Isolation and expression of cDNAs from rainbow trout (Oncorhynchus mykiss) that encode two novel basic helix-loop-Helix/PER-ARNT-SIM (bHLH/PAS) proteins with distinct functions in the presence of the aryl hydrocarbon receptor. Evidence for alternative mRNA splicing and dominant negative activity in the bHLH/PAS family.

cDNAs encoding two distinct basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins with similarity to the mammalian aryl hydrocarbon nuclear translocator (ARNT) protein were isolated from RTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human ARNT. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence of an additional 373 base pairs of sequence that have the characteristics of an alternatively spliced exon. The presence of the 373-base pair region causes a shift in the reading frame. rtARNTa lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNTb contains the sequence and has a COOH-terminal domain of 190 residues rich in glutamine and asparagine. mRNAs for both rtARNT splice variants were detected in RTG-2 gonad cells, trout liver, and gonad tissue. rtARNTa and rtARNb protein were identified in cell lysates from RTG-2 cells. Transfection of rtARNT expression vectors into murine Hepa-1 cells that are defective in ARNT function (type II) result in rtARNT protein expression localized to the nucleus. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appears to be due to inefficient binding of rtARNTa.AHR complexes to DNA. In addition, the presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro.

Pharmacokinetics of the anticancer agent sulofenur in mice, rats, monkeys, and dogs.

The absorption and pharmacokinetics of sulofenur [N-(indan-5-sulfonyl)-N'-(4-chlorophenyl)urea, LY186641] and its major metabolites were examined in mice, rats, monkeys, and dogs. The compound is a diarylsulfonylurea currently being evaluated as an oncolytic agent in phase I and II trials. In all species, sulofenur was well absorbed after an oral dose, but over a prolonged period, and sulofenur exhibited a fairly long half-life of elimination from plasma. These values ranged from 6 h in rats up to 30, 110, and 200 h in mice, monkeys, and dogs, respectively, at doses (240-1000 mg/m2) within the range of those used in clinical trials. Experiments describing the high degree of binding of sulofenur to plasma proteins (consistently > 99%) help to explain these relatively long half-lives. There is, however, a large difference between these plasma half-lives in the species studied. Sulofenur was previously found to be extensively metabolized to products that are excreted primarily into the urine. In this study, its major metabolites, which are found mainly in the urine, were also minor components of the drug-related material (< 10% of the sulofenur concentrations) in the plasma of rats treated with sulofenur. The absorption, binding characteristics, and elimination of these major metabolites after their administration to rats were also compared with sulofenur.(ABSTRACT TRUNCATED AT 250 WORDS)

A species comparison of the toxicity of nabilone, a new synthetic cannabinoid.

Acute, subchronic, and chronic studies were conducted in various species to evaluate and compare the toxicity of nabilone, a new synthetic 9-ketocannabinoid that is orally effective for the treatment of nausea and vomiting induced by cancer chemotherapy agents. The oral LD50 in mice and rats for nabilone formulated as a polyvinylpyrrolidone (PVP) codispersion was in excess of 1000 mg/kg. Among nonrodents, rhesus monkeys had a higher tolerance to the CNS depression induced by single oral doses of nabilone-PVP than did dogs. Rats fed dietary mixtures of nabilone-PVP which provided approximate daily nabilone doses of 1 to 93 mg/kg tolerated treatment for 3 months with no deaths. Treatment-related changes (at doses greater than or equal to 5 mg/kg) were limited to reduced body temperature, slight-to-moderate decreases in weight gain, and behavioral changes (e.g., hyperactivity, hyperirritability to touch, and hypoactivity). All dogs treated for 3 months with daily oral doses of up to 1.0 mg/kg survived; treatment-related effects were limited to transient episodes of ataxia and anorexia. Nabilone treatment of rats and dogs for 3 months produced no evidence of systemic toxicity in the clinical chemistry, hematology, or pathology parameters examined. Chronic treatment of dogs with daily oral doses of nabilone-PVP equal to 0.5, 1.0, or 2.0 mg of nabilone/kg produced cumulative toxicity; by the end of 7 months, 2, 6, and 7 dogs in the respective dose groups had died. In a number of instances, death was preceded by one or more convulsive episodes. In contrast to the dog, the toxic potential of nabilone was minimal in rhesus monkeys treated with nabilone-PVP for 1 year at daily oral nabilone doses of up to 2.0 mg/kg. The enzymatic reduction of the 9-keto group of nabilone to form carbinol metabolites was a major metabolic pathway for nabilone in dogs but not in rhesus monkeys. The carbinols were long-lived metabolites in the plasma of dogs and accumulated in the plasma compartment with time. Furthermore, the carbinol metabolites were found to concentrate in the brain tissues of treated dogs. Although the precise mechanism for this marked species difference in chronic toxicity is not known, the metabolic differences responsible for the presence of the carbinol metabolites at high concentrations in the plasma and brain over time may play a role in the toxicity observed in the dog.

Species specificity in the metabolism of nabilone. Relationship between toxicity and metabolic routes.

The disposition of 14C-nabilone has been examined in dogs and monkeys; it is rapidly absorbed and extensively metabolized in both species. A metabolic pathway initiated by the stereoselective enzymic reduction of the 9-keto moiety of nabilone was of major importance in the biotransformation of nabilone in the dog but was only a minor pathway in the monkey. The resulting long half-lived carbinol metabolites accounted for 77% of the circulating 14C in dog but for only 19% in monkey, 48 h after drug administration. Accumulation of carbinol metabolites in brain tissue was observed in dogs, with 24 h brain concentrations being five to six times higher than the plasma concentrations. No accumulation of carbinol metabolites, nabilone or of 14C occurred in the brain of monkeys. Accumulation of the carbinol metabolites in brain tissue has been implicated as the causative factor for the CNS toxicity observed in dogs treated chronically with nabilone. Comparison of nabilone pharmacokinetics in dog and humans showed the dog to be unique in producing high levels of carbinol metabolites, so that monkey appeared to be a more appropriate model than dog for pre-clinical toxicological and safety studies. Studies with liver 15,000 g supernatants indicated that the enzymic reduction of nabilone to its carbinol metabolite was a viable metabolic pathway in rat, dog and monkey, so that a distinct species difference exists between dog and monkey in the subsequent metabolism and/or elimination of these carbinol metabolites.

Absorption and metabolism of anitrazafen, a topically effective anti-inflammatory agent, in the rat.

1. The metabolism and pharmacokinetics of anitrazafen, a topically effective anti-inflammatory agent, have been investigated in the rat after oral, subcutaneous and topical administration. 2. [14C]Anitrazafen is rapidly absorbed from the gastro-intestinal tract and subsequent metabolism is rapid and extensive; biliary excretion is the major route of elimination. After subcutaneous or topical administration, elimination of [14C]anitrazafen was delayed due to a slower rate of systemic absorption. 3. Pharmacokinetic studies confirmed these results. After oral administration peak concn. of parent drug were attained within 1 h; plasma concn. of 14C were an order of magnitude greater than those of unchanged drug. The apparent volume of distribution of anitrazafen was high (112l/kg) consistent with observed tissue 14C concentrations. Subcutaneous administration resulted in delayed absorption but with maximum bioavailability of parent drug. Absorption was slowest following topical application. 4. Anitrazafen was extensively metabolized in rats. No unchanged drug was found in excreta. The most important mechanism of biotransformation was oxidative O-demethylation, with glucuronide or sulphate conjugates of two isomeric mono-O-demethylated and the di-O-demethylated analogues of anitrazafen, as metabolites.

Biotransformation of 4'-ethynl-2-fluorobiphenyl in the rat. In vitro and in vivo studies.

The absorption, metabolism, and plasma pharmacokinetics of a novel anti-inflammatory agent, 4'-ethynyl-2-fluorobiphenyl, was studied in the rat. 4'-[1-14C]Ethynyl-2-fluorobiphenyl was quantitatively absorbed from the gastrointestinal tract. Excretion of radiocarbon into urine was greater than excretion into bile. Appreciate amounts of radiocarbon remained in the carcass 24 hr after dosing. The only metabolite in plasma was (2-fluoro-4'-biphenylyl)acetic acid, which also possessed anti-inflammatory activity. Unchanged 4'-ethynyl-2-fluorobiphenyl was present after administration of higher doses. Peak plasma concentrations of (5-fluoro-4'-biphenylyl)acetic acid were observed within 1 hr of administration. The apparent plasma half-life of this acidic metabolite was 4 hr. The major eliminated metabolite was (4-hydroxy-2-fluoro-4'-biphenylyl)acetic acid. In vivo and in vitro metabolism studies suggest that the major metabolic pathway involves microsomal hydroxylation of the C-H bond of the ethynyl moiety to yield, after rearrangement, a highly reactive intermediate metabolite, 2-fluoro-4'-biphenylylketene.

Metabolism of the antimicrobial agent nibroxane, 5-bromo-2-methyl-5-nitro-m-dioxane, in the rat.

1. The metabolism of nibroxane, a topically effective antimicrobial agent has been studied in the rat after oral and dermal administrations. 2. Plasma level studies in vitro and in vivo showed nibroxane to be rapidly debrominated to 2-methyl-5-nitro-m-dioxane. 3. Nibroxane is rapidly absorbed and extensively metabolized in the rat regardless of the route of administration. 4. Enzymic hydrolysis of the m-dioxane ring was of major importance in the biotransformation of nibroxane. The major eliminated metabolite in the rat was 2-nitropropan-1,3-diol.

Pharmacokinetics of nabilone, a psychotropically active 9-ketocannabinoid, in the dog. Utilization of quantitative selected ion monitoring and deuterium labeling.

Quantitative selected ion monitoring methods for the determination of plasma concentrations of nabilone, a psychotropically active 9-ketocannabinoid, and two carbinol metabolites using deuterium labeled internal standards are described. These specific methods have a lower limit of sensitivity of about 2 pmol ml-1 with a coefficient of variation of less than 4%. The utility of these methods was demonstrated by in vivo studies of single dose and steady state pharmacokinetics of nabilone and its carbinol metabolites in the dog.

Metabolic fate of [14C]cefamandole, a parenteral cephalosporin antibiotic, in rats and dogs.

The biotransformation of the parenterally effective cephalosporin antibiotic cefamandole nafate (I) has been studied in rats and dogs. After rapid in vivo hydrolysis of the nafate pharmaceutical form to cefamandole (II), the antibiotic was found to be very resistant to metabolic degradation in both species. In dogs, cefamandole escaped metabolism and was eliminated as unaltered antibiotic almost exclusively by renal excretion. In rats, cefamandole was somewhat labile to metabolism; however, a major portion of the administered antibiotic was eliminated unchanged principally by renal excretion.

Reduction of levopropoxyphene N-oxide to propoxyphene by dogs in vivo and rat liver microsomal fraction in vitro.

1. When l-propoxyphene-N-oxide was given orally to dogs, reduction occurred in vivo resulting in substantial plasma levels of l-propoxyphene. 2. When l-propoxyphene-N-oxide was given intravenously, near peak plasma levels of propoxyphene occurred in 10 minutes. This suggests that reduction is occurring at least in part in mammalian tissue rather than in gut flora. 3. Levopropoxyphene oxide was also readily reduced anaerobically in a rat liver microsomal fraction. 4. Results from this study explain our early observation that while l-propoxyphene-N-oxide is demethylated in vivo, demethylation does not occur in vitro when the oxide is incubated in air with liver homogenate. Thus demethylation of the oxide can occur only after reduction of the oxide to the tertiary amine. While reduction occurs readily in vivo it is completely inhibited by oxygen present in the usual microsomal incubation. 5. These studies further confirm that the N-oxide is not an intermediate in the demethylation of propoxyphene.

Metabolism of 1,4-dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525) in rats and cats.

1. The metabolism of 1,4-dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525), a sedative hypnotic drug, was studied in rat and cat. 2. Plasma concentrations of Lilly 72525 were measured fluorometrically after oral and intravenous doses of the compound in rats. A comparison of the area under the two curves suggested that 84% of the oral dose was absorbed. 3. Studies with 14C-labelled material in both species confirmed that the drug was well absorbed after oral administration and revealed that the dione was mainly eliminated unchanged in the urine. Bile duct cannulation experiments suggested that biliary excretion accounted for most or all of the drug present in faeces of rats. 4. Metabolites isolated from urinary extracts by t.l.c. were identified by g.l.c.-mass spectrometry. The only metabolite detected in rat urine or bile extracts was a ring-hydroxylated compound. This metabolite plus two N-hydroxylated metabolites were identified in extracts of cat urine.

Biosynthesis of thromboxane B2: assay, isolation, and properties of the enzyme system in human platelets.

The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on the thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B2). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriproroporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E1 are inhibitors of thromboxane B2 formation. This enzyme preparation also transformed eicosa-8. 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.

Simultaneous estimation of propoxyphene-do and propoxyphene-d2 levels by quantitative mass fragmentography. Potential utility in multidose investigations.

A quantitative mass fragmentographic method for the simultaneous determination of labeled and unlabeled propoxyphene in plasma is described. Dogs treated daily with propoxyphene-do were treated with a pulse dose of propoxyphene-d2 at day 20. It was found that following an initial rapid equilibrium phase levels of propoxyphene-d2 fell more rapidly than those of propoxyphene-do. This result suggests that 'deep' pools of tissue bound propoxyphene exist which exchange very slowly with drug present in the central compartment. Experimental evidence is presented which demonstrates that the difference in behavior of propoxyphene-do and -d2 is not due to unanticipated secondary isotope effects.