Special Considerations for Individual Biomarkers P gp

Special Considerations for Individual Biomarkers: P-gp Deanna L. Kroetz, Ph.D. Department of Biopharmaceutical Sciences University of California San Francisco Role of Transporters in Drug Response • Absorption and efflux across the gastrointestinal tract • Biliary excretion • Urinary excretion • Movement across the blood-brain and blood-testes border • Uptake into target cells • Target for many commonly prescribed drugs P-glycoprotein Functions as a Drug Efflux Pump Apical side ATP ADP + P042- ATP ADP + P042- ATP ADP + P042- Basal side 1 Drugs Which Interact with P-glycoprotein Substrates Actinomycin D Colchicine Docetaxel Doxorubicin Etoposide Mitomycin C Digoxin Fexofenadine Mitoxantrone Paclitaxel Teniposide Topotecan Cortisol Morphine Loperamide Ivermectin Substrates/Inhibitors Daunorubicin Erythromycin Itraconazole Saquinavir Celiprolol Verapamil Gramicidin D Diltiazem Indinavir Nelfinavir Vinblastine Vincristine Terfenadine Inhibitors Ketoconazole Progesterone Nifedipine Midazolam Testosterone Inducers Dexamethasone Rifampin St. John’s Wort P-glycoprotein in Cancer • Intrinsic overexpression - colon, kidney, adrenal, hepatic, and pancreatic cancers and in some leukemias. • Acquired overexpression subsequent to chemotherapy - breast tumors, ALL, AML, sarcomas and multiple myeloma. • Increased drug efflux from the tumor cells is one mechanism of resistance to multiple structurally diverse chemotherapeutic agents. P-glycoprotein Expression in Normal Human Tissues Blood brain barrier Adrenal gland Liver Kidney Pancreas Colon Small Intestine Maternal -fetal barrier Lymphocytes 2 ABCB1 (MDR1) Variants 48 Variants 5 non-coding 19 coding 24 intronic 7 synonymous 12 non-synonymous 3 allele frequency ≥1% 7 allele frequency ≥1% Common ABCB1 Haplotypes Ethnic Variation in Haplotype Structure Caucasians African Americans 24 haplotypes 52 haplotypes 3 Major ABCB1 Haplotypes A61G G(-25)T Intron 4 A(-44)G Intron 9 C1236T C(+44)T Intron 12 C(+24)T Intron 13 A(+38)G Intron 14 G(+24)A Intron 20 G2677T C3435T *1 *13 *26 *21 *11 *14 Ethnic Variation in Haplotype Structure 40 Population Frequency (%) 35 30 25 20 15 10 5 0 *1 *13 *26 *21 *11 *14 CA AA AS ME PA MDR1 Haplotype Coding Changes in Haplotypes Reference Ser893 Other AAs 35 Haplotypes 60% of chromosomes 11 Haplotypes 30% of chromosomes 18 Haplotypes 10% of chromosomes 4 P-gp Polymorphisms Ala893Ser Variant Has no Functional Phenotype with Calcein-AM 250 200 150 100 50 0 vector Ala893 CGC Ser893 TTT control +GF120918 Ala893Ser Variant Has No Functional Phenotype with Rhodamine Ala893 8 6 4 2 0 Median Calcein Fluroescence (% Control) Rhodamine123 Fluorescence Accumulation:Efflux Ser893 vector TGT TGC TTT TTC MDR1 1236/2677/3435 Genotype 5 No Effect of P-gp Polymorphisms on Calcein-AM Transport Intracellular Calcein Fluorescence (relative to NBD mutant) 125 100 75 50 25 0 S4 00 N R6 69 C A8 93 S A8 93 T S1 14 1T V1 25 1I N A8 21D 93 + S calcein AM + GF120918 Functional Consequences of P-glycoprotein Variants Baculovirus Expression • Variants – – – – – Asn21Asp Phe103Leu Ser400Asn Ala893Ser Ala893Thr NB D re mu fe t re nc e A8 93 S N2 1D calcein AM • Substrates – – – – – – – – Bodipy-FL-forskolin Bodipy-FL-paclitaxel Bodipy-FL-verapamil Bodipy-FL-vinblastine Bodipy-FL-prazosin Daunorubicin Calcein-AM Bisantrene No functional differences between reference and variant P-glycoproteins Kimchi-Sarfaty et al. Mol. Pharmacol. 62: 1 -6, 2002 No Functional Effect of Common MDR1 Haplotypes Stable LLC-PK1 cell lines using Flp-In® 2677 A A G G T T 3435 C T C T C T Thr893 Thr893 Ala893 (ref) Ala893 Ser893 Ser893 No differences in transport of verapamil, digoxin, vinblastine or cyclosporin A Morita et al. Biochem. Pharmacol. 65:1843-1852, 2003 6 P-glycoprotein Ser893 Variant has Increased Digoxin Transport Digoxin Content (nM/0.5 x 106 cells) 20000 15000 10000 5000 0 Untransfected P-gp Ala893 P-gp Ser893 Kim et al. Clin Pharmacol Ther 2001; 70:189-199 Clinical Significance of P-gp Polymorphisms Bioavailability CNS Exposure Drug Efficacy and Toxicity Tumor Resistance Correlation Between In Vitro and In Vivo Transporter Function??? Correlation Between Genotype and Phenotype??? 7 C3435T Variation is Associated with Decreased Intestinal P-gp and Increased Digoxin Absorption 2500 Intestinal P-gp Level (OD) 2000 1500 1000 500 0 T/T C/T C/C MDR1 3435 Genotype Hoffmeyer et al. PNAS 97:3473-3478, 2000 3.5 Digoxin C max (mg/L) 3 2.5 2 1.5 1 0.5 0 C/C T/T MDR1 3435 Genotype * C3435T ABCB1 Variant and Digoxin Absorption • Increased absorption – Hoffmeyer et al. 2000 – Kurata et al. 2002 (2677+3435) – Verstuyft et al. (2003) • Decreased absorption – Sakaeda et al. 2001 – Horinouchi et al. 2002 (2677+3435) • No effect – Becquemont 2001 – Gerloff et al. 2002 – Johne et al. 2002 (2677+3435) Effect of C3435T ABCB1 Variant on Pharmacokinetics • Fexofenadine – êAUC (1236+2677+3435) – Kim et al. 2001 – No change – Drescher et al. 2002 • Antiretrovirals – êMedian drug levels and CD4 cell count – Fellay et al. 2002 – No effect – Brumme et al. 2003, Winzer et al. 2003 • ,Cyclosporine – No effect – von Ashen et al. 2001, Min et al. 2002 – No effect of 1236/2677/3435 haplotype – Anglicheau et al. 2004, Haufroid et al. 2004 – No effect of 2677/3435 haplotype – Mai et al. 2003 – ↑Conc/Dose ratio with 3435TT – Bonhomme-Favre et al. 2004 8 Effect of C3435T ABCB1 Variant on Pharmacokinetics • Tacrolimus – No effect – MacPhee et al. 2002, Goto et al. 2002 – 2677/3435 haplotype associated with dose requirement – Anglicheau et al. 2003 • • • Talinolol – No effect – Siegmund et al. 2002 Risperidone – No effect of 2677/3435 haplotype – Yasui-Furukori et al. 2004 – ↓ Reduction in LDL and in ↑ elevation in HDL – Kajinami et al. 2004 Atorvastatin Effect of ABCB1 Variation on Cancer Phenotype • 2677T and 3435T genotypes overexpressed in colorectal tumors with high microsatellite instability – Potocnik et al. 2002 • 1236CC/2677GG/3435CC associated with ê overall survival and é probability of relapse in adult AML patients – Illmer et al. 2002 • 3435TT genotype overexpressed in renal cell carcinomas – Siegsmund et al. 2002 • 3435 genotype not related to drug resistance or prognosis in ALL patients – Efferth et al. 2003 Effect of ABCB1 Variation on Blood-Brain Barrier Function • Loperamide – Pauli-Magnus et al. 2003 • 3435 not associated with respiratory depression • Nortriptyline – Roberts et al. 2002 – 3435TT increased risk of postural hypotension • Tacrolimus – Yamauchi et al. 2002 – 2677 genotype positive predictor of neurotoxicity – 3435 genotype negative predictor of neurotoxicity • Parkinson’s disease – Furono et al. 2002 – Increased frequency of 3435TT genotype in earlyonset Parkinson’s disease patients 9 What Alleles Should be Genotyped? • Intronic – Intron – Intron – Intron – Intron – Intron 4 –G(-25)T 12 – C(+44)T 13 – C(+24)T 14 – A(+38)G 20 - G(+24)A • UTR – T-129C • Coding – – – – – A61G (Asn21Asp) C1236T G2677T/A (Ala893Ser/Thr) C3435T T3421A (Ser1141Thr) – African American specific Are There P-gp Poor Transporters??? SLCO1B1 (OATP1B1) • OATP1B1 (OATP-C) is an organic anion transporter highly expressed on the basolateral membrane of hepatocytes • Substrates include bile salts, conjugated and unconjugated bilirubin, steroid conjugates, methotrexate, pravastatin and rifampin 10 Genetic Polymorphisms in SLCO1B1 • At least 6 non-synonymous and two promoter SNPs have been identified • T521C (Val174Ala) and G1463C (Gly488Ala) have reduced function in vitro • A388G (Asn130Asp)/T521A (Val174Ala) haplotype associated with significant reductions in pravastatin CL NR Tirona et al. 2001, Nozawa et al. 2002, Nishizato et al. 2003, Niemi et al. 2004, Mwinyi et al. 2004 Acknowledgments Christiane Pauli-Magnus Trine Lillehammer Catherine Lacayo Laura Hodges Leslie Chinn Thy Giang Tan Nguyen Jason Gow Kathy Giacomini Ira Herskowitz Claire Brett Andy Clark Emil Lin John Feiner Bill Hyun Robert Black Charitable Foundation NIH GM61390 www.pharmgkb.org 11