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J S Harmatz

Publications and source records attributed to J S Harmatz.

At least 19 recordsLinked to original sources
PubMed1998

In vitro approaches to predicting drug interactions in vivo.

In vitro metabolic models using human liver microsomes can be applied to quantitative prediction of in vivo drug interactions caused by reversible inhibition of metabolism. One approach utilizes in vitro Ki, values together with in vivo values of inhibitor concentration to forecast in vivo decrements of clearance caused by coadministration of inhibitor. A critical limitation is the lack of a general scheme for assigning intrahepatic exposure of enzyme to inhibitor or substrate based only on plasma concentration; however, the assumption that plasma protein binding necessarily restricts hepatic uptake is not tenable. Other potential limitations include: flow-dependent hepatic clearance, "mechanism-based" chemical inhibition, concurrent induction, or a major contribution of gastrointestinal P450-3A isoforms to presystemic extraction. Nonetheless, the model to date has provided reasonably accurate forecasts of in vivo inhibition of clearance of several substrates (desipramine, terfenadine, triazolam, alprazolam, midazolam) by coadministration of selective serotonin reuptake-inhibitor antidepressants and azole antifungal agents. Such predictive models deserve further evaluation, since they may ultimately yield more cost-effective and expeditious screening for drug interactions, with reduced human drug exposure and risk.

Animals↗
PubMed1997

Alprazolam hydroxylation by mouse liver microsomes in vitro: the effect of age and phenobarbital induction.

The effects of age on hepatic microsomal enzyme induction were studied in male CD-1 mice. Six week old and 1 year old animals were treated with either phenobarbital (80 mg kg-1) or saline once daily for 3d. Twenty-four hours after the last treatment, animals were sacrificed and livers were harvested. Hepatic microsomal fractions were isolated and incubated with alprazolam, a triazolobenzodiazepine metabolized by cytochrome P-450-3A isoforms in humans. Metabolites were identified and quantitated by HPLC. All microsomal preparations produced two principal metabolites (alpha-OH- and 4-OH-alprazolam) while microsomes from phenobarbital-treated animals also produced a third metabolite (alpha, 4-dihydroxyalprazolam). Vmax, K(m), and intrinsic clearance (Vmax/K(m) ratio) for both alpha-OH- and 4-OH-alprazolam in the saline-treated control animals were not significantly different between age groups. Vmax and intrinsic clearance for both metabolites were more than three times greater in phenobarbital-treated animals than in the control mice (p < 0.001). Age did not influence the extent of induction, and both pathways were induced to approximately an equal extent. Thus the present in vitro study of liver microsomal preparations from male CD-1 mice does not delineate a mechanism for impaired alprazolam clearance in aging organisms in vivo. There is no evidence that age alters susceptibility to induction by phenobarbital.

Aging↗
PubMed1997

Influence of oral contraceptive use and cigarette smoking, alone and together, on antipyrine pharmacokinetics.

The pharmacokinetics of antipyrine following a single 1-g intravenous dose was determined in 63 healthy women. Subjects were divided into 4 groups as follows: 1) cigarette smokers using low-dose oral contraceptives (n = 15); 2) nonsmokers using low-dose oral contraceptives (n = 12); 3) cigarette smokers not using oral contraceptives (n = 10); and 4) controls, neither cigarette smokers nor oral contraceptive users. Plasma antipyrine concentrations during 24 to 48 hours after dosage were measured by high-performance liquid chromatography. Mean kinetic variables in the nonsmoking, non-oral contraceptive using control group were: volume of distribution, 37.7 L; elimination half-life, 13.2 hours; and clearance, 34.4 mL/min. In cigarette smoking, non-oral contraceptive users versus controls, elimination half-life was reduced (8.0 vs. 13.2 hours, P < 0.05) and clearance increased (56.0 vs. 34.4 mL/min, P < 0.05). In nonsmoker oral contraceptive users, the reverse was true (elimination half-life was significantly increased: 16.6 vs. 13.2 hours, P < 0.05; and clearance was significantly decreased: 24.8 vs. 34.4 mL/min, P < 0.05). In smokers who were using oral contraceptives, values were not significantly different from controls (elimination half-life, 11.2 hours; clearance, 39.5 mL/min). Volume of distribution did not differ among the four groups. Thus the opposing effects on antipyrine clearance of the induction of metabolism by cigarette smoking and the inhibition due to low dose oral contraceptive use in effect negate each other when combined in humans.

Adolescent↗
PubMed1997

Dose-dependent pharmacokinetics and psychomotor effects of caffeine in humans.

Twelve healthy volunteers received oral placebo, 250 mg of caffeine, and 500 mg of caffeine in a randomized, double-blind, single-dose crossover study. Caffeine kinetics were nonlinear, with clearance significantly reduced and elimination half-life prolonged at the 500-mg compared to the 250-mg dose. The lower dose of caffeine produced more favorable subjective effects than the higher dose (elation, peacefulness, pleasantness), whereas unpleasant effects (tension, nervousness, anxiety, excitement, irritability, nausea, palpitations, restlessness) following the 500-mg dose exceeded those of the 250-mg dose. The lower dose of caffeine enhanced performance on the digit symbol substitution test and a tapping speed test compared to placebo; high-dose caffeine produced less performance enhancement than the lower dose. The plasma concentration versus response relationship revealed concentration-dependent increases in anxiety and improvements in cognitive and motor performance at low to intermediate concentrations. Both caffeine doses reduced electroencephalographic amplitude over the 4 Hz to 30 Hz spectrum, as well as in the alpha (8-11 Hz) and beta (12-30 Hz) ranges; however, effects were not dose-dependent. While favorable subjective and performance-enhancing stimulant effects occur at low to intermediate caffeine doses, the unfavorable subjective and somatic effects, as well as performance disruption, from high doses of caffeine may intrinsically limit the doses of caffeine used in the general population.

Adult↗
PubMed1997

Human cytochromes mediating N-demethylation of fluoxetine in vitro.

Biotransformation of the selective serotonin reuptake inhibitor antidepressant, fluoxetine, to its principal metabolite, norfluoxetine, was evaluated in human liver microsomes and in microsomes from transfected cell lines expressing pure human cytochromes. In human liver microsomes, formation of norfluoxetine from R,S-fluoxetine was consistent with Michaelis-Menten kinetics (mean K(m) = 33 microM), with evidence of substrate inhibition at high substrate concentrations in a number of cases. The reaction was minimally inhibited by coincubation with chemical probes inhibitory for P450-2D6 (quinidine), -1A2 (furafylline, alpha-naphthoflavone), and -2E1 (diethyldithiocarbamate). Substantial inhibition was produced by coincubation with sulfaphenazole (Ki = 2.8 microM), an inhibitory probe for P450-2C9, and by ketoconazole (Ki = 2.5 microM) and fluvoxamine (Ki = 5.2 microM). However, ketoconazole, relatively specific for P450-3A isoforms only at low concentrations, reduced norfluoxetine formation by only 20% at 1 microM, and triacetyloleandomycin (> or = 5 microM) reduced the velocity by only 20-25%. Microsomes from cDNA-transfected human lymphoblastoid cells containing human P450-2C9 produced substantial quantities of norfluoxetine when incubated with 100 microM fluoxetine. Smaller amounts of product were produced by P450-2C19 and -2D6, but no product was produced by P450-1A2, -2E1, or 3A4. Cytochrome P450-2C9 appears to be the principal human cytochrome mediating fluoxetine N-demethylation. P450-2C19 and -3A may make a further small contribution, but P450-2D6 is unlikely to make an important contribution.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

Midazolam hydroxylation by human liver microsomes in vitro: inhibition by fluoxetine, norfluoxetine, and by azole antifungal agents.

Biotransformation of the imidazobenzodiazepine midazolam to its alpha-hydroxy and 4-hydroxy metabolites was studied in vitro using human liver microsomal preparations. Formation of alpha-hydroxy-midazolam was a high-affinity (Km = 3.3 mumol/L) Michaelis-Menten process coupled with substrate inhibition at high concentrations of midazolam. Formation of 4-hydroxy-midazolam had much lower apparent affinity (57 mumol/L), with minimal evidence of substrate inhibition. Based on comparison of Vmax/Km ratios for the two pathways, alpha-hydroxy-midazolam formation was estimated to account for 95% of net intrinsic clearance. Three azole antifungal agents were inhibitors of midazolam metabolism in vitro, with inhibition being largely consistent with a competitive mechanism. Mean competitive inhibition constants (Ki) versus alpha-hydroxy-midazolam formation were 0.0037 mumol/L for ketoconazole, 0.27 mumol/L for itraconazole, and 1.27 mumol/L for fluconazole. An in vitro-in vivo scaling model predicted inhibition of oral midazolam clearance due to coadministration of ketoconazole or itraconazole; the predicted inhibition was consistent with observed interactions in clinical pharmacokinetic studies. The selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine and its principal metabolite, norfluoxetine, also were inhibitors of both pathways of midazolam biotransformation, with norfluoxetine being a much more potent inhibitor than was fluoxetine itself. This finding is consistent with results of other in vitro studies and of clinical studies, indicating that fluoxetine, largely via its metabolite norfluoxetine, may impair clearance of P450-3A substrates.

Anesthetics, Intravenous↗
PubMed1996

Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine.

Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-Km (mean Km1 = 68 microM) and high-Km (mean Km2 = 7691 microM) components. The low-K(m) enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 microM. Among index inhibitor probes, alpha-naphthoflavone was a highly potent inhibitor of the low-Km enzyme (Ki1 = 0.013 microM); furafylline also was a moderately active inhibitor (Ki1 = 4.4 microM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (Ki1 = 32 microM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean Ki1 = 0.24 microM). The other SSRIs were more than tenfold less potent. Mean Ki1 values were: fluoxetine, 4.4 microM; norfluoxetine, 15.9 microM; sertraline, 8.8 microM; desmethylsertraline, 9.5 microM; paroxetine, 5.5 microM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity.

Antidepressive Agents, Second-Generation↗
PubMed1996

Inhibition of cytochrome P450 by nefazodone in vitro: studies of dextromethorphan O- and N-demethylation.

Nefazodone (NEF), a 5-HT2A/2C antagonist antidepressant, is extensively metabolized in the human body to hydroxy NEF (OH-NEF), p-hydroxy NEF (pOH-NEF), a dione metabolite, and via cleavage of the molecule to m-chlorophenyl-piperazine (mCPP) and BMY-33604. The latter is further metabolized to BMS-183695-01 (BMSa) and BMS-183562-01 (BMSb). To investigate the potential of NEF and its metabolites to interfere with the metabolism of other drugs, we tested these compounds for their ability to alter dextromethorphan (DMO) O-demethylation to dextrorphan (DOP; an index reaction for CYP2D6) and N-demethylation to 3-methoxy morphinan (MEM, a recently proposed index reaction of CYP3A3/4). The assay was performed in an in vitro system with human liver microsomes from three different donors. NEF, OH-NEF, pOH-NEF, mCPP and BMSb were weak inhibitors of DMO O and N-demethylation, with average Ki values ranging from 18 to 50 microM for DOP formation, and from 21 to > 200 microM for MEM formation. The dione metabolite and BMSa did not produce detectable inhibition of either pathway. The findings for DMO O-demethylation, well-established as a CYP2D6-mediated reaction, indicate that NEF and metabolites are weak inhibitors of this reaction, with Ki values at least 100 times higher than fluoxetine (Ki = 0.1 microM +/- 0.09). The implications of results on DMO N-demethylation are not clear. In vivo data, as well as in vitro data based on "pure' CYP3A3/4 substrates, provide evidence for clinically relevant CYP3A3/4 inhibition by NEF, OH-NEF, and pOH-NEF. Thus, formation of MEM by N-demethylation of DMO may not constitute a suitable index reaction to probe CYP3A3/4 activity.

Antidepressive Agents, Second-Generation↗
PubMed1996

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation.

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.

Amitriptyline↗
PubMed1996

Inhibition of terfenadine metabolism in vitro by azole antifungal agents and by selective serotonin reuptake inhibitor antidepressants: relation to pharmacokinetic interactions in vivo.

Biotransformation of the H-1 antagonist terfenadine to its desalkyl and hydroxy metabolites was studied in vitro using microsomal preparations of human liver. These metabolic reactions are presumed to be mediated by Cytochrome P450-3A isoforms. The azole antifungal agent ketoconazole was a highly potent inhibitor of both reactions, having mean inhibition constants (Ki) of 0.037 and 0.34 microM for desalkyl- and hydroxy-terfenadine formation, respectively. Itraconazole also was a potent inhibitor, with Ki values of 0.28 and 2.05 microM, respectively. Fluconazole, on the other hand, was a weak inhibitor. Six selective serotonin reuptake inhibitor antidepressants tested in this system were at least 20 times less potent inhibitors of terfenadine metabolism than was ketoconazole. An in vitro-in vivo scaling model used in vitro Ki values, typical clinically relevant plasma concentrations of inhibitors, and presumed liver:plasma partition ratios to predict the degree of terfenadine clearance impairment during coadministration of terfenadine with these inhibitors in humans. The model predicted a large and potentially hazardous impairment of terfenadine clearance by ketoconazole and, to a slightly lesser extent, by itraconazole. However, fluconazole and the six selective serotonin reuptake inhibitors (SSRIs) at usual clinical doses were not predicted to impair terfenadine clearance to a degree that would be of clinical importance. Caution is nonetheless warranted with the coadministration of SSRIs and terfenadine when high doses of SSRIs (particularly fluoxetine) are administered. Also, some individuals may be unusually susceptible to metabolic inhibition for a variety of reasons.

Antifungal Agents↗
PubMed1996

Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies.

The identification of cytochrome P450 (CYP) isozymes mediating metabolic pathways of drugs has become increasingly important in anticipating pharmacokinetic drug interactions. The activity of individual CYPs can be monitored in vitro with human liver microsomes by means by relatively specific metabolic reactions: for CYP1A1/2, phenacetin O-deethylation; for CYP2C9, phenytoin 4-hydroxylation; for CYP2C19, S-mephenytoin 4'-hydroxylation; for CYP2D6, dextromethorphan O-demethylation; for CYP3A3/4, alprazolam 4-hydroxylation, and for CYP2E1, chlorzoxazone 6-hydroxylation. We determined the kinetic parameters (Vmax, Km) of these reactions and utilized them to test a monoclonal rat liver CYP1A1 antibody, a monoclonal rat liver CYP2E1 antibody, a polyclonal rabbit anti-rat liver CYP3A1 antibody, and a polyclonal goat anti-rat liver CYP2C11 antibody for their specificity and inhibitory capacity. The CYP1A1 monoclonal antibody (MAb), the CYP2E1 MAb, and the CYP3A1 polyclonal antibody (PAb) inhibited only their respective index reactions. The CYP2C11 PAb inhibited both phenytoin 4-hydroxylation and S-mephenytoin 4'-hydroxylation. At a microsomal versus antibody protein mass ratio of 1:15, 4-hydroxyalprazolam formation was reduced by 73.4% of control with the CYP3A1 PAb; 4'-hydroxymephenytoin formation decreased by 66.3% and 4-hydroxyphenytoin formation by 43.4% with the CYP2C11 PAb; phenacetin O-deethylation was reduced by 39.7% with the CYP1A1 MAb, and 6-hydroxychlorzoxazone formation decreased by 30.0% with the CYP2E1 MAb. Thus, all antibodies tested are at least CYP subfamily specific. The PAbs exhibited greater than 60% inhibition versus the CYP3A3/4- and the CYP2C19-mediated reactions, whereas the MAbs produced less than 50% inhibition for their respective index reactions. Because of their limited inhibitory capacity, MAbs may be correspondingly limited as tools for identification of human CYP enzymes via immunoinhibition studies.

Animals↗
PubMed1996

Triazolam biotransformation by human liver microsomes in vitro: effects of metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole.

Biotransformation of the triazolobenzodiazepine triazolam to its hydroxylated metabolites, alpha-hydroxy (OH)- and 4-OH-triazolam, was studied in vitro using microsomal preparations of human liver. Mean values of Vmax (10.3 nM/min/mg of protein) and Km (304 microM) for the 4-OH pathway exceeded values for the alpha-OH pathway (2.4 and 74, respectively). However the mean Vmax/Km ratios for the two pathways were nearly identical, indicating that both contribute approximately equally to intrinsic clearance. Ketoconazole was a powerful inhibitor of triazolam biotransformation, having mean competitive Ki values of 0.006 and 0.023 microM for the alpha-OH and 4-OH pathways. This is consistent with the role of P450-3A isoforms in mediating triazolam biotransformation. The serotonin2 antagonist antidepressant nefazodone inhibited the alpha-OH and 4-OH pathways (Ki = 0.6 and 1.7 microM, respectively), but with considerably less activity than ketoconazole. Among six selective serotonin reuptake-inhibitor antidepressants, norfluoxetine was the most potent inhibitor (Ki = 2.7 and 8.0 microM) and fluoxetine itself was the weakest (Ki = 7.0 and 44.3 microM). In a double-blind clinical pharmacokinetic-pharmacodynamic study, administration of triazolam (0.125 mg) preceded by ketoconazole, compared to triazolam preceded by placebo, produced a nearly 9-fold reduction in apparent oral clearance of triazolam (41 vs. 337 ml/min) and a 4-fold prolongation of half-life (13.5 vs. 3.4 hr). Pharmacodynamic testing indicated enhancement of electroencephalographic beta activity, and enhanced decrements in digit-symbol substitution test performance, attributable to coadministration of ketoconazole. Plasma ketoconazole concentrations measured in the clinical study ranged from 0.02 microgram/ml (projected minimum) to 4.95 micrograms/ml (maximum). An in vitro-in vivo scaling model, using these plasma ketoconazole concentrations together with liver partition ratios and the in vitro Ki values, predicted a decrement of triazolam clearance due to ketoconazole coadministration that was consistent with the 88% decrement in clearance actually observed in vivo.

Adult↗
PubMed1995

Inhibition of alprazolam and desipramine hydroxylation in vitro by paroxetine and fluvoxamine: comparison with other selective serotonin reuptake inhibitor antidepressants.

In vitro preparations of human liver microsomes were used to study the inhibiting effects of two selective serotonin reuptake inhibitor (SSRI) antidepressants, paroxetine and fluvoxamine, on metabolism via hydroxylation of alprazolam and of desipramine. These reactions are mediated by Cytochromes P450-3A4 and P450-2D6, respectively. Paroxetine was a highly potent inhibitor of desipramine hydroxylation; the inhibition constant (Ki) value of 2.0 microM indicated greater inhibiting potency than fluoxetine or norfluoxetine. The in vitro data predicted in vivo impairment of desipramine clearance by coadministration of paroxetine which was in the same range as observed in a clinical study. Fluvoxamine, by contrast, was a much weaker inhibitor of desipramine hydroxylation, having a Ki value (16.6 microM) similar to those of sertraline and desmethylsertraline. For hydroxylation of alprazolam, paroxetine was a relatively weak inhibitor, approximately comparable to fluoxetine, whereas fluvoxamine showed inhibiting capacity similar to that of norfluoxetine. The in vitro data predicted the degree of impairment of alprazolam clearance observed in vitro model can therefore provide clinically relevant data on prediction of potential drug interactions with SSRIs.

Alprazolam↗
PubMed1995

Metabolism of drugs by cytochrome P450 3A isoforms. Implications for drug interactions in psychopharmacology.

Members of the P450 3A subfamily are the most abundant of the human hepatic cytochromes. CYP3A isoforms mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. Activity of CYP3A in humans is variable among individuals, but there is no evidence of genetic polymorphism. Significant amounts of CYP3A are present in the gastrointestinal tract, and may contribute to presystemic extraction of drugs such as cyclosporin. The azole antifungal agents ketoconazole and itraconazole are potent inhibitors of human CYP3A isoforms. Selective serotonin reuptake inhibitor (SSRI) antidepressants are also CYP3A inhibitors, but much less potent than ketoconazole or itraconazole. In vitro models can provide important information on the qualitative and quantitative activity of potential inhibitors of human cytochromes. However, in vitro inhibition constant (Ki) values alone do not predict the magnitude of an in vivo interaction, nor whether an interaction will be of clinical importance. For example, SSRIs are predicted to impair clearance of the antihistamine terfenadine in humans. However, the magnitude of this effect is much less than would be associated with a pharmacokinetic interaction of clinical importance.

Animals↗