Quantitative drug interactions prediction system (Q-DIPS): a dynamic computer-based method to assist in the choice of clinically relevant in vivo studies.

Metabolic drug interactions are a major source of clinical problems, but their investigation during drug development is often incomplete and poorly specific. In vitro studies give very accurate data on the interactions of drugs with selective cytochrome P450 (CYP) isozymes, but their interpretation in the clinical context is difficult. On the other hand, the design of in vivo studies is sometimes poor (choice of prototype substrate, doses, schedule of administration, number of volunteers), with the risk of minimising the real potential for interaction. To link in vitro and in vivo studies, several authors have suggested using extrapolation techniques, based on the comparison of in vitro inhibition data with the active in vivo concentrations of the inhibitor. However, the lack of knowledge of one or several important parameters (role of metabolites, intrahepatocyte accumulation) often limits the possibility for safe and accurate predictions. In consequence, these methods are useful to complement in vitro studies and help design clinically relevant in vivo studies, but they will not totally replace in vivo investigation in the future. We have developed a computerised application, the quantitative drug interactions prediction system (Q-DIPS), to make both qualitative deductions and quantitative predictions on the basis of a database containing updated information on CYP substrates, inhibitors and inducers, as well as pharmacokinetic parameters. We also propose a global approach to drug interactions problems--'good interactions practice--to help design rational drug interaction investigations, sequentially associating in vitro studies, in vitrolin vivo extrapolation and finally well-designed in vivo clinical studies.

Quantitative drug interactions prediction system (Q-DIPS): a computer-based prediction and management support system for drug metabolism interactions.

OBJECTIVE: Drug biotransformation and interactions are a major source of variability in the response to drugs. The superfamily of cytochromes P450 plays a key role in this phenomenon but, because of the complexity of interactions between drugs and isozymes, it becomes more and more difficult for clinicians to master the knowledge required to predict the occurrence of such drug interactions. To predict and help manage the occurrence of cytochrome P450-dependent interactions, we developed an original computer application: Q-DIPS (quantitative drug interactions prediction system). METHODS: A multidisciplinary work team was created, associating clinical pharmacologists, pharmacists and a computer scientist. Major steps of investigation were: (1) the creation of a database to collect qualitative and quantitative data describing substrates, inhibitors and inducers of specific cytochrome P450 isozymes, with quality assessments; (2) the development of multi-access to these data and (3) their incorporation into extrapolation systems allowing the prediction of in vivo drug interactions on the basis of in vitro data. As an example, prediction and validation studies of CYP3A4 inhibition by ketoconazole and fluconazole will be discussed. RESULTS: Q-DIPS gives up-to-date information, in dynamic tables, describing which specific P450 isozymes metabolise a given drug, as well as which drugs may inhibit or induce a given isozyme. To better answer common clinical questions and help to rapidly evaluate the risk of interactions, it is possible to obtain an overview of substances causing interactions with a specific drug or to focus on drugs taken by a patient ("clinical case"). For each question, key references, relevant quantitative data and quality indices are easily accessible. Two modules allowing input with commercial names and the anatomical therapeutic chemical classification were also included. On the basis of enzymatic and pharmacokinetic data generated in vitro or collected in vivo, the extrapolation module integrates quantitative models to predict the impact of a treatment on enzymatic activities. The simplest model predicted a strong but fluctuating inhibition of CYP3A4 by ketoconazole, whereas the impact of fluconazole was lower. Validations with published in vivo data suggested an appropriate prediction of the risk. CONCLUSION: The current Q-DIPS prototype shows promising potential for helping to improve the management of drug interactions involving metabolism. Validation of extrapolation techniques need to be completed, in view of including important factors such as intrahepatocyte drug accumulation, contribution of metabolites to inhibition as well as in vitro non-specific binding to microsomal proteins. The final goal will be to help select the most judicious clinical studies to be performed so as to avoid useless, expensive and unethical investigations in man.

Stereoselective interaction between piroxicam and acenocoumarol.

1. An open-label study was performed to assess the effect of piroxicam on the pharmacokinetics of acenocoumarol enantiomers. 2. Eight healthy male volunteers received an oral dose of 4 mg rac-acenocoumarol on days 1 and 8, plus 40 mg piroxicam orally 2 h before the anticoagulant on day 8. R- and S-acenocoumarol, piroxicam and their metabolites were measured in plasma over a 24 h interval. 3. The pharmacokinetics of R-acenocoumarol were markedly modified by piroxicam: Cmax+28.0% (s.d.23.8), P < 0.05; AUC(0, 24 h)+47.2% (21.5), P < 0.005; and t1/2 +38.0% (34.5), P < 0.01. A concomitant decrease of CL/F was observed: -30.8% (10.0), P < 0.0001. A similar, but statistically non-significant trend, was observed on the S-enantiomer: Cmax: +9.5% (s.d.36.6), AUC(0, 24 h): + 15.4% (23.4), t1/2: +19.9% (42.0), and CL/F: -9.8% (20.5). V/F remained unchanged for both enantiomers. 4. Piroxicam plasma AUC(0, 24 h) correlated closely with R- and S-acenocoumarol AUCs on day 1 (r = 0.901, P < 0.005 and r = 0.797, P < 0.05, respectively), as well as with the difference of AUC between days 1 and 8 for R-acenocoumarol (r = 0.903, P < 0.001) and S-acenocoumarol (r = 0.711, P < 0.05). 5. Piroxicam markedly reduced acenocoumarol enantiomer clearance, with a greater effect on the more active R-isomer. This interaction, which occurs in addition to the well documented pharmacodynamic one (effect on platelets), is expected to result in increased anticoagulant effect.

Role of human liver microsomal CYP2C9 in the biotransformation of lornoxicam.

OBJECTIVE: The nature of the enzyme(s) catalysing the biotransformation of lornoxicam to one of its major metabolites, 5'-hydroxy-lornoxicam, has been investigated in human liver microsomes. The reaction kinetics were characterised, the affinity of lornoxicam for three major human drug metabolising cytochrome P-450 isozymes (CYP2C9, CYP2D6 and CYP3A4) was determined, and inhibition of the reaction by known substrates (diclofenac, ibuprofen, mefenamic acid, phenytoin, tolbutamide and warfarin) and the prototype inhibitor (sulphaphenazole) of CYP2C9 was investigated. RESULTS: Lornoxicam 5'-hydroxylation displayed single enzyme Michaelis-Menten kinetics, with a KM of 3.6 mu mol center dot l-1 and a Vmax of 2.6 nmol center dot h-1 center dot mg-1 microsomal protein. The apparent affinity of lornoxicam was high for CYP2C9, but negligible for CYP3A4 and CYP2D6. Inhibition of lornoxicam 5'-hydroxylation by CYP2C9 substrates and sulphaphenazole competitively and completely inhibited lornoxicam 5'-hydroxylation (Ki = 0.31 mu mol center dot l-1 as well as lornoxicam clearance (Ki = 0.33 mu mol center dot l-1), partial metabolic clearance (fm) = 0.95). CONCLUSION: 5'-Hydroxylation appears to be the only cytochrome P-450 catalysed metabolic reaction of lornoxicam by human liver microsomes and this major in vivo biotransformation pathway is catalysed virtually exclusively by CYP2C9.

Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes.

OBJECTIVE: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies). METHODS: Diclofenac 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6. RESULTS: Diclofenac 4'-hydroxylation exhibited Michaelis-Menten kinetics with kM = 3.4 mumol.l-1 and Vmax = 45 nmole.mg-1 P.h-1. Relative immunoreactivity of CYP2C9 was correlated with Vmax and CL(int). Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with kM = 4.4 mumol.l-1 and Vmax = 5.0 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP2D6 was correlated with Vmax and CL(int). Midazolam 1'-hydroxylation also exhibited Michaelis-Menten kinetics with kM = 3.3 mumol.l-1 and Vmax = 35 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP3A4 was correlated with Vmax and CL(int). Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes. CONCLUSION: The velocity of metabolite formation (Vmax) by the three major human drug metabolising P450 monoxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for Vmax than kM. Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers.

In vitro comparative inhibition profiles of major human drug metabolising cytochrome P450 isozymes (CYP2C9, CYP2D6 and CYP3A4) by HMG-CoA reductase inhibitors.

OBJECTIVE: The affinity of (+)-, (-)- and (+/-)- fluvastatin, a new synthetic HMG-CoA reductase inhibitor developed as a racemate, for specific human P450 monooxygenases in liver microsomes was compared with that of the pharmacologically active acidic forms of lovastatin, pravastatin and simvastatin. METHODS: Affinity was determined as the inhibitory potency for prototype reactions for 3 major drug metabolising enzymes: diclofenac 4'-hydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), and midazolam 1'-hydroxylation (CYP3A4). RESULTS: Lovastatin acid, pravastatin and simvastatin acid displayed moderate affinity for all three P450 isozymes (estimated Ki > 50 micromol.1(-1)). Racemic and (+)- and (-)-fluvastatin showed moderate affinity (estimated Ki > 50 micromol.1(-1)) for CYP2D6 and CYP3A4, whereas their affinity for CYP2C9 was high (estimated Ki < 1 micromol.1(-1)). Diclofenac 4'-hydroxylation was competitively and stereoselectively inhibited, with measured Ki's of 0.06 and 0.28 micromol.1(-1) for (+)- and (-)- fluvastatin, respectively. CONCLUSION: Fluvastatin selectively inhibits a major drug metabolising enzyme (CYP2C9), the (+)-isomer (pharmacologically more active) showing 4-5 fold higher affinity. As already reported for lovastatin and simvastatin, in vivo drug interactions by inhibition of liver oxidation of CYP2C9 substrates (e.g. hypoglyceamic sulphonylureas and oral anticoagulants) may be expected.

In vivo inhibition profile of cytochrome P450TB (CYP2C9) by (+/-)-fluvastatin.

BACKGROUND: (+/-)-Fluvastatin is a synthetic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that selectively and competitively inhibits P450TB (CYP2C9) in vitro. The potential for kinetic interactions in vivo between fluvastatin and P450TB substrates was therefore investigated in healthy volunteers. METHODS: Diclofenac (25 mg orally) oxidation was used as a marker of P450TB activity on days 0, 1, and 8 of fluvastatin treatment (40 mg/day). RESULTS: Diclofenac peak concentration (Cmax) increased over time (0.28 [SD, 0.12], 0.38 [0.20], and 0.45 [0.4] mg/L on days 0, 1, and 8, respectively). Oral clearance was reduced on days 1 and 8 (14% and 15%, respectively). A time-dependent decrease in urinary metabolic ratio (MR, 4'-hydroxydiclofenac/diclofenac) was noted (1.07 [0.34], 0.90 [0.23] and 0.70 [0.18] on days 0, 1, and 8, respectively [p < 0.0001]) for the first 4 hours. The interaction was clear in only some individuals; MR reduction was related to baseline MR and it was more pronounced in subjects with a higher baseline MR (p < 0.01). Fluvastatin Cmax (0.18 [0.11] and 0.32 [0.1] mg/L on days 1 and 8, respectively) and area under the curve (0.28 [0.12] and 0.43 [0.15] hr.mg/L on days 1 and 8, respectively; p < 0.001) increased over time. Diclofenac MR reduction was correlated with fluvastatin concentrations. CONCLUSIONS: Interactions between fluvastatin and P450TB substrates (phenytoin, oral anticoagulants, oral hypoglycemic agents, and nonsteroidal antiinflammatory drugs) may occur, at least in some patients.

Single-dose pharmacokinetics of oral fleroxacin in bacteremic patients.

Fleroxacin is a new broad-spectrum quinolone which can be given by the oral route. The present study was designed to assess the influence of bacteremia on the pharmacokinetics of a single oral dose of fleroxacin. Thirteen patients with proven bacteremia (one or more pairs of positive blood cultures, no hypotension) were given a single 400-mg fleroxacin dose orally on two occasions while also receiving standard antibiotic therapy. The first dose was administered 12 to 36 h after the last positive blood culture was drawn (day 1), and a second dose was administered 7 days later (day 7 +/- 2) to compare the pharmacokinetics between the acute and the convalescent phases of the disease. Following each administration of fleroxacin, serial plasma samples were collected for up to 72 h and were analyzed for unchanged drug by a reversed phase high-pressure liquid chromatography technique. There were no significant changes in the following pharmacokinetic parameters (mean standard deviation) the maximum concentration of drug in serum (6.4 +/- 1.5 versus 6.7 +/- 1.9 mg/liter), the minimum concentration of drug in serum, defined as the concentration of drug in serum at 24 h postdose (3.0 +/- 1.7 versus 2.5 +/- 1.2 mg/liter), the time to the maximum concentration of drug in serum (2.3 +/- 1.4 versus 2.0 +/- 1.2 h), and the elimination half-life (19.7 +/- 8.0 versus 17.9 +/- 6.9 h). Fleroxacin clearances were compared for each individual patient. A positive correlation (R2 = 0.787) was found between the values measured on day 1 and day 7. Oral clearance of fleroxacin (CL = CL/F, where F is bioavailability was slightly, but not significantly, reduced during the bacteremic phase (oral clearance, 43.8+/- 23.5 versus 48.5 +/- 17.5 ml/min.). When compared with previous results obtained in healthy young subjects, longer times to the maximum concentration of drug in serum and elimination half-lives and higher areas under the curve were observed. This could be due to the bacteremic state, the old age of the patients (mean, 66 years), and the low renal clearance (mean calculated creatinine clearance, 71.1 ml/min). A single oral dose of 400 mg of fleroxacin provides sufficient levels in serum to cover susceptible microorganisms for at least 24 h in bacteremic patients. Renal function appeared to be the key element that had to be taken into consideration to adapt fleroxacin dosage profiles in our patient population. Bacteremia itself appeared to amplify that phenomenon, but to a much lesser extent than renal function did.

Selective inhibition of major drug metabolizing cytochrome P450 isozymes in human liver microsomes by carbon monoxide.

The selectivity of carbon monoxide binding to specific human cytochrome P450 isozymes was investigated by studying its inhibition of prototype reactions for 3 major drug metabolizing P450s in liver microsomes: dextromethorphan O-demethylation and (+)-bufuralol 1'-hydroxylation (P450DB1, CYP2D6), diclofenac 4'-hydroxylation (P450TB, CYP2C subfamily), and midazolam 1'-hydroxylation (P450NF, CYP3A subfamily). The affinity of carbon monoxide is different for each P450 isozyme. Warburg partition coefficients were 0.35, 1.1 and 3.9 microM for P450DB1, P450TB and P450NF, respectively. Differential inhibition by carbon monoxide may be a useful tool to identify specific human cytochrome P450 isozymes in the early screening of drug biotransformation catalysts. Further studies involving other P450 isozymes and substrates should extend our understanding of the phenomena and their implications.

Influence of rifampin on fleroxacin pharmacokinetics.

Staphylococcus aureus infections have been successfully treated in animal models with the combination of fleroxacin and rifampin. We studied the influence of rifampin, a potent cytochrome P-450 inducer, on the pharmacokinetics and biotransformation of fleroxacin in 14 healthy young male volunteers. Subjects were given 400 mg of fleroxacin orally once a day for 3 days to reach steady state. After a wash-out period of 2 days, the same subjects received 600 mg of rifampin orally once daily for 7 days. On days 5 to 7 of rifampin treatment, 400 mg of fleroxacin was again administered once daily. Concentrations of fleroxacin as well as its two major urinary metabolites, N-demethyl- and N-oxide-fleroxacin, in plasma and urine were determined by reverse-phase high-performance liquid chromatography. The extent of hepatic enzyme induction by rifampin was confirmed by a significant increase of 6-beta-hydroxycortisol urinary output from 160.8 +/- 41.4 to 544.8 +/- 120.7 micrograms/4 h. There were no significant changes in the peak fleroxacin concentration in plasma (6.3 +/- 1.2 versus 6.2 +/- 1.9 mg/liter), time to maximum concentration of fleroxacin in plasma (1.1 +/- 0.9 versus 1.3 +/- 1.1 h), or renal clearance (58.3 +/- 16.4 versus 61.9 +/- 19.2 ml/min). The area under the curve AUC (71.4 +/- 15.8 versus 62.2 +/- 13.7 mg.h/liter) and the terminal half-life of fleroxacin (11.4 +/- 2.2 versus 9.2 +/- 1.1 h) decreased (P < 0.05), while the total plasma clearance increased from 97.7 +/- 21.6 to 112.3 +/- 25.8 ml/min (P < 0.01). Despite being statistically significant, this 15% increase in total plasma clearance does not appear to be clinically relevant. Metabolic clearance by N demethylation was increased ( 6.9 +/- 2.4 versus 12.5 +/- 3.2 ml/min; P < 0.01), whereas clearance by N oxidation did not change (5.8 +/- 1.1 versus 5.8 +/- 1.5 ml/min). Fleroxacin elimination was slightly increased (about 15%) through induction of metabolic clearance to N-demethyl-fleroxacin. Since fleroxacin levels remained above the MIC for 90% of the tested isolates of methicillin-susceptible S. aureus for at least 24 h, dose adjustment does not appear necessary, at least for short-term treatments.

A major role for cytochrome P450TB (CYP2C subfamily) in the actions of non-steroidal antiinflammatory drugs.

Most non-steroidal antiinflammatory drugs (NSAIDs) are extensively metabolized by liver oxidation with broad interindividual variability, but little is known about the nature of the enzyme(s) catalysing these reactions. The role of specific cytochrome P450 isozymes in the formation of the major oxidized metabolites of phenylacetic acid (diclofenac), propionic acid (ibuprofen), fenamate (mefenamic acid) and oxicam (piroxicam and tenoxicam) derivatives was studied in human liver microsomes using mostly selective inhibition by known substrates and inhibitors of specific cytochrome P450 monooxygenases. A common isozyme (P450TB, CYP2C subfamily) controls the major elimination pathways of these NSAIDs. The authors have also determined, in two in vitro models of P450TB activity, the affinity for this isozyme of other NSAIDs (acetylsalicylic acid, indomethacin, pirprofen). The NSAIDs tested displayed a high affinity (5-500 microM): diclofenac approximately mefenamic acid > ibuprofen approximately indomethacin approximately piroxicam approximately tenoxicam > acetylsalicylic acid approximately pirprofen. Cytochrome P450TB therefore plays a key role in the oxidation by human liver of major NSAIDs from various chemical classes. Inhibition data and chemical structure similarities suggest that many other NSAIDs may be substrates of this isozyme as well. P450TB appears to be a common site both for the control of interindividual differences in the capacity to oxidize major NSAIDs and for interactions involving NSAIDs as well as other known substrates (oral anticoagulants, hypoglycaemic sulfonylureas, phenytoin) or inhibitors (antifungals, antibacterial sulfonamides, calcium channel blockers) of P450TB. Consequently this P450 isozyme is likely to be a major determinant of NSAIDs action.

Similar effect of oxidation deficiency (debrisoquine polymorphism) and quinidine on the apparent volume of distribution of (+/-)-metoprolol.

The influence of phenotype (debrisoquine type of oxidation polymorphism) and quinidine on (+/-)-metoprolol distribution parameters was investigated in 7 young male volunteers (4 extensive and 3 poor metabolisers). (+/-)-Metoprolol tartrate 20 mg was administered as a 20 min infusion i) alone, ii) 12 h after an oral 50 mg quinidine sulphate capsule, and iii) on the last day of 3 days of treatment with 250 mg quinidine sulphate b.d. as a slow-release tablet. No stereoselectivity was apparent in either poor or extensive metabolizers. When (+/-)-metoprolol was administered alone the apparent volume of distribution at steady-state (Vss) was higher in extensive than in poor metabolisers (4.84 vs 2.83 l.kg-1, respectively). Pre-treatment with low or multiple high doses of quinidine decreased Vss in extensive metabolisers to values comparable to those in poor metabolisers (3.50 and 3.18 l.kg-1, respectively), but had no significant effect in poor metabolisers (3.24 and 3.42 l.kg-1, respectively). Estimation of Vss by noncompartmental analysis or assuming elimination exclusively from the peripheral compartment yielded similar, although somewhat higher, estimates. Despite the small number of subjects, (+/-)-metoprolol distribution appeared to be different both in genetically and environmentally (quinidine)-determined poor metabolisers, and quinidine inhibition was a good, reversible in vivo model of the genetic deficiency in handling (+/-)-metoprolol. Differences both in first pass pulmonary elimination or in tissue binding are logically consistent with these observations, but the amplitude of the effect exceeds expectations from available biological evidence on selective pulmonary metabolic activity and on specific tissue binding sites.

Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4'-hydroxylation in human liver.

The nature of the enzyme(s) catalyzing the major metabolic pathway of diclofenac, 4'-hydroxylation, was investigated in human liver microsomes. Inhibition studies were performed with tolbutamide and sulfaphenazole (respectively the prototype substrate and a selective inhibitor of cytochrome P450TB--CYP2C subfamily), and with phenytoin and (+/-)-warfarin, other proposed substrates of P450TB. Diclofenac 4'-hydroxylation displayed single enzyme Michaelis-Menten kinetics and was similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6), with a Km of 5.6 +/- 1.5 microM (mean +/- sd) and a Vmax of 60.6 +/- 23.5 nmol/mgP/h. Inhibition by tolbutamide, sulfaphenazole, phenytoin and (+/-)-warfarin was comparable in all livers, with values predicted from their Km or Ki for cytochrome P450TB determined in separate studies and a competitive inhibition model. Sulfaphenazole competitively inhibited diclofenac 4'-hydroxylation (Ki = 0.11 +/- 0.08 microM, n = 3). Diclofenac 4'-hydroxylation is predominantly catalyzed by a cytochrome P450 isozyme of the CYP2C subfamily, most likely CYP2C9. This particular isozyme therefore appears to be responsible for the oxidation of polar acidic substances such as non-steroidal anti-inflammatory drugs from different chemical classes. It also constitutes a common site for drug interactions involving these compounds, as well as tolbutamide, phenytoin and warfarin.

[The biotransformation of NSAIDs: a common elimination site and drug interactions]. / La biotransformation des AINS: un site commun d'élimination et d'interactions médicamenteuses.

Many NSAIDs are eliminated predominantly through hepatic biotransformation in man. We have studied, in human hepatic microsomes, the role of specific cytochrome P450 isozymes in the formation of the major metabolites of oxicam (piroxicam and tenoxicam), phenylacetic (diclofenac) and propionic acid (ibuprofen) derivatives. A common isozyme (P450TB, CYP2C subfamily) controls the major elimination pathway of these NSAIDs. We have also determined, in two in vitro models of P450TB, the affinity for this isozyme of NSAIDs from other chemical classes (acetylsalicylic acid, mefenamic acid and indomethacin). All NSAIDs tested displayed a high affinity (3-300 microM) for cytochrome P450TB. Cytochrome P450TB plays a major role in the elimination of several NSAIDs with different chemical structures. NSAIDs are substrates as well as potential inhibitors of cytochrome P450TB. Their elimination can therefore be reduced by concomitant administration of known inhibitors of P450TB (antifungals, antibacterial sulfonamides, calcium channel blockers).

[Q-DIPS: current approach to prediction of drug interactions]. / Q-DIPS: une approche nouvelle de la prédiction des interactions médicamenteuses.

Interactions are an important cause of adverse drug effects. Because of the large number of substances and the complexity of the mechanisms involved, computers can be of great help in the detection, prediction and management of interactions. We propose a new complementary approach to the prediction of interactions through a key mechanism, hepatic drug biotransformation. The originality of the approach consists in integrating in vitro enzymatic data and pharmacokinetic data to make in vivo predictions. An Inhibition Index (II) is defined to characterize the interaction potential of an inhibitor for a specific isozyme independently of the isozyme's substrates. It is thus possible, even in the absence of prior clinical observations, to make individualized qualitative as well as quantitative predictions of potential in vivo interactions. Q-DIPS is a prototype computer system under development on a Macintosh to manage the large amount of multi-dimensional data and facilitate the investigation and validation of extrapolations. The kinetics of II for two antifungal drugs, fluconazole and ketoconazole, are simulated and compared in order to illustrate the potential of the approach.

In vitro oxidation of oxicam NSAIDS by a human liver cytochrome P450.

The nature of the enzyme(s) catalyzing the major metabolic pathway (5'-hydroxylation) of oxicam NSAIDs was investigated in subcellular preparations of human liver tissue. Microsomal, but not cytosolic, fractions catalyzed the 5'-hydroxylation of tenoxicam. This reaction required NADPH and was inhibited by various nonselective P450 inhibitors (CO, SKF-525A, ketoconazole), but not by the peroxidase inhibitor NaN3. Tenoxicam 5'-hydroxylation exhibited simple Michaelis-menten kinetics compatible with catalysis by a single enzyme, but it strongly inhibited its own oxidation at concentrations higher than 100-150 microM. Piroxicam competitively inhibited tenoxicam 5'-hydroxylation and, conversely, tenoxicam competitively inhibited piroxicam 5'-hydroxylation. Tenoxicam 5'-hydroxylation kinetics were similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6). Dextromethorphan (CYP2D6 prototype substrate) and midazolam (CYP3A prototype substrate) had no influence on tenoxicam 5'-hydroxylation, whereas mephenytoin, tolbutamide and sulfaphenazole (Ki = 0.1 microM) inhibited it. This indicates that the 5'-hydroxylation of both piroxicam and tenoxicam is predominantly catalyzed by at least one cytochrome P450 isozyme of the CYP2C subfamily.

The role of lipophilicity in the inhibition of polymorphic cytochrome P450IID6 oxidation by beta-blocking agents in vitro.

The importance of lipophilicity as a determinant of the affinity of beta-adrenoceptor blocking agents for a specific human hepatic monooxygenase--cytochrome P450IID6 (responsible for the debrisoquine-type of oxidation polymorphism)--was investigated in vitro by estimating the inhibition constants of a series of compounds in a microsomal system with monitoring of the kinetics of dextromethorphan O-demethylation. Lipophilicity is a key predictor of the affinity of beta-blocking drugs for cytochrome P450IID6 and of their potential to cause specific competitive drug interactions, but more complex structural factors appear to be important as well. A high lipophilicity is also a necessary, but not a sufficient condition for these compounds to be metabolized by cytochrome P450IID6.

Semi-quantitative simulation for reasoning about physiological models of drug kinetics and effects.

Inter- and intra-individual pharmacokinetic or pharmacodynamic variability is a major cause of adverse drug reactions or ineffective therapy. We are developing a computer-based tool for predicting the consequences of different physiological and pathological states and for reasoning about the possible causes of observed variability that may be useful both in a clinical decision support environment for drug monitoring and as a research aid in the investigation of the influence of physiological factors on drug response. It is based on a physiological approach to pharmacokinetic modeling in which actual anatomical or physiological entities, such as organs, tissues or blood flows, are represented. These models serve as the basis for semi-quantitative simulation, a method linking classical quantitative simulation (by numerical integration of differential equations) with artificial intelligence-based qualitative simulation techniques. This approach retains the mathematical power of the Systems Dynamics method for solving complex, time-varying systems containing feed-back loops, which are intractable for current qualitative knowledge representation techniques, and extends it with the causal reasoning and explanation power of symbolic inference techniques used in expert systems. It also allows problem solving in situations, so common in medicine, where initial values of variables and parameters cannot be estimated precisely. Simulation outputs are intended to be qualitatively, but not necessarily quantitatively, correct. The semi-quantitative simulation method was originally developed in MacLisp on a DEC 2060 and applied to modeling cardio-vascular physiology. We are porting the code to Common Lisp on a Macintosh and adapting the approach to pharmacology, concentrating on drug metabolism issues, with lidocaine pharmacokinetics as a test case.

Dextromethorphan O-demethylation in liver microsomes as a prototype reaction to monitor cytochrome P-450 db1 activity.

Liver dextromethorphan O-demethylation to dextrorphan is associated with the debrisoquin type of oxidation phenotype in humans. We studied dextromethorphan oxidation in vitro using human liver microsomes to investigate the kinetics of the polymorphic monooxygenase (cytochrome P-450 db1) and factors that may influence its activity. In microsomal preparations from six extensive metabolizers the reaction parameters were: Michaelis-Menten constant = 3.4 +/- SD 1.0 mumol/L and maximum rate of metabolism = 10.2 +/- 5.3 nmol x mg P-1 x hr-1, vs 48 mumol/L and 2.2 nmol x mg P-1 x hr-1, respectively in microsomes prepared from the liver of one poor metabolizer. The reaction was inhibited by nonspecific monooxygenase inhibitors such as SKF 525-A (Ki = 100 nmol/L) and cimetidine (Ki = 40 mumol/L), by known substrates of the polymorphic isozyme such as [+]-bufuralol (Ki = 7.5 mumol/L), debrisoquin (Ki = 25 mumol/L), and sparteine (Ki = 45 mumol/L), and by the selective cytochrome P-450 db1 inhibitor quinidine (Ki = 15 nmol/L). This assay permits in vitro screening for candidate substrates or inhibitors of the polymorphic isozyme.