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Strategies for in vitro Transporter Testing and Alignment with FDA Guidance Elke S. Perloff, Ph.D. BD Biosciences – Discovery Labware www.bdbiosciences.com/admetox Presentation Overview • Guidance documents and position papers • Transporter expression and function – Role in drug-drug interactions – In vitro models to study transporter interactions • Focus on assessment of P-glycoprotein (P-gp) Interactions – Identification of P-gp substrates and inhibitors – Technical considerations (cell lines, positive controls, assay conditions, parameter calculations) – Decision trees Guidance Documents and Position Papers Guidance and Position Papers • FDA DRAFT Guidance for Industry (Sept 2006) Drug Interaction Studies – Study Design, Data Analysis and Implications for Dosing and Labeling (http://www.fda.gov/cder/guidance/index.htm) • Tucker, et al. Optimizing drug development: strategies to assess drug metabolism/ transporter interaction potential – toward a consensus. Basel Conference 2001, sponsored by FDA, EUFEPS, AAPS: Clin. Pharmacol. Ther. 70:103-14 (2001) Br. J. Clin. Pharmacol. 52:107-17 (2001) Eur. J. Pharm. Sci. 13:417-28 (2001) Pharm. Res. 18:1071-80 (2001) • Zhang, et al. Scientific Perspectives on Drug Transporters and Their Role in Drug Interactions. Mol. Pharm. 3:62-69 (2006) • Zhang, et al. A Regulatory Viewpoint on Transporter-based Drug Interactions. Xenobiotica 38:709-724 (2008) • Huang, et al. New Era in Drug Interaction Evaluation: US FDA Update on CYP Enzymes, Transporters, and the Guidance Process. J. Clin. Pharmacol. 48:662-670 (2008) FDA Guidance Documents • Over 400 draft or final guidance documents http://www.fda.gov/cder/guidance/index.htm • Represent the Agency's current thinking • Do not bind the FDA or the public, but provide pharmaceutical companies with assurance – An alternative approach may be used if it satisfies requirements of any applicable statutes, or regulations – If in doubt, contact the originating office (e.g. CDER) Transporter Expression and Function Role in drug-drug interactions In vitro models to study transporter interactions Transporters • Membrane-bound proteins with asymmetric distribution in polarized cells of various tissues – e.g., intestinal enterocytes, hepatocytes, proximal tubules, blood-brain barrier capillary endothelial cells – Function as uptake and efflux pumps – Transport a variety of solutes: nutrients, cellular by-products, environmental toxins and drugs into and out of cells • Estimate: > 400 human transporters • Active (ATP-dependent, Na+ or H+ gradient driven) or passive (concentration gradient driven) transport mechanisms Major Human Transporters (ABC) Gene ABCB1 Aliases P-gp, MDR1 Tissue Drug Substrate intestine, liver, kidney, brain, digoxin, fexofenadine, placenta, adrenal, testes indinavir, vincristine, colchicine, topotecan, paclitaxel ABCB4 MDR3 liver ABCB11 ABCC1 BSEP MRP1 ABCC2 MRP2, CMOAT MRP3, CMOAT2 MRP4 MRP5 MRP6 BCRP ABCC3 ABCC4 ABCC5 ABCC6 ABCG2 Inhibitor ritonavir, cyclosporine, verapamil, erythromycin, ketocoanzole, itraconazole, quinidine, elacridar (GF120918) LY335979, valspodar (PSC833) digoxin, paclitaxel, vinblastine liver vinblastine intestine, liver, kidney, brain adefovir, indinavir intestine, liver, kidney, brain indinavir, cisplatin, intestine, liver, kidney, placenta, adrenal etoposide, methotrexate, tenoposide liver, kidney intestine, liver, breast, placenta cisplatin, daunorubicin daunorubicin, doxorubicin, topotecan, rosuvastatin, sulfasalazine FDA DRAFT Guidance for Industry (Sept 2006) cyclosporine elacridar (GF120918), gefitinib Major Human Transporters (SLC) Gene SLCO1B1 SLCO1B3 SLCO2B1 SLC10A1 SLC10A2 SLC15A1 Aliases OATP1B1, OATP-C OATP2 OATP1B3, OATP8, SLC21A9, OATP-B NTCP ASBT PEPT1 Tissue liver intestine, liver, kidney, brain liver, pancreas ileum, kidney, biliary tract intestine, kidney SLC15A2 PEPT2 kidney SLC22A1 OCT1 liver SLC22A2 OCT2 kidney, brain SLC22A3 OCT3 SLC22A4 OCTN1 SLC22A5 OCTN2 SLC22A6 OAT1 SLC22A7 SLC22A8 OAT2 OAT3 liver Drug Substrate rifampin, rosuvastatin, methotrexate, pravastatin, thyroxine digoxin, methotrexate, rifampin, pravastatin rosuvastatin ampicillin, amoxicillin, captopril, valacyclovir ampicillin, amoxicillin, captopril, valacyclovir acyclovir, amantadine, desipramine, ganciclovir, metformin amantadine, cimetidine, memantine skeletal muscle, liver, placenta, cimetidine kidney, heart kidney, skeletal muscle, quinidine, verapamil placenta, prostate, heart kidney, skeletal muscle, quinidine, verapamil prostate, lung, pancreas, heart, small intestine, liver acyclovir, adefovir, kidney, brain methotrexate, zidovudine liver, kidney zidovudine kidney, brain cimetidine, methotrexate, zidovudine FDA DRAFT Guidance for Industry (Sept 2006) Inhibitor cyclosporine, rifampin disopyramide, midazolam, phenformin, phenoxy-benzamine quinidine, ritonavir, verapamil desipramine, phenoxy-benzamine, quinine desipramine, prazosin, phenoxybenzamine probenecid, cefadroxil, cefamandole, cefazolin, probenecid, cefadroxil, cefamandole, cefazolin, Transporter Expression Liver Sinusoidal Transport Uptake: OCT1, OATP-C, OATP-B, OATP8, NTCP, OAT2 Secretion: MRP1, MRP3 Intestinal Lumen Absorption: PEPT1 Secretion: P-gp, OATP3 Blood-Brain Barrier P-gp (MDR1), OAT3, OATP-A, MRP1, MRP3 Liver Canalicular (Biliary) Transport Secretion: P-gp, MRP2, BCRP, BSEP, MDR3 Kidney Basolateral Transport: OCT1, OCT2, OAT1, OAT2, OAT3, MRP1 Kidney Apical Transport: Secretion: P-gp, OAT4 Reabsorption: PEPT2 From: Zhang, L. et al, Mol Pharm. 3(1):62-69 (2006). Drug-Drug Interactions Due to Transporters • Currently considered most important transporters for Drug-Drug Interactions (DDI): – ABC: P-gp, BCRP, BSEP – SLC: OATP1B1, OATP1B3 , OATP2B1, OCT1, OCT3, OAT2 • Currently considered less important: – MRP transporters • Most critical for drugs with a narrow therapeutic index (e.g., digoxin) where co-administration of a transporter inhibitor might require dose adjustment. • Depending on the expression pattern of the affected transporter, DDI can result in changes to absorption, tissue distribution (CNS, tumors), or elimination of the victim drug. Clinical Examples for Transporter-Related Drug-Drug Interactions • Cerivastatin (HMG-CoA reductase inhibitor) – Substrate of hepatic uptake transporter OATP1B1 – Increased plasma concentrations with inhibition of OATP1B1 by co-administered cyclosporine (JPET 304:610 [2003]) – (Withdrawn from market in 2001 after 52 deaths due to kidney failure) • Digoxin (cardiac glycoside) – Substrate of efflux transporter P-glycoprotein – Elimination of unchanged digoxin is by renal and biliary excretion. – AUC increased and renal clearance decreased with co-administration of P-gp inhibitor itraconazole (Ther. Drug Monit. 10:609 [1997]) – AUC decreased with co-administration of P-gp inducer rifampin (J. Clin. Invest. 104:147 [1999]). • Cephalosporin antibiotics – Substrates of uptake transporter OAT – Co-administration of OAT inhibitor probenecid resulted in decreased renal clearance, increased Cmax and AUC (Clin. Pharmacokinet. 24:289 [1993]). In Vitro Models for Transporter Interaction In vitro Model Description Membranes from insect cells expressing transporter cDNA ATPase assay; Discovery screen; Indirect assay; does not differentiate substrates/inhibitors “Inside-out” Vesicles from insect cells expressing transporter cDNA Direct, functional uptake assay with vesicles to identify substrates and inhibitors Polarized cell monolayers Caco-2 Direct, functional efflux assay; Substrate and/or inhibitor testing; IC50, Ki determination Transfected cell lines (LLC-PK1, MDCK) Transfection of single (e.g. MDR1) or multiple transporters Xenopus leavis oocytes expressing SLC transporters (e.g. OAT, OATP, OCT, NTCP, PEPT) Direct, functional uptake assay; Substrate and/or inhibitor screening; Affinity determination Km/Vmax, IC50, Ki Hepatocytes Fresh hepatocytes plated or in suspension Transporter Characterized BD Gentest™ Cryopreserved Human Hepatocytes (OATP, NTCP, OCT1) Sandwich cultured human hepatocytes Assess inter-individual variability Direct, functional uptake assay; Substrate and/or inhibitor screening; Affinity determination Km/Vmax, IC50, Ki Assess biliary excretion Assessment of P-gp Interactions Identification of P-gp substrates and inhibitors Technical considerations Decision trees FDA’s Current Thinking • “…in vitro studies can frequently serve as a screening mechanism to rule out the importance of a metabolic pathway and the drugdrug interactions that occur through this pathway so that subsequent in vivo testing is unnecessary.” • “...not every drug-drug interaction is metabolism-based, but may arise from changes in pharmacokinetics caused by absorption, distribution, and excretion interactions. Drug-drug interactions related to transporters are being documented with increasing frequency and are important to consider in drug development. “ • “Of the various transporters, P-gp is the most well understood and may be appropriate to evaluate during drug development.” FDA DRAFT Guidance for Industry (Sept 2006) P-gp • Apical side (e.g. intestinal lumen) P-glycoprotein (P-gp) commonly refers to MDR1 – P-glycoprotein (MDR1) is encoded by the ABCB1 gene. – MDR3 (encoded by ABCB4) is present in various human tissues, but there is little evidence of a major role in drug transport. • 170 kDa glycoprotein • ATP-binding cassette transporter Filaria Journal 2(Suppl 1):S8 (2003). P-gp consists of 2 sections, each containing 6 transmembrane domains and a large cytoplasmic domain with an ATP-binding site P-gp (MDR1) • Mainly present on the apical side of epithelial cells: – – – – brush border membrane of small intestine enterocytes proximal tubule cells in the kidney canalicular membrane of hepatocytes blood brain barrier capillary endothelial cells • Efflux pump with broad substrate specificity. Substrates tend to be large, lipophilic, positively charged or neutral molecules • Likely evolved as a defense mechanism against harmful substances • Modulation of P-gp activity and/or expression may affect oral bioavailability, biliary and renal clearance, and tissue distribution (e.g. tumors, placenta, CNS) of drugs potentially resulting in DDIs after co-administration of drugs that interact with P-gp Models to Study P-gp Interactions Assay Type Bi-Directional Transport Tissues Caco-2 cells MDCK-MDR1 cells LLC-PK1-MDR1cells Parameters Net drug flux ratio of B to A and A to B Comments Directly measure efflux across cell barrier Evaluation of P-gp transport and inhibition Uptake/efflux ATPase Allow for localization/identification of the transporters within the apical or basolateral side of the membrane Inhibition of uptake or efflux of Cannot distinguish substrate from tumor cells fluorescent probe inhibitor cDNA transfected cells oocytes injected with cRNA (Calcein-AM, rhodamine-123) Tends to fail to identify substrate and/or inhibitor with low permeability of transporters membrane vesicles from ATPase stimulation Same comments as uptake/efflux tissues or cells expressing Passay gp, Reconstituted P-gp “The bi-directional transport assay is regarded as the definitive assay for identifying P-gp substrates and inhibitors because it measures drug efflux in a more direct manner than other methods.” FDA DRAFT Guidance for Industry (Sept 2006) Bi-directional P-gp Transport Assays • Cell monolayers grown on filters and placed in cluster plates • Filters are typically PET or PC membranes with 0.4-1 μm pores • Transport is measured in two directions: –Apical (A) to Basolateral (B), i.e. test compound added to apical side –Basolateral (B) to Apical (A), i.e. test compound added to basolateral side Drug 1 = P-gp-mediated transport 2 = Passive diffusion A (apical) B (basolateral) 1 ATP 2 Cell monolayer Filter membrane Materials and Equipment Needed • Filter plate system (available in 6-, 12-, 24-, 48-, and 96-well formats) Example: BD Falcon™ 24-Multiwell HTS Insert Plate (1 µm PET) with feeder tray (for culture), 24-well cluster plate (for assay), and lid • 37°C incubator • TEER (Trans-Epithelial Electrical Resistance) meter to confirm presence of functionally polarized monolayers prior to the experiment – TEER [Ω·cm2] = resistance [Ω] x filter surface area [cm2] – varies with cell line, 100-800 Ω·cm2 • Liquid scintillation counter or LC/MS/MS for sample analysis • Pipettors (manual or automated) for liquid transfers Cell Lines for P-gp Transport Assays • Cells used for bi-directional transport studies should form a functionally polarized cell monolayer, complete with tight junctions (verified by preexperimental TEER of 100-800 Ω·cm2) • At present, preferred cells lines include – Caco-2 cells – MDR1 transfected LLC-PK1 cells – MDR1 transfected MDCK cells • LLC-PK1 and MDCK wild type cells should be used as negative controls • Cells should be allowed to grow to confluence (typically 3-7 days for LLC-PK1 or MDCK, 18-21 days for Caco-2, however accelerated 3-5 day Caco-2 models are available and produce similar results) • A paracellular marker such as [14C] mannitol may be used as an additional integrity marker (typical permeability: < 0.2-2 x 10-6 cm/sec) Cell Lines for P-gp Transport Assays • Caco-2 – Human colon carcinoma cell line – Morphologically similar to small intestinal epithelial cells – Most extensively characterized human cell-based model for investigating permeability and P-gp transport of drugs – Various uptake and efflux transporters are expressed in Caco-2 cells, however, P-gp is functionally the most predominant – No wild-type cells to run alongside • LLC-PK1-MDR1 – Transfected porcine kidney cell line – Low transporter background, especially for P-gp • MDCK-MDR1 – Transfected canine kidney cell line – High background dog P-gp activity Bi-Directional P-gp Transport Assays • The transcellular transport assay should be used as a definitive method for identifying P-gp substrates and inhibitors • Bi-directional transport assays may fail to identify highly permeable compounds as P-gp substrates, however for such compounds, P-gp is not likely to be a significant barrier to cross membranes • These experiments require the use of known P-gp probe substrates and P-gp inhibitors P-gp Probe Substrate Characteristics • Ideally – – – – – • Selective for P-gp Low to moderate passive permeability (2-30x10-6 cm/sec) May be used as an in vivo P-gp probe substrate (optional) No significant metabolism of the substrate (optional) Commercially available (optional) Realistically – A P-gp substrate that meets all of the above criteria has not been identified, due to overlapping substrate selectivity between different transporters as well as transporters and enzymes – Acceptable P-gp substrates meet the majority of the above mentioned criteria. These P-gp substrates serve as positive controls to ensure the cell systems have functional P-gp expression. FDA DRAFT Guidance for Industry (Sept 2006) Acceptable P-gp Probe Substrates Efflux Ratio Drug Concentration [µM] Caco-2 MDR1-MDCK MDR1-LLCPK1 Digoxin 0.01-10 4-14 4 4 Loperamide 1-10 2-5 3.4 Quinidine 0.05 3 5 Vinblastine 0.004 -10 2-18 Talinolol 30 26 >9 3 • Acceptable P-gp substrates are not limited to compounds listed • Selection of other compounds as probe P-gp substrates may be appropriate based on scientific justification FDA DRAFT Guidance for Industry (Sept 2006) P-gp Inhibitor Characteristics • Ideally – – – – – • Selective for P-gp Inhibit P-gp with low Ki or IC50 values (e.g., IC50 < 10 µM) No significant metabolism of the inhibitor occurs in the cells (optional) Commercially available (optional) May be used as an in vivo P-gp inhibitor (optional) Realistically – A P-gp inhibitor that meets all of the above criteria has not been identified, due to substantial overlap between different transporters (e.g., cyclosporine is also a potent MRP2 and OATP-C inhibitor; quinidine and verapamil also inhibits various organic cation transporters) – Because of the lack of specificity, the use of multiple inhibitors is recommended to determine whether the efflux activity observed in vitro is related to P-gp FDA DRAFT Guidance for Industry (Sept 2006) Acceptable P-gp Probe Inhibitors IC50 (µM) Ki (µM) Caco-2 Caco-2 MDCKMDR1 LLC-PK1MDR1 Cyclosporine A 1.3 0.5 2.2 1.3 Ketoconazole 1.2 Inhibitor LY335979 0.024 Nelfinavira 1.4 Quinidine 2.2 Ritonavir 3.8 Saquinavir 6.5 Tacrolimus 0.74 Valspodar (PSC833) 0.11 Verapamil 2.1 5.3 3.2 8.6 • Acceptable P-gp inhibitors are not limited to compounds listed • Selection of other compounds as probe P-gp inhibitors may be appropriate based on scientific justification • Use of multiple inhibitors is recommended 8 15 Elacridar (GF120918) (GG 918) 0.4 0.4 Reserpine 1.4 11.5 FDA DRAFT Guidance for Industry (Sept 2006) 23 Assay Design – P-gp Substrate Testing • Test a range of concentrations (e.g., 1, 10, and 100 µM) • Test multiple time points (30 min up to 4 hours) • Include a P-gp substrate as a positive control – Acceptable cell systems produce net flux ratios of probe substrates similar to values reported in the literature (net flux ratios of ≥ 2 are recommended) • Include parental LLC-PK1 and MDCK cells, respectively, side-by-side as negative controls • Test in presence and absence of 2-3 potent P-gp probe inhibitors to determine if efflux can be inhibited • Triplicate monolayers for each condition • Assess recovery of substrate, to assess metabolism and non-specific binding Time- and Concentration-Dependence of Digoxin Transport in MDR1-LLC-PK1 cells 1200 1400 digoxin 5.0 uM digoxin 50 uM digoxin 30 min digoxin 60 min 1200 P-gp transport [pmol] 1000 P-gp transport [pmol] P-gp facilitated Transport Concentration dependence P-gp facilitated Transport Time dependence digoxin 0.5 uM 800 600 400 200 digoxin 90 min digoxin 120 min 1000 800 600 400 200 0 0 0 50 100 min 150 0 10 20 30 40 50 60 uM Transport of P-gp probe substrate digoxin is linear over the concentration range of 0.5 - 50 µM and incubation times of 30 -120 min. Calculation of Papp and Efflux Ratios Apparent Permeability (Papp) Papp [cm/sec] = Vr/C0 x 1/S x dC/dt Vr is the volume in the receiver chamber [cm3] C0 is the concentration in the donor chamber at t=0 [mM] S is the filter surface area [cm2] dC/dt is the is the linear slope of the drug concentration in the receiver chamber with time after correcting for dilution [mM/sec] Efflux Ratio (RE) RE = Papp (B to A) / Papp (A to B) Papp (B-A) is the Papp value measured in the B to A direction Papp (A-B) is the Papp value measured in the A to B direction Net Efflux Ratio (R) – for use with transfected cell models only NER = RET / REW RET is the efflux ratio obtained in the P-gp transfected cells REW is the efflux ratio obtained in the wild-type control cells FDA DRAFT Guidance for Industry (Sept 2006) Decision Tree for P-gp Substrates Bidirectional Bidirectional transport transport assay assay Is Is efflux efflux ratio ratio ≥≥ 22 ?? ** Is Is efflux efflux ratio ratio << 22 ?? ** Is Is efflux efflux inhibited inhibited by by P-gp P-gp inhibitors? inhibitors? ** ** Unlikely Unlikely to to be be P-gp P-gp substrate substrate YES NO Likely Likely to to be be P-gp P-gp substrate substrate Transporters Transporters other other than than P-gp P-gp might might be be involved involved In In vivo vivo drug drug interaction interaction study study with with aa P-gp P-gp inhibitor inhibitor may may be be warranted warranted Further Further in in vitro vitro studies studies to to identify identify transporters transporters may may be be warranted warranted * There is concern that this value is too liberal and will lead to too may positive results. Alternatively, use a % value relative to a probe substrate, such as digoxin ** Reduces the efflux ratio significantly (> 50% or to unity) FDA DRAFT Guidance for Industry (Sept 2006) Example: Identification of a Test Article as a P-gp Substrate in Caco-2 Cells 1.1 3.6 2.8 2.3 1.3 1.4 1.3 1.3 1.1 1.2 TA 10 uM TA 1.0 uM + keto 25 uM TA 3.0 uM + keto 25 uM TA 10 uM + keto 25 uM TA 1.0 uM + csa 10 uM TA 3.0 uM + csa 10 uM TA 10 uM + csa 10 uM test article with 10 uM cyclosporine TA 3.0 uM Efflux Ratios: 14 1.4 test article with 25 uM ketoconazole TA 1.0 uM 25 test article without inhibitors digoxin 5 uM + csa 10 uM positive control +/- inhibitors Papp [10-6 cm/s] 20 15 10 5 B-A Papp digoxin 5 uM + keto 25 uM A-B Papp digoxin 5 uM 0 Data are for 120 min incubation time; similar results were obtained at 30 and 60 min Test article shows active efflux, which is inhibited by P-gp inhibitors Æ likely a P-gp substrate Assay Design – P-gp Inhibition Testing • Use a P-gp probe substrate that has been confirmed as a positive control in the test system – Acceptable cell systems produce net flux ratios of probe substrates similar to values reported in the literature (net flux ratios of ≥ 2 are recommended) • Initially, test a high concentration (e.g. >100 µM, solubility permitting) of the compound – If inhibition is observed, follow up with an IC50 determination • Include parental LLC-PK1 and MDCK cells, respectively, side-byside as negative controls • Use 2-3 potent P-gp probe inhibitors as positive controls • Triplicate monolayers for each condition Calculations for P-gp Inhibition IC50 Value REi/REa = 1 – [(Imax* Ic) / ( Ic + IC50c )] REi/REa is the efflux ratio of the probe P-gp substrate in the presence of inhibitor relative to that without inhibitor I is the inhibitor concentration [µM] Imax is the maximal inhibitory effect [fraction of control activity] c is the Hill exponent IC50 is the inhibitor concentration achieving half maximal inhibition effect [µM] • There is considerable discussion in the scientific community regarding the most appropriate way to calculate P-gp inhibition • A DIA initiative to assess inter-laboratory variability in P-gp inhibition testing and to evaluate various calculation methods is ongoing • Stay tuned for more information (white paper, final guidance) FDA DRAFT Guidance for Industry (Sept 2006) Decision Tree for P-gp Inhibitors Bidirectional Bidirectional transport transport assay assay with with P-gp P-gp probe probe substrate substrate Net Net flux flux ratio ratio of of probe probe substrate substrate decreases with increasing decreases with increasing concentrations concentrations of of test test compound compound Net Net flux flux ratio ratio of of probe probe substrate substrate is is not affected by increasing not affected by increasing concentrations concentrations of of test test compound compound P-gp P-gp inhibitor inhibitor Poor Poor or or non-inhibitor non-inhibitor of of P-gp P-gp Determine Determine IC IC5050 (or (or KKi)i) [I]/IC [I]/IC5050 >> 0.1 0.1 In In vivo vivo drug drug interaction interaction study study with with aa P-gp P-gp probe probe substrate substrate (e.g. (e.g. digoxin) digoxin) is is recommended recommended [I]/IC [I]/IC5050 << 0.1 0.1 In In vivo vivo drug drug interaction interaction study study with a P-gp probe substrate with a P-gp probe substrate is is not needed not needed FDA DRAFT Guidance for Industry (Sept 2006) [I] = total steady-state Cmax at highest dose Example: Identification of a Test Article as a P-gp Inhibitor in Caco-2 Cells 1.2 1.1 12.2 8.4 4.9 3.5 1.6 digoxin 5 uM + TA 0.5 uM digoxin 5 uM + TA 1 uM digoxin 5 uM + TA 2.5 uM digoxin 5 uM + TA 5 uM digoxin 5 uM + TA 10 uM Efflux Ratios: 14.8 digoxin 5 uM + csa 10 uM 14 increasing concentation of the test article digoxin 5 uM + keto 25 uM no inhibition probe P-gp inhibitors as positive controls Papp [10-6 cm/s] 12 10 8 6 4 2 A-B Papp B-A Papp digoxin 5 uM 0 Test article shows concentration dependent inhibition of digoxin efflux Æ P-gp inhibitor Æ determine IC50 Example: Identification of a Test Article as a P-gp Inhibitor in Caco-2 Cells Calculations performed: Decrease in Efflux Ratio (or Net Efflux Ratio, respectively) % Inhibition = (1-(REi – 1) / (REa – 1))*100 REi is the efflux ratio of the probe P-gp substrate in the presence of inhibitor REa is the efflux ratio of the probe P-gp substrate in the absence of inhibitor IC50 Value (SigmaPlot v8.0) f = 1 – [(Imax* Ic) / ( Ic + IC50c )] f is the fraction of control activity ((REi – 1) / (REa – 1)) I is the inhibitor concentration [uM] Imax is the maximal inhibitory effect [fraction of control activity] c is the Hill exponent IC50 is the inhibitor concentration achieving half maximal inhibition effect [µM] Example: Identification of a Test Article as a P-gp Inhibitor in Caco-2 Cells % Inhibition of digoxin efflux 120 [I] / IC50 > 0.1 is the recommended cut-off for in vivo testing 100 80 60 Æ If [I] (steady-state total Cmax after highest dose) is >0.12 uM, an in vivo P-gp interaction study might be warranted 40 20 IC50 = 1.2 µM 0 0 2 4 6 8 Test article concentration [uM] 10 12 Inhibition of Digoxin Efflux by Ketoconazole in Caco-2 and LLC-PK1 Cells MDR1-LLC-PK1 cells % Inhibition of digoxin efflux 100 80 60 40 20 IC50 = 2.6 uM 0 -20 0 10 20 Ketoconazole [uM] 30 % Inhibition of digoxin net efflux Caco-2 cells 120 (using both P-gp expressing and control cells) 100 80 60 40 20 IC50 = 1.8 uM 0 0 20 40 60 80 Ketoconazole [uM] 100 120 Non-Specific Binding / Recovery • Amount of compound recovered in the donor and receiver chambers at the end of the assay relative to the amount added to the donor chamber at t=0 • Low recovery may significantly underestimate Papp values • • – Always consider recovery when interpreting Papp values and efflux ratios – Typical cut-offs used in the industry are 50-80%, depending on the stage of the compound in the development process Factors contributing to low recovery include: – Non-specific binding to the outside of the cells – Uptake into the cells – Non-specific binding to the plastic plate – Metabolism or non-metabolic instability Addition of protein may improve recovery, however, the impact on Papp and efflux ratios is not fully understood Evaluation of P-gp Induction • • Expression of P-gp is inducible (e.g. by rifampin, hypericin, ritonavir) Methods for in vitro evaluation of P-gp induction are not well established – Caco-2 cells are not suitable for P-gp induction testing (lack of PXR expression) – As for CYPs, species differences in inductive response limit the use of animal models – Models that have been used successfully include: • Human hepatocytes (mRNA induction) • Human colon adenocarcinoma cells (LS180), and their adriamycin or vinblastine resistant sublines (mRNA, protein, activity assessment) • Co-induction of P-gp and CYP3A (both are regulated by PXR) – Because of similar mechanisms of CYP3A and P-gp induction, data on CYP3A inducibility can inform decisions about P-gp • If no CYP3A induction is observed in vitro, no further tests of CYP3A and P-gp induction in vivo are necessary • If CYP3A induction is observed in vitro, but not in vivo, no further tests of P-gp induction in vivo is necessary • If CYP3A induction is observed in vitro and in vivo, an additional study of the drug’s effect on a P-gp probe substrate in vivo is recommended Comments on Draft Guidance Document • Draft guidance has been commented on extensively, especially the transporter section: http://www.fda.gov/ohrms/dockets/dockets/06d0344/06d0344.htm • Main issues raised: – Degree of experimental detail prescribed – Use of control cells with transfected cell lines – Efflux ratio >2 as criteria for P-gp substrates – Method of calculating inhibition and IC50 values – Trigger for in vivo study and definition of [I] Expectations for Future Guidance • Our expectations: – Experimental detail for P-gp interaction testing may come into line with other sections – Clarification on calculation methods for P-gp inhibition – More detail around triggers for in vivo studies, and requirements for those – Number of transporters which require testing expected to increase (e.g. BCRP, BSEP, OAPT2, NTCP) • Recommendation on experimental systems and probes for transporters other than P-gp • Decision trees for other transporters – Standardized language for labeling regarding transporter interactions Recommendations • Try to generally follow recommendations in the draft FDA guidance – Until document is final, consider the docket comments – Use your scientific judgment to decide how to approach controversial issues – Consider “consensus” documents / White Papers published by representatives from industry and/or government • In vitro transport studies are not required to be GLP compliant – However, using validated methods and test systems, positive control tracking processes, SOPs, etc. are good practice • Use well-accepted reagents from reputable suppliers – Radiochemical purity of probe substrates and test compounds is critical Contact Information Elke S. Perloff e-mail: [email protected] tel: 781-935-5115 x2273