Anti-IL-7 receptor α monoclonal antibody (GSK2618960) in healthy subjects - a randomized, double-blind, placebo-controlled study.

AIM: Interleukin (IL)-7 signalling modulates T cell activity and is implicated in numerous autoimmune diseases. The present study investigated the safety, pharmacokinetics, target engagement, pharmacodynamics and immunogenicity of GSK2618960, an IL-7 receptor-α subunit (CD127) monoclonal antibody. METHODS: A double-blind (sponsor-unblind) study of a single intravenous infusion of either GSK2618960 (0.6 mg kg-1 or 2.0 mg kg-1 ) or placebo was carried out in 18 healthy subjects over 24 weeks. RESULTS: GSK2618960 was well tolerated; there were no serious or significant adverse events. The observed half-life was 5 (±1) days (2.0 mg kg-1 ), with nonlinear pharmacokinetics. Full receptor occupancy (>95%) was observed until day 8 (0.6 mg kg-1 ) and day 22 (2.0 mg kg-1 ). Maximal inhibition of IL-7-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation was observed in 5/6 subjects until day 22 (2.0 mg kg-1 ). Mean circulating IL-7 and soluble receptor (CD127) levels were increased above baseline during days 2 and 15 (0.6 mg kg-1 ) and days 2 and 22 (2.0 mg kg-1 ). No meaningful changes were observed in absolute numbers or proportions of immune cell populations or inflammatory cytokine profiles (IL-6, tumour necrosis factor-α, interferon-γ, IL-2). Persistent antidrug antibodies (ADAs) were detected in 5/6 subjects administered a dose of 0.6 mg kg-1 (neutralizing in 2/6) and in 6/6 subjects administered 2.0 mg kg-1 (neutralizing in 5/6). CONCLUSION: GSK2618960 was well tolerated and blocked IL-7 receptor signalling upon full target engagement. Although there was no discernible impact on peripheral T cell subsets in healthy subjects, GSK2618960 may effectively modulate the autoinflammatory activity of pathogenic T cells in diseased tissue. A relatively short half-life is likely the result of target-mediated rather than ADA-mediated clearance.

Adjustment of the area under the concentration curve by terminal rate constant for bioequivalence assessment in a parallel-group study of lamotrigine.

AIM: A new strength of lamotrigine extended-release formulation unexpectedly failed to show bioequivalence with the existing strengths at the same dose in a parallel-group study. We report the post-hoc analyses conducted to identify the cause and propose an approach for future evaluations in similar situations. METHODS: A seemingly bimodal distribution of the half-life among the study participants prompted the use of terminal-phase-rate-constant-adjusted area under the concentration curve as the endpoint for bioequivalence assessment. Population pharmacokinetic modelling was also performed to assess the bimodal distribution of apparent clearance and the potential treatment effects on bioavailability. RESULTS: The cause for failing to achieve bioequivalence appeared to be a biased representation of a bimodal clearance distribution between the groups. The pharmacokinetic modelling with a mixture routine identified two subpopulations: 88% had a mean clearance of 1.99 l h-1 ; 12% had a mean clearance of 0.64 l h-1 . The low-clearance population was unequally represented by 13% and 4% of subjects in the reference and test groups, respectively, and treatment appeared to have no significant effect on oral bioavailability. The bioequivalence comparison using the adjusted area concluded with a 90% confidence interval of 0.91-1.06, suggesting that treatment had no significant effect on bioavailability and the formulations would meet regulatory criteria for bioequivalence. CONCLUSIONS: The adjustment of the area under the concentration curve adjusted by terminal-phase rate constant should be considered for situational application in bioequivalence assessment when there are multiple clearance subpopulations in a parallel-group study.

Model-based Evaluation of the Clinical and Microbiological Efficacy of Vancomycin: A Prospective Study of Chinese Adult In-house Patients.

Background: Our aims in this prospective study were to evaluate the correlations between pharmacokinetic/pharmacodynamic (PK/PD) indices and the clinical/microbiological efficacy of vancomycin and to identify an appropriate PK/PD target in the Chinese population to guide vancomycin treatment in the clinic. Methods: Adult patients from 11 hospitals in China with gram-positive infections who received vancomycin therapy for ≥5 days and who were under therapeutic drug monitoring (TDM) were enrolled in this study. A 1-compartment population PK model was established and validated. The correlations between PK/PD indices (Cmin, Cmax, 0-24 hour area under the curve (AUC0-24), and AUC0-24/minimum inhibitory concentration (MIC) and clinical outcomes (clinical efficacy and bacterial eradication) were evaluated. Results: In total, 402 adult Chinese patients were enrolled. Among them, 380 patients were evaluable for PK analysis, and 334 were evaluable for PK/PD analysis. In the final population PK model, creatinine clearance (CLCR) was the significant covariate on CL (typical value, 3.87 L/hour; between-subject variability (BSV), 12.5%), and age was the significant covariate on volume of distribution (V) (typical value, 45.1 L; BSV, 24.8%). The univariate analysis showed that Cmax, AUC0-24, and AUC0-24/MIC were significantly different or marginally significantly different (P values were 0.009, 0.0385, and 0.0509, respectively) between microbiological outcome groups with coagulase-negative Staphylococcus infections. However, there were no significant differences (P > .05) in the above PK parameters by multivariate logistic regression analysis, indicating there was no independently associated factor. Conclusions: No significant correlations were identified between PK/PD indices and the clinical or microbiological efficacy of vancomycin in Chinese patients. The necessity of vancomycin TDM based on trough concentration and the current treatment target of AUC0-24/MIC ≥400 need to be further evaluated and confirmed in additional prospective studies.

The Constraints, Construction, and Verification of a Strain-Specific Physiologically Based Pharmacokinetic Rat Model.

The use of in vitro-in vivo extrapolation (IVIVE) techniques, mechanistically incorporated within physiologically based pharmacokinetic (PBPK) models, can harness in vitro drug data and enhance understanding of in vivo pharmacokinetics. This study's objective was to develop a user-friendly rat (250 g, male Sprague-Dawley) IVIVE-linked PBPK model. A 13-compartment PBPK model including mechanistic absorption models was developed, with required system data (anatomical, physiological, and relevant IVIVE scaling factors) collated from literature and analyzed. Overall, 178 system parameter values for the model are provided. This study also highlights gaps in available system data required for strain-specific rat PBPK model development. The model's functionality and performance were assessed using previous literature-sourced in vitro properties for diazepam, metoprolol, and midazolam. The results of simulations were compared against observed pharmacokinetic rat data. Predicted and observed concentration profiles in 10 tissues for diazepam after a single intravenous (i.v.) dose making use of either observed i.v. clearance (CLiv) or in vitro hepatocyte intrinsic clearance (CLint) for simulations generally led to good predictions in various tissue compartments. Overall, all i.v. plasma concentration profiles were successfully predicted. However, there were challenges in predicting oral plasma concentration profiles for metoprolol and midazolam, and the potential reasons and according solutions are discussed.

Effect of tamsulosin on the pharmacokinetics of dutasteride in Chinese male healthy volunteers.

The purpose of this study was to evaluate the effect of tamsulosin (0.2 mg) on the pharmacokinetics of dutasteride (0.5 mg) in a group of healthy Chinese male volunteers. This was an open-label, single-sequence, 3-period, drug-drug interaction phase 1 study. Twenty-four healthy Chinese male volunteers were enrolled and administered a single dose of 0.5 mg dutasteride and, following a 28- to 30-day washout period, 0.2 mg tamsulosin once daily for 7 days. On day 5, subjects received 0.2 mg tamsulosin coadministered with 0.5 mg dutasteride. Serum dutasteride and tamsulosin concentrations were monitored. In the presence or absence of tamsulosin, there were no apparent changes in dutasteride AUC and Cmax . Adverse events reported were mild to moderate in intensity and resolved by the end of the study. In healthy Chinese male volunteers, tamsulosin 0.2 mg at steady state had no apparent effect on dutasteride pharmacokinetics. Dutasteride and tamsulosin when administered alone or in combination were well tolerated.

Safety, pharmacokinetics, pharmacodynamics, and bioavailability of GSK2018682, a sphingosine-1-phosphate receptor modulator, in healthy volunteers.

The tolerability, pharmacokinetics, and pharmacodynamics of single (SD) and repeat (RD) doses of GSK2018682, a selective S1P1 receptor modulator, were evaluated in healthy volunteers. The bioavailability (BA) of different formulations and effects of food were also evaluated. SD of up to 24 mg and RD of up to 6 mg/day for 28 days were reasonably tolerated, despite higher incidences of gastrointestinal and cardiovascular adverse events compared to placebo. There was a linear relationship between dose and systemic exposure with a dose-independent half-life (t1/2 ) between 44.9 and 63.3 hours. GSK2018682 induced acute, transient and non-symptomatic decreases in heart rate and blood pressure. Dose-dependent reduction in absolute lymphocyte count (ALC), and all tested subsets, was observed to various degrees, up to a nadir of over 70% reduction from baseline. There was no difference in major pharmacokinetic parameters among three formulations of GSK2018682 and between fasted and fed subjects. However, there was a reduction in the extent of bradycardia following dosing in the fed state. Additionally, exercise induced robust increase in heart rate in subjects who had bradycardia following RD of GSK2018682 up to 6 mg, suggesting possible physiological methods of reducing the extent of S1P mediated bradycardia and subsequent AV-block.

Discovery of novel 1,2,4-thiadiazole derivatives as potent, orally active agonists of sphingosine 1-phosphate receptor subtype 1 (S1P(1)).

A novel series of 1,2,4-thiadiazole compounds was discovered as selective S1P(1) agonists. The extensive structure-activity relationship studies for these analogues were reported. Among them, 17g was identified to show high in vitro potency with reasonable free unbound fraction in plasma (F(u) > 0.5%), good brain penetration (BBR > 0.5), and desirable pharmacokinetic properties in mouse and rat. Oral administration of 1 mg/kg 17g resulted in significant peripheral lymphocytes reduction at 4 h after dose and rapid lymphocytes recovery at 24 h. 17g showed a transient lymphopenia profile in the repeated dose study in mouse. In addition, 17g also demonstrated efficacy comparable to that of FTY720 (1) in the mouse EAE model of MS.

[Near infrared reflectance spectroscopy (NIRS) and its application in the determination for the quality of animal feed and products].

Near-infrared reflectance spectroscopy (NIRS) has been the most rapidly developing and noticeable spectrographic analytical technique in recent years. The determining principle and progresses of near-infrared reflectance spectroscopy are presented briefly. It mainly includes the progresses in pre-processing technique and analyzing model of near-infrared reflectance spectroscopy. Two pre-processing techniques, including differential coefficient-dealt with technique, the signal-smoothing technique, and four analyzing models of near-infrared spectroscopy, including the multiplied lined regression (MLR), principal component analysis (PCA), partial least squares (PLS), and artificial nerve network (ANN). The application of near-infrared reflectance spectroscopy to the first time. The investigation of reviewed papers shows that the near-infrared reflectance spectroscopy is widely applied in feed analysis and animal products analysis because of its rapidness, non-destruction and non-pollution. The near infrared reflectance spectroscopy has been used to determine the feed common ingredient, such as dry matter, crude protein, crude fiber, crude fat and so on, micro-components including amino acid, vitamin, and noxious components, and to determine the physical and chemical properties of animal products which including egg, mutton, beef and pork. Details of the analytical characteristics of feed and animal products described in the reviewed papers are given. New trends and limits to the application of near-infrared reflectance spectroscopy in these fields are also discussed.

[Rapid evaluation of beef quality by NIRS technology].

The aim of the present study was to develop a near-infrared reflectance (NIR) spectroscopy rapid method for evaluation of beef quality. Partial least squares (PLS) prediction model for the physic-chemical characteristics such as moisture, fat, protein, pH, color and WBSF in beef was established with good veracity. One hundred fourteen samples from five different parts of beef carcass (tenderloin, ribeye, topside, shin, striploin) were collected from meat packer after 48 h aging. Spectra were obtained by scanning sample from 950 to 1 650 nm and pretreated the model by MSC, SNV and first derivative. Predictive correlation coefficients of physic-chemical parameters in beef were 0.947 2 (moisture), 0.924 5 (fat), 0.934 6 (protein), 0.620 2 (pH), 0.820 3 (L), 0.864 6 (a*), 0.753 0 (b*) and 0.475 9 (WBSF) respectively. Root mean square errors of calibration (RMSEC) were 0.313 3 (moisture), 0.221 0 (fat), 1.243 2 (protein), 0.744 6 (pH), 1.778 3 (L*), 1.394 2 (a*), 1.763 9 (b*) and 1.0743 (WBSF). They were externally validated with additional 30 beef samples. Statistics showed that there was no significant difference between predicted value and those obtained with conventional laboratory methods. The results showed that NIRS is a rapid, effective technique for evaluating beef quality. The predictions for chemical characteristics gave higher accuracy than prediction for physical characteristics.

Berberine suppresses intestinal disaccharidases with beneficial metabolic effects in diabetic states, evidences from in vivo and in vitro study.

Clinical reports have demonstrated that berberine is a potential antidiabetic agent, but the underlying mechanism is unclear. The purpose of this study was to investigate if berberine exerts its hypoglycemic action via inhibiting intestinal disaccharidases using in vivo and in vitro experiments. Streptozotocin-induced diabetic rats received berberine (100 or 200 mg/kg) orally once daily or acarbose (40 mg/kg) orally twice daily for 5 weeks. Disaccharidase activities and sucrase-isomaltase (SI) complex messenger RNA (mRNA) expression in intestinal regions were assessed. The same treatment was operated in normal rats. Sucrose and maltose loading tests were also documented. In addition, Caco-2 cells were cultured in medium containing berberine or berberine plus chelerythrine. Compound C or H-89 for 5 days, disaccharidase activities, and SI complex mRNA levels were measured. The animal experiments showed that berberine significantly decreased the disaccharidase activities and SI complex mRNA expression both in diabetic rats and normal rats. Berberine can also significantly lower postprandial blood glucose levels induced by sucrose or maltose loading in normal rats. The cellular results showed that berberine may suppress disaccharidase activities and downregulate SI complex mRNA expression in a concentration-dependent manner. Only H-89, an inhibitor of protein kinase A (PKA), may reverse the decrease in disaccharidase activities and SI complex mRNA expression induced by berberine. In conclusion, berberine suppresses disaccharidase activities and SI complex mRNA expression with beneficial metabolic effects in diabetic states. The inhibitory effect, at least partly, involves the PKA-dependent pathway.

Physiologically based mechanistic modelling to predict complex drug-drug interactions involving simultaneous competitive and time-dependent enzyme inhibition by parent compound and its metabolite in both liver and gut - the effect of diltiazem on the time-course of exposure to triazolam.

AIM: To predict the magnitude of metabolic drug-drug interaction (mDDI) between triazolam and diltiazem and its primary metabolite N-desmethyldiltiazem (MA). METHODS: Relevant in vitro metabolic and inhibitory data were incorporated into a mechanistic physiologically based pharmacokinetic model within Simcyp (Version 9.1) to simulate the time-course of changes in active CYP3A4 content in gut and liver and plasma concentrations of diltiazem, MA and triazolam in a virtual population with characteristics related to in vivo studies. RESULTS: The predicted median increases in AUC(0,infinity) of triazolam, which ranged from 3.9 to 9.5 for 20 simulated trials (median 5.9), were within 1.5-fold of the observed median value (4.4) in 14 of the trials. Considering the effects of diltiazem only and not those of MA, and ignoring auto-inhibition of MA metabolism and inhibition of its metabolism by diltiazem, resulted in lower increases in triazolam exposure (AUC ratios of 1.5-2.0 (median 1.7) and 2.7-5.3 (median 3.4), respectively). CONCLUSION: Prediction of mDDIs involving diltiazem requires consideration of both competitive and time-dependent inhibition in gut and liver by both diltiazem and MA, as well as the complex interplay between the two moieties with respect to mutual inhibition of parent compound and its metabolite.

Increased oral AUC of baicalin in streptozotocin-induced diabetic rats due to the increased activity of intestinal beta-glucuronidase.

The purpose of the study was to investigate the pharmacokinetics of baicalin, a major bioactive component of Scutellariae radix, in diabetic conditions. The 4-week diabetic rats were induced by intraperitoneal administration of streptozotocin. Plasma concentrations of baicalin were measured following oral (200 mg/kg) or intravenous (12 mg/kg) administration. Everted intestinal transport, intestinal mucosal metabolism of baicalin and intestinal beta-glucuronidase activity were also investigated. It was found that the diabetic condition significantly increased the exposure of baicalin following oral doses (AUC 100.77 +/- 4.16 microg x h/mL in diabetic rats vs. 48.48 +/- 7.94 microg x h/mL in normal rats). In contrast, the diabetic condition significantly decreased the exposure of baicalin following intravenous doses (AUC 11.20 +/- 2.28 microg x h/mL in diabetic rats vs. 18.02 +/- 3.45 microg x h/mL in normal rats). We also found lower apparent permeability coefficients of baicalin in the ileum of diabetic rats (8.43 x 10 (-6) +/- 2.40 x 10 (-6) cm/s in diabetic rats vs. 5.21 x 10 (-5) +/- 1.55 x 10 (-5) cm/s in normal rats). Further studies showed that the diabetic condition enhanced the hydrolysis of baicalin to baicalein in intestinal mucosal, accompanied by an increase of beta-glucuronidase activity. All these results suggested that the higher oral exposure of baicalin in diabetic rats did not result from the decreased hepatic metabolism or increased intestinal absorption of baicalin. The enhancement of intestinal beta-glucuronidase activity may partly account for the higher exposure of baicalin in diabetic rats after oral administration.

[Pith decompression of the femoral head and allograft fibula grafting for treatment of avascular necrosis of femoral head].

OBJECTIVE: To investigate the clinical efficacy of the pith decompression of the femoral head and fibular allograft transplantation in the treatment of early stage avascular necrosis. METHODS: From January 2004 to November 2008, 32 hips of 25 patients with avascular necrosis of femoral head of Ia-IIIb period were treated by the pith decompression of the femoral head and fibular allograft transplantation, hollow lag screw fixation, included 17 males and 8 females, aged from 20 to 55 years old (39.1 years on average). Preoperative pain was from 2 to 14 months (means 5.5 months). All patients were applied on conventional X-ray films, MRI examination, Harris score. RESULTS: The patients were followed up for 12 to 48 months (means 36.4 months). X-ray film showed 21 hips of 18 cases improved,6 hips of 4 cases unchanged, no collapse of articular surface, 3 hips of 2 cases deterioration, 2 hips of 1 case failed. Preoperative Harris score was (77.0 +/- 8.0) and postoperative (90.6 +/- 2.5), there was a significant difference (t = 1.67, P < 0.05). CONCLUSION: Pith decompression of the femoral head and fibular allograft transplantation in the treatment of early stage avascular necrosis of femoral head has advantage of joint function in bed-ridden after a short time, quick recovery,clinical symptoms improved. Its short-term efficacy is certain but long-term efficacy is still need further observation.

Population-based mechanistic prediction of oral drug absorption.

The bioavailability of drugs from oral formulations is influenced by many physiological factors including gastrointestinal fluid composition, pH and dynamics, transit and motility, and metabolism and transport, each of which may vary with age, gender, race, food, and disease. Therefore, oral bioavailability, particularly of poorly soluble and/or poorly permeable compounds and those that are extensively metabolized, often exhibits a high degree of inter- and intra-individual variability. While several models and algorithms have been developed to predict bioavailability in an average person, efforts to accommodate intrinsic variability in the component processes are less common. An approach that incorporates such variability for human populations within a mechanistic framework is described together with examples of its application to drug and formulation development.

The consequences of 3,4-methylenedioxymethamphetamine induced CYP2D6 inhibition in humans.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused substituted amphetamine. MDMA is predominantly O-demethylenated in humans by cytochrome P450 isoforms 2D6 and 1A2 (CYP2D6 and CYP CYP1A2, respectively). MDMA is also a mechanism-based inhibitor of CYP2D6. A controlled clinical trial was conducted in 15 healthy male subjects whereby a probe drug, dextromethorphan (DEX), was administered after an oral dose of 1.5 mg/kg MDMA. The pharmacokinetics of DEX and its metabolites were used to evaluate changes in CYP2D6 activity. The urinary metabolic ratio of DEX and dextrorphan was used to calculate a recovery half-life of CYP2D6. After MDMA, DEX Cmax and area under the curve increased approximately 10-fold with corresponding decreases in dextrorphan pharmacokinetic parameters. The metabolic ratio increased almost 100-fold from 0.0061 +/- 0.0056 to 0.4322 +/- 0.2848 after MDMA administration, with 67% of the subjects having a value greater than the antimode of 0.3 for assigning the poor metabolizer phenotype. CYP2D6 activity recovered after 10 days with a recovery half-life of 46.6 hours. In addition to the possible long-term serotonergic effects of MDMA, users must be warned of the consequences of such an inhibition.

Cytochrome p450 turnover: regulation of synthesis and degradation, methods for determining rates, and implications for the prediction of drug interactions.

In vivo enzyme levels are governed by the rates of de novo enzyme synthesis and degradation. A current lack of consensus on values of the in vivo turnover half-lives of human cytochrome P450 (CYP) enzymes places a significant limitation on the accurate prediction of changes in drug concentration-time profiles associated with interactions involving enzyme induction and mechanism (time)-based inhibition (MBI). In the case of MBI, the full extent of inhibition is also sensitive to values of enzyme turnover half-life. We review current understanding of CYP regulation, discuss the pros and cons of various in vitro and in vivo approaches used to estimate the turnover of specific CYPs and, by simulation, consider the impact of variability in estimates of CYP turnover on the prediction of enzyme induction and MBI in vivo. In the absence of consensus on values for the in vivo turnover half-lives of key CYPs, a sensitivity analysis of predictions of the pharmacokinetic effects of enzyme induction and MBI to these values should be an integral part of the modelling exercise, and the selective use of values should be avoided.

Prediction of intestinal first-pass drug metabolism.

Despite a lower content of many drug metabolising enzymes in the intestinal epithelium compared to the liver (e.g. intestinal CYP3A abundance in the intestine is 1% that of the liver), intestinal metabolic extraction may be similar to or exceed hepatic extraction. Modelling of events on first-pass through the intestine requires attention to the complex interplay between passive permeability, active transport, binding, relevant blood flows and the intrinsic activity and capacity of enzyme systems. We have compared the predictive accuracy of the "well-stirred" gut model with that of the "Q(Gut)" model. The former overpredicts the fraction escaping first-pass gut metabolism; the latter improves the predictions by accounting for interplay between permeability and metabolism.

Increased P-glycoprotein expression and decreased phenobarbital distribution in the brain of pentylenetetrazole-kindled rats.

The purposes of this study were to investigate whether P-glycoprotein (P-GP) is overexpressed in the brain of pentylenetetrazole (PTZ)-kindled rats, and to investigate the effects of P-GP up-regulation on the distribution of phenobarbital (PB) in brain and its antiepileptic effects. Kindled rats were developed using a subconvulsive dose of PTZ (30 mg/kg, once every 2 days, i.p.) for 24 days. P-GP expression and function were measured by Western blot analysis and rhodamine 123 (Rho 123) distribution in brain. Kindled rats received 10 mg/kg of PB alone or co-administration of cyclosporine A (CsA, 5 mg/kg). At 60 min after administration of PB, concentrations of PB in brain and plasma were measured and the tissue-to-plasma concentration ratios of PB were calculated. Anticonvulsive effects of PB (13.2 mg/kg) alone or co-administration of CsA on the kindled rats were observed. The results showed that kindling resulted in 1.7-fold increase of P-GP level in brain, accompanied by significant decrease of tissue-to-plasma concentration ratios of Rho 123 and PB in hippocampus and cortex without affecting their concentrations in plasma. Co-administration of CsA reversed the decrease of PB concentration in brain without affecting PB level in plasma and significantly potentiated the anticonvulsive effects of PB. The present study demonstrated that chronic PTZ-kindling might increase P-GP expression and function in brain of rat, resulting in decrease of Rho 123 and PB levels in brain tissues. Co-administration of CsA increased PB levels in brain and enhanced anticonvulsive effects of PB by inhibiting P-GP function.

Theoretical assessment of a new experimental protocol for determining kinetic values describing mechanism (time)-based enzyme inhibition.

We have shown previously that the conventional experimental protocol (CEP) used to characterise mechanism-based enzyme inhibition (MBI) of drug metabolism in vitro may introduce substantial bias in estimates of the relevant kinetic parameters. The aim of this study was to develop and assess, by computer simulation, an alternative, mechanistically-based experimental protocol (MEP). This protocol comprises three parts viz. assessment of the metabolism of the mechanism-based enzyme inactivator (MBEI), of its ability to participate in competitive inhibition and its ability to cause time-dependent inhibition. Thus, values of the maximum inactivation rate constant (k(inact)), the inactivator concentration associated with half-maximal rate of inactivation (K(I)), the partition ration (r), and the reversible inhibition constant (K(i)) of the MBEI are determined by nonlinear optimization of the experimental data using a model that allows for metabolism of both probe substrate and MBEI, the time-course of inactivation of the enzyme, and reversible inhibition of the metabolism of both probe substrate and MBEI. Sensitivity analysis is used to estimate the degree of confidence in the final parameter values. Virtual experiments using the MEP and the CEP were simulated, applying starting kinetic parameters reported for 16 known MBEIs. In the presence of simulated experimental error (5% CV), the MEP recovered accurate estimates of the kinetic values for all compounds, while estimates using the CEP were less accurate and less precise. The MEP promises to improve consistency in the determination of in vitro measures of MBI and, thereby, the quantitative assessment of its in vivo consequences.