Toxicokinetics and toxicity of atorvastatin in dogs.

HMG-CoA reductase inhibitors (e.g., statins) are an important clinical option to lower cholesterol and treat co-morbidities. Atorvastatin is the most prescribed statin and has obtained generic status. We recently had a clinical development program evaluating a combination of atorvastatin with a GPR119 agonist as a treatment for dyslipidemia, where toxicological evaluations in dogs were completed. There were several challenges related to selecting doses for atorvastatin, including understanding the dose-exposure relationship from different drug forms used by the innovator in their general toxicology studies, bioanalytical assays that did not separate and quantify parent from metabolites, and high variability in the systemic exposures following oral dosing. The studies in this report characterized the toxicokinetics and toxicity of atorvastatin in the dog for up to 13-weeks. Overall, there were no notable differences in the toxicokinetics of atorvastatin or the two active hydroxylated metabolites between the sexes at Week 13. However, systemic exposures were markedly lower at Week 13 compared to that observed at Week 4, suggesting induction of metabolism or reduced absorption from the gastrointestinal tract following oral dosing. Changes in laboratory chemistries included increased liver enzyme levels and lower cholesterol levels. Histopathologic evaluation revealed multifocal minimal to slight hemorrhages in the submucosa of the gallbladder; all findings were reversible. The information from these studies along with the existing clinical experience with atorvastatin can be used to design robust toxicology studies in dogs and reduce animal use.

GSK2374697, a novel albumin-binding domain antibody (AlbudAb), extends systemic exposure of exendin-4: first study in humans--PK/PD and safety.

GSK2374697 is a genetically engineered fusion protein of a human domain antibody to exendin-4. This molecule binds with a high affinity to human serum albumin, creating a long-duration glucagon-like peptide-1 (GLP-1) receptor agonist. This study is the first evaluation of the albumin-binding domain antibody (AlbudAb) drug delivery platform in humans. The aim of this randomized clinical study was to determine the pharmacokinetics, pharmacodynamics, safety, and tolerability of GSK2374697. The pharmacokinetic profile was prolonged, with estimated half-lives ranging from 6 to 10 days. Postprandial glucose and insulin were reduced, and gastric emptying was delayed in healthy subjects, confirming anticipated GLP-1 receptor agonist pharmacology. The safety and tolerability were as expected for a potent GLP-1 agonist. Gradual titration of doses greatly improved tolerability. Rapid tolerance to nausea was observed. Study results support further investigation in type 2 diabetes and for weight loss.

Allocare patterns among cercopithecines.

Cercopithecines show two general patterns of allocare. Many guenons permit extensive contact with young infants, while most baboons and macaques restrict early contact. Here social, ecological and life history variables among cercopithecines are examined for evidence of relationships with allocare patterns. This analysis suggests that in this group early allocare is most extensive in species with relatively relaxed social relationships among females, marked seasonal breeding and rapid rates of reproduction.

Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats.

The relationship between biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats was examined. The biliary excretion of seven model substrates in 96-h sandwich-cultured rat hepatocytes was determined by differential cumulative uptake of substrate in the monolayers preincubated in standard buffer (intact bile canaliculi) and Ca2+-free buffer (disrupted bile canaliculi). Biliary excretion in vivo was quantitated in bile duct-cannulated rats. The biliary excretion index of model substrates, equivalent to the percentage of retained substrate in the canalicular networks, was consistent with the percentage of the dose excreted in bile from in vivo experiments. The in vitro biliary clearance of inulin, salicylate, methotrexate, [D-pen2,5]enkephalin, and taurocholate, calculated as the ratio of the amount excreted into the bile canalicular networks and the area under the incubation medium concentration-time profile ( approximately 0, approximately 0, 4.1 +/- 1.0, 12.6 +/- 2.2, and 56. 2 +/- 6.0 ml/min/kg, respectively), correlated with their intrinsic in vivo biliary clearance (0.04, 0, 17.3, 34.4, and 116.9 ml/min/kg, respectively; r2 = 0.99). The model compound 264W94 was not excreted in bile either in vivo or in vitro. The glucuronide conjugate of 2169W94, the O-demethylated metabolite of 264W94, was excreted into bile in vitro when 2169W94, but not 264W94, was incubated with the monolayers; 2169W94 glucuronide undergoes extensive biliary excretion after administration of 264W94 or 2169W94 in vivo. Biliary excretion in long-term sandwich-cultured rat hepatocytes correlates with in vivo biliary excretion. The study of biliary excretion of metabolites in the hepatocyte monolayers requires consideration of the status of metabolic activities.

The pharmacokinetics and extra-hepatic clearance of remifentanil, a short acting opioid agonist, in male beagle dogs during constant rate infusions.

Remifentanil is a mu opioid receptor agonist, structurally related to fentanyl, being developed for use in anesthesia. Remifentanil was designed to be cleared rapidly by ester hydrolysis. To determine the pharmacokinetics of remifentanil in conscious beagle dogs, venous blood was collected at various times during and after the end of a 25 min intravenous infusion of the compound (0.36 or 36.0 micrograms [free base]/kg/min). In a separate set of studies designed to measure tissue clearance of remifentanil, catheters were implanted in various blood vessels of anesthetized beagle dogs to sample blood entering and leaving selected tissues. Approximately 40 min (steady state achieved) after initiation of remifentanil infusions at the same rates as described above, and while infusions were still in progress, blood was collected from the series of catheters. In a third set of experiments, blood obtained from either untreated dogs or from a dog that had been anesthetized, was incubated with remifentanil (either 10 or 1000 ng (free base)/ml). The observed half-life was used to provide an estimate of in vivo blood metabolic clearance of the compound. Extracts of the blood from all experiments were analyzed by either an HPLC or mass spectrometry assay for remifentanil concentration. There were no differences in systemic clearance (approximately 45 ml/kg/min), volume of distribution at steady state, mean residence time, dose-normalized normalized concentration at steady state, or dose-normalized AUC between the doses administered to conscious dogs; the t1/2 alpha was 3-5 min. In the anesthetized animals, muscle and intestine had the highest tissue clearance rates, but liver, kidneys, and blood each accounted for 1% or less of systemic clearance. The results indicate that, within the range of doses studied, the pharmacokinetics of remifentanil during infusion to steady state in dogs were not dependent on dose, and that the liver contributed very minimally to the overall clearance of the compound.

Diminished drug metabolism due to hormonal effects of cobalt mesoporphyrin on flavoproteins.

Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 mumol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by > 50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 mumol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 mumol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 mumol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 mumol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.

In vitro activation of 2-aminobenzyl alcohol and 2-amino-6-nitrobenzyl alcohol, metabolites of 2-nitrotoluene and 2,6-dinitrotoluene.

Previous results have suggested that key intermediates in the activation of 2-nitrotoluene and 2,6-dinitrotoluene are 2-aminobenzyl alcohol and 2-amino-6-nitrobenzyl alcohol, respectively. In order to determine the metabolic pathway(s) involved in the activation steps, calf thymus DNA and [14C]-2-aminobenzyl alcohol or [14C]-2-amino-6-nitrobenzyl alcohol were incubated with male Fischer-344 rat hepatic cytosol and PAPS, microsomes and NADPH, or microsomes and cytosol with PAPS, NADPH, and acetyl coenzyme A. DNA was isolated and analyzed for radiolabel bound covalently. Analysis of the incubations containing [14C]-2-aminobenzyl alcohol revealed radiolabel bound covalently to DNA, as well as one major metabolite labile in both sulfatase and acid. The appearance of each required the presence of PAPS and cytosol and was inhibited by the sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol. Neither NADPH nor acetyl coenzyme A played a role in the generation of detectable 14C bound to nucleic acids. 2-Amino-6-nitrobenzyl alcohol was converted to metabolites capable of binding to calf thymus DNA when incubated with cytosol and PAPS or with microsomes and NADPH. However, when cytosol and microsomes were incubated together, activation of 2-amino-6-nitrobenzyl alcohol appeared to require only PAPS, suggesting a minor role for NADPH-dependent enzymes in its activation. The results suggest that the metabolite of 2-nitrotoluene responsible for binding covalently to DNA is 2-aminobenzyl sulfate. There may be more than one pathway involved in the formation of metabolite(s) of 2,6-dinitrotoluene that bind covalently to DNA.

Pregnancy outcome in medically complicated and uncomplicated patients aged 40 years or older.

Patients (183) who were delivered at age greater than or equal to 40 years were studied to ascertain the nature and frequency of maternal and fetal complications at a single institution in a recent time period. These patients were further grouped into those of low parity, those who began pregnancy without underlying disease, and those who began pregnancy with underlying medical disorders. For the entire group preeclampsia, premature labor, precipitate labor, and malpresentation were significantly more common. The rate of vaginal delivery was substantially decreased, and serious postpartum morbidity was relatively common. The incidence of stillbirth, perinatal mortality, and abnormal birth weight was significantly increased. There were some differences in the nature and frequency of complications encountered among the subgroups, but no subgroup had a complication rate comparable to our general obstetric population.

Isomer- and sex-specific bioactivation of mononitrotoluenes. Role of enterohepatic circulation.

2-Nitrotoluene (2NT) induces DNA repair in the in vivo-in vitro hepatocyte unscheduled DNA synthesis assay in male, but not female, Fischer-344 rats. 3-Nitrotoluene (3NT) and 4-nitrotoluene (4NT) are inactive in both sexes. The structurally related rat hepatocarcinogen, 2,6-dinitrotoluene, which also displays a sex-specific toxicity, requires biliary excretion for bioactivation. Therefore, the role of enterohepatic circulation in the development of the isomer- and sex-specific hepatic bioactivation of the mononitrotoluenes was studied in male and female Fischer-344 rats. Male rats excreted 28.6, 10.8, and 9.8% of a dose (200 mg/kg) of 2NT, 3NT, or 4NT, respectively, in the bile 12 hr after the dose. Female rats excreted 9.6, 4.3, and 1.3% of a dose of 2NT, 3NT, or 4NT, respectively, in the bile 12 hr after the dose. Of the 2NT-related material excreted in the bile, 77% in males (22.0% of the dose) and 86% in females (8.3% of the dose) was 2-nitrobenzyl glucuronide. Of the 3NT-related material excreted in the bile, 28% in males (2.8% of the dose) and 16% in females (0.7% of the dose) was 3-nitrobenzyl glucuronide. Of the 4NT-related material excreted in bile, 9% in males (0.9% of the dose) and 7% in females (0.1% of the dose) was 4-nitrobenzyl glucuronide. Inhibition of enterohepatic circulation by bile duct cannulation resulted in a decrease in hepatic macromolecular covalent binding by 98, 75, and 78% in male rats, and by 85, 44, and 45% in female rats after 2NT, 3NT, or 4NT, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism and excretion of mononitrotoluenes by Fischer 344 rats.

The metabolism and excretion of 2-, 3-, and 4-nitrotoluene (2NT, 3NT, and 4NT, respectively) were studied in male Fischer 344 rats. Excreta were collected for 72 hr after an oral dose (200 mg/kg) of radiolabeled 2NT, 3NT, or 4NT. Radiolabel from each NT isomer was rapidly excreted (86, 73 and 74% of the dose for 2NT, 3NT, and 4NT, respectively in 24 hr). The urine was the major route of excretion with 70-85% of the dose being excreted by that route in 72 hr. Five to 13% and 0.0 to 0.1% of the dose was excreted in the feces and expired air, respectively, in 72 hr. The major metabolites excreted in urine in 72 hr after administration of 2NT were 2-nitrobenzoic acid (29% of the dose), an unidentified metabolite (16% of the dose), 2-nitrobenzyl glucuronide (14% of the dose), and S-(2-nitrobenzyl)-N-acetylcysteine (12% of the dose). The major metabolites excreted in urine in 72 hr after administration of 3NT were 3-nitrohippuric acid (24% of the dose), 3-nitrobenzoic acid (21% of the dose), and 3-acetamidobenzoic acid (12% of the dose). The major metabolites excreted in urine in 72 hr after administration of 4NT were 4-nitrobenzoic acid (28% of the dose), 4-acetamidobenzoic acid (27% of the dose), and 4-nitrohippuric acid (13% of the dose). Thus, the major urinary metabolites of 3NT and 4NT differed only quantitatively while those of 2NT were also qualitatively different.

Influence of dietary pectin on intestinal microfloral metabolism and toxicity of nitrobenzene.

Intestinal microfloral metabolism of nitrobenzene is essential for the production of methemoglobin. Since dietary pectin alters intestinal microflora, these studies were designed to examine the effects of dietary pectin on nitrobenzene-induced methemoglobinemia. Male Fischer-344 rats were fed either AIN-76A (purified diet containing 5% cellulose), AIN-76A with 5% pectin replacing the cellulose, or NIH-07 (cereal-based diet containing 8.4% pectin) for 28 days. Following this period, nitrobenzene (200 mg/kg) was administered by gastric intubation, and methemoglobin concentrations were determined after 1, 2, 4, 8, and 24 hr. Nitrobenzene-induced methemoglobinemia was evident as early as 1 hr, peaked at 4 hr, and diminished thereafter in rats fed NIH-07 diet. In contrast, nitrobenzene-induced methemoglobinemia was not detectable in rats fed AIN-76A; however, inclusion of 5% pectin in this diet resulted in methemoglobinemia comparable to that of NIH-07-fed animals at 4, 8, and 24 hr. Administration of 400 or 600 mg/kg nitrobenzene resulted in significant diet-related differences in methemoglobinemia. Administration of 600 mg/kg nitrobenzene to animals fed NIH-07 resulted in the highest methemoglobin concentrations (64 +/- 1%); those fed AIN-76A had the lowest (20 +/- 5%), and those fed AIN-76A containing pectin had intermediate methemoglobin concentrations (44 +/- 6%). No diet-related differences in the microbial population of the stomach or small intestine were observed. However, the number of anaerobes present in the ceca of rats fed AIN-76A containing pectin was 2 to 2.5 times greater than that of rats fed AIN-76A. In vitro reductive metabolism of [14C]nitrobenzene was significantly greater in the cecal contents of rats fed NIH-07 than that in the cecal contents of either of the groups fed the AIN-76A-based diets. These studies indicate that intestinal microfloral metabolism and red blood cell toxicity of nitrobenzene is markedly different in animals fed cereal-based versus purified diets. Furthermore, since inclusion of pectin into the purified diet diminishes the magnitude of these effects, differences in dietary composition of fermentable carbohydrates in cereal-based and purified diets may mediate differences in metabolism and toxicity of nitrobenzene.

Induction of hepatic and testicular lesions in Fischer-344 rats by single oral doses of nitrobenzene.

Since the acute toxicity of nitrobenzene (NB) in rats has not been characterized, experiments were performed to ascertain the possible deleterious effects of NB in different tissues of the male Fischer-344 rat. Rats were given single oral doses of NB (50-450 mg/kg) and at the time of sacrifice, 25 tissues were removed and examined histologically by light microscopy. Histopathological changes induced by a single oral dose of NB consistently involved only the liver and testes. One rat receiving 450 mg NB/kg had a microscopic cerebellar lesion. Hepatic centrolobular necrosis appeared inconsistently in rats given various doses of NB, while hepatocellular nucleolar enlargement was consistently detected in rats given doses of NB as low as 110 mg/kg. These data suggest that nucleolar enlargement was independent of cell death and subsequent regeneration. Testicular lesions were confined to the seminiferous tubules and consisted of necrosis of the primary and secondary spermatocytes with the appearance of multinucleated giant cells between one and four days after administration of NB 300 mg/kg. Necrotic debris and decreased numbers of spermatozoa were seen in the epididymis as early as three days after NB administration. The NB-induced methemoglobinemia does not appear to be solely responsible for the formation of early lesions in the rat liver, testes, or brain, since sodium nitrite administration, at dosages which produced methemoglobinemia equivalent to that of NB, did not produce any histopathological changes. Thus, the observed liver and testicular damage are probably due to a direct effect of NB or its metabolites.

Analytical approaches to the study of the disposition of myelotoxic agents.

A correlation of myelotoxic effect with concentration or a foreign compound of its metabolite at the site of action may provide useful insights into the mechanism of toxic action and/or its amelioration. This correlation requires sensitive and specific assay methods. This communication describes useful methods for the study of benzene disposition in rodents. The assays are sensitive, specific, and rapid. They rely on gas chromatography-mass spectrometry and on high performance liquid chromatography. These methods have allowed measurement of catechol, phenol, and hydroquinone in samples of rodent bone marrow following inhalation exposure to benzene. Their application to the study of benzene metabolism in rat bone marrow in situ is also described.