Effects of non-covalent self-association on the subcutaneous absorption of a therapeutic peptide.

PURPOSE: To utilize an acylated peptide as a model system to investigate the relationships among solution peptide conformation, non-covalent self-association, subcutaneous absorption and bioavailability under pharmaceutically relevant solution formulation conditions. METHODS: CD spectroscopy, FTIR spectroscopy, equilibrium sedimentation, dynamic light scattering, and size exclusion chromatography were employed to characterize the effects of octanoylation on conformation and self-association of the 31 amino acid peptide derivative des-amino-histidine(7) arginine(26) human glucagon-like peptide (7-37)-OH (IP(7)R(26)GLP-1). Hyperglycemic clamp studies were performed to compare the bioavailability, pharmacokinetics, and pharmacodynamics of solution formulations of oct-IP(7)R(26)GLP-1 administered subcutaneously to normal dogs. RESULTS: Octanoylation of IP(7)R(26)GLP-1 was shown to confer the propensity for a major solvent-induced conformational transition with an accompanying solvent- and temperature-dependent self-association behavior. Formulations were characterized that give rise to remarkably different pharmacodynamics and pharmacokinetics that correlate with distinct peptide conformational and self-association states. These states correspond to: (i) a minimally associated alpha-helical form (apparent molecular weight = 14 kDa), (ii) a highly associated, predominantly beta-sheet form (effective molecular diameter 20 nm), and (iii) an unusually large, micelle-like soluble beta-sheet aggregate (effective molecular diameter 50 nm). CONCLUSIONS: Bioavailability and pharmacokinetics of a self-associating peptide can be influenced by aggregate size and the ease of disruption of the non-covalent intermolecular interactions at the subcutaneous site. Hydrophobic aggregation mediated by seemingly innocuous solution formulation conditions can have a dramatic effect on the subcutaneous bioavailability and pharmacokinetics of a therapeutic peptide and in the extreme, can totally preclude its absorption. A size exclusion chromatographic method is identified that distinguishes subcutaneously bioavailable aggregated oct-IP(7)R(26)GLP-1 from non-bioavailable aggregated oct-IP(7)R(26)GLP-1.

An attempt to prevent anaphylaxis by aerosolized antigen in guinea pigs.

In an attempt to provide an alternative and safer technique of specific hyposensitization for those patients for whom the conventional treatment presents a high risk for anaphylaxis, we used a guinea pig model for extracorporeal hyposensitization. We sensitized three groups of guinea pigs with an olive pollen extract. Two of the groups were sensitized before removal of their buffy coat and the third group was sensitized after the removal of their buffy coat. The buffy coat cells were incubated with the same antigen and intracardially reinjected into their respective donors. Subsequent challenges by aerosolized antigen and intracardiac injections were performed. We did a basophil degranulation on random samples of buffy coat of tested and control animals. We skin tested the animals and we examined the histopathology of their lungs. The animals reinjected with the incubated buffy coat prior to sensitization showed a greater protection from aerosolized antigen and the onset of their anaphylactic symptoms was significantly delayed when compared with controls. The results of basophil degranulation studies showed that animals sensitized before removal of their buffy coat had approximately a threefold increase of degranulated basophils as compared with animals that were sensitized after removal of buffy coat and approximately a fourfold increase when compared with controls. This degranulation did not affect the specific skin test. Histopathologic observations did not reveal any characteristics typical of anaphylaxis.

Extracorporeal hyposensitization.

Sensitized guinea pigs were reinjected with autologous cells of the buffy coat or T-cell enriched populations of lymphocytes following incubation with sensitizing antigen. The incubated cells of the buffy coat appeared to give the animal protection from severe signs and lesser manifestations of anaphylaxis.

Hepatitis B surface antigen carriers--to biopsy or not to biopsy.

In order to assess the frequency of significant liver disease in hepatitis B surface antigen carriers with normal liver tests, 54 such individuals were identified and prospectively followed for 4 to 48 months with monthly liver tests. Upon testing, 4 were found to carry e antigen and 14 carried e antibody (anti-e). During follow-up, only 4 patients, none of whom were e antigen-positive, developed persisting abnormalities in liver tests. Of the 23 patients who underwent percutaneous liver biopsies, normal histologies were found in 2, nonspecific changes (ground glass hepatocytes, focal necrosis, fatty changes, etc.) in 18, and chronic persistent hepatitis (with or without other nonspecific changes) in 3. Chronic active hepatitis and/or cirrhosis, lesions which may carry more serious prognostic implications, were not seen in any biopsies. Two of the 4 e antigen-positive patients consented to biopsy, both of whom had chronic persistent hepatitis. All 6 patients with anti-e who underwent biopsy had ground glass hepatocytes, which were found in only about 50% of the remaining patients. It is concluded that hepatitis B surface antigen carriers should be followed with serial liver tests, and those whom tests remain normal should not be considered for liver biopsy.

Alteration of the response of guinea pigs to freund's complete adjuvant by ascaris extract: a preliminary histologic study.

Freund's adjuvant has been asserted to cause systemic complications in man similar to those occurring with B.C.G. vaccine. In search of factors predisposing to such complications of this vaccine, the potential role of a coexisting parasitic infestation was considered. A potentiating effect of the ascaris antigen on the pathological changes produced in guinea pigs by intraperitoneally injected Freund's complete adjuvant was noted. The pertinency of this observation to immunotherapy of cancer with B.C.G. is purely speculative.