J. Cory Kalvass

Use of Plasma and Brain Unbound Fractions to Assess the Extent of Brain Distribution of 34 Drugs: Comparison of Unbound Concentration Ratios to in Vivo P-Glycoprotein Efflux Ratios

The P-glycoprotein (P-gp)-deficient mouse model is used to assess the influence of P-gp-mediated efflux on the central nervous system (CNS) distribution of drugs. The steady-state unbound plasma/unbound brain concentration ratio ([plasma],u/[brain],u) is an alternative method for assessing CNS distribution of drugs independent of the mechanism(s) involved. The objective of this study was to compare the degree of CNS distributional impairment determined from the in vivo P-gp efflux ratio with that determined from the [plasma],u/[brain],u ratio. CNS distribution of 34 drugs, including opioids, triptans, protease inhibitors, antihistamines, and other clinically relevant drugs with either poor CNS distribution or blood-brain barrier efflux, was studied. Plasma and brain unbound fractions were determined by equilibrium dialysis. Kp,brain and the P-gp efflux ratio were obtained from the literature or determined experimentally. The P-gp efflux ratio and the [plasma],u/[brain],u ratio were in concurrence (<3-fold difference) for 21 of the 34 drugs. However, the [plasma],u/[brain],u ratio exceeded the P-gp efflux ratio substantially (>4-fold) for 10 of the 34 drugs, suggesting that other, non-P-gp-mediated mechanism(s) may limit the CNS distribution of these drugs. The P-gp efflux ratio exceeded the [plasma],u/[brain],u ratio by more than 3-fold for three drugs, suggesting the presence of active uptake mechanism(s). These observations indicate that when mechanisms other than P-gp affect CNS distribution (non-P-gp-mediated efflux, poor passive permeability, cerebrospinal fluid bulk flow, metabolism, or active uptake), the P-gp efflux ratio may underestimate or overestimate CNS distributional impairment. The [plasma],u/[brain],u ratio provides a simple mechanism-independent alternative for assessing the CNS distribution of drugs.

In vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through P-glycoprotein up-regulation.

The ATP-driven drug export pump, P-glycoprotein, is a primary gatekeeper of the blood-brain barrier and a major impediment to central nervous system (CNS) pharmacotherapy. Reducing P-glycoprotein activity dramatically increases penetration of many therapeutic drugs into the CNS. Previous studies in rat showed that brain capillary P-glycoprotein was transcriptionally up-regulated by the pregnane X receptor (PXR), a xenobiotic-activated nuclear receptor. Here we used a transgenic mouse expressing human PXR (hPXR) to determine the consequences of increased blood-brain barrier P-glycoprotein activity. P-glycoprotein expression and transport activity in brain capillaries from transgenic mice was significantly increased when capillaries were exposed to the hPXR ligands, rifampin and hyperforin, in vitro and when the mice were dosed with rifampin in vivo. Plasma rifampin levels in induced mice were comparable with literature values for patients. We also administered methadone, a CNS-acting, P-glycoprotein substrate, to control and rifampin-induced transgenic mice and measured the drugs antinociceptive effect. In rifampin-induced mice, the methadone effect was reduced by approximately 70%, even though plasma methadone levels were similar to those found in transgenic controls not exposed to rifampin. Thus, hPXR activation in vivo increased P-glycoprotein activity and tightened the blood-brain barrier to methadone, reducing the drugs CNS efficacy. This is the first demonstration of the ability of blood-brain barrier PXR to alter the efficacy of a CNS-acting drug.

Kinetic Considerations for the Quantitative Assessment of Efflux Activity and Inhibition: Implications for Understanding and Predicting the Effects of Efflux Inhibition

PurposeUnexpected and complex experimental observations related to efflux transport have been reported in the literature. This work was conducted to develop relationships for efflux activity (PSefflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), efflux ratio (ER), degree of efflux inhibition (ϕi), 50% inhibitory concentration (IC50), and Michaelis–Menten constant (Km)].MethodsA three-compartment model (apical, cellular, and basolateral) was used to derive flux equations relating the initial rate of flux and steady-state mass transfer in the presence or absence of active efflux. Various definitions of efflux ratio (ER) were examined in terms of permeability-surface area products. The efflux activity (PSefflux) was expressed in terms of ER and PS. The relationships between PSefflux and PS, ER, ϕi, IC50, and Km were solved mathematically. Simulations and examples from the literature were used to illustrate the resulting mathematical relationships.ResultsThe relationships derived according to a three-compartment model differed fundamentally from commonly accepted approaches for determining PSefflux, ϕi, IC50 and Km. Based on the model assumptions and mathematical derivations, currently used mathematical relationships erroneously imply that efflux activity is proportional to change in PS (i.e., flux or Papp) and thus underestimate PSefflux and ϕi, and overestimate IC50 and Km.ConclusionsAn understanding of the relationship between efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of efflux transport, and predicting the clinical significance of efflux inhibition.

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice.

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also ∼35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (≥37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.

Relationship between Drug/Metabolite Exposure and Impairment of Excretory Transport Function

The quantitative impact of excretory transport modulation on the systemic exposure to xenobiotics and derived metabolites is poorly understood. This article presents fundamental relationships between exposure and loss of a specific excretory process that contributes to overall clearance. The mathematical relationships presented herein were explored on the basis of hepatic excretory data for polar metabolites formed in the livers of various transporter-deficient rodents. Experimental data and theoretical relationships indicated that the fold change in exposure is governed by the relationship, 1/(1 – fe), where fe is the fraction excreted by a particular transport protein. Loss of function of a transport pathway associated with fe < 0.5 will have minor consequences (<2-fold) on exposure, but exposure will increase exponentially in response to loss of function of transport pathways with fe > 0.5. These mathematical relationships may be extended to other organs, such as the intestine and kidney, as well as to systemic drug exposure. Finally, the relationship between exposure and fe is not only applicable to complete loss of function of a transport pathway but also can be extended to partial inhibition scenarios by modifying the equation with the ratio of the inhibitor concentration and inhibition constant.

Microdialysis Evaluation of Atomoxetine Brain Penetration and Central Nervous System Pharmacokinetics in Rats

A comprehensive in vivo evaluation of brain penetrability and central nervous system (CNS) pharmacokinetics of atomoxetine in rats was conducted using brain microdialysis. We sought to determine the nature and extent of transport at the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCB) and to characterize brain extracellular and cellular disposition. The steady-state extracellular fluid (ECF) to plasma unbound (uP) concentration ratio (CECF/CuP = 0.7) and the cerebrospinal fluid (CSF) to plasma unbound concentration ratio (CCSF/CuP = 1.7) were both near unity, indicating that atomoxetine transport across the BBB and BCB is primarily passive. On the basis of the ratios of whole brain concentration to CECF (CB/CECF = 170), brain cell (BC) concentration to CECF (CBC/CECF = 219), and unbound brain cell concentration to CECF (CuBC/CECF = 2.9), we conclude that whole brain concentration does not represent the concentration in the biophase and atomoxetine primarily partitions into brain cells. The distributional clearance at the BBB (QBBB = 0.00110 l/h) was estimated to be 12 times more rapid than that at the BCB (QBCB = 0.0000909 l/h) and similar to the clearances across brain parenchyma (CLECF-BC = 0.00216 l/h; CLBC-ECF = 0.000934 l/h). In summary, the first detailed examination using a quantitative microdialysis technique to understand the brain disposition of atomoxetine was conducted. We determined that atomoxetine brain penetration is high, movements across the BBB and BCB occur predominantly by a passive mechanism, and rapid equilibration of ECF and CSF with plasma occurs.

Fexofenadine Brain Exposure and the Influence of Blood-Brain Barrier P-Glycoprotein After Fexofenadine and Terfenadine Administration

P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administration demonstrated that fexofenadine brain penetration was 48-fold higher in mdr1a(–/–) mice than in mdr1a(+/+) mice. In contrast, the P-gp efflux ratio at the blood-brain barrier (BBB) for fexofenadine was only ∼4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp efflux ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetration after terfenadine administration was ∼25- to 27-fold higher than after fexofenadine administration in both mdr1a(+/+) and mdr1a(–/–) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitro incubation. The fexofenadine formation rate increased ∼5-fold during a 2-h brain tissue homogenate incubation with hydroxyl-terfenadine, suggesting that the hydroxylation of terfenadine is the rate-limiting step in fexofenadine formation. Moreover, regional brain metabolism seems to be an important factor in terfenadine brain disposition and, consequently, fexofenadine brain exposure. Taken together, these results indicate that the fexofenadine BBB P-gp efflux ratio has been underestimated previously due to the lack of complete equilibration of fexofenadine across the blood-brain interface under typical experimental paradigms.

Hepatobiliary Disposition of a Drug/Metabolite Pair: Comprehensive Pharmacokinetic Modeling in Sandwich-Cultured Rat Hepatocytes

The hepatobiliary disposition of xenobiotics may involve passive and/or active uptake, metabolism by cytochromes P450, and excretion of the parent compound and/or metabolite(s) into bile. Although in vitro systems have been used to evaluate these individual processes discretely, mechanistic in vitro studies of the sequential processes of uptake, metabolism, and biliary or basolateral excretion are limited. The current studies used sandwich-cultured (SC) rat hepatocytes combined with a comprehensive pharmacokinetic modeling approach to investigate the hepatobiliary disposition of terfenadine and fexofenadine, a model drug/metabolite pair. The metabolism of terfenadine and the biliary excretion of terfenadine and fexofenadine were determined in control and dexamethasone-treated SC rat hepatocytes. Dexamethasone (DEX) treatment increased the formation rates of the terfenadine metabolites azacyclonol and fexofenadine ∼20- and 2-fold, respectively. The biliary excretion index (BEI) of fexofenadine, when generated by terfenadine metabolism, was not significantly different from the BEI of preformed fexofenadine (15 ± 2% versus 19 ± 2%, respectively). Pharmacokinetic modeling revealed that the rate constant for hepatocyte uptake was faster for terfenadine compared with preformed fexofenadine (2.5 versus 0.08 h-1, respectively), whereas the biliary excretion rate constant for preformed fexofenadine exceeded that of terfenadine (0.44 versus 0.039 h-1, respectively). Interestingly, the rate constants for basolateral excretion of terfenadine and fexofenadine were comparable (3.2 versus 1.9 h-1, respectively) and increased only slightly with DEX treatment. These studies demonstrate the utility of the SC hepatocyte model, coupled with pharmacokinetic modeling, to evaluate the hepatobiliary disposition of generated metabolites.

Assessment of Blood–Brain Barrier Permeability Using the In Situ Mouse Brain Perfusion Technique

PurposeTo assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake.MethodsThe BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared.ResultsThere was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD7.4 in the range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain.ConclusionsIn situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.

Pharmacokinetics and Pharmacodynamics of Alfentanil in P-Glycoprotein-Competent and P-Glycoprotein-Deficient Mice: P-Glycoprotein Efflux Alters Alfentanil Brain Disposition and Antinociception

Previous studies have indicated that P-glycoprotein (P-gp) attenuates the central nervous system penetration and central activity of some opioids. The impact of P-gp-mediated efflux on the disposition and efficacy of the synthetic opioid alfentanil currently is unknown. In this study, P-gp-competent [mdr1a(+/+)] and P-gp-deficient [mdr1a(–/–)] mice were used to investigate the impact of P-gp-mediated efflux on the systemic pharmacokinetics, brain disposition, and central activity of alfentanil. Equipotent doses of alfentanil were administered to mdr1a(+/+) and mdr1a(–/–) mice (0.2 and 0.067 mg/kg, respectively), and the time course of brain and serum concentrations as well as antinociception were determined. A pharmacokinetic-pharmacodynamic (PK-PD) model was fit to the data and used to assess the impact of P-gp on parameters associated with alfentanil disposition and action. The mdr1a(+/+) mice were less sensitive to alfentanil than mdr1a(–/–) mice, requiring a 3-fold higher dose to produce similar antinociception. PK-PD modeling revealed no differences in alfentanil systemic pharmacokinetics between P-gp expressers and nonexpressers. However, the steady-state brain-to-serum concentration ratio (Kp,brain,ss) was ∼3-fold lower in mdr1a(+/+) mice compared with mdr1a(–/–) mice (0.19 ± 0.01 versus 0.54 ± 0.04, respectively). Consistent with the ∼3-fold lower Kp,brain,ss, the antinociception versus serum concentration relationship in mdr1a(+/+) mice was shifted ∼3-fold rightward compared with mdr1a(–/–) mice. However, there was no difference in the antinociception versus brain concentration relationship, or in the brain tissue EC50 (11 ± 1.8 versus 9.2 ± 1.7 ng/g), between mdr1a(+/+) and mdr1a(–/–) mice. These results indicate that alfentanil is an in vivo P-gp substrate and are consistent with the hypothesis that P-gp-mediated efflux attenuates antinociception by reducing alfentanil Kp,brain,ss.