Gert Fricker

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and — when barrier integrity is impaired — in the pathology of many common CNS disorders such as Alzheimers disease, Parkinsons disease and stroke.

Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo.

Paclitaxel concentrations in the brain are very low after intravenous injection. Since paclitaxel is excluded from some tumors by p-glycoprotein (p-gp), the same mechanism may prevent entry into the brain. In vitro, paclitaxel transport was examined in capillaries from rat brains by confocal microscopy using BODIPY Fl-paclitaxel. Western blots and immunostaining demonstrated apical expression of p-gp in isolated endothelial cells, vessels, and tissue. Secretion of BODIPY Fl-paclitaxel into capillary lumens was specific and energy-dependent. Steady state luminal fluorescence significantly exceeded cellular fluorescence and was reduced by NaCN, paclitaxel, and SDZ PSC-833 (valspodar), a p-gp blocker. Leukotriene C(4) (LTC(4)), an Mrp2-substrate, had no effect. Luminal accumulation of NBDL-cyclosporin, a p-gp substrate, was inhibited by paclitaxel. In vivo, paclitaxel levels in the brain, liver, kidney, and plasma of nude mice were determined after intravenous injection. Co-administration of valspodar led to increased paclitaxel levels in brains compared to monotherapy. Therapeutic relevance was proven for nude mice with implanted intracerebral human U-118 MG glioblastoma. Whereas paclitaxel did not affect tumor volume, co-administration of paclitaxel (intravenous) and PSC833 (peroral) reduced tumor volume by 90%. Thus, p-gp is an important obstacle preventing paclitaxel entry into the brain, and inhibition of this transporter allows the drug to reach sensitive tumors within the CNS.

Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

1 . The interaction of cyclosporin A (CyA) with p‐glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco‐2 cells and an intubation study in healthy volunteers. 2 . CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco‐2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 μm. At higher concentrations permeation increased over‐proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 μl min−1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 μm and a Jmax of 6.5 picomol min−1 per filter. 3 . CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomcyin and a non‐immunosuppressive CyA‐derivative. All compounds inhibit p‐glycoprotein‐mediated transport processes. Basolateral to apical permeation of CyA showed a dose‐dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco‐2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4 . Western blot analysis of Caco‐2 cells with the monoclonal antibody C219 confirmed the presence of p‐glycoprotein in the used cell system. 5 . When the absorption of CyA in the gastrointestinal (G***I)‐tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in absorption exhibited a marked correlation (r = 0.994) to the expression of mRNA for p‐glycoprotein over the G**I‐tract (stomach < jejunum < colon). 6 . All data provide evidence that CyA is a substrate of p‐glycoprotein in the G***I‐tract, which might explain the local differences and the high variability in cyclosporin absorption found in vivo.

Phospholipids and lipid-based formulations in oral drug delivery.

ABSTRACTPhospholipids become increasingly important as formulation excipients and as active ingredients per se. The present article summarizes particular features of commonly used phospholipids and their application spectrum within oral drug formulation and elucidates current strategies to improve bioavailability and disposition of orally administered drugs. Advantages of phospholipids formulations not only comprise enhanced bioavailability of drugs with low aqueous solubility or low membrane penetration potential, but also improvement or alteration of uptake and release of drugs, protection of sensitive active agents from degradation in the gastrointestinal tract, reduction of gastrointestinal side effects of non-steroidal anti-inflammatory drugs and even masking of bitter taste of orally applied drugs. Technological strategies to achieve these effects are highly diverse and offer various possibilities of liquid, semi-liquid and solid lipid-based formulations for drug delivery optimization.

HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833

The effect of P-glycoprotein inhibition on the uptake of the HIV type 1 protease inhibitor saquinavir into brain capillary endothelial cells was studied using porcine primary brain capillary endothelial cell monolayers as an in vitro test system. As confirmed by polymerase chain reaction and Western blot analysis, this system functionally expressed class I P-glycoprotein (pgp1A). P-Glycoprotein isoforms pgp1B or pgp1D could not be detected. The uptake of saquinavir into endothelial cells could be described as the result of a diffusional term of uptake and an oppositely directed saturable extrusion process. Net uptake of saquinavir into cultured brain endothelial cells could be increased significantly up to 2-fold by SDZ PSC 833 in a dose-dependent manner, with an IC(50) of 1.13 microM. In addition, the HIV protease inhibitor ritonavir inhibited p-glycoprotein-mediated extrusion of saquinavir with an IC(50) of 0.2 microM, indicating a high affinity of ritonavir for p-glycoprotein. In conclusion, we showed that the HIV protease inhibitor ritonavir is a more potent inhibitor of P-glycoprotein than the multidrug resistance (MDR)-reversing agent SDZ PSC 833. The inclusion of this drug in combination regimens may greatly facilitate brain uptake of HIV protease inhibitors, which is especially important in patients suffering from AIDS dementia complex.

Evidence for Different ABC-Transporters in Caco-2 Cells Modulating Drug Uptake

AbstractPurpose. Secretory systems contribute to drug absorption in the gastrointestinal tract. The purpose of this study was the identification of members of the ATP binding cassette superfamily of secretory transport proteins that may potentially modulate drug absorption in Caco-2 cells, which are an important cellular model predicting enteral absorption of drugs. Methods. Kinetic studies as well as PCR- and Western blot studies with confluent epithelial layers of human Caco-2 cells. Results. The study demonstrates functional expression of multidrug resistance related protein (MRP) and P-glycoprotein (P-gp) in Caco-2 cells: 1) Efflux studies with the MRP specific substrate glutathion-methylfluorescein (GS-MF) showed functional activity of MRP in Caco-2 cells preloaded with the metabolic precursor of GS-MF, chloro-methylfluoresceine-diacetate, CMFDA. Excretion of GS-MF was decreased in presence of the MRP-blocker MK-571.2) Transport experiments with cyclosporin A demonstrated the functional activity of P-gp. Cellular accumulation was increased in presence of the P-gp blocking agent SDZ-PSC 833.3) The expression of the 190 kDa protein MRP and the 170 kDa protein P-gp in Caco-2 cells was shown by Western blot analysis with specific monoclonal antibodies. 4) The expression of MRP-mRNA in Caco-2 cells was detected by RT-PCR and compared with the MRP over-expressing cell line H69AR. MRP primers recognize specifically human MRP1 (GenBank accession number L05628), but not all other published sequences of MRP (MRP2-MRP6). P-gp expression on mRNA-level was also confirmed by RT-PCR. Conclusions. The data demonstrate that besides P-gp, multidrug resistance related protein (MRP) is functionally expressed in Caco-2 cells and contributes to the active excretion of substrates in this cell line.

Evidence for P‐glycoprotein‐modulated penetration of morphine‐6‐glucuronide into brain capillary endothelium

1 Morphine‐6‐glucuronide is one of the major metabolites of morphine. The potent analgesic action of this compound together with its potential lower apparent toxicity in man, when compared with morphine, indicated its clinical importance. 2 Primary cultures of porcine brain capillary endothelial cells were used to study brain penetration of morphine‐6‐glucuronide. Biochemical characterization of the cell cultures revealed a marked enrichment in enzymatic activity of alkaline phosphatase (56 fold) and angiotensin converting enzyme (230 fold) as compared to whole brain tissue. By immunostaining the presence of vimentin, factor VIII, the tight junction associated protein ZO‐1, and P‐glycoprotein was shown. Functional characterization revealed that the carrier system responsible for transport of neutral amino acids was intact. 3 Uptake and transport of morphine‐6‐glucuronide was marginal and in the range of the extracellular marker sucrose. However, uptake of morphine‐6‐glucuronide was enhanced significantly (P<0.0001) in presence of the inhibitors of P‐glycoprotein, verapamil or vincristine. The finding that morphine‐6‐glucuronide may serve as a substrate for P‐glycoprotein was confirmed in multidrug‐resistant P388 tumour cells. 4 We conclude that penetration of the blood‐brain barrier by morphine‐6‐glucuronide may depend on the expression of the product of the multidrug‐resistance (MDR) gene in brain capillary endothelial cells.

Intracerebral accumulation of glutaric and 3‐hydroxyglutaric acids secondary to limited flux across the blood–brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl‐CoA dehydrogenase deficiency

Glutaric acid (GA) and 3‐hydroxyglutaric acids (3‐OH‐GA) are key metabolites in glutaryl co‐enzyme A dehydrogenase (GCDH) deficiency and are both considered to be potential neurotoxins. As cerebral concentrations of GA and 3‐OH‐GA have not yet been studied systematically, we investigated the tissue‐specific distribution of these organic acids and glutarylcarnitine in brain, liver, skeletal and heart muscle of Gcdh‐deficient mice as well as in hepatic Gcdh–/– mice and in C57Bl/6 mice following intraperitoneal loading. Furthermore, we determined the flux of GA and 3‐OH‐GA across the blood–brain barrier (BBB) using porcine brain microvessel endothelial cells. Concentrations of GA, 3‐OH‐GA and glutarylcarnitine were significantly elevated in all tissues of Gcdh–/– mice. Strikingly, cerebral concentrations of GA and 3‐OH‐GA were unexpectedly high, reaching similar concentrations as those found in liver. In contrast, cerebral concentrations of these organic acids remained low in hepatic Gcdh–/– mice and after intraperitoneal injection of GA and 3‐OH‐GA. These results suggest limited flux of GA and 3‐OH‐GA across the BBB, which was supported in cultured porcine brain capillary endothelial cells. In conclusion, we propose that an intracerebral de novo synthesis and subsequent trapping of GA and 3‐OH‐GA should be considered as a biochemical risk factor for neurodegeneration in GCDH deficiency.

Modulation of p-glycoprotein transport function at the blood-brain barrier

The central nervous system (CNS) effects of many therapeutic drugs are blunted because of restricted entry into the brain. The basis for this poor permeability is the brain capillary endothelium, which comprises the blood-brain barrier. This tissue exhibits very low paracellular (tight-junctional) permeability and expresses potent, multispecific, drug export pumps. Together, these combine to limit use of pharmacotherapy to treat CNS disorders such as brain cancer and bacterial or viral infections. Of all the xenobiotic efflux pumps highly expressed in brain capillary endothelial cells, p-glycoprotein handles the largest fraction of commonly prescribed drugs and thus is an obvious target for manipulation. Here we review recent studies focused on understanding the mechanisms by which p-glycoprotein activity in the blood-brain barrier can be modulated. These include (i) direct inhibition by specific competitors, (ii) functional modulation, and (iii) transcriptional modulation. Each has the potential to specifically reduce p-glycoprotein function and thus selectively increase brain permeability of p-glycoprotein substrates.

Potent and Selective Inhibitors of Breast Cancer Resistance Protein (ABCG2) Derived from the p-Glycoprotein (ABCB1) Modulator Tariquidar

The efflux pumps ABCB1 (p-gp, MDR1) and ABCG2 (BCRP) are expressed to a high extent by endothelial cells at the blood-brain barrier (BBB) and other barrier tissues and are involved in drug resistance of tumor (stem) cells. Whereas numerous ABCB1 inhibitors are known, only a few ABCG2 modulators with submicromolar activity have been published. Starting from tariquidar (4) analogues as ABCB1 modulators, minimal structural modifications resulted in a drastic shift in favor of ABCG2 inhibition. Highest potency was found when the 3,4-dimethoxy-2-(quinoline-3-carbonylamino)benzoyl moiety in 4 was replaced with a 4-methoxycarbonylbenzoyl moiety bearing a hetarylcarboxamido group in 3-position, e.g., quinoline-3-carboxamido (5, IC(50): 119 nM) or quinoline-2-carboxamido (6, IC(50): 60 nM, flow cytometric mitoxantrone efflux assay, topotecan-resistant MCF-7 breast cancer cells); the selectivity for ABCG2 over ABCB1 was about 100-500 fold and the compounds were inactive at ABCC2 (MRP2). Chemosensitivity assays against MCF-7/Topo cells revealed that the nontoxic inhibitor 6 completely reverted ABCG2-mediated topotecan resistance at concentrations >100 nM, whereas 5 showed ABCG2 independent cytotoxicity. ABCG2 inhibitors might be useful for cancer treatment with respect to reversal of multidrug resistance, overcoming the BBB and targeting of tumor stem cells.