Passive drug permeation through membranes and cellular distribution.

Although often overlooked, passive mechanisms can lead to significant accumulation or restriction of drugs to intracellular sites of drug action. These mechanisms include lipoidal diffusion of ionized species and pH partitioning according to the electrochemical potential and to pH gradients that exist across subcellular compartments, respectively. These mechanisms are increasingly being exploited in the design of safe and effective drugs for the treatment of a wide variety of diseases. In this work, the authors review these efforts and the associated passive mechanisms of cellular drug permeation. A generic mathematical model of the cell is provided and used to illustrate concepts relevant to steady-state intracellular distribution. Finally, the authors review methods for estimating determinant parameters and measuring the net effect at the level of unbound intracellular drug concentrations.

Model-Based Assessment of Plasma Citrate Flux Into the Liver: Implications for NaCT as a Therapeutic Target.

Cytoplasmic citrate serves as an important regulator of gluconeogenesis and carbon source for de novo lipogenesis in the liver. For this reason, the sodium-coupled citrate transporter (NaCT), a plasma membrane transporter that governs hepatic influx of plasma citrate in human, is being explored as a potential therapeutic target for metabolic disorders. As cytoplasmic citrate also originates from intracellular mitochondria, the relative contribution of these two pathways represents critical information necessary to underwrite confidence in this target. In this work, hepatic influx of plasma citrate was quantified via pharmacokinetic modeling of published clinical data. The influx was then compared to independent literature estimates of intracellular citrate flux in human liver. The results indicate that, under normal conditions, <10% of hepatic citrate originates from plasma. Similar estimates were determined experimentally in mice and rats. This suggests that NaCT inhibition will have a limited impact on hepatic citrate concentrations across species.

A Mechanistic Pharmacokinetic Model for Liver Transporter Substrates Under Liver Cirrhosis Conditions.

Liver cirrhosis is a disease characterized by the loss of functional liver mass. Physiologically based pharmacokinetic (PBPK) modeling was applied to interpret and predict how the interplay among physiological changes in cirrhosis affects pharmacokinetics. However, previous PBPK models under cirrhotic conditions were developed for permeable cytochrome P450 substrates and do not directly apply to substrates of liver transporters. This study characterizes a PBPK model for liver transporter substrates in relation to the severity of liver cirrhosis. A published PBPK model structure for liver transporter substrates under healthy conditions and the physiological changes for cirrhosis are combined to simulate pharmacokinetics of liver transporter substrates in patients with mild and moderate cirrhosis. The simulated pharmacokinetics under liver cirrhosis reasonably approximate observations. This analysis includes meta-analysis to obtain system-dependent parameters in cirrhosis patients and a top-down approach to improve understanding of the effect of cirrhosis on transporter-mediated drug disposition under cirrhotic conditions.

Toward Prospective Prediction of Pharmacokinetics in OATP1B1 Genetic Variant Populations.

Physiologically based pharmacokinetic (PBPK) models are increasingly being used to provide human pharmacokinetic (PK) predictions for organic anion-transporting polypeptide (OATP) substrates based on in vitro assay data. As a natural extension in the application of these models, in this study, we incorporated in vitro information of three major OATP1B1 genetic variants into a previously reported PBPK model to predict the impact of OATP1B1 polymorphisms on human PK. Using pravastatin and rosuvastatin as examples, we showed that the predicted plasma concentration-time profiles in groups carrying different OATP1B1 genetic variants reasonably matched the clinical observations from multiple studies. This modeling and simulation approach may aid decision making in early pharmaceutical research and development as well as patient-specific dose adjustment in clinical practice.

Pharmacokinetics, disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid, a potent and subtype-selective peroxisome proliferator-activated receptor alpha agonist in preclinical species and human.

5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-alpha). In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague-Dawley rats (3.2 +/- 1.4 ml min(-1) kg(-1)) and cynomolgus monkeys (6.1 +/- 1.6 ml min(-1) kg(-1)) resulting in plasma half-lives of 7.1 +/- 0.7 h and 9.4 +/- 0.8 h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50 mg kg(-1)). In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50's > 30 microM) was discerned with 1. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

Influence of microsomal concentration on apparent intrinsic clearance: implications for scaling in vitro data.

The influence of microsomal concentration on unbound fraction (fu(mic)), half-life (t(1/2)), apparent intrinsic clearance (CL(int,app)) and apparent Michaelis-Menten constant (K(m,app)) was examined for two compounds, one representative of high nonspecific binding to microsomes (compound A) and one representative of low (compound B). Kinetic parameters were estimated for the two probe compounds at two human microsomal protein concentrations (0.46 and 2.3 mg/ml) and cytochrome P450 concentrations (0.20 and 1.0 microM), representing a 5-fold difference in microsomal concentration. For compound A, fu(mic) and CL(int,app) were inversely proportional to microsomal concentration. Conversely, the K(m,app) of compound A was proportional to microsomal concentration and the half-life was unchanged. For compound B, half-life was inversely proportional to microsomal concentration. In this case, fu(mic), CL(int,app), and K(m,app) were not proportionally influenced. The experimental observations were entirely consistent with that predicted by a mathematical relationship between microsomal concentration, fu(mic), t(1/2), CL(int,app), and K(m,app). These results demonstrate that when nonspecific binding is extensive, CL(int,app) is dependent on the arbitrary choice of microsomal concentration included in the incubation.

Impact of mechanism-based enzyme inactivation on inhibitor potency: implications for rational drug discovery.

Mechanism-based enzyme inactivators (MBEIs) have unique kinetic actions that make predictions of potency, selectivity, and potential for metabolic drug interactions more complex than for competitive antagonists. We have derived a mathematical relationship that links the influence of substrate concentration and binding constant ([S] and K(m), respectively), inhibitor concentration and binding constant ([I] and K(I), respectively), and inactivation rate constant (k(inact)) to enzyme activity (v) and maximal activity (V(max)) at any time (t). The kinetic behavior of this relationship was validated in murine-macrophage cell cultures using MBEIs of nitric oxide synthase (NOS). This initial equation was also used in the derivation of a new relationship that directly links the kinetic parameters of mechanism-based inactivation to inhibitory potency at a particular time (IC((t))(50)). Using this direct relationship, we observed that the predicted rank inhibitory potency of a series of MBEIs was improved over that predicted by the K(I) parameter alone. These relationships offer a fundamental understanding of the kinetics of MBEI action and may be useful in the evaluation of these compounds during the discovery process.

Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence.

The binding affinity (K(I)) and inactivation rate (k(inact)) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.

Examination of N-hydroxylation as a prerequisite mechanism of nitric oxide synthase inactivation.

L-N5-(1-Hydroxyiminoethyl)-ornithine (L-NHIO) and L-N6-(1-hydroxyiminoethyl)-lysine (L-NHIL) were synthesized and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by L-N5-iminoethylornithine (L-NIO) and L-N6-iminoethyllysine (L-NIL). Although these compounds were determined to be competitive inhibitors, mechanism-based inactivation was not observed.

In vivo disposition of 3-nitro-L-tyrosine in rats: implications on tracking systemic peroxynitrite exposure.

In many pathological conditions such as inflammatory and neurodegenerative diseases, the in vivo toxicity of nitric oxide has been attributed to the toxic oxidant peroxynitrite. Interaction of peroxynitrite with biological molecules can modify tyrosine residues on the proteins at the ortho position resulting in the formation of the stable end-product, 3-nitro-L-tyrosine (3-NT). Recent investigations indicate that changes in the circulating concentrations of 3-NT in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. In the present study, we examined the in vivo disposition characteristics of 3-NT in rats after either a single i.v. bolus dose (10 mg/kg) or a loading and maintenance infusion at 10 or 30 mg/kg. Plasma concentrations of 3-NT were analyzed by a reversed-phase HPLC method. After a single bolus dose of 3-NT at 10 mg/kg, the average half-life of the elimination phase for the drug was 68.5+/-18.4 min (n = 5). Infusions of 3-NT at two different doses (10 and 30 mg/kg) indicated that the pharmacokinetic properties of 3-NT below plasma concentrations of 100 microM were both linear and stationary. Urinary excretion of unchanged 3-NT was minimal, but two distinct metabolites of 3-NT were identified in the urine collected throughout the study. These findings may be useful in the interpretation of the plasma and urine 3-NT concentrations as possible indices of systemic peroxynitrite exposure.

Nonlinear pharmacokinetics of L-N(G)-methyl-arginine in rats: characterization by an improved HPLC assay.

L-N(G)-methyl-arginine (L-NMMA) is an inhibitor of nitric oxide synthase (NOS) enzymes. We have characterized the pharmacokinetics of L-NMMA in rats using HPLC. The HPLC assay requires pre-column derivatization, gradient elution and ultraviolet detection. The limit of sensitivity in plasma was 3.0 microM (0.75 microg mL(-1)). Using this assay, the pharmacokinetics of L-NMMA were characterized following iv bolus doses of 25, 50 and 100 mg kg(-1). Compartmental and noncompartmental data analysis suggest that L-NMMA pharmacokinetics are nonlinear at these doses. From the nonlinear compartmental analysis, we estimated the K(m) and V(max) parameters of L-NMMA elimination to be 70.2 microM and 4.59 microM min(-1), respectively. This estimated K(m) value of L-NMMA elimination is consistent with its nonlinear elimination characteristics in humans and its saturable metabolism by the N(G), N(G)-dimethylarginine dimethylamino-hydrolase enzyme in isolated rat tissue.