CSF as a surrogate for assessing CNS exposure: an industrial perspective

For drugs that directly act on targets in the central nervous system (CNS), sufficient drug delivery into the brain is a prerequisite for drug action. Systemically administered drugs can reach CNS by passage across the endothelium of capillary vasculatures, the so-called blood-brain barrier (BBB). Literature data suggest that most marketed CNS drugs have good membrane permeability and relatively high plasma unbound fraction, but are not good P-glycoprotein (P-gp) substrates. Therefore, it is important to use the in vitro parameters of P-gp function activity, membrane permeability and plasma unbound fraction as key criteria for lead optimization during the early stage of drug discovery. Evidence from preclinical and clinical studies suggests that drug concentration in cerebrospinal fluid (CSF) appears to be reasonably accurate in predicting unbound drug concentration in the brain. Therefore, CSF can be used as a useful surrogate for in vivo assessment of CNS exposure and provides an important basis for the selection of drug candidates for entry into development. However, it is important to point out that CSF drug concentration is not always an accurate surrogate for predicting unbound drug concentration in the brain. Depending on the physicochemical properties of drugs and the site/timing of CSF sampling, the unbound drug concentration at the biophase within the brain could differ significantly from the corresponding CSF drug concentration.     

Microdialysis Studies of the Distribution of Stavudine into the Central Nervous System in the Freely-Moving Rat

Purpose. To study the extent and time course of distribution of stavudine (d4T) into the central nervous system (CNS) and to investigate the transport mechanisms of antiviral nucleosides in the CNS. Methods. Microdialysis with on-line HPLC analysis was used to measure drug concentrations in the brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) in the freely-moving rat. The in vivo recovery of d4T and zidovudine (AZT) was estimated by retrodialysis, which was validated by the zero-net flux method. The CNS distribution of d4T was investigated during iv and intracerebroventricular (icv) infusion. In the subsequent studies, the effect of AZT on CNS distribution of d4T was examined. Results. During iv infusion, d4T distributed rapidly into the CNS. Its brain ECF/plasma and CSF/plasma steady-state concentration ratios were 0.33 ± 0.06 and 0.49 ± 0.12, respectively (n = 15). During icv infusion, the steady-state d4T concentrations in the brain ECF were 23-fold higher than those during iv infusion, whereas its steady-state plasma levels were about the same for these two routes. Coadministration of AZT with d4T did not alter their respective brain distribution and systemic clearance at the concentrations examined. More importantly, the steady-state brain ECF/plasma and CSF/plasma concentration ratios of d4T were about 2-fold higher than those of AZT (0.15 ± 0.04 and 0.25 ± 0.08) determined in the same animals. Conclusions. d4T readily crosses the blood-brain barrier (BBB) and blood-CSF barrier. An active efflux transport system in the BBB and blood-CSF barrier may be involved in transporting d4T out of the CNS. Direct icv administration of d4T can be used to enhance its brain delivery. Moreover, d4T exhibits a more favorable penetration into the CNS than AZT and therefore may be useful in the treatment of AIDS dementia complex.     

P-glycoprotein-based loperamide-cyclosporine drug interaction at the rat blood-brain barrier: prediction from in vitro studies and extrapolation to humans

We have shown that the rat can quantitatively predict the verapamil-cyclopsorine A (CsA) drug-drug interaction (DDI) at the human blood-brain barrier (BBB). In addition, the potency (EC(50)) of CsA to inhibit rat BBB P-gp can be predicted from in vitro studies in MDRI-transfected cells. To assess if these excellent agreements extend to other substrates, we determined the magnitude of P-gp-based DDI at the rat BBB between loperamide (Lop) or its metabolite, N-desmethyl Lop (dLop), and escalating CsA blood concentrations. The percent increase in the brain:blood Lop concentration ratio was described by the Hill equation, E(max) = 2000%, EC(50) = 7.1 μM and γ = 3.7. The potency (EC(50)) of CsA to inhibit P-gp at the rat BBB was independent of the substrate used (verapamil, Lop, or dLop). Like the verapamil-CsA DDI, the potency (EC(50)) of CsA to inhibit rat BBB P-gp could be predicted from studies in MDRI-transfected cells. When (11)C-Lop was coadministered with a 10 mg/kg iv infusion of CsA (1) yielding ~5.6 uM CsA blood concentration to healthy volunteers, the brain distribution of (11)C-radioactivity was increased by 110%. (1) When corrected for diffusible Lop metabolite(s), this translates into an increase in (11)C-Lop brain distribution of 457%. Based on our rat data, we estimated a similar value at 5.6 μM blood CsA concentration, 588% increase in Lop brain distribution. These data support our conclusion that the rat is a promising model to predict P-gp based DDI at the human BBB.     

Effect of indinavir on the intestinal exsorption of amprenavir,saquinavir and nelfinavir after intravenous administration in rats

To elucidate drug interaction between human immunodeficiency virus (HIV) protease inhibitors (PIs), the effect of indinavir (IDV) on the intestinal exsorption of other HIV PIs, amprenavir (APV), saquinavir (SQV) and nelfinavir (NFV) was investigated in rats using an in situ single perfusion method. IDV in the intestinal perfusate inhibited the exsorption of rhodamine 123 (Rho123), a known P-glycoprotein (P-gp) substrate, from blood into intestinal lumen in a concentration-dependent manner, and the inhibitory potency of 10 micro M IDV in the perfusate was close to that of 10 micro M cyclosporin A (CsA) in the perfusate. Ten micro M of IDV in the intestinal perfusate also decreased significantly the exsorption clearance of Rho123 after intravenous administration. The IDV concentration in this system was not likely to cause hepatic interaction between HIV PIs, because the plasma IDV concentration was far below its inhibition constants for other HIV PIs in the liver microsomes. Thus, 10 micro M of IDV was chosen to investigate the effect of this inhibition on the exsorption of APV, SQV and NFV. IDV in the intestinal perfusate markedly increased the exsorbed amounts of SQV and NFV but not APV after intravenous administrations. Their exsorption clearances, however, showed only a slight increasing tendency or remained unchanged. These findings suggest that in addition to P-gp inhibition, other factors such as CYP3A inhibition might be important in the drug interaction of IDV with APV, SQV and NFV after intravenous administration in rat small intestine. The results obtained in this study will provide useful information to discuss the interactions among PIs when a double protease therapy is used for in HIV-infected patients.     

The impact of P-gp functionality on non-steady state relationships between CSF and brain extracellular fluid

In the development of central nervous system (CNS)-targeted drugs, the prediction of human CNS target exposure is a big challenge. Cerebrospinal fluid (CSF) concentrations have often been suggested as a ‘good enough’ surrogate for brain extracellular fluid (brain_ECF, brain target site) concentrations in humans. However, brain anatomy and physiology indicates prudence. We have applied a multiple microdialysis probe approach in rats, for continuous measurement and direct comparison of quinidine kinetics in brain_ECF, CSF, and plasma. The data obtained indicated important differences between brain_ECF and CSF kinetics, with brain_ECF kinetics being most sensitive to P-gp inhibition. To describe the data we developed a systems-based pharmacokinetic model. Our findings indicated that: (1) brain_ECF- and CSF-to-unbound plasma AUC_0–360 ratios were all over 100 %; (2) P-gp also restricts brain intracellular exposure; (3) a direct transport route of quinidine from plasma to brain cells exists; (4) P-gp-mediated efflux of quinidine at the blood–brain barrier seems to result of combined efflux enhancement and influx hindrance; (5) P-gp at the blood–CSF barrier either functions as an efflux transporter or is not functioning at all. It is concluded that in parallel obtained data on unbound brain_ECF, CSF and plasma concentrations, under dynamic conditions, is a complex but most valid approach to reveal the mechanisms underlying the relationship between brain_ECF and CSF concentrations. This relationship is significantly influenced by activity of P-gp. Therefore, information on functionality of P-gp is required for the prediction of human brain target site concentrations of P-gp substrates on the basis of human CSF concentrations.     

Blood-brain barrier permeation and efflux exclusion of anticholinergics used in the treatment of overactive bladder

Overactive bladder (OAB) is a common condition, particularly in the elderly. Anticholinergic agents are the mainstay of pharmacological treatment of OAB; however, many anticholinergics can cross the blood-brain barrier (BBB) and may cause central nervous system (CNS) effects, including cognitive deficits, which can be especially detrimental in older patients. Many anticholinergics have the potential to cause adverse CNS effects due to muscarinic (M(1)) receptor binding in the brain. Of note, permeability of the BBB increases with age and can also be affected by trauma, stress, and some diseases and medications. Passive crossing of a molecule across the BBB into the brain is dependent upon its physicochemical properties. Molecular characteristics that hinder passive BBB penetration include a large molecular size, positive or negative ionic charge at physiological pH, and a hydrophilic structure. Active transport across the BBB is dependent upon protein-mediated transporter systems, such as that of permeability-glycoprotein (P-gp), which occurs only for P-gp substrates, such as trospium chloride, darifenacin and fesoterodine. Reliance on active transport can be problematic since genetic polymorphisms of P-gp exist, and many commonly used drugs and even some foods are P-gp inhibitors or are substrates themselves and, due to competition, can reduce the amount of the drug that is actively transported out of the CNS. Therefore, for drugs that are preferred not to cross into the CNS, such as potent anticholinergics intended for the bladder, it is optimal to have minimal passive crossing of the BBB, although it may also be beneficial for the drug to be a substrate for an active efflux transport system. Anticholinergics demonstrate different propensities to cross the BBB. Darifenacin, fesoterodine and trospium chloride are substrates for P-gp and, therefore, are actively transported away from the brain. In addition, trospium chloride has not been detected in cerebrospinal fluid assays and does not appear to have significant CNS penetration. This article reviews the properties of anticholinergics that affect BBB penetration and active transport out of the CNS, discusses issues of increased BBB permeability in patients with OAB, and examines the clinical implications of BBB penetration on adverse events associated with anticholinergics.     

Regulation of P-glycoprotein by miR-27a-3p at the Brain Endothelial Barrier

Multi-drug resistance P-glycoprotein (P-gp/MDR1) is one of the most clinically relevant ABC transporters, highly enriched at the blood-brain barrier (BBB) with a broad substrate spectrum including therapeutic drugs and metabolic waste products. Altered P-gp transport function has been implicated in multi-drug resistance and in the pathogenesis and progression of neurological diseases. Recent studies have shown that P-gp expression is modulated by micro-RNAs in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multi-drug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in-vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a-3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms explored. Our results showed that transfection of hCMEC/D3 cells with miR-27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3ß) inhibition and activating Wnt/ß-catenin signaling. These findings shed light on miR-27a-3p/GSK3ß/ß-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection.     

Potential role of ABC transporters as a detoxification system at the blood-CSF barrier

Exchange of compounds between blood and brain occurs at two barriers, the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). The barrier function is mainly a result of the functionality of the cerebral endothelial cells and choroidal epithelial cells, respectively. These cell types have restricted permeability due to the presence of tight junctions between the cells. Furthermore, these cells express a broad range of transporters. So far, the BBB has been viewed as the most important barrier, especially as its surface is about 3 orders of magnitude larger than that of the BCSFB. Today, there is a shift in the appreciation of the contribution of the BCSFB. In a few recent studies, it has been shown that the BCSFB expresses two types of ATP-binding cassette (ABC) transporters, being the multidrug transporters P-glycoprotein (P-gp) and the multidrug resistance-related protein 1 (MRP1). The knowledge on the function of these transporters in the BCSFB is relatively scarce, but in general, it seems that MRP1 transport is directed towards the blood side, which makes this transporter helpful in elimination of harmful compounds from the CSF. Thereby MRP1 potentially contributes to detoxification of the brain, as a whole, as it is also expressed at the level of the BBB. P-gp, however, while also functional as an efflux pump at the BBB, has an opposite transport direction at the level of the BCSFB, towards the CSF. P-gp may therefore raise the concentration of neurotoxic P-gp substrates in the CSF. Whether this will have a significant contribution to the toxicity in the regions directly exposed to the CSF (periventricular organs) remains to be determined. Specifically, in the epithelial cells of the choroid plexus of the BCSFB, P-gp and MRP1 together serve a protective role by preventing the accumulation of their overlapping and often toxic substrates. A concerted action of P-gp and MRP1 at the choroid plexus might contribute to the maintenance of the role of the BCSFB in brain homeostasis.     

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs.

In recent years there has been increasing focus on the role of the drug transporter P-glycoprotein (P-gp) with regard to drug penetration into the brain. Studies using mice devoid of functional P-gp have revealed that P-gp at the blood-brain barrier (BBB) can exert a profound effect on the ability of some drugs to enter the brain, e.g. cardiovascular drugs (digoxin, quinidine), opioids (morphine, loperamide, methadone), HIV protease inhibitors, the new generation of antihistamines, and some antidepressants and antipsychotics. Among the latter group, risperidone is strongly influenced having about 10 times higher cerebral concentration in P-gp knock-out mice than in control mice. Taking into account that polytherapy is commonplace in psychiatry, theoretically there is a risk of drug-drug interactions with regard to P-gp at the BBB. Here we review the evidence for a role of P-gp with regard to psychoactive drugs from in vitro studies and experiments in knock-out mice devoid of functional P-gp. Moreover, the evidence for significant drug-drug interactions involving psychotropic drugs in rodents is considered. Clinical observations suggesting a role for P-gp in relation to drug-drug interactions at the BBB are sparse, and a definite conclusion awaits further studies. Also, the possible clinical relevance of P-gp genetic polymorphisms is questionable, and more investigations are needed on this subject.     

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid/plasma concentration ratio.

The ratio of drug levels in cerebrospinal fluid (CSF) to plasma (CSF/plasma) at equilibrium has been viewed as in vivo free fraction (fp) in plasma [CSF/plasma = fp], if no active transport is involved in brain penetration. We determined the CSF/plasma level following oral administration in rats and in vitro rat plasma protein binding for 20 compounds that were synthesized in our institute and have similar physicochemical properties. However, results indicated that the CSF/plasma was not only poorly correlated with fp but remarkably lower than fp in most of the compounds tested, suggesting that certain transporters such as P-glycoprotein (P-gp) located in blood-brain barrier (BBB) may decrease the unbound drug concentration in the brain. We evaluated P-gp-mediated transport activity of the 20 compounds with P-gp (mdr1a)-transfected LLC-PK1 cells and calculated P-gp efflux index (PEI), indicating the extent of P-gp-mediated transport. A plot of the CSF/plasma versus fp/PEI showed a strong correlation (r = 0.93), and the absolute values were almost identical [CSF/plasma = fp/PEI]. These results suggest that P-gp quantitatively shifts the equilibrium of unbound drugs across the BBB. Although we cannot rule out the possibility that endogenous transporters other than P-gp on BBB and/or blood-CSF barrier may affect CSF levels of compounds, the present study indicated that fp and PEI measurements may be useful in predicting in vivo CSF/plasma fractions for central nervous system-targeting drugs.     

Central nervous system pharmacokinetics of the Mdr1 P-glycoprotein substrate CP-615,003: intersite differences and implications for human receptor occupancy projections from cerebrospinal fluid exposures.

The central nervous system (CNS) distribution and transport mechanisms of the investigational drug candidate CP-615,003 (N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide) and its active metabolite CP-900,725 have been characterized. Brain distribution of CP-615,003 and CP-900,725 was low in rats and mice (brain-to-serum ratio < 0.2). Cerebrospinal fluid (CSF)-to-serum ratios of CP-615,003 were 6- to 8-fold lower than the plasma unbound fraction in rats and dogs. In vitro, CP-615,003 displayed quinidine-like efflux in MDR1-expressing Madin-Darby canine kidney II cells. The brain-to-serum ratio of CP-615,003 in mdr1a/1b (-/-) mice was approximately 7 times that in their wild-type counterparts, confirming that impaired CNS distribution was explained by P-gp efflux transport. In contrast, P-gp efflux did not explain the impaired CNS penetration of CP-900,725. Intracerebral microdialysis was used to characterize rat brain extracellular fluid (ECF) distribution. Interestingly, the ECF-to-serum ratio of the P-gp substrate CP-615,003 was 7-fold below the CSF-to-serum ratio, whereas this disequilibrium was not observed for CP-900,725. In a clinical study, steady-state CSF exposures were measured after administration of 100 mg of CP-615,003 b.i.d. The human CSF-to-plasma ratios of CP-615,003 and CP-900,725 were both approximately 10-fold below their ex vivo plasma unbound fractions, confirming impaired human CNS penetration. Preliminary estimates of CNS receptor occupancy from human CSF concentrations were sensitive to assumptions regarding the magnitude of the CSF-ECF gradient for CP-615,003 in humans. In summary, this case provides an example of intersite differences in CNS pharmacokinetics of a P-gp substrate and potential implications for projection of human CNS receptor occupancy of transporter substrates from CSF pharmacokinetic data when direct imaging-based approaches are not feasible.     

The impact of pharmacologic and genetic knockout of P-glycoprotein on nelfinavir levels in the brain and other tissues in mice

Insufficient concentrations of protease inhibitors such as nelfinavir may reduce the effectiveness of HIV dementia treatment. The efflux transporter mdr1 product P-glycoprotein (P-gp) has been demonstrated to play a role in limiting nelfinavir brain levels. The goal of this study was to compare the effect of GF120918 (10 mg/kg, IV), a P-gp inhibitor, on intravenous nelfinavir (10 mg/kg) in vivo disposition and tissue penetration in P-gp-competent mdr1a/1b (+/+) mice versus P-gp double knockout mdr1a/1b (-/-) mice. Intravenous administration with the P-gp inhibitor GF120918 to mdr1a/1b (+/+) mice increased nelfinavir concentrations over a range of 2.3- to 27-fold, whereas nelfinavir distribution in mdr1a/1b (-/-) mice was 2- to 16-fold higher than that in their wild counterparts. Nelfinavir levels after GF120918 coadministration were higher in the heart, liver, and kidneys than those detected with mdr1a/1b knockout mice. In contrast, mdr1a/1b knockout mice exhibited higher nelfinavir levels in the brain (16.1-fold vs. 8.9-fold increase) and spleen (4.1-fold vs. 2.3-fold increase) compared to pharmacological inhibition with GF120918 in wild mice. Most notably, GF120918 provided tissue-specific effects in mdr1a/1b knockout mice with enhanced (p < 0.05) drug accumulation in the brain ( approximately 21-fold) and heart (3.3-fold). Our results suggest mdr1a/1b-independant mechanisms may also contribute to nelfinavir tissue distribution in mice.     

Tools for eradicating HIV in the brain: prodrug dimeric inhibitors of P-gp

Despite positive developments with the use of combination antiretroviral therapy, a major impediment to limiting the neurocognitive effects of HIV and eradicating HIV brain reservoirs is the penetration of these therapies across the blood-brain barrier (BBB). The focus of our work, therefore, has been to develop tools to significantly improve the penetration of antiretroviral agents to sites of HIV reservoirs, with an emphasis on the CNS. To this end, we have developed an innovative chemical approach--dimeric prodrugs of the antiretroviral agents themselves with a traceless tether. These dimeric prodrugs were designed to serve two purposes: inhibition of P-gp, the major drug efflux protein at the BBB, by occupying two substrate binding sites in the transporter; and prodrug dimers that gain entry into the endothelial cells at the BBB would revert to their monomeric forms in the reducing environment of the cytosol due to breakdown of the traceless tether, thus delivering the therapy. We have demonstrated the feasibility of this design by dimerizing the P-gp substrate and antiviral agent abacavir with a traceless tether. Abacavir dimers displayed potent inhibition of P-gp in two different cellular settings and reverted to active abacavir in the reducing environment of HIV-infected T cells, also leading to antiviral activity. Overall, these experiments point to the excellent promise for future use of dimeric prodrug inhibitors of P-gp for brain penetration of a wide range of CNS-active agents that are substrates of P-gp.     

Enhanced brain accumulation of pazopanib by modulating P-gp and Bcrp1 mediated efflux with canertinib or erlotinib

Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ～5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2-2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib efflux modulation as a combination regimen for the treatment of recurrent brain tumors.     

Influence of P-Glycoprotein Inhibition or Deficiency at the Blood–Brain Barrier on 18F-2-Fluoro-2-Deoxy-d-glucose (18F-FDG) Brain Kinetics

The fluorinated d-glucose analog ¹⁸F-2-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein’s (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact ¹⁸F-FDG incorporation, suggesting that P-gp function at the blood–brain barrier may also modulate ¹⁸F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5 μM) on the uptake of ¹⁸F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for ¹⁸F-FDG brain kinetics were then assessed using (i) ¹⁸F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15 mg kg⁻¹ h⁻¹) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled d-glucose exposure to the brain. In vitro, the time course of ¹⁸F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of d-glucose but not in the presence of high d-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K₁, k₂, k₃, V_T, and CMR_Glc. The lack of P-gp effect on in vivo¹⁸F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates ¹⁸F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. ¹⁸F-FDG is not a P-gp substrate at the BBB and ¹⁸F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.KEY WORDS: ABC transporters, blood–brain barrier, cyclosporine, glucose, multidrug resistance, nonhuman primate, positron emission tomography     

Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier

1. The blood-brain barrier (BBB) contributes to brain homeostastis and fulfills a protective function by controlling the access of solutes and toxic substances to the central nervous system (CNS). The efflux transporter P-glycoprotein (P-gp) is a key element of the molecular machinery that confers special permeability properties to the BBB. 2. P-gp, which was initially recognized for its ability to expel anticancer drugs from multidrug-resistant cancer cells, is strongly expressed in brain capillaries. Its expression in the BBB limits the accumulation of many hydrophobic molecules and potentially toxic substances in the brain. 3. The purpose of this review is to summarize the current state of knowledge about the expression of P-gp, its cellular localization as well as its possible functions in the BBB.     

P-glycoprotein mediates brain-to-blood efflux transport of buprenorphine across the blood-brain barrier

The involvement of P-glycoprotein (P-gp) in buprenorphine (BNP) transport at the blood-brain barrier (BBB) in rats was investigated in vivo by means of both the brain uptake index technique and the brain efflux index technique. P-gp inhibitors, such as cyclosporin A, quinidine and verapamil, enhanced the apparent brain uptake of [3H]BNP by 1.5-fold. The increment of the BNP uptake by the brain suggests the involvement of a P-gp efflux mechanism of BNP transport at the BBB. [3H]BNP was eliminated with an apparent elimination half-life of 27.5 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [3H]BNP across the BBB was 0.154 ml/min/g brain, which was calculated from the elimination rate constant (2.52 x 10- 2 min- 1) and the distribution volume in the brain (6.11 ml/g brain). The efflux transport of [3H]BNP was inhibited by range from 32 to 64% in the presence of P-gp inhibitors. The present results suggest that BNP is transported from the brain across the BBB via a P-gp-mediated efflux transport system, at least in part.     

Interactions of pluronic block copolymers on P-gp efflux activity: experience with HIV-1 protease inhibitors

The objective was to examine the influence of Pluronic block-copolymers on the interaction between the drug efflux transporter, P-glycoprotein and HIV-1 protease inhibitors (PIs). The ATPase assay determined the effect of various Pluronics on PI-stimulated P-gp ATPase activity. Cellular accumulation studies were conducted using MDCKII and LLC-PK1 cells transfected with human MDR1 to assess Pluronic modulation of PI efflux. Pluronic P85 inhibited both basal and nelfinavir-stimulated P-gp ATPase activity, while Pluronic F127 had no effect. In cell accumulation studies, Pluronic P85 restored the accumulation of nelfinavir in MDCKII-MDR1 cells while Pluronic F127 and F88 had no effect. Pluronic P85 increased saquinavir accumulation in wild-type and MDR1-transfected cells in both the MDCKII and LLC-PK1 cell models, suggesting inhibition of multiple transporters, including MRPs. In conclusion, this study provides evidence that a block-copolymer, Pluronic P85, effectively inhibits the interaction of P-gp with nelfinavir and saquinavir. These data indicate that effective inhibition of HIV-1 PI efflux by Pluronic P85 may influence the distribution of antiretroviral agents to sites protected by efflux mechanisms, such as the blood-brain barrier, and possibly increase the brain exposure of these drugs resulting in suppression of viral replication and reduction in the incidence of drug resistant mutants.