg/dL and 3.1 mg/dL, or the person has a moderate associated adverse event, and this dose is maintained if the total bilirubin concentration remains greater than 2.0 mg/dL after the last dose administered; (c) the dose administered to the person is reduced or ASM is no longer administered if the total bilirubin concentration is greater than 3 mg/dL, or the person has a severe associated adverse event.

In a specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof a dose of 0.1 mg/kg ASM and after two weeks a dose of 0.3 mg/kg ASM every two weeks. In another specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof a dose of 0.1 mg/kg ASM, a dose of 0.3 mg/kg ASM two weeks after administration of the 0.1 mg/kg dose of ASM, and a dose of 0.6 mg/kg ASM two weeks after administration of the 0.3 mg/kg dose of ASM. In another specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof a dose of 0.1 mg/kg ASM, a dose of 0.3 mg/kg ASM two weeks after administration of the 0.1 mg/kg dose of ASM, a dose of 0.6 mg/kg ASM two weeks after administration of the 0.3 mg/kg dose of ASM, and a dose of 1 mg/kg ASM two weeks after administration of the 0.6 mg/kg dose of ASM. In certain embodiments, each dose may be repeated at least two times, and preferably two to four times, before increasing the dose to the next level. According to these embodiments, the dose is increased only if the total bilirubin value is equal to or less than 2.0 mg/dL and/or the subject experiences a mild associated adverse event. The dose is not increased if the total bilirubin value is between 2.1 and 3 mg/dL and/or the subject experiences a moderate associated adverse event. The dose is reduced to the previous tolerated dose if the total bilirubin value is greater than 3.0 mg/dL of total bilirubin and/or the subject experiences a serious associated adverse event.

In specific embodiments, the patient is treated for ASMD according to a protocol described in Section 8 or 9 et seq., below, or a similar protocol.

In a specific embodiment, a method of treating ASMD comprises administering to a subject in need thereof 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 0.1 mg/kg to 0.5 mg/kg, or 0.1 mg/kg to 1 mg/kg of ASM and, after a specified period of time (e.g., 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks), sequentially increasing the dose of ASM administered to the subject if the total bilirubin concentration is less than or equal to 2.1 mg/dL and the subject does not experience a moderate or severe associated adverse event. In some embodiments, the dose of ASM is sequentially increased until a maximum or highest dose tolerated by the subject that is therapeutically effective is reached. In certain embodiments, such highest or maximum tolerated dose is administered until the mass of accumulated sphingomyelin in the diseased organs is reduced, after which the subject is administered a maintenance dose that is below the maximum tolerated dose and is still therapeutically effective. In some embodiments, the maintenance dose is reduced over time as the patient's condition improves.

In a particular embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing one, two or more severe associated adverse events or associated adverse events that are defined using, for example, the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in total bilirubin concentration relative to the total bilirubin concentration in the patient before administration of the ASM, wherein the increase persists for more than two days, three days, five days, one week, two weeks, or three weeks. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a total bilirubin concentration that is greater than normal total bilirubin concentrations that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours, 5 days, one week, two weeks, or three weeks after administration of a dose of ASM. The normal total bilirubin concentration in a person without ASMD (e.g., a healthy person) is less than about 1.2 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a total bilirubin concentration of greater than 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, 2.75 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 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 that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, one week, two weeks, or three weeks after administration of a dose of ASM.

In another embodiment, the highest tolerated dose is the highest dosage that is effective in treating ASMD without resulting in a plasma ceramide concentration that is greater than the normal plasma ceramide concentration at 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. The normal plasma ceramide concentration in a person without ASMD (e.g., a healthy person) is approximately 1.5-8 mcg/dL. In certain embodiments, the highest tolerated dose is the highest dosage that is effective in treating ASMD without resulting in a plasma ceramide concentration of greater than about 8.2 mcg/dL, 8.3 mcg/dL, 8.4 mcg/dL, 8.5 mcg/dL, 8.75 mcg/dL, 9 mcg/dL, 9.5 mcg/dL, 10 mcg/dL, 11 mcg/dL, 12 mcg/dL, 13 mcg/dL, 14 mcg/dL, 15 mcg/dL, 16 mcg/dL, 17 mcg/dL, 18 mcg/dL, 19 mcg/dL, 20 mcg/dL, 25 mcg/dL, 30 mcg/dL, 35 mcg/dL, 40 mcg/dL, 45 mcg/dL, 50 mcg/dL, 55 mcg/dL, 60 mcg/dL, 65 mcg/dL, 70 mcg/dL, 75 mcg/dL, or 80 mcg/dL or in the range of 8.2 mcg/dL to 10 mcg/dL, 8.5 mcg/dL to 10 mcg/dL, 9 mcg/dL to 12 mcg/dL, 10 mcg/dL to 12 mcg/dL, 10 mcg/dL to 15 mcg/dL, 10 mcg/dL to 20 mcg/dL, 15 mcg/dL up to 20 mcg/dL or 20 mcg/dL to 30 mcg/dL at 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after the ASM dose.

In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an acute phase response. The acute phase response can be assessed by a change in the concentration of the acute phase agent, a change in prothrombin time, a change in partial thromboplastin time, or a change in the relative neutrophil count. For example, the acute phase response may be assessed by an increase in one or more of the following factors following administration of ASM to a subject relative to those factors before administration of ASM to the subject or relative to those factors in a person without ASMD (e.g., a healthy person): relative neutrophil count, prothrombin time (PT), partial thromboplastin time (PTT), total bilirubin concentration, C-reactive protein concentration (CRP/hs-CRP), 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 may also be assessed by a decrease in iron concentration or albumin concentration following administration of ASM to a subject relative to the iron concentration or albumin concentration in the subject before administration of ASM or relative to the iron concentration or albumin concentration in a person without ASMD (e.g., a healthy person).

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose relative to the CRP/hs-CRP concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma CRP/hs-CRP concentration greater than the normal plasma CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. The normal plasma CRP/hs-CRP concentration in a person without ASMD (e.g., a healthy person) is less than approximately 8 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma CRP/hs-CRP concentration of greater than about 8.1 mg/dL, 8.2 mg/dL, 8.3 mg/dL, 8.4 mg/dL, 8.5 mg/dL, 8.6 mg/dL, 8.7 mg/dL, 8.8 mg/dL, 8.8 mg/dL, 8.9 mg/dL, 9 mg/dL, 9.5 mg/dL, 10 mg/dL, 11 mg/dL, or 12 mg/dL, or in the range of 8.5 mg/dL to 10 mg/dL, or 8.5 mg/dL to 12 mg/dL, or 10 mg/dL to 12 mg/dL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after ASM dose administration.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the ferritin concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose relative to the ferritin concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma ferritin concentration greater than the normal plasma ferritin concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. The normal plasma ferritin concentration in a person without ASMD (e.g., a healthy person) is 10-30 ng/mL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing plasma ferritin concentrations of greater than about 300 ng/mL, 325 ng/mL, 350 ng/mL, 375 ng/mL, 400 ng/mL, 425 ng/mL, 450 ng/mL, 475 ng/mL, 500 ng/mL, 525 ng/mL, 550 ng/mL, 575 ng/mL, 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/ml, 800 ng/ml, 850 ng/ml, 900 ng/ml, 950 ng/ml, 1000 ng/ml, 1050 ng/ml, 1100 ng/ml, 1150 ng/ml, or 1200 ng/ml, or in the range of 600 ng/ml to 800 ng/ml, 650 ng/ml to 850 ng/ml, 600 ng/ml to 1000 ng/ml, 600 ng/ml to 1200 ng/ml, 800 ng/ml to 1000 ng/ml, 900 ng/ml to 1000 ng/ml, or 1000 ng/ml, or from 1000 ng/mL to 1200 ng/mL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after ASM dosing, and can be used as a measure of the acute phase response.

In a specific embodiment, an increase in the concentration of IL-8 in plasma or serum at 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 relative to the concentration of IL-8 in the patient before administration of ASM can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of IL-8 in plasma or serum that is greater than the normal concentration of IL-8 in plasma at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement 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 at 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 measurement of the acute phase response.

In a specific embodiment, an increase in the concentration of IL-6 in plasma or serum at 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 relative to the concentration of IL-6 in the patient before administration of ASM can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of IL-6 in plasma or serum that is greater than the normal concentration of IL-6 in plasma or serum at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement of the acute phase response. In certain embodiments, a plasma IL-6 concentration 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 at 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 measurement of the acute phase response.

In a specific embodiment, an increase in the concentration of calcitonin in plasma or serum at 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 relative to the concentration of calcitonin in the patient before administration of ASM can be used as a measurement of the acute phase response. In another specific embodiment, a concentration of calcitonin in plasma or serum that is greater than the normal concentration of calcitonin in plasma at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM can be used as a measurement 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 at 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 measurement of the acute phase response, 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/ml, 800 ng/ml, 850 ng/ml, 900 ng/ml, 950 ng/ml, 1000 ng/ml, 1050 ng/ml, 1100 ng/ml, 1150 ng/ml, or 1200 ng/ml or in the range of 600 ng/ml to 800 ng/ml, 650 ng/ml to 850 ng/ml, 600 ng/ml to 1000 ng/ml, 600 ng/ml to 1200 ng/ml, 800 ng/ml to 1000 ng/ml, 900 ng/ml to 1000 ng/ml, or 1000 ng/ml to 1200 ng/mL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after ASM dose administration.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in fibrinogen concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose relative to the fibrinogen concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma fibrinogen concentration that is greater than the normal plasma fibrinogen concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. A normal plasma fibrinogen concentration in a human without ASMD (e.g., a healthy human) is from 150 mg/dL to 300 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma fibrinogen concentration of greater than about 350 mg/dL, 375 mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, 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 at 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.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the relative content of neutrophils to the total number of white blood cells at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours after administration of the ASM dose compared to the relative content of neutrophils to the total number of white blood cells of the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a relative content of neutrophils to the total number of white blood cells that is greater than the normal relative content of neutrophils to the total concentration of white blood cells at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours after administration of the ASM dose. The normal relative content of neutrophils to the total number of white blood cells in a person without ASMD (e.g., a healthy person) is from 45% to 60%. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the relative content of neutrophils to the total number of white blood cells that is 70%, 75%, 80%, 85%, 90%, 95% or more at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM.

In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in antibodies to the administered ASM. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a hypersensitivity reaction. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing cytokine release syndrome.

In certain embodiments, the highest dose tolerated by a subject that is therapeutically effective is from 1 mg/kg to 2.5 mg/kg, from 2 mg/kg to 3 mg/kg, from 3 mg/kg to 5 mg/kg, from 5 mg/kg to 10 mg/kg, from 10 mg/kg to 15 mg/kg, from 15 mg/kg to 20 mg/kg, from 15 mg/kg to 25 mg/kg, from 20 mg/kg to 30 mg/kg, or from 25 mg/kg to 50 mg/kg. In some embodiments, the highest dose tolerated by a subject that is therapeutically effective 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 highest dose tolerated by a subject that is therapeutically effective is 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

In a particular embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing one, two or more severe associated adverse events or associated adverse events that are defined using, for example, the standard terminology of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model v.3.1.1. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in total bilirubin concentration relative to the total bilirubin concentration in the patient before administration of the ASM, wherein the increase persists for more than two days, three days, five days, one week, two weeks, or three weeks. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a total bilirubin concentration that is greater than normal total bilirubin concentrations and that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours, 5 days, one week, two weeks, or three weeks after administration of a dose of ASM. A normal total bilirubin concentration in a person without ASMD (e.g., a healthy person) is less than about 1.2 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a total bilirubin concentration that is greater than 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, 2.75 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 between 2.1 mg/dL and 2.5 mg/dL, 2.1 mg/dL and 3.0 mg/dL, or 2.1 mg/dL and 4 mg/dL, that persists for more than 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, one week, two weeks, or three weeks after administration of a dose of ASM.

In another embodiment, the highest tolerated dose is the highest dosage that is effective in treating ASMD without resulting in a plasma ceramide concentration greater than the normal plasma ceramide concentration at 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. The normal plasma ceramide concentration in a person without ASMD (e.g., a healthy person) is approximately 1.5-8 mcg/dL. In certain embodiments, the highest tolerated dose is the highest dosage that is effective in treating ASMD without resulting in a plasma ceramide concentration of greater than about 8.2 mcg/dL, 8.3 mcg/dL, 8.4 mcg/dL, 8.5 mcg/dL, 8.75 mcg/dL, 9 mcg/dL, 9.5 mcg/dL, 10 mcg/dL, 11 mcg/dL, 12 mcg/dL, 13 mcg/dL, 14 mcg/dL, 15 mcg/dL, 16 mcg/dL, 17 mcg/dL, 18 mcg/dL, 19 mcg/dL, 20 mcg/dL, 25 mcg/dL, 30 mcg/dL, 35 mcg/dL, 40 mcg/dL, 45 mcg/dL, 50 mcg/dL, 55 mcg/dL, 60 mcg/dL, 65 mcg/dL, 70 mcg/dL, 75 mcg/dL, or 80 mcg/dL or in the range of 8.2 mcg/dL to 10 mcg/dL, 8.5 mcg/dL to 10 mcg/dL, 9 mcg/dL to 12 mcg/dL, 10 mcg/dL to 12 mcg/dL, 10 mcg/dL to 15 mcg/dL, 10 mcg/dL to 20 mcg/dL, 15 mcg/dL up to 20 mcg/dL or 20 mcg/dL to 30 mcg/dL at 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after the ASM dose.

In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an acute phase response. The acute phase response can be assessed by a change in the concentration of the acute phase agent, a change in prothrombin time, a change in partial thromboplastin time, or a change in the relative neutrophil content. For example, the acute phase response can be assessed by an increase in one or more of the following factors after administration of ASM to a subject relative to these factors before administration of ASM to the subject or relative to these factors in a person without ASMD (e.g., a healthy person): relative neutrophil content, prothrombin time (PT), partial thromboplastin time (PTT), total bilirubin concentration, C-reactive protein concentration (CRP/hs-CRP), 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 the decrease in iron concentration or albumin concentration after ASM administration to a subject relative to the iron concentration or albumin concentration in the subject before ASM administration or relative to the iron concentration or albumin concentration in a person without ASMD (e.g., a healthy person).

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose, relative to the CRP/hs-CRP concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma CRP/hs-CRP concentration that is greater than the normal plasma CRP/hs-CRP concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. The normal plasma CRP/hs-CRP concentration in a person without ASMD (e.g., a healthy person) is less than approximately 8 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma CRP/hs-CRP concentration of greater than about 8.1 mg/dL, 8.2 mg/dL, 8.3 mg/dL, 8.4 mg/dL, 8.5 mg/dL, 8.6 mg/dL, 8.7 mg/dL, 8.8 mg/dL, 8.8 mg/dL, 8.9 mg/dL, 9 mg/dL, 9.5 mg/dL, 10 mg/dL, 11 mg/dL, or 12 mg/dL, or in the range of 8.5 mg/dL to 10 mg/dL, or 8.5 mg/dL to 12 mg/dL, or 10 mg/dL to 12 mg/dL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after ASM dose administration.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the ferritin concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose relative to the ferritin concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma ferritin concentration greater than the normal plasma ferritin concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. The normal plasma ferritin concentration in a person without ASMD (e.g., a healthy person) is 10-30 ng/mL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma ferritin concentration of greater than about 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL, 1050 ng/mL, 1100 ng/mL, 1150 ng/mL, or 1200 ng/mL, or in the range of 600 ng/mL to 800 ng/mL, 650 ng/mL to 850 ng/mL, 600 ng/mL to 1000 ng/mL, 600 ng/mL to 1200 ng/mL, 800 ng/mL to 1000 ng/mL, 900 ng/mL to 1000 ng/mL, or 1000 ng/mL to 1200 ng/mL at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after the ASM dose.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in fibrinogen concentration at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose relative to the fibrinogen concentration in the patient before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma fibrinogen concentration that is greater than the normal plasma fibrinogen concentration at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. A normal plasma fibrinogen concentration in a human without ASMD (e.g., a healthy human) is from 150 mg/dL to 300 mg/dL. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a plasma fibrinogen concentration of greater than about 350 mg/dL, 375 mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, 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 at 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.

In a specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the relative content of neutrophils to the total number of white blood cells at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose compared to the relative content of neutrophils in the patient to the total number of white blood cells before administration of the ASM. In another specific embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a relative content of neutrophils to the total number of white blood cells that is greater than the normal relative content of neutrophils to the total concentration of white blood cells at 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of the ASM dose. The normal relative content of neutrophils to the total number of white blood cells in a person without ASMD (e.g., a healthy person) is from 45% to 60%. In certain embodiments, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in the relative content of neutrophils to the total number of white blood cells that is 70%, 75%, 80%, 85%, 90%, 95% or more at 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after administration of a dose of ASM.

In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing an increase in antibodies to the administered ASM. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing a hypersensitivity reaction. In another embodiment, the highest tolerated dose that is therapeutically effective is the highest dosage that is effective in treating ASMD without causing cytokine release syndrome.

In certain embodiments, the highest dose tolerated by a subject that is therapeutically effective is from 1 mg/kg to 2.5 mg/kg, from 2 mg/kg to 3 mg/kg, from 3 mg/kg to 5 mg/kg, from 5 mg/kg to 10 mg/kg, from 10 mg/kg to 15 mg/kg, from 15 mg/kg to 20 mg/kg, from 15 mg/kg to 25 mg/kg, from 20 mg/kg to 30 mg/kg, or from 25 mg/kg to 50 mg/kg. In some embodiments, the highest dose tolerated by a subject that is therapeutically effective 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 highest dose tolerated by a subject that is therapeutically effective is 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

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

In some embodiments, a dose of ASM (e.g., a maintenance dose) is administered to the subject for a period of 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 an associated adverse event, a total bilirubin value greater than the bilirubin value for an individual without ASMD (e.g., a healthy individual), a plasma ceramide concentration greater than the plasma ceramide concentration for an individual without ASMD (e.g., a healthy individual), or an acute phase response.

In certain embodiments, the initial low non-toxic dose of ASM is administered to the subject every 3 days, every 4 days, every 5 days, every 6 days, every week, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, or every 5 weeks. In some embodiments, the dose of ASM described herein is administered to the subject every 3-5 days, every 3-7 days, every 5-7 days, every 5-10 days, every 5-14 days, every 7-14 days, every 2-4 weeks, or every 3-5 weeks. In certain embodiments, the initial low non-toxic dose of ASM is administered over a period of 4 weeks, 6 weeks, 8 weeks, 12 weeks, 14 weeks, or longer.

In certain embodiments, the subsequent increase in the ASM 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 subsequent increase in the ASM dose is from 0.1 to 0.5 mg/kg, from 0.1 mg/kg to 1 mg/kg, from 0.5 mg/kg to 1 mg/kg, from 0.5 mg/kg to 2 mg/kg, from 1 mg/kg to 2 mg/kg, from 2 mg/kg to 4 mg/kg, or from 2 mg/kg to 5 mg/kg above the previous dose.

The ASM doses described herein can be administered by any route that is suitable to achieve a therapeutic effect. Specific routes for administering ASM doses include, but are not limited to, intravenous, intraventricular, 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.

A dose of ASM enzyme that is effective in treating the somatic (non-central nervous system) manifestations of ASMD is not able to effectively cross the blood-brain barrier. Thus, in certain embodiments, ASM is administered to a patient intraventricularly or intrathecally into the brain of a patient with NPD. See, e.g., U.S. Patent Application Publication Nos. 2009/0130079 and 2009/0123451, which are incorporated herein by reference in their entireties, for methods of intraventricularly delivering lysosomally stored enzymes to the brain. In certain embodiments, ASM is administered to a patient intracerebroventricularly. See, e.g., Dodge et al., 2009, Experimental Neurology 215: 349-357, which is incorporated herein by reference in its entirety, for methods of intracerebroventricular infusion of ASM. In some embodiments, ASM is administered to a patient by indirect intraparenchymal injections. See, for example, Yang et al., 2007, Experimental Neurology 207: 258-266. In some embodiments, a modified form of ASM that targets the enzyme to the brain, as described in Section 5.2, below, is administered to a patient to treat ASMD.

Only a small percentage of the ASM enzyme administered to a patient reaches the lung. Thus, in certain embodiments, the ASM is administered to the lung of the patient. In certain embodiments, the ASM is administered to the patient via the intranasal route or by inhalation. See, for example, Ziegler et al., 2009, Molecular Genetics and Metabolism 97: 35-42, which is incorporated herein by reference in its entirety, for information regarding intranasal administration of ASM. In some embodiments, a modified form of ASM that directs the enzyme to the lung is administered to a patient for treatment of ASMD, as described in Section 5.2 below. In certain embodiments, the ASM is administered to the patient using a nebulizer. Delivery of ASM to the lung can occur via intrapulmonary injection via a bronchoscope, a metered-dose inhaler, or a nebulizer.

In certain embodiments, the ASM enzyme is administered to the patient systemically, and is also administered locally to specific organs, such as the brain and lung. In some embodiments, local administration of the ASM enzyme is supplemented by systemic administration of the enzyme. In certain embodiments, the ASM enzyme is administered locally, such as to the brain or lung, after the mass of accumulated sphingomyelin in the patient has been reduced by systemic administration (e.g., intravenous administration).

In certain embodiments, the patient is genotyped prior to administration of the ASM. In some embodiments, prior to administration of the ASM, the glycosylation pattern of the ASM expressed by the patient is determined. Administration of an ASM that is similar/compatible with that endogenously expressed by the patient may reduce the potential for immunogenicity.

In certain embodiments, endogenously expressed ASM activity is determined prior to administration of ASM. Endogenously expressed ASM activity can be measured in DBS and in cultured fibroblasts using techniques known to those skilled in the art.

In a specific embodiment, the methods for treating ASMD provided herein reduce spleen volume, as assessed by techniques known in the art, such as MRI. In another specific embodiment, the methods for treating ASMD provided herein reduce liver sphingomyelin levels, as assessed by techniques known in the art, such as biochemical analysis and/or histomorphometric analysis of liver samples. In another specific embodiment, the methods for treating ASMD provided herein increase exercise capacity, as assessed by techniques known in the art, such as maximal workload using bicycle ergometry, including percent estimated maximal workload, peak oxygen consumption, and carbon dioxide production. In another specific embodiment, the methods for treating ASMD provided herein increase pulmonary function, as assessed by techniques known in the art, such as DL _CO , FVC, FEV, and/or TLC. In another specific embodiment, the methods of treating ASMD provided herein reduce bronchoalveolar lavage (BAL) sphingomyelin. In another specific embodiment, the methods of treating ASMD provided herein reduce liver volume, as assessed by techniques known in the art, such as MRI. In another specific embodiment, the methods of treating ASMD provided herein improve lung appearance, as assessed by techniques known in the art, such as high-resolution CT imaging or chest x-ray. In another specific embodiment, the methods of treating ASMD provided herein improve lung clearance. In another specific embodiment, the methods of treating ASMD provided herein reduce sphingomyelin concentrations in liver, skin, plasma, and DBS. In another specific embodiment, the methods of treating ASMD provided herein reduce serum chitotriosidase levels. In another specific embodiment, the methods of treating ASMD provided herein reduce serum CCL18 levels. In another specific embodiment, the methods of treating ASMD provided herein improve the lipid profile of a patient (e.g., reduce cholesterol). In another specific embodiment, the methods of treating ASMD provided herein reduce or alleviate the severity of ASMD and/or a symptom associated therewith. In another specific embodiment, the methods of treating ASMD provided herein reduce the duration of a symptom associated with ASMD. In another specific embodiment, the methods of treating ASMD provided herein prevent the recurrence of a symptom associated with ASMD. In another specific embodiment, the methods of treating ASMD provided herein reduce hospitalization of a subject. In another specific embodiment, the methods of treating ASMD provided herein reduce the length of hospitalization. In another specific embodiment, the methods of treating ASMD provided herein increase the survival of a subject. In another specific embodiment, the methods of treating ASMD provided herein reduce mortality in a subject. In another specific embodiment, the methods of treating ASMD provided herein reduce the frequency of hospitalization of a subject. In another specific embodiment, the methods of treating ASMD provided herein reduce the number of symptoms associated with ASMD. In another specific embodiment, the methods of treating ASMD provided herein increase symptom-free survival in patients with ASMD. In another specific embodiment, the methods of treating ASMD provided herein improve neurological function (e.g., psychomotor function, social responsibility, etc.) of a subject.

In another specific embodiment, the ASMD treatment methods described herein improve quality of life in a patient. In certain specific embodiments, the method of treating ASMD provided herein improves quality of life in a patient as assessed by, 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 consists of the BFI, BPI-SF, and the Medical Examination Short Form 36, the Chronic Respiratory Questionnaire-SAS-Standardized Format (CRQ-SAS; Schunemann et al., 2005, Eur. Respir. J. 25: 31-40) for assessing dyspnea and fatigue, and the Ambulatory Activity Measure (AM-PAC). a computer-adapted test that assesses functional mobility (e.g., ambulation, self-care, and applied thinking).

Enzyme - acid sphingomyelinase

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 skilled in the art, such as those described in U.S. Patent Nos. 4,039,388, 4,082,781, 5,686,240, and 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, the acid sphingomyelinase enzyme has the ability to hydrolyze sphingomyelin to ceramide and phosphorylcholine in a fluorescence-based high performance liquid chromatography assay described in He et al., 2003, Analytical Biochemistry 314: 116-120. In a specific embodiment, the acid sphingomyelinase has at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95%, or 98%, or 30% to 50%, 40% to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%, or 85% to 95% of the activity of ASM-1 (SEQ ID NO: 1, below), as measured by, for example, the assay described in He et al., 2003, Analytical Biochemistry 314: 116-120.

In a specific embodiment, the ASM is human ASM. There are different isoforms of human ASM, which are produced by alternative splicing. One isoform of human ASM, human ASM isoform 1 (sometimes referred to as ASM-1), has the amino acid sequence listed under UniProtKB/Swiss-Prot accession number. P17405-1. Another isoform of human ASM, human isoform 2 (sometimes referred to as ASM-2), has the amino acid sequence listed under UniProtKB/Swiss-Prot accession number. P17405-2. A third isoform of human ASM, human isoform 3 (sometimes referred to as ASM-3), has the amino acid sequence listed under UniProtKB/Swiss-Prot accession number. P17405-3. The amino acid sequence of ASM-1, which is the most common isoform, is shown below:

(SEQ ID NO:1). ASM-2 differs from ASM-1 in that amino acid residues 363-374 of ASM-1 (i.e., IGGFYALSPYPG (SEQ ID NO:2)) are replaced by amino acids YLSSVETQEGKR (SEQ ID NO:3), and amino acid residues 375-418 of ASM-1 are missing from ASM-2. ASM-3 differs from ASM-1 in that amino acid residues 363-418 of ASM-1 are missing from ASM-3. Provided that ASM-2 and ASM-3 have enzymatic activity, as measured by, for example, 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 a specific embodiment, provided that ASM-2 and ASM-3 have at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95%, or 98%, or from 30% to 50%, 40% to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%, or 85% to 95% of the activity of ASM-1, as measured using, for example, 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 a specific embodiment, the human ASM has the amino acid sequence of human acid sphingomyelinase disclosed in SEQ ID NO:1, previously, the amino acid sequence of human acid sphingomyelinase disclosed in U.S. Patent No. 6,541,218 (e.g., SEQ ID NO:2 in U.S. Patent No. 6,541,218), or the amino acid sequence disclosed in Fig. 3 of Schuchman et al., 1991, J. Biol. Chem. 266:8531-8539, each of which is incorporated herein by reference in its entirety.

In particular embodiments, human ASM is a processed mature form. In other embodiments, human ASM is an immature, unprocessed form. Relative 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 a signal peptide and is from amino acid residue 47 to amino acid residue 629. Human ASM-1 contains a B-type saposin domain from amino acid residues 85 to 169. Relative to ASM-1, the following amino acid residues are glycosylated (particularly N-linked glycosylated): 86, 175, 335, 395, and 520.

In addition to the human ASM isoforms, there are various naturally occurring variants of human ASM. For example, naturally occurring variants of the human ASM gene have been identified with varying numbers of hexanucleotide repeat units occurring within the region of the gene encoding the putative ASM signal peptide. See, e.g., Wan and Schuchman, 1995, Biochimica et Biophysica Acta 1270: 207-210 (which is incorporated herein by reference in its entirety), which describes the identification of five alleles corresponding to nine, seven, six, five, and four hexanucleotide repeats. Additionally, naturally occurring variants of human ASM with individual amino acid changes have been identified. See, e.g., Schuchman et al., 1991, J. of Biol. Chem 266: 8531-8539 and Schuchman et al., 1991, Nucleic Acids Research 19(11): 3160, which are incorporated herein by reference in their entireties, for information regarding single nucleotide polymorphisms. Provided that any of the naturally occurring human ASM variants has enzymatic activity as measured by, for example, the assay described in He et al., 2003, Analytical Biochemistry 314: 116-120, they are included as ASM that can be administered to a subject. In a specific embodiment, provided that any of the naturally occurring human ASM variants has at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95%, or 98%, or 30% to 50%, 40% to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%, or 85% to 95% of the activity of ASM-1, as measured by, for example, the assay described in He et al., 2003, Analytical Biochemistry 314: 116-120, they are included as ASM that can be administered to a subject.

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

In one embodiment, the ASM is a modified form of human ASM that has enhanced enzymatic activity relative to unmodified human ASM, such as ASM-1. See, e.g., 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 with enzymatic activity greater than wild-type recombinant human ASM (such as, e.g., ASM-1). In some embodiments, the ASM has enhanced enzymatic activity relative to unmodified human ASM (such as, e.g., ASM-1) due to the addition of zinc cations. See, e.g., Schissel et al., 1998, J. Biol. Chem 273: 18250-18259, which is incorporated herein by reference in its entirety, for a discussion regarding the role of zinc in ASM activity. In another embodiment, the ASM is a modified form of human ASM that has increased affinity for a natural human ASM receptor (e.g., mannose 6-phosphate or high mannose) relative to unmodified human ASM (such as, for example, ASM-1). In a specific embodiment, the ASM is conjugated to an oligosaccharide as described in U.S. Patent No. 7,001,994 and International Patent Application Publication No. WO 2010/075010 and U.S. Patent Application Publication No. 2010/0173385 (which are incorporated herein by reference in their entireties) to enhance the targeting of the enzyme to its natural receptor. In another embodiment, the ASM is a modified form of human ASM that binds to an alternative receptor (e.g., intracellular adhesion molecule (ICAM)-1, which can increase targeting to organs such as the lung) rather than the natural receptor for ASM. In an embodiment, the ASM is a modified form of human ASM that has increased stability relative to unmodified human ASM (such as, for example, ASM-1).

In some embodiments, the ASM is conjugated or fused, directly or indirectly, to a targeting molecule 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). See, for example, U.S. Patent Application Publication No. 2009/0029467 and U.S. Patent No. 7,560,424 (which is incorporated herein by reference in its entirety) for a discussion of the use of IGF-I as a targeting molecule for lysosomally stored enzymes, and U.S. Patent No. 7,396,811 (which is incorporated herein by reference in its entirety) for a discussion of the use of IGF-II as a targeting molecule for lysosomally stored enzymes. The targeting molecules IGF-I and IGF-II can facilitate the targeting of the ASM enzyme to the lysosome. For a discussion of the use of hormones such as G-CSF and leptin as targeting molecules, see, for example, International Patent Application Publication No. WO 2007/091250 and U.S. Patent Application Publication No. 2010/0183577, which are incorporated herein by reference in their entireties. See, for example, U.S. Patent Application Publication No. 2004/0101904, which is incorporated herein by reference in its entirety, for a discussion of a humanized monoclonal antibody that binds to the human insulin receptor (HIR) attached to a pharmaceutical such as an enzyme. The targeting molecules G-CSF, leptin, and an antibody that binds to the HIR can facilitate the targeting of the ASM enzyme to the brain.

In certain embodiments, the ASM is a modified form of ASM (e.g., a modified form of human ASM) with decreased activity relative to unmodified ASM (such as unmodified human ASM, e.g., ASM-1). In some embodiments, the ASM is a modified form of ASM (e.g., a modified form of human ASM) with increased targeting to pathological sites and decreased enzymatic activity relative to unmodified ASM (such as unmodified human ASM, e.g., ASM-1).

In certain embodiments, the ASM is a highly phosphorylated form of ASM. See, for example, U.S. Patent Application Publication No. 2002/0150981, which is incorporated herein by reference, for a discussion of techniques for producing highly phosphorylated forms of lysosomal enzymes.

The ASM enzyme 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. Patent No. 5,773,278, which are incorporated by reference herein in their entireties for human ASM cDNA clones), genomic cloning, gene activation (see, e.g., U.S. Patent No. 5,641,670, which is incorporated by reference herein in its entirety for gene activation techniques), or selected cell lines (e.g., mammalian, yeast, prokaryotic, insect (e.g., Sf 9, Sf21, Trichoplusia ni, Spodoptera frugiperda, and Bombyx mori), and plant cells) that produce high levels of the ASM enzyme. ASM, as well as methods for producing ASM, are described, for example, in U.S. Patent Nos. 5,773,278; 6,541,218; 5,686,240; and 7,527,956; each of which is incorporated herein by reference in its entirety.

In a particular embodiment, the ASM is a recombinantly produced ASM (e.g., recombinantly produced human ASM). Preferred are cell expression systems that have cellular machinery and elements for proper processing, i.e., signal cleavage, glycosylation, phosphorylation, and protein sorting. For example, mammalian cell expression systems are preferred for expressing biologically active enzymes that are properly folded and processed; when administered to humans, such expression products should exhibit proper tissue targeting and not elicit an undesirable immunological response.

In certain embodiments, ASM (e.g., human ASM) is produced by overexpressing ASM cDNA in mammalian cells. In a particular embodiment, human ASM is produced by overexpressing human ASM cDNA in Chinese hamster ovary (CHO) cells. See, e.g., He et al., 1999, Biochimia et Biophsyica Acta 1432:251-264 for methods of overexpressing human ASM cDNA in CHO cells and U.S. Patent No. 6,541,218, each of which is incorporated by reference herein in its entirety. In certain embodiments, human ASM is produced by overexpressing human ASM cDNA in CHO cells that coexpress alpha-2,6-sialitransferase. See, for example, U.S. Patent No. 5,047,335, incorporated herein by reference in its entirety, which describes CHO cells engineered to express alpha 2,6-sialitransferase.

In another specific embodiment, the mammalian cells are human cells. Examples of human cells that can be used to recombinantly express ASM (e.g., human ASM) include, but are not limited to, 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 those human cell lines listed in the Immunopolymorphism Database of the European Molecular Biology Laboratory and the U.S. National Institutes of Health database, as well as those commercially available from, for example, the American Type Culture Collection, Manassas, VA, and Lifeline Cell Technology, Walkersville, MD. When using human cell lines to produce rhASM, rhASM can be produced using cDNA, gDNA for hASM, or gene activation techniques such as those described in U.S. Patent No. 5,641,670, which is incorporated by reference herein in its entirety.

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, 9B5 clone AF-2 cells, 9B5 clone AF-2 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.

In some embodiments, a plant cell culture system is used to express ASM. See, e.g., U.S. Patent 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 herein by reference in their entireties) for plant cells and methods of producing proteins using a plant cell culture system. In particular, U.S. Patent Application Nos. 2009/0208477, 2008/0038232, and 2006/0204487 describe the expression and production of enzymatically active high-mannose lysosomal enzymes using a transgenic plant root, particularly carrot cells. In a particular embodiment, the carrot cells are engineered to express ASM. In certain embodiments, algae (e.g., Chlamydomonas reinhardtii) can be engineered to express ASM (see, e.g., Rasala et al., 2010, Plant Biotechnology Journal (published online March 7, 2010, which is incorporated herein by reference in its entirety).

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 enhance drug distribution. For example, ASM can be produced using cells that have a mutation, e.g., a knockout, in at least one Golgi processing mannosidase. In one embodiment, the mutation reduces gene expression, reduces protein levels or activity, or alters distribution or other post-translational modifications of the mannosidase, e.g., carbohydrate chain processing. In a specific embodiment, the mutation reduces the level of Golgi processing mannosidase activity. The mutation can be in a class 1 processing mannosidase; a class 2 processing mannosidase; class 1 processing mannosidase and class 2 processing mannosidase. Class 1 processing mannosidase includes: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC. Class 2 processing mannosidase includes: Golgi mannosidase II. See, e.g., International Patent Application Publication No. WO 02/15927 and U.S. Patent No. 7,138,262, which are incorporated herein by reference in their entireties, for methods of producing a high mannose protein.

In certain embodiments, cells expressing ASM are cultured in the presence of a mannosidase inhibitor, such as an antibody, 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). Culturing ASM in the presence of such inhibitors can result in the production of high-mannose ASM. In some embodiments, cells expressing ASM include a nucleic acid sequence, such as an antisense molecule or a ribozyme, that can bind to or inactivate a cellular mannosidase nucleic acid sequence, such as mRNA, and inhibit protein expression. See, for example, International Patent Application Publication No. WO 02/15927 and U.S. Patent No. 7,138,262, which are incorporated herein by reference in their entireties, for methods of producing high-mannose protein.

In certain embodiments, the carbohydrate chains of the recombinantly expressed ASM enzyme are converted by sequential treatment with different enzymes, such as neuraminidase, galactosidase, and β-N-acetylglucosaminidase. See, for example, U.S. Patent No. 5,549,892, which is incorporated herein by reference in its entirety, for methods of converting the carbohydrate chains of a lysosomal enzyme.

Mannose-6-phosphate (M6P)-mediated uptake of ASM can be enhanced by modifying ASM to have highly phosphorylated mannose residues and M6P. For example, ASM can be modified using recombinant technology to introduce 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 combined with highly phosphorylated oligosaccharide derivatives containing 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, a yeast culture system can be used to express recombinant ASM that contains additional highly phosphorylated mannose-6-phosphate residues. See, for example, Akeboshi et al., 2009 Glycobiology 19(9):1002-1009, which is incorporated by reference herein in its entirety.

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

The ASM enzyme can be formulated for any route of administration (e.g., infusion, subcutaneous, intramuscular, intrathecal, intraventricular, intranasal, inhalation, or intradermal). The ASM enzyme can be supplied in a lyophilized form that is reconstituted prior to use, such as in sterile saline (e.g., 0.9% sodium chloride) or sterile water. Alternatively, the ASM enzyme can be supplied in aqueous form. In certain embodiments, the ASM is administered to the subject in a formulation containing zinc. In some embodiments, the ASM enzyme is administered to the patient by infusion, using, for example, a syringe pump or an infusion bag with a pump.

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

5.2 PATIENT POPULATIONS

In specific embodiments, a subject treated for ASMD according to the methods provided herein is a human who has or is diagnosed as having one or more mutations in a gene encoding acid sphingomyelinase. In specific embodiments, a subject treated for ASMD according to the methods provided herein is a human who has or is diagnosed as having Niemann-Pick disease (NPD). In one embodiment, a subject treated for ASMD according to the methods provided herein is a human who has or is diagnosed as having NPD type A. In another specific embodiment, a subject treated for ASMD according to the methods provided herein is a human who has or is diagnosed as having NPD type B.

In certain embodiments, a 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 that results in the deletion of one, two, or more amino acid residues. In specific embodiments, a subject treated for ASMD according to the methods provided herein has one or more mutations.

Table 1 Amino acid position of ASM-1 Mutation 49 D → V in NPDB 92 C → W in NPDB 103 L → P in NPDA and NPDB 130 V → A in NPDB 137 L → P in NPDB 157 C → R in NPDB 166 G → R in NPDB 176 I → N in NPDB 184 P → L in NPDA/NPDB 196 A → P in NPDB 200 R → C in NPDB 225 L → M in NPDB 225 L → P in NPDB 228 R → C in NPDB 228 R → H in NPDA/NPDB 232 G → D in NPDB 241 A → V in NPDA/NPDB 242 G → R in NPDB 244 W → C in NPDB 245 G → S in NPDA and NPDB 246 E → K in NPDA 246 E → Q in NPDA 248 S → R in NPDA and NPDB 251 D → E in NPDA/NPDB 278 D → A in NPDA/NPDB 281 A → T in NPDB 289 R → H in NPDB 292 Q → K in NPDA/NPDB 294 R → Q in NPDA 302 L → P in NPDA 313 Y → H in NPDA 319 H → Y in NPDA 323 P → A in NPDB. 330 P → R in NPDB 341 L → P in NPDA/NPDB 357 A → D in NPDB 367 Y → C in NPDA 371 P → S in NPDB 376 R → H in NPDB 376 R → L in NPDB 379 S → P in NPDB 382 M → I in NPDA 383 N → S in NPDB 389 N → T in NPDA 390 Missing in NPDA. 391 W → G in NPDB 413 A → V in NPDB 421 H → R in NPDA 421 H → Y in NPDB 431 C → R in NPDB 432 L → P in NPDB 435 W → C in NPDB 436 S → R in NPDB 446 Y → C in NPDA 450 L → P in NPDA 452 A → V in NPDB 456 G → D in NPDB 463 F → S in NPDA 467 Y → S in NPDA 474 R → W in NPDB 475 P → L in NPDA and NPDB 480 F → L in NPDB 482 A → E in NPDA 485 A → V in NPDB 486 T → A in NPDB 488 Y → N in NPDB 494 G → S in NPDB 496 R → C in NPDB 496 R → H in NPDA 496 R → L in NPDA 514 H → Q in NPDB 515 E → V in NPDB 517 Y → C in NPDA 533 W → R in NPDB 537 Y → H in NPDA 549 L → P in NPDB 563 D → Y in NPDB 576 K → N in NPDB 577 G → S in NPDA 592 Missing in NPDA. 600 R → H in NPDB 600 R → P in NPDB 608 Missing in NPDB

See, for example, Simonaro et al., 2002, Am. J. Hum. Genet. 71: 1413-1419, which is incorporated herein by reference, regarding mutations in the acid sphingomyelinase gene (designated SMPD1).

In certain embodiments, a subject treated for ASMD according to the methods provided herein endogenously expresses ASM with 2-5%, 5-10%, 5-15%, 5-20%, 5-30%, or 20-30% of the activity of normal human ASM, such as ASM-1. In some embodiments, a subject treated for ASMD according to the methods provided herein endogenously expresses ASM with less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the activity of normal human ASM, such as ASM-1. See, for example, U.S. Patent Nos. 4,039,388, 4,082,781, 5,686,240, and 7,563,591 and International Publication Nos. WO 2007/078806 and WO 2006/058385, which are incorporated herein by reference in their entireties, for techniques that can be used to measure ASM activity. In a particular embodiment, the fluorescence-based high performance liquid chromatography assay described in He et al., 2003, Analytical Biochemistry 314: 116-120 (which is incorporated herein by reference) is used to measure ASM activity.

In certain embodiments, a subject receiving treatment for ASMD in accordance with the methods described herein exhibits one or more symptoms of NPD. Symptoms of NPD include, but are not limited to, abdominal distension, hepatomegaly, splenomegaly, hepatosplenomegaly, neutropenia, lung disease, lymphadenopathy, presence of histochemically distinctive foam cells of NPD, anemia (eg, microcytic anemia), thrombocytopenia, recurrent vomiting, chronic constipation, growth failure (eg, decreased linear height and body weight), delayed puberty, recurrent bruising, recurrent bleeding, atherogenic lipid profile (high cholesterol, triglycerides, LDL, and low HDL), pain (headache, back pain, extremity pain, abdominal pain), fatigue, early satiety, low endurance, osteopenia, neurologic manifestations, and respiratory difficulties (eg, interstitial lung disease, dyspnea). Neurological manifestations of NPD include cherry pit syndrome, hypotonia, muscle weakness, psychomotor retardation, spasticity, social unresponsiveness, irritability, and seizures.

In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human subject that exhibits two or more clinical features characteristic of a non-neuropathic NPD. In particular embodiments, a subject treated for ASM according to the methods provided herein is a human subject that exhibits two or more clinical features characteristic of a non-neuropathic NPD: thrombocytopenia, anemia, neutropenia, hepatomegaly, splenomegaly, and lung disease. In other embodiments, a subject treated for ASMD according to the methods provided herein is a human subject that exhibits two or more clinical features characteristic of a neuropathic NPD.

In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human with a spleen volume that is two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times larger than the spleen volume of a healthy human, as assessed by techniques known in the art, such as, for example, magnetic resonance imaging (MRI). In specific embodiments, a subject treated for ASMD according to the methods provided herein is a human with a spleen volume that is eight times larger than the spleen volume of a healthy human, as assessed by techniques known in the art, such as, for example, MRI. In some embodiments, a subject treated for ASMD according to the methods provided herein is a human with a spleen volume eight to twelve, nine to twelve, ten to twelve, or twelve to fourteen times larger than the spleen volume of a healthy human, as assessed by techniques known in the art, such as, for example, MRI. In particular embodiments, a subject treated for ASMD according to the methods provided herein is a human with a spleen volume ≥8 times normal (MN) (i.e., 1.6% of body weight), as assessed by techniques known in the art, such as, for example, MRI.

In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human with a diffusing capacity of the lungs (DL _CO ) of 20% to 80%, 25% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, or 60% to 80% of the estimated DL _CO of a healthy human. In some embodiments, a subject treated for ASMD according to the methods provided herein is a human with a DL _CO of 20% to 90%, 25% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, or 70% to 90% of the estimated DL _CO of a healthy human. DL _CO measures the rate of diffusion of a diffusion-limited gas (e.g., carbon monoxide) per minute across the alveolar capillary membrane. DL _CO can be calculated by comparing the amount of carbon monoxide exhaled to a known amount of inhaled carbon monoxide. Techniques known in the art can be used to estimate DL _CO .

In certain embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human infant. In some embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human who is 2-3 months, 2-6 months, 3-6 months, 4-6 months, 5-8 months, or 6-9 months old. In other embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human child. In some embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human who is 1-3 years old, 2-3 years old, 3-5 years old, 4-5 years old, 5-7 years old, or 6-9 years old.

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

In particular embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human female. In other embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human male. In certain embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human female who is not pregnant or breastfeeding. In other embodiments, the subject receiving treatment for ASMD according to the methods provided herein is a human female who is pregnant, or will become pregnant, or is breastfeeding.

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

In certain embodiments, a subject treated for ASMD in accordance with the methods described herein is a human who does not have or has not been diagnosed with one or more of the following diseases: active hepatitis B, active hepatitis C, human immunodeficiency virus (HIV) infection, cirrhosis, or serious cardiac disease (e.g., moderate or severe pulmonary hypertension, or valvular dysfunction, or left ventricular ejection fraction by echocardiography less than 50%, less than 40%, less than 30%, or less than 20%). In other embodiments, a subject treated for ASMD according to the methods described herein is a human who has or has been diagnosed with one or more of the following diseases: active hepatitis B, active hepatitis C, human immunodeficiency virus (HIV) infection, cirrhosis, or significant cardiac disease (e.g., moderate or severe pulmonary hypertension, or valvular dysfunction, or left ventricular ejection fraction by echocardiography less than 50%, less than 40%, less than 30%, or less than 20%).

In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human who does not have one or more of the following: an international normalized ratio (INR) of greater than 1.25, 1.5, 1.75, or 2, a platelet count of less than 60× ¹⁰³ per μL, an alanine aminotransferase (ALT) of greater than 250 IU/L, an aspartate aminotransferase of greater than 250 IU/L, or a total bilirubin of greater than 1.5 mg/dL, 1.75 mg/dL, or 2 mg/dL. In other embodiments, a subject treated for ASMD according to the methods provided herein is a human who has one or more of the following: an international normalized ratio (INR) of greater than 1.25, 1.5, 1.75, or 2, a platelet count of less than 60× ¹⁰³ per μL, an alanine aminotransferase (ALT) of greater than 250 IU/L, an aspartate aminotransferase of greater than 250 IU/L, or a total bilirubin of greater than 1.5 mg/dL, 1.75 mg/dL, or 2 mg/dL.

In certain embodiments, a subject treated for ASMD according to the methods provided herein is a human who is not receiving one or more 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 who is receiving one or more of the following medications: chlorpromazine, imipramine, or desipramine.

In certain embodiments, a subject treated for ASMD in accordance with the methods provided herein is a human who is not receiving herbal supplements or medications that may cause or prolong bleeding (e.g., anticoagulants, ibuprofen, aspirin, garlic supplements, ginkgo, and ginseng) or have potential hepatotoxicity (e.g., 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors, erythromycin, valproic acid, antidepressants, kava, and echinacea). In other embodiments, the subject receiving treatment for ASMD in accordance with the methods provided herein is a human receiving one or more of the following herbal supplements or medications: anticoagulants, ibuprofen, aspirin, garlic supplements, ginkgo, ginseng, 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors, erythromycin, valproic acid, antidepressants, kava, and echinacea.

In specific embodiments, the subject receiving treatment for ASMD in accordance with the methods provided herein is a human who meets one, two, or more, or all of the criteria for subjects in the working examples in Sections 6, 8, and 9 et seq.

5.3. TREATMENT MONITORING

According to the method provided herein, a number of 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 and, if necessary or recommended, during the course of treatment with ASM. The physical examination can include the following assessments: general appearance, skin, head, ears, eyes, nose, and throat (HEENT), lymph nodes, heart, lungs, abdomen, extremities/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 rates can be monitored continuously via telemetry, starting, for example, 6 hours before administration of a dose of ASM until 72 hours after administration of a dose of ASM.

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

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 histological analysis using MetaMorph.

In certain embodiments, pulmonary function tests are performed before, during, and/or after administration of a dose of ASM. Calibration of pulmonary function testing equipment and test protocols may be standardized according to American Thoracic Society (ATS) guidelines (ATS, 1991, Am Rev Respir Dis 144: 1202-1218). In certain embodiments, relative estimated 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 expiratory maneuver. FVC can be measured using standard spirometric techniques.

In certain embodiments, the forced expiratory volume in 1 second (FEV1) may be measured before, during, and after the ASM dose is administered. FEV1 is the volume of air expelled during the first second of FVC. FEV1 may be measured using standard spirometric techniques.

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 maximal inspiratory effort. TLC can be measured using whole-body plethysmography.

In certain embodiments, DL _CO can be estimated before, during, and after administration of a dose of ASM. DL _CO measures the rate of diffusion of diffusion-limited gas (carbon monoxide, CO) per minute across the alveolar capillary membrane. DL _CO can be calculated by comparing the amount of carbon monoxide (CO) exhaled after a known amount of inspired CO. Helium, which does not diffuse across the alveolar capillary membrane, can be included as an isotope with inspired CO to monitor air entrainment.

In certain embodiments, chest radiography (posterior-anterior and lateral) may be obtained before, during, and/or after administration of the ASM dose. In certain embodiments, abdominal MRI is obtained before, during, and/or after administration of the ASM dose.

In certain embodiments, a blood sample is collected before, during, and/or after administration of a dose of ASM to assess biomarkers, bilirubin concentration, and the relative proportion of neutrophils to total white blood cells. In a particular embodiment, the concentration of one or more of the following biomarkers is assessed using techniques known to one of skill 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 certain embodiments, the concentration of one or more of the following cytokines is assessed using techniques known to one of skill in the art: IL-6 and IL-8.

In certain embodiments, the concentration of anti-ASM antibodies (e.g., IgG to recombinant human ASM and/or IgE to recombinant human ASM) is assessed before, during, and/or after administration of a dose of ASM. In some embodiments, the concentration of one, two, 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, a skin test for IgE-mediated reactions to ASM is assessed before, during, and/or after administration of a dose of ASM.

In certain embodiments, the pharmacokinetic profile for ASM is measured before, during, and/or after administration of a dose of ASM. In some embodiments, the BAL cell count is measured before, during, and/or after administration of a dose of ASM. See Section 8 et seq., below, for other parameters that may be assessed before, during, and/or after administration of a dose of ASM.

In certain embodiments, a factor or marker described herein is assessed 1 week, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes before the ASM dose is administered. In some embodiments, a factor or marker described herein is assessed at the time the ASM dose is administered. For example, a factor or marker described herein is assessed at the time the ASM dose is administered. In certain embodiments, a factor or marker described herein is assessed 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, two weeks, three weeks, four weeks, one month, two months, three months, or longer after the ASM dose is administered. In certain embodiments, a factor or marker described herein is assessed several hours or weeks after a certain number of doses of ASM. For example, a factor or marker described herein can be assessed every four weeks, a month, 2 months, 3 months, 4 months, 5 months, or 6 months after a certain number of doses of ASM. In certain embodiments, a factor or marker is assessed every 4 weeks, a 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 embodiments, the factor or marker is assessed every month to 2 months, every month to 4 months, every 2 months to 4 months, every 2 months to 4 months, every 3 months to 4 months, every 2 months to 5 months, every 3 months to 5 months, every 4 months to 5 months, every 2 months to 6 months, every 3 months to 6 months, every 4 months to 6 months, or every 5 months to 6 months. In certain embodiments, the factor or marker is assessed every 6 months to 8 months, every 6 months to 12 months, every 8 months to 12 months, every 9 months to 12 months, or every 10 months to 12 months. In some embodiments, one or more factors or markers described herein are not assessed before, during, and/or after administration of a dose of ASM.

In certain embodiments, the results from the assessment of one or more factors or markers indicate that the dosage of ASM should be adjusted.

5.4. BIOLOGICAL SAMPLES

According to the methods described herein, a biological sample is subjected to one or more pre-treatment steps prior to detecting and/or measuring a cell population, factor, or marker (e.g., a biomarker). In certain embodiments, a biological fluid is pre-treated by centrifugation, filtration, sedimentation, dialysis, or chromatography, or a combination of such pre-treatment steps. In other embodiments, a tissue sample is pre-treated by freezing, chemical fixation, paraffin immersion, dehydration, permeabilization, or homogenization, followed by centrifugation, filtration, sedimentation, dialysis, or chromatography, or a combination of such pre-treatment steps. In certain embodiments, a sample is pre-treated by removing a particular cell type from the sample or removing foreign matter from the sample prior to determining the number or amount of a particular cell type(s) in the sample according to the methods described herein.

The biological sample may be a tissue sample, a biological fluid, a secretion, or any other sample from a human subject. In some embodiments, the biological sample is a blood or 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 swab of the oral cavity.

Techniques known in the art can be used to assess the presence, number, quantity, or relative abundance of a particular cell type(s) present in a biological sample. For example, cells can be sorted using a fluorescence-activated cell sorter (FACS). Fluorescence-activated cell sorting (FACS) is a known method for separating particles, including cells, based on the fluorescence properties of the particles. See, for example, Kamarch, 1987, Methods Enzymol 151:150-165. Laser excitation of fluorescent molecules in individual particles results in a small electrical charge that allows electromagnetic separation of positive and negative particles from a mixture. An antibody or ligand used to detect an antigenic determinant present on the cell surface of particular cells is labeled with a fluorochrome such as FITC or phycoerythrin. The cells are incubated with a fluorescently labeled antibody or ligand for a period of time sufficient to allow binding of the labeled antibody or ligand to the cells. The cells are passed through a cell sorter, allowing the separation of the cells of interest from other cells. FACS-sorted particles can be directly dispensed into individual wells of microtiter plates to facilitate separation.

Magnetic beads can also be used to separate cells. For example, cells can be sorted using magnetically activated cell sorting (MACS), a method for separating particles based on their ability to bind to magnetic beads (0.5-100 μm in diameter). A variety of suitable modifications can be made to the magnetic beads, including a covalent addition to an antibody that specifically recognizes a cell-solid phase surface molecule or hapten. A magnetic field is then applied to physically manipulate the selected beads. In a particular embodiment, antibodies to a blood cell surface marker are coupled to magnetic beads. The beads are then mixed with a blood cell culture to ensure binding. The cells are then passed through a magnetic field to separate out cells having the blood cell surface markers of interest. These cells can then be isolated.

In some embodiments, the surface of a culture dish can be coated with antibodies and used to separate cells using a method called panning. Individual dishes can be coated with an antibody specific to particular cells. The cells can be added first to a dish coated with blood cell-specific antibodies of interest. After thorough washing, the cells that remain bound to the dish will be cells that express the cellular markers of interest. Examples of antigenic determinants or cell surface markers include, but are not limited to, CD2 for T lymphocytes and natural killer cells, CD3 for T lymphocytes, CD11a for leukocytes, CD28 for T lymphocytes, CD19 for B lymphocytes, CD20 for B lymphocytes, CD21 for B lymphocytes, CD22 for B lymphocytes, CD23 for B lymphocytes, CD29 for leukocytes, CD14 for monocytes. CD41 for platelets, CD61 for thrombocytes, CD66 for granulocytes, CD67 for granulocytes, and CD68 for monocytes and macrophages.

The presence, concentration or amount of a marker (including a biomarker) or factor can be assessed using techniques known in the art. The presence, concentration or amount of a marker (including a biomarker) or factor can be measured at the protein level and/or the RNA level using techniques known to those skilled in the art. At the protein level, immunoassays such as ELISA and immunoprecipitation and Western blotting assays can be used to measure the presence, concentration or amount of a marker (including a biomarker) or factor. In addition, FACS can be used to measure the presence, concentration or amount of a marker (including a biomarker) or factor. At the RNA level, real-time PCR (polymerase chain reaction) and Northern blotting assays can be used to measure the presence, concentration or amount of a marker (including a biomarker) or factor.

5.5. COMBINATION THERAPIES

In some embodiments, a method of treating ASMD comprises administering ASM in combination with one or more additional therapies. As used herein, the term "in combination" refers, in the context of administering ASM, to administering a dose of ASM prior to, in conjunction with, or following administration of one or more additional therapies (e.g., agents) for use in treating ASMD. Use of the term "in combination" does not limit the order in which ASM and one or more additional therapies are administered to a subject. In particular embodiments, the time intervals between administration of the ASM dose and administration of one or more additional therapies may be as follows: about 1-5 minutes, 1-30 minutes, 30 minutes - 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 period of time in between. In certain embodiments, the ASM dose and one or more additional therapies are administered less than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, 2 months, 3 months, 6 months, 1 year, 2 years, or 5 years apart.

In some embodiments, the combination therapies provided herein comprise administering a dose of ASM every 2-4 weeks, and administering one or more additional therapies once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 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, ASM and one or more additional therapies are administered cyclically to a subject. Cycling therapy comprises administering ASM for a period of time, followed by administering one or more additional therapies for a period of time, and repeating this sequential administration. In certain embodiments, the cyclic therapy may also include a rest period wherein the ASM or additional therapy 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 cycles administered is 1 to 12 cycles, 2 to 10 cycles, or 2 to 8 cycles.

In some embodiments, the methods for treating ASMD provided herein comprise administering ASM as a single agent for a period of time prior to administering ASM in combination with an additional therapy. In certain embodiments, the methods for treating ASMD provided herein comprise administering an additional therapy alone for a period of time prior to administering ASM in combination with an additional therapy.

In some embodiments, administration of ASM and one or more additional therapies according to the methods provided herein has an additive effect relative to administration of ASM or said one or more additional therapies alone. In some embodiments, administration of ASM and one or more additional therapies according to the methods provided herein has a synergistic effect relative to administration of ASM or said one or more additional therapies alone.

As used herein, the term "synergistic" refers to the effect of administering a dose of ASM in combination with one or more additional therapies (e.g., agents), wherein the combination is more effective than the additive effects of any two or more monotherapies (e.g., agents). In a particular embodiment, the synergistic effect of the combination therapy allows for lower dosages (e.g., suboptimal doses) of ASM or the additional therapy to be administered and/or less frequently administered to the subject. In certain embodiments, the ability to use lower dosages of ASM or the additional therapy and/or to administer ASM or the additional therapy less frequently reduces the toxicity associated with administering ASM or the additional therapy, respectively, to the subject without reducing the effectiveness of ASM or the additional therapy, respectively, in treating ASMD. In some embodiments, the synergistic effect results in improved effectiveness of ASM and each of the additional therapies in treating ASMD. In some embodiments, the synergistic effect of the combination of ASM and one or more additional therapies avoids or reduces the adverse or undesirable side effects associated with the use of either monotherapy.

In specific embodiments, one or more additional therapies are administered in combination with ASM to subjects to reduce or alleviate one or more of the following events that may be associated with administration of a specific dose of ASM: (i) a related adverse event, (ii) a total bilirubin value greater than the bilirubin value for an individual without ASMD (e.g., a healthy individual), (iii) a plasma ceramide concentration greater than the plasma ceramide concentration of an individual without ASMD (e.g., a healthy individual), or (iv) an acute phase response. In specific embodiments, one or more additional therapies are administered in combination with ASM to subjects to increase pulmonary function while minimizing one or more of the following events that may be associated with administration of a specific dose of ASM: (i) a related adverse event, (ii) a total bilirubin value greater than the bilirubin value for an individual without ASMD (e.g., a healthy individual), (iii) a plasma ceramide concentration greater than the plasma ceramide concentration of an individual without ASMD (e.g., a healthy individual), or (iv) an acute phase response.

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

In certain embodiments, chaperones, such as small molecule chaperones, are administered in combination with ASM. See, for example, U.S. Patent No. 7,750,050 and International Publication Nos. WO 2004/045574 and WO 2010/015816, which are incorporated herein by reference, for agents (e.g., small molecules) that can be administered in combination with ASM to a subject. In some embodiments, a chaperone (e.g., a small molecule chaperone) does one, two, or all of the following: increases the targeting of ASM to sites of pathology, stabilizes ASM activity, and enhances ASM activity.

In some embodiments, a glucocorticosteroid, such as dexamethasone, is administered in combination with ASM. See, for example, U.S. Patent No. 7,658,916, which is incorporated herein by reference in its entirety, for agents (e.g., glucocorticosteroids) that can be administered in combination with ASM.

In some embodiments, a substrate-reducing molecule is administered in combination with ASM to a subject. In particular embodiments, a molecule (e.g., a small molecule) that either reduces the amount of sphingomyelin, reduces the rate of sphingomyelin synthesis, or both reduces and reduces is administered in combination with ASM to a subject. See, e.g., Li et al. 2007, Biochim Biophys Act 1771(9):1186-1194 (which is incorporated herein by reference in its entirety) for sphingomyelin synthase inhibitors such as tricyclodecan-9-yl xanthate and sphingomyelin synthase small interfering RNAs.

In some embodiments, a therapy that reduces the potential immunogenicity of ASM is administered in combination with ASM to a subject. In certain embodiments, benedryl is administered in combination with ASM to a subject.

A combination of ASM and one or more additional therapies may be administered to a subject in a single pharmaceutical composition. Alternatively, ASM and one or more additional therapies may be administered simultaneously to a subject in separate pharmaceutical compositions. ASM and one or more additional therapies may be administered sequentially to a subject in separate pharmaceutical compositions. ASM and one or more additional therapies may also be administered to a subject by the same or different routes of administration.

5.6. PHARMACEUTICAL PRODUCTS

In one aspect described herein, finished packaged and labeled pharmaceutical products are provided. In one embodiment, the pharmaceutical product comprises a unit dosage form of ASM in an appropriate vessel or container (e.g., a glass ampoule or other container that is hermetically sealed). In some embodiments, the unit dosage form is a lyophilized form of ASM, and under these conditions, the pharmaceutical product may comprise a second container of sterile saline or sterile water for reconstituting the lyophilized form of ASM. In other embodiments, the unit dosage form is an aqueous form of ASM that does not require reconstitution prior to administration to a subject. In particular embodiments, the unit dosage form of ASM comprises ASM in an amount sufficient to administer a low, non-toxic 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 a particular embodiment, the unit dosage form is suitable for intravenous delivery to a subject.

In one embodiment, the pharmaceutical product comprises a unit dosage form of ASM in a suitable vessel or container (e.g., a glass ampoule or other container that is hermetically sealed) and an infusion pump or syringe pump for administering the ASM to a subject.

As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and transportation. Additionally, the pharmaceutical product includes instructions for use or other informational material that advises the physician, paramedic, or patient on how to appropriately treat ASMD. In other words, the pharmaceutical product includes means for instructions that prescribe or suggest a dosing regimen, including, but not limited to, actual doses (e.g., a dose escalation protocol), tracking procedures, and other monitoring information. In certain embodiments, the pharmaceutical product includes means for genotyping the patient (e.g., PCR primers for the SMPD1 gene).

6. CASE STUDY 1: A HUMAN STUDY OF ENZYME REPLACEMENT THERAPY WITH RECOMBINANT ACID SPHINGOMYELINASE (rhASM) IN ADULTS WITH ASM DEFICIENCY

6.1 INTRODUCTION

ASMD is an autosomal recessive, lysosomal storage disorder that occurs when sphingomyelin is unavailable to be normally catabolized to ceramide and phosphorylcholine. Consequently, sphingomyelin accumulates within cells primarily of the reticuloendothelial system, leading to hepatosplenomegaly, anemia, thrombocytopenia, and interstitial lung disease. Growth retardation and an atherogenic lipid profile are also frequently found. Patients who have little to no residual ASM activity exhibit the most severe symptoms with onset in infancy, developmental delay, neurodegeneration, and death by 3 years of age (Niemann-Pick disease type A, NPD A). Patients with higher amounts of residual ASM activity have variable ages of onset, heterogeneous manifestations, and somatic symptoms, little to no neurologic involvement, and typically survive into adulthood (NPD B). There is currently no treatment for patients with ASMD.

Enzyme replacement therapy (ERT) has been used successfully to treat several lysosomal storage disorders, including Gaucher disease, mucopolysaccharidoses types I, II, and VI, Fabry disease, and Pompe disease. Recombinant human lysosomal enzymes are administered intravenously and are taken up by cells via receptor-mediated endocytosis for subsequent targeting to lysosomes. The validity of the principle for treating ASMD was demonstrated by the laboratory of Dr. Edward Schuchman (Mount Sinai Medical Center) in the ASM knockout (ASMKO) mouse model, where intravenous injections of recombinant human ASM (rhASM) effectively reduced sphingomyelin levels in the liver and spleen, and to a lesser extent in the lung (Miranda, et al., FASEB 2000; 14:1988). However, sphingomyelin levels were not reduced in the brain due to the inability of rhASM to cross the blood-brain barrier. Because the ASMKO mouse has no residual ASM activity or protein and develops rapid and severe neurological disease, this animal has been considered the most relevant model of NPD type A (see Buccinna et al., 2009, J. Neurochem. 109:105-115).

Additional studies confirmed that biweekly doses of rhASM reduced sphingomyelin levels in ASMKO mice in a dose-dependent manner (0.3-3 mg/kg). No-observed-adverse-effect levels (NOAELs) for single and repeated dosing were determined to be 0.3 and 3 mg/kg, respectively, in ASMKO mice. Subsequent attempts to reduce lung sphingomyelin levels with higher doses of rhASM resulted in unexpected toxicity. At doses ≥10 mg/kg, ASMKO mice, but not normal animals, exhibited liver inflammation, adrenal hemorrhage, cardiovascular shock, and death accompanied by highly elevated cytokine levels consistent with a cytokine release syndrome. The toxicity and cytokine elevation observed with high doses of rhASM could be alleviated or prevented by pre-treatment of ASMKO mice with some lower doses of rhASM, suggesting that the rate and amount of sphingomyelin breakdown plays a key role.

In the ASMKO mouse, a model for NPD type A, no toxicity was observed following single doses of ≤0.3 mg/kg rhASM and repeated doses of ≤3.0 mg/kg rhASM; severe toxicity was not observed until single doses of ≥10 mg/kg rhASM were administered. Therefore, a conservative starting dose of 0.03 mg/kg rhASM was chosen for single-dose treatment in human subjects to provide 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 rhASM was chosen for single-dose treatment in human subjects to provide a 10-fold safety margin relative to the dose at which severe toxicity was observed in the ASMKO mouse (10 mg/kg). When performing the protocol outlined below, toxicity in human subjects was unexpectedly observed at doses up to 0.3 mg/kg rhASM.

6.2. MATERIALS AND METHODS

6.2.1. HUMAN PROTOCOL OUTLINE

This protocol was a single-center, single-dose, dose-escalating phase 1 study. The primary objectives of the study were to evaluate the safety and pharmacokinetics of single doses of rhASM in adults with non-neuropathy ASMD (Niemann-Pick B). Single doses of 0.03, 0.1, 0.3, 0.6, and 1.0 mg/kg rhASM were administered by infusion to sequential dose cohorts. The original study design required a minimum of 15 patients (5 cohorts of 3 patients each). Due to difficulties in enrolling patients, the protocol was modified so that the first 2 cohorts included 3 patients each and the last 3 cohorts included 2 patients each. An independent data monitoring committee oversaw the study, and all protocol procedures were approved by the IRB. Figure 1 depicts the protocol patient flow.

6.2.2. PATIENTS

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

A total of 13 patients were included, and 11 patients were infused with rhASM. rhASM was produced by overexpressing ASM cDNA in Chinese hamster ovary cells. The mean age of patients who were infused was 30.8 years, all were white (non-Hispanic/non-Latino), and the mean spleen volume was 10.8 times normal. One had a partial splenectomy; the remaining patients had intact spleens at the start of the protocol. Figure 2 displays the demographics and baseline characteristics of patients in this protocol.

Once screening was completed and eligibility confirmed, patients were admitted to the Cardiac Care Unit (CCU) overnight for baseline telemetry and the following morning infusion with rhASM. Patients were monitored for 72 hours post-dose on telemetry (24 hours in the CCU and 48 hours in the General Clinical Research Center). On day 14, patients were returned for overnight stay and on day 28 for an outpatient visit.

The following are the sets of medical assessments that were obtained during the protocol:

- Physical examination - days 0, 1, 2, 14 and 28

- Chemistry, hematology and urinalysis - before infusion, then 24, 48, 72 hours; days 14, 28

- Liver function tests - pre-infusion, then q12 hours for 72 hours, days 14, 28, aldosterone, cortisol, delta-4-androstenedione - pre-infusion, then q12 hours for 72 hours

- ACTH stimulation study - screening, day 14 telemetry - continuous 72 hours ECG, echocardiogram - before infusion, at the end of infusion and after infusion at 1, 2, 6, 12 and 4 hours, day 14

- Cardiac biomarkers (BNP, cardiac troponin-I, CPK-MB) - before infusion, then 2, 6, 12, 24 hours

- Plasma sphingomyelin, ceramide levels - pre-infusion, then 24, 48, 72 hours; days 14 and 28

- Pulmonary function test and CXR - screening, day 14

- Liver and spleen volume using MRI - screening, day 14

- Liver and skin biopsies - screening, day 14

- Biomarkers - Pre-dose, Days 14, 28

- Anti-rhASM IgG assay - Pre-infusion, Day 28

- Pharmacokinetics - within 30 minutes before infusion, 15 minutes after the start of infusion, at the end of infusion and at the following time intervals after infusion: 15 minutes, 30 minutes, 45 minutes; 1 hour, 2, 3, 4, 6, 8, 12, 18, 24, 48, 72 hours.

- Cytokines (IL-1a, IL-1b, IL-6, G-CSF, GM-CSF, MIP-1a, TNF-a) - pharmacokinetic time points (above) plus day 14.

6.3. RESULTS

As shown in Fig. 3A and 3B, plasma ceramide levels and plasma sphingomyelin levels, respectively, were determined at multiple time points. Plasma ceramide levels showed a dose-dependent increase at 6 hours and peaked at 18-72 hours. Plasma sphingomyelin levels were normal at baseline and showed no significant change over time. In patient #12112, who received the highest dose (1 mg/kg), plasma sphingomyelin levels increased post-dose and peaked at 72 hours.

Figure 4 depicts total bilirubin levels determined at several time points during the protocol. Total bilirubin showed a dose-related increase over 24 hours and peaked at 48-60 hours. The highest total bilirubin was 4.7 mg/dL in patient #12112, who received the highest dose (1 mg/kg). There were proportional increases in direct and indirect bilirubin. No increases were observed in ALT, AST, or alkaline phosphatase. There was a slight increase in GGT at 72 hours (not shown) in the two patients who received the highest (1 mg/kg, patient #12112) and lowest (0.03 mg/kg, patient #10503) doses. Hemoglobin and hematocrit levels were stable throughout the protocol, indicating that hemolysis was not responsible for the elevated bilirubin levels.

Figures 5A-5G depict levels of acute phase response (inflammatory) markers, CRP/hs-CRP, % neutrophils, fibrinogen, ferritin, IL-8, IL-6, and calcitonin, respectively, determined at specific time points in several patients. CRP/hs-CRP showed a transient dose-related increase at 24 hours, peaking at 48-72 hours, and returned to normal by day 14. Other acute phase agents also showed increases (% neutrophils, fibrinogen, ferritin, PT, and PTT) and decreases (iron, albumin). Certain inflammatory biomarkers, such as the cytokines IL-6 and IL-8, and calcitonin, also showed significant increases that were dose-dependent and peaked at 48 hours post-infusion. Among these inflammatory mediators, the largest changes in descending order occurred in IL-8 (peak 33.8× ULN), calcitonin (peak 33.4× ULN), CRP (peak 9.8× ULN), and ferritin (3.9× ULN). There was no change in platelet count or fibrin degradation product levels (not shown). Elevations in laboratory acute-phase reactants correlated with systemic clinical symptoms of fever, nausea, vomiting, headache, pain, and myalgias in some patients.

Figure 6 is a flow chart of treatment-emergent adverse events for four patients each on a different dose of rhASM, where events were considered to be related (possibly, probably, or definitely) to treatment. As shown in the flow chart, patient #11509, who received a dose of 0.3 mg/kg rhASM, experienced moderate adverse events beginning on day 2.

Of the protocol-reported adverse events, no significant cardiovascular changes were observed using telemetry, ECG, echocardiogram, or biomarkers (BNP, cardiac troponin-I, CPK-MB) or adrenal hormonal dysfunction. One patient (#12112, 1.0 mg/kg cohort) had elevated morning cortisol 24 hours post-dose, which most likely represented a normal physiologic stress response to some of the moderate associated adverse events that occurred. Additionally, four of six patients receiving ≥0.3 mg/kg rhASM experienced a total of 19 clinical and laboratory associated adverse events assessed as drug-related. Associated adverse events ranged in intensity from mild to severe, but most were moderate and did not require any intervention. These included the following: fever (n=2), pain [myalgia; abdominal, calf, and thigh pain] (n=2), nausea (n=2), scleral icterus and urinary urobilinogen (n=1), fatigue (n=1), vomiting (n=1), lymphocytic infiltrate/hepatocellular degradation on liver biopsy (n=1), acute phase reaction (n=4), elevated bilirubin (n=2), and increased fibrin D-dimer (n=1). Onset of clinical symptoms began 12 hours after infusion and resolved within 72 hours, except for hip pain in one patient, which began after 72 hours. On day 14, a liver biopsy in one patient (0.6 mg/kg rhASM, patient #12313) revealed two new foci of lymphocytic infiltrates: one was very small (0.1 mm in diameter) and the other was medium (0.5 mm in diameter), and was associated with hepatocellular degradation. The protocol was stopped when the first patient in cohort 5 (1 mg/kg rhASM, patient #12112) experienced dose-limiting hyperbilirubinemia toxicity (peak 4.7 mg/dL).

The results of this study showed an unexpected and delayed onset of dose-related clinical and laboratory adverse events in humans suffering from ASMD at rhASM doses of 0.3 mg/kg and higher. Additionally, two major laboratory safety observations were made in view of the results of this protocol. One concerned hyperbilirubinemia, where dose-proportional increases in direct and indirect bilirubin were observed without significant markers of liver injury (AST, ALT, AP) or an event of hemolysis (hemoglobin and hematocrit). Two patients had mildly elevated GGT at 48-72 hours, and one patient had two new foci of lymphocytic infiltrates in the liver, one of which was associated with hepatocellular degradation.

Another observation concerned the acute phase response, i.e. inflammation. The results showed an increase in the following: CRP/hs-CRP, % neutrophils, ferritin, IL-6, IL-8, calcitonin, fibrinogen, PT, PTT and a decrease in iron and albumin in response to increasing doses of rhASM. Systemic clinical symptoms of fever, nausea, vomiting, pain and myalgia were most likely related to the acute phase response. The results showed no evidence of cytokine release syndrome associated with cardiovascular changes and no evidence of adrenal hormonal dysfunction.

6.4. CONCLUSIONS

The protocol described above represents the first experience with enzyme replacement therapy in adult human patients with ASMD. At bioactive doses in humans, rhASM did not induce cytokine release syndrome associated with cardiovascular changes or induce adrenal hormonal dysfunction. The main safety observations were dose-related hyperbilirubinemia and acute phase response. Both adverse events were likely related to the breakdown of sphingomyelin to ceramide and phosphylcholine, but the precise molecular mechanisms are not fully understood. Several safety biomarkers were identified, including bilirubin, ceramide, CRP/hs-CRP, 1L-8, 1L-6, calcitonin, and ferritin. Additionally, based on the hyperbilirubinemia findings, the maximum tolerated starting dose of rhASM was 0.6 mg/kg.

Moreover, the occurrence of adverse events with clinical symptoms in patients observed at doses up to 0.3 mg/kg was unexpected given that the NOAEL (no-observed-adverse-effect-level) in ASMKO mice was 0.3 mg/kg, and clinical symptoms of toxicity were not observed in ASMKO mice until doses greater than or equal to 10 mg/kg were used.

7. EXAMPLE 2. TOXICITY PROTOCOL OF INTRAVENOUS INJECTION OF REPEAT DOSE AFTER THE SPHINGOMYELIN REDUCTION PHASE IN ACID SPHINGOMYELINASE KNOCKOUT MICE

This example describes a study of the potential toxicity of repeated intravenous administration of recombinant human acid sphingomyelinase (rhASM) following a sphingomyelin mass reduction phase in acid sphingomyelinase knockout (ASMKO) mice. Having established that toxic side effects became observable at initial doses of 10 mg/kg rhASM in ASMKO mice, a subsequent study was designed to determine whether administration of increasing doses of rhASM to ASMKO mice would sufficiently reduce accumulated sphingomyelin mass such that a higher dose of rhASM could be administered to ASMKO mice to target sites of pathology such as the lung and brain with minimal or no observable toxicity.

JK ASMKO mice were treated with 3 mg/kg rhASM on study days (SD) 1, 3, 5, 7 (sphingomyelin mass reduction phase). Beginning on SD 9 and continuing biweekly for 13 weeks (7 doses), mice received treatment doses of 3, 10, or 30 mg/kg rhASM (treatment phase).

rhASM was administered intravenously via a bolus injection into the lateral tail vein of male and female ASMKO mice. Groups 1–3 received 3 mg/kg rhASM during the sphingomyelin mass reduction phase (SD 1, 3, 5, and 7). Groups 1, 2, and 3 received 3, 10, and 30 mg/kg rhASM, respectively, during the treatment phase (SD 9, 23, 37, 51, 65, 79, and 93). One subset of mice in each group (the first 2 mice/sex in Group 1 and the first 4 mice/sex in Groups 2–3) was bled before the study and at 5 min and 4 h after doses of Treatment Phase 1, 4, and 7 for toxicokinetics (TK) (5 min) and ceramide levels (4 h). Another subset of mice in each group (the last 2 mice/sex in Group 1 and the last 4 mice/sex in Groups 2-3) were bled pre-study and at 4 and 24 hours after the Phase 1, 4, and 7 doses for rodent multi-analyte profiles (4 hours) and acute phase protein/liver function test (24 hours) levels. See Table 2 below for study design.

Because of the potential for anaphylactic reactions beginning with the first dose of the treatment phase and each dose thereafter, each mouse was closely observed for signs of anaphylaxis (e.g., restlessness, chewing, face rubbing, urticaria, edema, lethargy, and unkempt fur) following administration of test drug. Mice received diphenhydramine (DPH) beginning before the first dose of the treatment phase and each dose thereafter.

DPH at a concentration of 5 mg/mL was prepared by diluting a 50 mg/mL commercially available stock solution with sterile 0.9% saline. DPH was administered intraperitoneally (IP) at a dose of 20 mg/kg (4 mL/kg) based on the most recent body weight, 10-20 minutes before the rhASM dose to prevent potential anaphylactic reactions. If any animal demonstrated a hypersensitivity reaction after administration of study drug despite pretreatment with DPH, a second dose of DPH was administered IP at a dose of 10 mg/kg (2 mL/kg).

All mice were sacrificed by _CO2 asphyxiation at SD 100. After euthanasia, blood was collected for clinical pathology and immunogenicity analysis via cardiac puncture. Necropsy was performed on all mice after blood collection. All tissues were preserved in 10% neutral buffered formalin (NBF) for subsequent histopathological analysis. A piece of liver, spleen, kidney, and lung were preserved in 2% glutaraldehyde/2% paraformaldehyde for sphingomyelin load analysis. A piece of liver, spleen, kidney, and lung were also collected and stored at ≤-70°C for possible future analysis.

After dissection, all remaining tissues, including the carcass, were placed in 10% NBF.

Table 2
Study design Dose identification No. of animals Sphingomyelin mass reduction phase rhASM treatment dose level Treatment dose concentration (mg/ml) Treatment dose volume (ml/kg) Male Female 1 - rhASM* 4 4 All mice were administered 3 mg/kg rhASM on days 1, 3, 5 and 7 of the study. Doses were administered at a concentration of 0.39 mg/mL in a volume of 7.7 mL/kg. 3 mg/kg starting on day 9 of the study and every two weeks thereafter for 13 weeks 0.39 7.7 2 - rhASM* 8 8 10 mg/kg starting on day 9 of the study and every two weeks thereafter for 13 weeks 1.3 3 - rhASM* 8 8 30 mg/kg starting on day 9 of the study and every two weeks thereafter for 13 weeks 3.9 * Diphenylhydramine will be administered to all mice prior to the 1st dose of the treatment phase and every dose thereafter.

Observations during life

The first day of the study was considered SD1 (day 1 of dosing). Animals were measured for body weight once each week during the study period on SD-1 and on Mondays of each week thereafter. Cage observations were made once daily Monday through Friday. Any abnormalities and normal observations were recorded. Post-dose/scoring clinical observations were made immediately before, 10-20, and 50-70 minutes after each dose. Any abnormalities and normal observations were recorded. Adverse reactions were consulted with the treating veterinarian and/or study director and appropriate actions were taken based on their recommendations.

Euthanasia

If there was an adverse reaction that affected the health and welfare of the animal, the animal was euthanized by CO ₂ asphyxiation, opened through the thoracic, abdominal and cranial cavities and placed in 10% neutral buffered formalin (NBF) for possible further analysis. If the animal was found dead, the whole carcass was preserved in 10% NBF for possible further analysis. At the end of the study, all surviving animals were euthanized by CO ₂ asphyxiation.

Sample collection

Dose Assay: Approximately five hundred microliters (500 µL) of study drug from all dose levels were collected within 1-10 minutes of formulation on each dosing day and stored on dry ice until transfer to a freezer set to maintain a temperature of ≤-70°C. Dose assay samples were transferred to SDs 1, 3, 5, 7, 9, 23, 37, 51, 65, 79, and 93 and stored at ≤-70°C until analysis. Dose assay samples were measured using the A280 assay.

Blood Collection: To collect blood for TK analysis, rodent multianalyte profiles, and acute phase protein/liver function test levels, mice were anesthetized with a mixture of isoflurane and oxygen. Blood collections occurred at SD -1, 9, 51, and 93 (pre-test and at doses 1, 4, and 7 of the treatment phase, respectively) according to the blood collection tables (see Tables 4 and 5, below) and the following text.

rhASM Toxicokinetics : A blood sample was collected from the first 2 mice/sex in Group 1 and the first 4 mice/sex in Groups 2-3 for analysis of peak rhASM levels pre-study (SD -1) and 5 minutes after the 1st, 4th, and 7th doses of the treatment phase. Blood from all animals was collected from the retro-orbital plexus in unconscious mice. Approximately 60 µL of whole blood was collected into hematocrit tubes and allowed to clot at room temperature for at least 1 minute. Serum was prepared from these samples by centrifugation for 5 minutes at 10,000 rpm. Following centrifugation, serum was collected and stored on dry ice until transfer to a freezer set to maintain a temperature of ≤-70°C. Following transfer, all samples were stored at ≤-70°C until analysis. TK samples were transferred to SD 1, 9, 51 and 93. TK samples were measured by enzyme-linked immunosorbent assay (ELISA).

Ceramide Analysis : A blood sample was collected from the first 2 mice/sex in Group 1 and the first 4 mice/sex in Groups 2-3 for ceramide analysis pre-study (SD -1) and 4 hours after the 1st, 4th, and 7th doses of the treatment phase. Blood from all animals was collected from the retro-orbital plexus in unconscious mice. Approximately 240 µL of whole blood was collected into potassium EDTA tubes and placed on a Nutator rocker for up to 30 minutes to prevent clot formation. Plasma was prepared from these samples by centrifugation for 5 minutes at 10,000 rpm. Following centrifugation, plasma was collected and stored on dry ice until transfer to a freezer set to maintain a temperature of ≤-70°C. Once transferred, all samples were stored at ≤-70°C until analyzed by mass spectrometry. Ceramide samples were transferred no later than SD 94. Ceramide samples were measured by mass spectrometry.

Rodent Multi-Analyte Profile (MAP) Analysis : A blood sample was collected from the last 2 mice/sex in Group 1 and the last 4 mice/sex in Groups 2-3 for rodent multi-analyte profile analysis pre-study (SD -1) and 4 hours after the 1st, 4th, and 7th doses of the treatment phase. Blood from all animals was collected from the retro-orbital plexus in unconscious mice. Approximately 150 µL of whole blood was collected into serum preparative centrifuge tubes and allowed to clot for at least 30 minutes. Serum was prepared from these samples by centrifugation for 5 minutes at 10,000 rpm. Following centrifugation, serum was collected and stored on dry ice until transfer to a freezer set to maintain a temperature of ≤-70°C. Once transferred, all samples were stored at ≤-70°C until transported no later than SD 94 for rodent multi-analyte profiling. Samples were transported to Rules Based Medicine on dry ice. The rodent multi-analyte profiling table (see Table 3, below) lists the analytes that were measured.

Table 3
Multianalyte profiles of rodents Analytes to be investigated Apolipoprotein A1 Interleukin-2 MIP-3 beta C-reactive protein Interleukin-3 MMP-9 CD40 Interleukin-4 MCP-1 CD40 ligand Interleukin-5 MCP-3 Endothelin-1 Interleukin-6 MCP-5 Eotaxin Interleukin-7 Myeloperoxidase Epidermal growth factor Interleukin-10 Myoglobin Factor VII Interleukin-11 Oncostatin M Fibrinogen Interleukin-12p70 RANTES FGF-basic Interleukin-17 Serum amyloid P FGF-9 KC/GRO alpha SGOT GCP-2 Leukemia inhibitory factor Stem cell factor GM-CSF Lymphotactin Thrombopoietin GST-alpha M-CSF TIMP 1 Haptoglobin MDC Tissue factor Immunoglobulin A MIP-1 alpha Tumor necrosis factor alpha Inducible protein-10 MIP-1 beta VCAM-1 Interferon gamma MIP-1 gamma VEGF Interleukin-1 alpha MIP-2 Von Willebrand factor Interleukin-1 beta

Acute Phase Protein/Liver Function Assay Analysis: A blood sample was collected from the last 2 mice/sex in Group 1 and the last 4 mice/sex in Groups 2-3 for analysis of acute phase protein (serum amyloid-A and serum amyloid-P) and liver function (bilirubin and alanine aminotransferase (ALT)) levels before the study (SD -1) and 24 hours after the 1st, 4th, and 7th doses of the treatment phase. Blood from all animals was collected from the retro-orbital plexus in unconscious mice. Approximately 150 µl of whole blood was collected into serum preparative centrifuge tubes and allowed to clot for at least 30 minutes. Serum was prepared from these samples by centrifugation for 5 minutes at 10,000 rpm. Following centrifugation, serum was collected and stored on dry ice until transfer to a freezer set to maintain a temperature of ≤-70°C. Once transferred, all samples were stored at ≤-70°C until transport no later than SD 94 for acute phase protein/liver function assay analysis. Samples were transported to Analytics on dry ice.

Table 4
Blood collection table before the study (SD -1) Group Animal No. Floor Preliminary blood collection for TC analysis (60 µl) Pre-collection of blood for MAP analysis in rodents (150 µl) Preliminary blood collection for ceramide analysis (240 µl) Preliminary blood collection for APP/liver function analysis (150 µl) 1 1, 2 M X X 5, 6 AND X X 3, 4 M X X 7, 8 AND X X 2 9, 10, 11, 12 M X X 17, 18, 19, 20 AND X X 13, 14, 15, 16 M X X 21, 22, 23, 24 AND X X 3 25, 26, 27, 28 M X X 33, 34, 35, 36 AND X X 29, 30, 31, 32 M X X 37, 38, 39, 40 AND X X

Table 5
Blood collection table for SD 9, 51 and 93 Group Animal No. Floor Blood collection for TK analysis (60 µl) 5 minutes after dose administration Blood collection for rodent Map analysis (150 µl) 4 hours post-dose Blood collection for ceramide analysis (240 µl) 4 hours post-dose Blood collection for APP/liver function analysis (150 µL) 24 hours post-dose 1 1, 2 M X X 5, 6 AND X X 3, 4 M X X 7, 8 AND X X 2 9, 10, 11, 12 M X X 17, 18, 19, 20 AND X X 13, 14, 15, 16 M X X 21, 22, 23, 24 AND X X 3 25, 26, 27, 28 M X X 33, 34, 35, 36 AND X X 29, 30, 31, 32 M X X

Opening

Final Blood Collection for Clinical Pathology and Immunogenicity Analysis: Following euthanasia, all animals underwent cardiac puncture to collect whole blood (approximately ≥500 µL) from all primary study animals for clinical pathology and immunogenicity analysis. Approximately 150 µL of whole blood was placed into potassium EDTA tubes and gently inverted for analysis of hematological parameters. After gentle inversion, all samples were stored at room temperature with rocking on a Nutator until samples were maintained at 2-10°C until analysis. The remaining blood (from cardiac puncture collection after removal of 150 µL for hematology analysis) was placed in a serum preparative centrifuge tube, allowed to clot at room temperature for at least 30 min, spun down for 5 min at 10,000 rpm, and serum collected. Approximately 30 µL of serum was placed in an Eppendorf tube for immunogenicity analysis, while the remaining serum was placed in an Eppendorf tube for clinical chemistry. Immunogenicity samples were stored on dry ice until all samples were stored at ≤-70°C. Immunogenicity samples were measured by ELISA. Clinical chemistry samples were placed on dry ice until all samples were stored at ≤-20°C until analysis. The Hematology Analytes and Clinical Chemistry Analytes Table (see Table 6, below) lists the analytes that were measured.

Table 6
Table of hematological and clinical chemical analytes Hematological parameters Clinical chemical parameters Number of leukocytes (with total and absolute count of formed elements) alkaline phosphatase red blood cell count total bilirubin (with direct bilirubin if total bilirubin exceeds 1 mg/dL) hemoglobin aspartate aminotransferase hematocrit alanine aminotransferase mean corpuscular hemoglobin gamma glutamyl transferase mean corpuscular volume urine nitrogen mean corpuscular hemoglobin concentration (calculated) creatinine absolute reticulocyte count total protein platelet count albumen blood cell morphology globulin and A/G (albumin/globulin) ratio (calculated) blood smear glucose total cholesterol triglycerides electrolytes (sodium, potassium, chloride) calcium phosphorus

Results from hematology and clinical chemistry analyses from all animals were interpreted by a board-certified pathologist.

Tissue Collection: Following euthanasia at SD 100, all animals were necropsied for tissue collection. Necropsy included examination of external carcasses, external body orifices, abdominal and thoracic cavities, organs, and tissues. Macroscopic observations were recorded. The tissue collection chart (see Table 7, below) lists the tissues that were collected in 10% NBF. Macroscopic lesions were also collected and stored in 10% NBF. The remaining carcasses were preserved in 10% NBF.

Table 7
Fabric collection table Textile Textile adrenal gland (2) pancreas aorta pituitary brain prostate caecum rectum colon salivary gland [mandibular (2)] duodenum seminal vesicle (2) epididymus (2) sciatic nerve esophagus skeletal muscles (quadriceps) eye with optic nerve (2) leather femur with bone marrow (articular surface of the distal part) spinal cord (cervical, thoracic and lumbar) gall bladder spleen heart sternum with bone marrow ileum stomach jejunum testicles (2) kidney (2) thymus liver thyroid gland (2) with parathyroid gland lung with main bronchi language lymph node (mandibular) trachea lymph node (mesenteric) bladder mammary gland (in females) uterus with cervix ovary (2) macroscopic damage

Samples in bold will be weighed.

Organ Weights: At necropsy, the above bolded organs were weighed; paired organs were weighed together. Weights were recorded on the organ weight form. Organ weight to body weight and organ weight to brain weight percentages were calculated from pre-necropsy body weights.

Histopathology: Specimens from all animals, as well as macroscopic lesions from any animal in the study, were submitted for histology. The preserved tissues listed above from each animal were embedded in paraffin. All tissues were sectioned, stained with hematoxylin and eosin, and examined microscopically. All tissue sections were examined microscopically for histologic changes by a board-certified pathologist.

Liver, spleen, kidney, and lung samples from all animals were collected and fixed in 2% glutaraldehyde/2% paraformaldehyde for sphingomyelin load analysis. These tissues were then fixed in potassium dichromate/osmium tetroxide and embedded in Epon. One-micron sections were stained with tannic acid and toluidine blue for examination by light microscopy. Sphingomyelin load in each sample was quantified by computer-assisted morphometry with MetaMorph software by a board-certified pathologist.

Liver, spleen, kidney and lung samples from all animals were collected, frozen in liquid nitrogen and stored frozen at ≤-70°C for possible future analysis.

Sample analysis

Dose assay was measured by A280 assay and toxicokinetics, and samples for immunogenicity were analyzed by ELISA.

Ceramide analysis was measured by mass spectrometry.

Hematology and clinical pathology samples were measured using a Sysmex XTV 2000 IV hematology analyzer and a Radnox Daytona clinical chemistry analyzer, respectively.

Tissue samples were processed and histopathology was interpreted by a SOP-certified pathologist. Clinical pathology results were interpreted by a SOP-certified pathologist.

8. EXAMPLE 3. ENZYME REPLACEMENT THERAPY WITH INCREASING DOSE OF RECOMBINANT HUMAN ACID SPHINGOMYELINASE (rhASM)

8.1 INTRODUCTION

The protocol described below uses single-patient dose escalation as an optional means to achieve higher repeat doses of rhASM. The safety, efficacy, and pharmacokinetics (PK) of rhASM infusions at doses of 0.3, 0.6, and 1.0 mg/kg administered every 2 weeks (q2w) for 40 weeks, as well as the long-term safety and efficacy of rhASM infusions, are assessed.

8.2. MATERIALS and METHODS

8.2.1.PROTOCOL OUTLINE

The 12 patients with NP-type B to be treated will be of either sex. Patients will receive at least 1 dose of 0.1 mg/kg rhASM initially. Patients who tolerate 0.1 mg/kg rhASM infusion will then receive escalating doses of 0.3 mg/kg, 0.6 mg/kg, and 1 mg/kg every 2 weeks as tolerated. Patients who tolerate 1.0 mg/kg will then be stratified by spleen volume (<12 times or ≥12 times normal) and randomized to receive 1 of 2 target doses: 1.0 mg/kg rhASM or 3.0 mg/kg; patients randomized to 3.0 mg/kg will be titrated to 2.0 mg/kg and then to 3.0 mg/kg as tolerated. Patients who cannot tolerate the 0.1 mg/kg rhASM dose will be switched. Patients who cannot tolerate escalation to higher doses will be maintained on their maximum tolerated dose for a 26-week maintenance dose period.

All patients will have their doses titrated in increments of 0.1 mg/kg to their target dose. Dose adjustments during the escalation period will occur at 2-week intervals until the target dose is reached (if tolerated). Following the initial infusion (0.1 mg/kg rhASM) and subsequent doses, patient outcomes, i.e. adverse event (AE) occurrence/severity and serum bilirubin, hsCRP and other acute phase proteins, and ceramide, will be reviewed. The following criteria will determine the next dose administered during the escalation period:

1. total bilirubin value ≤2.0 mg/dL or mild AE → increase to next dose (if necessary);

2. total bilirubin value = 2.1-3.0 mg/dL or moderate AE → no dosage increase; repeat current dose;

3. total bilirubin value >3.0 mg/dL) or severe AE → dose reduction to the previously administered/tolerated dose.

Any serious adverse event (SAE) that is judged to be related to rhASM, a bilirubin value that does not resolve to <2.0 mg/dL by the next scheduled dose, or any AE that becomes a safety concern for rhASM at the dose being administered may be considered a dose-limiting toxicity (DLT). If a patient experiences a DLT, subsequent doses to the patient should be temporarily withheld. Patients may undergo a repeat dose trial to obtain the dose that results in the DLT, and if tolerated, treatment will proceed as originally planned.

If a patient cannot tolerate 2 doses of 0.1 mg/kg (i.e., initial infusion and one dose for the repeat dose trial), they will not receive escalation therapy, will not be able to continue treatment, and will be replaced in the study. Patients who tolerate the 0.3 mg/kg dose will receive an initial infusion of rhASM (0.1 mg/kg) and after 2 weeks, 0.3 mg/kg rhASM (provided that the dose escalation criteria after the 0.1 mg/kg dose are met). Subsequent doses of rhASM will be administered during the dose escalation period q2w for a maximum of 18 weeks. If a patient is successfully escalated to 0.3 mg/kg and therefore meets criteria #2 or #3 (above), the patient may undergo the repeat dose trial twice. If the repeat dose trial is unsuccessful (i.e. the target dose cannot be achieved), the patient will continue to receive rhASM at 0.1 mg/kg for the remainder of the 40-week treatment period.

Patients treated with the 0.6 mg/kg dose regimen will receive an initial infusion of rhASM (0.1 mg/kg) followed by 1 dose of 0.3 mg/kg rhASM, followed by 0.6 mg/kg rhASM q2w for the remainder of the 40-week treatment period (provided that the previous 0.1 mg/kg and 0.3 mg/kg rhASM infusions are well tolerated). Patients eligible for treatment with the 1.0 mg/kg regimen will receive an initial infusion of rhASM (0.1 mg/kg) followed by 1 dose of 0.3 mg/kg rhASM, followed by 1 dose of 0.6 mg/kg rhASM, followed by 1.0 mg/kg rhASM q2w for the remainder of the 40-week treatment period (provided that the previous 0.1 mg/kg, 0.3 mg/kg, and 0.6 mg/kg rhASM infusions are well tolerated). If a patient has been successfully titrated from 0.3 to 0.6 mg/kg or 0.6 to 1.0 mg/kg and therefore meets criteria #2 or #3 (above), the patient may undergo a repeat dose trial twice. If the re-dose trial is unsuccessful (i.e. the target dose cannot be achieved), the patient will continue to receive the lower tolerated dose for the remainder of the 40-week treatment period.

After the 40-week treatment phase, patients may be allowed to continue treatment at their maintenance dose level.

8.2.2. PATIENTS

Each patient must meet the following inclusion criteria to receive treatment according to these regimens:

1. documented ASM deficiency consistent with Niemann-Pick (N-P) disease;

2. diffusion capacity of the lungs (DL _CO ) >20% and ≤80% of the calculated normal value;

3. spleen volume ≥8 times normal (≥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 contraceptive method for the duration of the protocol.

8.2.3. TREATMENT

Patients will receive a single dose of 0.1 mg/kg rhASM IV over an approximately 35-minute period. Guidelines for administration are provided in Table 8, below. Patients who tolerate the initial 0.1 mg/kg rhASM infusion will have their dose escalated to the target treatment dose (rhASM 0.3, 0.6, or 1.0 mg/kg). All patients will receive doses escalated by 0.1 mg/kg increments to their target dose. Dosage adjustments during the escalation period will occur at 2-week intervals until the target dose is achieved.

Table 8
Introduction to rhASM Target dose (mg/kg) Approximate infusion rate (ml/hour) Approximate infusion rate (mg/kg/hour) Duration of administration (approximate time in minutes) 0,1 Step 1: 20 ml/hour over 20 min (+/- 5 min) if no IAR
Step 2: 60 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 2: 0.3 mg/kg/hr for remainder of infusion if no IAR 35 0.3 Stage 1: 17 ml/hour over 20 min (+/- 5 min), if no IAR
Step 2: 50 ml/hour over 20 min (+/- 5 min) if no IAR
Step 3: 100 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 3: 0.6 mg/kg/hr for remainder of infusion if no IAR 60 0.6 Stage 1: 17 ml/hour over 20 min (+/- 5 min), if no IAR
Step 2: 50 ml/hour over 20 min (+/- 5 min) if no IAR
Step 3: 100 ml/hour over 20 min (+/- 5 min) if no IAR
Step 4: 167 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 4: 1.0 mg/kg/hr for remainder of infusion if no IAR 80 1.0 Step 1: 10 ml/hour over 20 min (+/- 5 min) if no IAR
Step 2: 30 ml/hour over 20 min (+/- 5 min) if no IAR
Step 3: 60 ml/hour over 20 min (+/- 5 min) if no IAR
Step 4: 100 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (+/- 5 min) if no IAR
Step 4: 1.0 mg/kg/hr for remainder of infusion if no IAR 100

Each degree of splenomegaly of the patient from the baseline can be recorded as a multiple of normal (× normal) as an indicator of the severity of the disease. The normal volume of the spleen is 0.2% of body weight.

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

Clinical endpoints may be measured as % change from baseline to week 26 of the maintenance dose period. The primary efficacy endpoint is reduction in spleen volume as measured by MRI. Secondary efficacy endpoints include the following: reduction in liver sphingomyelin levels; increase in exercise capacity as measured by % estimated maximal workload using cycle ergometry; increased pulmonary function as % estimated DL _CO ; increased lung clearance as measured by cell count and bronchoalveolar lavage (BAL) profile, sphingomyelin, ceramide, cytokine, and chitotriosidase levels. Tertiary efficacy endpoints may include the following: reduction in liver volume as measured by MRI; increased pulmonary function as measured by % estimated FVC, FEV1, TLC; improved lung appearance as measured by high-resolution CT, chest radiography; improved lipid profile as measured by HDL, LDL, total cholesterol, triglycerides, and total cholesterol to HDL cholesterol ratio; improved platelet count; hemoglobin; reduced sphingomyelin levels in skin, plasma, DBS; and improvement in other biomarkers such as CCL18, ACE.

9. EXAMPLE 4: ENZYME REPLACEMENT THERAPY WITH A GROWING DOSE OF HUMAN RECOMBINANT ACID SPHINGOMYELINASE (rhASM)

9.1 INTRODUCTION

The protocol described below uses a repeat dose, dose comparison to evaluate the safety, efficacy, and pharmacokinetics of recombinant human acid sphingomyelinase (rhASM) in adult patients with ASMD. The objectives of the protocol 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 until protocol completion (at least 182 weeks in duration).

9.2 MATERIALS and METHODS

9.2.1. PATIENTS

Each patient must meet the following inclusion criteria to receive treatment according to these regimens:

1. documented acid sphingomyelinase (ASM) deficiency consistent with Niemann-Pick disease (NPD).

2. two or more characteristic clinical features including thrombocytopenia, anemia, neutropenia, hepatomegaly, splenomegaly, and pulmonary disease consistent with non-neuronopathic NPD.

3. diffusion capacity of the lungs for carbon monoxide (DL _CO )>20% and ≤80% of the calculated normal value;

4. spleen volume ≥8 times normal (MN) (i.e. ≥1.6% of body weight); partial splenectomy may be allowed if performed ≥1 year after screening/baseline and residual splenic volume is ≥8 MN;

5. Female patients of childbearing potential must have a negative serum pregnancy test for β-hCG and agree to use a medically acceptable contraceptive method for the duration of the protocol.

9.2.2. PROTOCOL SCHEME

Patients who tolerate rhASM at 0.1 mg/kg may be included in the protocol. For each patient, participation in the protocol may consist of 3 periods:

1. screening/baseline (-60 to -1 days);

2. The primary treatment period (approximately 32 to 46 weeks).

Dose Escalation Phase (DE) (initial DE - approximately 6-16 weeks).

- With safe escalation from 0.1 to 0.3 to 0.6 to 1.0 mg/kg rhASM, patients can be stratified by spleen volume (<12 and ≥12 MN) and randomized to either continuous dosing at 1.0 mg/kg or continuous dosing with dose escalation at 2.0 and 3.0 mg/kg, then receive 3.0 mg/kg rhASM or the maximum tolerated dose (secondary LOI of 4 weeks) for the remainder of the protocol.

- Non-randomized patients may remain on their maximal tolerated dose (<1.0 mg/kg rhASM).

Maintenance dose (MD) phase (26 weeks at maximum tolerated or randomized dose).

3. Long-term treatment period (at least 182 weeks at maximum tolerated or randomized dose)

Screening/baseline assessments may be completed at least 24 hours prior to the initial rhASM infusion. Entry into the protocol is contingent on the patient being able to tolerate 2 doses of 0.1 mg/kg rhASM. Initially, all eligible patients may receive a single dose of 0.1 mg/kg rhASM on Day 1 of the dose escalation (DE). Patients unable to tolerate this dose will undergo a repeat dose challenge 2 weeks later. Patients unable to tolerate 2 doses of 0.1 mg/kg rhASM (i.e., initial dose and repeat dose challenge dose) may not continue in the study and will be replaced to ensure inclusion of approximately 12 patients (who can tolerate rhASM at the 0.1 mg/kg dose).

rhASM doses can be escalated every 2 weeks from 0.1 to 0.3 to 0.6 to 1.0 mg/kg during the primary UD phase. During the primary UD phase, patients can receive rhASM doses safely escalated every 2 weeks from 0.1 to 1.0 mg/kg (as shown in Fig. 7).

During the primary and secondary UD phases, the following dose escalation criteria may determine the next dose of rhASM to be administered:

1. If total bilirubin (highest value prior to next scheduled rhASM dose, and includes pre-infusion blood draw) is ≤2.0 mg/dL, or no adverse event/mild related adverse event (AE) → Escalate to next dose.

2. If total bilirubin is >2.0 to <3.0 mg/dL, or moderate associated AE → Repeat the same dose.

- If total bilirubin remains >2.0 mg/dL until next dose → Temporarily stop further dosing of the patient (potential dose-limiting toxicity [DLT]).

3. If total bilirubin is ≥3.0 mg/dL, or severe associated AE → Reduce dose.

- If total bilirubin remains >2.0 mg/dL until next dose → Temporarily stop further doses to the patient (potential DLT).

4. Patients may be subjected to a re-challenge trial (at a given dose level) as many times as necessary during the primary LE phase only (re-challenge trials are not permitted during the secondary LE phase) before patients are randomized or maintained on their maximum tolerated dose by the end of the 16-week primary LE phase.

5. Included patients (i.e., those who can tolerate rhASM at 0.1 mg/kg) may be substituted if they are not escalated to 1.0 mg/kg and randomized, or cannot tolerate their target randomized dose (i.e., 1.0 or 3.0 mg/kg rhASM). These patients may remain in the protocol at their highest tolerated dose.

If an infusion is missed, the patient may receive the same dose as the previous infusion. If the interval between visits is exceeded ±5 days, then this infusion cannot be given and the patient must receive their next infusion at the scheduled time for this infusion.

For the purposes of this protocol, if any of the following criteria are met, the results may be considered to indicate potential dose-limiting toxicity (DLT) for rhASM at a given dose:

- any serious adverse event (SAE) confirmed by a physician as related to rhASM, or

- total bilirubin remains >2.0 mg/dL until the next dose, or

- any adverse event (AE) that, in the opinion of the physician, raises a concern regarding the safety of rhASM at the dose administered.

The severity of the associated adverse event will be assessed by the physician. A mild associated adverse event is usually transient and may require only minimal treatment or therapeutic intervention. A moderate associated adverse event is usually relieved by additional specific therapeutic intervention. The event interferes with normal activities, causing discomfort, but does not have a significant or persistent risk of harm to the subject. A severe adverse event interferes with normal activities or significantly affects clinical status, or may require intensive therapeutic intervention.

All patients who received escalated doses and safely tolerated 1.0 mg/kg rhASM at week 16 could be stratified by spleen volume (<12 and ≥12 MN) and randomized in a 1:1 ratio to any of the following:

- continue receiving 1.0 mg/kg rhASM (or maximum tolerated dose) every 2 weeks for 26 weeks; or

- continue dose escalation (DE) through 2.0 and 3.0 mg/kg, and then receive 3.0 mg/kg rhASM (or maximum tolerated dose) every 2 weeks for 26 weeks.

During the secondary UD phase, patients randomized to the 3.0 mg/kg group may have only 4 weeks to escalate their rhASM dose to 3.0 mg/kg before entering the 26-week PD phase at 3.0 mg/kg (or maximum tolerated dose; see Fig. 7).

The primary UD phase may range from approximately 6 to 16 weeks in duration for each patient. This may depend on the individual patient's tolerance limitations, if any, that the patient encounters during rhASM dose escalation. During the primary UD phase, each patient may receive rhASM dose escalated every 2 weeks from 0.1 to 1.0 mg/kg (as shown in Fig. 7).

The primary UD phase may be completed when the patient receives and tolerates a dose of 1.0 mg/kg rhASM or reaches week 16, whichever occurs sooner. If none of the criteria leading to a repeat or dose reduction trial are met, the patient may receive a single infusion of each dose (i.e., 4 rhASM infusions total), resulting in a primary UD phase of 6 weeks. However, if the criteria leading to a repeat or dose reduction trial are met, the primary UD phase may be longer than 6 weeks, but will not exceed 16 weeks.

Patients who received escalating doses and safely tolerated 1.0 mg/kg by week 16 may be allocated to 1 of 2 target dose groups (i.e., 1.0 or 3.0 mg/kg rhASM).

Patients randomized to the 3.0 mg/kg rhASM group may enter a secondary LD phase (see Fig. 7) consisting of 2 infusions: the first infusion (i.e., 2.0 mg/kg) will be 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) will be 4 weeks after the first tolerated dose of 1.0 mg/kg. Therefore, the secondary LD phase can be completed 4 weeks after the first tolerated dose of 1.0 mg/kg. A repeat dose trial is not allowed. If the patient does not tolerate the first 2.0 mg/kg dose, the second rhASM dose can be reduced to the maximum tolerated dose (i.e., 1.0 mg/kg) or temporarily withheld in case of potential dose-limiting toxicity (DLT).

Patients may continue to receive rhASM at the maximum tolerated or randomized dose administered IV every 2 weeks during the 26-week PD phase of the primary treatment period. This may include both randomized and non-randomized patients.

As shown in Fig. 7, patients in the 1.0 mg/kg dose group can begin the PD phase at their first 1.0 mg/kg dose after randomization, whereas patients in the 3.0 mg/kg group can begin the PD phase at their third rhASM dose after randomization. Subsequent rhASM doses can be administered IV every 2 weeks for 26 weeks in the PD phase for all randomized patients.

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

If a randomized patient experiences a moderate/severe related AE (i.e., infusion-related reaction [IAR]) during an infusion in the PD phase, the patient may have the rhASM infusion stopped and restarted (i.e., interrupted) or the infusion rate reduced. Provided that a potential DLT does not occur, the patient may continue on their rhASM dose for the remainder of the PD phase.

Once the entire 26-week protocol assessments are completed, all patients (including nonrandomized patients) may enter the long-term treatment period and continue rhASM treatment (at the rhASM dose administered during the PD phase) IV every 2 weeks for at least 182 weeks [3.5 years] in duration or until protocol termination. The long-term treatment period includes a protocol completion (or patient withdrawal/early termination) visit and a safety monitoring call (30-37 days after the last rhASM dose). Individual patient rhASM doses may be adjusted during the long-term treatment period based on the results of the primary treatment period analyses. Any necessary dose escalation may occur as described previously.

9.3. TREATMENT

Patients will receive intravenous infusions of rhASM at doses of 0.1, 0.3, 0.6, and 1.0 mg/kg every 2 weeks during the primary LE phase. Patients randomized to 3.0 mg/kg rhASM will receive 2.0 mg/kg up to 3.0 mg/kg (or maximum tolerated dose) 2 weeks later during the secondary LE phase. During the 26-week PD phase, patients will receive 1.0 or 3.0 mg/kg (or maximum tolerated dose) rhASM every 2 weeks after the LE phase. During the long-term rhASM treatment period, the dose given during the PD phase will be given IV every 2 weeks.

Patients will receive each intravenous infusion of rhASM over a period of approximately 35-135 minutes (min) depending on the dose (see Table 9 below). Protocol treatment (rhASM) will be administered by infusion 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 weight for patients with a body mass index (BMI) ≤30 (weight in kg)/(height in meters); for patients with a BMI>30, calculate the weight corresponding to a BMI of 30 using the patient's height.

Table 9
Introduction to rhASM rhASM dose (mg/kg) Total volume of 0.9% sodium chloride for injection to be mixed (ml) Approximate infusion rate (ml/hour) Approximate infusion rate (mg/kg/hour) Duration of administration (approximate time [min]) 0,1 20 Step 1: 20 ml/hour over 20 min (±5 min) if no IAR
Step 2: 60 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr for remainder of infusion if no IAR 35 0.3 50 Stage 1: 17 ml/hour over 20 min (±5 min) if no IAR
Step 2: 50 ml/hour over 20 min (±5 min) if no IAR
Step 3: 100 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (±5 min) if no IAR
Step 3: 0.6 mg/kg/hr for remainder of infusion if no IAR 60 0.6 100 Stage 1: 17 ml/hour over 20 min (±5 min) if no IAR
Step 2: 50 ml/hour over 20 min (±5 min) if no IAR
Step 3: 100 ml/hour over 20 min (±5 min) if no IAR
Step 4: 167 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (±5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (±5 min) if no IAR
Step 4: 1.0 mg/kg/hr for remainder of infusion if no IAR 80 1.0 100 Step 1: 10 ml/hour over 20 min (±5 min) if no IAR
Step 2: 30 ml/hour over 20 min (±5 min) if no IAR
Step 3: 60 ml/hour over 20 min (±5 min) if no IAR
Step 4: 100 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (±5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (±5 min) if no IAR
Step 4: 1.0 mg/kg/hr for remainder of infusion if no IAR 100 2.0 200 Step 1: 10 ml/hour over 20 min (±5 min) if no IAR
Step 2: 30 ml/hour over 20 min (±5 min) if no IAR
Step 3: 60 ml/hour over 20 min (±5 min) if no IAR
Step 4: 100 ml/hour over 20 min (±5 min) if no IAR
Step 5: 200 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (±5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (±5 min) if no IAR
Step 4: 1.0 mg/kg/hr over 20 min (±5 min) if no IAR
Step 5: 2.0 mg/kg/hr for remainder of infusion if no IAR 120 3.0 300 Step 1: 10 ml/hour over 20 min (±5 min) if no IAR
Step 2: 30 ml/hour over 20 min (±5 min) if no IAR
Step 3: 60 ml/hour over 20 min (±5 min) if no IAR
Step 4: 100 ml/hour over 20 min (±5 min) if no IAR
Step 5: 200 ml/hour over 20 min (±5 min) if no IAR
Step 6: 300 ml/hour for the remainder of the infusion if no IAR Step 1: 0.1 mg/kg/hr over 20 min (±5 min) if no IAR
Step 2: 0.3 mg/kg/hr over 20 min (±5 min) if no IAR
Step 3: 0.6 mg/kg/hr over 20 min (±5 min) if no IAR
Step 4: 1.0 mg/kg/hr over 20 min (±5 min) if no IAR
Step 5: 2.0 mg/kg/hr over 20 min (±5 min) if no IAR
Step 6: 3.0 mg/kg/hr for remainder of infusion if no IAR 135 h = hour; IAR = infusion-related reaction; min = minutes.

Clinical endpoints can be measured as the percentage (%) change from baseline in spleen volume increased compared with normal (as measured by magnetic resonance imaging) at 26 weeks after receiving the maximum tolerated or randomized dose of rhASM. Secondary efficacy endpoints include liver volume (as measured by MRI); lung imaging (using high-resolution computed tomography imaging and chest radiography); pulmonary function tests including percent estimated diffusing capacity of the lungs for carbon monoxide, percent estimated forced vital capacity, forced expiratory volume in 1 second, and total lung capacity; exercise capacity using cycle ergometry including percent estimated maximal workload, peak oxygen consumption, and carbon dioxide production; physician's global assessment of change; performance of biomarkers such as serum chitotriosidase, CCL18, and ACE; and hematological parameters such as platelet count, hemoglobin level. Efficacy assessments may also include fasting lipids, coagulation studies, disease-related biomarkers, measures of general health, and patient photographs.

Safety assessments may be performed before, during, and/or after dosing. Safety assessments that may be performed include the following: clinical examination with neurological evaluation, vital signs (e.g., systolic and diastolic blood pressure, temperature, heart rate, respiratory rate, and oxygen saturation), ECG, ultrasound with Doppler, blood chemistry (e.g., sodium, potassium, calcium, chloride, blood urea 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 testing for cancer-related biomarkers, hormones, cytokines (e.g., IL-8 and IL-6), cardiovascular risk, acute phase reactants and other cellular processes, serum concentrations anti-rhASM IgG, serum anti-rhASM IgE concentration, serum tryptase activity, and skin hypersensitivity testing. In addition, pharmacokinetic and pharmacodynamic assessments can be performed before, during, and/or after dosing. For example, sphingomyelin levels can be assessed in liver and skin by biopsy and in plasma and DBS by tandem mass spectrometry.

All references cited in this document are hereby 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 for all purposes.

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

--->

list of sequences

<110> SCHUCHMAN, Edward H.

DESNICK, Robert J.

COX, Gerald F.

ANDREWS, Laura P.

MURRAY, James M.

<120> ENZYME REPLACEMENT THERAPY WITH DOSE INCREASE FOR TREATMENT

ACID SPHINGOMYELINASE DEFICIENCY

<130> 6923-159-999

<140>

<141>

<150> 61/238113

<151> 2009-08-28

<160> 3

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 629

<212> PRT

<213> Homo sapiens

<220>

<223> human ASM isoform 1 (ASM-1), UniProtKB/Swiss-Prot

inventory number P17405-1

<400> 1

Met Pro Arg Tyr Gly Ala Ser Leu Arg Gln Ser Cys Pro Arg Ser Gly

1 5 10 15

Arg Glu Gln Gly Gln Asp Gly Thr Ala Gly Ala Pro Gly Leu Leu Trp

20 25 30

Met Gly Leu Val Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu Ser

35 40 45

Asp Ser Arg Val Leu Trp Ala Pro Ala Glu Ala His Pro Leu Ser Pro

50 55 60

Gln Gly His Pro Ala Arg Leu His Arg Ile Val Pro Arg Leu Arg Asp

65 70 75 80

Val Phe Gly Trp Gly Asn Leu Thr Cys Pro Ile Cys Lys Gly Leu Phe

85 90 95

Thr Ala Ile Asn Leu Gly Leu Lys Lys Glu Pro Asn Val Ala Arg Val

100 105 110

Gly Ser Val Ala Ile Lys Leu Cys Asn Leu Leu Lys Ile Ala Pro Pro

115 120 125

Ala Val Cys Gln Ser Ile Val His Leu Phe Glu Asp Asp Met Val Glu

130 135 140

Val Trp Arg Arg Ser Val Leu Ser Pro Ser Glu Ala Cys Gly Leu Leu

145 150 155 160

Leu Gly Ser Thr Cys Gly His Trp Asp Ile Phe Ser Ser Trp Asn Ile

165 170 175

Ser Leu Pro Thr Val Pro Lys Pro Pro Pro Lys Pro Pro Ser Pro Pro

180 185 190

Ala Pro Gly Ala Pro Val Ser Arg Ile Leu Phe Leu Thr Asp Leu His

195 200 205

Trp Asp His Asp Tyr Leu Glu Gly Thr Asp Pro Asp Cys Ala Asp Pro

210 215 220

Leu Cys Cys Arg Arg Gly Ser Gly Leu Pro Pro Ala Ser Arg Pro Gly

225 230 235 240

Ala Gly Tyr Trp Gly Glu Tyr Ser Lys Cys Asp Leu Pro Leu Arg Thr

245 250 255

Leu Glu Ser Leu Leu Ser Gly Leu Gly Pro Ala Gly Pro Phe Asp Met

260 265 270

Val Tyr Trp Thr Gly Asp Ile Pro Ala His Asp Val Trp His Gln Thr

275 280 285

Arg Gln Asp Gln Leu Arg Ala Leu Thr Thr Val Thr Ala Leu Val Arg

290 295 300

Lys Phe Leu Gly Pro Val Pro Val Tyr Pro Ala Val Gly Asn His Glu

305 310 315 320

Ser Thr Pro Val Asn Ser Phe Pro Pro Pro Phe Ile Glu Gly Asn His

325 330 335

Ser Ser Arg Trp Leu Tyr Glu Ala Met Ala Lys Ala Trp Glu Pro Trp

340 345 350

Leu Pro Ala Glu Ala Leu Arg Thr Leu Arg Ile Gly Gly Phe Tyr Ala

355 360 365

Leu Ser Pro Tyr Pro Gly Leu Arg Leu Ile Ser Leu Asn Met Asn Phe

370 375 380

Cys Ser Arg Glu Asn Phe Trp Leu Leu Ile Asn Ser Thr Asp Pro Ala

385 390 395 400

Gly Gln Leu Gln Trp Leu Val Gly Glu Leu Gln Ala Ala Glu Asp Arg

405 410 415

Gly Asp Lys Val His Ile Ile Gly His Ile Pro Pro Gly His Cys Leu

420 425 430

Lys Ser Trp Ser Trp Asn Tyr Tyr Arg Ile Val Ala Arg Tyr Glu Asn

435 440 445

Thr Leu Ala Ala Gln Phe Phe Gly His Thr His Val Asp Glu Phe Glu

450 455 460

Val Phe Tyr Asp Glu Glu Thr Leu Ser Arg Pro Leu Ala Val Ala Phe

465 470 475 480

Leu Ala Pro Ser Ala Thr Thr Tyr Ile Gly Leu Asn Pro Gly Tyr Arg

485 490 495

Val Tyr Gln Ile Asp Gly Asn Tyr Ser Gly Ser Ser His Val Val Leu

500 505 510

Asp His Glu Thr Tyr Ile Leu Asn Leu Thr Gln Ala Asn Ile Pro Gly

515 520 525

Ala Ile Pro His Trp Gln Leu Leu Tyr Arg Ala Arg Glu Thr Tyr Gly

530 535 540

Leu Pro Asn Thr Leu Pro Thr Ala Trp His Asn Leu Val Tyr Arg Met

545 550 555 560

Arg Gly Asp Met Gln Leu Phe Gln Thr Phe Trp Phe Leu Tyr His Lys

565 570 575

Gly His Pro Pro Ser Glu Pro Cys Gly Thr Pro Cys Arg Leu Ala Thr

580 585 590

Leu Cys Ala Gln Leu Ser Ala Arg Ala Asp Ser Pro Ala Leu Cys Arg

595 600 605

His Leu Met Pro Asp Gly Ser Leu Pro Glu Ala Gln Ser Leu Trp Pro

610 615 620

Arg Pro Leu Phe Cys

625

<210> 2

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> ASM-1 amino acid residues 363 to 374

<400> 2

Ile Gly Gly Phe Tyr Ala Leu Ser Pro Tyr Pro Gly

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> ASM-2 amino acid residues 363 to 374 (see

UniProtKB/Swiss-Prot inventory number P17405-2)

<400> 3

Tyr Leu Ser Ser Val Glu Thr Gln Glu Gly Lys Arg

1 5 10

<---

Claims (49)

1. A method of treating acid sphingomyelinase deficiency (ASMD) in a human subject, comprising administering to the human subject recombinant human acid sphingomyelinase (rhASM) in a dose escalation regimen comprising: (a) administering a dose of 0.025 mg/kg to 0.05 mg/kg rhASM to a human subject; (b) sequentially, administration of a single dose of 0.1 mg/kg rhASM to a human subject; (c) sequentially, administration of a dose of 0.3 mg/kg rhASM to a human subject; and (d) sequentially, administration of two doses of 0.6 mg/kg rhASM to a human subject; Moreover, each dose of rhASM is administered two weeks after the previous dose. 2. The method of claim 1, wherein the dose in (a) is 0.03 mg/kg rhASM. 3. The method according to claim 1 or 2, wherein the weight of the subject is calculated using the height of the subject if the subject has a BMI >30. 4. The method according to any one of claims 1-3, wherein the dose escalation regimen further comprises a dose of 1 mg/kg rhASM. 5. The method of claim 4, wherein the dose escalation regimen further comprises a dose of 2 mg/kg rhASM. 6. The method of claim 5, wherein the dose escalation regimen further comprises a dose of 3 mg/kg rhASM. 7. A method of treating acid sphingomyelinase deficiency (ASMD) in a human subject, comprising administering to the human subject recombinant human acid sphingomyelinase (rhASM) in a dose escalation regimen comprising: (a) administering a dose of 0.075 mg/kg to 0.1 mg/kg rhASM to a human subject; (b) sequentially, administration of two doses of 0.3 mg/kg rhASM to a human subject; (c) sequentially, administration of two doses of 0.6 mg/kg rhASM to a human subject; and (d) sequentially, administration of two doses of 1 mg/kg rhASM to a human subject; Moreover, each dose of rhASM is administered two weeks after the previous dose. 8. The method of claim 7, wherein the subject's weight is calculated using the subject's height if the subject has a BMI >30. 9. The method of claim 7 or 8, wherein the dose escalation regimen further comprises administering to the human subject a dose of 2 mg/kg rhASM. 10. The method of claim 9, wherein the dose escalation regimen further comprises administering to the human subject a dose of 3 mg/kg rhASM. 11. The method of any one of claims 1-10, wherein the concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in a blood sample from a human subject is assessed within 72 hours prior to administration of a dose of rhASM. 12. The method according to any one of claims 1-11, wherein the doses are administered to the human subject intravenously. 13. The method according to any one of claims 1-12, wherein ASMD is Niemann-Pick disease (NPD) type B. 14. The method according to any one of paragraphs 1-13, wherein the human subject is under 18 years of age. 15. The method of any one of claims 1-14, which further comprises a maintenance dose regimen comprising administering to the human subject a dose of rhASM equal to or less than the highest dose tolerated by the human subject as a maintenance dose. 16. The method according to claim 15, wherein the maintenance dose is 3 mg/kg. 17. The method of claim 15 or 16, wherein the maintenance dose is administered to the human subject every one to four weeks. 18. The method according to any one of claims 15-17, wherein the maintenance dose is administered to the human subject every two weeks. 19. The method of any one of claims 1-18, wherein the ASMD is not neuropathic. 20. The method of any one of claims 1-19, wherein the spleen volume in the human subject decreases following administration of rhASM in a dose escalating fashion. 21. The method of claim 20, wherein the subject has a spleen volume ≤8 times normal (MN) prior to administration of rhASM in a dose escalation regimen. 22. A method of treating acid sphingomyelinase deficiency (ASMD) in a human subject, comprising administering to the human subject in a dose escalation regimen recombinant human acid sphingomyelinase (rhASM) in a liposomal carrier, polycationic carrier, or ICAM-1 targeting nanocarrier, wherein the dose escalation regimen comprises: (a) administration of one or two doses of 0.1 mg/kg rhASM to a human subject; (b) sequentially, administration of two doses of 0.3 mg/kg rhASM to a human subject; and (c) sequentially, administration of two doses of 0.6 mg/kg rhASM to a human subject; where each dose of rhASM is administered two weeks after the previous dose. 23. A method of treating acid sphingomyelinase deficiency (ASMD) in a human subject, comprising administering to the human subject in a dose escalating regimen recombinant human acid sphingomyelinase (rhASM) in a liposomal carrier, polycationic carrier, or ICAM-1 targeting nanocarrier, wherein the dose escalating regimen comprises: (a) administering a dose in the range of 0.001 mg/kg to 0.05 mg/kg rhASM to a human subject; and (b) sequentially, administration of a dose of 0.1 mg/kg rhASM to a human subject; (c) sequentially, administration of two doses of 0.3 mg/kg rhASM to a human subject; and (d) sequentially, administration of two doses of 0.6 mg/kg rhASM to a human subject; Moreover, each dose of rhASM is administered two weeks after the previous dose. 24. The method of claim 22 or 23, wherein the ASMD is Niemann-Pick disease (NPD) type B. 25. The method of any one of claims 22-24, wherein the ASMD is not neuropathic. 26. The method of any one of claims 22-25, wherein the subject's weight is calculated using the subject's height if the subject has a BMI >30. 27. The method of any one of claims 22-26, which further comprises a maintenance dose regimen comprising administering to the human subject a dose of rhASM equal to or less than the highest dose tolerated by the human subject as a maintenance dose. 28. The method according to claim 27, wherein the maintenance dose is 3 mg/kg. 29. The method of claim 27 or 28, wherein the maintenance dose is administered to the human subject every one to four weeks. 30. The method according to any one of claims 27-29, wherein the maintenance dose is administered to the human subject every two weeks.