Multivessel coronary artery spasm.

Coronary spasm is increasingly recognised as an important aetiological mechanism causing myocardial ischaemia. Occasionally cases present with evidence of ST segment elevation myocardial infarction, usually secondary to spasm confined to a solitary coronary artery. We present the rare and life-threatening case of severe coronary spasm afflicting all three major epicardial arteries simultaneously. It describes the difficult emergency scenario and ongoing management dilemmas encountered by physicians confronted with multivessel coronary spasm. Moreover we discuss the malignant prognosis associated with this ailment and describe the potential insights provided by cardiac magnetic resonance imaging that might identify those at greatest risk after the index event.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Importance of mechanistic drug metabolism studies in support of drug discovery: A case study with an N -sulfonylated dipeptide VLA-4 antagonist in rats.

N-(1-(3,5-dichlorobenzenesulfonyl)-2S-methyl-azetidine-2-carbonyl)-L-4-(2',6'-dimethoxyphenyl)phenylalanine (1) is a potent antagonist of the very late activating (VLA) antigen-4. During initial screening, 1 exhibited an apparent plasma clearance (CL) of 227 ml min(-1) kg(-1) in Sprague-Dawley rats following an intravenous bolus dose formulated in an aqueous solution containing 40% polyethylene glycol. Such a high CL value led to speculation that the elimination of compound 1 involved extra-hepatic tissues. However, the apparent plasma CL was reduced to 97 ml in(-1) kg(-1) when a 2-min time point was added to sample collections, and further decreased to 48 ml min(-1) kg(-1) after the dose was formulated in rat plasma. The lung extraction of 1 in rats was negligible whereas the hepatic extraction was > or =90%, based on comparison of area under the curve (AUC) values derived from intra-artery, intravenous, and portal vein administration. In rats dosed intravenously with [(14)C]-1, approximately 91% of the radioactivity was recovered in bile over 48 h, with 85% accounted for in the first 4-h samples. The biliary radioactivity profile consisted of approximately 30% intact parent compound, 20% 1-glucuronide, and 50% oxidation products resulting from O-demethylation or hydroxylation reactions. When incubated with rat liver microsomes, oxidative metabolism of 1 was inhibited completely by 1-aminobenzotriazole (ABT), whereas the oxidation and glucuronidation reactions were little affected in the presence of cyclosporin A (CsA). In contrast, the hepatic extraction of 1 in rats was unperturbed in animals pre-dosed with ABT, but was reduced approximately 60% following treatment with CsA. In vitro, 1 was a substrate of the rat organic anion transporter Oatp1b2, and its cellular uptake was inhibited by CsA. In addition, the hepatic extraction of 1 was approximately 30% lower in Eisai hyperbilirubinaemic rats which lack functional multidrug resistant protein-2 (MRP2). Collectively, these data suggest that the clearance of 1 in rats likely is a result of the combined processes of hepatic oxidation, glucuronidation and biliary excretion, all of which are facilitated by active hepatic uptake of parent compound and subsequent active efflux of both unchanged parent and its metabolites into bile. It was concluded, therefore, that multiple mechanisms contribute to the clearance of 1 in rats, and suggest that appropriate pharmacokinetic properties might be difficult to achieve for this class of compounds. This case study demonstrates that an integrated strategy, incorporating both rapid screening and mechanistic investigations, is of particular value in supporting drug discovery efforts and decision-making processes.

Hans Christian Andersen's visit to James Young Simpson.

Preparations are currently underway in Denmark for the celebration in 2005 of the 200th anniversary of the birth of that country's illustrious son Hans Christian Andersen. The renowned author of fairy tales had a passion for travel and recorded in his diary and in the several editions of his autobiography his encounters with the dignitaries and celebrities of his day. On this anniversary we recall the little-known occasion on which Hans Andersen was entertained to dinner and to a demonstration of the effects of inhalation of ether at the home of James Y. Simpson.

Influence of different recombinant systems on the cooperativity exhibited by cytochrome P4503A4.

1. The in vitro cooperativity exhibited by cytochrome P450 (CYP) 3A4 is influenced by the nature of the recombinant system in which the phenomenon is studied. Diclofenac, piroxicam and R-warfarin were used as model substrates, and quinidine was the effector. 2. The 5-, 5'- and 10-hydroxylation of diclofenac, piroxicam and R-warfarin, respectively, were enhanced five- to sevenfold by quinidine in human liver microsomal incubations. Whereas these cooperative drug interactions were apparent in incubations with CYP3A4 expressed in human lymphoblast cells, similar phenomena were not observed with the enzyme expressed in insect cells. 3. Insect cell microsomes were treated with a detergent and CYP3A4 was solubilized into a buffer medium. In incubations with CYP3A4 'freed' from its host membrane, the 5-hydroxylation of diclofenac increased with increasing quinidine concentrations, reaching a maximal eightfold elevation relative to controls. The metabolism of piroxicam and warfarin was similarly enhanced by quinidine. 4. Kinetically, enhancement by quinidine of the 5-hydroxylation of diclofenac in incubations with solubilized CYP3A4 was characterized by increases in the rate of metabolism with little change in the substrate-binding affinity. Conversely, the 3-hydroxylation of quinidine was not affected by diclofenac. 5. The data suggest that certain properties of CYP3A4 are masked by expression of the protein in insect cells and reinforce the concept that the enzyme possesses multiple binding domains. The absence of cooperative drug interactions with quinidine when CYP3A4 was expressed in insect cells might be due to an absence of enzyme conformation changes on quinidine binding, or the inability of quinidine to gain access to a putative effector-binding domain. 6. Caution should be exercised when comparing models for CYP3A4 cooperativity derived from different recombinant preparations of the enzyme.

Robert Halliday Gunning and the Victoria Jubilee prizes.

More than a century after the death of Robert Halliday Gunning, a large number of lectureships and prizes bearing his name continue to be awarded by scientific bodies and learned institutions in Scotland. Most of these awards were endowed in HM Queen Victoria s Jubilee year (1887-88) and bear the additional qualification 'Victoria Jubilee'. An account of the life of Robert Gunning and his various endowments is complemented by an analysis of the factors which determined the nature of his benefactions.

Mechanistic studies on the reversible metabolism of rofecoxib to 5-hydroxyrofecoxib in the rat: evidence for transient ring opening of a substituted 2-furanone derivative using stable isotope-labeling techniques.

Rofecoxib is a potent and highly selective cyclooxygenase-2 inhibitor used for the treatment of osteoarthritis and pain. Following administration of [4-(14)C]rofecoxib to intact rats, the plasma C(max) (at approximately 1 h) was followed by a secondary C(max) (at approximately 10 h), which was not observed in bile duct-cannulated rats. Following administration of [4-(14)C]5-hydroxyrofecoxib to intact or bile duct-cannulated rats, radiolabeled rofecoxib was detected in plasma, and once again a secondary C(max) for rofecoxib was observed (at approximately 10 h), which occurred only in the intact animals. These results indicate that reversible metabolism of rofecoxib to 5-hydroxyrofecoxib occurs in the rat and that the process is dependent upon an uninterrupted bile flow. Studies on the contents of the gastrointestinal tract of rats showed that conversion of 5-hydroxyrofecoxib to parent compound occurs largely in the lower intestine. Treatment of rats with [5-(18)O]5-hydroxyrofecoxib, followed by liquid chromatography-tandem mass spectrometry analyses of plasma samples, confirmed that 5-hydroxyrofecoxib undergoes metabolism to the parent drug, yielding [1-(18)O]rofecoxib, [2-(18)O]rofecoxib, and unlabeled rofecoxib. Similarly, treatment with [1,2-(18)O(2)]rofecoxib afforded the same three isotopic variants of rofecoxib. These findings are consistent with a metabolic sequence involving 5-hydroxylation of rofecoxib, biliary elimination of the corresponding glucuronide, and deconjugation of the glucuronide in the lower gastrointestinal tract. Reduction of the 5-hydroxyrofecoxib thus liberated yields a hydroxyacid that cyclizes spontaneously to regenerate rofecoxib, which is reabsorbed and enters the systemic circulation. This sequence represents a novel form of enterohepatic recycling and reflects the susceptibility of 5-hydroxyrofecoxib, as well as rofecoxib itself, to reversible 2-furanone ring opening under in vivo conditions.

Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4.

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin, marker substrates for cytochrome P450 3A4 are commonly used within the pharmaceutical industry to screen new chemical entities as inhibitors of CYP3A4 in a high-throughput manner to predict the potential for drug-drug interactions. However, it has been observed that inhibition data obtained with a given CYP3A4 probe substrate may not correlate well with results from a different probe. As a consequence, the choice of the probe compound becomes an important consideration in such screens. In the present study, kinetic interactions between either two of the above three substrates were evaluated, and three-dimensional nonlinear regression analysis was performed to understand the kinetic mechanisms of drug interaction. Our results demonstrate that the kinetic interaction between each pair of substrates does not appear to be competitive and that the interactions are characterized by an unchanged or a decrease in both apparent K(m) (a = 0.21-0.72, a change of K(m) in the absence of the effector) and V(max) (alpha and beta = 0.09-0.75, changes of V(max) in the absence of the effector). These data suggest that 1) the three substrates bind to different domains; 2) at least two substrates can coexist in the active site of CYP3A4; and 3) the two bound substrates interact kinetically with each other (e.g., through steric hindrance), thereby leading to a change in both apparent kinetic parameters and partial inhibition. Selection of multiple substrates, which are shown not to be competitive, is necessary to accurately predict CYP3A4 inhibition and the potential for drug-drug interaction.

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

In vitro stimulation of warfarin metabolism by quinidine: increases in the formation of 4'- and 10-hydroxywarfarin.

It has been demonstrated that the activity of cytochrome P450 (CYP)3A4 in certain cases is stimulated by quinidine (positive heterotropic cooperativity). We report herein that the 4'- and 10-hydroxylation of S- and R-warfarin are enhanced in human liver microsomal incubations containing quinidine. These reactions were catalyzed by CYP3A4, based on data derived from immunoinhibitory studies, with 4'-hydroxylation being preferentially associated with S-warfarin and 10-hydroxylation with R-warfarin. The 4'-hydroxylation of S-warfarin and 10-hydroxylation of R-warfarin increased with increasing quinidine concentrations and maximized at ~3- and 5-fold the values of controls, respectively. Stimulatory effects of quinidine also were observed with recombinant CYP3A4, suggesting that increases in warfarin metabolism were due to quinidine-mediated enhancement of CYP3A4 activity. This positive cooperativity of CYP3A4 was characterized by a 2.5-fold increase in V(max) for the 4'-hydroxylation of S-warfarin and a 5-fold increase in V(max) for the 10-hydroxylation of R-warfarin, with little change in K(m) values. Conversely, V(max) for the 3-hydroxylation of quinidine was not influenced by the presence of warfarin. These results are consistent with previous findings suggesting the existence of more than one binding site in CYP3A4 through which interactions may occur between substrate and effector at the active site of the enzyme. Such interactions were subsequently illustrated by a kinetic model containing two binding domains, and a good regression fit was obtained for the experimental data. Finally, stimulation of warfarin metabolism by quinidine was investigated in suspensions of human hepatocytes, and increases in the formation of 4'- and 10-hydroxywarfarin again were observed in the presence of quinidine, indicating that this type of drug-drug interaction occurs in intact cells.

Studies on the metabolism of troglitazone to reactive intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission.

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic alpha-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.

A kinetic model for the metabolic interaction of two substrates at the active site of cytochrome P450 3A4.

In many cases, CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrates that coexist at the active site. In the present study, we observed that alpha-naphthoflavone (alpha-NF) exhibited a differential effect on CYP3A4-mediated product formation as shown by an increase and decrease, respectively, of the carboxylic acid (P(2)) and omega-3-hydroxylated (P(1)) metabolites of losartan, while losartan was found to be an inhibitor of the formation of the 5,6-epoxide of alpha-NF. Thus, to address this problem, a kinetic model was developed on the assumption that CYP3A4 can accommodate two distinct and independent binding domains for the substrates within the active site, and the resulting velocity equations were employed to predict the kinetic parameters for all possible enzyme-substrate species. Our results indicate that the predicted values had a good fit with the experimental observations. Therefore, the kinetic constants can be used to adequately describe the nature of the metabolic interaction between the two substrates. Applications of the model provide some new insights into the mechanism of drug-drug interactions at the level of CYP3A4.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

Cytochrome P450 3A4-mediated interaction of diclofenac and quinidine.

The metabolism of diclofenac to its 5-hydroxylated derivative in humans is catalyzed by cytochrome P450 (CYP)3A4. We report herein that in vitro this biotransformation pathway is stimulated by quinidine. When diclofenac was incubated with human liver microsomes in the presence of quinidine, the formation of 5-hydroxydiclofenac increased approximately 6-fold relative to controls. Similar phenomena were observed with diastereoisomers of quinidine, including quinine and the threo epimers, which produced an enhancement in the formation of 5-hydroxydiclofenac in the order of 6- to 9-fold. This stimulation of diclofenac metabolism was diminished when human liver microsomes were pretreated with a monoclonal inhibitory antibody against CYP3A4. In contrast, neither cytochrome b(5) nor CYP oxidoreductase appeared to mediate the stimulation of diclofenac metabolism by quinidine, suggesting that the effect of quinidine is mediated through CYP3A4 protein. Further kinetic analyses indicated that V(max) values for the conversion of diclofenac to its 5-hydroxy derivative increased 4.5-fold from 13.2 to 57.6 nmol/min/nmol of CYP with little change in K(m) (71-56 microM) over a quinidine concentration range of 0 to 30 microM. Conversely, the metabolism of quinidine was not affected by the presence of diclofenac; the K(m) value estimated for the formation of 3-hydroxyquinidine was approximately 1.5 microM, similar to the quinidine concentration required to produce 50% of the maximum stimulatory effect on diclofenac metabolism. It appears that the enhancement of diclofenac metabolism does not interfere with quinidine's access to the ferriheme-oxygen complex, implicating the presence of both compounds in the active site of CYP3A4 at the same time. Finally, a approximately 4-fold increase in 5-hydroxydiclofenac formation was observed in human hepatocyte suspensions containing diclofenac and quinidine, demonstrating that this type of drug-drug interaction occurs in intact cells.

Metabolism of A dopamine D(4)-selective antagonist in rat, monkey, and humans: formation of A novel mercapturic acid adduct.

3-([4-(4-Chlorophenyl)piperazin-1-yl]-methyl)-1H-pyrrolo-2, 3-beta-pyridine (L-745,870) is a dopamine D(4) selective antagonist that has been studied as a potential treatment for schizophrenia, with the expectation that it would not exhibit the extrapyramidal side effects often observed with the use of classical antipsychotic agents. The metabolism of L-745,870 in vivo was investigated in the rat, rhesus monkey, and human using liquid chromatography-tandem mass spectrometry and/or NMR techniques in conjunction with radiochemical detection. In all three species, two major metabolic pathways were identified, namely N-dealkylation at the substituted piperazine moiety and the formation of a novel mercapturic acid adduct. It is proposed that the latter biotransformation process involves the formation of an electrophilic imine methide intermediate, analogous to that produced from 3-methyl indole. This report appears to represent the first example of metabolic activation of a 3-alkyl-7-azaindole nucleus.

Patterns of differentiation and proliferation in intraductal carcinoma of the prostate: significance for cancer progression.

BACKGROUND: Cribriform prostatic intraepithelial neoplasia (C-PIN) identifies a unique histological pattern: dysplastic cells line ductal/acinar walls but also span across gland lumens. C-PIN is distinct from other forms of dysplasia; it is seldom seen except within invasive cancer, it is more frequent in larger/higher-grade cancers; and it appears to contribute independently to aggressive behavior. Hence, C-PIN may represent a separate, more aggressive entity: intraductal carcinoma of the prostate (IDC-P). Here, support for that distinction stems from a histologic/biologic subclassification of IDC-P, whose elements are linked to features of invasive cancer. METHODS: Histologic criteria were tested against 26 radical prostatectomies, using immunostains for prostate-specific antigen, MUC-2, androgen receptor (differentiation), and Ki-67 (proliferation). Invasive cancer grade, stage, and follow-up were compared. RESULTS: Architecture of the central (luminal) cell compartment defined three subclasses of IDC-P: A (trabecular), B (cribriform), and C (solid/comedo), which represented progressive dedifferentiation with a reciprocal increase in proliferation. The IDC-P subpattern correlated with cancer stage, grade, and clinical course. CONCLUSIONS: IDC-P is a separate entity, distinct from PIN; cancers associated with IDC-P are more aggressive than those associated with only PIN. It comprises a spectrum of histological patterns which appear to be determined in concert with invasive cancer, whose prognosis it worsens.

Identification of S-(n-butylcarbamoyl)glutathione, a reactive carbamoylating metabolite of tolbutamide in the rat, and evaluation of its inhibitory effects on glutathione reductase in vitro.

Tolbutamide (TOLB), a widely used hypoglycemic agent in the therapy of non-insulin-dependent diabetes mellitus, has been reported to be teratogenic and/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are linked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductase (GR), although the mechanism by which this inhibition occurs remains unknown. In the study presented here, rats were injected with TOLB (200 mg/kg ip), and bile was collected for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarbamoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl isocyanate, as a minor metabolite of TOLB in bile. Upon incubation of SBuG (0.25-1.0 mM) with GR from either yeast or bovine intestinal mucosa in the presence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentration-dependent manner. No inhibition was observed when NADPH was omitted from incubations, or when the natural substrate for the enzyme, glutathione disulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over the concentration range of 0.8-2.0 mM. It is concluded that metabolic activation of TOLB in vivo leads to the generation of reactive intermediates (n-butyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At critical periods of organogenesis, the resulting perturbation of GSH homeostasis in exposed tissues may play a key role in the teratogenic and/or embryotoxic effects of TOLB.