Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates.

CD4-IgG2 is a novel fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. This tetrameric protein is being developed as an immunoprophylactic agent to reduce the probability of infection following HIV-1 exposure, in settings such as occupational or perinatal exposure to the virus. CD4-IgG2 has been expressed in Chinese hamster ovary cells and is secreted as a fully assembled heterotetramer. The protein binds with nanomolar affinity to purified gp120 from both a laboratory-adapted strain and a primary isolate of HIV-1. Pharmacokinetic studies in rabbits demonstrated that CD4-IgG2 has a plasma terminal half-life greater than 1 day, compared with 15 min for soluble CD4 (sCD4). CD4-IgG2 does not bind to Fc receptors on the surface of U937 monocyte/macrophage cells. Compared to molecules that incorporate the Fc portion of IgG1, CD4-IgG2 has less potential to mediate functions such as antibody-dependent enhancement of infection or transplacental transmission of HIV-1. When tested in a virus-free HIV-1 envelope glycoprotein-mediated cell fusion assay, the tetrameric CD4-IgG2 molecule inhibited syncytium formation more effectively than monomeric sCD4 or a dimeric CD4-gamma 2 fusion protein. This suggests the protein will block cell-to-cell transmission of HIV-1. Moreover, CD4-IgG2 effectively neutralized a panel of laboratory-adapted strains and primary isolates of HIV-1, including strains with different tropisms and isolated from different stages of the disease, at concentrations that should be readily achieved in vivo.

Inhibition of ligand binding to thromboxane A2/prostaglandin H2 receptors by diethylpyrocarbonate. Protection by receptor ligands and reversal by hydroxylamine.

The potential of histidines to modulate the binding of agonists and antagonists to human platelet thromboxane A2 (TXA2) receptors was investigated. TXA2 receptors were purified from crude platelet membranes via affinity and wheat germ lectin chromatography. Radioligand binding studies were conducted using the TXA2, mimetic [125I]BOP (I-BOP (I-BOP = [1S-(1 alpha,2 beta(5Z),3 alpha(1E, 3R*),4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)7-oxabicyclo- [2.2.1]heptan-2-yl]-5-heptenoic acid) and the TXA2 receptor antagonist [125I]SAP (I-SAP = 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-iodobenzene- sulfonylamino)-bicyclo-[3.1.1]hept-2-yl]-(5Z)-heptenoic acid). The histidine modifying reagent diethyl-pyrocarbonate (DEPC) produced a concentration (30-100 microM) dependent inhibition of binding of both [125I]BOP and [125I]SAP. DEPC treatment significantly (P < 0.05, N = 6) decreased the affinity of the receptor for [125I]SAP (Kd = 2.4 +/- 0.4 and 5.4 +/- 0.4 nM, control and DEPC, respectively) without significantly decreasing the Bmax. The effects of DEPC were reversed by hydroxylamine. The inhibition of [125I]BOP and [125I]SAP binding produced by DEPC was reduced significantly by prior incubation of the purified receptors with the TXA2 receptor agonist U-46619 or the TXA2 receptor antagonist SQ 29548. The results strongly support the notion that one or more histidines reside in a domain that can modulate ligand binding to the TXA2 receptor.