Jean-Michel Scherrmann

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.

CNS Delivery Via Adsorptive Transcytosis

Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity—like the cationization strategy—as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

ABC transporters, cytochromes P450 and their main transcription factors: expression at the human blood–brain barrier

We have established the expression patterns of the genes encoding ATP‐binding cassette (ABC) transporters and cytochromes P450 (CYPs) at the adult human blood–brain barrier (BBB) using isolated brain microvessels and cortex biopsies from patients with epilepsia or glioma. Microves synaptophysin (neurons) and neuron‐glial antigen 2 (NG2) (pericytes). ABCG2 [breast cancer resistance protein (BCRP)] and ABCB1 (MDR1) were the main ABC transporter genes expressed in microvessels, with 20 times more ABCG2 and 25 times more ABCB1 in microvessels than in the cortex. The CYP1B1 isoform represented over 80% of all the CYPs genes detected in microvessels. There were 14 times more CYP1B1 in microvessels than in the cortex, showing that CYP1B1 is mainly expressed at the BBB. p‐glycoprotein (ABCB1), BCRP (ABCG2) and CYP1B1 proteins were found in microvessels by western blotting. The expression of genes encoding three transcription factors [pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR)] was also investigated. The AhR gene, involved in the regulation of CYP1B1 expression, was highly expressed in brain microvessels, whereas PXR and CAR genes were almost undetected. This detailed pattern of ABC and CYPs gene expression at the human BBB provides useful information for understanding how their substrates enter the brain.

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.

Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells

We investigated the expression of genes encoding ABC transporters, cytochromes P450 (CYPs) and some transcription factors in the hCMEC/D3 immortalized human cerebral microvascular endothelial cell line, a promising in vitro model of the human BBB, and we compared these expressions to a non-brain endothelial cell line (HUVEC) and freshly human brain microvessels. qRT-PCR showed that the MDR1, BCRP, MRP1, MRP3, MRP4 and MRP5 genes were expressed and that the main CYP gene was CYP2U1 in hCMEC/D3. The pattern of ABC and CYPs gene expression in hCMEC/D3 differed from HUVEC which did not express MDR1. Moreover, expression of P-gp and BCRP was lower in hCMEC/D3 than in human brain microvessels but remain functional as shown by rhodamine 123 efflux assay. The gene encoding the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of some ABC and CYPs was highly expressed in hCMEC/D3 and HUVEC, while the pregnane-X-receptor (PXR) and the constitutive androstane receptor (CAR) were barely detected. We investigated the function of the AhR-mediated regulatory pathway in hCMEC/D3 by treating them with the AhR agonist TCDD. The expressions of two AhR-target genes, CYP1A1 and CYP1B1, were increased 26-fold and 28-fold. But the expressions of ABC transporter genes were not significantly altered. We have thus determined the pattern of expression of the genes encoding ABC transporters, CYPs and three transcription factors in hCMEC/D3 and shown that the AhR pathway might afford an original functional transport and metabolic pattern in cerebral endothelial cells that is different from other peripheral endothelial cells.

Doxorubicin-peptide conjugates overcome multidrug resistance.

A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein (P-gp), which is capable of lowering intracellular drug concentrations. To overcome this problem, we tested the capability of two peptide vectors that are able to cross cellular membranes to deliver doxorubicin in P-gp-expressing cells. The antitumor effect of peptide-conjugated doxorubicin was tested in human erythroleukemic (K562/ADR) resistant cells. The conjugate showed potent dose-dependent inhibition of cell growth against K562/ADR cells as compared with doxorubicin alone. Doxorubicin exhibited IC50 concentrations of 65 μM in the resistant cells, whereas vectorized doxorubicin was more effective with IC50 concentrations of 3 μM. After treatment of the resistant cells with verapamil, the intracellular levels of doxorubicin were markedly increased and consequent cytotoxicity was improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement in the cell uptake nor in the cytotoxic effect of the conjugated doxorubicin, indicating that the conjugate bypasses the P-gp. Finally, we show by the in situ brain perfusion method in P-gp-deficient and competent mice that vectorized doxorubicin bypasses the P-gp present at the luminal site of the blood-brain barrier. These results indicate that vectorization of doxorubicin with peptide vectors is effective in overcoming multidrug resistance.

Colchicine nonresponsiveness in familial mediterranean fever: clinical, genetic, pharmacokinetic, and socioeconomic characterization

OBJECTIVES To identify the ethnic, clinical, genetic, and pharmacokinetic correlates of colchicine treatment failure in patients with familial Mediterranean fever (FMF). METHODS Fifty-nine FMF patients, unresponsive to a daily dose of > or =2 mg colchicine, were compared with 51 colchicine-responsive patients by clinical, demographic, and socioeconomic assessment, FMF gene (MEditerranean FeVer [MEFV]) mutation and serum amyloid A1 (SAA1) gene polymorphism analysis, and plasma and white blood cell colchicine level determination. RESULTS Colchicine responders and nonresponders were comparable with respect to gender, age, duration and onset of the disease, and various demographic parameters. The 2 cohorts were found to carry mainly the M694V MEFV mutation and had a similar number of homozygotes or compound heterozygotes. Predominance of the alpha/beta alleles of SAA1 and comparable plasma and polymorphonuclear colchicine concentrations characterized both groups. Nonresponders were from lower socioeconomic backgrounds, had less education, and a more severe form of disease. A statistically significant 2-fold elevation of colchicine concentration in the mononuclear cells (MNC) of responders was found. CONCLUSIONS Colchicine treatment failure in FMF is associated with inadequate colchicine MNC concentration, probably resulting from a genetic defect unrelated to the underlying FMF.

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.

Expression of drug transporters at the blood–brain barrier using an optimized isolated rat brain microvessel strategy

Quantitative RT-PCR (qRT-PCR) and Western blotting studies on transporters at the blood-brain barrier (BBB) of isolated brain microvessels have produced conflicting data on their cellular distribution. A major problem is identifying cells expressing the genes of interest, since isolated brain microvessels are composed of several cell types and may be contaminated with mRNA or proteins from astrocytes and neurons. We isolated rat brain microvessels and examined microscopically samples at each step of isolation to evaluate microvessel purity. The expression of specific markers of endothelial cells (Glut-1, Flk-1), pericytes (Ng2), neurons (synaptophysin, Syn) and astrocytes (Gfap) was measured by qRT-PCR in order to select the protocol giving the least astrocyte and neuron mRNAs and the most endothelial mRNAs. We also evaluated the gene expression of drug transporters (Mdr1a, Mdr1b, Mrp1-5, Bcrp and Oatp-2) at each step to optimize their location in cells at the BBB. The Mdr1a, Mrp4, Bcrp and Oatp-2 gene profiles were similar to those of endothelium markers. The profiles of Mrp2 and Mrp3 closely resembled that of Ng2. Mrp5 and Mrp1 expression was not increased in the microvessel-enriched fraction, suggesting that they are ubiquitously expressed throughout the cortex parenchyma. We also evaluated by Western blotting the expression of P-gp, Mrp2, Gfap and Syn in the cortex and in the purest obtained microvessel fraction. Our results showed that P-gp expression strongly increased in microvessels whereas Mrp2 was not detected in any of the fraction. Surprisingly, Gfap expression increased in isolated microvessels whereas Syn was not detected. Our results showed that the strategy consisting of identifying gene expression at different steps of the protocol is useful to identify cells containing mRNA at the BBB and give overall similar results with protein expression.

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.