In Vitro Transporter Testing Alignment with Regulatory Agency Guidelines and ITC Recommendations. Elke S. Perloff, Ph.D.

Transcription

1 In Vitro Transporter Testing Alignment with Regulatory Agency Guidelines and ITC Recommendations Elke S. Perloff, Ph.D. BD Biosciences February 23, 2011

2 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-gp and BCRP Interactions Identification of substrates and inhibitors using the bidirectional transport assay Technical considerations (cell lines, positive controls, assay conditions, parameter calculations, data interpretation) Decision trees

3 Guidance Documents and Position Papers

4 Guidance Documents and Position Papers on Transporter DDI Testing Tucker et al. (2001) Basel conference, sponsored by FDA, EUFEPS, and AAPS US FDA (2006) Draft Guidance for Industry Zhang et al. (2006) Mol Pharmacol 3:62 Huang et al. (2008) J. Clin Pharmacol 48:662 Zhang et al. (2008) Xenobiotica 38:709 International Transporter Consortium White Paper (2010) Nature Rev Drug Discovery 9:215 EMA (2010) Draft Guideline on the Investigation of Drug Interactions

5 FDA Guidance Documents Over 400 draft or final guidance documents 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).

6 Transporter Expression and Function Role in drug-drug interactions In vitro models to study transporter interactions

7 Transporters Membrane-bound proteins with asymmetric distribution in polarized cells of various tissues e.g. intestinal enterocytes, hepatocytes, proximal tubules, bloodbrain 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

8 Major Human Transporters (ABC) Gene Aliases Tissue Substrate Inhibitor ABCB1 P-gp, MDR1 intestine, liver, kidney, brain, placenta, adrenal, testes digoxin, fexofenadine, indinavir, vincristine, colchicine, topotecan, paclitaxel, loperamide, doxorubicin, vinblastine ritonavir, cyclosporine, verapamil, erythromycin, ketocoanzole, itraconazole, quinidine, elacridar (GF120918), LY335979, valspodar (PSC833) ABCB4 MDR3 liver digoxin, paclitaxel, vinblastine verapamil, cyclosporine ABCB11 BSEP liver pravastatin, taurocholic acid, bile acids cyclosporine, rifampicin, glibenclamide ABCC1 MRP1 intestine, liver, adefovir, indinavir kidney, brain ABCC2 MRP2, CMOAT intestine, liver, kidney, brain methotrexate, glucuronides, valsartan, indinavir, cisplatin, cyclosporine, delavirdine, efavirenz, emtricitabine ABCC3 MRP3, CMOAT2 intestine, liver, kidney, placenta, etoposide, methotrexate, tenoposide, glucoronides, fexofenadine delavirdine, efavirenz, emtricitabine adrenal ABCC4 MRP4 liver, brain adeforvir, tenofovir, methotrexate, topotecan, furosemide, camp, ABCC5 MRP5 brain ABCC6 MRP6 liver, kidney cisplatin, daunorubicin ABCG2 BCRP intestine, liver, daunorubicin, doxorubicin, topotecan, mammary glands, rosuvastatin, sulfasalazine, imatinib, placenta methotrexate celecoxib, diclofenac elacridar (GF120918), gefitinib, fumitremorgin C, novobiocin FDA DRAFT Guidance (2006) and ITC White Paper (2010)

9 Major Human Transporters (SLC) Gene Aliases Tissue Substrate Inhibitor SLCO1A2 OATP1A2, OATP-A brain, liver, kidney E3S, statins, fexofenadine, bile salts, digoxin, methotrexate naringin, ritonavir, lopinavir, saquinavir, rifampicin SLCO1B1 OATP1B1, OATP- C, OATP2 liver rifampin, rosuvastatin, methotrexate, pravastatin, thyroxine, bilirubin, E17BG, cyclosporine, rifampicin, saquinavir, ritonavir, lopinavir SLCO1B3 OATP1B3, OATP8 liver digoxin, fexofenadine, telmisartan, E17BG, rifampicin, cyclosporine, ritonavir, bile acids, statins, lopinavir SLCO2B1 OATP2B1, OATP-B intestine, liver, kidney, brain E3S, statins, taurocholate, fexofenadine, glyburide rifampicin, cyclosporine SLC10A1 NTCP liver, pancreas rosuvastatin, chlorambucil taurocholate SLC15A1 PEPT1 intestine, kidney gly-sar, cephalexin, amoxicillin, captopril, gly-pro SLC15A2 PEPT2 kidney, brain, lung gly-sar, cephalexin, amoxicillin, captopril, fosinopril, zofenopril SLC22A1 OCT1 liver, intestine tetraethylammonium, oxaliplatin, metformin, N-methylpyridinium disopyramide, disopyraminde, quinidine, ritonavir, verapamil SLC22A2 OCT2 kidney, brain tetraethylammonium, oxaliplatin, metformin, N-methylpyridinium, pindolol, cimetidine, cetirizine, testosterone, quinine SLC22A4 OCTN1 kidney, muscle, lung quinidine, verapamil, ipatriopium SLC22A5 OCTN2 kidney, muscle, lung quinidine, verapamil, ipatriopium SLC22A6 OAT1 kidney, brain para-aminohippurate, ciprofloxacine, probenecid, cefadroxil, novobiocin acyclovir, adefovir, methotrexate, SLC22A8 OAT3 kidney, brain E3S, NSAIDs, cefaclor, furosemide, probenecid, novobiocin bumetanide SLC47A1 MATE1 kidney, liver, muscle metformine, N-methylpyridinium, quinidine, cimetidine, procainamide SLC47A2 MATE2-K kidney metformine, N-methylpyridinium, quinidine, cimetidine, procainamide FDA DRAFT Guidance (2006) and ITC White Paper (2010)

10 Transporter Expression Liver Sinusoidal Transport Uptake: OCT1, OATP1B1, OATP2B1, OATP1B3, NTCP, OAT2 Secretion: MRP3, MRP4 Liver Canalicular (Biliary) Transport Secretion: P-gp, BCRP, BSEP, MRP2, MATE1 Blood-Brain Barrier Uptake: OATP1A2, OATP2B1 Efflux: P-gp (MDR1), BCRP, MRP4, MRP5 Kidney Basolateral Transport Uptake: OCT2, OAT1, OAT2, OAT3, OATP4C1 Intestinal Lumen Absorption: PEPT1, OATP Secretion: P-gp, BCRP, MRP2 Kidney Apical Transport P-gp, MRP2, MRP4, MATE, OCTN1, OCTN2, OAT4, Reabsorption: PEPT1, PEPT2 From: Zhang L et al. 2006, Mol Pharm.; 3:62, ITC paper 2010, Nature Rev Drug Disc 9:215

11 Transporter Drug-Drug Interactions Depending on the expression pattern of the affected transporter, DDI can result in changes to Absorption, Tissue Distribution (e.g. CNS, tumors), and/or Elimination of the victim drug. PK changes that can result in toxicity or inactivity for drugs with a narrow therapeutic index (e.g. digoxin). Tissue distribution changes may not be reflected in PK alone, but may lead to organ specific toxicity (e.g. liver). Therefore, the inclusion of pharmacodynamic markers to reflect altered distribution to the organs expressing the transporter should be considered whenever possible.

12 Clinical Examples for Transporter Related Drug-Drug Interactions From: ITC paper (2010) Nature Rev Drug Disc 9:215

13 Relevant Transporters for DDI Currently considered most important transporters for DDI: FDA 2006 Draft Guidance: P-gp ITC 2010 White Paper: P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2 EMA 2010 Draft Guideline: P-gp, BCRP, BSEP, OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2 Currently considered less important: MRP transporters

14 In Vitro Models for Transporter Interaction In Vitro Model Membranes from cells expressing transporter cdna Inside-out Vesicles from cells expressing transporter cdna Polarized cell monolayers Caco-2 Transfected cell lines (LLC-PK1, MDCK) Xenopus leavis oocytes expressing SLC transporters (e.g. OAT, OATP, OCT, NTCP, PEPT) Hepatocytes Fresh hepatocytes plated or in suspension Transporter Characterized Cryopreserved Hepatocytes (OATP, NTCP, OCT1) Sandwich cultured human hepatocytes Notes ATPase assay; Discovery screen; Indirect assay; does not differentiate substrates/inhibitors Direct, functional uptake assay with vesicles to identify substrates and inhibitors Direct, functional efflux assay; Substrate and/or inhibitor testing; IC 50, K i determination Transfection of single (e.g. MDR1) or multiple transporters Direct, functional uptake assay; Substrate and/or inhibitor screening; Affinity determination K m /V max, IC 50, K i Assess inter-individual variability Direct, functional uptake assay; Substrate and/or inhibitor screening; Affinity determination K m /V max, IC 50, K i Assess biliary excretion

15 Considerations for In Vitro Transport Studies Evaluate NME as a substrate and inhibitor of transporters In line with the practices for enzyme identification, if renal and biliary secretion account for more than 25% of systemic clearance, attempt to identify the transporter(s) involved Investigate drug transport with and without presence of the specific transporter activity Include positive controls (selective substrates/inhibitors) to verify specific transporter activity Test a concentration range of the investigational drug expected to be relevant for the site of interaction For systemic transporter interaction, consider expected unbound C max For local transporter interaction, consider concentration at active site Use a eukaryotic system where the physiological functions of the transporter are preserved EMA Draft Guideline (2010)

16 Assessment of P-gp and BCRP Interactions Identification of P-gp/BCRP substrates and inhibitors using the bidirectional transport assay

17 P-gp and BCRP P-gp (MDR1, ABCB1) Expressed in intestine, kidney proximal tubule, hepatocytes (canalicular), brain endothelia Involved in absorption, disposition, excretion Known clinical DDI Resistance to cancer chemotherapy Substrates are generally hydrophobic molecules, cationic or neutral MW 200 to >1000 Multiple substrate binding sites BCRP (ABCG2) Expressed in intestine, kidney proximal tubule, hepatocytes (canalicular), brain endothelia, placenta, stem cells, mammary glands Involved in absorption, disposition, excretion Known clinical DDI Resistance to cancer chemotherapy Clinically relevant polymorphisms Substrates overlap with P-gp, but also include acids and conjugates Physiological substrates include porphyrins, riboflavin and potentially other vitamins

18 Bidirectional Transport Assay Design Cell monolayers grown on filters and placed in cluster plates Filters are typically PET or PC membranes with 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 Inhibition testing: Same as transport, but with inhibitor added to both A and B Drug 1 = Active transport A (apical) B (basolateral) 1 2 ATP 2 = Passive diffusion Cell monolayer Filter membrane

19 Materials & Equipment Filter plate system (available in 6-, 12-, 24-, 96-well formats) Example: BD Falcon TM 24-multiwell HTS insert plate (1 µm PET) with feeder tray (for culture), 24-well cluster plates (for assay), lid 37 C incubator Pipettes (manual or automated) for liquid transfers TEER (Trans-Epithelial Electrical Resistance) meter to confirm presence of functionally polarized monolayers prior to the experiment TEER [Ω cm 2 ] = resistance (cells-blank) [Ω] x filter area [cm 2 ] Varies with cell line, Ω cm 2 Liquid scintillation counter or LC/MS/MS for analysis

20 Cell Lines for Bidirectional 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/BCRP transport of drugs Expression of various uptake and efflux transporters P-gp and BCRP are functionally the most predominant, allows testing for both transporters in the same system and assay. No wild-type cells to run alongside LLC-PK 1 Transfected porcine kidney cell line Low transporter background, especially for P-gp MDCK Transfected canine kidney cell line High background of dog transporters, especially for P-gp

21 Cell Monolayers & QC Cells used for bidirectional transport studies should form a functionally polarized cell monolayer, complete with tight junctions. Cells should be allowed to grow to confluence on filter plates typically 3-7 days for LLC-PK 1 or MDCK days for Caco-2 accelerated 3-5 day Caco-2 models are available Verify monolayer integrity by pre-experimental TEER measurements (e.g. > 150 Ω cm 2 for LLC-PK 1 cells, >250 Ω cm 2 for Caco-2 cells) A paracellular marker (e.g. mannitol, lucifer yellow) is commonly used as an additional integrity marker Concurrently with the test article, or after the end of the assay Detects toxicity or physical damage to monolayers during the assay Validate effects of organic solvents in your model (<1% organic is typically ok, keep constant)

22 Positive Control Substrate Characteristics Ideally: Selective for the transporter of interest Low to moderate passive permeability May be used as an in vivo probe substrate No significant metabolism in the cells Commercially available Realistically: A P-gp or BCRP substrate that meets all these criteria has not been identified, due to overlapping substrate selectivity between different transporters as well as transporters and enzymes. Acceptable probe substrates meet the majority of the criteria and can confirm that the cell systems have functional P-gp and BCRP activity. Examples: P-gp: digoxin, vinblastine, fexofenadine, saquinavir BCRP: estrone-3-sulfate, prazosin, sulfasalazine Select based on guidance and scientific justification

23 Positive Control Inhibitor Characteristics Ideally: Selective for the transporter of interest Low K i or IC 50 values (e.g. IC50 < 10 µm) No significant metabolism in the cells May be used as an in vivo inhibitor Commercially available Realistically: A P-gp or BCRP inhibitor that meets all these criteria has not been identified, due to substantial overlap between different transporters. Because of the lack of specificity, use multiple inhibitors to determine whether the efflux activity observed in vitro is related to P-gp or BCRP. Examples: P-gp: quinidine, verapamil, ketoconazole BCRP: novobiocin, FTC, sulfasalazine Select based on guidance and scientific justification

24 P-gp/BCRP Substrate Testing Test a range of concentrations for intestinal transport: 0.1 to 50 x dose/250 ml for systemic transport: 0.1 to 50 fold unbound C max Use triplicate monolayers for each condition Include wild-type LLC-PK 1 or MDCK cells as negative controls Include a probe substrate as a positive control Test in presence and absence of at least 2 potent P-gp/BCRP positive control inhibitors to determine if efflux can be inhibited Determine compound recovery from the test system at the end of the assay sample donor compartments at t=0 min and at the last time point

25 P-gp/BCRP Inhibition Testing Use a P-gp probe substrate that has been validated as a positive control in the test system. Select a concentration and incubation time within the linear range of transport for the probe substrate Efflux ratio should be high enough to provide sufficient dynamic range for inhibition (the >2 cutoff may be too low) Run in absence and presence of positive control inhibitors Use triplicate monolayers for each condition Initially, test a high concentration (e.g. 100 µm, solubility permitting). If inhibition is observed, follow up with an IC 50 determination. Include 2 potent P-gp/BCRP positive control inhibitors. Single concentration of positive controls typically sufficient In IC 50 assays a full IC 50 curve of the positive control might be valuable

26 Probe Substrate Efflux Screen Probe substrate Probe substrate + inhibitor 20 Caco-2 20 MDR1 -LLC-PK Digoxin (5uM) ± Verapamil (50uM) E3S (5uM) ± Novobiocin (10uM) Prazosin (5 um) ± Novobiocin (10uM) LTC4 (0.1uM) ± MK571 (50uM) E17BG (5uM) ± MK571 (10uM) Efflux ratio (B-A/A-B) Control-LLC-PK1 Digoxin (5uM) ± Verapamil (50uM) E3S (5uM) ± Novobiocin (10uM) Prazosin (5 um) ± Novobiocin (10uM) Quinidine (100 um) LTC4 (0.1uM) ± MK571 (50uM) E17BG (5uM) ± MK571 (10uM) Efflux ratio (B-A/A-B) Efflux ratio (B-A/A-B) Digoxin (5uM) ± Verapamil (50uM) E3S (5uM) ± Novobiocin (10uM) Prazosin (5 um) ± Novobiocin (10uM) LTC4 (0.1uM) ± MK571 (50uM) E17BG (5uM) ± MK571 (10uM) Perloff et al., 2010 AAPS Annual Meeting

27 Probe Substrate Validation: Digoxin Transport in MDR1-LLC-PK 1 Cells P-gp facilitated Transport Time dependence P-gp facilitated Transport Concentration dependence 1200 digoxin 0.5 um digoxin 5.0 um 1400 digoxin 30 min 1000 digoxin 50 um 1200 digoxin 60 min digoxin 90 min P-gp transport [pmol] P-gp transport [pmol] digoxin 120 min min um Transport of the P-gp probe substrate digoxin is linear over the concentration range of µm and incubation times of min. Standard assay conditions: 5 90 min

28 Probe Substrate Validation: E3S Transport in Caco-2 Cells Net E3S transport [pmol] um 1.0 um 10 um Time dependence Net E3S transport [pmol] Concentration dependence 45 min 90 min 120 min 180 min min um Transport of the BCRP probe substrate estrone-3-sulfate is linear over the concentration range of µm and incubation times of min. Standard assay conditions: 5 90 min

29 Positive Control Inhibitor Validation Caco-2 cells Perloff et al., 2010 AAPS Annual Meeting

30 Positive Control Inhibitor Selectivity P-gp and BCRP inhibitors tested in Caco-2 cells P-gp Inhibition (digoxin efflux) BCRP inhibition (E3S efflux) Relative Selectivity IC 50 [um] SE* IC 50 [um] SE* P-gp BCRP Ketoconazole Quinidine >300 n/a ++ - Verapamil Elacridar Novobiocin >100 n/a Fumitremorgin C Sulfasalazine >100 n/a * Standard Error of the parameter fit, SigmaPlot v8.0 Perloff et al., 2010 AAPS Annual Meeting

31 Calculations: P app and Efflux Ratios Apparent Permeability (P app ) P app [cm/s] = V r /C 0 x 1/S x dc/dt V r is the volume in the receiver chamber [cm 3 ] C 0 is the concentration in the donor chamber at t=0 [mm] S is the filter surface area [cm 2 ] dc/dt is the is the linear slope of the drug concentration in the receiver chamber with time after correcting for dilution [mm/s] Efflux Ratio Efflux Ratio = P app (B to A) / P app (A to B) P app (B-A) P app (A-B) is the P app value measured in the B to A direction is the P app value measured in the A to B direction

32 Calculations: IC50 Value IC 50 Value F= 1 [(I max * I c ) / ( I c + IC 50c )] (Hill equation) F is the fraction of control activity remaining in presence of the inhibitor I is the inhibitor concentration [um] I max c IC 50 is the maximal inhibitory effect [fraction of control activity] is the Hill exponent is the inhibitor concentration achieving half maximal inhibition [um] If I max = 0 (i.e. there is 100% inhibition) IC 50 is the inhibitor concentration achieving 50% inhibition Constraining I max to 100% inhibition in the non-linear regression is advisable in most cases (lack of complete inhibition is most commonly due to solubility and/or toxicity issues of the inhibitor at higher concentration)

33 Calculations: Inhibition (fraction of control) There is considerable discussion in the scientific community regarding the most appropriate way to calculate the degree of P-gp inhibition An industry initiative to assess inter-laboratory variability in P-gp inhibition testing and evaluate various calculation methods is ongoing Approaches include assessment of the inhibitors effect on probe substrate s Efflux ratio (B-to-A / A-to-B) Net flux ([B-to-A] [A-to-B]) Unidirectional permeability (B-to-A or A-to-B Papp only) This gets further complicated by the option of factoring in the effect of a positive control inhibitor (assumed to cause maximum effect) vs. measuring against baseline (no inhibitor) activity alone Choice of calculation method has significant impact on IC 50 Preliminary findings to be presented at the AAPS Transporter Workshop in March 2011 (Caroline Lee, Pfizer) Publications, updated guidance to follow

34 Decision Tree for P-gp / BCRP Substrates Bidirectional transport assay Is Is efflux ratio 2? * Is Is efflux inhibited by P-gp/BCRP inhibitors? ** ** YES NO Is Is efflux ratio < 2? * Unlikely to to be a P-gp/BCRP substrate Likely to be P-gp/BCRP substrate In In vivo drug interaction study may be warranted Transporters other than P-gp or or BCRP might be involved Further in in vitro studies to to identify transporters may be warranted * There is concern that this value is too liberal and will lead to too many 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 (2006) and ITC White Paper (2010)

35 Example: Identification of a Test Article as a P-gp Substrate in Caco-2 Cells 25 positive control +/- inhibitors test article without inhibitors test article with 25 um ketoconazole test article with 10 um cyclosporine Efflux Ratios: Papp [10-6 cm/s] A-B Papp B-A Papp digoxin 5 um digoxin 5 um + keto 25 um digoxin 5 um + csa 10 um TA 1.0 um TA 3.0 um 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 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 likely a P-gp substrate

36 Decision Tree for P-gp/BCRP Inhibitors Bidirectional transport assay with P-gp/BCRP probe substrate Net flux ratio of of probe substrate decreases with increasing concentrations of of test compound P-gp/BCRP inhibitor Net flux ratio of of probe substrate is is not affected by increasing concentrations of of test compound Poor or or non-inhibitor of of P-gp/BCRP Determine IC50 (or Ki) [I]/IC50 > 0.1, [I [I 2 ]/IC50>10 In In vivo drug interaction study may be advisable [I]/IC50 < 0.1, [I [I 2 ]/IC50>10 In In vivo drug interaction study likely not needed [I] = total steady-state C max at highest dose [I 2 ] = intestinal concentration FDA DRAFT Guidance (2006) and ITC White Paper (2010)

37 Example: Identification of a Test Article as a P-gp Inhibitor in Caco-2 Cells no inhibition probe P-gp inhibitors as positive controls increasing concentation of the test article 14 Efflux Ratios: Papp [10-6 cm/s] A-B Papp B-A Papp digoxin 5 um digoxin 5 um + keto 25 um digoxin 5 um + csa 10 um 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 Test article shows concentration dependent inhibition of digoxin efflux P-gp inhibitor determine IC 50

38 Example: Identification of a Test Article as a P-gp Inhibitor in Caco-2 Cells % Inhibition of digoxin efflux IC 50 = 1.2 µm [I] / IC 50 > 0.1 is the recommended cut-off for in vivo testing If [I] (steady-state total C max after highest dose) is >0.12 um, an in vivo P-gp interaction study might be warranted Test article concentration [um]

39 Interpretation of Results Highly permeable compounds may not be identified as substrates, however for such compounds, basolateral to apical efflux is not likely to be a significant barrier to cross membranes. Consider the following factors when interpreting bidirectional transport data: Mass balance (can impact P app and efflux ratio) Limited solubility (can underestimate P app or % inhibition) Toxicity (can mimic inhibition or mask efflux) Other transporters (probe substrates/inhibitors are not 100% selective) Radiochemical purity (impurity may impact results)

40 Mass Balance (Compound Recovery) Amount of compound recovered in the donor and receiver chambers at the end of the assay relative to the amount in the donor chamber at T=0 min Low recovery may significantly underestimate P app values and overestimate 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 cells Uptake into the cells Non-specific binding to the plastic plate Metabolism or non-metabolic instability Addition of protein (e.g. 2% BSA) may improve recovery, however, the impact on P app and efflux ratios is not fully understood Preincubation may help by saturating binding sites

41 Recommendations Wherever possible, follow recommendations in the draft regulatory guidance documents Be prepared to explain/defend alternate approaches Until documents are final, consider the docket comments and consensus documents / White Papers Contact the agency early for input on complex interaction scenarios 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 expectations Use well accepted reagents from reputable suppliers Engage experienced CRO partners to help meet your transport needs!

42 Questions? Contact information: Elke Perloff, PhD Technical Support: tel: bdbiosciences.com/webinars For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are property of Becton, Dickinson and Company BD

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