CHoical Trials of the Recombioaot Factor VIII Product, Kogeoate Martin Inwood and Jeanne lusher
ORTY PATIENTS with hemophilia A, ageą 4 to 72 years, have received the recombinant factor VIII product, Kogenate, for 1 year on home infusions and clinic visits, at doses ranging from 20 to 60 U/kg. In approximately 3,000 infusions, very few minor and no major side effects have occurred. Mi1d reactions inc1uded light headedness, nausea, and urticaria. The low incidence and mi1d nature of acute adverse reactions indicates that the product is safe for outpatient and home infusion use. Following the demonstration of the safety and efficacy of Cutter' s recombinant factor VIII (rFVIII) in previously treated patients, 1 an international multicenter trial in previously untreated patients (PUPs) was begun in January 1989. This report will describe the study design and laboratory methods of the ongoing study with Kogenate in PUPs. To be eligible for the Kogenate PUP study, patients had to have hemophilia A and no prior exposure to blood or blood products. Once a potentia1ly eligible subject was identified, the study was discussed with the patient's parents and informed consent was obtained. A baseline blood sample was obtained for a battery of tests and the patient was entered in the study and given an identification number. If the patient had not been immunized with hepatitis B vaccine, immunization was begun. Patients with mild-to-moderate hemophilia A were not exc1uded from participation, nor were those with a family history of FVIII inhibitor antibodies. Once an enrolled patient was treated with Kogenate (considered time zero), follow-up laboratory tests and observations were obtained at specific intervals. Patients received Kogenate for treatment of all bleeding episodes and for prophylaxis against bleeding (eg, surgery). According to the study design, blood sampies were obtained for inhibitor assays every 3 months from the time a patient first received Kogenate. Patients found to have positive inhibitor assays were recalled for repeat testing. This inc1uded the collection of another blood sample for inhibitor assay to be performed not only at the treating center' s coagulation laboratory but also to be sent to the reference laboratory of the study for confirma-
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Transfusion Medicine Reviews, Vol VI, No 4 (October), 1992: pp 261-262
tion. An inhibitor assay value of ~0.6 Bethesda Units (BU) was considered positive. Additional plasma aliquots were obtained for other studies to characterize the inhibitors (heavy and light chain specificity, epitope mapping, and FVIII gene analysis). Whenever the presence of an inhibitor was confirmed by the reference laboratory, FVIII recovery studies were performed whenever possible and subsequent inhibitor assays were performed more frequent1y. Data collected were reviewed periodically (four times annually) by a six member study overseeing committee with international representation. The study overseeing committee was also to be notified of any adverse events between such formal meetings. In the initial study design, if a study patient developed an inhibitor, further therapy was left to the discretion of the treating physician. Study patients could remain on episodic ("on demand") treatment with Kogenate, or they could be put on an immune tolerance regimen of the treating physician's choice. However, in December 1991, a standardized protocol was developed for attempted immune tolerance induction using rFVIII; or they could be taken off study and treated with another product (such as a Factor IX complex concentrate, porcine FVIII, or rFVIIa). lABORATORY METHODS FOR INHIBITOR DETECTION
Plasma sampies for FVIII assay were collected by standard techniques: 4.5 mL venous blood was drawn into a plastic syringe and was immediately added to 0.5 mL buffered 3.8% sodium citrate and mixed. Sampies were centrifuged at 4°C for 30 minutes at lO,OOOg to 15,000g. Plasma aliquots were transferred to plastic snap-top tubes, quick frozen in alcohol and dry ice, and kept at - 70°C From the Department oj Medicine, University oj Western Ontario, London, Ontario, Canada and the Department ojPediatrics. Wayne State University, Detroit, MI. Address reprint requests to Martin Inwood, MD, Hemophilia Program, St Joseph' s Hospital, 268 Grosvenor St, London, Ontario, N6A 4V2 Canada. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0604-0005$3.0010
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until assayed (or until shipped on dry ice to the reference laboratory). Plasma FVIII levels were measured in the research laboratories of Cutter Biological (Berkeley, CA) by a one-stage clotting assay based on the activated partial thromboplastin time. 2 Diluted standard, control, and test sample assays were run in duplicate. Test results were expressed as the percentage of FVIII present in pooled normal human plasma. FVIII inhibitor assays were measured at each participating center's coagulation laboratory by the Bethesda method. 3 As previously noted, any sample found to have ~0.6 BU was considered positive for FVIII inhibitor. Such positive samples were also assayed at the study's reference laboratory (University ofCA) (C.F. Abildgaard, J. Harrison, Davis, CA). Inhibitor assays were run against human FVIII and rFVIII. Once an inhibitor was confirmed in any patient, alI subsequent plasma samples from that patient were sent to the reference laboratory for inhibitor assay. ADDITIONAL STUDIES
Plasma sampies from patients with confirmed FVIII inhibitors were also sent to the laboratories of Dr Leon Hoyer (American Red Cross, Rockville, MD), for heavy and light chain specificity of the antibody, 4,5 and to Dr Dorothea ScandelIa at the American Red Cross laboratory for epitope mapping. 6 CelIs from patients with confirmed inhibitors were also sent to Dr Ted Tuddenham at the London, United Kingdom, laboratory for analysis of the patient's FVIII gene. 7 Antibodies to murine
immunoglobulin G(lgG) , baby hamster kidney protein, and rFVIII were assayed at Cutter Biological by enzyme-linked immunosorbent assay, and were reported as negative, borderline, or positive (expressed as 1 +, 2 +, 3 + ). .The se1eet group of 66 PUPs and two minimal prior treatment (MTX) patients were studied separately. An additional 26 PUPs have enrolIed in the study but have not yet required treatment. The median age at first treatment with rFVIII was 13.2 months (range Odays to 55 years); among 30 PUPs with severe hemophilia A (defined as FVIII < 0.01 U/mL), the median age at first treatment was 9 months. The median 1ength of treatment for all PUPs was 292.5 days (range 68 to 965 days). To date, 8 PUPs and 2 MTX of 59 tested have developed inhibitor antibodies to rFVIII (after 3 to 16 exposure days to rFVIII). Age at first detection of the inhibitor ranged from 1 to 2.4 years (median 1.4 years). In 7 of 10 patients, the peak inhibitor eoncentration was
ORTY PATIENTS with hemophilia A, ageą 4 to 72 years, have received the recombinant factor VIII product, Kogenate, for 1 year on home infusions and clinic visits, at doses ranging from 20 to 60 U/kg. In approximately 3,000 infusions, very few minor and no major side effects have occurred. Mi1d reactions inc1uded light headedness, nausea, and urticaria. The low incidence and mi1d nature of acute adverse reactions indicates that the product is safe for outpatient and home infusion use. Following the demonstration of the safety and efficacy of Cutter' s recombinant factor VIII (rFVIII) in previously treated patients, 1 an international multicenter trial in previously untreated patients (PUPs) was begun in January 1989. This report will describe the study design and laboratory methods of the ongoing study with Kogenate in PUPs. To be eligible for the Kogenate PUP study, patients had to have hemophilia A and no prior exposure to blood or blood products. Once a potentia1ly eligible subject was identified, the study was discussed with the patient's parents and informed consent was obtained. A baseline blood sample was obtained for a battery of tests and the patient was entered in the study and given an identification number. If the patient had not been immunized with hepatitis B vaccine, immunization was begun. Patients with mild-to-moderate hemophilia A were not exc1uded from participation, nor were those with a family history of FVIII inhibitor antibodies. Once an enrolled patient was treated with Kogenate (considered time zero), follow-up laboratory tests and observations were obtained at specific intervals. Patients received Kogenate for treatment of all bleeding episodes and for prophylaxis against bleeding (eg, surgery). According to the study design, blood sampies were obtained for inhibitor assays every 3 months from the time a patient first received Kogenate. Patients found to have positive inhibitor assays were recalled for repeat testing. This inc1uded the collection of another blood sample for inhibitor assay to be performed not only at the treating center' s coagulation laboratory but also to be sent to the reference laboratory of the study for confirma-
F
Transfusion Medicine Reviews, Vol VI, No 4 (October), 1992: pp 261-262
tion. An inhibitor assay value of ~0.6 Bethesda Units (BU) was considered positive. Additional plasma aliquots were obtained for other studies to characterize the inhibitors (heavy and light chain specificity, epitope mapping, and FVIII gene analysis). Whenever the presence of an inhibitor was confirmed by the reference laboratory, FVIII recovery studies were performed whenever possible and subsequent inhibitor assays were performed more frequent1y. Data collected were reviewed periodically (four times annually) by a six member study overseeing committee with international representation. The study overseeing committee was also to be notified of any adverse events between such formal meetings. In the initial study design, if a study patient developed an inhibitor, further therapy was left to the discretion of the treating physician. Study patients could remain on episodic ("on demand") treatment with Kogenate, or they could be put on an immune tolerance regimen of the treating physician's choice. However, in December 1991, a standardized protocol was developed for attempted immune tolerance induction using rFVIII; or they could be taken off study and treated with another product (such as a Factor IX complex concentrate, porcine FVIII, or rFVIIa). lABORATORY METHODS FOR INHIBITOR DETECTION
Plasma sampies for FVIII assay were collected by standard techniques: 4.5 mL venous blood was drawn into a plastic syringe and was immediately added to 0.5 mL buffered 3.8% sodium citrate and mixed. Sampies were centrifuged at 4°C for 30 minutes at lO,OOOg to 15,000g. Plasma aliquots were transferred to plastic snap-top tubes, quick frozen in alcohol and dry ice, and kept at - 70°C From the Department oj Medicine, University oj Western Ontario, London, Ontario, Canada and the Department ojPediatrics. Wayne State University, Detroit, MI. Address reprint requests to Martin Inwood, MD, Hemophilia Program, St Joseph' s Hospital, 268 Grosvenor St, London, Ontario, N6A 4V2 Canada. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0604-0005$3.0010
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until assayed (or until shipped on dry ice to the reference laboratory). Plasma FVIII levels were measured in the research laboratories of Cutter Biological (Berkeley, CA) by a one-stage clotting assay based on the activated partial thromboplastin time. 2 Diluted standard, control, and test sample assays were run in duplicate. Test results were expressed as the percentage of FVIII present in pooled normal human plasma. FVIII inhibitor assays were measured at each participating center's coagulation laboratory by the Bethesda method. 3 As previously noted, any sample found to have ~0.6 BU was considered positive for FVIII inhibitor. Such positive samples were also assayed at the study's reference laboratory (University ofCA) (C.F. Abildgaard, J. Harrison, Davis, CA). Inhibitor assays were run against human FVIII and rFVIII. Once an inhibitor was confirmed in any patient, alI subsequent plasma samples from that patient were sent to the reference laboratory for inhibitor assay. ADDITIONAL STUDIES
Plasma sampies from patients with confirmed FVIII inhibitors were also sent to the laboratories of Dr Leon Hoyer (American Red Cross, Rockville, MD), for heavy and light chain specificity of the antibody, 4,5 and to Dr Dorothea ScandelIa at the American Red Cross laboratory for epitope mapping. 6 CelIs from patients with confirmed inhibitors were also sent to Dr Ted Tuddenham at the London, United Kingdom, laboratory for analysis of the patient's FVIII gene. 7 Antibodies to murine
immunoglobulin G(lgG) , baby hamster kidney protein, and rFVIII were assayed at Cutter Biological by enzyme-linked immunosorbent assay, and were reported as negative, borderline, or positive (expressed as 1 +, 2 +, 3 + ). .The se1eet group of 66 PUPs and two minimal prior treatment (MTX) patients were studied separately. An additional 26 PUPs have enrolIed in the study but have not yet required treatment. The median age at first treatment with rFVIII was 13.2 months (range Odays to 55 years); among 30 PUPs with severe hemophilia A (defined as FVIII < 0.01 U/mL), the median age at first treatment was 9 months. The median 1ength of treatment for all PUPs was 292.5 days (range 68 to 965 days). To date, 8 PUPs and 2 MTX of 59 tested have developed inhibitor antibodies to rFVIII (after 3 to 16 exposure days to rFVIII). Age at first detection of the inhibitor ranged from 1 to 2.4 years (median 1.4 years). In 7 of 10 patients, the peak inhibitor eoncentration was
