ABSTRACT
BACKGROUND: Reconstitution of CroFab(®) (Crotalidae Polyvalent Immune Fab [ovine]) lyophilized drug product was previously performed using 10 mL sterile water for injection followed by up to 36 min of gentle swirling of the vial. CroFab has been clinically demonstrated to be most effective when administered within 6 h of snake envenomation, and improved clinical outcomes are correlated with quicker timing of administration. An alternate reconstitution method was devised, using 18 mL 0.9% saline with manual inversion, with the goal of shortening reconstitution time while maintaining a high quality, efficacious product. METHODS: An analytical study was designed to compare the physicochemical properties of 3 separate batches of CroFab when reconstituted using the standard procedure (10 mL WFI with gentle swirling) and a modified rapid procedure using 18 mL 0.9% saline and manual inversion. The physical and chemical characteristics of the same 3 batches were assessed using various analytic methodologies associated with routine quality control release testing. In addition further analytical methodologies were applied in order to elucidate possible structural changes that may be induced by the changed reconstitution procedure. RESULTS: Batches A, B, and C required mean reconstitution times of 25 min 51 s using the label method and 3 min 07 s (a 88.0% mean decrease) using the modified method. Physicochemical characteristics (color and clarity, pH, purity, protein content, potency) were found to be highly comparable. Characterization assays (dynamic light scattering, analytical ultracentrifugation, LC-MS, SDS-PAGE and circular dichroism spectroscopy were also all found to be comparable between methods. DISCUSSION: When comparing CroFab batches that were reconstituted using the labeled and modified methods, the physicochemical and biological (potency) characteristics of CroFab were not significantly changed when challenged by the various standard analytical methodologies applied in routine quality control analysis. Additionally, no changes in the CroFab molecule regarding degradation, aggregation, purity, structure, or mass were observed. CONCLUSION: The analyses performed validated the use of the more rapid reconstitution method using 18 mL 0.9% saline in order to allow a significantly reduced time to administration of CroFab to patients in need.
Subject(s)
Antivenins/pharmacology , Drug Compounding/methods , Immunoglobulin Fab Fragments/pharmacology , Immunologic Factors/pharmacology , Animals , Chromatography, Liquid , Circular Dichroism , Crotalid Venoms/toxicity , Electrophoresis, Polyacrylamide Gel , Female , Mice , Snake Bites/drug therapy , Sodium Chloride , Treatment OutcomeABSTRACT
Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease. We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro. Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons. Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha. Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.
Subject(s)
Brain Tissue Transplantation , Corpus Striatum/metabolism , Fetal Tissue Transplantation , Interleukin-1/metabolism , Neurons/transplantation , Animals , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Corpus Striatum/surgery , Culture Media/pharmacology , Enzyme-Linked Immunosorbent Assay , Graft Survival/physiology , In Vitro Techniques , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/pharmacology , Neurons/cytology , Parkinson Disease/surgery , Rats , Rats, Wistar , Sialoglycoproteins/pharmacologyABSTRACT
Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD). We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1. Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250%. Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD.
