Orally active inhibitors of human leukocyte elastase. I. Disposition of L-683,845 in rats and rhesus monkeys.

L-683,845 is an orally active inhibitor of human leukocyte elastase. Its disposition was studied in rats and rhesus monkeys after dosing with a 3H- or 14C-labeled compound intravenously at 5 mg/kg and orally at 10 mg/kg. L-683,845 exhibited different pharmacokinetics in these two species. In rats, L-683,845 was well-absorbed after oral dosing, with a maximum concentration of 6 microg/ml at 2 hr and bioavailability of approximately 100%. After intravenous dosing, it was cleared slowly at approximately 3 ml/min/kg, with a terminal half-life of approximately 7 hr and a volume of distribution at steady-state of 1 liter/kg. After both intravenous and oral dosing, L-683,845 comprised 50-95% of plasma radioactivity. About 75% of the intravenous and 87% of the oral dose were recovered in the feces as parent and/or conjugates, with the remaining fraction recovered in the urine as polar components. In rhesus monkeys, maximum concentration after oral dosing was only 0.25 microg/ml, and bioavailability was 50%. Plasma clearance was 8-fold higher, at 23 ml/min/kg, and volume of distribution at steady-state larger, at 2 liters/kg, than in rats. The terminal half-life of L-683,845 could not be determined accurately after intravenous dosing, but seemed to be long in orally dosed animals, approximately 13 hr. Intact L-683,845 was a minor component in plasma comprising only approximately 20% of the radioactivity at most time points. Moreover, persistent levels of radioactivity were detected in plasma and urine of rhesus monkeys even at 1-month postdose, and > or = 25% of the radioactivity in plasma was irreversibly bound to proteins at the later time points. Recovery of the radioactivity was incomplete, with only 77% of the intravenous and 43% of the oral dose recovered over a 4-day period. L-683,845-derived radioactivity distributed to all major rat tissues, with highest levels in the liver followed by the small intestine, adrenals, kidneys, and lungs. Radioactivity concentrations in the liver were high even at 24 hr, 22.7 microg eq/g. A large portion of the intravenous dose was recovered in the small intestine, approximately 40% at 2 hr, indicating rapid and extensive biliary excretion. L-683,845 was metabolized primarily to the acyl glucuronide, which was very unstable in rat plasma, and was subject to hydrolysis to L-683,845 and rearrangement. The glucuronide and L-683,845 were degraded in rat plasma by opening the beta-lactam ring and loss of the C4 substituent followed by decarboxylation to give an olefin and/or decomposition to the monosubstituted urea. Based on inhibition by organophosphorus compounds, it is speculated that their degradation is catalyzed by a type B esterase.

Absorption and glucuronidation of the angiotensin II receptor antagonist losartan by the rat intestine.

The absorption and metabolism by the rat intestine of the tetrazole-containing angiotensin II receptor antagonist losartan were determined using in vitro, in situ and in vivo models of absorption. The permeability coefficient of losartan was similar at mucosal concentrations of 0.5 to 2 mM when assayed using segments of jejunum in the Sweetana/Grass diffusion cell. The compound was conjugated during transport to form a glucuronide at the N2-position of the tetrazole group; the structure was confirmed by LC/MS/MS. Approximately 12% to 20% of losartan transported across the duodenum and jejunum was conjugated to the glucuronide. The glucuronide was not detected when sections of the ileum or colon were used. In the in situ intestinal loop model, 18% to 23% of the losartan injected into the lumen was recovered in the mesenteric vein by 1 hr. As in the in vitro model, 11% to 15% of the compound was conjugated on the tetrazole group during absorption. EXP-3174, the pharmacologically active carboxylic acid metabolite of losartan, was not detected in either the serosal buffer from the in vitro study or the mesenteric plasma from the in situ intestinal loop. In conscious rats, N2-glucuronide was detected in plasma samples from the portal vein soon after oral administration of losartan. It was detected at low concentrations in only a few of the arterial samples assayed. In conclusion, losartan is conjugated with glucuronic acid at the N2-position of the tetrazole group during absorption by the rat upper gastrointestinal tract.

Disposition and metabolism of the hypoglycemic agent pioglitazone in rats.

The disposition and metabolism of [3H]pioglitazone was determined in male rats after oral administration. The peak plasma concentration of 10 micrograms/ml occurred 1 hr after dosing at 10 mg/kg p.o.; the apparent plasma terminal half-life was 7.5 hr. Most of the radioactivity in plasma up to 8 hr after dosing was due to the parent drug. Pioglitazone was highly protein-bound in plasma; only 1-2% was free at concentrations of 0.1-10 micrograms/ml. Within 3 days after oral administration to bile duct-cannulated rats, 36% and 15% of the oral dose was recovered in the bile and urine, respectively. The pattern of biliary and urinary metabolites was similar. A total of eight metabolites were isolated and identified on the basis of NMR spectroscopy and MS. Metabolites resulting from hydroxylation of either carbon adjacent to the pyridine ring were conjugated with glucuronic acid (M7) or sulfuric acid (M6). The metabolite hydroxylated on the terminal carbon of the ethyl side chain was further oxidized to the carboxylic acid derivative (M3). Oxidative loss of the terminal carbon led to a nicotinic acid derivative (M2) and loss of both carbon atoms to the corresponding 3-hydroxypyridine (M9) derivative that was excreted as the sulfate conjugate (M8). The two carboxylic acid metabolites were also conjugated with taurine (M4 and M5).

Disposition of the angiotensin II receptor antagonist L-158,809 in rats and rhesus monkeys.

The disposition of the angiotensin II receptor antagonist L-158,809 was studied in male rats and female rhesus monkeys. Rats were dosed either intravenously or orally with 0.3 mg [3H]L-158,809/kg. The terminal half-life of L-158,809 was 7.6 +/- 3.1 hr. Plasma clearance was 45.5 +/- 15.9 ml/hr/kg, and the volume of distribution at steady state was 0.37 +/- 0.11 liter/kg. The drug was completely bioavailable in rats. After oral administration, the peak plasma concentration of L-158,809 was 0.70 +/- 0.21 micrograms/ml at 15 min; only the parent drug was observed in plasma. After an oral dose of 3.0 mg [3H]L-158,809/kg, the peak plasma concentration of 4.9 +/- 0.6 micrograms/ml occurred at 1 hr. By 24 hr, 53.3 +/- 9.9% of an intraduodenal dose of 0.3 mg [3H]L-158,809/kg was excreted into bile. L-158,809 and its tetrazole-N2-beta-glucuronide were the major biliary components. Rhesus monkeys were dosed orally and intravenously at 1.0 mg and 0.5 mg [3H]L-158,809/kg, respectively. The plasma concentrations of L-158,809 varied considerably between monkeys after oral administration. The peak concentration was 42 +/- 42 ng/ml at 30 min, and the bioavailability was 32.3 +/- 12.6%. Plasma clearance was 839 +/- 364 ml/hr/kg; the volume of distribution at steady state was 1.42 +/- 0.73 liter/kg. Besides the parent, the major metabolite in monkey plasma was the tetrazole-N2-glucuronide of L-158,809. Both species excreted > 10% of the dose in the urine after intravenous or oral dosing; most of the dose was excreted in the feces, indicating extensive biliary excretion.

Highly potent, orally active diester macrocyclic human renin inhibitors.

Replacing one amide bond in macrocyclic renin inhibitors of the general structure 1 and 2 with an ester linkage gave glutamate-derived inhibitors 3 and serine-derived inhibitors 4. While this oxygen-for-nitrogen exchange had little effect on potency in the glutamate series, potency was dramatically increased in the serine series. In this series, the 14-membered ring compounds proved to be more potent than the corresponding 13-membered ring derivatives. Substitution of the ring at the position corresponding to P2' generally increased potency. The absolute configuration at this center was shown to be R for the 4-morpholinomethyl derivative (4o), both by asymmetric synthesis and X-ray crystallography. Replacing the "Boc-Phe" moiety of inhibitor 4o with a variety of substituents led to subnanomolar inhibitors, one of which (the "3(S)-quinuclidinyl-Phe" derivative 33) lowered blood pressure 20 mmHg and completely inhibited plasma renin activity for 6 h in sodium-depleted rhesus monkeys. This compound proved to have limited bioavailability (1% in rats) due to cleavage of the serine ester bond and rapid hepatic extraction.

In vivo metabolism of atrial natriuretic peptide: identification of plasma metabolites and enzymes responsible for their generation.

The in vivo metabolism of atrial natriuretic peptide (ANP) has been studied in the rat after i.v. administration of either [106Phe-14C]- or [126Tyr-125I]-ANP(103-126). Plasma samples containing radioactive peptides were separated by reverse-phase high-performance liquid chromatography. The major plasma metabolites were [125I]Tyr and [14C]Phe for the iodinated and 14C-labeled peptides, respectively. Both peptides had ANP(104/5-126) as a metabolite. Administration of labeled peptide by either bolus or infusion produced the same metabolite profile. To determine which enzymes were responsible for generating these initial metabolites, animals were first dosed with various protease inhibitors before the infusion of [14C]ANP(103-126). The amino-peptidase inhibitor bestatin and the angiotensin converting enzyme inhibitor captopril caused 54 and 66% increases in plasma ANP(103-126), respectively, but no other effects. Administration of the endopeptidase 24.11 inhibitor thiorphan led to a 158% increase of ANP(103-126) in plasma and an 11-fold increase in ANP(104/5-126). The latter metabolite could be selectively decreased by pretreatment with bestatin in combination with thiorphan. The results demonstrate that the initial plasma metabolites of ANP(103-126) are due to the activity of endopeptidase 24.11, a bestatin-sensitive aminopeptidase, and a carboxypeptidase. The plasma clearance of the peptide is probably also due to cellular binding and uptake in combination with glomerular filtration as very few plasma metabolites were observed even at very high rates of ANP(103-126) infusion.

Specific inhibitors of endopeptidase 24.11 inhibit the metabolism of atrial natriuretic peptides in vitro and in vivo.

Atrial natriuretic peptides (ANPs) are degraded rapidly by renal brush border membranes in vitro. Here, we report that thiorphan, a specific inhibitor of endopeptidase 24.11, afforded almost complete protection against inactivation of ANPs by a renal brush border membrane preparation. The diastereoisomers of [3-(N-hydroxy)carboxamido-2-benzylpropanoyl]-L-alanine (HCBA) are potent inhibitors of endopeptidase 24.11 and were also tested for their abilities to inhibit ANP-(103-126) degradation. The (S,S)-diastereoisomer was more effective than the (R,S)-diastereoisomer (kelatorphan), but both were less potent than thiorphan. To determine if endopeptidase inhibitors could decrease ANP metabolism in in vivo, thiorphan and (S,S)-HCBA were given to rats with or without a continuous infusion of ANP-(103-126). Both inhibitors induced rapid increases in plasma ANP concentration in rats administered exogenous ANP-(103-126), but had no effect on endogenous ANP levels. Thus, specific inhibitors of endopeptidase 24.11 decrease the degradation of ANPs in vitro, and are effective in reducing the metabolism of ANP-(103-126) in vivo.

Metabolism of atrial natriuretic peptide. Extraction by organs in the rat.

The pharmacokinetics and metabolism of atrial natriuretic peptide (ANP24) were investigated in male Sprague-Dawley rats. Animals were instrumented with arterial and venous catheters to infuse ANP24 and sample blood at various systemic locations; ANPir concentrations were determined by radioimmunoassay. Total clearances (TC) were 150 +/- 15 and 90 +/- 3 ml of blood/min/kg at infusion rates of 137 and 833 ng/min/kg and the half-life of ANPir was 35 +/- 5 seconds. The volume of distribution was 87 +/- 8 ml/kg at the high infusion rate. The kidneys' extraction ratios (E) of ANPir at the two rates of infusion were 0.55 and 0.61, respectively, whereas the intestines' were 0.44 and 0.27. E values of the muscle/sex organs were 0.43 and 0.54 at the two infusion rates. In contrast, no significant degree of extraction was observed for the liver or the heart and lungs. Taking regional blood flow into account, the kidneys were responsible for one sixth to one third of TC while the intestines accounted for one sixth of TC. We conclude that ANP24 is cleared from the blood at a relatively high rate under steady state conditions and that the kidneys, intestines, and muscle/sex organs contribute to its metabolic fate.

Immunoquantitation of cytochrome b5 and methylcholanthrene-induced cytochromes P-450.

The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes b5 and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.

Assessment of internal primary structure of polypeptides newly translated in vitro by reticulocyte lysate: a study with cytochrome b5.

A modified peptide mapping technique is described that allows the survey of the primary structure of proteolytic fragments of particular newly translated proteins in reticulocyte lysate. The technique is demonstrated with rat liver cytochrome b5.

Studies on the biliary efflux of GSH from rat liver due to the metabolism of aminopyrine.

The biliary efflux of GSH and GSSG due to aminopyrine was studied using perfused rat livers. The infusion of 0.8 mM aminopyrine led to a rapid rise in the amount of GSH released into the bile with only a small increase in the amount of GSSG released; caval GSH + GSSG efflux was unaffected. N-Benzylimidazole, an inhibitor of cytochrome P-450, completely blocked the response while phenobarbital pretreatment of the rats doubled the rate of GSH efflux. H2O2 and selenium-containing glutathione peroxidase were not involved since livers from selenium-deficient rats perfused with aminopyrine released GSH at the same rate as control livers. Aminopyrine injected i.p. into conscious rats also stimulated biliary GSH efflux to the same extent as with perfused livers. Biliary release of GSH in the perfused livers could be duplicated by infusing formaldehyde. It is proposed that formaldehyde produced during the N-demethylation of aminopyrine by cytochrome P-450 combines reversibly with GSH to form S-hydroxymethylglutathione which is oxidized by formaldehyde dehydrogenase to S-formylglutathione. Formaldehyde formed in excess of its capacity to be metabolized enzymatically is released into the bile as S-hydroxymethylglutathione which then dissociates to its initial reactants.

Increased biliary GSSG efflux from rat livers perfused with thiocarbamide substrates for the flavin-containing monooxygenase.

Thiourea, phenylthiourea, and methimazole perfused into rat liver stimulated the biliary efflux of GSSG without affecting the excretion of GSH into either the bile or the caval perfusate. The thiocarbamide moiety appears essential, since perfusion with urea, phenylurea, or N-methylimidazole did not stimulate GSSG release. Hydrogen peroxide is also not an obligatory intermediate, since thiocarbamide-induced GSSG efflux was undiminished in livers from selenium-deficient animals. The response was also not affected by N-benzylimidazole, a potent cytochrome P-450 inhibitor, which suggests that this monooxygenase is not involved. However, the results are consistent with a model based on S-oxygenation of thiocarbamides to formamadine sulfenates catalyzed exclusively by the flavin-containing monooxygenase. The resulting sulfenate is reduced by GSH, yielding GSSG and the parent thiocarbamide. Rapid cellular oxidation of GSH by this mechanism leads to biliary efflux of the disulfide.

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.

Cytochrome P-450 and halothane metabolism. Decrease in rat liver microsomal P-450 in vitro.

Anaerobic in vitro incubation of microsomes from phenobarbital(PB)-induced rats with halothane results in an irreversible decrease of measurable cytochrome P-450. There is a parallel decrease in heme content under the same incubation conditions. However, microsomes from 3-methylcholanthrene(3-MC)-induced or untreated animals do not show a reduction in cytochrome P-450 content. Aerobic incubation with halothane results in a decrease of cytochrome P-450 which can be completely reversed by dialysis or the addition of potassium ferricyanide. These latter treatments only partially restore the cytochrome P-450 levels following anaerobic incubations. The decrease in cytochrome caused by halothane is not associated with measureable heme N-alkyl adduct formation; lipid peroxidation does not play a role as indicated by the lack of effect of 1 mM EDTA or a decrease in glucose-6-phosphatase activity. Halothane metabolites are bound irreversibly to microsomal protein as determined by gel electrophoresis only when the oxygen concentration is very low. The mechanism of cytochrome P-450 decrease is consistent with the formation of a reactive metabolite which binds to the protein portion and also destroys heme.