Drug-protein conjugates--XII. A study of the disposition, irreversible binding and immunogenicity of penicillin in the rat.

The disposition, irreversible binding and immunogenicity of benzylpenicillin (BP) were studied in male Wistar rats. [3H]BP, administered i.v. to anaesthetized rats at two doses (27 mumol/kg, 2.7 mmol/kg), showed dose-dependent kinetics: plasma and tissue concentrations of total BP were disproportionately increased at the higher dose. BP was rapidly cleared from the plasma at both doses (less than 0.05% of administered dose/ml plasma after 3 hr). In spite of the disproportionately elevated levels of total BP after the higher dose, covalent binding to plasma proteins was quantitatively similar as a percentage of the dose at both doses. Three hours after i.v. injection of 27 mumol/kg and 2.7 mmol/kg of the drug, 5.6% +/- 1.7% and 3.3% +/- 1.1% respectively of circulating BP was covalently bound, representing less than 0.004% of the administered dose bound per ml of plasma in each case. Covalent binding of BP to rat plasma proteins in vitro was of a similar magnitude to that observed in vivo: 1.6% +/- 0.4% of BP was bound to 25% rat plasma after 3 hr incubation at 37 degrees. In a separate series of experiments the immunogenicity of BP was studied by chronic administration of the drug to rats. Following daily i.v. or i.m. administration of BP (27 mumol/kg, 270 mumol/kg, 2.7 mmol/kg) for 4 consecutive days at 4-week intervals (three series of injections) neither IgG nor IgM anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of the high dose of BP was discontinued after the first series of injections due to local necrosis. In contrast to free BP, BPO-keyhole limpet haemocyanin (BPO-KLH, 42 nmol BP bound/mg KLH) administered by single i.v. injection at 4-week intervals at two doses (20 and 200 micrograms conjugate/kg, corresponding to 0.84 and 8.4 nmol BPO/kg) readily induced IgG and IgM anti-BPO antibody responses (median IgG titres were 872 and 5470 one week after the third injection of the low and high dose of conjugate respectively; corresponding IgM titres were 4513 and 22,866). The specificity of the IgG and IgM antibodies for the BPO determinant was confirmed by ELISA inhibition with BPO-aminocaproate. These experiments show that BP binds irreversibly, but to a limited extent, to plasma proteins in vivo, and that such a degree of conjugation appears to be insufficient to elicit a detectable anti-BPO antibody response.