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 × 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.     

Blood–brain barrier transport of naloxone does not involve P-glycoprotein-mediated efflux

The blood–brain barrier (BBB) transport of naloxone, a potent and specific opioid antagonist, was investigated in rats using the brain uptake index method and the brain efflux index method. The apparent influx clearance of [³H]naloxone across the BBB was 0.305 mL/min/g brain. [³H]naloxone was eliminated from the brain with an apparent elimination half-life of 15.1 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [³H]naloxone across the BBB was 0.152 mL/min/g brain, which was calculated from the elimination rate constant (4.79 × 10^?2 min^?1) and the distribution volume in the brain (3.18 mL/g brain). The influx clearance across the BBB was two times greater than the efflux clearance. The elimination of [³H]naloxone from the brain was not inhibited in the presence of the typical P-glycoprotein (P-gp) inhibitors such as quinidine, verapamil, vinblastine, and vincristine, indicating that naloxone is not a P-gp substrate in the rat. In vitro experiments by using human multidrug resistance 1 (MDR1)/P-gp overexpressing HeLa cells showed that the uptake of naloxone by the cells did not change in the presence of the P-gp inhibitors. In conclusion, the present results obtained from in vivo and in vitro studies suggest that P-gp is not involved in the BBB transport of naloxone. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:413–421, 2010     

Involvement of P-glycoprotein in blood-brain barrier transport of pentazocine in rats using brain uptake index method

The involvement of P-glycoprotein (P-gp) in pentazocine (PTZ) transport at the blood-brain barrier (BBB) in rats was evaluated by means of an in vivo study using the brain uptake index (BUI) method. The amount of radioactivity in the brain was estimated at different intervals (up to 240 s) after carotid injection in rats. The apparent elimination rate constant (k(test)) due to efflux of PTZ from the brain was calculated as 0.22 min(-1). The observed BUI values of [(3)H]-PTZ (0.35 microM) were not significantly different between 5 and 15 s after the carotid injection. The concentration-dependent uptake of PTZ by the brain was increased gradually by increasing the concentration (0.01-1 mM) of PTZ in the injection solution. The apparent uptake of PTZ by the brain increased in the presence of P-gp inhibitors such as cyclosporin A, quinidine, verapamil and vinblastine after the carotid injection. These results suggest that the increment of PTZ uptake by the brain could be explained by the saturable efflux transport system involving a P-gp-mediated efflux mechanism of PTZ transport at the BBB.     

In vivo evidence for the efflux transport of pentazocine from the brain across the blood-brain barrier using the brain efflux index method

The efflux transport of pentazocine (PTZ) from the brain across the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method. PTZ was eliminated with the apparent elimination half-life of 13.0 min after microinjection into the parietal cortex area 2 region of the rat brain. The apparent efflux clearance of PTZ across the BBB was 137 microl/min/g brain, which was calculated from the elimination rate constant (5.35 x 10(-2) min(-1) and the distribution volume in the brain (2.56 ml/g brain). The efflux transport of PTZ was decreased in the presence of unlabeled PTZ, suggesting that PTZ is eliminated by a carrier-mediated transport system across the BBB. To characterize the efflux transport of PTZ from the brain in vivo, the effects of several compounds on the efflux transport of PTZ were investigated. P-glycoprotein (P-gp) inhibitors (verapamil and quinidine) reduced the PTZ efflux transport. In addition, the efflux transport of PTZ was inhibited by organic cations such as l-carnitine and tetraethylammonium (TEA), whereas organic anions such as p-aminohippuric acid, probenecid and taurocholate did not affect the PTZ efflux transport. The present results suggest that PTZ is transported from the brain across the BBB via l-carnitine/TEA-sensitive carrier-mediated efflux transport system(s) in addition to P-gp.     

Possible involvement of cationic‐drug sensitive transport systems in the blood‐to‐brain influx and brain‐to‐blood efflux of amantadine across the blood–brain barrier

The purpose of this study was to characterize the brain‐to‐blood efflux transport of amantadine across the blood–brain barrier (BBB). The apparent in vivo efflux rate constant for [³H]amantadine from the rat brain (k_eff) was found to be 1.53 × 10^‐2 min^‐1 after intracerebral microinjection using the brain efflux index method. The efflux of [³H]amantadine was inhibited by 1‐methyl‐4‐phenylpyridinium (MPP⁺), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P‐glycoprotein (P‐gp), did not affect the efflux transport of [³H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [³H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P‐gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [³H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [³H]amantadine was significantly decreased with the co‐perfusion of quinidine or verapamil, which are cationic P‐gp inhibitors, while MPP⁺ did not have a significant effect. It is thus concluded that while P‐gp is not involved, it is possible that rPMAT and the cationic drug‐sensitive transport system participate in the brain‐to‐blood efflux and the blood‐to‐brain influx of amantadine across the BBB, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

P glycoprotein regulated transport of glutamate at blood brain barrier.

AIM: To study whether efflux of glutamate (Glu) at blood brain barrier (BBB) was regulated by P-glycoprotein (P-gp). METHODS: 1) After intracerebral microinjection [3H]Glu 5 min, recoveries were determined in injected cerebrums in presence of multidrug-resistant(MDR) reversing agents verapamil (Ver), vincristine(VCR), an d cyclosporin A(CsA); 2) apparent transfer constants (Kin) of [3H]Glu from plasma to brain were determined after the in situ rat brain perfusion 2 min us ing solution containing MDR-reversing agents; 3) uptake amount of [3H]Glu by prima ry cultured bovine brain capillary endothelial cells(BCEC) was analyzed; and 4) In presence of MDR-reversing agents and antibody of P-gp, C(219), uptake amount of [3H]Glu by luminal membrane vesicles derived from BCEC was also determined. RESULTS: In control rats, remaining percentage of [3H]Glu in inject ed cerebrums was 25 %+/-16 % at 5 min after intracerebral injection. After pre-treating with CsA 10, 100 micromol/L, VCR 20 micromol/L and Ver 100 micromol/L, the remaining percentages of [3H]Glu were increased to about 2.2, 2.5, 2.3, a nd 2.7 folds of control, respectively. In the in situ rat brain per fusion experiment, VCR and CsA in perfusion medium concentration-dependently increased [3H]Glu BBB permeability to brain. Co-administration o f CsA 40 micromol/L mad e BBB permeability of [3H]Glu in cerebral cortex, hippocampus and striatum increase to about 9, 3, 7, and 4.6 folds of control, respectively. Steady-state uptake of [3H]Glu by BCEC was also increased up to 2.5 folds in presence of 100 micromol/L CsA. MDR-reversing agents and antibody of P-gp, C219, level-dependently inhibited the uptake of [3H]Glu by luminal membrane vesicles of BCEC. And this process is ATP-dependent. CONCLUSION: Efflux of Glu at BBB may be regulated by P-gp.     

Evaluation of blood-brain barrier transporters by co-culture of brain capillary endothelial cells with astrocytes

To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as gamma-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [(3)H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [(14)C]benzoic acid was saturable, and the K(m) value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [(14)C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [(3)H]dehydroepiandrosterone sulfate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.     

ATA2 is predominantly expressed as system A at the blood-brain barrier and acts as brain-to-blood efflux transport for L-proline

Although system A is present at the blood-brain barrier (BBB), the physiological roles of system A have not been clarified. The efflux transport of the substrates of system A, such as L-proline (L-Pro), glycine (Gly), and alpha-methylaminoisobutyric acid (MeAIB), across the BBB was investigated using the in vivo Brain Efflux Index method. Over a period of 40 min, L-[(3)H]Pro and [(3)H]Gly underwent efflux from the brain, whereas [(3)H]MeAIB did not. The efflux of L-[(3)H]Pro was inhibited by the presence of unlabeled L-Pro and MeAIB, suggesting that carrier-mediated efflux transport of L-Pro across the BBB is involved in system A. L-[(3)H]Pro uptake by TR-BBB cells, used as an in vitro BBB model, was Na(+)-dependent with high-affinity (K(m1) = 425 microM) and low-affinity (K(m2) = 10.8 mM) saturable processes. The manner of inhibition of L-[(3)H]Pro uptake for amino acids was consistent with system A. Although GlnT, ATA2, and ATA3 mRNA were all expressed in TR-BBB cells, ATA2 mRNA was predominant. Under hypertonic conditions, ATA2 mRNA in TR-BBB cells was induced by up to 373%, and it activated [(3)H]MeAIB uptake. In light of these observations, our results indicate that L-Pro and Gly are transported from the brain across the BBB, whereas MeAIB is retained in the brain. System A is involved in efflux transport for L-Pro at the BBB. The predominantly expressed ATA2 mRNA at the BBB may play a role in maintaining the concentration of small neutral amino acids and cerebral osmotic pressure in the brain under pathological conditions.     

Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [³H]naloxone exhibited saturability in a concentration‐dependent manner (concentration range 0.5 µM to 15 mM ) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier‐mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K_t, was 2.99±0.71 mM ; the maximum uptake rate, J_max, was 0.477±0.083 µmol/min/g brain; and the nonsaturable first‐order rate constant, K_d, was 0.160±0.044 ml/min/g brain. The uptake of [³H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H₁‐antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [³H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L ‐carnitine or substrates for organic anion transport systems such as p‐aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH‐dependent and saturable influx transport system that is a selective transporter for cationic H₁‐antagonists is involved in the BBB transport of naloxone in the rat. Copyright © 2010 John Wiley & Sons, Ltd.     

脑缺血再灌注后（^3H）GABA通过大鼠血脑屏障的外排转运

目的 研究脑缺血／再灌后（＾３Ｈ）ＧＡＢＡ通过大鼠血脑屏障的外排运是否增强及其机制。方法 将（＾３Ｈ）ＧＡＢＡ或ＧＡＢＡ（或丙磺舒）与其联合注射到缺血／再灌大在脑皮层顶二区后,测定（＾３Ｈ）ＧＡＢＡ的脑外排指数（ＢＥＩ）及ｉｖ依文思蓝（ＥＢ）后ＥＢ的脑摄取量。结果：１０ｍｉｎ缺血／再灌３０ｍｉｎ,２ｈ,６ｈ和２４ｈ大鼠的ＢＥＩ分别为６７％,８３％,９２％和８７％,显著高于对照值（５８％）,ＥＢ脑报     

Blood-brain barrier transport of H1-antagonist ebastine and its metabolite carebastine

The transport mechanism of the non-sedative H1-antagonist ebastine and its first-pass carboxylic acid metabolite carebastine at the blood-brain barrier (BBB) was studied. In rats, the brain uptake index (BUI) value of [14 C]carebastine was significantly lower than that of [14 C]ebastine. The BUI value of [14 C]carebastine was greatly increased by the addition of non-labeled carebastine. The steady-state uptake of [14 C]carebastine by P-glycoprotein-overexpressing K562/ADM cells was significantly lower than that by their parental drug-sensitive cell line K562. The decreased steady-state uptake of [14 C]carebastine by K562/ADM cells was reversed by verapamil. Steady-state uptake of [14 C]carebastine by primary cultured bovine brain capillary endothelial cells (bovine BCECs) was increased in the presence of metabolic inhibitors and verapamil. Non-labeled carebastine increased the steady-state uptake of a P-glycoprotein substrate, [3 H]vincristine, by bovine BCECs. The initial uptake of [3 H]mepyramine by bovine BCECs and RBEC1 (an immortalized cell line from rat brain capillary endothelial cells) was strongly inhibited by ebastine, while zwitterionic carebastine was slightly inhibitory. The values of brain-to-plasma unbound concentration ratio (Kp,f) in mdr1a(-/-) mice were increased 5.3-fold and 4.2-fold for [14 C ebastine and for [14 C]carebastine, respectively, compared with those in mdr1a(+/+) mice. Non-radiolabeled carebastine increased the Kp,f values of [14 C]carebastine in both types of mice. In conclusion, carebastine was shown to be a substrate for P-glycoprotein-mediated efflux from the brain at the BBB. A second efflux system may also be involved. The relatively low affinity of the uptake transport system for carebastine also limits the brain distribution of ebastine/carebastine.     

In-vivo and in-vitro evidence of a carrier-mediated efflux transport system for oestrone-3-sulphate across the blood-cerebrospinal fluid barrier

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.     

Altered Brain Uptake of Therapeutics in a Triple Transgenic Mouse Model of Alzheimer’s Disease

Purpose

The purpose of this study was to systematically assess the impact of Alzheimer’s disease (AD)-associated blood–brain barrier (BBB) alterations on the uptake of therapeutics into the brain.

Methods

The brain uptake of probe compounds was measured in 18–20 month old wild type (WT) and triple transgenic (3×TG) AD mice using an in situ transcardiac perfusion technique. These results were mechanistically correlated with immunohistochemical and molecular studies.

Results

The brain uptake of the paracellular marker, [¹⁴C] sucrose, did not differ between WT and 3×TG mice. The brain uptake of passively diffusing markers, [³H] diazepam and [³H] propranolol, decreased 54–60% in 3×TG mice, consistent with a 33.5% increase in the thickness of the cerebrovascular basement membrane in 3×TG mice. Despite a 42.4% reduction in P-gp expression in isolated brain microvessels from a sub-population of 3×TG mice (relative to WT mice), the brain uptake of P-gp substrates ([³H] digoxin, [³H] loperamide and [³H] verapamil) was not different between genotypes, likely due to a compensatory thickening in the cerebrovascular basement membrane counteracting any reduced efflux of these lipophilic substrates.

Conclusion

These studies systematically assessed the impact of AD on BBB drug transport in a relevant animal model, and have demonstrated a reduction in the brain uptake of passively-absorbed molecules in this mouse model of AD.     

Involvement of P-Glycoprotein in the Transport of Saquinavir and Indinavir in Rat Brain Microvessel Endothelial and Microglia Cell Lines

PURPOSE: Membrane-bound efflux transporters, such as P-glycoprotein (P-gp), may limit the brain entry and distribution of HIV-1 protease inhibitors and be in part responsible for HIV-1-associated dementia treatment failure. The purpose of this study was to characterize the transport properties of saquinavir and indinavir in a brain microvessel endothelial cell line and in microglia, the immune cells of the brain and primary HIV-1 cellular target. METHODS: Biochemical and transport studies were performed in an immortalized rat brain endothelial cell line (RBE4), a rat microglia cell line (MLS-9), and a P-gp overexpressing Chinese hamster ovary cell line (CHRC5). RESULTS: Western blot analysis using the P-gp monoclonal antibody C219 detected a single band at approximately 170 to 180 kDa (a size previously reported for P-gp) in all cell lines. Cellular accumulation of [14C]saquinavir and [3H]indinavir by RBE4, MLS-9, and CHRC5 monolayers was significantly enhanced in the presence of P-gp inhibitors, HIV-1 protease inhibitors, the ATPase inhibitor sodium azide, and the ATP depleting agent 2',4'-dinitrophenol respectively. [14C]Saquinavir and [3H]indinavir efflux from both cell systems was rapid and significantly reduced in the presence of PSC833. CONCLUSIONS: These results provide evidence for P-gp mediated transport of saquinavir and indinavir in RBE4 and MLS-9 and suggest that this transporter can restrict, at least in part, the permeation of HIV-1 protease inhibitors at both the brain barrier site and in brain parenchyma.     

Co-administration strategy to enhance brain accumulation of vandetanib by modulating P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) mediated efflux with m-TOR inhibitors

The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors.     

In Situ Transport of Vinblastine and Selected P-glycoprotein Substrates: Implications for Drug-Drug Interactions at the Mouse Blood-Brain Barrier

PURPOSE: To study the intrinsic parameters of P-glycoprotein (P-gp) transport and drug-drug interactions at the blood-brain barrier (BBB), as few quantitative in vivo data are available. These parameters could be invaluable for comparing models and predicting the in vivo implications of in vitro studies. METHODS: The brains of P-gp-deficient mice mdr1a(-/-) and wild-type mice were perfused in situ using a wide range of colchicine, morphine, and vinblastine concentrations. The difference between the uptake by the wild-type and P-gp-deficient mice gave the P-gp-linked apparent transport at the BBB. Drug-drug interactions were examined using vinblastine and compounds that bind to P-gp sites (verapamil, progesterone, PSC833) other than the vinblastine site to take into account the multispecific drug P-gp recognition. RESULTS: P-gp limited the brain uptake of morphine and colchicine in a concentration-independent way up to 2 mM. In contrast, vinblastine inhibited its own P-gp transport with an IC50 of approximately 56 microM and a Hill coefficient of approximately 4. The vinblastine efflux by P-gp was described by a Km at 16 microM and a maximal efflux velocity, Jmax, of approximately 8 pmol s(-1) g(-1) of brain. Similarly, vinblastine brain transport was increased by inhibiting P-gp as shown by the IC50 ranking, which was PSC833 < verapamil < vinblastine < progesterone. CONCLUSIONS: P-gp is responsible for both capacity-limited and -unlimited transport of P-gp substrates at the mouse BBB. In situ perfusion of mdr1a(-/-) and wild-type mouse brains could be used to predict drug-drug interactions for P-gp at the mouse BBB.     

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.     

Validation and application of Caco-2 assays for the in vitro evaluation of development candidate drugs as substrates or inhibitors of P-glycoprotein to support regulatory submissions

1.?An understanding of the role that transporters, in particular P-glycoprotein (P-gp), can play in the absorption, distribution, metabolism and excretion (ADME) of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug–drug interactions in the clinic, is required to support drug development and registration. It is therefore necessary to validate preclinical assays for the in vitro evaluation of candidate drugs as substrates or inhibitors of human P-gp.

2.?The present study has characterized a Caco-2 cell monolayer model by determining the bi-directional apparent permeabilities and efflux ratios of the known P-gp substrates ([³H]-digoxin, [³H]-ketoconazole, [³H]-verapamil, [³H]-quinidine, dipyridamole and loratidine; 1–100 µM) a non-substrate ([³H]-propranolol; 10 µM), or by determining the inhibitory potencies (IC₅₀) of inhibitors (verapamil, ketoconazole, quinidine, dipyridamole and probenecid; 0.1–100 µM) on the basolateral-to-apical transport of [³H]-digoxin (5 µM), in order to validate methodologies for the identification of substrates or inhibitors of P-gp, respectively.

3.?The reproducibility of the [³H]-digoxin or verapamil data determined from replicate monolayers across different cell passages indicates that the functional expression of P-gp is consistent across the range of passages (25–40) utilized for transport experiments and that the determination of bi-directional apparent permeability, or IC₅₀ for inhibition of P-gp, respectively, need only be performed on one occasion for a test compound. [³H]-digoxin and [³H]-propranolol or verapamil and probenecid were considered to be appropriate positive and negative controls of P-gp-mediated transport, or inhibition of P-gp, respectively, to ensure performance of the assays when assessing candidate drugs. Additionally, the low IC₅₀ values determined for ketoconazole and quinidine indicated that these inhibitors were suitable to use to confirm the role of P-gp in the efflux of a test compound.

4.?These validated Caco-2 assays are robust, reproducible and suitable for routine in vitro evaluation of candidate drugs. They have been successfully applied to development projects resulting in the identification of two candidate drugs as substrates and inhibitors of P-gp, whereas a third was neither a substrate nor an inhibitor of this transporter.     

Circumventing P-glycoprotein-mediated cellular efflux of quinidine by prodrug derivatization

The objective of this study is to investigate whether transporter-targeted prodrug derivatization of quinidine, a model P-glycoprotein (P-gp) substrate, can circumvent P-gp-mediated efflux. The L-valine ester of quinidine (val-quinidine) was synthesized in our laboratory. Uptake and transport studies were carried out using the MDCKII-MDRI cell line at 37 degrees C for 10 min and 3 h, respectively. [3H]Ritonavir and cyclosporine were also used as model P-gp substrates to delineate the kinetics of translocation of val-quinidine across the MDCKII-MDRI cell monolayer. The rate of uptake of [3H]ritonavir by MDCKII-MDRI cells exhibited a 2-fold increase in the presence of 75 microM quinidine, but 75 microM val-quinidine did not demonstrate any effect on [3H]ritonavir uptake. The rate of transport of quinidine from the basolateral to the apical membrane [(18.3 +/- 1.25) x 10(-6) cm s(-1)] and from the apical to the basolateral membrane [(6.5 +/- 0.66) x 10(-6) cm s(-1)] exhibited a 3-fold difference. However, transport of val-quinidine from the apical to the basolateral membrane [(5.13 +/- 0.49) x 10(-6) cm s(-1)] and from the basolateral to the apical membrane [(6.17 +/- 1.28) x 10(-6) cm s(-1)] did not demonstrate any statistically significant difference. Moreover, cyclosporine, a potent P-gp substrate and/or inhibitor, did not alter the transport kinetics of val-quinidine. The rates of uptake of [3H]Gly-Sar and various amino acid model substrates were reduced in the presence of 200 microM val-quinidine. Results from this study clearly indicate that prodrug derivatization of quinidine into val-quinidine can overcome P-gp-mediated efflux. Val-quinidine once bound to a peptide or amino acid transporter is probably not recognized and cannot be accessed by the P-gp efflux pump. Transporter-targeted prodrug derivatization seems to be a viable strategy for overcoming P-gp-mediated efflux.     

Endomorphins, Met-enkephalin, Tyr-MIF-1, and the P-glycoprotein efflux system.

The P-glycoprotein (P-gp) transport system, responsible for the efflux of many therapeutic drugs out of the brain, recently has been shown to transport the endogenous brain opiate endorphin. We used P-gp knockout mice (Mdr1a) and their controls to determine where P-gp is involved in the saturable efflux systems of four other endogenous opiate-modulating peptides across the blood-brain barrier (BBB). After injection of endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)), endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)), Met-enkephalin (Tyr-Gly-Gly-Phe-Met-OH), and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) into the lateral ventricle of the mouse brain, residual radioactivity was measured at 0, 2, 5, 10, and 20 min later. The results showed no difference in the disappearance of any of these peptides from the brains of knockout mice compared with their controls. This demonstrates that unlike endorphin and morphine, P-gp does not seem to be required for the brain-to-blood transport of the endomorphins, Met-enkephalin, or Tyr-MIF-1 across the BBB.