Gary M. Pollack

Pharmacokinetic and Pharmacodynamic Implications of P-glycoprotein Modulation

P‐glycoprotein (P‐gp) is a cell membrane—associated protein that transports a variety of drug substrates. Although P‐gp has been studied extensively as a mediator of multidrug resistance in cancer, only recently has the role of P‐gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined. P‐glycoprotein is present in organ systems that influence drug absorption (intestine), distribution to site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues. Many marketed drugs inhibit P‐gp function, and several compounds are under development as P‐gp inhibitors. Similarly, numerous drugs can induce P‐gp expression. While P‐gp induction does not have a therapeutic role, P‐gp inhibition is an attractive therapeutic approach to reverse multidrug resistance. Clinicians should recognize that P‐gp induction or inhibition may have a substantial effect on the pharmacokinetics and pharmacodynamics of concomitantly administered drugs that are substrates for this transporter.

Development of an In Situ Mouse Brain Perfusion Model and its Application to mdr1a P-Glycoprotein-Deficient Mice:

An in situ mouse brain perfusion model predictive of passive and carrier-mediated transport across the blood-brain barrier (BBB) was developed and applied to mdr1a P-glycoprotein (Pgp)-deficient mice [mdr1a(−/−)]. Cerebral flow was estimated from diazepam uptake. Physical integrity of the BBB was assessed with sucrose/inulin spaces; functional integrity was assessed with glucose uptake, which was saturable with a Km of ∼17 mmol/L and Vmax of 310 mmol · 100 g−1 · min−1. Brain uptake of a Pgp substrate (colchicine) was significantly enhanced (two- to fourfold) in mdr1a(−/−) mice. These data suggest that the model is applicable to elucidating the effects of efflux transporters, including Pgp, on brain uptake.

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.

(Section B: Integrated Function of Drug Transporters In Vivo) Drug Transport at the Blood-Brain Barrier and the Choroid Plexus

The blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) represent the main interfaces between the central nervous system (CNS) and the peripheral circulation. Drug exposure to the CNS is dependent on a variety of factors, including the physical barrier presented by the BBB and the BCSFB and the affinity of the substrate for specific transport systems located at both of these interfaces. It is the aggregate effect of these factors that ultimately determines the total CNS exposure, and thus pharmacological efficacy, of a drug or drug candidate. This review discusses the anatomical and biochemical barriers presented to solute access to the CNS. In particular, the important role played by various efflux transporters in the overall barrier function is considered in detail, as current literature suggests that efflux transport likely represents a key determinant of overall CNS exposure for many substrates. Finally, it is important to consider not only the net delivery of the agent to the CNS, but also the ability of the agent to access the relevant target site within the CNS. Potential approaches to increasing both net CNS and target-site exposure, when such exposure is dictated by efflux transport, are considered.

Coordinated Nuclear Receptor Regulation of the Efflux Transporter, Mrp2, and the Phase-Ii Metabolizing Enzyme, GSTπ, at the Blood—Brain Barrier

Xenobiotic efflux pumps at the blood—brain barrier are critical modulators of central nervous system pharmacotherapy. We previously found expression of the ligand-activated nuclear receptor, pregnane X receptor (PXR), in rat brain capillaries, and showed increased expression and transport activity of the drug efflux transporter, P-glycoprotein, in capillaries exposed to PXR ligands (pregnenolone-16α-carbonitrile (PCN) and dexamethasone) in vitro and in vivo. Here, we show increased protein expression and transport activity of another efflux pump, multidrug resistance-associated protein isoform 2 (Mrp2), in rat brain capillaries after in vitro and in vivo exposure to PCN and dexamethasone. The phase-II drug-metabolizing enzyme, glutathione S-transferase-π (GSTπ), was found to be expressed in brain capillaries, where it colocalized to a large extent with Mrp2 at the endothelial cell luminal plasma membrane. Like Mrp2, GSTπ protein expression increased with PXR activation. Colocalization and coordinated upregulation suggest functional coupling of the metabolizing enzyme and efflux transporter. These findings indicate that, as in hepatocytes, brain capillaries possess a regulatory network consisting of nuclear receptors, metabolizing enzymes, and efflux transporters, which modulate blood—brain barrier function.

Increased brain P-glycoprotein in morphine tolerant rats.

The objective of this study was to determine whether chronic morphine exposure increased P-glycoprotein in rat brain. Male Sprague-Dawley rats were treated with morphine, saline, or dexamethasone for 5 days. On day 6, antinociceptive effect was measured to evaluate the extent of functional tolerance to morphine. Brain P-glycoprotein was detected by Western blot analysis of whole brain homogenate. Morphine- and dexamethasone-treated rats exhibited decreased antinociceptive response when compared to saline-treated controls. Brain P-glycoprotein was approximately 2-fold higher in morphine-treated rats compared to saline controls based on Western blot analysis. Chronic morphine exposure appears to increase P-glycoprotein in rat brain. P-glycoprotein induction may enhance morphine efflux from the brain, thus reducing morphines pharmacologic activity. Induction of P-glycoprotein may be one mechanism involved in the development of morphine tolerance.

Circulating concentrations of di(2-ethylhexyl) phthalate and its de-esterified phthalic acid products following plasticizer exposure in patients receiving hemodialysis

The degree of exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) was assessed in 11 patients undergoing maintenance hemodialysis for the treatment of renal failure. The amount of DEHP leached from the dialyzer during a 4-hr dialysis session was estimated by monitoring the DEHP blood concentration gradient across the dialyzer. Circulating concentrations of the biologically active products of DEHP de-esterification, viz., mono(2-ethylhexyl) phthalate (MEHP) and phthalic acid, were also determined during the dialysis session. On the average, an estimated 105 mg of DEHP was extracted from the dialyzer during a single dialysis session, with a range of 23.8 to 360 mg. The rate of extraction of DEHP from the dialyzer was correlated with serum lipid content as expressed by the sum of serum cholesterol and triglyceride concentrations (r = +0.65, p less than 0.05). Time-averaged circulating concentrations of MEHP during dialysis (1.33 +/- 0.58 micrograms/ml) were similar to those of DEHP (1.91 +/- 2.11 micrograms/ml). Blood concentrations of phthalic acid (5.22 +/- 3.94 micrograms/ml) were higher than those of the esters. The length of time patients had been receiving regular dialysis treatment was not a determinant of circulating concentrations of DEHP or MEHP. In contrast, time-averaged circulating concentrations of phthalic acid correlated strongly with the duration (in years) of dialysis treatment (r = +0.92, p less than 0.001). The results indicated substantial exposure to DEHP during hemodialysis and that the de-esterified products of DEHP are present in significant concentrations in the systemic circulation. Further study is needed to assess the contribution of these metabolites to the biological actions of DEHP in man.

P-glycoprotein-mediated transport of morphine in brain capillary endothelial cells

Cell accumulation, transendothelial permeability, and efflux studies were conducted in bovine brain capillary endothelial cells (BBCECs) to assess the role of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) transport of morphine in the presence and absence of P-gp inhibitors. Cellular accumulation of morphine and rhodamine 123 was enhanced by the addition of the P-gp inhibitors N-{4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9- carboxamide (GF120918), verapamil, and cyclosporin A. Positive (rhodamine 123) and negative (sucrose and propranolol) controls for P-gp transport also were assessed. Morphine glucuronidation was not detected, and no alterations in the accumulation of propranolol or sucrose were observed. Transendothelial permeability studies of morphine and rhodamine 123 demonstrated vectorial transport. The basolateral to apical (B:A) fluxes of morphine (50 microM) and rhodamine (1 microM) were approximately 50 and 100% higher than the fluxes from the apical to the basolateral direction (A:B), respectively. Decreasing the extracellular concentration of morphine to 0.1 microM resulted in a 120% difference between the B:A and A:B permeabilities. The addition of GF120918 abolished any significant directionality in transport rates across the endothelial cells. Efflux studies showed that the loss of morphine from BBCECs was temperature- and energy-dependent and was reduced in the presence of P-gp inhibitors. These observations indicate that morphine is transported by P-gp out of the brain capillary endothelium and that the BBB permeability of morphine may be altered in the presence of P-gp inhibitors.

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.