Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies.

The fraction of substrate metabolized (fm ) can be used to estimate drug interactions and can be determined by comparison of the intrinsic clearances (CLint ) of victim drugs obtained from inhibited and uninhibited hepatic enzymes. Commercially available human liver microsomes were recently developed in which one cytochrome P450 (P450) isoform is selectively inactivated. These inactivated liver microsomes were used to evaluate the roles of P450 2C isoforms in the depletion and oxidation of probe substrates. Determination of CLint with sets of control and P450 2C9-inactivated liver microsomes yielded fm,P450 2C9 values of 0.69-1.0 for celecoxib, diclofenac and warfarin. Apparent minor contributions of P450 1A2/2C8/3A4 were seen in depletion assays, yielding ~1 for the sum of the fm values. Selectively inactivated liver microsomes were thereby shown to be potentially useful for determining the in vitro fm values for major P450 2C9 contributions to substrate oxidations. Metabolite formations from diclofenac and warfarin were suppressed by 62-84% by the replacement of control liver microsomes with P450 2C9-inactivated liver microsomes. R-, S- and racemic omeprazole and troglitazone oxidation activities by liver microsomes at multiple substrate concentrations were suppressed by 26-36% and 22-50%, respectively, when P450 2C19- and 2C8-inactivated liver microsomes were used in place of control liver microsomes. This study provides important information to help elucidate the different roles of P450 isoforms in metabolite formation at different substrate concentrations. The data obtained allow the fractions metabolized to be calculated for victim drugs.

Development of an in vitro system with human liver microsomes for phenotyping of CYP2C9 genetic polymorphisms with a mechanism-based inactivator.

Polymorphisms in cytochrome P450 enzymes can significantly alter the rate of drug metabolism, as well as the extent of drug-drug interactions. Individuals who homozygotically express the CYP2C9*3 allele (I359L) of CYP2C9 exhibit ∼70 to 80% reductions in the oral clearance of drugs metabolized through this pathway; the reduction in clearance is ∼40 to 50% for heterozygotic individuals. Although these polymorphisms result in a decrease in the activity of individual enzyme molecules, we hypothesized that decreasing the total number of active enzyme molecules in an in vitro system (CYP2C9*1/*1 human liver microsomes) by an equivalent percentage could produce the same net change in overall metabolic capacity. To this end, the selective CYP2C9 mechanism-based inactivator tienilic acid was used to reduce irreversibly the total CYP2C9 activity in human liver microsomes. Tienilic acid concentrations were effectively titrated to produce microsomal preparations with 43 and 73% less activity, mimicking the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, respectively. With probe substrates specific for other major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), no apparent changes in the rate of metabolism were noted for these enzymes after the addition of tienilic acid, which suggests that this model is selective for CYP2C9. In lieu of using rare human liver microsomes from CYP2C9*1/*3 and CYP2C9*3/*3 individuals, a tienilic acid-created knockdown in human liver microsomes may be an appropriate in vitro model to determine CYP2C9-mediated metabolism of a given substrate, to determine whether other drug-metabolizing enzymes may compensate for reduced CYP2C9 activity, and to predict the extent of genotype-dependent drug-drug interactions.

Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (+/-)-suprofen: a comparison of kinetics and probe substrate selection.

In vitro experiments were conducted to compare k(inact), K(I) and inactivation efficiency (k(inact)/K(I)) of cytochrome P450 (P450) 2C9 by tienilic acid and (+/-)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. Although the inactivation of P450 2C9 by tienilic acid when (S)-flurbiprofen and diclofenac were used as substrates was similar (efficiency of approximately 9 ml/min/micromol), the inactivation kinetics were characterized by a sigmoidal profile. (+/-)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (approximately 1 ml/min/micromol). In contrast, inactivation of P450 2C9-mediated (S)-warfarin 7-hydroxylation by tienilic acid and (+/-)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/micromol) and approximately 3-fold higher (3 ml/min/micromol), respectively, relative to that of the other probe substrates, which argues for careful consideration of reporter substrate when mechanism-based inactivation of P450 2C9 is assessed in vitro. Further investigations into the increased inactivation seen with tienilic acid relative to that with (+/-)-suprofen revealed that tienilic acid is a higher affinity substrate with a spectral binding affinity constant (K(s)) of 2 microM and an in vitro half-life of 5 min compared with a K(s) of 21 microM and a 50 min in vitro half-life for (+/-)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs.

Markers of electrophilic stress caused by chemically reactive metabolites in human hepatocytes.

The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites.

Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

Effects of diuretics on urate and calcium excretion.

Forty-nine patients with gout, many with hypertension and/or renal calculi, were given hydrochlorothiazide, furosemide, or ticrynafen. Diuresis and increased clearances of sodium (Na), potassium (K), chloride (Cl), and calcium (Ca) occurred after a single dose of hydrochlorothiazide, 100 mg, or furosemide, 40 mg, orally. There was very slight change in urate and phosphorus clearances. With prolonged use of hydrochloride or furosemide, diuresis and increased electrolyte excretion disappeared. Urate and Ca excretion fell with hydrochlorothiazide. With long-term use of furosemide, urate excretion was suppressed, but Ca excretion was sustained. Ticrynafen produced diuresis and increased clearances of Na, K, and Cl. Calcium excretion was increased after a single dose and minimally decreased after long-term use. Most striking was the severe and rather sustained uricosuria. Though ticrynafen is an effective uricosuric, natriuretic, and antihypertensive agent, its hepatotoxicity and nephrotoxicity mitigate against its clinical use.

Ticrynafen in anephric patients: effects on uric acid and pharmacokinetics.

Ticrynafen was given to 6 anephric patients undergoing maintenance hemodialysis. Ticrynafen was given daily for the 3 days between hemodialyses. Ticrynafen had no effect on the interdialysis rise in serum uric acid levels. Ticrynafen did not accumulate in serum, but levels of metabolites continued to rise over the 3 days. Hemodialysis (5 hr) reduced levels of ticrynafen by 38% but had less effect on metabolite levels. There was no effect on serum cholesterol or triglycerides.

Ticrynafen-racemic warfarin interaction: hepatotoxic or stereoselective?

To examine the ticrynafen-warfarin interaction, normal subjects received large single doses of 1.5 mg/kg racemic warfarin with and without daily oral doses of 250 mg ticrynafen beginning 3 days before warfarin and continuing for the duration of hypoprothrombinemia. Daily blood samples were analyzed for one-stage prothrombin time (Quick method) and warfarin concentrations (high-pressure liquid chromatography). Ticrynafen induced augmentations of both prothrombin time and warfarin concentration (P less than 0.001). The interaction was evaluated further with separated warfarin enantiomorphs. Ticrynafen induced augmentation of prothrombin times and warfarin concentrations of S-warfarin, but had little effect on R-warfarin. Thus, ticrynafen probably augments the hypoprothrombinemia of racemic warfarin by reducing metabolic clearance of S-warfarin. The lack of effect of ticrynafen on R-warfarin suggest that the interaction is stereoselective rather than hepatotoxic.

Effects of diuretic and propranolol on plasma lipoprotein lipids.

Ticrynafen (TCNF), a nonthiazide diuretic, has been reported to be nonhyperlipidemic. To define the effects of these drugs on plasma lipoproteins, experiments were performed in hypertensive subjects after placebo therapy, 4 wk after therapy with either hydrochlorothiazide (HCTZ) or TCNF, 3 mo after diuretic with propranolol, and 1 mo after therapy with propranolol alone. Plasma lipoproteins were separated by ultracentrifugation and the lipid fractions isolated by extraction and silicic acid thin-layer chromatography. Plasma low-density lipoprotein (LDL) total cholesterol fell and high-density lipoprotein (HDL) total cholesterol rose in subjects receiving TCNF. TCNF had no effect on plasma low-density lipoprotein (VLDL) triglyceride or phospholipid. There was no significant changes in LDL or HDL total cholesterol in subjects on HCTZ. HCTZ tended to increase plasma VLDL triglyceride and phospholipid. The addition of propranolol to either diuretic had no effect on LDL or HDL total cholesterol but increased VLDL triglyceride, especially in subjects on HCTZ. Propranolol alone had no effect on any of the lipids measured.

Studies on uricosuric diuretics. 4. Three-dimensional structure-activity relationships and receptor mapping of (aryloxy)acetic acid diuretics.

Attempts to develop new (aryloxy)acetic acids with a better profile of diuretic and uricosuric activities as well as with fewer side effects have produced a series of compounds in which the ring system has been varied. Diuretic screening of these analogues in rats indicated that the great difference in the activity between these compounds might be ascribed to a difference in the ring system rather than that in the substituent effect and that the annulation hypothesis described before is not necessarily applicable to all of these compounds. This prompted us to study the relationship between the structure and the diuretic activity of the (aryloxy)acetic acids. An active model (receptor model) was created with the indanone moiety of R-(-)-3 and the dihydrobenzofuran-2-carboxylic acid moiety of S-(+)-4. The three-dimensional structure-activity study of known compounds 2-4, and 5a using the active model showed that the degree of fitting to the model is related to the diuretic activity of these compounds. This was also confirmed for compounds 6a, 6b, 9a, 10a, 11a, 12a, 13a, 14a, 15a, and 16a, and the relation between the structure and the diuretic activity was rationalized qualitatively. With these insights in mind, a modified receptor model was constructed. We believe that this model is useful for a prediction of the activity of compounds not yet synthesized as well as for designing new (aryloxy)acetic acid diuretics.

Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility.

PURPOSE: The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro. In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity. To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, inadcrinone and ticrynafen, and the MT-disrupting drug vinblastine. METHODS: Excised bovine and porcine eyes were perfused and outflow facility measured. Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment. RESULTS: Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility. In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine. Although indacrinone and ticrynafen produced changes in cell shape in vitro, the beta-tubulin staining pattern of treated cells was not altered. Vinblastine caused cell shape change and the expected MT disruption. CONCLUSIONS: Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA). These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM. An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function.

Effect of ticrynafen on aqueous humor dynamics in monkeys.

OBJECTIVE: To determine the effect of ticrynafen, a nonsulfhydryl-reactive compound similar to ethacrynic acid, on outflow facility in normotensive monkey eyes and on intraocular pressure (IOP) in monkey eyes with laser-induced glaucoma. METHODS: In normotensive eyes, facility (perfusion) was measured shortly before and after bolus or exchange intracameral infusion of ticrynafen or vehicle in opposite eyes, and 3.5 to 4.5 hours after 5 days of twice-daily 2% ticrynafen or vehicle ointment. In glaucomatous eyes, baseline and vehicle diurnal IOP curves were established, 2% ticrynafen ointment was given twice daily for 5 days, and IOP was measured immediately before and 0.5 to 6 hours after each morning treatment. RESULTS: In normotensive eyes, exchange 2-mL influsion of 0.2-, 1-, or 4-mmol/L ticrynafen increased facility by 33% +/- 6% (mean +/- SEM), 73% +/- 18%, and 60% +/- 11%, respectively. Day 5 posttreatment facility was higher in the ticrynafen group than in controls by 28% +/- 9%. In glaucomatous eyes, maximum IOP decline, from approximately 35 mm Hg, was 7.5 +/- 2.0 mm Hg on day 4 and 9.8 +/- 2.4 mm Hg on day 5 of twice-daily ticrynafen treatment. CONCLUSION: The facility-increasing, IOP-lowering action of ticrynafen, ethacrynic acid, and derivatives may not depend entirely on sulfhydryl reactivity. CLINICAL RELEVANCE: Whether such drugs as ethacrynic acid and ticrynafen prove valuable for glaucoma therapy, at the least they are useful probes to study aqueous outflow mechanisms.

Tubular secretion and effects of tienilic acid in the hen.

The relationship between renal tubular secretion of the uricosuric diuretic tienilic acid (10 or 30 microgram/kg per min) and its saluretic effects was determined using a modified Sperber technique. A true tubular excretion fraction (TTEF) or 29.9 +/- 4.1% (mean +/- S.D.) was found for tienilic acid, a value significantly (P less than 0.001) reduced by novobiocin to 3.3 +/- 2.8%. This demonstrates active tubular secretion of the diuretic by an organic anion transport system in the hen kidney. Infusion of the diuretic into one leg vein caused a marked ipsilateral excess excretion of chloride, sodium and potassium; these effects were significantly reduced by novobiocin. Thus, in the hen a significant part of the saluretic effect of tienilic acid depends on the active secretion of the drug by tubular cells, as has previously been found for three loop diuretics i.e. ethacrynic acid, furosemide and piretanide. In analogy with these, it is suggested that the saluretic effect of tienilic acid is evoked mainly from the luminal side of the avian nephron. At an infusion rate of 20 microgram/kg per min of tienilic acid, a marked saluresis occurred; there was, however, no effect on the urinary urate excretion.

Effect of the glutathione S-transferase inhibitor, tienilic acid, on biliary excretion of sulphobromophthalein.

Tienilic acid, a phenoxyacetic acid diuretic, reduces the amount of total sulphobromophthalein (BSP) excretion in the isolated perfused rat liver (IPRL). This reduction was primarily by reduction in excretion of conjugated BSP, with excretion of unchanged BSP being relatively unaffected. TA also reduces the amount of conjugated BSP formed in vitro, indicating that its effect in the IPRL may be by means of inhibiting the glutathione S-transferase enzymes involved in the formation of the conjugate. It would appear that a reduction in the biliary excretion of BSP cannot be taken to be an indication of reduced liver function in a general sense.

Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells.

Several pharmaceutical drugs show strong hepatotoxicity during therapeutic use. We have studied 6 of them: aminophenazone, clofibrate, nifuroxazide, oxamniquine, perhexiline maleate, tienilic acid. Their mutagenicity was assessed in the Ames test on 6 strains of Salmonella typhimurium, and in V79 Chinese hamster lung cells using a rat-hepatocyte-mediated metabolic activation system and the HGPRT and Na+/K+ ATPase assay. Nifuroxazide was positive in the Ames test in two Salmonella strains (TA100, and TA100 Fr1). In the hepatocyte-mediated mammalian V79 cell system, nifuroxazide, clofibrate and aminophenazone were negative; oxamniquine and tienilic acid were positive with and without metabolic activation in tests looking for ouabain and 6-thioguanine resistance. Perhexiline maleate was negative for the direct induction of 6-thioguanine resistance without metabolic activation, and positive after metabolisation mediated by primary rat's hepatocytes. These results suggest the need for some caution in the use of some pharmaceutical drugs because of hepatotoxicity and because 3 out of 6 drugs were shown to be slightly mutagenic in mammalian cells.

Indirect inhibition of vitamin K epoxide reduction by salicylate.

Salicylate antagonizes the vitamin K-dependent biosynthesis of clotting factors in the rat and produces an elevation of the ratio of vitamin K epoxide to vitamin K in the liver. Vitamin K epoxide is reduced to vitamin K by a vitamin K epoxide reductase, and 1 mM salicylate was required to cause a 50% inhibition of the dithiothreitol-dependent in-vitro reduction of vitamin K epoxide by this enzyme. This enzyme was, however, inhibited 50% by as little as 70-80 microM salicylate when reducing equivalents for the reaction were furnished by endogenous cytosolic reductants. This effect on the cytosolic reductant supply was shown to be unrelated to a previously demonstrated inhibition of DT-diaphorase by salicylate. The concentrations of salicylate at which significant inhibitory effects are exerted in-vitro (50-100 microM) are below the 200 microM levels observed in the livers of rats given an anticoagulating dose of salicylate.

Stop-flow studies on tubular transport of uric acid in rats.

A stop-flow technique using pyrazinoic acid(PZO)-treated and -untreated rats was devised to evaluate drug effects on bi-directional transport of uric acid in the tubules. Constant venous infusion of test drugs to PZO-untreated rats was used to estimate their inhibitory effects on urate secretion, while their inhibitory effects on urate reabsorption was studied by intravenous administration as a bolus to PZO-treated rats. Probenecid, tienilic acid and R-(+)-enantiomer of S-8666, which is the uricosuric component of a new uricosuric diuretic, decreased the (Tua/Pua)/(Tin/Pin) value in the distal and proximal tubules by inhibiting urate secretion in PZO-untreated rats. On the other hand, all of these drugs increased the (Tua/Pua)/(Tin/Pin) value in the tubules in PZO-treated rats, which suggested that they also inhibited the reabsorptive flux of urate. This stop-flow technique in rat kidneys showed the possibilities of bi-directional inhibition by these drugs of urate transport in the tubules.