Christophe Rousselle

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.

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.

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.

Improved Brain Delivery of Benzylpenicillin with a Peptide-vector-mediated Strategy

Previous studies from our laboratory have demonstrated that the coupling of doxorubicin with SynB1 vector dramatically increases its brain uptake. In the present study, we have evaluated the broad application of this approach using another molecule: benzylpenicillin (B-Pc). We, therefore, have coupled the β -lactam antibiotic B-Pc with SynB1 and assessed its ability to cross the blood-brain barrier (BBB) using the in situ rat brain perfusion method. We first confirmed the very low brain uptake of free radiolabeled B-Pc. When B-Pc was coupled to SynB1, its uptake in brain was increased by a factor of 7, without compromising the BBB integrity. The vectorised B-Pc was distributed in all the gray areas assessed (frontal, parietal, and occipital cortex, thalamus, hippocampus, and striatum). Moreover, using a wash-out procedure and a capillary depletion method, we have shown that the radiolabeled B-Pc was associated mainly with brain parenchyma. In summary, this study demonstrates the successful application of the use of SynB1 vector for the transport of B-Pc across the BBB.

In situ transport of vinblastine and selected P-glycoprotein substrates: implications for drug-drug interactions at the mouse blood-brain barrier.

AbstractPurpose. 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 ∼56 μM and a Hill coefficient of ∼4. The vinblastine efflux by P-gp was described by a Km at 16 μM and a maximal efflux velocity, Jmax, of ∼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.

Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors

Biological membranes normally restrict the passage of hydrophilic molecules. This impairs the use of a wide variety of drugs for biomedical applications. To overcome this problem, researchers have developed strategies that involve conjugating the molecule of interest to one of a number of peptide entities that are efficiently transported across the cell membranes. In the past decade, a number of different peptide families with the ability to cross the cell membranes have been identified. Certain of these families enter the cells by a receptor-independent mechanism, are short (10–27 amino acid residues), and can deliver successfully various cargoes across the cell membrane into the cytoplasm or nucleus. Surprisingly, some of these vectors, the SynB vectors, have also shown the ability to deliver hydrophilic molecules across the blood-brain barrier, one of the major obstacles to the development of drugs to combat diseases affecting the CNS.

Vector-mediated drug delivery to the brain.

As a consequence of the growing ageing population, many neurodegenerative diseases, cancer and infections of the brain will become more prevalent. Despite major advances in neuroscience, many potential therapeutic agents are denied access to the central nervous system (CNS) because of the existence of the blood-brain barrier (BBB). This barrier is formed by the endothelial cells of the brain capillaries and its primary characteristic is the impermeability of the capillary wall due to the presence of complex tight junctions and a low endocytic activity. The BBB behaves as a continuous lipid bilayer and prevents the passage of polar and lipid-insoluble substances. The BBB is, therefore, the major obstacle to drugs that are potentially useful for combating diseases affecting the CNS. Extensive efforts have been made to develop CNS drug delivery strategies in order to enhance delivery of therapeutic molecules across the BBB. The current challenge is to develop drug-delivery strategies that will allow the passage of therapeutic drugs through the BBB in a safe and effective manner. This review focuses specifically on the strategies developed to enhance drug delivery across the BBB with an emphasis on the vector-mediated strategy.

In vivo saturation of the transport of vinblastine and colchicine by P-glycoprotein at the rat blood-brain barrier.

AbstractPurpose. To determine concentration-dependent P-gp-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in rats. Methods. The transport of radiolabeled colchicine and vinblastine across the rat BBB was measured with or without PSC833, a well known P-gp inhibitor, and within a wide range of colchicine and vinblastine concentration by an in situ brain perfusion. Thus, the difference of brain transport achieved with or without PSC833 gives the P-gp-mediated efflux component of the compound transported through the rat BBB. Cerebral vascular volume was determined by coperfusion with labeled sucrose in all experiments. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. P-gp limited the uptake of both colchicine and vinblastine, but the compounds differ in that vinblastine inhibited its own transport. Vinblastine transport was well fitted by a Hill equation giving IC50 at ∼71 μM, a Hill coefficient (n) ∼2, and a maximal efflux velocity Jmax of ∼9 pmol s−1 g−1 of brain. Conclusions. P-gp at the rat BBB may carry out both capacity-limited and capacity-unlimited transport, depending on the substrate, with pharmacotoxicologic significance for drug brain disposition and risk of drug-drug interactions.