Aspects of the relationship between the metabolism and toxicity of cyclosporin A
The fungal metabolite cyclosporin A (CsA) is the first of a new order of immunosuppressant drugs. Unlike conventional immunosuppressive therapy, which produces "blanket suppression", CsA has a selective inhibitory effect on T cell activation. Although CsA does not cause the serious side-effects, such as myelotoxicity, normally associated with cytotoxic agents, the drug is not without its problems, the most clinically important of which are nephro- and hepatotoxicity. The primary aim of the work presented in this thesis was to study mechanisms of CsA- induced toxicity in the rat. In an initial study a toxic oral dose of CsA was administered daily along with the diuretic frusemide (Fr) for a period of 14 days. Although CsA had no effect on Fr-induced diuresis, Fr did enhance the nephrotoxicity associated with the immunosuppressant. The nature of this interaction was unclear but it was postulated that Fr might inhibit the metabolism of CsA by the hepatic cytochrome P-450 (cyt P-450) dependent mono-oxygenase enzyme system, thus producing toxic circulating levels of CsA. To further investigate this important role of the cyt P-450 system, the effect of inhibition, suppression or induction of hepatic drug metabolism on the toxicity of CsA was investigated. The co-administration of either an inhibitor (SKF-525A) or a suppressor (CoCl2) of hepatic mono-oxygenase activity with a toxic dose of CsA produced inconclusive results. When, however, CsA was given along with known inducers of both cyt P-450 and the conjugating enzyme glucuronyl transferase (GT), namely either Aroclor 1254 or low doses of phenobarbitone (PB: 40 or 60 mg/kg/ 24 hr), the trough serum CsA levels were reduced and the nephrotoxicity associated with CsA either ameliorated (when assessed by serum and urinary biochemical criteria) or abolished (when assessed histologically). The co-administration of 3-methylcholanthrene (3-MC), a compound which induces cyt P-450 and GT isozymes distinct from those induced by PB, with CsA had no effect on either the trough serum levels or toxicity of the immunosuppressant. Since Aroclor 1254 combines the inducing properties of PB and 3-MC, it was concluded that the amelioration of the nephrotoxicity associated with CsA by Aroclor 1254 was due to its PB-type induction of either cyt P-450 or GT. None of the inducers had any effect on the CsA-evoked changes in hepatic biochemical function, although the co-administration of CsA and Aroclor 1254 resulted in a quite severe fatty change in the liver. This was in contrast to the relatively mild fatty changes seen when CsA was co-administered with either PB or 3-MC. No inducer had any effect on either the immunosuppressive property of CsA or on CsA- induced changes in lymphoid and bone marrow tissue morphology. As a result of these studies, an experiment was designed to determine whether the spontaneous remission in CsA-induced renal damage noted previously in this laboratory could be related to changes in the activity of the hepatic mono-oxygenase enzyme system and in the possible hepatic metabolism of CsA. A toxic dose of CsA was administered to rats for a seven-week period, during which time groups of 4 rats were killed at various times. Nephrotoxicity appeared within 4 days of starting treatment and continued until day 28. There was then a one-week period of remission followed by the return of renal damage. Hepatic mono-oxygenase activity, as indicated by aminopyrine N-demethylation (AD) and NADPH-cytochrome c (P-450) reductase activity in vitro, fell during the first 28 days but as the rats entered the remission period AD activity rose to above pretreatment levels, while NADPH-cyt c reductase activity returned to a level similar to its pretreatment value. During the same remission period the serum CsA level fell to its lowest concentration. With subsequent relapse, hepatic enzyme activity and serum CsA levels both returned to their pre-remission values. These results were consistent with cyclical changes in CsA induced nephrotoxicity being caused by corresponding variations in circulating drug levels. The changes in serum levels of CsA were, it was suggested, best explained by cyclical changes in the drugs metabolic detoxification by the hepatic mono-oxygenase enzyme system, due to the production of a hepatic CsA metabolite which "suicide inactivated" the hepatic mono-oxygenase system. The results presented in this thesis indicate that, in the rat, either the parent CsA, molecule or a, non-PB inducible metabolite recognised, in the CsA radioimmunoassay is responsible for the CsA-induced nephrotoxicity. In addition, these results, taken together with recent published case reports, suggest that the co-administration of CsA, with drugs which either induce or inhibit cyt P-450 should, whenever possible, be avoided.