Dan Combs

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.

Most lymphomas that involve the central nervous system are B-cell neoplasms that express the cell surface molecule CD20. After intravenous administration, rituximab can be reproducibly measured in the cerebrospinal fluid (CSF) in patients with primary central nervous system lymphoma; however, the CSF levels of rituximab are approximately 0.1% of serum levels associated with therapeutic activity in patients with systemic non-Hodgkin lymphoma. Because lymphomatous meningitis is a frequent complication of non-Hodgkin lymphoma, we have conducted an analysis of the safety and pharmacokinetics of direct intrathecal administration of rituximab using cynomolgus monkeys. No significant acute or delayed toxicity, neurologic or otherwise, was detected. Pharmacokinetic analysis suggests that drug clearance from the CSF is biphasic, with a terminal half-life of 4.96 hours. A phase 1 study to investigate the safety and pharmacokinetics of intrathecal rituximab in patients with recurrent lymphomatous meningitis will be implemented based on these findings.

Rituximab is a B cell-depleting anti-CD20 chimeric IgGkappa monoclonal antibody being investigated for the treatment of rheumatoid arthritis. The purpose of this study was to develop a population pharmacokinetic model in rheumatoid arthritis patients. In addition, the final pharmacokinetic model was used to assess the variability in drug exposure (AUC0-infinity) for fixed versus body surface area-based dosing. A total of 102 patients were included in this population pharmacokinetic analysis. A 2-compartment pharmacokinetic model described the data reasonably well. Body surface area and gender were the most significant covariates for both CL and Vc. Body surface area alone only explained about 19.7% of the total interindividual variability of CL. In a simulation study, body surface area-based dosing normalized drug exposure over a wide range of body surface area but did not seem to improve the predictability of rituximab AUC0-infinity in rheumatoid arthritis patients. Therefore, no rationale for body surface area-based dosing for rituximab in rheumatoid arthritis patients was found.

PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.