Acid-Catalyzed Peptide Bond Hydrolysis of Recombinant Human Interleukin 11

Recombinant human interleukin 11 (rhIL-11) is a multispectrum cytokine that plays an important role in megakaryocytopoiesis and platelet production. Probing rhIL-11 chemical reactivity in aqueous solution is an important initial step in developing a dosage form for rhIL-11 clinical trials. This report documents rhIL-11 degradation kinetics at 50°C in solutions adjusted to pH 3.0 to 9.5. Stressed samples were analyzed by reverse-phase HPLC and degradation product peaks were isolated for structural characterization. The results show maximal stability in the region pH 6.5 to 7.0. Degradation product identification shows that the major reaction pathway in acidic solution involves peptide cleavage at aspartate₁₃₃–proline₁₃₄. In alkaline solution, protein disappearance proceeds via nonspecific loss to container surfaces. Degradation products at alkaline pH have not been identified.     

Characterization of lymphocyte subpopulations in warthin's tumor

Cell suspensions from six Warthin's tumors (WTs) were characterized with fluorescence-labeled cell cytometry. WT lymphocyte subsets were identified with monoclonal antibodies directed against lymphocyte-associated cell antigens including T lymphocyte subsets, B lymphocytes, and natural killer (NK) cells. Results showed that T cell proportions were 58% and B cell proportions were 39%. The T cell helper:cytotoxic-suppressor ratio was 5.7:1 and the B to T cell ratio was 0.8:1. NK cells represented 1.3% of cells. When compared to peripheral blood lymphocytes (PBLs) in the same patients, statistically significant differences were noted between PBLs and WT lymphocytes in the percentage of B lymphocytes (P<.01), T cytotoxic-suppressor lymphocytes (P<.02), NK cells (P<.01), and in the ratios of B to T lymphocytes (P<.01) and T helper to T cytotoxic-suppressor lymphocytes (P<.03). Comparing these data to retrospective data on lymphocyte distribution in normal and reactive lymph nodes, the epithelial component does not appear to exert a local effect on the lymphoid component of WT.     

Payment under public and private insurance and access to cochlear implants

BACKGROUND: Cochlear implants are expensive, yet often cost-effective. However, among hundreds of thousands of potential US candidates, only about 3000 received implants in 1999. To analyze whether insurance reimbursement levels may contribute to low access rates. DESIGN: Surveys were performed during 1999 and 2000 of physicians and audiologists at clinics providing cochlear implant services, selected hospitals where surgery is performed, and state Medicaid agencies. Secondary data were obtained on Medicare payment rates and hourly incomes of otolaryngologists and audiologists. PARTICIPANTS: One hundred thirty-one physicians (response rate 67.9%), 111 audiologists (74.0%), 60 hospitals (73.2%), and 44 Medicaid agencies (86.3%). OUTCOME MEASURES: Reimbursement rates for selected Current Procedural Terminology codes and for cochlear implant systems (devices); time required to perform services; additional time not reimbursed; and device purchase prices. RESULTS: Medicare and Medicaid payment rates often fail to cover costs of aural rehabilitation. Medicare sometimes and Medicaid often fails to cover surgeon costs. Sometimes private insurance does not cover hospitals' device costs. Under Medicare, in 1999 hospitals lost more than $10 000 per device for inpatient surgery and about $5000 per device for each outpatient surgery. Device reimbursement in 2002 for outpatient surgery under Medicare is about $3773 higher than in 1999. Medicaid device payment policies vary greatly and fail to cover costs in at least 18 states, accounting for 44% of national Medicaid enrollment. CONCLUSIONS: Efforts to expand access to cochlear implants may be impeded by financial incentives. Facilitating access for Medicare and Medicaid patients could require changes in payment policies.     

Temperature and pH Dependence of Fluocinolone Acetonide Degradation in a Topical Cream Formulation

We investigated the degradation of fluocinolone acetonide (FA) incorporated into an oil-in-water cream base. The study examined the influence of temperature (23 to 80 degrees C) and cream pH (pH 2.3 to 6) on FA degradation rates. FA degradation followed pseudo-first-order kinetics and adhered to the Arrhenius expression over the entire temperature range investigated. At all temperatures, the pH strongly influenced the observed degradation rate constant (kobs) values, with rate minima observed near pH 4. The FA log(degradation rate)-pH profiles were consistent with a reaction mechanism requiring drug hydrolysis catalyzed by hydroxide and hydrogen ions. Taking into account both the temperature and the pH dependence of FA degradation permits calculating kobs values from the following equation: kobs = exp[22.5 - (17,200/RT)] + exp[38.7 - (22,200/RT)] x [H+] + exp[49.5 - (21,100/RT)] x [OH-] where the three bracketed terms represent Arrhenius expressions for neutral, acid-catalyzed, and base-catalyzed hydrolysis reactions. FA degradation in the cream base parallels the degradation of a related steroid (triamcinolone acetonide) in an aqueous alcohol solution. The equivalence between FA and triamcinolone acetonide kinetics in the different reaction media suggests that in the cream base, FA degradation is limited to an aqueous phase largely unperturbed by the presence of nonaqueous constituents that comprise the cream formulation.     

Characterization and resolution of reversed phase HPLC chromatography failure attributed to sulfobutylether-β-cyclodextrin in a pharmaceutical sample preparation

A reversed phase HPLC method developed for a drug product formulation using hydroxypropyl-β-cyclodextrin (HPCD) was rendered ineffective for analyzing a similar formulation containing sulfobutylether-β-cyclodextrin (SBECD). The active pharmaceutical ingredient (API) and the majority of its impurities became more strongly retained, eluting as an incoherent conglomerate of peaks. Furthermore, this phenomenon was reproduced in subsequent injections of the API reference standard. Based on HPLC and LC–ESI-MS studies, the chromatography failure was attributed to the accumulation of SBECD on the HPLC column. The subsequent interaction of the API with bound SBECD resulted in the aberrant chromatography. An anion-exchange solid-phase extraction treatment was developed and qualified to selectively remove SBECD from sample solutions, thereby allowing the same HPLC method to be used. The sample treatment procedure exhibited suitable accuracy and precision for quantitating the API and its impurities, and resulted in typical chromatographic profiles.     

Nonquaternary cholinesterase reactivators. 2. Alpha-heteroaromatic aldoximes and thiohydroximates as reactivators of ethyl methylphosphonyl-acetylcholinesterase in vitro

We prepared six pairs of alpha-heteroaromatic aldoximes, RC(= NOH)H, and thiohydroximates, RC(= NOH)S-(CH2)2N(C2H5)2, where R represents various oxadiazole and thiadiazole rings. Each compound was characterized with respect to the following: structure, (hydroxyimino)methyl acid dissociation constant, nucleophilicity toward trigonal carbon and tetrahedral phosphorus, octanol-buffer partition coefficient, reversible inhibition of eel acetylcholinesterase (AChE), and in vitro reactivation of AChE inhibited by ethyl p-nitrophenyl methylphosphonate. Eight of the twelve compounds significantly reactivate ethyl methylphosphonyl-AChE, but inherent reactivities are moderate to low: the most potent nonquaternary reactivator, 3-phenyl-5-[(hydroxyimino)methyl]-1,2,4-oxadiazole, is 17 times less reactive than the well-known reactivator 2-[(hydroxyimino)methyl]-1-methylpyridinium iodide (2-PAM). One of the nonquaternary compounds, 3-phenyl-1,2,4-oxadiazole-5-thiohydroximic acid 2-(diethylamino)ethyl S-ester, is a powerful reversible inhibitor of AChE (I50 = 7.5 microM). The observed relationships between nonquaternary compound structure, reactivation potency, and anti-AChE activity reveal important molecular requirements for high reactivity toward phosphonylated AChE.     

Innovative Approaches in Drug Development

This is the white paper on Innovative Approaches in Drug Development developed by the Biotechnology Industry Organization's (BIO) Clinical Trial Designs Workgroup. As recognized by the Food and Drug Administration's Critical Path Opportunities Report, the need to develop and apply innovative approaches to create new trial designs and clinical development programs is rapidly on the rise. Such novel approaches hold tremendous potential in the ability to refine mechanisms lying at the fundamental core of product safety and efficacy. The paper addresses the opportunities, challenges in pre-clinical and clinical trial design innovations; emphasizes the importance of safety database; and makes recommendations for carrying out the innovative approaches. The views expressed here in are solely those of the authors and do not reflect the official views of the Biotechnology Industry Organization.