Transporter-Mediated Hepatic Uptake Plays an Important Role in the Pharmacokinetics and Drug-Drug Interactions of Montelukast.

Montelukast, a leukotriene receptor antagonist commonly prescribed for treatment of asthma, is primarily metabolized by cytochrome P450 (CYP)2C8, and has been suggested as a probe substrate for investigating CYP2C8 activity in vivo. We evaluated the quantitative role of hepatic uptake transport in its pharmacokinetics and drug-drug interactions (DDIs). Montelukast was characterized with significant active uptake in human hepatocytes, and showed affinity towards organic anion transporting polypeptides (OATPs) in transfected cell systems. Single-dose rifampicin, an OATP inhibitor, decreased montelukast clearance in rats and monkeys. Clinical DDIs of montelukast were evaluated using physiologically based pharmacokinetic modeling; and simulation of the interactions with gemfibrozil-CYP2C8 and OATP1B1/1B3 inhibitor, clarithromycin-CYP3A and OATP1B1/1B3 inhibitor, and itraconazole-CYP3A inhibitor, implicated OATPs-CYP2C8-CYP2C8 interplay as the primary determinant of montelukast pharmacokinetics. In conclusion, hepatic uptake plays a key role in the pharmacokinetics of montelukast, which should be taken into account when interpreting clinical interactions.

Should the incorporation of structural alerts be restricted in drug design? An analysis of structure-toxicity trends with aniline-based drugs.

Certain idiosyncratic adverse drug reactions (IADRs) can be triggered by electrophilic protein-reactive metabolites that are formed in the process of drug metabolism. While methodologies (e.g., structural alert concept in drug design, glutathione (GSH) trapping, and protein covalent binding) for examining reactive metabolite (RM) formation are available, predicting the IADR potential applying these parameters remains a significant challenge. The present work examines toxicity trends associated with the aniline structural alert in the top 200 prescribed drugs of 2011 and recently approved (2009-2013) small molecule drugs, in relation with 30 aniline-based drugs withdrawn from commercial use or associated with a black box warning for IADRs. The aniline sub-structure was found in several drugs from the toxic, most prescribed, and recently approved category. RMs resulting from the bioactivation of the aniline alert was also noted in the three categories chosen for comparison. A major discriminator between the toxic drugs and the majority of drugs in the most-prescribed list, however, was the daily dose--drugs most frequented associated with IADRs were the ones with higher daily doses (exceeding hundreds of milligrams). A greater tolerance for IADRs was also noted with certain drugs intended to treat rare, unmet medical needs (e.g., cancer). Overall, the analysis suggests that optimization of pharmacologic potency and pharmacokinetics that would lead to a lower daily dose, and therefore, a lower body burden of parent drug/metabolites, should be taken into consideration in drug discovery.

Utility of the carboxylesterase inhibitor bis-para-nitrophenylphosphate (BNPP) in the plasma unbound fraction determination for a hydrolytically unstable amide derivative and agonist of the TGR5 receptor.

The potent, functional agonist of the bile acid Takeda G-protein-coupled receptor 5 (TGR5), (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (3), represents a useful tool to probe in vivo TGR5 pharmacology. Rapid degradation of 3 in both rat and mouse plasma, however, hindered the conduct of in vivo pharmacokinetic/pharmacodynamic investigations (including plasma-free fraction (f(u plasma)) determination) in rodent models of pharmacology. Studies were therefore initiated to understand the biochemical basis for plasma instability so that appropriate methodology could be implemented in in vivo pharmacology studies to prevent the breakdown of 3. Compound 3 underwent amide bond cleavage in both rat and mouse plasma with half-lives (T(1/2)) of 39 + or - 7 and 9.9 + or - 0.1 min. bis(p-nitrophenyl) phosphate (BNPP), a specific inhibitor of carboxylesterases, was found to inhibit hydrolytic cleavage in a time- and concentration-dependent manner, which suggested the involvement of carboxylesterases in the metabolism of 3. In contrast with the findings in rodents, 3 was resistant to hydrolytic cleavage in both dog and human plasma. The instability of 3 was also observed in rat and mouse liver microsomes. beta-Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent metabolism of 3 occurred more rapidly (T(1/2) approximately 2.22-6.4 min) compared with the metabolic component observed in the absence of the co-factor (T(1/2) approximately 89-130 min). Oxidative metabolism dominated the NADPH-dependent decline of 3, whereas NADPH-independent metabolism of 3 proceeded via simple amide bond hydrolysis. Compound 3 was highly bound (approximately 95%) to both dog and human plasmas. Rat and mouse plasma, pre-treated with BNPP to inhibit carboxylesterases activity, were used to determine the f(u plasma) of 3. A BNPP concentration of 500 microM was determined to be optimal for these studies. Higher BNPP concentrations (1000 microM) appeared to displace 3 from its plasma protein-binding sites in preclinical species and human. Under the conditions of carboxylesterases-inhibited rat and mouse plasma, the level of protein binding displayed by 3 was similar to those observed in dog and human. In conclusion, a novel system has been devised to measure f(u plasma) for a plasma-labile compound. The BNPP methodology can be potentially applied to stabilize hydrolytic cleavage of structurally diverse carboxylesterase substrates in the plasma (and other tissue), thereby allowing the characterization of pharmacology studies on plasma-labile compounds if and when they emerge as hits in exploratory drug-discovery programmes.

Utility of multivariate analysis in support of in vitro metabolite identification studies: retrospective analysis using the antidepressant drug nefazodone.

The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile. The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities. Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites. A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in beta-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported. Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.

Pharmacokinetics, disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid, a potent and subtype-selective peroxisome proliferator-activated receptor alpha agonist in preclinical species and human.

5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-alpha). In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague-Dawley rats (3.2 +/- 1.4 ml min(-1) kg(-1)) and cynomolgus monkeys (6.1 +/- 1.6 ml min(-1) kg(-1)) resulting in plasma half-lives of 7.1 +/- 0.7 h and 9.4 +/- 0.8 h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50 mg kg(-1)). In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50's > 30 microM) was discerned with 1. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

Disposition of CP-671, 305, a selective phosphodiesterase 4 inhibitor in preclinical species.

1. The disposition of (+)-2-[4-({[2-(benzo[1,3] dioxol-5-yloxy)-pyridine-3-carbonyl]-amino)-methyl)-3-fluoro-phenoxyl-propionic acid (CP-671,305), a potent and selective inhibitor of phosphodiesterase 4 (subtype D), was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 2. CP-671,305 demonstrates generally favourable pharmacokinetic properties in all species examined. Systemic plasma clearance after intravenous administration was low in Sprague-Dawley rats (9.60+/-1.16 ml min(-1) kg(-1)), beagle dogs (2.90+/-0.81 ml min(-1) kg(-1)) and cynomolgus monkeys (2.94+/-0.87ml min(-1) kg(-1)) resulting in plasma half-lives > 5 h. Moderate to high bioavailability in rats (43-80%), dogs (45%) and monkeys (26%) was observed after oral dosing. In rats, oral pharmacokinetics were dose dependent over the dose range studied (10 and 25 mgkg(-1)). 3. CP-671,305 was > 97% bound to plasma proteins in rat, dog, monkey and human. 4. The principal route of clearance of CP-671,305 in rats and dogs was by renal and biliary excretion of unchanged drug. This finding was consistent with CP-671,305 resistance towards metabolism in hepatocytes and NADPH-supplemented liver microsomes from preclinical species and human. 5. CP-671,305 did not exhibit competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, 2C9, 2C19, 2D6 and 3A4 (IC50's > 50 microM). Likewise, no time-dependent inactivation of the five major cytochrome P450 enzymes was discernible with CP-671,305. 6. Overall, the results indicate that the absorption, distribution, metabolism and excretion (ADME) profile of CP-671,305 is relatively consistent across preclinical species and predict potentially favourable pharmacokinetic properties in humans, supporting its selection for toxicity/safety assessment studies and possible investigations in humans.

Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.

Discovery and design of selective cyclooxygenase-2 inhibitors as non-ulcerogenic, anti-inflammatory drugs with potential utility as anti-cancer agents.

The recent marketing of two selective cyclooxygenase-2 (COX-2) inhibitors, celecoxib and rofecoxib is remarkable considering that COX-2 was only discovered eight years ago as a growth factor- and cytokine-inducible gene. Concomitant with these pharmaceutical successes is the advances in our understanding of the molecular and structural basis for selective COX-2 inhibition. This review provides a perspective on the ongoing structure-activity relationship (SAR) efforts in the search of COX-2-specific inhibitors with particular reference to their structural basis for isozyme-specific inhibition. In addition to the existing inhibitor classes, this review will also highlight many novel structural classes which have recently emerged due to a better understanding of the active site differences between the two isozymes with a special emphasis on the modification of the well-established non-steroidal anti-inflammatory drug (NSAID) scaffold. In addition to its role in inflammation, recent studies suggest that COX-2-derived prostaglandins may play a pivotal part in the maintenance of tumor viability, growth, and metastasis. In this review, we summarize the NSAID epidemiological evidence, studies demonstrating overexpression of COX-2 in multiple human tumors and pharmacological evidence in animal models, which indicate that COX-2 inhibitors could be used in the prevention or treatment of a broader range of disease.

Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors.

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.

Biochemically based design of cyclooxygenase-2 (COX-2) inhibitors: facile conversion of nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors.

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isozymes to different extents, which accounts for their anti-inflammatory and analgesic activities and their gastrointestinal side effects. We have exploited biochemical differences between the two COX enzymes to identify a strategy for converting carboxylate-containing NSAIDs into selective COX-2 inhibitors. Derivatization of the carboxylate moiety in moderately selective COX-1 inhibitors, such as 5,8,11,14-eicosatetraynoic acid (ETYA) and arylacetic and fenamic acid NSAIDs, exemplified by indomethacin and meclofenamic acid, respectively, generated potent and selective COX-2 inhibitors. In the indomethacin series, esters and primary and secondary amides are superior to tertiary amides as selective inhibitors. Only the amide derivatives of ETYA and meclofenamic acid inhibit COX-2; the esters are either inactive or nonselective. Inhibition kinetics reveal that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Site-directed mutagenesis of murine COX-2 indicates that the molecular basis for selectivity differs from the parent NSAIDs and from diarylheterocycles. Selectivity arises from novel interactions at the opening and at the apex of the substrate-binding site. Lead compounds in the present study are potent inhibitors of COX-2 activity in cultured inflammatory cells. Furthermore, indomethacin amides are orally active, nonulcerogenic, anti-inflammatory agents in an in vivo model of acute inflammation. Expansion of this approach can be envisioned for the modification of all carboxylic acid-containing NSAIDs into selective COX-2 inhibitors.

Cyclooxygenase 2 inhibitors: discovery, selectivity and the future.

The recent marketing of two selective cyclooxygenase 2 (COX-2) inhibitors climaxes the first phase of an exciting and fast-paced effort to exploit a novel molecular target for nonsteroidal anti-inflammatory drugs (NSAIDs). Much has been written in the lay and scientific press about the potential of COX-2 inhibitors as anti-inflammatory and analgesic agents that lack the gastrointestinal side-effects of traditional NSAIDs. Although research on COX-2 inhibitors has focussed mainly on inflammation and pain, experimental and epidemiological data suggest that COX-2 inhibitors could be used in the treatment or prevention of a broader range of diseases. In this review, some key points and unresolved issues related to the discovery of COX-2 inhibitors, the kinetic and structural basis for their selectivity, and possible complications in their development and use will be discussed.

Covalent modification of cyclooxygenase-2 (COX-2) by 2-acetoxyphenyl alkyl sulfides, a new class of selective COX-2 inactivators.

All of the selective COX-2 inhibitors described to date inhibit the isoform by binding tightly but noncovalently at the substrate binding site. Recently, we reported the first account of selective covalent modification of COX-2 by a novel inactivator, 2-acetoxyphenyl hept-2-ynyl sulfide (70) (Science 1998, 280, 1268-1270). Compound 70 selectively inactivates COX-2 by acetylating the same serine residue that aspirin acetylates. This paper describes the extensive structure-activity relationship (SAR) studies on the initial lead compound 2-acetoxyphenyl methyl sulfide (36) that led to the discovery of 70. Extension of the S-alkyl chain in 36 with higher alkyl homologues led to significant increases in inhibitory potency. The heptyl chain in 2-acetoxyphenyl heptyl sulfide (46) was optimum for COX-2 inhibitory potency, and introduction of a triple bond in the heptyl chain (compound 70) led to further increments in potency and selectivity. The alkynyl analogues were more potent and selective COX-2 inhibitors than the corresponding alkyl homologues. Sulfides were more potent and selective COX-2 inhibitors than the corresponding sulfoxides or sulfones or other heteroatom-containing compounds. In addition to inhibiting purified COX-2, 36, 46, and 70 also inhibited COX-2 activity in murine macrophages. Analogue 36 which displayed moderate potency and selectivity against purified human COX-2 was a potent inhibitor of COX-2 activity in the mouse macrophages. Tryptic digestion and peptide mapping of COX-2 reacted with [1-14C-acetyl]-36 indicated that selective COX-2 inhibition by 36 also resulted in the acetylation of Ser516. That COX-2 inhibition by aspirin resulted from the acetylation of Ser516 was confirmed by tryptic digestion and peptide mapping of COX-2 labeled with [1-14C-acetyl]salicyclic acid. The efficacy of the sulfides in inhibiting COX-2 activity in inflammatory cells, our recent results on the selectivity of 70 in attenuating growth of COX-2-expressing colon cancer cells, and its selectivity for inhibition of COX-2 over COX-1 in vivo indicate that this novel class of covalent modifiers may serve as potential therapeutic agents in inflammatory and proliferative disorders.

Design of selective inhibitors of cyclooxygenase-2 as nonulcerogenic anti-inflammatory agents.

The discovery of a second isoform of cyclooxygenase (cyclooxygenase-2) that is expressed in inflammatory cells and the central nervous system, but not in the gastric mucosa, offers the possibility of developing anti-inflammatory and analgesic agents that lack the gastrointestinal side effects of currently available nonsteroidal anti-inflammatory drugs. Lead compounds from several different structural classes have been identified and shown to be slow, tight-binding inhibitors that express their selectivity for cyclooxygenase-2 in the time-dependent step. The determination of structures of enzyme-inhibitor co-crystals along with site-directed mutagenesis experiments reveal the molecular basis for selectivity of some, but not all, inhibitors. Preclinical and clinical studies suggest cyclooxygenase-2 inhibitors are highly promising new agents for the treatment of pain and inflammation, and for the prevention of cancer.

Aspirin-like molecules that covalently inactivate cyclooxygenase-2.

Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.

Kinetics of the interaction of nonsteroidal antiinflammatory drugs with prostaglandin endoperoxide synthase-1 studied by limited proteolysis.

Many nonsteroidal antiinflammatory agents (NSAIDs) bind to prostaglandin endoperoxide synthase (PGHS) and induce a conformational change in the PGHS apoprotein that renders it resistant to cleavage by trypsin at Arg277. In the present study, the trypsin protection assay was modified to permit detection of conformational changes at times as short as 5 s after the addition of inhibitor. The kinetics of the induction and reversal of trypsin resistance in apoPGHS-1 by a series of NSAIDs and isozyme-specific PGHS-1 and PGHS-2 inhibitors were determined. All compounds induced resistance to trypsin cleavage at a rapid rate. The conformational change induced by competitive inhibitors was reversed on prolonged incubation with trypsin (approximately 5 min). In contrast, the resistance induced by irreversible inhibitors was not lost during a 5 min incubation with trypsin. All of the selective PGHS-2 inhibitors protected against tryptic cleavage of apoPGHS-1 but did not inhibit the protein's cyclooxygenase activity. The results suggest that induction of trypsin resistance is a reflection of the initial association of reversible as well as irreversible inhibitors with the apoprotein.

Design, synthesis, and biochemical evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases.

N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibitors of prostaglandin endoperoxide synthase (PGHS). The corresponding N-alkylmaleimides were only time-dependent inactivators of PGHS, suggesting that the carboxylate is critical for rapid inhibition. Several N-substituted maleimide analogs containing structural features similar to those of the nonsteroidal anti-inflammatory drug aspirin were synthesized and evaluated as inhibitors of PGHS. Most of the aspirin-like maleimides inactivated the cyclooxygenase activity of purified ovine PGHS-1 in a time- and concentration-dependent manner similar to that of aspirin. The peroxidase activity of PGHS was also inactivated by the maleimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The corresponding succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition of PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-14C]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to the chromatographic conditions. Furthermore, hematin-reconstituted PGHS-1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displayed spontaneous regeneration of about 50% of the cyclooxygenase and peroxidase activities, suggesting that the adduct responsible for the inhibition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited by N-(carboxyheptyl)maleimide, did not display regeneration of enzyme activity, but addition of hematin to the inhibited apoenzyme led to spontaneous recovery of about 50% of cyclooxygenase activity. These results suggest that addition of heme leads to a conformational change in the protein which increases the susceptibility of the adduct toward hydrolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimide, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cyclooxygenase active site is proposed.