The effects of antirhino- and enteroviral vinylacetylene benzimidazoles on cytochrome P450 function and hepatic porphyrin levels in mice
Introduction
The antipicornaviral benzimidazoles, which were originally discovered at Lilly Research Laboratories over 20 years ago, are a unique class of compounds with potent, broad-spectrum activity against both rhino- and enteroviruses (DeLong et al., 1978, DeLong and Reed, 1980, Wikel et al., 1980, Herrmann et al., 1981 Victor et al., 1997b). In the early 1980’s, two candidates, 1 and 2, from this series were tested in clinical trials (Fig. 1). Enviroxime (1) suffered from poor plasma concentrations on oral dosing (Cmax=14 ng, 100 mg dose) and marginal clinical efficacy in challenge studies as well as demonstrating some undesirable side effects in both dogs and humans (DeLong, 1984, Miller et al., 1985). Enviradene (2) showed improved pharmacokinetics in dogs (46% oral bioavailability) and showed no adverse toxicological events in dogs or humans. In humans, however, it gave unacceptably low plasma concentrations (Cmax=69 ng, 500 mg dose) and was, therefore, never advanced to challenge studies (Enviradene-IND, 1981a, Enviradene-IND, 1981b). Further work on this compound class was not pursued for over a decade at which time attempts were made to improve the bioavailability of the antiviral benzimidazoles by reducing metabolic clearance rates.
The initial discovery of vinylacetylene benzimidazoles (3) evolved from efforts to find a metabolic obstacle for the allylic oxidation that was seen at the vinyl methyl position of Enviradene (Fig. 1). The acetylene modification did indeed appear to inhibit the oxidative metabolism at the vinyl methyl position as evidenced by the improvement in oral bioavailability observed in rhesus monkeys (Enviradene-IND, 1984, Bopp and Quay, 1985). Additionally, in vitro metabolism studies carried out using hepatocytes from several different animal species, including human, demonstrated that the extent of oxidative metabolism observed in this new series was substantially less than that seen with Enviroxime or Enviradene (Victor et al., 1997a). Based on these preliminary results as well as on the resolution of several synthetic issues (Tebbe et al., 1997), efforts to synthesize new vinylacetylene benzimidazoles were renewed with an emphasis on optimizing antiviral activity while maximizing oral plasma concentrations in mice (Tebbe et al., 1997). As with their predecessors, many of these new compounds are potent antivirals, and they consistently demonstrate broad-spectrum antiviral activity against a range of both rhino- and enteroviruses. Additionally, compounds dosed orally to mice during initial discovery studies demonstrated significant exposure (Tebbe et al., 1997). This research effort culminated in the selection of four compounds for further investigation (Fig. 2).
The major structural difference between this series of compounds and the earlier series studied is the incorporation of an acetylene moiety. Since the acetylene functionality is known in some cases to be metabolically labile and potentially hepatotoxic, a study was designed to assess the propensity of this series to produce these undesirable effects. The major concern with alkynes is their interactions with cytochromes P450. The cytochrome P450 monooxygenases (P450s) are a group of hemoproteins that catalyze the oxidative metabolism of endogenous substrates such as steroids and vitamins as well as xenobiotics. A variety of compounds containing an acetylene moiety have been shown to produce destruction of the heme sub-unit of several P450 isoforms leading to irreversible mechanism-based inhibition of enzymatic activity and decreases in the measurable P450 content (Oritz de Montellano and Kunze, 1980, Oritz de Montellano and Correia, 1983, Hopkins et al., 1992, Chan et al., 1993, Foroozesh et al., 1997). Further, the destruction of the heme moiety may cause depletion of the free heme pool and accumulation of porphyrin intermediates in the liver (Marks et al., 1988). Thus, repeated exposure to porphyrinogenic drugs can produce sufficient porphyrin accumulation to induce hepatic porphyria, which may ultimately lead to hepatic necrosis. Given the anticipated use of these compounds to treat a self-limiting viral infection, even a hint of hepatotoxicity would be unacceptable. The mere presence of an alkyne in a molecule is not sufficient to produce these unwanted P450 interactions, however, since molecule-enzyme interactions are dependent on the compound as a whole. In fact, there are many marketed drugs that contain alkynes and do not appear to have any detrimental effects on P450, its function, or hepatic porphyrin levels (Ross, 1997).
The current studies were conducted to assess the potential for the vinylacetylene antivirals to inactivate hepatic cytochromes P450 and cause porphyrin accumulation in mice following single or multiple oral doses of the compounds. In a single dose study, plasma concentrations of the drugs were determined to confirm adequate oral exposure in the mouse, and hepatic porphyrin levels were measured. In a more intensive multiple dose study, the animals were dosed for seven days, and porphyrin levels were determined. In addition, the possible mechanism-based destruction of hepatic cytochromes P450 was evaluated by determining the cytochrome P450-dependent 7-ethoxyresorufin O-deethylase and benzphetamine N-demethylase activities as well as the total cytochrome P450 content. Limited clinical chemistry was also performed in order to monitor for early signs of hepatotoxicity.
Section snippets
Antiviral agents
Compounds 4, 5, 6 and 7 were synthesized at Lilly Research Laboratories as previously described (Tebbe et al., 1997).
Antiviral activity
The antiviral activity and cellular toxicity of the compounds was measured as previously described (Tebbe et al., 1997). In general, susceptible Hela cells were grown in 6-well tissue culture cluster plates. When confluent monolayers were formed, growth medium was removed and an appropriate dilution of virus was added. After adsorption for 1–2 h at room temperature, the infected
In vitro antiviral activity
The four vinylacetylenes selected for the current investigation are the result of optimization of antiviral activity and plasma concentrations in mice using a screening model. The spectrum and potency of the compounds are illustrated in Table 1. These compounds all demonstrated good antiviral potency as well as good broad-spectrum activity in the viruses selected to test against.
Single dose study
In order to examine the effects of the acetylene functionality on liver enzymes and hepatic porphyrin levels as well
Discussion
The antirhino- and enteroviral benzimidazoles have undergone extensive evaluations in efforts to define a series of compounds to take into the clinic to test the efficacy of an oral drug for treatment of the common cold. Two compounds were advanced into the clinic in the early 1980’s to try to address this question; however, both compounds showed poor oral bioavailability in humans. A series of vinylacetylene benzimidazoles was found to produce improved oral bioavailability which subsequently
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