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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- Milan, It., 27-28 April 1 Discovery of drug transporters • anticancer drugs • antibiotics • antifungals ISAP post- Milan, It., 27-28 April 2 Anticancer drugs transporters : discovery • Decreased retention of actinomycin D as the basis for crossresistance 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- Milan, It., 27-28 April 3 Anticancer drugs transporters : inbition by chemically-unrelated other 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- Milan, It., 27-28 April 4 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 Semisynthetic Antibiotic Tiamulin. L.G. Baggetto, M. Dong, J. Bernaud, L. Espinosa, D. Rigal, R., & E. Marthinet. Biochem. Pharmacol (1998) 56 12191228. ISAP post- Milan, It., 27-28 April 5 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. AntimicrobAgents-Chemother. 1995 Nov; 39(11): 2378-8 Fluconazole-resistant C. albicans isolates failed to accumulate 3Hlabelled 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- Milan, It., 27-28 April 6 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- Milan, It., 27-28 April 7 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. McMurryL; 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- Milan, It., 27-28 April 8 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- Milan, It., 27-28 April 9 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- Milan, It., 27-28 April 10 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 Yeast E. coli % of total Yeast E. coli 0 4364 2861 70.8 66.8 soluble proteins 1 937 655 15.3 15.3 signal peptides 2-3 4-6 7-9 > 10 390 185 144 121 220 211 153 82 6.5 3.1 2.3 2.0 5.1 4.9 3.6 4.3 potential transport proteins (14 %) Paulsen et al FEBS Lett (1998) 430:116-125 ISAP post- Milan, It., 27-28 April 11 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: amphiphilicity... ISAP post- Milan, It., 27-28 April 12 Why amphiphilic compounds ? ISAP post- Milan, It., 27-28 April 13 Because they are the only ones that can easily pass across membrane bilayers ... ISAP post- Milan, It., 27-28 April 14 Why should cells extrude amphiphilic compounds ? Efflux Detoxifying metabolism toxicity Concerted barrier against invasion ... ISAP post- Milan, It., 27-28 April 15 But why drugs ? Most if not all of our drugs are amphiphilic ... because we selected or designed them to be so…. in order to penetrate cells and tissues ... ISAP post- Milan, It., 27-28 April • Oral bioavailability • tissue distribution • penetration in difficult-toreach compartments, etc... 16 Transporters - data bases http://www-biology.ucsd.edu/~msaier/transport/titlepage.html main drug transporters Classification page Transport analysis page Phylogenetic page ISAP post- Milan, It., 27-28 April combination of phylogenetic and functional information comparison of transporters in complete genomes phylogenetic trees of transporters families 17 Most frequent antibiotic-pumps in procaryotes (1/2) • Resistance Nodulation Division (Gram - ) TOPOLOGY MECHANISM ANTIBIOTICS tetracyclines fluoroquinolones erythromycin rifampicin H+ a b -lactams fluoroquinolones fusidic acid c H+ d NH 2 chloramphenicol COOH proton antiport ISAP post- Milan, It., 27-28 April + aminoglycosides 18 Most frequent antibiotic-pumps in procaryotes (2/2) • Major Facilitator Superfamily (Gram + and - ) TOPOLOGY MECHANISM ANTIBIOTICS 12 TMS b d2 c g tetracyclines fluoroquinolones macrolides lincosamides rifampicin pristinamycin H+ NH2 a COOH 14 TMS c d1 chloramphenicol H+ b h e + f a COOH NH2 ISAP post- Milan, It., 27-28 April aminoglycosides proton antiport 19 Most frequent antibiotic-pumps in eucaryotes (1/2) • Multiple Drug Resistance (P-gp) TOPOLOGY MECHANISM ANTIBIOTICS tetracyclines fluoroquinolones erythromycin lincosamides rifampicin ? COOH NH2 ATP ATP-driven efflux ISAP post- Milan, It., 27-28 April chloramphenicol ADP + Pi + aminoglycosides 20 Most frequent antibiotic-pumps in eucaryotes (2/2) • Multidrug Resistance Proteins (MRP) TOPOLOGY MECHANISM ANTIBIOTICS fluoroquinolones NH2 tetracyclines macrolides COOH ? GSH ATP ADP + Pi ATP-driven efflux with GSH as co-factor ISAP post- Milan, It., 27-28 April 21 Antibiotic classes recognized by efflux pumps in different types of organisms Antibiotic class bacteria Gram (+) Gram(-) fungi superior eucaryotes -lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones ISAP post- Milan, It., 27-28 April 22 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- Milan, It., 27-28 April 23 Do efflux pumps act as vacuum cleaners ? Structural evidence for P-glycoprotein: Rosenberg et al, JBC (1997) 272:10685-94 ISAP post- Milan, It., 27-28 April 24 Two probable mechanisms of drug transport by eucaryotic efflux pumps... • “vacuum cleaner” • “flippases” • the drug gets in the cell, and is sucked from the cyplasmic domain • most effects are on the efflux ISAP post- Milan, It., 27-28 April 25 Do efflux pumps act as flippases ? Functional evidences for MDR & MRP: Smith et al, J Biol Chem (2000) 275:23530-9 ISAP post- Milan, It., 27-28 April Translocation of phosphatidylcholine at the apical (closed bars) and basolateral (open bars) membranes of MDR-3 (left) and MRP-1 (right) transfected LLCPK1cells, in the absence of in the presence of inhibitors of the corresponding pump. 26 Drugs and physiological substrates for transporters in eucaryotic cells superfamily transporter ABC MDR1 phospholipids MRP1 phospholipids leukotrienes conjugates conjugates MRP2 MFS OAT NPT1 OATP1 ISAP post- Milan, It., 27-28 April physiol. antibiotics substrates phosphates bile salts steroids fluoroquinolones macrolides -lactams tetracyclines streptogramins fluroquinolones macrolides rifamycins fluoroquinolones -lactams -lactams -lactams 27 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- Milan, It., 27-28 April 28 Efflux and multi / cross-resistance in pathogenic bacteria 1 bacteria several pumps multiresistance 1 pump several classes of antibiotics crossresistance 1 class of antibiotics several pumps efficacy of inhibitors ? ISAP post- Milan, It., 27-28 April 29 Efflux and selection of resistance to FQ 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 Gyrase/ Topoisomerase ISAP post- Milan, It., 27-28 April 30 Efflux and selection of resistance to FQ Get a AUC ! Get a peak ! Gyrase/ Topoisomerase ISAP post- Milan, It., 27-28 April 31 Efflux and selection of resistance to FQ Oh! Efflux pumps will reduce the concentration at the level of the target and thereby favor the selection of target mutants! Crazy !! Gyrase/ Topoisomerase ISAP post- Milan, It., 27-28 April 32 Efflux and selection of resistance Frequency of Levofloxacin-resistant mutants in Pseudomonas aeruginosa with deletions of the efflux pump operons Pump status WT mexAB-oprM mexCD-oprJ mexEF-oprN mexAB-oprM; mexEF-oprN mexCD-oprJ; mexEF-oprN mexAB-oprM; mexCD-oprJ mexAB-oprM; mexCD-oprJ; mexEF-oprN Lomovskaya et al, AAC (1999) 43:1340-1346 ISAP post- Milan, It., 27-28 April LVX MIC Frequency of LVXresistant mutants 0.25 0.015 0.25 0.25 0.015 0.25 0.015 0.015 2 × 107 - 4 × 107 2 × 107 - 4 × 107 2 × 107 - 4 × 107 2 × 107 - 4 × 107 2 × 107 - 107 2 × 106 1 × 109 <1 × 1011 Selection of mutants in FQ target undetectable if ALL pumps are disrupted 33 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- Milan, It., 27-28 April 34 Tissue PK : combined action of drug transporters for drug elimination / absorption efflux / reuptake of -lactams in kidney proximal tubular cell urine Mrp2 Npt-1 Mct1 Oat1/3 blood PepT2 OctN2 Mrp1 distal tubular cell ISAP post- Milan, It., 27-28 April 35 Tissue PK: combined action of drug transporters for drug elimination / absorption efflux / reuptake of -lactams in kidney proximal tubular cell Mrp2 cloxacilline Npt-1 urine cephalexine cephaloridine Mct1 Oat1/3 PepT2 OctN2 cephaloridine cephalexine carbenicilline bloodG penicilline ... probenecid Mrp1 distal tubular cell ISAP post- Milan, It., 27-28 April 36 Tissue PK: efflux cooperates with other mechanisms of drug clearance hepatocyte CONJUGATION CYP bile GS-D’ D’ D’ D Phase II Phase I blood D Mdr1 Mrp2 Phase III Ishikawa, Trends Biochem Sci (1992) 17:463-468. ISAP post- Milan, It., 27-28 April 37 Tissue PK: Efflux may also contribute to poor oral absorption enterocyte D D CYP lumen blood D’ D Mdr1 Mrp2 ISAP post- Milan, It., 27-28 April 38 Cellular PK: ciprofloxacin efflux in macrophages 1. Probenecid (PRB) increases ciprofloxacin accumulation and decreases ciprofloxacin efflux Michot et al., ICAAC 2000 Residual fraction (%) Ce = 50 µM (17µg/ml) Cc/Ce 10.0 7.5 5.0 2.5 0.0 Control 5 mM PRB 2h incubation at 37°C ISAP post- Milan, It., 27-28 April 100 PRB 5 mM PRB 0 mM 75 50 25 0 0 10 20 30 Time (min) 39 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) Residual fraction (%) 10.0 Cc/Ce 7.5 5.0 2.5 75 50 25 0 0.0 Control ATP depletion 2h incubation at 37°C ISAP post- ATP depletion Control 100 Milan, It., 27-28 April 0 5 10 15 Time (min) 40 Cellular PK : handling of macrolides macrophages Influence of P-gp inhibitors on azithromycin accumulation (5µg/ml; 3 h) Seral et al., unpublished ISAP post- Milan, It., 27-28 April 41 Towards the future ... Inhibitors of transporters should help you to keep your stuff in ... ISAP post- Milan, It., 27-28 April 42 They contributed to this review and/or gave us unpublished data... • • • • • • 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 !! ISAP post- Milan, It., 27-28 April 43