In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration.

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

Biotransformation of sulindac in end-stage renal disease.

In normal humans sulindac, a prodrug, undergoes two major biotransformations: irreversible oxidation to the inactive sulfone metabolite and reversible reduction to the pharmacologically active sulfide metabolite. To assess any effect of end-stage renal failure on sulindac biotransformation, six patients were given 200 mg sulindac orally. Plasma was sampled over 24 hours. Protein binding of sulindac and metabolites was determined by equilibrium dialysis. Results were compared with historic controls. AUC(0-12) for sulindac and the sulfone were similar to controls. AUC(0-12) for the sulfide was significantly reduced to 4.85 micrograms X hr/ml from 13.1 micrograms X hr/ml (P less than 0.02). Protein binding of all three compounds was significantly reduced by renal failure. When corrected for protein binding, the AUC(0-12) for sulindac and the sulfone was twice that of controls whereas that of the sulfide was 42 ng X hr/ml compared with 83 ng X hr/ml in normal individuals (P less than 0.001). This suggests that end-stage renal failure impairs the reduction of sulindac to the active sulfide whereas oxidation to the sulfone is intact.

Error dependent on renal function when monoexponential equation assumed. Serum clearances of 125I-iothalamate and 131I-o-iodohippurate.

An example from the literature has been used to demonstrate errors involved in calculating drug clearance by inappropriate use of the apparent drug distribution volume Vdext. The Vdext is always an overestimate of the true volume of distribution in a multicompartment system, and the degree of overestimation in using it to calculate clearance for such a system will increase as renal function increases. Drug dosages calculated on the basis of overestimated clearance values may give rise to overdosage in normal individuals, or therapeutic failure in severely uremic patients. Problems associated with the use of an oversimplified pharmacokinetic model for clearance calculations are discussed, together with the concept of model-independent calculations.

Enterohepatic circulation of sulindac and metabolites.

Four subjects were studied by continuous intraduodenal sampling to establish the existence and determine the extent of enterohepatic recirculation of sulindac and its sulfide and sulfone metabolites. Sulindac, 200 mg by mouth, was given every 12 hr for 7 days. After the last dose was given intraduodenally, constant duodenal infusion of a nutrient mixture and sampling of duodenal contents were performed through a triple-lumen intraduodenal tube for 12 hr. Calculation of nonabsorbed drug in the samples and quantitation of drug and metabolite levels in the biliary secretions were made possible by nonabsorbable markers in the drug solution and in the infusate. Interindividual variations in the absolute values for each of the chemical species were over a 200% range, but for each subject relative clearances were in a remarkably constant ratio, averaging 1:12:12 for sulfide:sulindac:sulfone. Total biliary excretions of the prodrug (sulindac) and active pharmacophore (sulfide) calculated from these biliary clearances and historic mean plasma AUCs were 136% and 22% of dose. Thus, there is a correlation between data in man and those in five other species and the data established that, after sulindac, the contribution of enterohepatic circulation to conservation of the active pharmacophore is achieved predominantly at the level of inactive prodrug.

3-Hydroxy-alpha-methyltyrosine progenitors: synthesis and evaluation of some (2-oxo-1,3-dioxol-4-yl)methyl esters.

The (5-methyl-2-oxo-1,3- dioxol -4-yl)methyl and (5-tert-butyl-2-oxo-1, 3- dioxol -4-yl)methyl esters of 3-hydroxy-alpha-methyltyrosine (methyldopa) were prepared and evaluated as progenitors of the amino acid. 1H NMR experiments reveal that the esters are converted cleanly to methyldopa and the corresponding alpha-diketone at pH 7.4, with the 5-methyl derivative undergoing hydrolysis faster than the 5-tert-butyl analogue. Bioavailability studies in dogs show that the esters, particularly the 5-methyl derivative, yield significant plasma levels of methyldopa. Both esters are orally effective antihypertensive agents in spontaneously hypertensive (SH) rats. These studies indicate that (2-oxo-1,3- dioxol -4-yl)methyl esters are viable prodrugs for the latentiation of methyldopa.

Concomitant use of cytochrome P450 3A4 inhibitors and simvastatin.

The long-term safety profile of simvastatin, established over 10 years of clinical use, is excellent. The principal adverse effect of all inhibitors of hydroxymethylglutarate co-enzyme A (HMG-CoA) reductase, myopathy, is infrequent. Simvastatin is a substrate for cytochrome P450 3A4 (CYP3A4). CYP3A4 inhibitors can elevate the plasma concentration of HMG-CoA reductase inhibitory activity derived from simvastatin. Clinical experience has shown that concomitant use of potent inhibitors of CYP3A4 increase the risk for myopathy. Evaluation of data from clinical trials and postmarketing surveillance allows assessment of whether concomitant use of weaker CYP3A4 inhibitors, as represented by calcium channel blockers, has any effect on the risk of myopathy. Cases of myopathy in long-term clinical megatrials and in analyses of postmarketing adverse event reports have been surveyed. In megatrials with simvastatin, the overall incidence of myopathy was 0.025%. The proportion of patients developing myopathy who were taking a calcium channel blocker with simvastatin (1 of 3) was similar to the proportion of patients taking a calcium channel blocker overall. Among marketed-use adverse event reports, concomitant medication with a potent CYP3A4 inhibitor was more frequent among reports of myopathy than among reports of nonmusculoskeletal adverse events. No excess use of calcium channel blockers among myopathy reports was observed. We conclude that the overall risk of myopathy during treatment with simvastatin is very low. Potent CYP3A4 inhibitors, especially cyclosporine, significantly increase the risk. There is no evidence that weaker CYP3A4 inhibitors such as calcium channel blockers increase the risk.

The efficacy and six-week tolerability of simvastatin 80 and 160 mg/day.

The hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin is the most effective of the currently approved hypolipidemic drugs and has been shown to reduce mortality and coronary morbidity in patients with coronary artery disease. For these patients the United States National Cholesterol Education Program advocates reducing low-density lipoprotein (LDL) cholesterol to <100 mg/dl. However, in some patients this cannot be achieved using monotherapy with simvastatin 40 mg/day, the current maximal recommended dose. To evaluate the effectiveness of extending the dosage range, 156 subjects with LDL cholesterol >160 mg/dl and triglycerides (TG) <350 mg/dl were randomized to simvastatin at doses of 40, 80, and 160 mg/day in a 26 week, double-blind, 3-period, complete block crossover study. Each active treatment period was 6 weeks in duration with intervening 2 week washout periods. Median reductions from baseline in LDL cholesterol were 41%, 47%, and 53% in the 40-, 80-, and 160-mg groups, respectively. The corresponding reductions in plasma TG were 21%, 23%, and 33%. High-density lipoprotein (HDL) cholesterol increased by 6% to 8% in each group. One patient (0.7%) taking 160 mg developed myopathy; 1 patient (0.7%) taking 80 mg, and 3 (2.1%) taking 160 mg had transaminase elevations > 3 times the upper limit of normal. No new or unexpected adverse effects were observed. We conclude that simvastatin at doses of 80 and 160 mg/day provides additional efficacy with a low short-term incidence of adverse effects; our results support the continued investigation of simvastatin at these doses.

Use of in vitro drug metabolism data to evaluate metabolic drug-drug interactions in man: the need for quantitative databases.

It has become widely accepted that metabolic drug-drug interactions can be forecast using in vitro cytochrome P450 (CYP) data. For any CYP form-inhibitor pair, the magnitude of the interaction will depend on the potency of the inhibitor (inhibition constant, Ki) the concentration of the inhibitor available for inhibition ([I]), the fraction of the substrate dose metabolized by CYP (fm), and the fraction of the CYP-dependent metabolism catalyzed by the inhibited CYP form (e.g., fm,CYP3A4). While progress is being made toward our understanding of the factors necessary for predictions of [I]/Ki in vivo, it is evident that there is a need for quantitative databases that contain in vitro (e.g., Ki, fm,CYP3A4) and in vivo pharmacokinetic/absorption-distribution-metabolism-excretion (PK/ADME) data (e.g., fm) for a large number of marketed drugs. Ultimately, such databases would allow one to integrate all of the data necessary for the prediction of drug-drug interactions and permit the rational evaluation of new drug entities.

Pharmacokinetics of 125I-iothalamate and 131I-o-iodohippurate in man.

The pharmacokinetics of 125I-iothalamate (IOT125I) and 131I-o-iodohippurate (OIH131I) have been studied in patients with varying degrees of renal insufficiency. Both compounds have been shown to obey two-compartment model kinetics after intravenous administration. The clearances are closely related to renal function, but poor correlations were obtained between renal function and compartmental distribution characteristics. This study provides further evidence that IOT125I and OIH131I may be used as indicators of renal function in patients after a single intravenous injection.

Pharmacokinetics of methsuximide and a major metabolite in dogs.

The pharmacokinetics of methsuximide and its major metabolite 2-methyl-2-phenylsuccinimide were studied in dogs after single intravenous doses. Plasma methsuximide levels were described by a two-compartment open model, and those of the metabolite were described by a one-compartment open model. An expression was derived that describes both methsuximide and metabolite plasma levels after methsuximide administration. Excellent fits were obtained between observed data and those predicted from the model. The metabolite accounted for 40% of the overall elimination of methsuximide, and the half-life of the metabolite (15 hr) was much greater than that of the parent drug (1--3.5 hr). The results suggest that pharmacological effects after methsuximide administration may be due primarily to the metabolite, which may accumulate in the body during repeated doses.

An automated sample preparation and high performance liquid chromatographic method for the determination of MK-591, a novel leukotriene biosynthesis inhibitor, in human plasma.

A high performance liquid chromatography assay utilizing an automated sample preparation procedure for the determination of a novel leukotriene biosynthesis inhibitor, (MK-591), in human plasma has been developed. After aliquoting plasma samples and adding internal standard manually, the BenchMate Workstation executed protein precipitation and solid-phase extraction. Following evaporation to dryness, the residue was reconstituted and chromatographed isocratically on a cyano-phase analytical column. MK-591 and the internal standard were separated from each other and from endogenous plasma substances and detected with an absorbance detector. The assay has been validated in the concentration range 10-1000 ng ml-1 and has the sensitivity and specificity necessary to quantify plasma concentrations from several clinical studies.

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine.

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology Robot and BenchMate Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid-liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate performed buffer and organic solvent addition, liquid-liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2-100 ng ml-1 in plasma and 0.1-20 micrograms ml-1 in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.

Sensitive high-performance liquid chromatographic assay using electrochemical detection for a novel prodrug ester of methyldopa, pivaloyloxyethyl 3-(3,4-dihydroxyphenyl)-2-methylalaninate, in plasma and urine.

The pivaloyloxyethyl ester of methyldopa is an antihypertensive prodrug possessing improved bioavailability properties over methyldopa. A sensitive cation-exchange, high-performance liquid chromatographic assay using electrochemical detection has been developed for the ester in plasma and urine in order to determine the extent of its hydrolysis after oral administration. The chromatographic conditions involve two Altex Partisil 10 SCX columns (25 cm X 4.6 mm) in series; a mobile phase consisting of methanol, potassium phosphate buffer, pH 3.0, and EDTA disodium dihydrate; and an electrochemical detector set at 0.5 V. The pivaloyloxyethyl ester in plasma or urine is extracted into ethyl acetate, back-extracted into 0.1 M sulfuric acid, and analyzed directly by high-performance liquid chromatography. For urine, the ethyl acetate extract is washed with a buffer (pH 8.0) prior to the back-extraction step. The assay gives a linear response over the concentration range of 10-160 ng/ml in plasma and 20-400 ng/ml in urine.

Bioavailability of fluvoxamine given with and without food.

The influence of concomitant food intake on plasma concentrations of the antidepressant drug fluvoxamine maleate was investigated in a two-way, crossover study design. Eight male and four female healthy, young volunteers received a single oral dose of fluvoxamine maleate (50 mg, tablet) on two occasions: after an overnight fast and immediately after a breakfast. Food did not affect maximum fluvoxamine plasma levels (Cmax), or the time to reach Cmax (tmax). The plasma AUC of fluvoxamine was on average 7 per cent lower in the fed than in the fasted state. It is concluded that the effect of food on the pharmacokinetics of fluvoxamine is negligible.

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection.

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(-)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.

The use of stable isotope labeling and liquid chromatography/tandem mass spectrometry techniques to study the pharmacokinetics and bioavailability of the antimigraine drug, MK-0462 (rizatriptan) in dogs.

MK-0462 (rizatriptan) is a 5HT1D agonist being developed for the treatment of migraine. The assay for this substance in plasma and urine is based on HPLC with tandem mass spectrometry (MS/MS) detection. The procedure has been modified to include the simultaneous determination of the [triazole-13C2, 15N3-] stable-isotope-labelled analogue for which the lower quantifiable limit was 0.1 ng mL-1. The assay has been applied to study the pharmacokinetics of MK-0462 after simultaneous oral and intravenous administration of the drug and its stable-isotope-labelled analogue to dogs. The experiment afforded an estimate of plasma clearance concomitant with a precise measurement of the drug's oral bioavailability. The increasing use of LC-MS/MS in quantitative experiments may renew interest in stable isotopes as tools for pharmaceutical research.

Pharmacokinetics of the pivaloyloxyethyl (POE) ester of methyldopa, a new prodrug of methyldopa.

A prodrug of methyldopa, the pivaloyloxyethyl (POE) ester, was administered orally to healthy human volunteers at doses equivalent to 500 and 1000 mg of methyldopa and was compared to oral and intravenous doses of methyldopa. The time courses of availability of methyldopa to the general circulation were compared and contrasted with the model-independent estimates of total systemic availability. The POE ester of methyldopa is completely hydrolyzed on the first pass; delivery of methyldopa to the general circulation was faster, more uniform, and more extensive compared to orally administered methyldopa. The systemic availability of methyldopa averaged 64% of the dose with a coefficient of variation (CV) of 15% for the prodrug treatments compared to 27% of the dose with a CV of 63% for methyldopa. First-pass metabolism of drug to the mono-O-sulfate conjugate of methyldopa was lower for the POE ester than for methyldopa.

Stereoselectivity in the disposition and metabolism of the uricosuric-diuretic agent, indacrinone, in Rhesus monkeys.

The physiological disposition following intravenous dosing of the separate enantiomers of indacrinone-14C (I), and of their major metabolite, 4'-hydroxyindacrinone-14C (M), was studied in the rhesus monkey. Pharmacokinetic analysis indicated that the disposition of I and M was stereoselective. In the case of the enantiomers of I, the areas under the curves of plasma concentration vs. time were about sevenfold greater for the (S)(+)- as compared to the (R)(-)-enantiomer. Renal and plasma clearances of (R)(-)-I were five to seven times greater than those of (S)(+)-I. Total urinary recovery of unchanged drug and metabolite accounted for 70% of the administered dose of either enantiomer. The systemic availability of (R)(-)-M from (R)(-)-I was approximately 21% of the dose, whereas that of (S)(+)-M from (S)(+)-I was only 4%. More pronounced differences were noted in the kinetics of metabolite disposition. The AUC values were about 27 times greater for (S)(+)-M than (R)(-)-M, and the renal and plasma clearances were approximately 25-fold higher for (R)(-)-M as compared to (S)(+)-M. The volume of distribution of (S)(+)-M was only 12% of that observed with (R)(+)-M. There was no evidence of glucuronide or sulfate conjugates of any of the enantiomers. These findings are consistent with the pharmacological activity attributed to the different enantiomers.