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PK/PD in an Era of Drug Transporters

PK/PD in an Era of Drug Transporters. Paul M. Tulkens & Françoise Van Bambeke Unité de pharmacologie cellulaire et moléculaire Université catholique de Louvain Brussels, Belgium. http://www.md.ucl.ac.be/facm. Discovery of drug transporters. anticancer drugs antibiotics antifungals.

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PK/PD in an Era of Drug Transporters

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  1. PK/PD in an Era of Drug Transporters Paul M. Tulkens & Françoise Van Bambeke Unité de pharmacologie cellulaire et moléculaire Université catholique de Louvain Brussels, Belgium http://www.md.ucl.ac.be/facm ISAP post-ECCMID

  2. Discovery of drug transporters • anticancer drugs • antibiotics • antifungals ISAP post-ECCMID

  3. Anticancer drugs transporters : discovery • Decreased retention of actinomycin D as the basis for cross-resistance in anthracycline-resistant sublines of P388 leukemia. Inaba M & Johnson RK Cancer-Res. 1977 Dec; 37(12): 4629-34 • Active efflux of daunorubicin and adriamycin in sensitive and resistant sublines of P388 leukemia.Inaba M, Kobayashi H, Sakurai Y, Johnson RK, Cancer-Res. 1979 Jun; 39(6 Pt 1): 2200-3 There is an active outward transport mechanism for anthracyclines in P388 leukemia cells and an enhanced activity of this efflux process renders cells highly resistant to the cytostatic and cytotoxic effects of ADR and DAU. ISAP post-ECCMID

  4. Anticancer drugs transporters : inbition by chemically-unrelatedother anticancer drugs • Alteration of methotrexate uptake in human leukemia cells by other agents. Bender RA, Bleyer WA, Frisby SA, Oliverio VT. Cancer-Res. 1975 May; 35: 1305-8 Vincristine sulfate enhanced MTX uptake by inhibiting MTX efflux, thus increasing the level of intracellular drug in excess of the tightly bound fraction. The potential clinical implications of using MTX in commbination with the aforementioned drugs for cancer chemotherapy are discussed. ISAP post-ECCMID

  5. Anticancer drug efflux inhibited by an antibiotic ... The semi-synthetic antibiotic tiamulin, … overpasses the capacities of reference products such as verapamil and cyclosporin-A in terms of in vitro reversal effect [of anthracycline resistance]. In Vitro and In Vivo Reversal of Cancer Cell Multidrug Resistance by the Semi-synthetic Antibiotic Tiamulin. L.G. Baggetto, M. Dong, J. Bernaud, L. Espinosa, D. Rigal, R., & E. Marthinet. Biochem. Pharmacol (1998) 56 1219-1228. ISAP post-ECCMID

  6. Antifungal drugs transporters : discovery • A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins. Nishi-K; Yoshida-M; Nishimura-M; Nishikawa-M; Nishiyama-M; Horinouchi-S; Beppu-T. Mol-Microbiol. 1992 Mar; 6(6): 761-9 • Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Sanglard-D; Kuchler-K; Ischer-F; Pagani-JL; Monod-M; Bille-J. Antimicrob-Agents-Chemother. 1995 Nov; 39(11): 2378-8 Fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. ISAP post-ECCMID

  7. Antifungal drug efflux Molecular mechanisms of azole resistance. Drugs enter the cell through an unknown mechanism (passive diffusion ?). Two types of efflux pumps are expressed at low levels. In a "model" resistant cell, the drugs are less effective because … the azoles are effluxed through overexpression of the CDR (ABCT) and MDR (MF) genes.Adapted from White, T. C. 1997. Antifungal drug resistance in Candida albicans. ASM News 63:427-433 ISAP post-ECCMID

  8. Antibiotic transporters in bacteria : discovery • Plasmid-mediated tetracycline resistance in Escherichia coli involves increased efflux of the antibiotic. Ball-PR; Shales-SW; Chopra-I.Biochem-Biophys-Res-Commun. 1980 Mar 13; 93(1): 74-81 • Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. McMurry-L; Petrucci-RE Jr; Levy-SB. Proc-Natl-Acad-Sci-U-S-A. 1980 Jul; 77(7): 3974-7 The decrease in tretracycline intrabacterial accumulation was attributable to an active efflux ... … Active export of tetracycline is a common component of the mechanism for tetracycline resistance encoded by different plasmid-borne determinants in bacteria. ISAP post-ECCMID

  9. Why drug transporters ? • because cells like to build up specific resistance mechanism ? • because cells have been exposed to drugs in their history ? • because drugs have special properties ? ISAP post-ECCMID

  10. There is much more than drugs ... mid-90’s : The genomic analysis of Saccharomyces cerevisiae and of Pseudomonas aeruginosa reveals a very large number of potential “drug” transporters ISAP post-ECCMID

  11. Inventory of transporters in the complete genomes of S. cerevisiae and E. coli Abundance of proteins of differing predicted membrane topologies nb TMS nb proteins % of total Yeast E. coli Yeast E. coli 0 4364 2861 70.8 66.8 1 937 655 15.3 15.3 2-3 390 220 6.5 5.1 4-6 185 211 3.1 4.9 7-9 144 153 2.3 3.6 > 10 121 82 2.0 4.3 soluble proteins signal peptides potential transport proteins (14 %) Paulsen et al FEBS Lett (1998) 430:116-125 ISAP post-ECCMID

  12. amphiphilicity... There is indeed much more than drugs ... mid-90’s : Proteomic and functional analyses reveals that the potential transporters identified by genomics are responsible for the efflux of a very large variety of substances with a common biophysical property: ISAP post-ECCMID

  13. Why amphiphilic compounds ? ISAP post-ECCMID

  14. Because they are the only ones that can easily pass across membrane bilayers ... ISAP post-ECCMID

  15. Efflux Detoxifying metabolism toxicity Why should cells extrude amphiphilic compounds ? Concerted barrier against invasion ... ISAP post-ECCMID

  16. Oral bioavailability • tissue distribution • penetration in difficult-to-reach compartments, etc... because we selected or designed them to be so…. in order to penetrate cells and tissues ... But why drugs ? Most if not all of our drugs are amphiphilic ... ISAP post-ECCMID

  17. main drug transporters Transporters - data bases http://www-biology.ucsd.edu/~msaier/transport/titlepage.html Classification page combination of phylogenetic and functional information Transport analysis page comparison of transporters in complete genomes Phylogenetic page phylogenetic trees of transporters families ISAP post-ECCMID

  18. + + + - - - + a H b c d + H NH 2 COOH proton antiport Most frequent antibiotic-pumps in procaryotes (1/2) • Resistance Nodulation Division (Gram - ) TOPOLOGY MECHANISM ANTIBIOTICS tetracyclines fluoroquinolones erythromycin rifampicin -lactams fluoroquinolones fusidic acid chloramphenicol aminoglycosides + ISAP post-ECCMID

  19. + + + b c g + d2 H a NH 2 COOH + H b c h d1 e f proton antiport a COOH NH 2 Most frequent antibiotic-pumps in procaryotes (2/2) • Major Facilitator Superfamily (Gram + and - ) TOPOLOGY MECHANISM ANTIBIOTICS 12 TMS tetracyclines fluoroquinolones macrolides lincosamides rifampicin pristinamycin 14 TMS chloramphenicol aminoglycosides + ISAP post-ECCMID

  20. + + + + + + COOH NH 2 ADP + Pi ATP ATP-driven efflux Most frequent antibiotic-pumps in eucaryotes (1/2) • Multiple Drug Resistance (P-gp) TOPOLOGY MECHANISM ANTIBIOTICS tetracyclines fluoroquinolones erythromycin lincosamides rifampicin ? chloramphenicol aminoglycosides + ISAP post-ECCMID

  21. - + - - - - - NH 2 COOH GSH ADP + Pi ATP ATP-driven efflux with GSH as co-factor Most frequent antibiotic-pumps in eucaryotes (2/2) • Multidrug Resistance Proteins (MRP) TOPOLOGY MECHANISM ANTIBIOTICS fluoroquinolones tetracyclines macrolides ? ISAP post-ECCMID

  22. Antibiotic classes recognized by efflux pumps in different types of organisms Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes b-lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones ISAP post-ECCMID

  23. Two probable mechanisms of drug transport by eucaryotic efflux pumps ... • “vacuum cleaner” • “flippases” • the drug never really penetrates the cell • all effects are on the influx ISAP post-ECCMID

  24. Do efflux pumps act as vacuum cleaners ? Structural evidence for P-glycoprotein: Rosenberg et al, JBC (1997) 272:10685-94 ISAP post-ECCMID

  25. the drug gets in the cell, and is sucked from the cyplasmic domain • most effects are on the efflux Two probable mechanisms of drug transport by eucaryotic efflux pumps... • “vacuum cleaner” • “flippases” ISAP post-ECCMID

  26. Do efflux pumps act as flippases ? Functional evidences for MDR & MRP: Translocation of phosphatidylcholine at the apical (closed bars) and basolateral (open bars) membranes of MDR-3 (left) and MRP-1 (right) transfected LLC-PK1cells, in the absence of in the presence of inhibitors of the corresponding pump. Smith et al, J Biol Chem (2000) 275:23530-9 ISAP post-ECCMID

  27. Drugs and physiological substrates for transporters in eucaryotic cells superfamily transporter physiol. antibiotics substrates ABC MDR1 phospholipids fluoroquinolones macrolides b-lactams tetracyclines streptogramins MRP1 phospholipids fluroquinolones leukotrienes macrolides conjugates rifamycins MRP2 conjugates fluoroquinolones b-lactams MFS NPT1 phosphates b-lactams OAT OATP1 bile salts b-lactams steroids ISAP post-ECCMID

  28. Biological significance of antibiotic transport ... • at the level of the bacteria (procaryotes) • multi / cross-resistance • role in emergence of resistance through exposure to subinbitory levels • at the level of the host (eucaryotes) • modulation of transport • modulation of intracellular activity ISAP post-ECCMID

  29. 1 bacteria several pumps multiresistance 1 pump several classes crossresistance of antibiotics 1 class several pumps efficacy of of antibiotics inhibitors ? Efflux and multi / cross-resistance in pathogenic bacteria ISAP post-ECCMID

  30. A certain serum AUC and a certain serum peak will determine the drug concentration at the target level which may prevent the selection of first and second mutation resistants Efflux and selection of resistance to FQ Gyrase/ Topoisomerase ISAP post-ECCMID

  31. Efflux and selection of resistance to FQ Get a AUC ! Get a peak ! Gyrase/ Topoisomerase ISAP post-ECCMID

  32. Oh! Crazy !! Efflux and selection of resistance to FQ Efflux pumps will reduce the concentration at the level of the target and thereby favor the selection of target mutants! Gyrase/ Topoisomerase ISAP post-ECCMID

  33. Selection of mutants in FQ target undetectable if ALL pumps are disrupted Efflux and selection of resistance Frequency of Levofloxacin-resistant mutants in Pseudomonas aeruginosa with deletions of the efflux pump operons Pump status LVX MIC Frequency of LVX- resistant mutants WT 0.25 2 × 107 - 4 × 107  mexAB-oprM 0.015 2 × 107 - 4 × 107  mexCD-oprJ 0.25 2 × 107 - 4 × 107  mexEF-oprN 0.25 2 × 107 - 4 × 107  mexAB-oprM; mexEF-oprN 0.015 2 × 107 - 107  mexCD-oprJ;  mexEF-oprN 0.25 2 × 106  mexAB-oprM;  mexCD-oprJ 0.015 1 × 109  mexAB-oprM;  mexCD-oprJ; 0.015 <1 × 1011  mexEF-oprN Lomovskaya et al, AAC (1999) 43:1340-1346 ISAP post-ECCMID

  34. Biological significance of antibiotic transport ... • at the level of the bacteria (procaryotes) • at the level of the host (eucaryotes) • modulation of tissue pharmacokinetics • modulation of cellular pharmacokinetics ISAP post-ECCMID

  35. Tissue PK : combined action of drug transporters for drug elimination / absorption efflux / reuptake of b-lactams in kidney proximal tubular cell Mrp2 Mct1 Oat1/3 Npt-1 blood urine PepT2 OctN2 Mrp1 distal tubular cell ISAP post-ECCMID

  36. probenecid Tissue PK: combined action of drug transporters for drug elimination / absorption efflux / reuptake of b-lactams in kidney proximal tubular cell cephaloridine cephalexine carbenicilline penicilline G ... Mrp2 Mct1 Oat1/3 Npt-1 cloxacilline blood PepT2 cephalexine urine OctN2 cephaloridine Mrp1 distal tubular cell ISAP post-ECCMID

  37. CONJUGATION CYP GS-D’ D’ D’ D Phase III Phase II Phase I Tissue PK: efflux cooperates with other mechanisms of drug clearance hepatocyte blood bile D Mdr1 Mrp2 Ishikawa, Trends Biochem Sci (1992) 17:463-468. ISAP post-ECCMID

  38. D CYP D’ D Tissue PK: Efflux may also contribute to poor oral absorption enterocyte D lumen blood Mdr1 Mrp2 ISAP post-ECCMID

  39. Cellular PK: ciprofloxacin efflux in macrophages 1. Probenecid (PRB) increases ciprofloxacin accumulation and decreases ciprofloxacin efflux Michot et al., ICAAC 2000 Ce = 50 µM (17µg/ml) 100 PRB 5 mM 10.0 PRB 0 mM 75 7.5 Cc/Ce Residual fraction (%) 50 5.0 25 2.5 0.0 0 Control 5 mM PRB 0 10 20 30 2h incubation at 37°C Time (min) ISAP post-ECCMID

  40. ATP depletion ATP depletion Cellular PK: ciprofloxacin efflux in macrophages 2. ATP depletion also increases ciprofloxacin accumulation and decreases ciprofloxacin efflux Michot et al., ICCAC 2000 Ce = 50 µM (17µg/ml) 10.0 100 Control 7.5 75 Residual fraction (%) Cc/Ce 5.0 50 2.5 25 0 0.0 0 5 10 15 Control Time (min) 2h incubation at 37°C ISAP post-ECCMID

  41. Cellular PK : handling of macrolides macrophages Influence of P-gp inhibitors on azithromycin accumulation (5µg/ml; 3 h) Seral et al., unpublished ISAP post-ECCMID

  42. Inhibitors of transporters Towards the future ... should help you to keep your stuff in ... ISAP post-ECCMID

  43. Elisabetha Balzi • Jean-Michel Michot • Cristina Seral • Hugues Chanteux • Marie-Paule Mingeot-Leclercq • and many others... And thank you to Franco Scaglione for such an excellent meeting !! They contributed to this review and/or gave us unpublished data... ISAP post-ECCMID

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