Preclinical safety profile of sildenafil.

Sildenafil citrate, marketed as Viagra, for the treatment of erectile dysfunction, has a proven record of safety in humans as predicted by the results of extensive pharmacological and toxicological testing in animals and in vitro, and confirmed by pharmacokinetic exposure data. The aim of this paper is to review succinctly the main findings resulting from these experiments. Daily doses of sildenafil, within and far beyond the human therapeutic range, were given to dogs and rodents for up to 1 and 2 y, respectively. Plasma analyses were conducted to determine the exposure to sildenafil. We found species-specific effects in dogs (Beagle pain syndrome), mice (marked intestinal dilatation) and rats (adaptive reversible hepatocellular hypertrophy associated with secondary thyroid hypertrophy). All these effects in rodents and dogs have no relevance to humans. Morphometric thickness measurements of the retinal layers carried out in response to clinical observations of visual disturbances in humans indicated no difference between treated and control rats and dogs after up to 24 months of treatment. There was no evidence of histopathologic damage to any structures of the visual pathway. Sildenafil had no effects on fertility, no teratogenic potential, was not genotoxic and has no carcinogenic potential. In rats and dogs, safety ratios were 40:1 and 28:1, respectively, in terms of exposure over 24 h (AUC24 h) and 19:1 and 8:1, respectively, in terms of peak plasma concentration (Cmax). These safety ratios illustrate the separation between exposure to sildenafil of animals at large nontoxic doses and the much smaller human therapeutic exposure. This profile highlights the very low risk of human toxicity for sildenafil. The favourable results of the nonclinical safety evaluation of sildenafil in established animal models have been confirmed by many years of clinical experience during the development and marketing of sildenafil.

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures.

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.

Establishment of a rat Sertoli cell line that displays the morphological and some of the functional characteristics of the native cell.

Primary rat Sertoli cells are widely used as a model for mechanistic and toxicological studies, since they are often the target of toxicants in vivo. However, their isolation from testicular homogenates is tedious and requires the regular use of numerous immature animals. It is therefore of great interest to have available established cell lines that are usable in vitro for unlimited periods and closely similar to native cells. To this end, we have established a line of Wistar rat Sertoli cells (SerW3) by immortalization of fresh primary cells with the T antigens of the Simian virus (SV40). When plated on Matrigel, this cell line presents many of the functional characteristics of Sertoli cells in vivo. In addition, they are sensitive to cisplatin and secrete transferrin, although they do not show a clear response to follicle-stimulating hormone. They also present many morphological similarities, including the presence of tight junctions which mimic the natural epithelial barrier. Like Sertoli cells in vivo, they show extensive phagocytic activity. Finally, they display all the characteristics of immortalized, but not transformed, cells, i.e., topo-inhibition and apoptosis at confluence or under serum deprivation.

Diurnal exposure profile in rats from dietary administration of a chemical (doxazosin) with a short half-life: interplay of age and diurnal feeding pattern.

Doxazosin, an alpha-adrenergic blocking agent, has a plasma half-life in male rats of 1-2 h after i.v. administration. Plasma concentrations of doxazosin were measured in male rats receiving the drug mixed in the diet at dose levels from 5 to 40 mg kg-1. Samples taken at 4-h intervals during the light (0700-1900) and dark phases revealed peak concentrations at 0400 which were only about three times higher than the trough concentrations observed ca. 12 h later. The 24-h area under the curve (AUC) values increased disproportionately with dose and with age from 2 months up to 8 months of age; thereafter they were fairly stable to 24 months of age. This age-related effect may have been due to a reduction in clearance and/or a change in the feeding pattern of the rats. Young rats consumed ca. 84% and old rats only 45% of their daily feed during the nocturnal (active) phase. Given the known diurnal rhythms in absorption, protein binding and enzyme metabolising activity, such a change in feeding pattern with age may have wider toxicokinetic implications.

Design of toxicokinetic studies.

1. Toxicokinetics is defined as pharmacokinetic studies in animals during actual toxicity studies or under conditions mimicking them (species, duration, dose level, etc.). 2. Toxicology studies require toxicokinetics to check whether systemic exposure reflects administered dose. In particular, it is important to know whether the absence of toxicity at a given dose is due to the innocuousness of the compound or to its poor bioavailability. 3. Pivotal toxicology studies may require different toxicokinetic support than that of early studies, as more is learned of the compound and its metabolites. Considerations need to be placed on such factors as the choice of biological matrix for drug assay, the relevance of metabolites, and which dose levels require the most pharmacokinetic investigation.

A comparative study of the effects of ketoconazole and fluconazole on 17-beta estradiol production by rat ovaries in vitro.

In this study we have compared the effects of ketoconazole and fluconazole, a novel triazole antifungal agent, on 17-beta estradiol production in rat ovaries in vitro. For both compounds there was a lag phase, immediately after addition to the test system, during which the rate of oestradiol synthesis remained at control values. This may have been due to the time required for uptake of the compound and transfer to its site of action or for depletion of endogenous pools of intermediates. After the lag phase both compounds produced a reduction in the rate of estradiol synthesis. At any given concentration, fluconazole produced a reduction which was substantially less than that observed with ketoconazole. Indeed 2 microM ketoconazole reduced the rate of oestradiol production by greater than 90% while 10 microM fluconazole caused only a 70% reduction. These findings are consistent with reports that these compounds are inhibitors of cytochrome P450 and with the reduced sensitivity of mammalian cytochrome P450 to fluconazole as compared with ketoconazole.

The effect of luteinizing hormone on parturition in rats; imidazole antifungals may affect parturition via luteinizing hormone.

Studies were carried out to investigate the mechanism whereby the imidazole anti-fungal, tioconazole, affects parturition in rats. Administration s.c. of luteinizing hormone (LH) to pregnant rats on days 15-17 post-insemination (p.i.) or days 15-21 p.i. delayed the onset of parturition by a day and markedly reduced the ovarian production of 17 beta-estradiol, but not of progesterone. Administration of LH on days 18 and 19 p.i., which was already known to advance birth, reduced ovarian production of progesterone, but not of 17 beta-estradiol. The similarity of these results to those for tioconazole administered from days 15 p.i. or on days 18 and 19 p.i. suggests that tioconazole affects parturition in rats, at least in part, via LH.

Dazoxiben, a prototype inhibitor of thromboxane synthesis, has little toxicity in laboratory animals.

Dazoxiben, an orally active specific inhibitor of thromboxane synthetase, was administered by mouth daily to dogs and rats for 6 months. Dogs showed no evidence of toxicity up to 300 mg day-1 kg-1, the highest dose level used. Rats showed no evidence of toxicity after 100 mg day-1 kg-1, but at 300 mg day-1 kg-1 there were slight increases in plasma calcium and urea concentrations and a moderate incidence of focal nephrosis; males showed a slightly increased platelet count. Studies in rats and rabbits at dose levels up to 400 mg day-1 kg-1, by mouth, revealed no adverse effects on male or female fertility, embryogenesis, parturition or postnatal development. As dazoxiben is well absorbed after oral administration, the generally negative outcome to these toxicity studies suggests that selective inhibitors of thromboxane synthesis may be largely free of adverse effects which might impede their therapeutic or prophylactic use in clinical medicine.

A second peak in uptake of gentamicin by rat kidney after cessation of treatment.

Gentamicin was administered s.c. to rats once daily for 7 days at 5 mg kg-1. At the end of this period renal concentrations of gentamicin declined from day 1 to day 5 postdosing, but showed a second peak on day 8, after which they declined again through days 16 and 22. This second peak coincided with a peak of mitotic activity in the kidney, suggesting that this organ, while recovering from gentamicin-induced damage, passes through a temporary phase during which it has increased affinity for gentamicin.