US20110027254A1 - Compositions and methods for treating gaucher disease - Google Patents

Compositions and methods for treating gaucher disease Download PDF

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US20110027254A1
US20110027254A1 US12/845,653 US84565310A US2011027254A1 US 20110027254 A1 US20110027254 A1 US 20110027254A1 US 84565310 A US84565310 A US 84565310A US 2011027254 A1 US2011027254 A1 US 2011027254A1
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velaglucerase
enzyme replacement
replacement therapy
treatment
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Peter Francis Daniel
Michael W. Heartlein
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Shire Human Genetics Therapies Inc
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Shire Human Genetics Therapies Inc
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Priority to US14/169,628 priority patent/US11571466B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • Gaucher disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in the lysosomal enzyme, glucocerebrosidase (GCB).
  • GCB glucocerebrosidase hydrolyzes the glycolipid glucocerebroside that is formed after degradation of glycosphingolipids in the membranes of white blood cells and red blood cells.
  • the deficiency in this enzyme causes glucocerebroside to accumulate in large quantities in the lysosomes of phagocytic cells located in the liver, spleen and bone marrow of Gaucher patients.
  • Treatments for patients suffering from this disease include administration of analgesics for relief of bone pain, blood and platelet transfusions and, in some cases, splenectomy. Joint replacement is sometimes necessary for patients who experience bone erosion.
  • Enzyme replacement therapy with GCB has been used as a treatment for Gaucher disease.
  • Current treatment of patients with Gaucher disease includes administration of a carbohydrate remodeled GCB derived from human placenta or Chinese hamster ovary (CHO) cells transfected with a GCB expression construct and known as alglucerase or imiglucerase, respectively.
  • the disclosure is based, in part, on the discovery that velaglucerase elicits less of an immune response (e.g., less production of antibody, e.g., less production of neutralizing antibody) than imiglucerase upon administration to a subject (e.g., a subject with Gaucher disease).
  • the invention relates, inter alia, to compositions and methods for selecting a treatment for a subject with Gaucher disease, selecting subjects for treatment with velaglucerase (e.g., alone or in combination with another therapy), methods for reducing injection site reaction in subjects undergoing treatment for Gaucher disease, and methods of treating Gaucher disease.
  • the invention also relates, inter alia, to methods of determining antibody production, e.g., neutralizing antibody production, in a subject being treated for Gaucher disease.
  • the disclosure features a method of treating a subject with Gaucher disease (e.g., type I Gaucher disease), the method comprising
  • glucocerebrosidase enzyme replacement therapy e.g., velaglucerase, imiglucerase, or uplyso
  • intravenous infusion e.g., velaglucerase, imiglucerase, or uplyso
  • a glucocerebrosidase enzyme replacement therapy e.g., velaglucerase, imiglucerase, or uplyso
  • the glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase, uplyso) is administered more than once and each additional dose of the glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase, or uplyso) is administered by intravenous administration over a period of less than 2 hours, e.g., 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes), to the subject.
  • the glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase, uplyso) is administered at a dose of 15 to 60 U/kg (e.g., 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg), at a dose equal to or below 22.5 U/kg, at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U/kg.
  • 15 to 60 U/kg e.g., 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg
  • a dose equal to or below 22.5 U/kg at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U
  • glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase, uplyso) is administered at a dose of 2.5 U/kg to 60 U/kg.
  • the glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase, uplyso) is administered every other week.
  • the glucocerebrosidase enzyme replacement therapy e.g., velaglucerase, imiglucerase, uplyso
  • the velaglucerase is administered three times a week by intravenous infusion, e.g., at a dose of 2.5 U/kg.
  • the infusion of one or more dose occurs over 60 minutes.
  • the glucocerebrosidase enzyme replacement therapy is velaglucerase and the method includes: reconstituting lyophilized velaglucerase with a pharmaceutically acceptable carrier such as Sterile Water for Injection (e.g., reconstituting a 200 unit vial with 2.2 mL of Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection), thereby forming a solution, e.g., wherein the vial is not shaken after addition of the Sterile Water for Injection; optionally, inspecting the solution in the vials (and, e.g., optionally determining if the solution is discolored or if particulate matter is present, and optionally deciding not to use the solution if the solution is discolored or if particulate matter is present);
  • a pharmaceutically acceptable carrier such as Sterile Water for Injection
  • Sterile Water for Injection e.g., reconstituting a 200 unit vial with 2.2
  • a volume of solution to provide a pre-selected dose (e.g., a dose described herein such as 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg);
  • administering the diluted solution to the subject by intravenous infusion e.g., wherein the diluted solution is administered over one hour or at a rate of 1 U/kg/minute.
  • the glucocerebrosidase enzyme replacement therapy is administered at an infusion rate of 2 U/kg/minute, 1.5 U/kg/minute, 1 U/kg/minute, or 0.5 U/kg/minute.
  • the subject has measured antibody values that are greater than the value measured for the negative control (e.g., a negative control in an ELISA)
  • the subject is identified as having antibodies to the glucocerebrosidase enzyme replacement therapy.
  • the subject is optionally identified as suitable for treatment with an alternative Gaucher disease treatment (e.g., treatment with velaglucerase), e.g., if the measured value is greater than the value for the standard, e.g., by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • an alternative Gaucher disease treatment e.g., treatment with velaglucerase
  • the sample is a blood or serum sample.
  • the sample has been modified.
  • the measured antibody values were obtained by contacting the sample with an analytical reagent, e.g., a labeled reagent (e.g., labeled glucocerebrosidase), and/or a substrate or cell, e.g., a substrate or cell that binds to an antibody to the glucocerebrosidase enzyme replacement therapy.
  • the measured antibody values were obtained from a sample that has been enriched for antibodies, e.g., a concentrated portion of a blood or serum sample.
  • the evaluation was obtained by a method described herein.
  • the method includes measuring the measured antibody value, e.g., by a method described herein.
  • the disclosure provides a method for selecting a subject with Gaucher disease for treatment with a glucocerebrosidase enzyme replacement therapy.
  • the method includes selecting a subject for such treatment on the basis that the subject has tested positive for the production of antibodies (e.g., neutralizing antibodies) to the treatment the subject is currently taking for Gaucher disease.
  • antibodies e.g., neutralizing antibodies
  • the subject if the subject has measured antibody values that are greater than the value measured for the negative control (e.g., negative control in an ELISA), the subject is identified as having antibodies to the glucocerebrosidase enzyme replacement therapy.
  • the methods optionally include, selecting an alternative Gaucher disease treatment for the subject (e.g., treatment with velaglucerase), e.g., if the measured value is greater than the value for the standard (e.g., negative control), e.g., by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the sample is a blood or serum sample.
  • the sample has been modified.
  • the measured antibody values were obtained by contacting the sample with an analytical reagent, e.g., a labeled reagent (e.g., labeled glucocerebrosidase), and/or a substrate or cell, e.g., a substrate or cell that binds to an antibody to the glucocerebrosidase enzyme replacement therapy.
  • the measured antibody values were obtained from a sample that has been enriched for antibodies, e.g., a concentrated portion of a blood or serum sample.
  • the evaluation was obtained by a method described herein.
  • the method includes measuring the antibody value, e.g., by a method described herein.
  • the subject tested positive for the production of IgE antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • the subject tested positive for the production of IgM antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • the subject tested positive for the production of IgG and/or IgA antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • the disclosure provides a method for treating a subject with Gaucher disease.
  • the method includes selecting a subject on the basis that the subject has tested positive for the production of antibodies (e.g., neutralizing antibodies) to the treatment the subject is currently taking for Gaucher disease, e.g., by a method described herein, or on the basis that the subject is at risk for developing antibodies (e.g., neutralizing antibodies) to a treatment for Gaucher disease (e.g., imiglucerase or uplyso) and administering velaglucerase to the subject.
  • antibodies e.g., neutralizing antibodies
  • a treatment for Gaucher disease e.g., imiglucerase or uplyso
  • the subject tested positive for the production of IgE antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • the subject tested positive for the production of IgM antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • the subject tested positive for the production of IgG and/or IgA antibodies to the treatment the subject is currently taking for Gaucher disease (e.g., imiglucerase or uplyso).
  • Gaucher disease e.g., imiglucerase or uplyso.
  • velaglucerase is administered at a dose of 15 to 60 U/kg (e.g. 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg), at a dose equal to or below 22.5 U/kg, at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U/kg.
  • velaglucerase is administered at a dose of 2.5 U/kg to 60 U/kg.
  • the velaglucerase is administered every other week by intravenous infusion.
  • the velaglucerase is administered every week by intravenous infusion. In some embodiments, the velaglucerase is administered three times a week by intravenous infusion, e.g., at a dose of 2.5 U/kg.
  • the infusion of the dose occurs over less than 2 hours, e.g., 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes. In preferred embodiments, the infusion of the dose occurs over 60 minutes.
  • the method includes:
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier such as Sterile Water for Injection (e.g., reconstituting a 200 unit vial with 2.2 mL of Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection), thereby forming a solution, e.g., wherein the vial is not shaken after addition of the Sterile Water for Injection; optionally, inspecting the solution in the vials (and, e.g., optionally determining if the solution is discolored or if particulate matter is present, and optionally deciding not to use the solution if the solution is discolored or if particulate matter is present);
  • a pharmaceutically acceptable carrier such as Sterile Water for Injection
  • a volume of solution to provide a pre-selected dose (e.g., a dose described herein, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg);
  • velaglucerase is administered at a rate of 2 U/kg/minute, 1.5 U/kg/minute, 1 U/kg/minute, or 0.5 U/kg/minute.
  • the administering comprises home therapy (e.g., in the subject's home, workplace, or other non-clinical (e.g., non-hospital) setting).
  • the administering e.g., via infusion
  • a health care professional e.g., nurse or physician's assistant.
  • AE adverse event
  • the subject if the subject has not experienced an adverse event (AE) (e.g., a drug-related serious AE or an infusion-related AE, e.g., an event described herein), e.g., after one, two, or three administrations (e.g., via infusion) of velaglucerase, the subject is eligible to receive home therapy for subsequent administrations.
  • AE adverse event
  • the disclosure provides a method for identifying a subject as suitable for (e.g., being a candidate for) treatment with glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase).
  • the method includes evaluating (e.g., measuring) the subject for the presence of infusion site reaction (i.e., infusion-related adverse event) (e.g., during or within 12 hours of infusion of glucocerebrosidase enzyme replacement therapy) (e.g., to the therapy currently being administered to the subject, e.g., imiglucerase or uplyso), e.g., and comparing the measured level of the site reaction to a standard (e.g., a negative control).
  • infusion site reaction i.e., infusion-related adverse event
  • glucocerebrosidase enzyme replacement therapy e.g., to the therapy currently being administered to the subject, e.g., imiglucerase or uplyso
  • a standard
  • the subject has a measured value of site reaction to the treatment the subject is currently taking for Gaucher disease that is greater than the value measured for the negative control (e.g., the site reaction observed after placebo infusion in the subject), the subject is identified as suitable for treatment with an alternative Gaucher disease treatment (e.g., treatment with velaglucerase), e.g., if the measured value of site reaction to the treatment the subject is currently taking for Gaucher disease is greater than the value for the standard, e.g., by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • an alternative Gaucher disease treatment e.g., treatment with velaglucerase
  • the method includes determining the infusion site value, e.g., by a method described herein.
  • the disclosure provides a method for selecting a subject with Gaucher disease for treatment with glucocerebrosidase enzyme replacement therapy.
  • the method includes selecting a subject for such treatment on the basis that the subject is in need of reduced infusion site reaction (e.g., reduced as compared to the reaction associated with or caused by the treatment the subject is currently taking for Gaucher disease, e.g., imiglucerase or uplyso).
  • the method includes evaluating (e.g., measuring) or obtaining an evaluation of the subject for the presence of infusion site reaction (i.e., infusion-related adverse event) (e.g., during or within 12 hours of infusion of glucocerebrosidase enzyme replacement therapy) (e.g., to the therapy currently being administered to the subject, e.g., imiglucerase or uplyso), e.g., and comparing the measured level of the site reaction to a standard (e.g., a negative control).
  • infusion site reaction i.e., infusion-related adverse event
  • glucocerebrosidase enzyme replacement therapy e.g., to the therapy currently being administered to the subject, e.g., imiglucerase or uplyso
  • a standard e.g., a negative control
  • the subject has a measured value of site reaction to the treatment the subject is currently taking for Gaucher disease that is greater than (e.g., greater by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) the value measured for the negative control (e.g., the site reaction observed after placebo infusion in the subject), the subject is selected for an alternative Gaucher disease treatment (e.g., treatment with velaglucerase), e.g., if the measured value of site reaction to the treatment the subject is currently taking for Gaucher disease is greater than the value for the standard (e.g., negative control), e.g., by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the standard e.g., negative control
  • the method includes measuring the antibody value, e.g., by a method described herein.
  • the disclosure provides a method for treating a subject with Gaucher disease.
  • the method includes selecting a subject for such treatment on the basis that the subject is in need of reduced infusion site reaction (e.g., reduced as compared to the reaction associated with or caused by the treatment the subject is currently taking for Gaucher disease, e.g., imiglucerase or uplyso), e.g., by a method described herein, and administering velaglucerase to the subject.
  • reduced infusion site reaction e.g., reduced as compared to the reaction associated with or caused by the treatment the subject is currently taking for Gaucher disease, e.g., imiglucerase or uplyso
  • velaglucerase is administered at a dose of 15 to 60 U/kg (e.g. 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg), at a dose equal to or below 22.5 U/kg, at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U/kg.
  • velaglucerase is administered at a dose of 2.5 U/kg to 60 U/kg.
  • the velaglucerase is administered every other week by intravenous infusion.
  • the velaglucerase is administered every week by intravenous infusion. In some embodiments, the velaglucerase is administered three times a week by intravenous infusion, e.g., at a dose of 2.5 U/kg.
  • the infusion of the dose occurs over less than 2 hours, e.g., 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes.
  • the method includes:
  • a pharmaceutically acceptable carrier e.g., Sterile Water for Injection (e.g., reconstituting a 200 unit vial with 2.2 mL of Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection), thereby forming a solution, e.g., wherein the vial is not shaken after addition of the Sterile Water for Injection; optionally, inspecting the solution in the vials (and, e.g., optionally determining if the solution is discolored or if particulate matter is present, and optionally deciding not to use the solution if the solution is discolored or if particulate matter is present);
  • a pharmaceutically acceptable carrier e.g., Sterile Water for Injection
  • Sterile Water for Injection e.g., reconstituting a 200 unit vial with 2.2 mL of Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection
  • a volume of solution to provide a pre-selected dose (e.g., a dose described herein such as 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg);
  • the velaglucerase is administered at a rate of 2.0 U/kg/minute, 1.5 U/kg/minute, 1.0 U/kg/minute, or 0.5 U/kg/minute.
  • the administering comprises home therapy (e.g., in the subject's home, workplace, or other non-clinical (e.g., non-hospital) setting).
  • the administering e.g., via infusion
  • a health care professional e.g., nurse or physician's assistant.
  • AE adverse event
  • the subject if the subject has not experienced an adverse event (AE) (e.g., a drug-related serious AE or an infusion-related AE, e.g., an event described herein), e.g., after one, two, or three administrations (e.g., via infusion) of velaglucerase, the subject is eligible to receive home therapy for subsequent administrations.
  • AE adverse event
  • the disclosure features a method of selecting a subject with Gaucher disease (e.g., type I Gaucher disease) for treatment with velaglucerase, the method comprising
  • identifying a subject with Gaucher disease that has received a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase or uplyso
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase or uplyso
  • the method includes evaluating (e.g., measuring platelet count or obtaining an evaluation of platelet count of a sample from the subject, and comparing the measured value of the platelet count to the standard (e.g., negative control).
  • evaluating e.g., measuring platelet count or obtaining an evaluation of platelet count of a sample from the subject, and comparing the measured value of the platelet count to the standard (e.g., negative control).
  • the sample is a blood or serum sample. In some embodiments, the sample has been modified. In some embodiments, the measured antibody values were obtained from a sample that has been enriched for platelets, e.g., a concentrated portion of a blood sample. In some embodiments, the evaluation was obtained by a method described herein.
  • the method includes measuring the platelet count, e.g., by a method described herein.
  • the methods further includes administering velaglucerase to the subject, e.g., at a dose and/or dosing schedule described herein.
  • the velaglucerase is administered by intravenous infusion over a course of less than 2 hours, e.g., 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes. In some embodiments, the infusion occurs at a rate of 2 U/kg/minute, 1.5 U/kg/minute, 1 U/kg per minute or 0.5 U/kg/minute.
  • the standard is a platelet count below or equal to 80 ⁇ 10 3 platelets/mm 3 . In some embodiments, the standard is based on the percentage increase in mean platelet count after 6, 9, or 12 months of treatment with the glucocerebrosidase enzyme replacement therapy.
  • a subject in which the mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months
  • glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months
  • a subject in which the mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months
  • glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months
  • the disclosure features a method of treating a subject with Gaucher disease (e.g., type I Gaucher disease), the method comprising
  • a subject with Gaucher disease that has received a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or uplyso) and has a platelet count less than a standard, e.g., a subject identified by a method described herein; and
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase or uplyso
  • velaglucerase is administered at a dose of 15 to 60 U/kg (e.g. 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg), at a dose equal to or below 22.5 U/kg, at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U/kg.
  • velaglucerase is administered at a dose of 2.5 U/kg to 60 U/kg.
  • the velaglucerase is administered every other week by intravenous infusion.
  • the velaglucerase is administered every week by intravenous infusion. In some embodiments, the velaglucerase is administered three times a week by intravenous infusion, e.g., at a dose of 2.5 U/kg.
  • the infusion of the dose occurs over less than 2 hours, e.g., 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes.
  • the method includes:
  • a pharmaceutically acceptable carrier such as Sterile Water for Injection (e.g., reconstituting a 200 unit vial with 2.2 mL of Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection), thereby forming a solution, e.g., wherein the vial is not shaken after addition of the Sterile Water for Injection; optionally, inspecting the solution in the vials (and, e.g., optionally determining if the solution is discolored or if particulate matter is present, and optionally deciding not to use the solution if the solution is discolored or if particulate matter is present);
  • a pharmaceutically acceptable carrier such as Sterile Water for Injection
  • a volume of solution to provide a pre-selected dose (e.g., a dose described herein such as 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg);
  • the velaglucerase is administered at a rate of 2 U/kg/minute, 1.5 U/kg/minute, 1 U/kg/minute or 0.5 U/kg/minute.
  • the administering comprises home therapy (e.g., in the subject's home, workplace, or other non-clinical (e.g., non-hospital) setting).
  • the administering e.g., via infusion
  • a health care professional e.g., nurse or physician's assistant.
  • AE adverse event
  • the subject if the subject has not experienced an adverse event (AE) (e.g., a drug-related serious AE or an infusion-related AE, e.g., an event described herein), e.g., after one, two, or three administrations (e.g., via infusion) of velaglucerase, the subject is eligible to receive home therapy for subsequent administrations.
  • AE adverse event
  • the disclosure provides a method for evaluating a subject, e.g., a subject to whom a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso) is currently being administered.
  • the method includes evaluating (e.g., measuring) or obtaining an evaluation of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following parameters of the subject:
  • hemoglobin concentration e.g., platelet count, liver volume (e.g., as a percentage of total body weight), spleen volume (e.g., as a percentage of total body weight), infusion site reaction, a skeletal parameter, or presence of antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., the mean value of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of these parameters).
  • antibodies e.g., neutralizing antibodies
  • glucocerebrosidase enzyme replacement therapy e.g., the mean value of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of these parameters).
  • the skeletal parameter can be, e.g., bone mineral density (BMD) (e.g., as measured by a change (e.g., improvement) in Z score).
  • BMD can be evaluated e.g., by dual energy X-ray absorptiometry (DEXA).
  • Other skeletal parameters that can be evaluated include, e.g., T-score (e.g., to determine WHO classification of normal bone, osteopenia, and osteoporosis), growth of a subject (e.g., a pediatric subject); skeletal age of a subject (e.g., a pediatric subject); and bone marrow burden (BMB) (e.g., in an adult subject).
  • T-score e.g., to determine WHO classification of normal bone, osteopenia, and osteoporosis
  • growth of a subject e.g., a pediatric subject
  • skeletal age of a subject e.g., a pediatric subject
  • BMB bone marrow burden
  • the evaluating can be performed, e.g., about every week, about every two weeks, about every three weeks, about every four weeks, about every two months, about every three months, about every four months, about every five months, about every six months, about every seven months, about every eight months, about every nine months, about every ten months, about every eleven months, or about every twelve months during the course of treatment.
  • the evaluating can also be performed prior to commencing treatment (e.g., to establish a baseline value).
  • the evaluating can include comparing the value of the parameter from the subject to a standard (e.g., a standard described herein, e.g., a negative control), and optionally determining if a difference exists between the value of the parameter from the subject and the value from the standard.
  • a standard e.g., a standard described herein, e.g., a negative control
  • the standard can be, e.g., a value of the parameter measured in a subject with Gaucher disease being treated with a different therapy for Gaucher disease (e.g., a treatment described herein), or a mean value for a cohort of such subjects (e.g., after the same length of therapy), or the baseline value for the subject (or a mean baseline value for a cohort of subjects with Gaucher disease) prior to commencing therapy (e.g., prior to commencement of a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or velaglucerase)).
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase or velaglucerase
  • a skeletal parameter e.g., bone mineral density
  • a skeletal parameter can be evaluated for long term changes, e.g., after 1, 2, 3, 4, or more years of glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso).
  • glucocerebrosidase enzyme replacement therapy e.g., imiglucerase, velaglucerase or uplyso.
  • the evaluating can include determining if one or more of the following are present:
  • the evaluating can include determining if an infusion site reaction is present (e.g., during or within 12 hours after infusion) and/or if antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or uplyso) are present.
  • antibodies e.g., neutralizing antibodies
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase or uplyso
  • the evaluation can be of one or more of hemoglobin concentration, platelet count and determining if antibodies (e.g., neutralizing antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or uplyso) are present.
  • the evaluation for one or more of these parameters is performed on a sample from the subject, e.g., a blood or serum sample.
  • the sample has been modified. For example, the values were obtained by contacting the sample with an analytical reagent and/or a substrate or cell, e.g., a substrate or cell that binds to an antibody to the glucocerebrosidase enzyme replacement therapy.
  • the values were obtained from a sample that has been enriched for, e.g., hemoglobin, platelets and/or antibodies, e.g., a concentrated portion of a blood or serum sample.
  • the evaluation was obtained by a method described herein.
  • a treatment decision can be made for the subject. For example, if a subject receiving a treatment for Gaucher disease, such as a glucocerebrosidase enzyme replacement therapy, e.g., imiglucerase or uplyso, has a value for one or more of the parameters that differs from the value for a standard (e.g., a subject with Gaucher disease who is receiving a different therapy (velaglucerase)), a decision can be made to transfer the subject currently receiving a glucocerebrosidase enzyme replacement treatment (e.g., imiglucerase treatment) to a different glucocerebrosidase enzyme replacement treatment (e.g., velaglucerase treatment).
  • a glucocerebrosidase enzyme replacement treatment e.g., imiglucerase treatment
  • a different glucocerebrosidase enzyme replacement treatment e.g., velaglucerase treatment
  • antibodies e.g., neutralizing antibodies
  • IgE, IgM, IgG and/or IgA antibodies antibodies to imiglucerase
  • the subject can be transferred to treatment with velaglucerase.
  • an infusion site reaction is detected during or within 12 hours of administration of imiglucerase to a subject undergoing treatment with imiglucerase, the subject can be transferred to treatment with velaglucerase.
  • the mean platelet count in a subject undergoing treatment with imiglucerase is lower (e.g., 5%, 10%, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower) than the mean platelet count value obtained for a cohort of subjects with Gaucher disease treated with velaglucerase for the same duration at the same dose and same frequency of treatment administration, the subject can be transferred to velaglucerase treatment.
  • the disclosure provides a method of selecting a treatment for administration to a subject with Gaucher disease, the method comprising
  • antibodies e.g., neutralizing antibodies
  • the method can include providing the treatment to the subject, e.g., wherein providing includes administering the treatment or transferring the treatment to the subject's possession.
  • the treatment can be administered at a dose and/or dosing schedule described herein.
  • the method can include evaluating (e.g., measuring) or obtaining an evaluation of one or more of these parameters, e.g., by a method described herein.
  • the disclosure provides a method of selecting a treatment for administration to a subject in need of an increase in hemoglobin concentration, an increase in platelet level, a decrease in liver volume, a decrease in spleen volume, a decreased likelihood of injection site reaction, a change in a skeletal parameter (e.g., an increase in bone mineral density), and/or a decreased likelihood of production of antibodies to the treatment, the method comprising
  • antibodies e.g., neutralizing antibodies
  • the method can include providing the treatment to the subject, e.g., wherein providing includes administering the treatment or transferring the treatment to the subject's possession.
  • the treatment can be administered at a dose and/or dosing schedule described herein.
  • the method can include evaluating (e.g., measuring) or obtaining an evaluation of one or more of these parameters, e.g., by a method described herein.
  • the disclosure provides a method for evaluating a subject, e.g., a subject who has been selected to receive treatment with a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso).
  • the method includes evaluating (e.g., measuring) one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following parameters of the subject:
  • hemoglobin concentration e.g., platelet count, liver volume (e.g., as a percentage of total body weight), spleen volume (e.g., as a percentage of total body weight), infusion site reaction, a skeletal parameter, or presence of antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., the mean value of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of these parameters).
  • antibodies e.g., neutralizing antibodies
  • glucocerebrosidase enzyme replacement therapy e.g., the mean value of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of these parameters).
  • the skeletal parameter can be, e.g., bone mineral density (BMD) (e.g., as measured by a change (e.g., improvement) in Z score).
  • BMD can be evaluated e.g., by dual energy X-ray absorptiometry (DEXA).
  • Other skeletal parameters that can be evaluated include, e.g., T-score (e.g., to determine WHO classification of normal bone, osteopenia, and osteoporosis), growth of a subject (e.g., a pediatric subject); skeletal age of a subject (e.g., a pediatric subject); and bone marrow burden (BMB) (e.g., in an adult subject).
  • T-score e.g., to determine WHO classification of normal bone, osteopenia, and osteoporosis
  • growth of a subject e.g., a pediatric subject
  • skeletal age of a subject e.g., a pediatric subject
  • BMB bone marrow burden
  • the evaluating can include comparing the value of the parameter from the subject to a standard (e.g., a standard described herein, e.g., a negative control), and optionally determining if a difference exists between the value of the parameter from the subject and the value from the standard.
  • the standard can be, e.g., a value of the parameter measured in a subject with Gaucher disease being treated with a different therapy for Gaucher disease (e.g., a treatment described herein), or a mean value for a cohort of such subjects (e.g., after the same length of therapy), or the baseline value for the subject (or a mean baseline value for a cohort of subjects with Gaucher disease) prior to commencing therapy.
  • the evaluating can include determining if:
  • the difference between one or more of the hemoglobin concentration, platelet count, liver volume, spleen volume, or a skeletal parameter e.g., measured as bone mineral density (BMD)
  • a skeletal parameter e.g., measured as bone mineral density (BMD)
  • BMD bone mineral density
  • the evaluation can be of one or more of hemoglobin concentration, platelet count and determining if antibodies (e.g., neutralizing antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or uplyso) are present.
  • the evaluation for one or more of these parameters is performed on a sample from the subject, e.g., a blood or serum sample.
  • the sample has been modified. For example, the values were obtained by contacting the sample with an analytical reagent and/or a substrate or cell, e.g., a substrate or cell that binds to an antibody to the glucocerebrosidase enzyme replacement therapy.
  • the values were obtained from a sample that has been enriched for, e.g., hemoglobin, platelets and/or antibodies, e.g., a concentrated portion of a blood or serum sample.
  • the evaluation was obtained by a method described herein.
  • the disclosure provides a method of prescribing a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), the method comprising:
  • the subject is in need of one or more of an increase in hemoglobin concentration, an increase in platelet count and decrease in the likelihood of the production of antibodies (e.g., neutralizing antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or uplyso).
  • a determination that a subject is in need of one or more of these changes is based upon an evaluation of one or more of these parameters performed on a sample from the subject, e.g., a blood or serum sample. In some embodiments, the sample has been modified.
  • the values were obtained by contacting the sample with an analytical reagent and/or a substrate or cell, e.g., a substrate or cell that binds to an antibody to the glucocerebrosidase enzyme replacement therapy.
  • the values were obtained from a sample that has been enriched for, e.g., hemoglobin, platelets and/or antibodies, e.g., a concentrated portion of a blood sample.
  • the evaluation was obtained by a method described herein.
  • the disclosure provides a method of providing a recipient with information about, or with guidelines for, the use of a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or velaglucerase), the method comprising:
  • the disclosure provides a method of providing a recipient with information about a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), or with guidelines for, the use of a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase or velaglucerase), the method comprising:
  • the disclosure provides a method of providing a recipient with information about a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), or with guidelines for the use of a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), the method comprising:
  • the disclosure provides a database, medium, or computer containing or programmed to contain:
  • the disclosure provides a method of making a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso) available to a subject, the method comprising:
  • the disclosure provides a method of causing a subject to request a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), the method comprising:
  • the disclosure features a method of selecting a payment class for a course of treatment with a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso) for a subject with Gaucher disease (e.g., type I Gaucher disease).
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase, velaglucerase or uplyso
  • Gaucher disease e.g., type I Gaucher disease
  • the method includes providing (e.g., receiving) an evaluation of whether or not the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy; and performing at least one of (1) if the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy selecting a first payment class, and (2) if the subject does not experience an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or does not produce antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and
  • assignment of the subject is to the first class and the assignment authorizes payment for a course of treatment (e.g., velaglucerase).
  • a course of treatment e.g., velaglucerase
  • assignment of the subject is to the second class and the assignment authorizes payment for a course of treatment (e.g., imiglucerase, velaglucerase or uplyso).
  • a course of treatment e.g., imiglucerase, velaglucerase or uplyso
  • the evaluation is whether or not a subject produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy and the evaluation was obtained by a method described herein.
  • antibodies e.g., neutralizing antibodies
  • the disclosure features a method of selecting a payment class for a course of treatment with a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso) for a subject with Gaucher disease (e.g., type I Gaucher disease).
  • a glucocerebrosidase enzyme replacement therapy e.g., imiglucerase, velaglucerase or uplyso
  • Gaucher disease e.g., type I Gaucher disease
  • the method includes providing (e.g., receiving) an evaluation of whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months); and performing at least one of (1) if the subject's mean platelet count increased by less than 80%, 7
  • assignment of the subject is to the first class and the assignment authorizes payment for a course of treatment (e.g., velaglucerase).
  • a course of treatment e.g., velaglucerase
  • assignment of the subject is to the second class and the assignment authorizes payment for a course of treatment (e.g., imiglucerase, velaglucerase or uplyso).
  • a course of treatment e.g., imiglucerase, velaglucerase or uplyso
  • the evaluation was obtained by a method described herein.
  • the disclosure features a method of providing information on which to make a decision about a subject with Gaucher disease (e.g., type I Gaucher disease), or making such a decision.
  • the method includes providing (e.g., by receiving) an evaluation of a subject, wherein the evaluation was made by a method described herein, e.g., by optionally, administering a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), to the subject; providing a determination post administration of whether or not the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy, thereby providing a post administration determination; providing a comparison of the post administration determination with a standard (e.g., a standard
  • the method includes making the decision.
  • the method also includes communicating the information to another party (e.g., by computer, compact disc, telephone, facsimile, email, or letter).
  • another party e.g., by computer, compact disc, telephone, facsimile, email, or letter.
  • the decision includes selecting a subject for payment, making or authorizing payment for a first course of action (e.g., treatment with velaglucerase) if the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy and a second course of action (e.g., treatment with imiglucerase, velaglucerase or uplyso) if the subject does not experience an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or does not produce antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement.
  • a first course of action e.g., treatment with velagluce
  • the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy and the course of action is authorization of a course of therapy (e.g., treatment with velaglucerase).
  • antibodies e.g., neutralizing antibodies
  • the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy and the course of action is assigning the subject to a first class.
  • assignment to the first class will enable payment for a treatment (e.g., velaglucerase) provided to the subject.
  • payment is by a first party to a second party.
  • the first party is other than the subject.
  • the first party is selected from a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is an insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment.
  • the subject does not experience an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or does not produce antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement and the course of action is authorization of a course of therapy (e.g., imiglucerase, velaglucerase or uplyso).
  • antibodies e.g., neutralizing antibodies
  • the subject does not experience an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or does not produce antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement and the course of action is assigning the subject to a second class.
  • assignment to the second class will enable payment for a treatment (e.g., imiglucerase, velaglucerase or uplyso) provided to the subject.
  • payment is by a first party to a second party.
  • the first party is other than the subject.
  • the first party is selected from a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is an insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment.
  • the disclosure features a method of providing information on which to make a decision about a subject with Gaucher disease (e.g., type I Gaucher disease), or making such a decision.
  • the method includes providing (e.g., by receiving) an evaluation of a subject, wherein the evaluation was made by a method described herein, e.g., by optionally, administering a glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase, velaglucerase or uplyso), to the subject; providing a determination post administration of whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or
  • the method includes making the decision.
  • the method also includes communicating the information to another party (e.g., by computer, compact disc, telephone, facsimile, email, or letter).
  • another party e.g., by computer, compact disc, telephone, facsimile, email, or letter.
  • the decision includes selecting a subject for payment, making or authorizing payment for a first course of action (e.g., treatment with velaglucerase) if the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or if the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months),
  • a course of therapy e.g., treatment with ve
  • assignment of the subject is to the first class and the assignment authorizes payment for a course of treatment (e.g., velaglucerase).
  • a course of treatment e.g., velaglucerase
  • assignment of the subject is to the second class and the assignment authorizes payment for a course of treatment (e.g., imiglucerase, velaglucerase or uplyso).
  • a course of treatment e.g., imiglucerase, velaglucerase or uplyso
  • the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment,
  • assignment to the first class will enable payment for a treatment (e.g., velaglucerase) provided to the subject.
  • payment is by a first party to a second party.
  • the first party is other than the subject.
  • the first party is selected from a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is an insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment.
  • a course of therapy e.g
  • the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.
  • assignment to the second class will enable payment for a treatment (e.g., imiglucerase, velaglucerase or uplyso) provided to the subject.
  • payment is by a first party to a second party.
  • the first party is other than the subject.
  • the first party is selected from a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is an insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment.
  • the disclosure features a method of selecting a payment class for a course of treatment with a glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase) for a subject with Gaucher disease (type I Gaucher disease).
  • a glucocerebrosidase enzyme replacement therapy e.g., velaglucerase
  • Gaucher disease type I Gaucher disease
  • the method includes determining that an infusion site reaction during or within 12 hours after infusion of the therapy is present in the subject or that antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to the therapy are present in the subject, e.g., by a method described herein, and approving, making, authorizing, receiving, transmitting or otherwise allowing payment of a selected course of treatment, e.g., velaglucerase.
  • antibodies e.g., neutralizing antibodies
  • the method includes determining that an infusion site reaction during or within 12 hours after infusion of the therapy is not present in the subject or that antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to the therapy are not present in the subject, e.g., by a method described herein, and approving, making, authorizing, receiving, transmitting or otherwise allowing payment of a selected course of treatment, e.g., imiglucerase, velaglucerase or uplyso.
  • antibodies e.g., neutralizing antibodies
  • IgE IgE, IgM, IgG and/or IgA antibodies
  • the disclosure features a method of making a data record.
  • the method includes entering the result of a method described herein into a record, e.g., a computer readable record.
  • the record is available on the world wide web.
  • the record is evaluated by a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity, or a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment, or is otherwise relied on in a method described herein.
  • the disclosure features a data record (e.g., computer readable record), wherein the record includes results from a method described herein.
  • the record is available on the world wide web.
  • the record is evaluated and/or transmitted to a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity, or a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the disclosure features a method of providing data.
  • the method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, for determining if a payment will be provided.
  • the data is provided by computer, compact disc, telephone, facsimile, email, or letter.
  • the data is provided by a first party to a second party.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the second party is a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment and the second party is a governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment and the second party is an insurance company.
  • the disclosure features a method of transmitting a record described herein.
  • the method includes a first party transmitting the record to a second party, e.g., by computer, compact disc, telephone, facsimile, email, or letter.
  • the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is an insurance company or government entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the treatment.
  • the first party is a governmental entity or insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the treatment.
  • information e.g., about whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy (e.g., wherein the information is obtained as described herein) is provided (e.g., communicated, e.g., electronically communicated) to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • choice of medical procedure, payment for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information.
  • the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc. based on the information.
  • information e.g., whether or not the mean platelet count of a subject with Gaucher disease increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months) (e.g., wherein the information is obtained as described herein) is provided (e.g., wherein the
  • choice of medical procedure, payment for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information.
  • the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc. based on the information.
  • a premium for insurance (e.g., life or medical) is evaluated as a function of information about whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy.
  • antibodies e.g., neutralizing antibodies
  • premiums can be increased (e.g., by a certain percentage) if the subject experiences an infusion site reaction to a glucocerebrosidase enzyme replacement or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy.
  • antibodies e.g., neutralizing antibodies
  • a premium for insurance (e.g., life or medical) is evaluated as a function of information about whether or not the mean platelet count of a subject with Gaucher disease increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months) (e.g., wherein
  • premiums can be increased (e.g., by a certain percentage) if the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months).
  • the glucocerebrosidase enzyme replacement therapy e.g., imiglu
  • Information about whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy can be used, e.g., in an underwriting process for life insurance.
  • the information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth.
  • An insurance policy can be recommended as a function of the information on whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy.
  • antibodies e.g., neutralizing antibodies
  • An insurance premium or risk assessment can also be evaluated as function of whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy.
  • antibodies e.g., neutralizing antibodies
  • Information about whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months
  • the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months
  • the information can be incorporated into a profile about a subject.
  • Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth.
  • An insurance policy can be recommended as a function of the information on whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglu
  • An insurance premium or risk assessment can also be evaluated as function of whether or not whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months) or whether the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy (e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months).
  • imiglucerase treatment e.g., imiglucerase at a dose of 60 U
  • information about whether or not a subject with Gaucher disease experiences an infusion site reaction to a glucocerebrosidase enzyme replacement therapy or produces antibodies (e.g., neutralizing antibodies) (e.g., IgE, IgM, IgG and/or IgA antibodies) to a glucocerebrosidase enzyme replacement therapy is analyzed by a function that determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein).
  • the results may indicate that a subject is suitable for treatment (e.g., velaglucerase), suggesting that a treatment course (e.g., with velaglucerase) is needed, thereby triggering an outcome that indicates or causes authorization to pay for a service or treatment (e.g., velaglucerase) provided to a subject.
  • a subject is suitable for treatment (e.g., velaglucerase)
  • a treatment course e.g., with velaglucerase
  • an entity e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses
  • an entity can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient.
  • services e.g., a particular therapy
  • a first entity e.g., an insurance company
  • a first entity e.g., an insurance company
  • information about whether or not the subject's mean platelet count increased by less than 80%, 75%, 70%, 65%, 60%, or 55% after 9 or 12 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 9 months
  • the subject's mean platelet count increased by less than 40%, 35%, 30%, or 25% after 6 months of treatment as compared to their baseline mean platelet count prior to initiating the glucocerebrosidase enzyme replacement therapy e.g., imiglucerase treatment, e.g., imiglucerase at a dose of 60 U/kg, e.g., administered every other week for 6 months
  • a function determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision
  • the results may indicate that a subject is suitable for treatment (e.g., velaglucerase), suggesting that a treatment course (e.g., a subject is suitable for treatment (e.g., velaglucerase), suggesting that a treatment course (e.g., a subject is suitable for treatment (e.g., velaglucerase), suggesting that a treatment course (e.g., a subject is suitable for treatment (e.g., velaglucerase), suggesting that a treatment course (e
  • an entity e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses
  • an entity can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient.
  • services e.g., a particular therapy
  • a first entity e.g., an insurance company
  • a first entity e.g., an insurance company
  • the disclosure features a method of providing data.
  • the method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, for determining if a payment will be provided.
  • the data is provided by computer, compact disc, telephone, facsimile, email, or letter.
  • the data is provided by a first party to a second party.
  • the first party is selected from the subject, a healthcare provider, a treating physician, a health maintenance organization (HMO), a hospital, a governmental entity, or an entity which sells or supplies the drug.
  • HMO health maintenance organization
  • the second party is a third party payer, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.
  • the disclosure provides the use of a glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase, imiglucerase or uplyso), alone or in combination with another agent(s) described herein (e.g., isofagomine tartrate, miglustat, or Genz112638), for use in treatment.
  • a glucocerebrosidase enzyme replacement therapy e.g., velaglucerase, imiglucerase or uplyso
  • another agent(s) described herein e.g., isofagomine tartrate, miglustat, or Genz112638
  • the disclosure provides the use of a glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase or imiglucerase), alone or in combination with another agent(s) described herein (e.g., isofagomine tartrate, miglustat, or Genz112638), for the preparation of a medicament, e.g., for treating Gaucher disease.
  • a glucocerebrosidase enzyme replacement therapy e.g., velaglucerase or imiglucerase
  • another agent(s) described herein e.g., isofagomine tartrate, miglustat, or Genz112638
  • the disclosure provides a pharmaceutical composition of velaglucerase.
  • the composition comprises: velaglucerase, a lyoprotectant (e.g., a carbohydrate (e.g., sucrose)), a buffer salt (e.g., citrate and/or citric acid, e.g., sodium citrate and citric acid), and a stabilizing agent (e.g., polysorbate, e.g., polysorbate 20).
  • a lyoprotectant e.g., a carbohydrate (e.g., sucrose)
  • a buffer salt e.g., citrate and/or citric acid, e.g., sodium citrate and citric acid
  • a stabilizing agent e.g., polysorbate, e.g., polysorbate 20.
  • the composition can be lyophilized.
  • the moisture content of the lyophilized composition is 1% to 6%, e.g., 1.3% to 6.2%.
  • the moisture content of the lyophilized composition is 1% to 5%.
  • the moisture content of the lyophilized composition is 3% to 5%.
  • the moisture content is greater than or equal to 3%. In some embodiments, the moisture content is 3%.
  • the lyophilized composition can be evaluated.
  • the secondary structure of the lyophilized composition can be evaluated, e.g., by FT-IR.
  • the composition can be a reconstituted solution.
  • the composition is a reconstituted solution in a pharmaceutically acceptable carrier such as Sterile Water for Injection (e.g., a 200 unit vial with 2.2 mL Sterile Water for Injection or a 400 unit vial with 4.3 mL Sterile Water for Injection).
  • the composition can further comprise, or consist of, sodium chloride solution suitable for intravenous administration (e.g., 0.9% sodium chloride solution suitable for intravenous administration).
  • the reconstituted solution can be evaluated, e.g., for degradation.
  • the reconstituted solution can be evaluated by SE-HPLC and/or RP-HPLC, e.g., for the presence of degradation products.
  • the reconstituted solution can be evaluated for oxidation.
  • the reconstituted solution can be evaluated by peptide mapping.
  • the disclosure features an assay (e.g., method) for detecting an anti-glucocerebrosidase antibody in a sample (e.g., a patient sample, e.g., blood or serum).
  • a sample e.g., a patient sample, e.g., blood or serum.
  • the method includes:
  • a glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • a surface e.g., a microwell
  • the surface can be coated with a coupling agent such as strepavidin and the glucocerebrosidase (e.g., velaglucerase, imiglucerase or uplyso) can be bound to an agent (e.g., biotin) which associates with, e.g., binds to, the coupling agent, e.g., the glucocerebrosidase (e.g., velaglucerase, imiglucerase or uplyso) is immobilized to the surface via the biotin binding to the strepavidin;
  • a coupling agent such as strepavidin
  • the glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • the immobilized glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • the immobilized glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • an anti-glucocerebrosidase antibody in the sample if present, to bind to the immobilized glucocerebrocidase, thereby forming a mixture
  • optionally performing a wash step to remove from the mixture any material in the sample that is not bound to the immobilized glucocerebrosidase;
  • labeled glucocerebrosidase e.g., velaglucerase or imiglucerase
  • labeled glucocerebrosidase is labeled with a detectable label (e.g., ruthenium-labeled glucocerebrosidase)
  • the label is not the same as the coupling agent and/or the agent that binds to the coupling agent, e.g., if biotin is used to immobilize glucocerebrosidase to the surface, the label is not biotin
  • a detectable label e.g., ruthenium-labeled glucocerebrosidase
  • the label is not the same as the coupling agent and/or the agent that binds to the coupling agent, e.g., if biotin is used to immobilize glucocerebrosidase to the surface, the label is not biotin
  • detecting and optionally quantifying the label in the mixture, e.g., wherein detection of the label indicates that an anti-glucocerebrosidase antibody is present in the sample.
  • the detected label is quantified to a value and compared to a control, e.g., a negative control, wherein if the value of detected label is greater than the negative control, the sample contains anti-glucocerebrosidase antibody.
  • the negative control is the average negative control value (e.g., background) for a plurality of negative controls.
  • the negative control can be normal human serum (NHS), and the average negative control value can be the average for a plurality of NHS lots, or the average of the negative control values obtained from a plurality of assays.
  • the negative control value can be a value of 1, 2, 3, 4, 5 or 6 ng/ml of antibody, e.g., a value of greater than that number for a sample indicates that an anti-glucocerebrosidase antibody is present in the sample.
  • the negative control value can be 200, 250, 300 (e.g., 306), 350, or 400, e.g., a value of greater than that number for a sample indicates that an anti-glucocerebrosidase antibody is present in the sample.
  • the assay is for detecting anti-velaglucerase antibodies. In another embodiment, the assay is for detecting anti-imiglucerase antibodies. In some embodiments, the assay is for detecting anti-uplyso antibodies.
  • the disclosure features an assay for detecting an anti-glucocerebrosidase antibody (e.g., IgG anti-glucocerebrosidase antibody) in a sample (e.g., a patient sample, e.g., blood or serum).
  • a sample e.g., a patient sample, e.g., blood or serum.
  • the method includes:
  • glucocerebrosidase e.g., velaglucerase or imiglucerase
  • a detectable label e.g., the glucocerebrosidase is 125 I labeled
  • a resin e.g., Protein G, Protein A, Protein A/G, or Protein L resin
  • a resin e.g., a Protein G spin column
  • the label in the mixture (e.g., on the resin), e.g., wherein detection of the label indicates that anti-glucocerebrosidase antibody is present in the sample.
  • the assay is for detecting anti-velaglucerase antibodies. In another embodiment, the assay is for detecting anti-imiglucerase antibodies. In some embodiments, the assay is for detecting anti-uplyso antibodies.
  • the detected label is quantified to a value and compared to a control, e.g., a negative control, wherein if the value of detected label is greater than the negative control, the sample contains anti-glucocerebrosidase antibody.
  • the negative control is the average negative control value (e.g., background) for a plurality of negative controls.
  • the negative control can be normal human serum (NHS), and the average negative control value can be the average for a plurality of NHS lots, or the average of the negative control values obtained from a plurality of assays.
  • the disclosure features an assay for detecting a human anti-glucocerebrosidase antibody in a sample (e.g., a patient sample, e.g., blood or serum).
  • a sample e.g., a patient sample, e.g., blood or serum.
  • the method includes:
  • glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • a surface e.g., a microwell
  • the surface can be coated with a coupling agent such as strepavidin and the glucocerebrosidase can be bound to an agent (e.g., biotin) that associates with, e.g., binds to, the coupling agent, e.g., the glucocerebrosidase is immobilized to the surface via the biotin binding to the strepavidin);
  • an agent e.g., biotin
  • an antibody that binds to the human anti-glucocerebrosidase antibody to the mixture, wherein the antibody that binds to the human anti-glucocerebrosidase antibody is labeled with a detectable label (e.g., ruthenium or biotin), under conditions that allow the labeled antibody that binds to the human anti-glucocerebrosidase antibody to bind to the human anti-glucocerebrosidase antibody (e.g., that is bound to the immobilized glucocerebrosidase), if present, (preferably, the label is not the same as the coupling agent and/or the agent that binds to the coupling agent, e.g., if biotin is used to immobilize glucocerebrosidase to the surface, the label is not biotin);
  • a detectable label e.g., ruthenium or biotin
  • detecting (and optionally quantifying) the label in the mixture e.g., wherein detection of the label indicates that human anti-glucocerebrosidase antibody is present in the sample.
  • the assay detects anti-velaglucerase antibodies. In some embodiments, the assay detects anti-imiglucerase antibodies. In some embodiments, the assay detects anti-uplyso antibodies.
  • the detected label is quantified to a value and compared to a control, e.g., a negative control, wherein if the value of detected label is greater than the negative control, the sample contains anti-velaglucerase antibody.
  • the negative control is the average negative control value (e.g., background) for a plurality of negative controls.
  • the negative control can be normal human serum (NHS), and the average negative control value can be the average for a plurality of NHS lots, or the average of the negative control values obtained from a plurality of assays.
  • the antibody that binds to the human anti-glucocerebrosidase antibody is isotype specific, wherein the isotype specific antibody that binds to the human anti-glucocerebrosidase antibody binds specifically to a human antibody of the isotype to which it is specific.
  • the antibody that binds to the human anti-glucocerebrosidase antibody is an IgA specific antibody and binds to an IgA human anti-glucocerebrosidase antibody in the sample.
  • the antibody that binds to the human anti-glucocerebrosidase antibody is an IgE specific antibody and binds to an IgE human anti-glucocerebrosidase antibody in the sample.
  • the antibody that binds to the human anti-glucocerebrosidase antibody is an IgM specific antibody and binds to an IgM human anti-glucocerebrosidase antibody in the sample.
  • the antibody that binds to the human anti-glucocerebrosidase antibody is an IgG specific antibody and binds to an IgG human anti-glucocerebrosidase antibody in the sample.
  • the disclosure features a method of determining if an anti-glucocerebrosidase antibody (e.g., in a sample) neutralizes (e.g., inhibits) glucocerebrosidase (e.g., velaglucerase or imiglucerase) activity.
  • the method includes:
  • a cell e.g., a human cell, e.g., a human fibroblast cell
  • MMR human macrophage mannose receptor
  • labeled glucocerebrosidase e.g., velaglucerase, imiglucerase or uplyso
  • the glucocerebrosidase is labeled with a detectable label
  • the glucocerebrosidase is labeled with a fluorescent label, e.g., a green fluorescent dye, such as Alexa FLUOR® 488 or fluorescein isothiocyanate (FITC)
  • a detectable label e.g., the glucocerebrosidase is labeled with a fluorescent label, e.g., a green fluorescent dye, such as Alexa FLUOR® 488 or fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothiocyanate
  • the levels of labeled glucocerebrosidase are compared to a control, e.g., the level of labeled glucocerebrosidase detected in the absence of the anti-glucocerebrosidase antibody under identical conditions.
  • the cells do not express an Fc receptor (e.g., human Fc receptor).
  • Fc receptor e.g., human Fc receptor
  • the method detects whether neutralizing anti-velaglucerase antibodies are present. In some embodiments, the method detects whether neutralizing anti-imiglucerase antibodies are present. In some embodiments, the method detects whether neutralizing anti-uplyso antibodies are present.
  • the method detects whether an anti-imiglucerase antibody neutralizes imiglucerase activity. In some embodiments, the method detects whether an anti-velaglucerase antibody neutralizes velaglucerase activity. In some embodiments, the method detects whether an anti-uplyso antibody neutralizes uplyso activity.
  • the method detects whether an anti-imiglucerase antibody neutralizes velaglucerase and/or uplyso activity. In some embodiments, the method detects whether an anti-velaglucerase antibody neutralizes imiglucerase and/or uplyso activity. In some embodiments, the method detects whether an anti-uplyso antibody neutralizes imiglucerase and/or velaglucerase activity.
  • the disclosure features a method of determining if an anti-velaglucerase antibody (e.g., in a sample) neutralizes (e.g., inhibits) imiglucerase activity.
  • the method includes:
  • a cell e.g., a human cell, e.g., a human fibroblast cell
  • MMR human macrophage mannose receptor
  • the imiglucerase is labeled with a detectable label (e.g., the imiglucerase is labeled with a fluorescent label, e.g., a green fluorescent dye, such as Alexa FLUOR® 488 or fluorescein isothiocyanate (FITC)), under conditions that allow the labeled imiglucerase to bind to the MMR in the absence of an anti-velaglucerase antibody (e.g., wherein binding of imiglucerase to MMR allows cellular uptake of the imiglucerase);
  • a detectable label e.g., the imiglucerase is labeled with a fluorescent label, e.g., a green fluorescent dye, such as Alexa FLUOR® 488 or fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothiocyanate
  • the levels of labeled imiglucerase are compared to a control, e.g., the level of labeled imiglucerase detected in the absence of the anti-velaglucerase antibody under identical conditions.
  • the cells do not express an Fc receptor (e.g., human Fc receptor).
  • Fc receptor e.g., human Fc receptor
  • the disclosure features a method of determining if an anti-imiglucerase antibody (e.g., in a sample) neutralizes (e.g., inhibits) velaglucerase activity.
  • the method includes:
  • a cell e.g., a human cell, e.g., a human fibroblast cell
  • MMR human macrophage mannose receptor
  • the velaglucerase is labeled with a detectable label
  • a detectable label e.g., the velaglucerase is labeled with a fluorescent label, e.g., a green fluorescent dye, such as Alexa FLUOR® 488 or fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothiocyanate
  • the levels of labeled velaglucerase are compared to a control, e.g., the level of labeled velaglucerase detected in the absence of the anti-imiglucerase antibody under identical conditions.
  • the cells do not express an Fc receptor (e.g., human Fc receptor).
  • Fc receptor e.g., human Fc receptor
  • the disclosure features a hybrid antibody, wherein the hybrid antibody comprises a non-human anti-drug antibody and a human immunoglobulin (Ig).
  • the hybrid antibody comprises a non-human anti-drug antibody and a human immunoglobulin (Ig).
  • the non-human anti-drug IgG antibody is a sheep anti-drug IgG antibody.
  • the anti-drug antibody binds to velaglucerase.
  • the anti-drug antibody binds to imiglucerase.
  • the anti-drug antibody binds to uplyso.
  • the human Ig is IgA.
  • the human Ig is IgE.
  • the human Ig is IgM.
  • the human Ig is IgG.
  • the non-human anti-drug antibody is an IgG antibody.
  • the non-human anti-drug antibody and the human Ig are conjugated together by a chemical crosslinker, e.g., a long spacer arm cross linker, e.g., succinimidyl 6-[3′-2-pyridyldithio-propionamido]hexanoate (LC-SPDP).
  • a chemical crosslinker e.g., a long spacer arm cross linker, e.g., succinimidyl 6-[3′-2-pyridyldithio-propionamido]hexanoate (LC-SPDP).
  • the hybrid antibody is used as a positive control in assays that detect and/or measure levels and/or isotypes of anti-drug antibody in a sample, e.g., in a method described herein.
  • the hybrid antibody is used as a positive control in assays for determining if an anti-glucocerebrosidase antibody (e.g., in a sample) neutralizes (e.g., inhibits) glucocerebrosidase (e.g., velaglucerase or imiglucerase) activity, e.g., in a method described herein.
  • an anti-glucocerebrosidase antibody e.g., in a sample
  • glucocerebrosidase e.g., velaglucerase or imiglucerase
  • the hybrid antibody is used as an anti-glucocerebrosidase antibody (e.g., in a sample) in the assay.
  • the disclosure features a method of measuring cellular uptake (e.g., internalization) of glucocerebrosidase (e.g., velaglucerase or imiglucerase) into a cell.
  • the method includes:
  • glucocerebrosidase e.g., velaglucerase or imiglucerase
  • a cell e.g., a cell of a human leukemic monocyte lymphoma cell line (e.g., U937) or a cell of a murine macrophage cell line (e.g., J774)
  • glucocerebrosidase e.g., velaglucerase or imiglucerase
  • glucocerebrosidase e.g., velaglucerase or imiglucerase
  • the amount of uptake is measured by measuring glucocerebrosidase enzymatic activity in the cell.
  • a synthetic substrate that fluoresces upon cleavage e.g., 4-MU-glc is used.
  • the amount of uptake is measured by measuring intracellular glucocerebrosidase protein levels.
  • Western blot analysis is used.
  • immunohistochemistry analysis is used (e.g., immunohistochemistry on permeabilized cells).
  • the cell is washed one or more times prior to the measuring step.
  • the pH of the mixture is 7.5.
  • mannan is present in the mixture.
  • calcium is present in the mixture.
  • the glucocerebrosidase is velaglucerase.
  • the glucocerebrosidase is imiglucerase.
  • the amount of uptake of velaglucerase is compared to the amount of uptake of imiglucerase (e.g., under the same conditions).
  • subject refers to any mammal, including but not limited to, any animal classified as such, including humans, non human primates, primates, baboons, chimpanzees, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.
  • rodents e.g., mice, rats
  • rabbits cats, dogs, horses, cows, sheep, goats, pigs, etc.
  • patient can be used interchangeably with the term “patient.”
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived.
  • the term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably, at least 80% 90% (w/w) pure, even more preferably, 90 to 95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.
  • treatment refers to treatment of an existing disorder and/or prophylactic/preventative measures.
  • Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk or having, or who may ultimately acquire the disorder.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder.
  • Treatment may include slowing or reversing the progression of a disorder.
  • treating refers to administering a therapy in an amount, manner, and/or mode effective to improve or prevent a condition, symptom, or parameter associated with a disorder (e.g., a disorder described herein) or to prevent onset, progression, or exacerbation of the disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art. Accordingly, treating can achieve therapeutic and/or prophylactic benefits.
  • An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
  • the term “combination” refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time.
  • the agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order.
  • FIG. 1 ( a )-( f ) depicts mean % change in hematological values, organ values, and biomarkers in phase I/II trial.
  • FIG. 5 depicts TKT025 and TKT025EXT temporal change in mean Z-scores of lumbar spine from baseline.
  • FIG. 6 depicts TKT025 and TKT025EXT temporal change in mean Z-score of femoral neck from baseline.
  • FIG. 15 depicts TKT034 mean change from baseline in hemoglobin concentration.
  • FIG. 16 depicts TKT034 mean n percent change from baseline in platelet count.
  • FIG. 18 depicts TKT034 mean percent change from baseline in normalized spleen volume.
  • FIG. 19 depicts TKT034 mean percent change from baseline in plasma chitotriosidase.
  • FIG. 21 depicts an immunogenicity evaluation of patients in velaglucerase alfa clinical studies.
  • FIG. 22 depicts an anti-drug antibody screening by electro-chemiluminescence (ECL) immunoassay.
  • FIG. 23 depicts a screening assay dose response curve.
  • FIG. 24 depicts an IgG ADA confirmatory assay.
  • FIG. 25 depicts an RIP assay dose response curve.
  • the disclosure is based, in part, on the discovery that velaglucerase elicits less of an immune response (e.g., less production of antibody, e.g., less production of neutralizing antibody) than imiglucerase upon administration to a subject (e.g., a subject with Gaucher disease). It was discovered that velaglucerase elicits less infusion site reaction upon administration to a subject (e.g., a subject with Gaucher disease) than imiglucerase and velaglucerase can result in an increase in platelet count when administered to a subject (e.g., a subject having Gaucher disease) than imiglucerase.
  • an immune response e.g., less production of antibody, e.g., less production of neutralizing antibody
  • velaglucerase elicits less infusion site reaction upon administration to a subject (e.g., a subject with Gaucher disease) than imiglucerase and velaglucerase can result in an increase in platelet count when administered to a
  • the invention relates, inter alia, to compositions and methods for selecting a treatment for a subject with Gaucher disease, selecting subjects for treatment with velaglucerase (e.g., alone or in combination with another therapy), and methods for reducing injection site reaction in subjects undergoing treatment for Gaucher disease.
  • Velaglucerase is human ⁇ -glucocerebrosidase produced by gene-activation in a human cell line. Gene activation refers to targeted recombination with a promoter that activates the endogenous ⁇ -glucocerebrosidase gene in the selected human cell line. Velaglucerase is secreted as a monomeric glycoprotein of approximately 63 kDa and is composed of 497 amino acids with a sequence identical to the natural human protein. The amino acid sequence of velaglucerase is described in Zimran et al. (2007) Blood Cells Mol Dis, 39: 115-118.
  • Glycosylation of velaglucerase alfa is altered by using kifunensine, a mannosidase I inhibitor, during cell culture, which results in the secretion of a protein containing primarily high-mannose type glycans having 6-9 mannose units per glycan.
  • kifunensine a mannosidase I inhibitor
  • Glycosylation Site Predominant Glycan Other Glycans Asn19 High-mannose High-mannose (Man) 9 (GlcNAc) 2 (Man) 6-8 (GlcNAc) 2 Phosphorylated high-mannose (Phos) 1 (Man) 8-9 (GlcNAc) 2 GlcNAc capped phosphate (Phos) 1 (Man) 8-9 (GlcNAc) 3 Hybrid (Hex) 2 (Man) 3 (GlcNAc) 3 (Fuc) 1 Asn59 High-mannose High-mannose (Man) 9 (GlcNAc) 2 (Man) 5-8 (GlcNAc) 2 Phosphorylated high-mannose (Phos) 1 (Man) 7-9 (GlcNAc) 2 GlcNAc capped phosphate (Phos) 1 (Man) 8-9 (GlcNAc) 3 Hybrid (NeuAc) 1 (Gal) 1 (Man) 5 (GlcNAc) 3 (Fu
  • Velaglucerase has three non-contiguous domains, with the catalytic site located in domain III (residues 76-381 and 416-430), a ( ⁇ / ⁇ ) 8 (TIM) barrel.
  • Velaglucerase (VPRIVTM) is commercially available from Shire Human Genetics Therapies, Inc. Methods of making velaglucerase are described, for example, in U.S. Pat. No. 7,138,262.
  • Velaglucerase (also referred to herein as velaglucerase alfa) is a sterile, white to off-white, preservative-free lyophilized powder for solution in single-use vials for intravenous (IV) infusion after reconstitution with Sterile Water for Injection.
  • each vial Upon reconstitution with Sterile Water for Injection, each vial contains approximately 2.5 mg/mL (40 U/mL) of velaglucerase alfa, 50 mg/mL sucrose, 12.9 mg/mL sodium citrate dihydrate, 1.3 mg/mL citric acid monohydrate and 0.11 mg/mL polysorbate 20. Each vial contains an extractable volume of 2.0 mL for the 200 U vial and 4.0 mL for the 400 U vial.
  • Velaglucerase is supplied in a 200 U/vial (5 mg) or 400 U/vial (10 mg) of velaglucerase alfa, one unit (U) of enzyme activity being defined as the quantity of enzyme required to convert one micromole of p-nitrophenyl ⁇ -D-glucopyranoside to p-nitrophenol per minute at 37° C.
  • Velaglucerase is a sterile, lyophilized powder for solution supplied in either a 5 mL (200 U/vial presentation) or 20 mL (400 U/vial presentation) type I glass vial. Each vial contains either 200 U (5 mg) or 400 U (10 mg) of velaglucerase alfa. The vials are closed with a butyl rubber stopper with a fluoro-resin coating and are sealed with an aluminum overseal with a flip-off plastic cap.
  • Velaglucerase is a lyophilized powder for solution intended for intravenous infusion. Vials are single-use vials. Velaglucerase is not infused with other products in the same infusion. The total volume of infusion is delivered over a period of 60 minutes. Velaglucerase should be handled as follows:
  • Velaglucerase is administered at doses between (and including) 2.5 U/kg and 60 U/kg of subject body weight, e.g., 15 U/kg to 60 U/kg (e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg). Velaglucerase can be administered at a rate of 2 U/kg/minute, 1.5 U/kg/minute, 1 U/kg/minute or 0.5 U/kg/minute. The dose of velaglucerase is administered to the subject every other week.
  • Velaglucerase can be administered at a dose of 15 to 60 U/kg (e.g. 30 U/kg to 60 U/kg, e.g., 15 U/kg, 30 U/kg, 45 U/kg, or 60 U/kg), at a dose equal to or below 22.5 U/kg, at a dose between 22.5 and 37.5 U/kg, at a dose between 37.5 and 52.5 U/kg, or at a dose equal to or above 52.5 U/kg.
  • velaglucerase can be administered at a dose of 2.5 U/kg to 60 U/kg.
  • the velaglucerase can be administered every other week by intravenous infusion.
  • the velaglucerase can be administered every week by intravenous infusion. In some embodiments, the velaglucerase can be administered three times a week by intravenous infusion, e.g., at a dose of 2.5 U/kg.
  • the infusion of the dose occurs over less than 2 hours, e.g., less than 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes or 45 minutes.
  • Recombinantly produced human glucocerebrosidase Other forms of recombinantly-produced human glucocerebrosidase that can be used in the compositions, assays and methods described herein.
  • imiglucerase (Cerazyme®) is recombinately produced in Chinese Hamster Ovary (CHO) cells and is commercially available.
  • prGCD a recombinant glucocerebrosidase expressed in plant cells.
  • Plant recombinant glucocerebrosidase can be obtained by methods described, e.g., in U.S. Publication Nos: US 20090208477 and US 20080038232 and PCT Publication Nos.: WO 2004/096978 and WO 2008/132743.
  • velaglucerase can be used as an alternative treatment, e.g., for subjects who were previously treated with another therapy (i.e., a therapy other than velaglucerase, e.g., imiglucerase, alglucerase, uplyso, isofagomine tartrate, miglustat, or Genz112638).
  • a therapy other than velaglucerase e.g., imiglucerase, alglucerase, uplyso, isofagomine tartrate, miglustat, or Genz112638.
  • a subject who is undergoing treatment for Gaucher disease with another therapy can be transferred to treatment with velaglucerase, e.g., if the subject is experiencing a side effect or adverse effect from the other therapy.
  • a subject who is undergoing treatment for Gaucher disease with imiglucerase can be transferred to treatment with velaglucerase, e.g., velaglucerase can be administered at the same dose and with the same frequency at which the imiglucerase was administered.
  • the subject may have experienced an infusion site reaction upon or after administration of imiglucerase and/or developed anti-imiglucerase antibodies (e.g., neutralizing antibodies to imiglucerase).
  • a subject who has Gaucher disease can be administered a therapy that includes velaglucerase in an amount and for a time to provide an overall therapeutic effect.
  • the velaglucerase can be administered alone or in combination with an additional agent(s).
  • the amounts and times of administration can be those that provide, e.g., a synergistic therapeutic effect, or an additive therapeutic effect.
  • velaglucerase can be used in combination with another therapy for Gaucher disease, e.g., a therapy other than enzyme replacement therapy, e.g., isofagomine tartrate, miglustat, or Genz112638.
  • a therapy other than enzyme replacement therapy e.g., isofagomine tartrate, miglustat, or Genz112638.
  • Isofagomine tartrate (AT-2101, HGT-34100, PLICERA®) ((3R,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)piperidine L-(+)-tartrate; CAS No. 957230-65-8) selectively binds to and stabilizes glucocerebrosidase and facilitates proper trafficking of the enzyme to the lysosomes, the compartments in the cell where it is needed to break down glucocerebroside. See also U.S. Pat. No. 7,501,439.
  • Miglustat is an N-alkylated imino sugar, a synthetic analogue of D-glucose and a white to off-white crystalline solid that has a bitter taste.
  • Miglustat exhibits a large volume of distribution and has the capacity to access deep organs such as the brain, bone and lung.
  • Miglustat inhibits glucosylceramide synthase an essential enzyme for the synthesis of most glycosphingolipids.
  • Miglustat is a glucosylceramide synthase inhibitor. It works by blocking an enzyme that reduces the formation of certain chemicals in the body (glucosylceramide-based glycosphingolipids). Miglustat is used to treat adults with mild to moderate type 1 Gaucher disease.
  • Genz112638 is glucosylceramide analog given orally, and is designed to partially inhibit glucosylceramide synthase, which results in reduced production of glucosylceramide.
  • small molecules referred to as pharmacological chaperones can be used to selectively bind to a target macromolecule and increase its stability.
  • the binding of a pharmacological chaperone can help a target molecule, such as a protein (e.g., GCB) or other macromolecule (e.g., glucocerebroside), to fold into its correct three-dimensional shape. This can facilitate the activity and cellular function of the molecule (e.g., enzymatic activity in the case of GCB).
  • the pharmacological chaperone can facilitate trafficking of the chaperoned molecule from the ER to the appropriate location in a cell. One or both of these functions can reduce stress on cells.
  • Pharmacological chaperones also can decrease aggregation and/or accumulation of misfolded macromolecules.
  • restoring trafficking of misfolded macromolecules e.g., proteins
  • reducing their retention in the ER can have the added benefit of alleviating the toxic effects (e.g., proteotoxic effects) associated with mutant macromolecule accumulation and/or aggregation.
  • combination therapies can include one, two, or more glucocerebrosidase enzyme replacement therapies, optionally in combination with one or more small molecule therapies, such as isofagomine tartrate, miglustat, or Genz112638, and/or optionally in combination with one or more pharmacological chaperones and/or other agents.
  • small molecule therapies such as isofagomine tartrate, miglustat, or Genz112638
  • the combination when velaglucerase is administered in combination with an additional agent, the combination can result in a lower dose of the additional agent or velaglucerase being needed, such that side effects are reduced.
  • the combination may result in enhanced delivery and efficacy of one or both agents.
  • the agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks.
  • the use of velaglucerase can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered, e.g., to reduce the side-effects of a therapy other than enzyme replacement therapy. Accordingly, a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of velaglucerase.
  • a combination therapy can include administering an agent that reduces the side effects of other therapies.
  • a corticosteroid can be administered to a subject prior to administration of the treatment for Gaucher disease to decrease infusion site reaction.
  • iron supplement therapy can be given during the course of velaglucerase therapy.
  • Gaucher disease is the most common of the lysosomal storage diseases. It is caused by a hereditary deficiency of the enzyme glucocerebrosidase (also known as acid ⁇ -glucosidase). The enzyme acts on a fatty substance glucocerebroside (also known as glucosylceramide). When the enzyme is defective, the substance accumulates, particularly in cells of the mononuclear cell lineage. Fatty material can collect in the spleen, liver, kidneys, lungs, brain and bone marrow.
  • Symptoms may include enlarged spleen and liver, liver malfunction, skeletal disorders and bone lesions that may be painful, severe neurologic complications, swelling of lymph nodes and (occasionally) adjacent joints, distended abdomen, a brownish tint to the skin, anemia, low blood platelets and yellow fatty deposits on the white of the eye (sclera). Persons affected most seriously may also be more susceptible to infection.
  • the disease is caused by a recessive gene on chromosome 1 and affects both males and females.
  • Gaucher disease has three common clinical subtypes:
  • Type I (or non-neuropathic type) is the most common form of the disease, occurring in approximately 1 in 50,000 live births. It occurs most often among persons of Ashkenazi Jewish heritage. Symptoms may begin early in life or in adulthood and include enlarged liver and grossly enlarged spleen (together hepatosplenomegaly); the spleen can rupture and cause additional complications. Skeletal weakness and bone disease may be extensive. Spleen enlargement and bone marrow replacement cause anemia, thrombocytopenia and leucopenia. The brain is not affected, but there may be lung and, rarely, kidney impairment. Patients in this group usually bruise easily (due to low levels of platelets) and experience fatigue due to low numbers of red blood cells. Depending on disease onset and severity, type 1 patients may live well into adulthood. Many patients have a mild form of the disease or may not show any symptoms. In some embodiments, the methods and compositions described herein are used to treat type I Gaucher disease.
  • Type II typically begins within 6 months of birth and has an incidence rate of approximately 1 in 100,000 live births. Symptoms include an enlarged liver and spleen, extensive and progressive brain damage, eye movement disorders, spasticity, seizures, limb rigidity, and a poor ability to suck and swallow. Affected children usually die by age 2.
  • Type III (the chronic neuropathic form) can begin at any time in childhood or even in adulthood, and occurs in approximately 1 in 100,000 live births. It is characterized by slowly progressive but milder neurologic symptoms compared to the acute or type 2 version. Major symptoms include an enlarged spleen and/or liver, seizures, poor coordination, skeletal irregularities, eye movement disorders, blood disorders including anemia and respiratory problems. Patients often live into their early teen years and adulthood.
  • bone density refers to the amount of matter per square centimeter of bones. Bone density can be used in clinical medicine as an indirect indicator of osteoporosis and/or fracture risk.
  • BMD can be measured by a number of procedures, e.g., dual energy X-ray absorptiometry (DXA or DEXA), quantitative computed tomography (QCT), qualitative ultrasound (QUS), single photon absorptiometry (SPA), dual photon absorptiometry (DPA), digital X-ray radiogrammetry (DXR), and single energy X-ray absorptiometry (SEXA). Measurements can be made, e.g., over the lumbar spine, the upper part of the hip, or the forearm.
  • Densitometry results can be reported in, e.g., measured density in g cm ⁇ 3 , z-score, and t-score. Negative scores indicate lower bone density, and positive scores indicate higher.
  • Z-score refers to the number of standard deviations above or below the mean for the patient's age, sex and ethnicity.
  • T-score refers to the number of standard deviations above or below the mean for a healthy 30 year old adult of the same sex and ethnicity as the patient.
  • the criteria of the World Health Organization are:
  • the glucocerebrosidase enzyme replacement therapy described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously.
  • the glucocerebrosidase enzyme replacement therapy is administered invtravenously, with a dosage ranging from about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 U/kg, administered every other week, or according to the requirements of the particular compound.
  • the methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect.
  • the glucocerebrosidase enzyme replacement therapy can be administered as a continuous infusion, e.g., a continuous infusion over 60 minutes, 90 minutes, 120 minutes, or 150 minutes.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from 5% to 95% active compound (w/w).
  • such preparations contain from 20% to 80% active compound.
  • a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • a compound, composition or combination of this invention may be administered as a home therapy (e.g., in the subject's home, workplace, or other non-clinical (e.g., non-hospital) setting). It can be administered (e.g., via infusion) by a health care professional (e.g., nurse or physician's assistant). For example, if the subject has not experienced an adverse event (AE) (e.g., a drug-related serious AE or an infusion-related AE, e.g., an event described herein), e.g., after one, two, or three administrations (e.g., via infusion) of the compound, composition or combination, the subject is eligible to receive home therapy for subsequent administrations.
  • AE adverse event
  • a glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase) can be incorporated into a pharmaceutical composition for administration to a subject.
  • Such compositions typically include the glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase) and a pharmaceutically acceptable carrier.
  • the language “pharmaceutically acceptable carrier or adjuvant” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • pharmaceutically acceptable carrier or adjuvant refers to a carrier or adjuvant that may be administered to a subject, together with glucocerebrosidase, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the glucocerebrosidase.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal and subcutaneous.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vial
  • suitable carriers include Sterile Water for Injection, physiological saline, bacteriostatic water, CREMOPHOR ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars such as sucrose, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical composition can include, for example, sterile water for injection, sucrose, sodium citrate, citric acid and polysorbate.
  • Sterile injectable solutions can be prepared by incorporating the glucocerebrosidase in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a glucocerebrosidase enzyme replacement therapy (e.g., velaglucerase) can be provided in a kit.
  • the kit includes (a) the glucocerebrosidase enzyme replacement therapy, e.g., a composition that includes the glucocerebrosidase enzyme replacement therapy, and (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the glucocerebrosidase enzyme replacement therapy for the methods described herein.
  • the informational material describes methods for administering the glucocerebrosidase enzyme replacement therapy to treat Gaucher disease.
  • the informational material can include instructions to administer the glucocerebrosidase enzyme replacement therapy in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein).
  • the informational material can include instructions for identifying a suitable subject, e.g., a human.
  • the informational material of the kits is not limited in its form.
  • the informational material, e.g., instructions is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet.
  • the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording.
  • the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the modulator and/or its use in the methods described herein.
  • the informational material can also be provided in any combination of formats.
  • the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating Gaucher disease.
  • the other ingredients can be included in the kit, but in different compositions or containers than the glucocerebrosidase enzyme replacement therapy.
  • the kit can include instructions for admixing the glucocerebrosidase enzyme replacement therapy and the other ingredients (e.g., reconstituting a lyophilized therapy and/or diluting the reconstituted therapy prior to administration), or for using the glucocerebrosidase enzyme replacement therapy together with the other ingredients.
  • the glucocerebrosidase enzyme replacement therapy can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the glucocerebrosidase enzyme replacement therapy be substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the glucocerebrosidase enzyme replacement therapy is provided as a dried form, reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing the glucocerebrosidase enzyme replacement therapy.
  • the kit contains separate containers, dividers or compartments for the glucocerebrosidase enzyme replacement therapy (e.g., in a composition) and informational material.
  • the glucocerebrosidase enzyme replacement therapy e.g., in a composition
  • the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the glucocerebrosidase enzyme replacement therapy (e.g., in a composition) is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the glucocerebrosidase enzyme replacement therapy (e.g., in a composition).
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the glucocerebrosidase enzyme replacement therapy.
  • the containers of the kits can be air tight and/or waterproof.
  • the glucocerebrosidase enzyme replacement therapy (e.g., in a composition) can be administered to a subject with Gaucher disease.
  • the method can include evaluating a subject, e.g., as described herein and thereby identifying a subject being in need of treatment with the glucocerebrosidase enzyme replacement therapy.
  • Velaglucerase alfa is a novel enzyme replacement therapy (ERT) with unique characteristics (wild type amino acid sequence and high ⁇ -mannosyl content) that distinguishes it from imiglucerase.
  • This example describes a nine-month Phase I/II open-label, single center trial of velaglucerase alfa (TKT025) and ongoing extension (TKT025EXT) study conducted to evaluate safety and efficacy of velaglucerase alfa.
  • the primary objective of the Phase I/II trial was to assess the safety of velaglucerase alfa administered intravenously at a dose of 60 U/kg every-other-week for nine months in adult patients with symptomatic type 1 (non-neuronopathic) Gaucher disease (GD1).
  • the secondary objective of this trial was to assess the clinical activity of velaglucerase alfa on key disease features (Barton N W et al. N Engl J. Med. 1991; 324:1464-1470).
  • the extension study was similarly designed to evaluate the long-term safety and assess the effects of velaglucerase alfa on four disease measures, hemoglobin concentration, platelet count, liver volume, and spleen volume (Barton N W et al. N Engl J. Med. 1991; 324:1464-1470).
  • Patients Adult, symptomatic, enzymatically confirmed patients with GD1 were screened. Eligibility criteria included age ⁇ 18 years-old, an intact spleen, disease-related anemia (hemoglobin values at least 1 g/dL below lower limit of normal (LLN) for gender), thrombocytopenia (platelet counts below the LLN), and a negative result for hepatitis B and C antigen and human immunodeficiency virus. Patients were eligible if they were na ⁇ ve to ERT or had not received imiglucerase within the 12 months prior to enrollment and were imiglucerase antibody-negative. Patients were excluded if they had received an investigational therapy for any other indication ⁇ 30 days prior to enrollment or if they could not comply with the protocol for either medical or non-medical reasons.
  • Eligibility criteria included age ⁇ 18 years-old, an intact spleen, disease-related anemia (hemoglobin values at least 1 g/dL below lower limit of normal (LLN) for gender), thrombocyto
  • Velaglucerase alfa was supplied by Shire HGT as a lyophilized product and shipped at 2°-8° C. The product was reconstituted with preservative-free, sterile water for injection. The appropriate amount of velaglucerase alfa (based on body weight) was slowly mixed with normal saline to a final volume of 100 mL. The diluted velaglucerase alfa was administered intravenously across a 0.2 ⁇ m filter for a period of 60 minutes (maximum rate of 1.5 mg/kg/hour; 1 U/kg/min).
  • the first three patients received velaglucerase alfa on an every-other-week schedule at the trial site. Dose escalation was undertaken for the first three patients whereby dosing doubled from an initial dose of 15 U/kg until a final dose of 60 U/kg was achieved.
  • the second and third patients received their initial 15 U/kg infusion only after a 7-day observation period was completed for the first and second patients respectively.
  • the third patient received a single dose of 60 U/kg and was observed for a period of 7 days, nine additional patients were enrolled and received infusions of 60 U/kg every-other-week for a total of 20 doses.
  • Safety Assessments were evaluated throughout the study by every-other-week assessments of adverse events (including infusion-related reactions), concomitant medications, and vital signs performed before, during, and after infusions. Additional safety assessments were conducted approximately every 12 weeks and included physical examinations, clinical laboratory tests (hematology, serum chemistry, urinalysis, and pregnancy test), 12-lead electrocardiograms and echocardiograms at the trial site. Determination of the presence of anti-velaglucerase alfa antibodies were conducted at 3-month intervals at Shire HGT.
  • Antibody Assays All participants were screened for circulating anti-velaglucerase alfa antibodies using a validated indirect ELISA. Microwell plates were coated with velaglucerase alfa, washed, and blocked with bovine serum albumin to limit non-specific antibody binding. They were incubated with patient serum samples diluted 100-fold in phosphate buffered saline containing 0.05% Tween 20 for 60 minutes at 37° C. The microwells were washed and then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody.
  • HRP horseradish peroxidase
  • HRP-antibody, isotype-specific conjugates 1) goat anti-human IgG Fc, 2) goat anti-human IgA ⁇ -chain, 3) goat anti-human IgM ⁇ -chain, or 4) goat anti-human IgE ⁇ -chain secondary antibodies.
  • the microwells were washed one final time and incubated with the HRP chromogenic substrate 3,3′,5,5′ tetramethyl benzidine. The reaction was stopped by the addition of 2N sulfuric acid, and the absorbance of each well was quantified at 450 nm (A 450 ) using a Molecular Devices SPECTRAmax Plus384 plate reader and SOFTMax PRO software. Antibody-positive serum samples were obtained from patients receiving imiglucerase.
  • the ELISA antibody positive cutoff was established as a ratio ⁇ 2.0 and an A 450 ⁇ 0.040, where ratio is the A 450 of a patient sample taken at any time point, divided by the A 450 of the patient sample taken at baseline prior to the first ERT treatment. Any sample exceeding the ELISA positive cutoff would have been confirmed by a quantitative radioimmunoprecipitation assay and tested for neutralizing antibodies; however no sample achieved the established cutoff criteria (Mire-Sluis A R et al., J Immunol Methods. 2004; 289:1-16).
  • Clinical Activity The main efficacy assessments of hemoglobin concentration and platelet counts were evaluated at pre-determined tri-monthly intervals. Liver and spleen volumes were measured using quantitative abdominal MRI (on the same model apparatus) performed at baseline, six- and nine-months (at the Hadassah-Hebrew University Medical Center), and at 24, 33, and 45 months (at the MOR-MAR Imaging unit) during the extension study. Liver and spleen volumes were assessed at the end of the trial by a radiologist blinded to the patient's identity and the sequence by which the quantitative abdominal MRIs were performed. Chitotriosidase and CCL18 were measured at the Academic Medical Center; Amsterdam, the Netherlands.
  • the safety population which was also the intent-to-treat population, was defined as all enrolled patients receiving at least one infusion (partial or full) of velaglucerase alfa, and was used for all clinical activities. No imputation was utilized.
  • the null hypothesis is that there is no difference between the baseline value and end-of-the study value (nine months), and baseline value and end-of-48 month value. (i.e., the difference between the members of each pair of observation has median value zero.) Comparisons were performed using two-tailed hypothesis testing at the 5% level of significance. Differences between baseline and end of period values were analyzed using the Wilcoxon signed-rank test. Changes from baseline were calculated and the percentage changes from baseline are summarized using descriptive statistics.
  • Velaglucerase alfa was well tolerated and no patient developed antibodies. Clinically and statistically significant improvements in hematological parameters and organ volumes were observed as early as 3 to 6 months (Table 1).
  • Demographics and disposition A total of thirteen patients were screened and all consented to participate in this study; one patient (0004) was excluded because of imiglucerase antibodies. All the patients were treatment-na ⁇ ve at advent according to the protocol by virtue of not having been exposed to any Gaucher-specific therapy in the 12 months prior to enrolment although in the more distant past, 2 patients (0008 and 0009) had each received 3 infusions of imiglucerase, one patient (0003) had been exposed to miglustat, and two patients (0005 and 0007) had been exposed to both miglustat and imiglucerase.
  • the intent-to-treat population (Table 2) included twelve patients who received at least one dose of velaglucerase alfa; of these, 11 patients (92%) completed the Phase I/II study (one patient, 0006, withdrew after a sudden death in the family withdrew consent after receiving three infusions).
  • Normalized liver and spleen volumes are defined as 2.5% and 0.2% respectively of total body weight in kilograms (Pastores G M et al., Semin Hematol. 2004; 41:4-14). Multiples of normal is the observed organ volume divided by the normalized organ volume. By 24 months, all patients demonstrated normalization of hemoglobin concentrations and all but one patient demonstrated platelet counts greater than 100*10 9 /L.
  • BL baseline; Mo, month; M, male; F, Female; *Spleen volume not interpretable due to technical artifact.
  • the study was comprised of 2 phases, a dose escalation phase and a continuous dose phase.
  • IV Intravenous
  • the first patient was enrolled and administered Intravenous (IV) velaglucerase alfa 15 U/kg, then was followed for 7 days for assessment of safety.
  • 2 additional patients were enrolled and administered IV velaglucerase alfa 15 U/kg and followed for 7 days for assessment of safety.
  • the next two higher doses (30 and 60 U/kg) were administered in the same fashion.
  • an additional 9 patients were enrolled and administered the 60 U/kg dose. All patients then received the 60 U/kg dose every other week for a total of 20 doses of velaglucerase alfa.
  • PK Pharmacokinetics in humans were evaluated at Weeks 1, 3, 5, and 37/39 in TKT025. Additional PK studies were conducted at Week 65 of Study TKT025EXT. Velaglucerase alfa was rapidly cleared from blood with first-order elimination kinetics at each evaluation. Elimination half-life values for patients following initial exposure to velaglucerase alfa and following repeat administration of velaglucerase alfa were similar. In contrast, clearance and apparent volume of distribution values were reduced following repeat administration of velaglucerase alfa.
  • FIGS. 1A-1F presents the mean percent change in hematological parameters, organ volumes, and biomarkers for the Phase I/II study.
  • a marked increase in (a) hemoglobin concentration and (b) platelet count is observed during weeks 25 and 37 along with a marked reduction in (c) liver and (d) spleen volumes.
  • biomarker sampling for (e) chitotriosidase and (f) CCL18 was incomplete, a general decrease in both biomarkers relative to baseline is observed per patient over time.
  • TKT025EXT is an open-label extension study of velaglucerase alfa therapy in patients with type 1 Gaucher disease who completed study TKT025.
  • the primary objective of TKT025EXT is to evaluate the long-term safety of velaglucerase alfa when administered IV at a dose of 30 or 60 U/kg every other week for a total of 4 years.
  • the secondary objective is to continue to assess the effects of velaglucerase alfa on clinical activity in these patients as measured in hematological parameters and reductions in liver and spleen volumes. Plasma chitotriosidase and CCL18 as well as PFTs, MRI of the femoral neck and lumbar spine, skeletal survey, and bone densitometry are also being evaluated.
  • FIG. 3 shows the increase of mean hemoglobin concentration and platelet count from baseline in the patients treated with velaglucerase alfa for 60 months.
  • FIG. 4 shows the decrease of mean liver and spleen volumes from baseline in patients treated with velaglucerase alfa for 60 months.
  • Demographics and disposition Upon completion of Week 41 evaluations in TKT025, patients were eligible to participate in TKT025EXT. Of the 11 patients who completed Study TKT025, 10 patients entered Study TKT025EXT. One patient (0012) did not consent to enter the extension study because of the inconvenience of every-other-week hospital attendance, and one patient (0005) withdrew from the extension study because of pregnancy. Table 2 provides the demographic, genotypic, and clinical characteristics at baseline, as well as the clinical findings of each intent-to-treat patient at key data collection points within the extension studies.
  • Liver and spleen volumes normalized for percent of body weight were analyzed through Month 33; all 9 patients had liver data available and 8 patients had spleen data available for this assessment.
  • the mean (SE) change from pretreatment baseline in normalized liver and spleen volumes at Month 33 was ⁇ 1.5 (0.22) and ⁇ 2.8 (0.37), respectively.
  • FIG. 2 presents the mean percent change in hemoglobin concentration, platelet counts, liver volume and spleen volume that is plotted across time and demarcated for the extension studies.
  • a statistically significant change is observed from baseline to 48 months (p ⁇ 0.004) for each parameter. The most marked changes were observed for platelet count and spleen volume.
  • Hemoglobin values normalized for all patients by 24 months. Liver volumes approached normal. Continuous improvement in these clinical parameters was noted throughout the extension study ( FIG. 2 ) and normalization of hemoglobin was observed in all patients by 24 months (Table 2).
  • the mean percent change from baseline to 48 months was statistically significant (p ⁇ 0.004) for hemoglobin concentration (+21.7%), platelet counts (+157.8%), liver volume ( ⁇ 42.8%), and spleen volume ( ⁇ 79.3%).
  • influenza 8 patients, 24 events
  • arthralgia 8 patients, 21 events
  • headache (6 patients, 13 events); back pain (6 patients, 10 events); pharyngolaryngeal pain (5 patients, 7 events); abdominal pain upper (5 patients, 7 events); and gingival bleeding, pyrexia and fatigue (each 4 patients, 4 events).
  • BMD bone mineral density
  • T-scores were used to compare bone density to a “young, normal” healthy 30-year-old adult with peak bone density. Clinical bone status at baseline and 69 months was characterized according to the WHO criteria for T-scores: ⁇ 1 is normal; > ⁇ 2.5 and ⁇ 1 is osteopenia; ⁇ 2.5 is osteoporosis.
  • ITT intent-to-treat
  • Temporal changes in mean Z-scores of lumber spine and femoral neck are also shown in FIGS. 5 and 6 , respectively.
  • BMD for the intent-to-treat population improved significantly by Months 24 (LS: 0.39 (0.06, 0.72)) and Months 33 (FN: 0.39 (0.16, 0.62).
  • BMD for the patients who did not receive concomitant bisphosphonates improved significant by Months 24 (LS: 0.58 (0.08, 1.09)) and Months 33 (FN: 0.48 (0.10, 0.87)).
  • a lumbar spine slope of 0.013 increase per month corresponds to a 0.158 increase per year, and a femoral neck slope of 0.009 increase per month corresponds to a 0.103 increase per year.
  • a lumbar spine slope of 0.009 increase per month corresponds to a 0.111 increase per year, and a femoral neck slope of 0.004 increase per month corresponds to a 0.048 increase per year.
  • Skeletal pathology was measured as improvement in bone mineral density (BMD) at year 4 relative to baseline.
  • Velaglucerase alfa demonstrated efficacy in the four disease parameters studied with statistically significant and clinically meaningful improvements from baseline observed within the first six months of treatment and throughout the course of the trial and extension study.
  • all patients achieved normalization of hemoglobin level, all but one patient achieved platelet counts of greater than 100,000/mm 3 , all patients achieved near normalization in liver volumes, and all patients but one exhibited a reduction of more than 50% in spleen volume.
  • these improvements were observed throughout the duration of the studies, including the dose-reduction phase. The only patient who was returned to the original dose of 60 U/kg/every-other-week did so at 39 months secondary to bone pain following an initial dose reduction at 15 months.
  • This patient had boney destructive lesions in both her ankles at enrollment (imaging pathology could not rule out AVN) and had a prior history of osteomyelitis.
  • the principle investigator (AZ) attributed the worsening pain to the preexisting destructive lesion and prior pathology, and likely not related to the dose reduction or treatment failure.
  • FIGS. 7 and 8 show the increase of mean hemoglobin concentration and platelet count, respectively, from baseline in the patients treated with 45 U/kg or 60 U/kg velaglucerase alfa for 12 months.
  • FIGS. 9 and 10 show the decrease of mean normalized spleen volume and liver volume, respectively, from baseline in patients treated with 45 U/kg or 60 U/kg velaglucerase alfa for 12 months.
  • the primary objective of this study is to determine the efficacy of every other week dosing of velaglucerase alfa at a dose of 60 U/kg in patients with type 1 Gaucher disease as measured by increases in hemoglobin concentration.
  • the secondary objectives of this study are to evaluate the safety of every other week dosing of velaglucerase alfa at doses of 60 and 45 U/kg; to evaluate the efficacy of every other week dosing of velaglucerase alfa at a dose of 45 U/kg as measured by increases in hemoglobin concentration; to evaluate the efficacy of every other week dosing of velaglucerase alfa at doses of 60 and 45 U/kg by assessing increases in platelet counts, decreases in spleen and liver volumes, and decreases in levels of plasma chitotriosidase and Chemokine (C-C motif) ligand 18 (CCL18); to evaluate the effect of every other week dosing of velaglucerase alfa at doses of 60 and 45 U/kg on overall quality of life (QoL); and to evaluate the single- and repeat-dose pharmacokinetics of every other week dosing of velaglucerase alfa when administered at doses of 60 and 45 U/kg
  • the tertiary objectives of this study are to determine the time from Baseline to achieve a hemoglobin response, defined as an increase in hemoglobin concentration of 1 g/dL, after every other week dosing with velaglucerase alfa at doses of 60 or 45 U/kg; to evaluate the effect of every other week dosing of velaglucerase alfa at doses of 60 and 45 U/kg on pulmonary function tests (PFTs) in patients ⁇ 18 years-old; to evaluate growth velocity and Tanner staging in patients between 2 and 17 years-old; to evaluate changes in skeletal age in patients between 2 and 17 years-old by radiography of the left hand and wrist; to establish a Baseline from which to evaluate bone disease in patients between 2 and 17 years-old by magnetic resonance imaging (MRI) of the lumbar spine and femoral neck; and to establish a Baseline from which to evaluate the long-term effect of velaglucerase alfa therapy on Gaucher-related local and systemic bone disease in patients ⁇ 18 years-old by:
  • This study was comprised of 5 phases as follows: (1) Screening: Day ⁇ 21 through Day ⁇ 4; Baseline: Day ⁇ 3 through Day 0 (prior to first dose); Treatment Phase: Week 1 (Day 1; first dose) through Week 51 (a total of 26 infusions were administered per patient); End of Study Visit: Week 53; Follow-up: 30 days after the final infusion (for patients who discontinue/withdraw prior to the Week 53 evaluation, or for patients who complete this study but do not elect to enroll in the subsequent long-term clinical study).
  • Safety and efficacy assessments were made at regular intervals during the treatment period. The final assessments of safety and efficacy were made at the Week 51 and Week 53 visits. Safety was assessed throughout the study by assessments of adverse events (including infusion-related adverse events), concomitant medications, and vital signs. Additional safety assessments, including, 12-lead electrocardiograms, physical examinations, clinical laboratory tests (hematology, serum chemistry, and urinalysis), were made at Weeks 13, 25, 37, and 53. Determination of the presence of anti-velaglucerase alfa antibodies and enzyme neutralizing antibodies were conducted approximately every 6 weeks until Week 53.
  • Efficacy was assessed via hemoglobin concentration and platelet count, liver and spleen volume, and plasma chitotriosidase and CCL18 level. Additional efficacy assessments included growth velocity and Tanner staging, QoL indicators, skeletal growth and pulmonary function testing.
  • Eligible participants were males or females age ⁇ 2 years with diagnosed type 1 Gaucher disease (deficient glucocerebrosidase activity in leukocytes, or by genotype analysis), and disease-related anemia (hemoglobin levels ⁇ 1 g/dL below the local laboratory's lower limit of normal for age and gender). Participants also had 1 or more of the following: at least moderate splenomegaly (2 to 3 cm below the left costal margin) by palpation; disease-related thrombocytopenia (platelet count ⁇ 90 ⁇ 10 3 platelets/mm 3 ); or readily palpable enlarged liver. Participants could not have received treatment for Gaucher disease within 30 months prior to study entry.
  • Participants were excluded if they had a splenectomy; had (or were suspected of having) type 2 or 3 Gaucher disease; were antibody-positive or had experienced an anaphylactic shock to imiglucerase.
  • Other exclusion criteria included treatment with any non-Gaucher disease-related investigational drug or device within 30 days prior to study entry; positive test for HIV, or hepatitis B or C; exacerbated anemia (vitamin B12, folic acid, or iron deficiency-related), or any significant co-morbidity that could affect study data.
  • Pregnant or lactating women were excluded and women of child-bearing potential were required to use a medically acceptable method of contraception at all times.
  • Treatment Assignment Patients were randomized in a 1:1 ratio to receive: velaglucerase alfa 60 U/kg every other week for 51 weeks (12 patients, 26 infusions); or velaglucerase alfa 45 U/kg every other week for 51 weeks (12 patients, 26 infusions).
  • Randomization Following the completion of Baseline evaluations and confirmation of eligibility, patients were randomized in a ratio of 1:1 via a computer generated randomization schedule to receive either velaglucerase alfa 45 U/kg or 60 U/kg every other week infusions for 51 weeks.
  • Treatment Schedule Patients received their first infusion at Week 1. All patients were treated every other week for 12 months (51 weeks); therefore, a total of 26 infusions of velaglucerase alfa were administered.
  • Dose Calculation The actual dose of study drug was calculated based on the patient's weight at Baseline. A change in weight of ⁇ 5% noted at Weeks 13, 25, or 37 from the prior assessment would have required recalculation of the dose of study medication.
  • Velaglucerase alfa Administration Velaglucerase alfa was administered as a continuous IV infusion at both the 60 and 45 U/kg dose levels. All infusions were administered over a 1-hour duration. All infusions were reconstituted in 4.3 mL of preservative-free, Sterile Water for Injection, and then diluted in normal saline (0.9% sodium chloride) to yield a 100 mL total volume. Study drug infusions occurred on approximately the same day of the week but might occur every 14 days ( ⁇ 3 days) of the scheduled day in order to facilitate patient scheduling.
  • Genotyping All patients provided a blood sample at Screening for Gaucher disease genotyping and plasma chitotriosidase genotyping.
  • Vital Signs Vital signs parameters that were recorded included pulse, blood pressure, respiration rate, and temperature. The following schedule was followed for recording vital signs at infusion visits: start of infusion (within 10 minutes prior to starting the infusion), during infusion (30 minutes ( ⁇ 5 minutes)), after infusion (within 5 minutes, 30 minutes ( ⁇ 5 minutes), and 60 minutes ( ⁇ 5 minutes) after completing the infusion). At Screening, Baseline, and Week 53, vital signs were collected at one time point only.
  • Physical Examinations were performed at Screening, Baseline and at Study Weeks 13, 25, 37, and 53. Physical examinations included the following: general appearance, endocrine, head and neck, cardiovascular, eyes, abdomen, ears, genitourinary, nose, skin, throat, musculoskeletal, chest and lungs, and neurological. In addition, liver and spleen palpations were performed during Screening to confirm that the patient had moderate splenomegaly (2 to 3 cm below the left costal margin) and a Gaucher disease-related enlarged liver.
  • Height and Weight Height and weight were recorded at Baseline and at Study Weeks 13, 25, 37, and 53.
  • a 12-Lead ECG was performed at Baseline and Study Weeks 13, 25, 37, and 53.
  • Each 12-lead ECG included assessment of PR, QRS, QT, and QTc intervals, and heart rate.
  • CBC complete blood count
  • APPT activated partial thromboplastin time
  • reticulocyte count analyzed and reported by the clinical site's local laboratory
  • platelet count analyzed and reported by the clinical site's local laboratory
  • prothrombin time PT
  • Serum Chemistry Blood samples were collected for serum chemistry testing at Screening, Baseline, and at Study Weeks 13, 25, 37, and 53. The following serum chemistry parameters were evaluated: sodium, alanine aminotransferase, potassium, aspartate aminotransferase, glucose, lactate dehydrogenase, total calcium, gammaglutamyltransferase, total protein, creatinine phosphokinase, albumin, NTx*, creatinine, CTx*, urea nitrogen, folic acid (screening only), total bilirubin, vitamin B 12 (screening only), alkaline phosphatase* (* results were used for assessments of bone biomarkers).
  • Urinalysis Urine samples were collected for urinalysis at Screening, Baseline, and at Study Weeks 13, 25, 37, and 53. The following urinalysis parameters were evaluated: pH, macroscopic evaluation, microscopic evaluation.
  • Serum Anti-imiglucerase Antibody Determination All patients had a blood sample collected during Screening only for determination of serum anti-imiglucerase antibodies. Patients with a positive result were excluded from the study.
  • Serum Anti-velaglucerase alfa Antibody Determination Patients provided blood samples to measure anti-velaglucerase alfa antibodies in serum at Baseline and approximately every 6 weeks during the treatment phase (Weeks 7, 13, 19, 25, 31, 37, 43, and 49), and at Week 53. During the treatment phase, these blood samples were collected prior to the infusion.
  • Adverse Events Adverse events were monitored throughout the study from informed consent/assent through 30 days after the last infusion for patients who completed the study and did not elect to enroll in the long-term clinical study from the study prior to the Week 53 visit. For patients who completed this study and elected to enroll in the long-term clinical study, adverse events were monitored from informed consent/assent through the Week 53 visit.
  • Liver and Spleen MRI Patients had MRI of the liver and spleen at Baseline, Week 25 and Week 51. Liver and spleen size were measured using quantitative abdominal MRI.
  • Plasma Chitotriosidase Levels Blood samples (approximately 2.5 mL) were collected for the evaluation of plasma chitotriosidase levels at Baseline, Weeks 13, 25, 37, and at Week 53.
  • Plasma CCL18 Levels Blood samples (approximately 2.5 mL) were collected for the evaluation of plasma CCL18 levels at Baseline, Week 13, 25, 37 and at Week 53.
  • Skeletal Growth Patients between 2 and 17 years-old underwent radiography of the left hand and wrist at Baseline and Week 51 for evaluation of skeletal age.
  • Pulmonary Function Testing At Baseline and Week 53, patients ⁇ 18 years-old who were enrolled at study sites with the capability to perform spirometry had PFTs.
  • Lung volume and Diffusion Capacity (DL CO ) determinations were performed at the same time as spirometric testing.
  • Lung volume measurements were include Total Lung Capacity (TLC), and Residual Volume (RV), which were recorded as absolute values and % predicted of normal based on published reference values.
  • DL CO was also expressed as absolute values and % predicted of normal based on published reference values.
  • Serum samples were evaluated for the presence of administered velaglucerase alfa using a glucocerebrosidase antigen immunoassay.
  • the following PK parameters were evaluated: AUC (Area under the curve), C max (Maximum serum concentration), T max (Time to maximum serum concentration), CL (mL/min/kg) (Serum clearance, normalized for body weight), V ss (mL) (Apparent volume of distribution at steady-state), V ss (% BW) (V ss normalized for body weight), MRT (Mean residence time), and T 1/2 (Elimination half-life (analyzed with appropriate PK models)
  • Bone Biomarkers At Baseline only, patients ⁇ 18 years-old underwent DXA of the lumbar spine and femoral neck, including coronal imaging, to determine Gaucher-related local and systemic bone disease. Bone loss and demineralization were also evaluated by measuring serum alkaline phosphatase, NTx, and CTx. Results for these parameters were obtained from blood samples collected for clinical laboratory testing at Baseline only.
  • Adverse Event Definition An adverse event (AE) is any noxious, pathologic, or unintended change in anatomical, physiologic, or metabolic function as indicated by physical signs, symptoms, and/or laboratory changes occurring in any phase of a clinical trial, and whether or not considered study drug-related. This includes an exacerbation of a pre-existing condition. Adverse events were collected from informed consent/assent until 30 days after the last dose of study medication and/or until the event had been resolved/stabilized or an outcome was reached, whichever came first. For patients who completed this study and elected to enroll in the subsequent long-term clinical study, adverse events were monitored from the time the patient provided informed consent through the Week 53 visit of TKT032.
  • AEs include: worsening (change in nature, severity, or frequency) of conditions present at the onset of the study; intercurrent illnesses; drug interactions; events related to or possibly related to concomitant medications; abnormal laboratory values (this includes significant shifts from Baseline within the range of normal that the Investigator considers to be clinically important); clinically significant abnormalities in physical examination, vital signs, weight, and ECG.
  • AEs might also include unexpected laboratory values that became significantly out of range and determined to be clinically significant by the Investigator.
  • An infusion-related adverse event is defined as an adverse event that 1) begins either during or within 12 hours after the start of the infusion, and 2) is judged as possibly or probably related to study medication.
  • SAE Serious Adverse Event Definition: A serious AE (SAE) is any AE occurring at any dose that results in any of the following outcomes: death, is life-threatening, requires inpatient hospitalization, requires prolongation of existing hospitalization, a persistent or significant disability/incapacity, and a congenital anomaly/birth defect.
  • SAEs Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered as SAEs when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent one of the outcomes listed above.
  • a life-threatening AE is defined as an AE that placed the patient, in the view of the initial reporter, at immediate risk of death from the AE as it occurred (i.e., it does not include an AE that, had it occurred in a more severe form, might have caused death).
  • NCI CTC National Cancer Institute Common Toxicity Criteria
  • Clarification between Serious and Severe The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as “serious,” which is based on the outcome or action criteria usually associated with events that pose a threat to life or functioning. Seriousness (not severity) and causality serve as a guide for defining regulatory reporting obligations.
  • Adverse Event Monitoring and Period of Observation For the purposes of this study, the period of observation extended from informed consent/assent until the patient's final evaluation of the study. For safety purposes, the final evaluation was defined as the post-study safety evaluation performed approximately 30 days after the last infusion for patients who completed the study and did not elect to enroll in the long-term study. For patients who completed this study and elected to enroll in the long-term clinical study, adverse events were monitored from the time the patient provides informed consent through the Week 53 visit of TKT032.
  • Demographic and Baseline characteristics were summarized as frequencies and percentages, and data were presented using descriptive statistics. Additional analyses were conducted specifically for patients between 2 and 17 years old.
  • sample Size justification The sample size for this study was chosen to have a high power to detect a clinically significant difference in mean hemoglobin concentrations from Baseline to 12 months. A total of 12 patients per treatment arm were required for the primary analysis. This number was based on results from the Phase I/II Study TKT025, examining the within patient change from Baseline results. It was observed that at Week 25, the average hemoglobin increase from Baseline was 1.92 g/dL with a standard deviation of 0.824. The assumption was that the standard deviation of the mean change was approximately the same.
  • the primary population for analyses of efficacy data was the ITT patient population, defined as all enrolled and treated patients who receive at least one velaglucerase alfa infusion (or partial infusion). It was anticipated that attrition from the original random sample due to lack of post-Baseline data would be sufficiently small (5% or less) so as to minimize concerns regarding bias due to the exclusion of such patients.
  • the primary clinical activity variable is hemoglobin concentrations in the patients randomized to 60 U/kg of velaglucerase alfa.
  • the primary objective is to demonstrate efficacy by showing a mean change in hemoglobin from Baseline to 12 months in patients randomized to 60 U/kg of velaglucerase alfa.
  • hemoglobin values collected at Screening and Baseline were averaged to establish the Baseline used to calculate change.
  • the null hypothesis is that there will be no change from Baseline in hemoglobin concentrations to 12 months.
  • the mean difference from Baseline to 12 months was tested using a paired t-test or Wilcoxon signed rank test. A 95% confidence interval for the mean difference was also presented.
  • Secondary and tertiary clinical activity variables are: hemoglobin concentrations (change from Baseline to 12 months (Week 53) was assessed for the 45 U/kg group); platelet counts (change from Baseline to 12 months (Week 53) was assessed for both treatment groups); spleen volume (percent change from Baseline to 12 months (Week 51) was assessed for both treatment groups; in addition to observed values, spleen volumes were normalized by body weight and also presented by multiples of normal); liver volume (percent change from Baseline to 12 months (Week 51) was assessed for both treatment groups; in addition to observed values, liver volumes were normalized by body weight and also presented by multiples of normal); plasma chitotriosidase (change from Baseline to 12 months (Week 53) was assessed for both treatment groups; a statistically significant decrease expected after 12 months of treatment); plasma CCL18 (change from Baseline to 12 months (Week 53) was assessed for both treatment groups; a statistically significant decrease expected after 12 months of treatment); quality of life (SF-36 and CHQ) (
  • the null hypothesis is that there will be no change from Baseline to 12 months (Week 51 or Week 53) for each treatment group.
  • platelet values collected at Screening and Baseline were averaged to establish the Baseline used to calculate change for both the treatment groups.
  • the mean difference from Baseline to 12 months (Week 51 or Week 53) was tested using a paired t-test or Wilcoxon signed rank test. A 95% confidence interval for the mean difference was also presented.
  • Safety Analyses All patients who received at least one dose of study drug (or partial dose) were assessed for clinical safety and tolerability. No formal statistical tests were performed on the safety parameters. Vital signs, 12-lead ECG, clinical chemistry, hematology, and urinalysis safety monitoring were listed for each patient and abnormal values were flagged. For categorical variables, such as AEs, the number and percentage of patients experiencing each AE were tabulated. AEs were summarized by severity of event. The number and percentage of patients experiencing drug related AEs as well as AEs that were not considered related to study drug were also displayed.
  • Pharmacokinetic Analyses The single- and repeat-dose pharmacokinetic profiles for velaglucerase alfa were established by analyzing standard PK parameters at Week 1 and Week 37, respectively.
  • Velaglucerase alfa was generally well tolerated with no drug-related serious AEs, and no patient withdrew due to an AE.
  • the most common AEs were headache, nasopharyngitis, injury, arthralgia, cough, and pyrexia.
  • a single patient developed antibodies.
  • HGT-GCB-039 (9M, 60 U/kg Velaglucerase Alfa or Imiglucerase)
  • This example describes a multicenter, Phase III, randomized, double-blind, parallel-group study designed to compare the safety and efficacy of the enzyme replacement therapy velaglucerase alfa with imiglucerase in the treatment of patients with type 1 Gaucher disease.
  • splenectomized patient(s) excluded (10 velaglucerase alfa 60 U/kg; 10 imiglucerase 60 U/kg).
  • c Based on a mixed model adjusting for age at informed consent and baseline values.
  • d There are 13 patient(s) deficient in chitotriosidase activity excluded (7 velaglucerase alfa 60 U/kg; 6 imiglucerase 60 U/kg).
  • the primary objective of this study was to compare the effects of velaglucerase alfa and imiglucerase on hemoglobin concentration in patients with type 1 Gaucher disease.
  • the tertiary objectives of this study are: to evaluate the effects of velaglucerase alfa and imiglucerase on growth velocity and Tanner staging in patients between 2 and 17 years-old; to evaluate the effects of velaglucerase alfa and imiglucerase on changes in skeletal age in patients between 2 and 17 years-old by radiography of the left hand and wrist; to evaluate the effects of velaglucerase alfa and imiglucerase on changes in overall QoL, as measured by the SF-36 for patients ⁇ 18 years of age and the CHQ PF-50 for patients 5 to 17 years-old; to evaluate the effects of velaglucerase alfa and imiglucerase on immune and inflammatory responses in patients ⁇ 18 years of age as measured by selected cytokine assessments (TNF- ⁇ , IL6, IL1b, IL8, IL13, CD14, and GM-CSF); to establish a baseline from which to evaluate bone disease in patients between 2 and 17 years-old by MRI of the lumbar spine and
  • the primary endpoint of this study is to measure the mean change from Baseline to week 41/End of Study (EOS) in hemoglobin concentration between the two treatment groups.
  • EOS End of Study
  • the secondary endpoints of this study are: to evaluate the safety of velaglucerase alfa and imiglucerase, as assessed by adverse events and infusion-related adverse events (and the proportion of patients requiring premedication use to manage infusion-related adverse events), clinical laboratory values, vital signs, 12-lead electrocardiograms (ECG), antibody formation and enzyme neutralizing antibody activity; to compare the mean and percent changes from Baseline in platelet count between treatment groups; to compare the mean and percent changes from Baseline in liver and spleen volumes by MRI between treatment groups; to compare the mean and percent changes from Baseline in plasma chitotriosidase and plasma CCL18 levels between treatment groups; and to compare time to response for hemoglobin concentration (defined as a ⁇ 1 g/dL improvement in hemoglobin levels relative to Baseline) between treatment groups.
  • Efficacy was assessed via hemoglobin concentration and platelet count, liver and spleen volume, and plasma chitotriosidase and CCL18 level. Additional efficacy assessments included growth velocity and Tanner staging, QoL indicators, skeletal growth. Immune and inflammatory response (as measured by selected cytokine parameters) was measured in patients who are ⁇ 18 years of age at study entry. The duration of treatment in this study was 39 weeks and the duration of patient participation in this study was up to 11 months (from Screening through follow-up). Patients who completed this study were provided the opportunity to enroll in a subsequent open-label long-term clinical study, in which all patients would receive velaglucerase alfa.
  • Treatment Assignment Patients were randomized in a 1:1 ratio prior to administration of the first dose of: velaglucerase alfa 60 U/kg every other week for 39 Weeks (up to 16 patients, 20 infusions), or imiglucerase 60 U/kg every other week for 39 Weeks (up to 16 patients, 20 infusions). All study medication was administered by IV infusion over 1 hour to maintain the treatment blind.
  • Study Medication Infusions Double-blind study medication infusions were administered at the clinical site as a continuous 1-hour IV infusion to maintain the treatment blind. Study medication infusions occurred on approximately the same day of the week but occurred every 14 days ( ⁇ 3 days) in order to facilitate patient scheduling.
  • Study Entrance Criteria At Screening patients were reviewed for eligibility against the study entrance criteria. Patients who did not meet the study entrance criteria were considered Screen failures.
  • Genotyping All patients provided a blood sample at Screening for Gaucher disease genotyping and plasma chitotriosidase genotyping.
  • Vital Signs Vital signs parameters that were recorded included pulse, blood pressure, respiration rate, and temperature. The following schedule was followed for recording vital signs at infusion visits: start of infusion (within 10 minutes prior to starting the infusion), during infusion (30 minutes ( ⁇ 5 minutes)), after infusion (within 5 minutes, 30 minutes ( ⁇ 5 minutes), and 60 minutes ( ⁇ 5 minutes) after completing the infusion). At Screening, Baseline, and Week 41, vital signs were collected at one time point only.
  • Physical Examinations were performed at Screening, Baseline and at Study Weeks 13, 25, and 41. Physical examinations included the following: general appearance, endocrine, head and neck, cardiovascular, eyes, abdomen, ears, genitourinary, nose, skin, throat, musculoskeletal, chest and lungs, and neurological. Any abnormal change in physical findings was recorded as an adverse event on the appropriate CRF page(s).
  • Height and Weight Height and weight were recorded at Baseline and at Study Weeks 13, 25, and Week 41. For pediatric patients (i.e., 2 to 17 years-old), height and weight assessments were used to determine growth velocity.
  • a 12-lead Electrocardiograms A 12-lead ECG was performed at Baseline and at Study Weeks 13, 25, and 41, and included assessment of PR, QRS, QT, and QTc intervals, and heart rate.
  • Hematology Blood samples were collected during Screening and at Baseline to measure hemoglobin levels for statistical analysis. Blood samples were also collected at Screening, Baseline, and Weeks 13, 25, and 41 for complete hematology testing. The following hematology parameters were evaluated: complete blood count (CBC) with differential, platelet count, activated partial thromboplastin time (aPPT), reticulocyte count (analyzed and reported by the clinical site's local laboratory), and prothrombin time (PT). Blood samples were collected at Screening, Baseline, and at every study visit (except at the Week 1 visit) to measure hemoglobin concentration and platelet count.
  • CBC complete blood count
  • aPPT activated partial thromboplastin time
  • reticulocyte count analyzed and reported by the clinical site's local laboratory
  • PT prothrombin time
  • Serum Chemistry Blood samples were collected for serum chemistry testing at Screening, Baseline, and at Study Weeks 13, 25, and 41. The following serum chemistry parameters were evaluated: sodium, alanine aminotransferase, potassium, aspartate aminotransferase, glucose, lactate dehydrogenase, total calcium, gammaglutamyltransferase, total protein, creatinine phosphokinase, albumin, NTx*, creatinine, CTx*, urea nitrogen, folic acid (to determine study eligibility), total bilirubin, vitamin B 12 (screening only), alkaline phosphatase* (* results were used for assessments of bone biomarkers). Patients who at Screening had folic acid and/or vitamin B 12 deficiency-related anemia, and so did not meet study entry criteria were considered a screen failure.
  • Urinalysis Urine samples were collected for urinalysis at Screening, Baseline, and at Study Weeks 13, 25, and 41. The following urinalysis parameters were evaluated: pH, microscopic evaluation, and macroscopic evaluation.
  • Serum Anti-velaglucerase alfa Antibodies Patients provided blood samples to measure anti-velaglucerase alfa antibodies in serum at Screening and approximately every 6 weeks during the treatment phase (Weeks 7, 13, 19, 25, 31, and 37), and at Week 41. During the treatment phase, these blood samples were collected prior to the infusion of double-blind study medication.
  • Plasma samples collected for anti-velaglucerase alfa antibody determination were evaluated. These samples were screened using an enzyme-linked immunosorbence assay (ELISA), and all positive samples were confirmed positive using a radioimmunoprecipitaion assay (RIP). Positive samples were isotyped (IgG, IgA, IgM, or IgE). In addition, positive samples were tested for enzyme neutralizing activity using an in vitro assay.
  • ELISA enzyme-linked immunosorbence assay
  • RIP radioimmunoprecipitaion assay
  • Serum Anti-imiglucerase Antibody Determination Patients provided blood samples at Screening to measure anti-imiglucerase antibodies. The anti-imiglucerase antibody analyses were performed using the same samples obtained for anti-velaglucerase alfa antibody analyses.
  • Antibody cross-reactivity testing (to velaglucerase alfa) was conducted for patients who develop anti-imiglucerase antibodies during this study.
  • Immune and Inflammatory Response Testing Patients who were ⁇ 18 years of age provided blood samples for immune and inflammatory response testing at Baseline and at Weeks 13, 25, and 41. One sample was obtained at Baseline. At Weeks 13, 25, and 41, samples were obtained before, immediately following, and 1 hour after each infusion with study drug.
  • Adverse Events Adverse events were monitored throughout the study from the time the patient provided informed consent through 30 days after the last infusion for patients who completed the study and did not elect to enroll in the subsequent open-label long-term clinical study, or for patients who discontinue or withdraw from the study prior to the Week 41 visit. For patients who completed this study and elected to enroll in the subsequent open-label long-term clinical study, adverse events were monitored from informed consent through completion of the Week 41 visit.
  • Infusions of proteins can be associated with reactions to the infusion.
  • An infusion-related adverse event is defined as an adverse event that 1) begins either during or within 12 hours after the start of the infusion, and 2) is judged as possibly or probably related to blinded study medications.
  • Liver and Spleen MRI Patients underwent MRI of the liver and spleen at Baseline and at Weeks 25 and 41/EOS. Liver and spleen size were measured using quantitative abdominal MRI.
  • Plasma Chitotriosidase Levels Blood samples were collected for the evaluation of plasma chitotriosidase levels at Baseline and at Weeks 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, and Week 41. Chitotriosidase was analyzed using an enzyme activity assay.
  • Plasma CCL18 Levels Blood samples were collected for the evaluation of plasma CCL18 levels at Baseline and at Weeks 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, and Week 41. CCL18 levels were measured by an enzyme-linked immunosorbent assay (ELISA) in a commercially available kit.
  • ELISA enzyme-linked immunosorbent assay
  • Skeletal Growth Patients between 2 and 17 years-old underwent radiography of the left hand and wrist at Baseline and Week 41 for evaluation of skeletal age.
  • patients might receive corticosteroids as premedications to mitigate potential infusion-related adverse events.
  • Adverse Event Definition An adverse event (AE) is any noxious, pathologic, or unintended change in anatomical, physiologic, or metabolic function as indicated by physical signs, symptoms, and/or laboratory changes occurring in any phase of a clinical trial, and whether or not considered study drug-related. This includes an exacerbation of a pre-existing condition. Adverse events were collected from the time the patient provides signed informed consent until 30 days after the last dose of blinded study medication and/or until the event had been resolved/stabilized or an outcome was reached, whichever comes first. For patients who discontinued or were withdrawn prior to the Week 41 visit, AEs were followed up to 30 days after their last infusion. For patients who completed this study and elected to enroll in the long-term clinical study, adverse events were monitored from the time the patient provides informed consent through the Week 41 visit.
  • AEs include: worsening (change in nature, severity, or frequency) of conditions present at the onset of the study; intercurrent illnesses; drug interactions; events related to or possibly related to concomitant medications; abnormal laboratory values (this includes significant shifts from Baseline within the range of normal that the Investigator considers to be clinically important); clinically significant abnormalities in physical examination, vital signs, weight, and ECG.
  • AEs might also include unexpected laboratory values that became significantly out of range and determined to be clinically significant by the Investigator. In the event of an unexpected out-of-range value, the laboratory test was repeated until it returned to normal or could be explained and the patient's safety was not at risk.
  • An infusion-related adverse event was defined as an adverse event that 1) begins either during or within 12 hours after the start of the infusion, and 2) is judged as possibly or probably related to blinded study medication.
  • SAE Serious Adverse Event Definition: A serious AE (SAE) is any AE occurring at any dose that results in any of the following outcomes: death, is life-threatening, requires inpatient hospitalization, requires prolongation of existing hospitalization, a persistent or significant disability/incapacity, and a congenital anomaly/birth defect.
  • SAEs Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered as SAEs when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent one of the outcomes listed above.
  • a life-threatening AE is defined as an AE that placed the patient, in the view of the initial reporter, at immediate risk of death from the AE as it occurred (i.e., it does not include an AE that, had it occurred in a more severe form, might have caused death).
  • NCI CTC National Cancer Institute Common Toxicity Criteria
  • Probably related is defined as a clinical event/laboratory abnormality with a reasonable time sequence to administration of study drug, unlikely to be attributable to concurrent disease or other drugs and chemicals and which follows a clinically reasonable response on dechallenge.
  • the association of the clinical event/laboratory abnormality must also have some biologic plausibility, at least on theoretical grounds.
  • Clarification between Serious and Severe The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as “serious,” which is based on the outcome or action criteria usually associated with events that pose a threat to life or functioning. Seriousness (not severity) and causality serve as a guide for defining regulatory reporting obligations.
  • Adverse Event Monitoring and Period of Observation For the purposes of this study, the period of observation extended from the time the patient provided informed consent until the patient's final evaluation of the study. For safety purposes, the final evaluation was defined as the post-study safety evaluation performed approximately 30 days after the last infusion for patients who completed the study and did not elect to enroll in the long-term study, or for patients who discontinued or withdrew from the study prior to the Week 41 visit. For patients who completed this study and elected to enroll in the long-term clinical study, adverse events were monitored from the time the patient provides informed consent through the Week 41 visit. If the Investigator considered it necessary to report an AE in a study patient after the end of the observation period, he or she would contact the Sponsor to determine how the AE should be documented and reported.
  • tabular summaries consisted of n, mean, standard deviation, minimum, maximum, and median. Graphs of the key efficacy variables were presented by treatment groups. For categorical variables, tabular summaries consisted of presenting the frequency and the percentage in each category by treatment group.
  • the primary efficacy variable was presented by treatment group, and included: raw values: the untransformed value of the variable in the originally reported scale; the absolute change in the value from Baseline, i.e., X ⁇ B (where B is the Baseline value and X is a post-Baseline value); and the percent change in the value from Baseline, i.e., 100*(X ⁇ B)/B (where B is the Baseline value and X is a post-Baseline value).
  • the null hypothesis for the primary efficacy endpoint is that the mean change in hemoglobin concentration from Baseline to Week 41 for velaglucerase alfa is at least 1 g/dL inferior to the mean change in hemoglobin concentration from Baseline to Week 41 for imiglucerase.
  • the hypothesis to be tested can be stated as:
  • H 0 velaglucerase alfa is inferior with respect to the mean hemoglobin response
  • H A velaglucerase alfa is non-inferior with respect to the mean hemoglobin response
  • sample Size Justification When the sample size in each treatment group is 14, a two-group 0.025 one-sided t-test will have an 80% power to reject the null hypothesis that the difference in means for hemoglobin is ⁇ 1 g/dL in favor of the alternative hypothesis that the difference in means is greater than ⁇ 1, assuming that the expected difference in means is 0, and the common standard deviation is 0.90.
  • the primary efficacy endpoint is the mean change from Baseline to Week 41 in hemoglobin concentration between the two treatment groups.
  • the primary analysis was carried out using the ITT population. This is a non-inferiority randomized controlled trial designed to demonstrate that velaglucerase alfa is non-inferior to imiglucerase in terms of efficacy in treating patients with type 1 Gaucher disease.
  • a one-sided 97.5% confidence interval was used. Non-inferiority was demonstrated by either a one-sided confidence interval or a hypothesis test for testing the null hypothesis that the treatment difference is less than or equal to the lower equivalence margin in hemoglobin ( ⁇ 1 g/dL) versus the alternative that imiglucerase treatment difference is greater than the lower equivalence margin.
  • a one-sided 97.5% confidence interval [(a, ⁇ )]
  • Kaplan-Meier (product limit) survival curves were presented for each treatment group and a log-rank test were used to compare curves between treatment groups. The median time and 95% confidence interval were presented for each treatment group. Patients who did not experience the event by the end of the study (i.e., by Week 41) were censored at Week 41. Patients who withdrew or were discontinued prior to the Week 41 evaluation and who did not achieve a response at the time of withdrawal or discontinuation were censored at the time of the last known evaluation for that patient. In addition, the proportion of patients who responded versus non-responders were presented and compared between treatment groups using Fisher's Exact Test.
  • This example describes a global, open-label, 12-month study to examine the safety and efficacy of velaglucerase alfa in patients with type 1 Gaucher disease previously receiving imiglucerase. Patients aged ⁇ 2 years received velaglucerase alfa at a dose equal to their prior imiglucerase dose, with infusions administered over 1 hour every other week.
  • Velaglucerase alfa was generally well tolerated with most adverse events (AEs) of mild or moderate severity.
  • the primary objective of this study was to evaluate the safety of every other week dosing of velaglucerase alfa in patients with type 1 Gaucher disease who were previously treated with imiglucerase.
  • the secondary objectives were: to evaluate changes from Baseline in hemoglobin concentration after every other week dosing of velaglucerase alfa, to evaluate changes from Baseline in platelet count after every other week dosing of velaglucerase alfa, and to evaluate changes from Baseline in liver and spleen volume by abdominal MRI after every other week dosing of velaglucerase alfa.
  • the tertiary/exploratory objectives were: to evaluate changes from Baseline in levels of plasma chitotriosidase and Chemokine (C-C motif) ligand 18 (CCL18) after every other week dosing of velaglucerase alfa, to evaluate changes in skeletal age in patients between 2 and 17 years-old by radiography of the left hand and wrist after every other week dosing of velaglucerase alfa, to evaluate changes in growth velocity and Tanner staging in patients between 2 and 17 years-old after every other week dosing of velaglucerase alfa, to establish a Baseline from which to monitor the long term effect of velaglucerase alfa therapy on Gaucher-related local and systemic bone disease in patients 18 years-old, as measured by bone density (DXA) of the lumbar spine and femoral neck (including coronal imaging), serum alkaline phosphatase, N-telopeptide cross-links (NTx), and C-telopeptide cross links (
  • the study was comprised of 5 phases as follows: Screening (Day ⁇ 14 through Day ⁇ 4), Baseline (Day ⁇ 3 through Day 0 (prior to first dose)), Treatment Phase: Week 1 (Day 1; first dose) through Week 51 (a total of 26 infusions were administered per patient), End of Study Visit: Week 53, Follow-up: 30 days after the final infusion (for patients who discontinued/withdrew prior to the Week 53 evaluation, or for patients who completed this study but did not elect to enroll in the subsequent long-term clinical study).
  • Week 1 (Day 1).
  • Velaglucerase alfa infusions were administered every other week for 12 months (51 weeks) for a total of 26 infusions.
  • Patients received the same number of units of velaglucerase alfa as their imiglucerase dose.
  • Doses ranged between 15 U/kg and 60 U/kg.
  • Infusion time was 60 minutes (1 hour).
  • Increased infusion durations (e.g., 2 hours) were documented in the source documentation and appropriate CRF. Infusions were not less than 1 hour in duration.
  • the first 3 velaglucerase alfa infusions for each patient were administered at the clinical site. Patients who did not experience a treatment-related serious adverse event or a velaglucerase alfa infusion-related adverse event might receive their subsequent infusions at home by qualified and trained medical personnel, per the discretion and direction of the Investigator. Patients who experienced an infusion-related adverse event might be re-evaluated at a later time point during the study for consideration to transition to home infusions. Patients receiving velaglucerase alfa as home therapy were required to return to the clinical site at Weeks 7, 13, 19, 25, 31, 37, 45, and 51 and 53.
  • the study completion visit is defined as Week 53. Patients were considered to have completed this study once they have 1) completed the 51-week treatment period, and 2) complete the study visits at Week 51 and Week 53.
  • Eligible participants were males or females age ⁇ 2 years with diagnosed type 1 Gaucher disease (deficient glucocerebrosidase activity in leukocytes, or by genotype analysis), who had received consistent treatment with imiglucerase for a minimum of 30 consecutive months; one patient was allowed to participate having had 22 consecutive months of previous treatment with imiglucerase.
  • diagnosed type 1 Gaucher disease deficient glucocerebrosidase activity in leukocytes, or by genotype analysis
  • Participants were excluded if they had both hemoglobin concentration ⁇ 10 g/dL and platelet count ⁇ 80 ⁇ 10 3 platelets/mm 3 ; had unstable hemoglobin concentration (exceeded a range of ⁇ 1 g/dL of the screening value) or platelet count (exceeded ⁇ 20% of the screening value) during the 6 months prior to screening; had (or were suspected of having) type 2 or 3 Gaucher disease; had experienced an anaphylactic shock to imiglucerase; had inconsistent treatment with imiglucerase or had received miglustat in the 6 months prior to study entry; or had radiologically-confirmed active, clinically significant spleen infarction or worsening bone necrosis within 12 months of screening.
  • exclusion criteria included treatment with any investigational drug or device within 30 days prior to study entry; positive test for HIV, hepatitis B or C; non-Gaucher disease-related anemia at screening; or any significant co-morbidity that could affect study data.
  • Pregnant or lactating women were excluded and women of child-bearing potential were required to use a medically acceptable method of contraception at all times.
  • Treatment Assignment Patients received velaglucerase alfa infusions every other week at the same number of units of as their imiglucerase dose. The patient's current imiglucerase dose was recorded at Baseline. Velaglucerase alfa doses ranged between 15 U/kg and 60 U/kg.
  • Treatment Schedule Patients received their first infusion on Week 1 (Day 1). All patients received velaglucerase alfa once every other week for 12 months (51 weeks); therefore, a total of 26 infusions are to be administered.
  • velaglucerase alfa All doses of velaglucerase alfa were administered as continuous IV infusions at a maximum rate of 1 U/kg/minute. Infusion time was 60 minutes (1 hour). Increased infusion durations (e.g., 2 hours) were documented in the source documentation and appropriate CRF. Infusions might not be less than 1 hour in duration. Patients received their final imiglucerase dose a maximum of 30 days prior to study entry and a minimum of 14 days prior to study entry.
  • Dose Calculation A change in weight of >5% from Baseline or the previously recorded weight used to calculate dose at Weeks 13, 25, or 37 required recalculation of the dose of study medication.
  • Dose Adjustments Patients were monitored throughout the treatment period for changes in clinical parameters (i.e., hemoglobin concentration, platelet count, and liver and spleen volume). If a patient demonstrated a clinically significant change in these parameters the investigator evaluated the option of increasing the patient's dose by 15 U/kg.
  • clinical parameters i.e., hemoglobin concentration, platelet count, and liver and spleen volume.
  • a dose adjustment was considered if two or more of the following four criteria were met and consistent over two consecutive evaluations: decrease from Baseline in hemoglobin concentration of >1 g/dL; a decrease from Baseline in platelet count of >20%; an increase in liver volume as indicated by organ palpation and confirmed to be >15% relative to Baseline as measured by MRI; and an increase in spleen volume as indicated by organ palpation and confirmed to be >15% relative to Baseline as measured by MRI.
  • Velaglucerase alfa was administered intravenously. Study drug infusions occurred on approximately the same day of the week but might occur every 14 days ( ⁇ 3 days) of the target day in order to facilitate patient scheduling. If at all possible, missed infusions should be avoided. If a patient was not dosed within 17-days from their scheduled dose, the patient would receive the next infusion as soon as possible after approval for the patient to continue in the study. It might be acceptable to give the next infusion as early as 7 days after the previous infusion. Subsequent infusions would return to the original schedule.
  • the first three velaglucerase alfa infusions were administered at the clinical site. After the first three doses, patients who had not experienced a treatment-related serious adverse event or an infusion-related adverse event might receive their subsequent infusions at home. Patients who had experienced an infusion-related adverse event might be re-evaluated at a later time point during the study for consideration to transition to home infusions. Patients receiving velaglucerase alfa as home therapy were required to return to the clinical site at Weeks 7, 13, 19, 25, 31, 37, 45, 51, and 53.
  • An infusion-related adverse event was defined as an adverse event that 1) begins either during or within 12 hours after the start of the infusion, and 2) is judged as possibly or probably related to study drug.
  • Velaglucerase alfa is a lyophilized product that was supplied and shipped by a qualified distributor to the clinical study site to be stored at 2 to 8° C.
  • Gaucher Disease Specific Treatment History At Screening, all Gaucher disease-specific treatments, including the patient's current imiglucerase dose, were recorded. The patient's initial velaglucerase alfa dose was based on the current imiglucerase dose recorded.
  • Gaucher Disease and Chitotriosidase Genotyping At Screening only, all patients had a blood sample collected for Gaucher disease and plasma chitotriosidase genotyping.
  • Vital Signs Vital signs parameters recorded included pulse, blood pressure, respiration rate, and temperature.
  • Physical Examinations were performed at the Baseline visits, and at Weeks 13, 25, 37, 51 and 53. Physical examinations included the following: general appearance, endocrine, head and neck, cardiovascular, eyes, abdomen, ears, genitourinary, nose, skin, throat, musculoskeletal, chest and lungs, and neurological. Any abnormal change in physical findings was recorded as an adverse event on the appropriate CRF page(s).
  • Height and Weight Height and weight were recorded at the Baseline visit, and at Weeks 13, 25, 37 and 51. Growth velocity was calculated using height and weight measurements, and correlated with Tanner staging.
  • a 12-Lead ECG was performed at the Baseline visit, and at Weeks 13, 25, 37 and 51.
  • Each 12-lead ECG included assessment of PR, QRS, QT, and QTc intervals, and heart rate.
  • Hematology Blood samples were collected for hematology testing at the Screening and Baseline visits, and at Weeks 7, 13, 19, 25, 31, 37, 45, 51 and 53. The following hematology parameters were evaluated: complete blood count (CBC) with differential, activated partial thromboplastin time (aPPT), reticulocyte count (performed by the site's local laboratory), platelet count, and prothrombin time (PT).
  • CBC complete blood count
  • APPT activated partial thromboplastin time
  • reticulocyte count performed by the site's local laboratory
  • platelet count platelet count
  • prothrombin time prothrombin time
  • Serum Chemistry Blood samples were collected for serum chemistry testing at the Baseline visit, and at Weeks 13, 25, 37, 51 and 53.
  • the following serum chemistry parameters were evaluated: sodium, total bilirubin, potassium, alkaline phosphatase*, glucose, alanine aminotransferase, total calcium, aspartate aminotransferase, total protein, lactate dehydrogenase, albumin,
  • Urinalysis Urine samples were collected for urinalysis at the Baseline visit, and at Weeks 13, 25, 37, 51 and 53. The following urinalysis parameters were evaluated: pH, microscopic evaluation, and macroscopic evaluation.
  • Serum Anti-imiglucerase Antibody Determination All patients had a blood sample at Baseline only for determination of serum anti-imiglucerase antibodies. Patients were eligible for enrollment in this study regardless of their anti-imiglucerase antibody status. Patients who were anti-imiglucerase antibody positive would be allowed to enter this study. These blood samples were evaluated to determine the presence of anti-imiglucerase antibodies.
  • Serum Anti-velaglucerase alfa Antibody Determination Blood samples were collected for determination of anti-velaglucerase alfa antibodies at the Baseline visit, and at Weeks 7, 13, 19, 25, 31, 37, 45 and 51. Blood samples collected for anti-velaglucerase alfa antibody determination were evaluated. These samples were screened using an enzyme-linked immunosorbence assay (ELISA).
  • ELISA enzyme-linked immunosorbence assay
  • Adverse Events Adverse events were monitored throughout the study from the time the patient provides signed informed consent through 30 days after their last infusion for patients who completed the study and did not elect to enroll in the subsequent long-term open-label clinical study, or for patients who discontinued or withdrew from the study prior to the Week 53 visit. For patients who completed this study and elected to enroll in the subsequent long-term open-label clinical study, adverse events were monitored from informed consent through the Week 53 visit of this study (TKT034).
  • Hemoglobin concentration was measured at the time points described herein. The change from Baseline to 12 months in hemoglobin concentration was a secondary endpoint of this study.
  • Platelet count was measured at the time points described herein. The change from Baseline to 12 months in platelet count was a secondary endpoint of this study.
  • Liver and Spleen Volumes Measured by Abdominal MRI Patients underwent quantitative abdominal MRI of the liver and spleen at Baseline, Week 25, and Week 51. The change from Baseline to 12 months in liver and spleen volume was a secondary endpoint of this study.
  • Plasma Chitotriosidase and CCL19 Levels Blood samples were collected for the evaluation of plasma chitotriosidase and CCL18 levels at the Baseline visit, and at Weeks 13, 25, 37, 51 and 53. The change from Baseline to 12 months in chitotriosidase and CCL18 levels was a tertiary endpoint of this study.
  • Growth Velocity and Tanner Staging For patients 2 to 17 years-old, growth was assessed at the time points defined herein. Growth velocity was calculated using height and weight measurements recorded at regular time points during this study, and correlated with Tanner staging. The change from Baseline to 12 months in growth velocity and Tanner staging was a tertiary endpoint of this study.
  • Skeletal Growth Patients between 2 and 17 years-old underwent radiography of the left hand and wrist at Baseline and Week 51 for evaluation of skeletal age. The change from Baseline to 12 months in skeletal growth in patients 2 to 17 years of age was a tertiary endpoint of this study.
  • Bone Biomarkers At Baseline only, patients ⁇ 18 years-old underwent DXA of the lumbar spine and femoral neck, including coronal imaging, to determine Gaucher-related local and systemic bone disease. Bone loss and demineralization were also evaluated by measuring serum alkaline phosphatase, NTx, and CTx. Results for these parameters were obtained from blood samples collected for clinical laboratory testing at Baseline only.
  • Adverse Event Definition An adverse event (AE) is any noxious, pathologic, or unintended change in anatomical, physiologic, or metabolic function as indicated by physical signs, symptoms, and/or laboratory changes occurring in any phase of a clinical study, and whether or not considered study drug-related. This includes an exacerbation of a pre-existing condition. Adverse events were collected from informed consent until 30 days after the last dose of study drug and/or until the event had been resolved/stabilized or an outcome was reached, whichever came first. For patients who discontinued or were withdrawn prior to the Week 53 visit, AEs were followed up to 30 days after their last infusion of velaglucerase alfa.
  • AEs include: worsening (change in nature, severity, or frequency) of conditions present at the onset of the study; intercurrent illnesses; drug interactions; events related to or possibly related to concomitant medications; abnormal laboratory values (this includes significant shifts from Baseline within the range of normal that the Investigator considers to be clinically important); clinically significant abnormalities in physical examination, vital signs, weight, and ECG.
  • AEs might also include unexpected laboratory values that become significantly out of range and determined to be clinically significant by the Investigator. In the event of an unexpected out-of-range value, the laboratory test would be repeated until it returns to normal or can be explained and the patient's safety is not at risk.
  • Infusion-Related Adverse Event Definition An infusion-related adverse event was defined as an adverse event that 1) begins either during or within 12 hours after the start of the infusion, and 2) is judged as possibly or probably related to study drug. Other AEs which occurred prior to the infusion, along with AEs associated with protocol-defined testing and assessments (e.g., laboratory testing, ECGs, and physical examinations) which were performed prior to the infusion, was not be defined as infusion-related adverse events. Infusion-related adverse events were managed as defined above.
  • SAE Serious Adverse Event Definition: A serious AE (SAE) is any AE occurring at any dose that results in any of the following outcomes: death, is life-threatening, requires inpatient hospitalization, requires prolongation of existing hospitalization, a persistent or significant disability/incapacity, and a congenital anomaly/birth defect.
  • SAEs Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered as SAEs when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent one of the outcomes listed above.
  • a life-threatening AE is defined as an AE that placed the patient, in the view of the initial reporter, at immediate risk of death from the AE as it occurred (i.e., it does not include an AE that, had it occurred in a more severe form, might have caused death).
  • NCI CTC National Cancer Institute Common Toxicity Criteria
  • Clarification between Serious and Severe The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as “serious,” which is based on the outcome or action criteria usually associated with events that pose a threat to life or functioning. Seriousness (not severity) and causality serve as a guide for defining regulatory reporting obligations.
  • Adverse Event Monitoring and Period of Observation For the purposes of this study, the period of observation extended from the time the patient provides signed informed consent until the patient's final evaluation of the study. For safety purposes, the final evaluation was defined as the post-study safety evaluation performed approximately 30 days after the last infusion for patients who completed the study and did not elect to enroll in the subsequent long-term open-label clinical study, or for patients who discontinued or withdrew from the study prior to Week 53. For patients who elected to enroll in the subsequent long-term open-label clinical study, adverse events were monitored until Week 53; adverse events that had not resolved as of the Week 53 visit for this study were recorded in the patient's medical history for the subsequent long-term open-label clinical study.
  • ITT intent-to-treat
  • Statistical data analyses were performed on the ITT population. Continuous data collected at Baseline and at subsequent study visits were summarized using descriptive statistics (m, mean, median, minimum, maximum, and standard deviation). Categorical data were summarized as frequencies and percentages. Descriptive statistics were presented for all patients in the ITT population according to demographic and Baseline characteristics.
  • the inclusion of at least 26 patients provided basic information on safety and tolerability. From the TKT025 study, the average change at 6 months across patients does not indicate any worsening of adverse events (AEs) from Baseline. Some patients will have their AEs resolved, some patients a worsening of their AEs. Assuming the natural variability of the patients, then the chance of seeing a worsening for a single patient is 11%. This 11% failure rate is for one patient. The likelihood that all 26 patients in the trial will not have an SAE is equal to the likelihood of patient 1 not having an SAE multiplied by the likelihood of patient 2 not having an SAE multiplied by ( . . . etc. . . . )
  • the null hypothesis is that the mean changes from Baseline (i.e., the end of imiglucerase treatment) to Month 12 for each of the selected clinical parameters (hemoglobin concentration, platelet count, liver and spleen volumes) are within the pre-specified clinically significantly values.
  • Clinically significant changes from Baseline to Week 53 for hemoglobin was defined as a change of no less than 1 gm/dL, and a 20% change in platelet count.
  • the changes from Baseline to Week 53 were defined as being no more than 15% increase.
  • the sample size estimate of 26 patients was based on a paired t-test of means, with a standard deviation of 0.671 with a two-sided alpha level of 0.05, and 80% power.
  • the primary clinical variable was to evaluate the safety of velaglucerase alfa administered every other week to patients with type I Gaucher disease who were clinically stable on imiglucerase. Safety was evaluated by assessing vital signs and documenting adverse events (by type, frequency, and severity) at each study visit, as well as by performance of physical examinations and changes in laboratory assessments at required visits.
  • AEs were coded using MedDRA Coding Dictionary. AE summaries in general were based on all AEs occurring after the patient's first infusion of study drug (treatment-emergent).
  • Secondary Analyses Secondary endpoints of this study are: change from Baseline to 12 months in hemoglobin concentration; change from Baseline to 12 months in platelet count; change from Baseline to 12 months in spleen volume by abdominal MRI (evaluated as % change) (Spleen volumes were normalized by body weight); and change from Baseline to 12 months in liver volume by abdominal MRI (evaluated as % change) (Liver volumes were normalized by body weight).
  • the alternative hypothesis is that the mean change from Baseline (i.e., the end of imiglucerase treatment) to Month 12 was within the specified clinically significant levels for the parameters to be evaluated (where the population mean change from Baseline for hemoglobin is within 1 g/dL, the platelet count is within 20%, and the liver and spleen volumes are within 15%. This was evaluated using a 2-sided 90% confidence interval for the true difference from Baseline for these clinical parameters. For example, efficacy of velaglucerase alfa was concluded if the confidence interval for the change from Baseline of hemoglobin was within the interval ⁇ 1 to 1 g/dL.
  • the Sponsor's expectation is that the mean hemoglobin concentration was essentially constant over the 12-month period. For example, instead of using a 90% confidence interval for secondary efficacy analysis for hemoglobin, the following pair of statistical hypothesis tests, each at an alpha level of 0.05, could be used.
  • the sponsor considers the confidence interval method to be easier to interpret than the corresponding method using hypothesis tests. Therefore the confidence interval method will be used for the secondary inference.
  • the tertiary endpoints for this study are: change from Baseline to 12 months in plasma chitotriosidase and CCL18 levels; change from Baseline to 12 months in skeletal age in patients 2 to 17 years old; and change from Baseline to 12 months in growth velocity and Tanner staging.
  • the tertiary endpoints were summarized using descriptive statistics (mean, median, standard deviation, minimum and maximum) at each time point. For endpoints where data were collected at Baseline and other time points during the study, the within group changes were examined.
  • Velaglucerase alfa was generally well tolerated, with most adverse events (AEs) of mild or moderate severity (Table 21). The most frequently reported AEs were nasopharyngitis (8/40 patients), arthralgia (9/40 patients), and headache (12/40 patients). Overall, 11 of 40 patients (28%) experienced an AE considered possibly or probably related to study drug; the majority of these events were considered infusion related. No patient experienced a life-threatening AE. One severe adverse event was considered probably related to treatment and occurred in a patient who had a severe hypersensitivity reaction. This patient tested negative for all 4 isotypes (IgE, IgM, IgG, IgA), including neutralizing antibodies, both at the time of the infusion and 2 weeks later.
  • IgE isotypes
  • the first three infusions for each patient were administered at the clinical site, after which patients who had not experienced a drug-related serious AE or an infusion-related AE were eligible to receive subsequent infusions at home.
  • 25 (63%) of 40 eligible patients received home therapy at least once, ten patients (67%) in the 15 U/kg group, six (50%) in the 30 U/kg group, five (83%) in the 45 U/kg group, and four (57%) in the 60 U/kg group.
  • the mean change from Baseline was ⁇ 0.1 g/dL, with a 90% confidence interval of ⁇ 0.3 to 0.1 g/dL, within the predefined efficacy criterion of ⁇ 1 g/dL.
  • the percent change from Baseline was +7.0%, with a 90% confidence interval of 0.5 to 13.5%, within the predefined efficacy criterion of ⁇ 20%.
  • the percent change from Baseline was ⁇ 0.0%, with a 90% confidence interval of ⁇ 2.6 to 2.6% within the predefined efficacy criterion of ⁇ 15%.
  • the percent change from Baseline was ⁇ 5.6%, with a 90% confidence interval of ⁇ 10.8 to ⁇ 0.4% within the predefined efficacy criterion of ⁇ 15%.
  • Hemoglobin concentration, platelet counts, and liver and spleen volume were sustained at therapeutic levels through 1 year of velaglucerase alfa treatment, as demonstrated by pre-specified efficacy criteria for clinically significant change.
  • Mean change in hemoglobin and mean percent change in platelet count and organ volumes are shown in FIGS. 15-18 . For each parameter, similar results were seen across the four dose groups.
  • the percent change in plasma chitotriosidase and plasma CCL18 are shown in FIGS. 19 and 20 . Levels of both biomarkers were sustained and possibly reduced over the 12-month treatment period.
  • HGT-GCB-058 is a multicenter, open-label treatment study to observe the safety of velaglucerase alfa in patients with Gaucher disease type 1 who were newly diagnosed (treatment na ⁇ ve) or transitioned from imiglucerase to velaglucerase alfa.
  • the study design was for male or female patients two years old or older.
  • Velaglucerase was administered at a dose of 15-60 U/kg every other week (EOW) by 1-hour intravenous (IV) infusion—Patients received the same number of units of velaglucerase alfa as their prior imiglucerase dose (patients receiving ⁇ 15 U/kg imiglucerase EOW received 15 U/kg velaglucerase alfa).
  • the rate of infusion was a maximum of 1 U/kg/minute.
  • HGT-GCB-058 was initiated to provide an alternative treatment option for patients who would otherwise have limited or no access to imiglucerase due to supply constraints.
  • the primary endpoint was to observe the safety of velaglucerase alfa.
  • HGT-GCB-058 For HGT-GCB-058, within 3 months of the first site initiated, 20 clinical sites across the US were enrolling patients. Between Sep. 1, 2009 and Jan. 31, 2010 more than 150 patients enrolled onto HGT-GCB-058 and received at least one infusion of velaglucerase alfa. Only 3 patients were treatment na ⁇ ve; all others were previously treated with imiglucerase.
  • Previously treated with imiglucerase 35.8% at least one TE-AE; 18.2% at least one possibly/probably related TE-AE; 13.8% at least one infusion related reaction; ⁇ 1%—serious AE (a 69-year-old female experienced a severe cerebrovascular accident requiring hospitalization) not related; 3.1%—at least one severe AE: arthralgia (not related), fatigue (probably related), bone pain (not related), pain in extremity (not related), leucopenia (possibly related), cerebrovascular accident (not related).
  • the objective of this example is to compare the efficacy of Ceredase®, Cerezyme®, veluglucerase alfa, Genz-112638, and Zavesca® in treating type 1 Gaucher disease. Hemoglobin concentration, platelet count, liver volume, and spleen volumes were measured after 6, 9, or 12 months of treatment.
  • Enzyme replacement therapy 60 U/kg EOW; Genz112638: 50 & 100 mg BID; Zavesca: 100 mg TID
  • Ratio of Males to Females Cerezyme (8 to 7); Ceredase (3 to 2); Velaglucerase Ph I/II TKT025 study (5 to 7); Velaglucerase-TKT032 study-45 U/kg dose (8 to 5); Velaglucerase-TKT032 study-60 U/kg dose (7 to 5); Zavesca (1 to 1); Genz-112638 (3 to 4)
  • Velaglucerase TKT025 anemia and thrombocytopenia
  • Genz-112638 anemia, thromb, & splenomegaly
  • ADA anti-drug antibody
  • NAb neutralizing antibody
  • This assessment can be performed for any ERT.
  • FIG. 21 An example of such an assessment is shown in FIG. 21 .
  • Anti-velaglucerase alfa and anti-imiglucerase antibodies were evaluated identically using bridge immunoassays and immunoglobulin (Ig) subclass-specific indirect immunoassays, all based on an electrochemiluminescent platform, as well as RIP assays.
  • the bridge electrochemiluminescent immunoassay detected all immunoglobulin subclasses and was considered the antibody screening assay.
  • the Ig subclass electrochemiluminescent immunoassays were confirmatory assays for the presence of IgA, IgM and IgE antibodies, while the RIP assay was confirmatory for the presence of IgG antibodies.
  • the antibody screening assays and IgG assays were calibrated, quantitative and utilized human antibody positive controls.
  • the IgA, IgM and IgE assays were semi-quantitative and utilized hybrid (human-sheep) positive controls.
  • All anti-velaglucerase alfa and anti-imiglucerase immunoassays were identical, including positive cut off criteria, except that either velaglucerase alfa or imiglucerase were used to interrogate the sample. These assays are high throughput, provide increased surface area for detection, allow use of high serum sample concentration with minimum non-specific binding and detect all antibody subclasses.
  • anti-drug antibody screening can be performed using an electrochemilluminescence (ECL) immunoassay.
  • ECL electrochemilluminescence
  • Anti-velaglucerase alfa (anti-imiglucerase) antibodies were detected using immobilized biotin-conjugated velaglucerase alfa (or imiglucerase) on streptavidin-coated microwell plates. Immobilized biotinylated velaglucerase alfa (or imiglucerase) captured anti-velaglucerase alfa (anti-imiglucerase) antibodies present in patients' sera and unbound proteins were removed by washing. Ruthenium complex-labeled velaglucerase alfa (or imiglucerase) was added to each microwell resulting in the formation of a complex with the bound anti-velaglucerase alfa (anti-imiglucerase) antibodies. This was followed by a second wash step, in which unbound labeled proteins were removed.
  • Labeled molecules bound near the microwell surface emit light in a process triggered by the electrochemiluminescent reaction, measured by an MSD SECTOR
  • Imager 2400 instrument http://www.mesoscale.com/CatalogSystemWeb/WebRoot/products/imager — 2400.aspx).
  • a mouse monoclonal antibody with cross-reactivity to velaglucerase alfa and imiglucerase was used as a calibrator within each assay plate and a human anti-imiglucerase antibody cross-reactive with velaglucerase alfa was use as positive assay control.
  • the concentration of anti-velaglucerase alfa (anti-imiglucerase) antibodies in test samples was estimated by interpolating the unknown's measured electrochemiluminescent signal on the calibration curve.
  • a minimum of 67 Gaucher patient baselines were tested in order to set the antibody positive cut points for these assays.
  • the test design included at least three analysts testing replicate samples using different plate lot numbers over a period of at least 14 days. At least three different microwell plate lots were used. Two available MSD instruments were used randomly for a total of 1269 determinations for each assay.
  • the assay cut point for anti-velaglucerase alfa (anti-imiglucerase) antibodies was established as the mean plus 1.645 standard deviation of electrochemiluminescent values obtained as recommended in Mire-Sluis, A R et al. Journal of Immunological Methods 289 (2004), pp 1-16.
  • the assay sensitivity was estimated to be 33.4 and 65.6 ng/mL for anti-velaglucerase alfa and anti-imiglucerase antibodies, respectively.
  • the screening assay dose response curve for velaglucerase is shown in FIG. 23 .
  • IgG Immunoglobulin G
  • RIP radioimmunoprecipitation
  • anti-velaglucerase alfa anti-imiglucerase
  • IgG antibodies present in patient serum bound 125 I-velaglucerase alfa (or imiglucerase) in solution phase and formed antigen/antibody complexes that were captured using Protein G mini-columns.
  • the mini-columns were washed to remove free label and quantified directly in a gamma counter.
  • the radioactive counts retained in the mini-column were proportional to the concentration of anti-velaglucerase alfa (anti-imiglucerase) IgG antibodies in the test sample.
  • the concentration of anti-velaglucerase alfa (anti-imiglucerase) IgG antibodies in test samples was estimated from a calibration curve using the same monoclonal antibody calibrator discussed above. The same human antibody positive control described above was used in this assay.
  • the least squares line fit to the high purity, monoclonal antibody based calibration curve, using well characterized known concentrations of antibody, provided a reliable and consistent method for calculating uncertainty in assay determinations.
  • This tool allows for normalization of the cut point for inter-assay changes in counts that may occur from reagent radiolabel decay, radioautolysis and/or assay handling variability as well as allowing for changes in non specific binding and for changing assay readouts that may occur over time.
  • a total of 59 Gaucher patient baselines were tested in order to set the antibody positive cut point for this assay.
  • the assay cut points for anti-velaglucerase alfa (imiglucerase) IgG were established as described above and recommended in Mire-Sluis et al 2 .
  • Assay sensitivity was estimated to be 28.3 and 64.5 ng/mL for the anti-velaglucerase alfa IgG and anti-imiglucerase IgG assays, respectively.
  • FIG. 25 An example of a dose response curve obtained for a RIP assay using velaglucerase is shown in FIG. 25 .
  • assays are performed to screen for the presence of IgE antibodies. Assays can also be performed to detect the presence of IgA and IgM antibodies.
  • IgA, IgM, and IgE ADA were detected using ECL assays.
  • An example of such an assay is shown in FIG. 26 .
  • Anti-velaglucerase alfa (imiglucerase) antibodies were analyzed for their Ig subclass using indirect electrochemiluminescent immunoassays. Antibodies were detected in serum by immobilizing biotinylated velaglucerase alfa (imiglucerase) on streptavidin-coated microwell plates. Diluted serum samples were added to the immobilized velaglucerase alfa (imiglucerase), which captured any anti-velaglucerase alfa (anti-imiglucerase) antibodies present in the sample. After sample incubation, the microwells were washed to remove unbound proteins.
  • ruthenium complex-labeled anti-human secondary antibodies against IgA, IgM or IgE were added separately and further incubated resulting in the formation of an Ig class-specific complex with any bound anti-velaglucerase alfa or imiglucerase antibodies. This was followed by another wash step, in which unbound labeled secondary antibody was removed. The labeled molecules bound near the microwell surface then emit light as described above.
  • the IgA-, IgM- and IgE-antibody hybrids therefore bound to velaglucerase alfa (or imiglucerase) through the sheep antibody domain, and were detected using ruthenium complex-tagged anti-human secondary antibodies against the human IgA, IgM or IgE domains, respectively.
  • the assay cut points for anti-velaglucerase alfa (imiglucerase) IgA, IgM and IgE were established as described above and recommended in Mire-Sluis et al 2 . Assay sensitivity was estimated to be 10.6 and 11.0 ng/mL for the anti-velaglucerase alfa IgE and anti-imiglucerase IgE assays, respectively.
  • the assay positive cut points had to be met in addition to a ratio greater than or equal to 2.0 of the time point signal to the pre-infusion baseline signal 3 .
  • hybrid positive controls can be used.
  • hybrid positive controls can be used.
  • hybrid positive controls can be used. For example:
  • IgA, IgM and IgE confirmatory assay characteristics are shown in Table 28.
  • IgA, IgM, and IgE ADA isotyping assays were validated for evaluating antibody response in patients receiving velaglucerase alfa or imiglucerase.
  • Inhibition of in vitro enzymatic activity by anti-velaglucerase alfa (imiglucerase) antibodies was tested using an assay that detects and quantifies antibodies that inhibit velaglucerase alfa (imiglucerase) activity.
  • the method is based on a colorimetric activity assay that measures the ability of velaglucerase alfa (imiglucerase) to hydrolyze the synthetic substrate 4-nitrophenyl- ⁇ -D-glucopyranoside to p-nitrophenol and D-glucopyranoside.
  • Anti-velaglucerase alfa (imiglucerase) antibody positive serum samples were pre-incubated with a fixed amount of velaglucerase alfa or imiglucerase, respectively for 30 minutes at 37° C. Sheep polyclonal antibodies known to inhibit velaglucerase alfa and imiglucerase in vitro activity were used as positive controls.
  • the 4-nitrophenyl- ⁇ -D-glucopyranoside substrate solution was then added and incubated with the serum sample/enzyme mixture for one hour at 37° C. The enzymatic reaction was stopped by addition of glycine/sodium carbonate buffer (pH 10.7) and the product (p-nitrophenol) was measured at the absorbance wavelength of 405 nm.
  • velaglucerase alfa (imiglucerase) activity was defined as the amount of enzyme required to hydrolyze one ⁇ mole of the substrate 4-nitrophenyl- ⁇ -D-glucopyranoside in one minute at 37° C. Enzymatic activity was quantified by comparison of the released p-nitrophenol in test samples and assay controls to a p-nitrophenol calibration curve measured in the same assay plate. Results of the test samples were expressed relative to the activity of velaglucerase alfa (imiglucerase) measured in the absence of serum sample and reported as % inhibition.
  • the cut point for the velaglucerase alfa and imiglucerase neutralizing antibody assays was defined as inhibition >20.0% based on these 87 samples. Therefore, a patient sample was considered to be negative for inhibitory antibodies if the level of inhibition observed was ⁇ 20.0% and to be positive if inhibition >20.0%.
  • Table 29 shows the specification of assay characteristics for neutralizing levels of anti-drug antibody (velaglucerase was used as the drug).
  • Cell line MRC1-18 is derived from the line HT1080 and is stably-transfected with human CD206 (the macrophage mannose receptor; MMR, also referred to as MRC1, mannose receptor C type 1).
  • HT1080(saf) cells were transfected with an expression vector carrying the gene encoding the MMR (that was isolated from a human liver cDNA library) by electroporation and immediately plated into 96-well plates. Stable clones were selected using media containing 0.4 mg/mL G418.
  • MRC1 expression was analyzed using FITC anti-MRC1 staining and analysis by fluorescence shift. Expression of MMR on MRC1-18 was confirmed by surface staining with anti-MMR Ab. Additionally, MRC1-18 has been verified by immunostaining and flow cytometry to be negative for expression of Fc (gamma) receptors.
  • Alexa FLUOR® 488 conjugated velaglucerase alfa and imiglucerase velaglucerase alfa or imiglucerase was conjugated with Alexa FLUOR® 488 using the Alexa FLUOR®r 488 protein labeling kit following the manufacturer's protocol (Molecular Probes, catalog #A 10235).
  • PC Assay Positive Control
  • Alexa FLUORO 488 labeled velaglucerase alfa or imiglucerase was added, and incubated at 37° C. for an additional 2 hours.
  • a calibration curve of Alexa FLUORO 488 labeled velaglucerase alfa or imiglucerase was included in each experiment by incubation of MRC1-18 cells with Alexa FLUORO 488 labeled velaglucerase alfa or imiglucerase (0-10 nM) in 1:20 diluted NHS at 37° C. for 2 h.
  • the adjusted MFI was calculated by subtraction of the MFI of the background sample (cells with 0 nM drug) from the MFI of each sample. Inhibition of velaglucerase alfa and imiglucerase uptake by patient samples was determined relative to a normal human serum sample (NHS) or the patient's own naive baseline sample, when available. Percent inhibition can be calculated using the following equation:
  • the average baseline serum effect on the uptake of velaglucerase alfa is similar to the baseline serum effect on the uptake of imiglucerase. However, the variability, calculated as the CV %, is much greater for imiglucerase than for velaglucerase alfa (28% vs 15%) (data not shown).
  • Patient KM (039-167-0001) was naive at baseline and was then treated with imiglucerase in a subsequent study. Samples from all three visits showed a significant inhibition of imiglucerase uptake (24-52% inhibition relative to its baseline), relative to the equivalent inhibition of velaglucerase alfa uptake (0-15%, which is within the range of variability of the assay).
  • Velaglucerase alfa and imiglucerase differ structurally, with regard to both protein sequence (e.g., R495H mutation in imiglucerase) and carbohydrate structure.
  • Experiments were performed to address the hypotheses that antigenic differences exist between velaglucerase alfa and imiglucerase, and that these epitopes differentiate velaglucerase alfa from imiglucerase with respect to the ability of anti-drug antibodies (ADA) to block cell binding and/or cell internalization of each therapeutic.
  • ADA anti-drug antibodies
  • the inhibition of imiglucerase or velaglucerase alfa uptake by patient serum samples was measured relative to each other.
  • Each serum sample was tested in parallel for its ability to block imiglucerase uptake and/or velaglucerase alfa uptake, regardless of which enzyme preparation initially elicited the production of the antibodies.
  • Such comparisons of the inhibition of velaglucerase alfa uptake, versus inhibition of imiglucerase uptake, allow for crude “mapping” of epitopes to biologically-relevant portions of each therapeutic (with the caveat that the antisera tested herein are polyclonal, and thus each antiserum likely contains a mixture of antigen specificities, perhaps recognizing multiple epitopes).
  • MRC1-18 is a cell line engineered at Shire HGT from HT1080 cells that were stably-transfected with the human macrophage mannose receptor C, type 1.
  • anti-velaglucerase alfa (imiglucerase) antibody positive serum samples were pre-incubated with MRC1-18 cells in culture media in flat-bottom, 96-well plates at 37° C. for 15 minutes. Pooled normal human serum (NHS) was used as negative control. Sheep polyclonal antibodies known to inhibit velaglucerase alfa and imiglucerase uptake as well as mannan, the MMR-specific ligand (Sung S J et al. J. Cell Biol. 1983; 96:160-166) were used as positive controls to block enzyme uptake.
  • Alexa Fluor-488-labeled velaglucerase alfa or imiglucerase was added, and incubated at 37° C. for an additional 2 hours.
  • a calibration curve consisting of incubation of Alexa Fluor-488-labeled enzyme with MRC1-18 cells was included in each experiment. After incubation, media was removed by centrifugation, cells were treated with trypsin-EDTA for 3 minutes to remove surface-bound enzyme, and finally the reaction pH was returned to neutral with by addition of an equal volume of culture media. Cells were washed once and resuspended using phosphate buffered saline containing 0.5% BSA. Cells were analyzed using a Becton Dickinson FACS Canto II instrument calibrated before each analysis using the instrument's cytometer setup and tracking beads.
  • the assay cut point was determined by analysis of 25 individual sera collected from treatment-na ⁇ ve Gaucher patients. Each serum was tested on four separate days for a total of 100 values and the positive cut point was defined as inhibition greater than the mean of these values plus 1.645 standard deviations.
  • This assay was developed to compare uptake of velaglucerase alfa to imiglucerase.
  • DOE Design of Experiments
  • PMA phorbol myristate acetate
  • M6P mannose-6-phosphate
  • U937-derived macrophages were incubated for 3 hours with GCB at pH 7.5 with 10 mM calcium. Internalized drug was measured by an activity assay with a synthetic substrate (4-MU-glc) that fluoresces upon cleavage.
  • DOE assays revealed that: i) the interaction of calcium with pH greatly impacts uptake; and ii) bioassay sample comparisons required the presence of calcium, consistent with the known calcium-dependence of the MMR.
  • M6P mannose-6-phosphate
  • velaglucerase alfa is internalized more efficiently than imiglucerase. While both enzymes are primarily internalized via the MMR, a small portion, greater for imiglucerase than for velaglucerase alfa, is internalized by an alternative mechanism. These data may prove valuable in differentiating velaglucerase alfa, imiglucerase, and other future therapies.
  • the patient seroconversion summary is shown in Table 34.
  • 82 received velaglucerase alfa and 17 received imiglucerase.
  • One of 82 patients receiving velaglucerase alfa and four of 17 patients receiving imiglucerase developed antibodies during the trials.
  • the protein was formulated into a sucrose-containing solution and lyophilized using a FTS lab-scale lyophilizer (Lyostar II).
  • the lyophilized vials were removed at intervals after primary drying with a sample thief to yield samples with varying moisture content.
  • the secondary structure of the lyophilized protein was examined by FT-IR. The thermal stability of these samples was characterized by physical appearance, moisture content, size exclusion and reversed phase HPLC, and oxidation by peptide mapping methods.
  • the velaglucerase product Upon reconstitution with Sterile Water for Injection, the velaglucerase product contains approximately 2.5 mg/mL (40 U/mL) of velaglucerase alfa, 50 mg/mL sucrose, 12.9 mg/mL sodium citrate dihydrate, 1.3 mg/mL citric acid monohydrate and 0.11 mg/mL polysorbate 20.
  • TKT032 HGT-GCB-039, HGT-GCB-039 patients with intact spleen, HGT-GCB-039 splenectomized patients, TKT034, and TKT025 are described in Tables 35-40.

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