Development of an animal-human antibody complex for use as a control in ELISA.

In order to provide the equivalent of a human anti-human protein antibody as positive control in ELISAs, a goat-human antibody complex was created using chemical cross-linking. The resulting hybrid complex had a larger molecular size on HPLC and SDS-PAGE. In ELISA, the goat-human complex bound to human antigen and was detectable by a secondary anti-human conjugate. The method to make the hybrid complex is simple, cost-effective and can be used to make human-like antibodies to many human proteins.

Albugranin, a recombinant human granulocyte colony stimulating factor (G-CSF) genetically fused to recombinant human albumin induces prolonged myelopoietic effects in mice and monkeys.

PURPOSE: Albugranin fusion protein is recombinant granulocyte colony stimulating factor (rG-CSF) genetically fused at its N-terminus to the C-terminus of recombinant serum human albumin and is expected to have a relatively long half-life compared with rG-CSF alone. In this study, the pharmacodynamics and pharmacokinetics of Albugranin were evaluated in BDF1 mice and cynomolgus monkeys. METHODS: Single doses of Albugranin (0.25-5 mg/kg) or Filgrastim (methionyl rG-CSF, 0.25, or 1.25 mg/kg) were administered subcutaneously (SC) to mice and multiple doses of Albugranin (25-100 microg/kg every 4 or 7 days) or Filgrastim (5 microg/kg daily) were administered SC for 14 days to monkeys for hematologic evaluation. For pharmacokinetics studies, mice were injected intravenously (IV) or SC with single doses of Albugranin (0.25-1.25 mg/kg) or Filgrastim (0.25 mg/ kg) and monkeys were injected SC with multiple doses of Albugranin (100-1,000 microg/kg once weekly for 5 weeks). Plasma levels of Albugranin and Filgrastim were measured by enzyme-linked immunosorbent assay. RESULTS: In mice, administration of Albugranin effectively increased the number of peripheral granulocytes and mobilized hematopoietic progenitor cells for up to 5 days. The magnitude and duration of this effect were dose-dependent. In contrast, administration of Filgrastim resulted in a small increase in both cell types on day 1 only. Albugranin administered to cynomolgus monkeys caused an increase in peripheral neutrophils, with a less prominent increase in peripheral monocytes. Albugranin-induced neutrophilia peaked 24 h following each dose administration. Administration of Filgrastim daily in monkeys resulted in moderate increases in neutrophils that were maximal on days 8-12 during the course of treatment. Compared with Filgrastim, Albugranin had a longer terminal half-life (t(1/2,term)) and mean residence time (MRT), and slower clearance (CL/F) in mice. The t(1/2,term), MRT, and CL/F of Albugranin following SC administration to BDF1 mice were 5.6-5.7 h, 16.7-20.7 h, and 6.37-12.2 mL/h/kg, respectively, compared with 2.54 h, 4.9 h, and 164 mL/h/kg, respectively for Filgrastim. In cynomolgus monkeys, the corresponding values of t(1/2,term), MRT, and CL/F for Albugranin were 7.73-133 h, 19.4-27.3 h, and 7.90-27.5 mL/h/kg, respectively, for doses of 100-1000 microg/kg. An exposure-response relationship that could be empirically described with a simple Emax model with baseline was found between day 15 absolute neutrophil count and area under the curve following the first dose in cynomolgus monkeys. CONCLUSION: The sustained activity of Albugranin in mice and monkeys demonstrated in these studies suggests that this agent could be given less frequently than Filgrastim to achieve similar therapeutic effects in patients.

Pharmacologic and pharmacokinetic profile of repifermin (KGF-2) in monkeys and comparative pharmacokinetics in humans.

Repifermin (truncated, recombinant human keratinocyte growth factor-2, KGF-2) was evaluated in cynomolgus monkeys and healthy humans during a phase 1 trial. Monkeys received vehicle or repifermin at 20, 75, or 200 microg/kg IV or 750 microg/kg subcutaneous (SC) daily for 29 days. Clinical observations were made during the entire dosing period. Gross and microscopic changes were assessed at necropsy. Pharmacokinetic parameters and immunogenicity were evaluated in these monkeys and in humans, following a single or 7 daily IV bolus injections of 1, 5, 25, or 50 microg/kg repifermin. In monkeys, repifermin was well tolerated, and histologic evaluation demonstrated dose-dependent, reversible thickening of the mucosa throughout the alimentary tract, except for the stomach. In the alimentary tract tissues, nonepithelial tissues were not affected, indicating a specificity of repifermin for epithelial cells. Pharmacokinetics in both monkeys and humans were dose proportional, showed lack of drug accumulation with repeated daily dosing, and were characterized by high volumes of distribution and clearance rates, indicating substantial tissue binding and metabolism. Repifermin was not markedly immunogenic following multiple daily IV injections in either species. Serum repifermin concentrations in humans were comparable to those attained in monkeys that produced significant pharmacological effects on epithelial cells in the alimentary tract. These findings provide additional support for the ongoing clinical development of repifermin for diseases involving epithelial injury.