N-Alkylprotoporphyrin Formation and Hepatic Porphyria in Dogs After Administration of a New Antiepileptic Drug Candidate: Mechanism and Species Specificity

Porphyria is a group of heritable metabolic disorders where the enzymes of the heme biosynthetic pathway are partially or totally deficient (Balwani and Desnick, 2012; Hift et al., 2011; Nordmann and Puy, 2002; Sassa, 2006; Sassa and Kappas, 2000; Thadani et al., 2000). As a consequence, porphyrins and heme precursors may accumulate to toxic levels. Their increased concentration in excreta confirms the diagnosis of porphyria and helps in distinguishing among the different forms of the disease. Depending on the location and the nature of the enzyme deficiency, porphyrias are classified as hepatic or erythropoietic (Poh-Fitzpatrick, 1998). Hepatic porphyrias are characterized by neurovisceral complications whereas erythropoietic porphyrias manifest as photocutaneous problems. In addition, hepatic porphyria may lead to hepatobiliary impairment as porphyrin precursors precipitate in the canaliculi (Bloomer, 1998) and/or impair canalicular secretory function (Meerman, 2000). Most of the gene carriers of inherited porphyrias remain clinically asymptomatic. The disease mostly develops when the genetic defect is combined with precipitating factors such as infection, surgery, smoking, hormonal status, diet, and exposure to some drugs with cytochrome P-450 (CYP) inducing properties (Downey, 1999; Smith and De, 1980; Thadani et al., 2000). So far, very few xenobiotics were found to be porphyrogenic in nongene carrier individuals (Smith and De, 1980). This contrasts with laboratory animals where the disease can be induced by a number of chemicals. They include the herbicide 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenylcyclohex-2-enone (Brady et al., 1993), the allyl-substituted barbiturates secobarbital, sedormid, and the chemically related allylisopropylacetamide (AIA) (Ortiz de Montellano et al., 1984), griseofulvin (De Matteis et al., 1991; Holley et al., 1991), hexachlorobenzene (Wainstok de et al., 1989), dihydropyridine calcium antagonists (Schoenfeld et al., 1985), the antiarthritic sydnone 3-[(arylthio)ethyl]sydnone (TTMS) (McNamee and Marks, 1996; Sutherland et al., 1986), and the garlic derivative diallyl sulfone (Black et al., 2006). The mechanism of induced protoporphyria in laboratory animals involves mechanism-based inactivation of CYPs (De Matteis and Marks, 1996; Gamble et al., 2000; Marks et al., 1988; Marks et al.,1989). Reactive metabolites bind to the prosthetic heme of CYP through N-alkylation of one of the pyrrole moieties. The alkylated heme then dissociates from the apoprotein with the iron atom being liberated, yielding N-alkylprotoporphyrin IX (N-alkylPP). Once released, some N-alkylPPs have the potential to inhibit ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. After loss of alkylated heme, CYP apoprotein reconstitutes with fresh heme. This phenomenon and the impaired heme biosynthesis are responsible for free heme pool depletion, which in turn stimulates an up-regulation of δ-aminolevulinic acid synthase (ALAS). As a result of all these changes, protoporphyrin IX accumulates in tissues, as characteristic dark pigments. The present work describes a new synaptic vesicle protein 2a (SV2a) antiepileptic drug candidate that was tested in 4-week oral toxicity studies in rat and dog. Brown pigments were found in the liver of dogs treated with 200 mg/kg/day of the compound. The pigment's characteristic red birefringence under polarized light, with occasional “Maltese cross” configurations, suggested accumulated hepatic porphyrin. These findings were accompanied by increased plasma liver enzymes. Although exposed to much higher drug levels, rats did not show any liver toxicity. Assays were conducted to explore the mechanisms underlying the dog liver findings and evaluate their clinical relevance.