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Gary M. Pollack - One of the best experts on this subject based on the ideXlab platform.
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Fexofenadine brain exposure and the influence of blood–brain barrier P-glycoprotein after fexofenadine and terfenadine administRation. Drug Metab Dispos 2009; 37: 529–35. M. Farré et al
2016Co-Authors: Rong Zhao, Cory J Kalvass, Souzan B Yanni, Arlene S Bridges, Gary M. PollackAbstract:P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administRation demonstrated that fexofenadine brain penetRation was 48-fold higher in mdr1a(/) mice than inmdr1a(/) mice. In contrast, the P-gp Efflux Ratio at the blood-brain barrier (BBB) for fexofenadine was only 4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp Efflux Ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetRation after terfenadine adminis-tRation was 25- to 27-fold higher than after fexofenadine adminis-tRation in both mdr1a(/) and mdr1a(/) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitr
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fexofenadine brain exposure and the influence of blood brain barrier p glycoprotein after fexofenadine and terfenadine administRation
Drug Metabolism and Disposition, 2009Co-Authors: Rong Zhao, Cory J Kalvass, Souzan B Yanni, Arlene S Bridges, Gary M. PollackAbstract:P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administRation demonstrated that fexofenadine brain penetRation was 48-fold higher in mdr1a(–/–) mice than in mdr1a(+/+) mice. In contrast, the P-gp Efflux Ratio at the blood-brain barrier (BBB) for fexofenadine was only ∼4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp Efflux Ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetRation after terfenadine administRation was ∼25- to 27-fold higher than after fexofenadine administRation in both mdr1a(+/+) and mdr1a(–/–) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitro incubation. The fexofenadine formation rate increased ∼5-fold during a 2-h brain tissue homogenate incubation with hydroxyl-terfenadine, suggesting that the hydroxylation of terfenadine is the rate-limiting step in fexofenadine formation. Moreover, regional brain metabolism seems to be an important factor in terfenadine brain disposition and, consequently, fexofenadine brain exposure. Taken together, these results indicate that the fexofenadine BBB P-gp Efflux Ratio has been underestimated previously due to the lack of complete equilibRation of fexofenadine across the blood-brain interface under typical experimental paradigms.
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Kinetic ConsideRations for the Quantitative Assessment of Efflux Activity and Inhibition: Implications for Understanding and Predicting the Effects of Efflux Inhibition
Pharmaceutical Research, 2007Co-Authors: J. Cory Kalvass, Gary M. PollackAbstract:Purpose Unexpected and complex experimental observations related to Efflux transport have been reported in the literature. This work was conducted to develop relationships for Efflux activity (PS_Efflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), Efflux Ratio (ER), degree of Efflux inhibition (ϕ_i), 50% inhibitory concentRation (IC_50), and Michaelis–Menten constant (K_m)]. Methods A three-compartment model (apical, cellular, and basolateral) was used to derive flux equations relating the initial rate of flux and steady-state mass transfer in the presence or absence of active Efflux. Various definitions of Efflux Ratio (ER) were examined in terms of permeability-surface area products. The Efflux activity (PS_Efflux) was expressed in terms of ER and PS. The relationships between PS_Efflux and PS, ER, ϕ_i, IC_50, and K_m were solved mathematically. Simulations and examples from the literature were used to illustrate the resulting mathematical relationships. Results The relationships derived according to a three-compartment model differed fundamentally from commonly accepted approaches for determining PS_Efflux, ϕ_i, IC_50 and K_m. Based on the model assumptions and mathematical derivations, currently used mathematical relationships erroneously imply that Efflux activity is proportional to change in PS (i.e., flux or P_app) and thus underestimate PS_Efflux and ϕ_i, and overestimate IC_50 and K_m. Conclusions An understanding of the relationship between Efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of Efflux transport, and predicting the clinical significance of Efflux inhibition.
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Use of Plasma and Brain Unbound Fractions to Assess the Extent of Brain Distribution of 34 Drugs: Comparison of Unbound ConcentRation Ratios to in Vivo P-Glycoprotein Efflux Ratios
Drug Metabolism and Disposition, 2007Co-Authors: J. Cory Kalvass, Tristan S. Maurer, Gary M. PollackAbstract:The P-glycoprotein (P-gp)-deficient mouse model is used to assess the influence of P-gp-mediated Efflux on the central nervous system (CNS) distribution of drugs. The steady-state unbound plasma/unbound brain concentRation Ratio ([plasma],u/[brain],u) is an alternative method for assessing CNS distribution of drugs independent of the mechanism(s) involved. The objective of this study was to compare the degree of CNS distributional impairment determined from the in vivo P-gp Efflux Ratio with that determined from the [plasma],u/[brain],u Ratio. CNS distribution of 34 drugs, including opioids, triptans, protease inhibitors, antihistamines, and other clinically relevant drugs with either poor CNS distribution or blood-brain barrier Efflux, was studied. Plasma and brain unbound fractions were determined by equilibrium dialysis. Kp,brain and the P-gp Efflux Ratio were obtained from the literature or determined experimentally. The P-gp Efflux Ratio and the [plasma],u/[brain],u Ratio were in concurrence ( 4-fold) for 10 of the 34 drugs, suggesting that other, non-P-gp-mediated mechanism(s) may limit the CNS distribution of these drugs. The P-gp Efflux Ratio exceeded the [plasma],u/[brain],u Ratio by more than 3-fold for three drugs, suggesting the presence of active uptake mechanism(s). These observations indicate that when mechanisms other than P-gp affect CNS distribution (non-P-gp-mediated Efflux, poor passive permeability, cerebrospinal fluid bulk flow, metabolism, or active uptake), the P-gp Efflux Ratio may underestimate or overestimate CNS distributional impairment. The [plasma],u/[brain],u Ratio provides a simple mechanism-independent alternative for assessing the CNS distribution of drugs.
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Kinetic consideRations for the quantitative assessment of Efflux activity and inhibition: implications for understanding and predicting the effects of Efflux inhibition.
Pharmaceutical research, 2006Co-Authors: J. Cory Kalvass, Gary M. PollackAbstract:Purpose Unexpected and complex experimental observations related to Efflux transport have been reported in the literature. This work was conducted to develop relationships for Efflux activity (PSEfflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), Efflux Ratio (ER), degree of Efflux inhibition (ϕi), 50% inhibitory concentRation (IC50), and Michaelis–Menten constant (Km)].
Patrick J. Sinko - One of the best experts on this subject based on the ideXlab platform.
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membrane transport of camptothecin facilitation by human p glycoprotein abcb1 and multidrug resistance protein 2 abcc2
BMC Medicine, 2004Co-Authors: Anita Lalloo, Feng Roger Luo, Ailan Guo, Pankaj V Paranjpe, Sunghack Lee, Viral Vyas, Eric J Rubin, Patrick J. SinkoAbstract:The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentRation, inhibitors and temperature on CPT directional permeability were determined. The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentRations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent K m values were comparable in all three cell lines. The Efflux Ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the Efflux Ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT Efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family. The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.
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The Effect of Cell Culture Conditions on Saquinavir Transport Through, and Interactions with, MDCKII Cells Overexpressing hMDR1
Journal of pharmaceutical sciences, 2003Co-Authors: Gregory C. Williams, Gregory T. Knipp, Patrick J. SinkoAbstract:Abstract MDCK cells are cultured using wide‐ranging conditions and can produce variable results. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII‐PGP) and MDCKII wild‐type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 × 10 5 or 5 × 10 4 cells/cm 2 ), substratum (polycarbonate ± collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport. The saquinavir Efflux Ratio (Ratio of BL → AP/AP → BL permeability) for MDCKII‐PGP cells (6.9 × 10 5 cells/cm 2 ) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir Efflux Ratios were obtained with 5 × 10 4 cells/cm 2 on polycarbonate (78) or collagen‐coated polycarbonate (126). The MDCKII/wt saquinavir Efflux Ratio was 9. Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen‐coated membranes. Collagen coating caused increased Pgp expression and saquinavir Efflux Ratios correlated ( r 2 = 0.96) with Pgp expression levels [MDCKII‐PGP (on collagen‐coated polycarbonate) > MDCKII‐PGP (on polycarbonate) > MDCKII/wt (on collagen‐coated polycarbonate)]. These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1957–1967, 2003
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Direct Evidence that Saquinavir Is Transported by Multidrug Resistance-Associated Protein (MRP1) and Canalicular Multispecific Organic Anion Transporter (MRP2)
Antimicrobial agents and chemotherapy, 2002Co-Authors: Gregory C. Williams, Gregory T. Knipp, Angela Liu, Patrick J. SinkoAbstract:To determine if saquinavir mesylate (saquinavir) is a substrate of human multidrug resistance-associated protein 1 (hMRP1 [ABCC1]) or hMRP2 (cMOAT, or ABCC2), MDCKII cells that overexpress either hMRP1 (MDCKII-MRP1) or hMRP2 (MDCKII-MRP2) were used to investigate saquinavir's cytotoxicity and transport in comparison with those of control MDCKII wild-type (MDCKII/wt) cells. Cytotoxicity was assessed with the mitochondrial marker MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium], and saquinavir transport was measured directly through the cell monolayers. GF120918 (an inhibitor of P glycoprotein, but not of the MRP family) and MK-571 (an MRP family inhibitor) were used to delineate the specific contributions of these transporters to saquinavir cytotoxicity and transport. In the presence of GF120918 and increasing saquinavir concentRations, the MDCKII-MRP1 (50% lethal dose [LD50] = 10.5 μM) and MDCKII-MRP2 (LD50 = 27.1 μM) cell lines exhibited statistically greater viability than the MDCKII/wt cells (LD50 = 7.8 μM). Saquinavir Efflux was directional, not saturable, and was inhibited by MK-571 (35 and 75 μM) in all cell lines. The Ratios of saquinavir (3 μM) basolateral to apical permeability (i.e., Efflux Ratios) for the MDCKII/wt, MDCKII-MRP1, and MDCKII-MRP2 cell monolayers were 2.6, 1.8, and 6.8, respectively. The MDCKII-MRP1 cells have a significantly reduced saquinavir Efflux Ratio relative to MDCKII/wt cells, due to basolaterally directed transport by hMRP1 competing with endogenous, apically directed canine MRP2. The MDCKII-MRP2 cells have a significantly increased saquinavir Efflux Ratio relative to MDCKII/wt cells, due to the additive effects of the apically directed transport by hMRP2 and endogenous MRP2. Collectively, the cytotoxicity and transport results provide direct evidence that saquinavir is transported by MRP1 and MRP2.
J. Cory Kalvass - One of the best experts on this subject based on the ideXlab platform.
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Kinetic ConsideRations for the Quantitative Assessment of Efflux Activity and Inhibition: Implications for Understanding and Predicting the Effects of Efflux Inhibition
Pharmaceutical Research, 2007Co-Authors: J. Cory Kalvass, Gary M. PollackAbstract:Purpose Unexpected and complex experimental observations related to Efflux transport have been reported in the literature. This work was conducted to develop relationships for Efflux activity (PS_Efflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), Efflux Ratio (ER), degree of Efflux inhibition (ϕ_i), 50% inhibitory concentRation (IC_50), and Michaelis–Menten constant (K_m)]. Methods A three-compartment model (apical, cellular, and basolateral) was used to derive flux equations relating the initial rate of flux and steady-state mass transfer in the presence or absence of active Efflux. Various definitions of Efflux Ratio (ER) were examined in terms of permeability-surface area products. The Efflux activity (PS_Efflux) was expressed in terms of ER and PS. The relationships between PS_Efflux and PS, ER, ϕ_i, IC_50, and K_m were solved mathematically. Simulations and examples from the literature were used to illustrate the resulting mathematical relationships. Results The relationships derived according to a three-compartment model differed fundamentally from commonly accepted approaches for determining PS_Efflux, ϕ_i, IC_50 and K_m. Based on the model assumptions and mathematical derivations, currently used mathematical relationships erroneously imply that Efflux activity is proportional to change in PS (i.e., flux or P_app) and thus underestimate PS_Efflux and ϕ_i, and overestimate IC_50 and K_m. Conclusions An understanding of the relationship between Efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of Efflux transport, and predicting the clinical significance of Efflux inhibition.
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Use of Plasma and Brain Unbound Fractions to Assess the Extent of Brain Distribution of 34 Drugs: Comparison of Unbound ConcentRation Ratios to in Vivo P-Glycoprotein Efflux Ratios
Drug Metabolism and Disposition, 2007Co-Authors: J. Cory Kalvass, Tristan S. Maurer, Gary M. PollackAbstract:The P-glycoprotein (P-gp)-deficient mouse model is used to assess the influence of P-gp-mediated Efflux on the central nervous system (CNS) distribution of drugs. The steady-state unbound plasma/unbound brain concentRation Ratio ([plasma],u/[brain],u) is an alternative method for assessing CNS distribution of drugs independent of the mechanism(s) involved. The objective of this study was to compare the degree of CNS distributional impairment determined from the in vivo P-gp Efflux Ratio with that determined from the [plasma],u/[brain],u Ratio. CNS distribution of 34 drugs, including opioids, triptans, protease inhibitors, antihistamines, and other clinically relevant drugs with either poor CNS distribution or blood-brain barrier Efflux, was studied. Plasma and brain unbound fractions were determined by equilibrium dialysis. Kp,brain and the P-gp Efflux Ratio were obtained from the literature or determined experimentally. The P-gp Efflux Ratio and the [plasma],u/[brain],u Ratio were in concurrence ( 4-fold) for 10 of the 34 drugs, suggesting that other, non-P-gp-mediated mechanism(s) may limit the CNS distribution of these drugs. The P-gp Efflux Ratio exceeded the [plasma],u/[brain],u Ratio by more than 3-fold for three drugs, suggesting the presence of active uptake mechanism(s). These observations indicate that when mechanisms other than P-gp affect CNS distribution (non-P-gp-mediated Efflux, poor passive permeability, cerebrospinal fluid bulk flow, metabolism, or active uptake), the P-gp Efflux Ratio may underestimate or overestimate CNS distributional impairment. The [plasma],u/[brain],u Ratio provides a simple mechanism-independent alternative for assessing the CNS distribution of drugs.
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Kinetic consideRations for the quantitative assessment of Efflux activity and inhibition: implications for understanding and predicting the effects of Efflux inhibition.
Pharmaceutical research, 2006Co-Authors: J. Cory Kalvass, Gary M. PollackAbstract:Purpose Unexpected and complex experimental observations related to Efflux transport have been reported in the literature. This work was conducted to develop relationships for Efflux activity (PSEfflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), Efflux Ratio (ER), degree of Efflux inhibition (ϕi), 50% inhibitory concentRation (IC50), and Michaelis–Menten constant (Km)].
Richard D. Caldwell - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists.
Bioorganic & medicinal chemistry letters, 2019Co-Authors: Yufang Xiao, Andreas Goutopoulos, Karra Srinivasa R, Noune Tahmassian Morse, Susan Zhang, Mohanraj Dhanabal, Hui Tian, Jeyaprakashnarayanan Seenisamy, Jayadevan Jayashankaran, Richard D. CaldwellAbstract:Abstract The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1β) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low Efflux Ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (Kp,uu of 0.37).
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Optimization of the Efflux Ratio and permeability of covalent irreversible BTK inhibitors.
Bioorganic & medicinal chemistry letters, 2018Co-Authors: Hui Qiu, Lesley Liu-bujalski, Richard D. Caldwell, Ariele Viacava Follis, Anna Gardberg, Andreas Goutopoulos, Roland Grenningloh, Jared Head, Theresa L. Johnson, Christopher Charles Victor JonesAbstract:Abstract Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated Efflux Ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in Efflux Ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.
Peter Krajcsi - One of the best experts on this subject based on the ideXlab platform.
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Abcb1a (P-glycoprotein) limits brain exposure of the anticancer drug candidate seliciclib in vivo in adult mice
Brain Research Bulletin, 2017Co-Authors: Franciska Erdő, Ildikó Nagy, Beáta Tóth, Annamária Bui, Éva D. Molnár, Zoltán Tímár, Rémi Magnan, Peter KrajcsiAbstract:Seliciclib displayed limited brain exposure in vivo in adult rats with mature blood-brain barrier (BBB). Selicilib was shown to be a specific substrate of human ABCB1 in vitro. To demonstrate that ABCB1/Abcb1 can limit brain exposure in vivo in mice we are showing that seliciclib is a substrate of mouse Abcb1a, the murine ABCB1 ortholog expressed in the BBB as LLC-PK-Abcb1a cells displayed an Efflux Ratio (ER) of 15.31±3.54 versus an ER of 1.44±0.10 in LLC-PK1-mock cells. Additionally, in the presence of LY335979, an ABCB1/Abcb1a specific inhibitor, the observed ER for seliciclib in the LLC-PK1-mMdr1a cells decreased to 1.05±0.25. To demonstrate in vivo relevance of seliciclib transport by Abcb1a mouse brain microdialysis experiments were carried out that showed that the AUCbrain/AUCblood Ratio of 0.143 in anesthetized mice increased about two-fold to 0.279 in the presence of PSC833 another ABCB1/Abcb1a specific inhibitor. PSC833 also increased the brain exposure (AUCbrain) of seliciclib close to 2-fold (136 vs 242) in awake mice. In sum, Abcb1a significantly decreases seliciclib permeability in vitro and is partly responsible for limited brain exposure of seliciclib in vivo in mice.
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ATP-binding cassette B1 transports seliciclib (R-roscovitine), a cyclin-dependent kinase inhibitor.
Drug Metabolism and Disposition, 2010Co-Authors: Zsuzsanna Rajnai, Francis Lévi, Dóra Méhn, Erzsébet Beéry, Alper Okyar, Márton Jani, Gábor K. Tóth, Ferenc Fülöp, Peter KrajcsiAbstract:Seliciclib, a cyclin-dependent kinase inhibitor, is a promising candidate to treat a variety of cancers. Pharmacokinetic studies have shown high oral bioavailability but limited brain exposure to the drug. This study shows that seliciclib is a high-affinity substrate of ATP-binding cassette B1 (ABCB1) because it activates the ATPase activity of the transporter with an EC50 of 4.2 μM and shows vectorial transport in MDCKII-MDR1 cells, yielding an Efflux Ratio of 8. This interaction may be behind the drug9s limited penetRation of the blood-brain barrier. ABCB1 overexpression, on the other hand, does not confer resistance to the drug in the models tested. These findings should be considered when treatment strategies using seliciclib are designed.
