Salvatore Cisternino

Expression, Up-Regulation, and Transport Activity of the Multidrug-Resistance Protein Abcg2 at the Mouse Blood-Brain Barrier

The breast cancer resistance protein (BCRP/ABCG2) is, like P-glycoprotein (P-gp), a member of the ABC family of drug transporters. These proteins actively transport various anticancer drugs from cells, causing multidrug resistance. The physiological expression of P-gp/ABCB1 at the blood-brain barrier (BBB) effectively restricts the brain uptake of many antitumor drugs by mediating their active efflux from the brain to the blood vessel lumen. However, little is known about the function of Abcg2 at the BBB in vivo. We used in situ brain perfusion to measure the uptake of two known Abcg2 substrates, prazosin and mitoxantrone, and the nonsubstrate vinblastine by the brains of wild-type and P-gp-deficient mutant mdr1a(-/-) mice with or without the P-gp/Abcg2 inhibitor GF120918 or the P-gp inhibitor PSC833. P-gp had no effect on the brain transport of prazosin and mitoxantrone at the mouse BBB, but wild-type and P-gp-deficient mouse brains perfused with GF120918 or a high concentration of prazosin showed carrier-mediated effluxes of prazosin and mitoxantrone from the brain that did not involve P-gp. In contrast, the brain uptake of vinblastine was restricted only by P-gp and not by Abcg2 at the BBB. The amounts of abcg2 mRNA in cortex homogenates and capillary-enriched fractions of wild-type and mdr1a(-/-) mouse brains were measured by real-time quantitative reverse transcription-PCR. There was approximately 700-times more abcg2 mRNA in brain microvessels than in the cortex of the wild-type mice, confirming that Abcg2 plays an important role at the BBB. There was also approximately 3 times more abcg2 mRNA in the microvessels from P-gp-deficient mutant mouse brains than in the microvessels of wild-type mouse brains. These findings confirm that Abcg2 is a physiological transporter at the BBB that restricts the permeability of the brain to its substrates in vivo. Lastly, the defective P-gp in the mutant mdr1a(-/-) mice was associated with increased abcg2 mRNA at the BBB and a greater export of prazosin and mitoxantrone from the brain, as measured in the P-gp-deficient mice versus the wild-type mice.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters

Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

Deletion of Astroglial Connexins Weakens the Blood–Brain Barrier

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood–brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and β-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.

Evidence for an active transport of morphine-6-β-d-glucuronide but not P-glycoprotein-mediated at the blood–brain barrier

Morphine‐6‐β‐d‐glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood–brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P‐glycoprotein (P‐gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance‐associated protein mrp1 and the glucose transporter GLUT‐1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild‐type and deficient mice [mdr1a(–/–) and mrp1(–/–)], with and without probenecid, digoxin, PSC833 or d‐glucose. No difference was found between P‐gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co‐perfused with probenecid in wild‐type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co‐perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P‐gp and sensitive to digoxin and PSC833, may be involved. The co‐perfusion of [14C]M6G with d‐glucose revealed a threefold decrease in M6G uptake. In conclusion, P‐gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT‐1 and a digoxin‐sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.

Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood-brain barrier in mice and rats

TXD258, a new taxoid antitumor agent, is a poor substrate for the P‐glycoprotein (P‐gp) in Caco‐2 cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice under a variety of conditions (dose and infusion time, species and plasma concentration) using conventional in vivo pharmacokinetic techniques and in situ brain perfusion. Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m−2 for 45 s or 1 h and rats were infused with 15 and 60 mg m−2 over 2.3 min. The radioactivity in the plasma and brains was measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of TXD258 increased linearly in mice with the infused dose, the brain content increased more than proportionally with the dose between 15 and 90 mg m−2. This nonlinear uptake of TXD258 also occurred in the plasma and brain of the rat. These findings suggest that the protein‐mediated efflux across the blood–brain barrier (BBB) becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P‐gp substrate at the BBB of mice and rats. The P‐gp of both species was saturated at the half‐inhibitory concentration (∼13 μM) produced by i.v. infusion. Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation of the P‐gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P‐gp‐mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity.

Screening of multidrug-resistance sensitive drugs by in situ brain perfusion in P-glycoprotein-deficient mice

AbstractPurpose. This study was conducted to assess the influence of P-glycoprotein (P-gp) on brain uptake of multidrug resistance sensitive drugs using an in situbrain perfusion technique in P-gp-deficient (mdr1a[−/−]) and wild-type mice. Methods. The blood-brain transport of radiolabeled vinblastine, vincristine, doxorubicin, colchicine, and morphine was evaluated in mdr1a(−/−) and wild-type CF-1 mice with the in situ brain perfusion technique. Brain uptake of drugs after intravenous pretreatment with P-gp reversal agents, (PSC 833, GF 120918, or (±)-verapamil), or vehicle also was studied in wild-type mice. In all experiments, cerebral vascular volume was determined by co-perfusion of sucrose. Results. Cerebral vascular volume was preserved during perfusion, indicating maintenance of blood-brain barrier integrity in both types of mice within the concentration range of substrates in the perfusate. The apparent brain transport of colchicine, vinblastine, doxorubicin, and morphine was increased 3.0, 2.7, 1.5, and 1.4-fold, respectively, in mdr1a(−/−) mice compared with the wild-type; the brain uptake of vincristine was not affected by P-gp. Preadministration of PSC 833 or GF 120918 in wild-type mice led to a ∼3-fold increase in the brain transport of colchicine and vinblastine, but no effect was observed for the other compounds. Intravenous verapamil enhanced colchicine brain transport (1.8-fold), but failed to increase the brain uptake of vinblastine and morphine. Conclusion. The in situ brain perfusion technique appears to be a sensitive and powerful tool for medium throughput screening of the brain uptake of multidrug resistance sensitive drugs. The effect of P-gp is characterized more efficiently with mdr1a(−/−) mice than by using modulators of P-gp in wild-type mice.

Apparent Lack of Mrp1-Mediated Efflux at the Luminal Side of Mouse Blood-Brain Barrier Endothelial Cells

AbstractPurpose. The purpose of this work was to determine mrp1-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in mice. Methods. The transport of radiolabeled etoposide, 17β-estradiol-D-17β-glucuronide (E217βG), vincristine, and doxorubicin across the BBB of mrp1(–/–) and wild-type mice was evaluated by in situ brain perfusion. Etoposide transport was also determined in P-glycoprotein-deficient mdr1a(–/–) mice perfused with both etoposide and mrp1 inhibitors like probenecid or MK571. Cerebral vascular volume was determined by co-perfusion with labeled sucrose. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. The transport of etoposide, E217βG, vincristine, and doxorubicin into the brain was not affected by the lack of mrp1. Trans-efflux studies in mrp1-deficient mice with etoposide and E217βG confirmed that mrp1 was not involved in the efflux of these substrates across the BBB. There was also a significant P-gp-mediated efflux of etoposide in studies with P-glycoprotein-deficient mdr1a(–/–) mice. Perfusion of mdr1a(–/–) mice etoposide plus probenecid or MK571 did not affect the brain transport of etoposide. Conclusion. Efflux mediated by mrp1 does not seem to occur across the luminal membrane of the endothelial cells forming the mouse BBB.

Heterogeneity in the rat brain vasculature revealed by quantitative confocal analysis of endothelial barrier antigen and P-glycoprotein expression

While phenotypic endothelial heterogeneity is well documented in peripheral organs, it is only now being explored in the brain. We used confocal imaging of thick sections of rat brain to qualitatively and quantitatively examine the expression of two key markers of the blood—brain barrier (BBB) in the rat, P-glycoprotein (P-gp), and endothelial barrier antigen (EBA). We found that these markers were not uniformly distributed throughout the whole vasculature of the cortex and hippocampus. P-glycoprotein displayed a gradient of expression from an almost undetectable level in large penetrating arterioles to a high and uniform level in capillaries and venules. While EBA was lacking in all cerebral arterioles, regardless of their size, its expression varied greatly among endothelial cells in capillaries and venules, yielding a striking mosaic pattern. A detailed quantitative analysis of the distribution of these markers at the single cell level in capillaries is provided. These results challenge the view of a uniform BBB and suggest that regulatory mechanisms might differentially modulate BBB features not only among arterioles/capillaries/venules but also at the single cell level within the capillaries. Hypotheses are made regarding the underlying mechanisms and physiopathological consequences of this heterogeneity.

Opioid Transport by ATP-Binding Cassette Transporters at the Blood-Brain Barrier: Implications for Neuropsychopharmacology

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.

Clonidine transport at the mouse blood-brain barrier by a new H + antiporter that interacts with addictive drugs

Identifying drug transporters and their in vivo significance will help to explain why some central nervous system (CNS) drugs cross the blood-brain barrier (BBB) and reach the brain parenchyma. We characterized the transport of the drug Clonidine at the luminal BBB by in situ mouse brain perfusion. Clonidine influx was saturable, followed by Michaelis–Menten kinetics (Km = 0.62 mmol/L, Vmax = 1.76 nmol/sec per g at pH 7.40), and was insensitive to both sodium and trans-membrane potential. In vivo manipulation of intracellular and/or extracellular pH and Trans-stimulation showed that Clonidine was transported by an H+-coupled antiporter regulated by both proton and Clonidine gradients, and that diphenhydramine was also a substrate. Organic cation transporters (Oct1–3), P-gp, and Bcrp did not alter Clonidine transport at the BBB in knockout mice. Secondary or tertiary amine CNS compounds such as oxycodone, morphine, diacetylmorphine, methylenedioxyamphetamine (MDMA), cocaine, and nicotine inhibited Clonidine transport. However, cationic compounds that interact with choline, Mate, Octn, and Pmat transporters did not. This suggests that Clonidine is transported at the luminal mouse BBB by a new H+-coupled reversible antiporter.