Assessing oral drug absorption and metabolism in human intestinal tissues


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Presentation from "Addressing Biological Barriers" Conference organised by Molecular Profiles on 22 Jan 2014 in Nottingham, UK. Fresh functional human tissues are increasingly recognised as an effective way to model oral drug absorption and metabolism; Biopta is a contract research organisation that offers services in DMPK.

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Assessing oral drug absorption and metabolism in human intestinal tissues

  1. 1. Assessing oral drug permeability and absorption using ex vivo human tissues © Biopta Ltd 2014
  2. 2. Biopta- Leaders in Fresh Human Tissue Research Glasgow, UK • • • • • • HQ and lab in Glasgow, UK Lab facility in Maryland Founded 2002 First, and currently only, GLP compliant functional human tissue CRO Experts in human tissue research Pharma and Biotech customers worldwide © Biopta Ltd 2014 Beltsville, Maryland
  3. 3. Biopta – How we work Client Scientific Question Biopta Scientific Experience Fresh Human Tissue Pharmacology Techniques Protocol design Functional data 5 min 10 min % Constriction 0 min Analysed data 100 Interim and final reports 75 50 15 min 20 min Constriction to Phenylephrine 25 25 min 0 -9 Constriction ↑ blood pressure © Biopta Ltd 2014 % Relaxation 0 -8 -7 -6 -4 Relaxation to Acetylcholine 25 50 Relaxation 75 100 -10 -5 log M [phenylephrine] -9 ↓ blood pressure -8 -7 log M [acetylcholine] -6 -5 Decision-making data
  4. 4. Fresh Functional Tissues: Wide Range of Endpoints • Biopta possess all the necessary specialist equipment and expertise for ex vivo pharmacology studies Tissue baths Ussing Perfusion Wire Myographs Ussing Chambers Wire myographs (Small smooth / cardiac (Mucosal membrane chambers myographs • • Smooth muscle and cardiac muscle contractility • Nerve-muscle interaction © Biopta Ltd 2014 • transport) Smoothmuscle and muscle contractility) • GI drug absorption, • Pressure and cardiac muscle transporters and flow studies in contractility metabolism tubular tissues Nerve-muscle • Ion channel • Vascular interaction function permeability Ex vivo cultures • Mediator release assays (e.g. cytokines)
  5. 5. Biopta Human Tissues Blood Vessels: Gastrointestinal: Others: - resistance arteries - large intestine - skin - coronary arteries - small intestine - skeletal muscle - renal arteries - oesophagus - pulmonary arteries - stomach - cerebral arteries Cardiovascular: Genitourinary: Respiratory: - atrial appendage - bladder - bronchus - ventricular muscle - urethra - trachea - uterus From both healthy and diseased patients: heart failure, asthma, COPD, diabetes, atherosclerotic tissue, ischaemic limbs, psoriatic and atopic dermatitis skin biopsies Comparative studies also available across most preclinical models © Biopta Ltd 2014
  6. 6. Human Test Systems for Drug Permeability Biopta is able to conduct experiments on human healthy and diseased tissues including gastrointestinal tract Isolate intact mucosal layer of stomach, small intestine or large intestine Human colon Measure drug absorption and metabolism © Biopta Ltd 2014
  7. 7. Why Use Fresh Human Tissue to Predict In Vivo Drug Absorption and Metabolism? • Represents the closest possible model to patients • Biopta uses the actual site of absorption that is relevant in vivo e.g. fresh small intestine for oral drugs or skin for topically-applied drugs. • Avoids species differences animal tissues and cell-based models do not fully reflect human biology, with differences in transporters, metabolic enzymes and diet © Biopta Ltd 2014
  8. 8. Species differences in oral bioavailability rat oral absorption (%) 120 R2 = 0.97 100 80 60 40 20 0 0 20 40 60 80 100 120 human oral absorption (%) • Rat intestinal permeability similar to human intestine, but human bioavailability is not predicted by rat bioavailability • Passive absorption and expression of transporters is similar in rats and humans but no correlation between rat and human metabolizing enzymes in GI tract © Biopta Ltd 2014 Figure taken from Grass & Sinko, (2002). Adv. Drug Delivery Reviews, 54, 433-451.
  9. 9. The Prediction of Human Bioavailability • Oral drugs require an estimation of the fraction reaching the systemic circulation (F). • This is estimated by F = Fa x Fg x Fh Fa = fraction absorbed into the portal circulation Fg = fraction escaping gut clearance Fh = fraction escaping hepatic clearance. • No other model reflects both human native tissue Fa and Fg in the same system (e.g. differences in transporter expression in Caco-2 cells) • Conduct comparative studies between species © Biopta Ltd 2014
  10. 10. Ussing Chamber Set-Up Addition of test substance to apical or basolateral surface of membrane Samples can be collected from each chamber at various time-points Heated, gassed physiological saline solution Voltage and current electrodes: electrical parameters can be measured to monitor ion © Biopta Ltd 2014 flow, cell integrity etc Bi-directional membrane transport can be measured Tissue forms a barrier between right and left chambers
  11. 11. How is the rate of absorption measured in vitro? Permeability Co-efficient (Papp) The following formula is used to calculate the permeability co-efficient of a compound: Papp = VR/(A.C0).dC/dt (cm/s) Where: VR = Volume of receiver chamber A = Area of tissue exposed to compound C0 = Initial concentration of compound dC/dt = Slope of concentration versus time This is a standard measure of the rate of absorption, which can be used to compare to human in vivo fraction absorbed © Biopta Ltd 2014
  12. 12. Permeability Human Duodenum Reference Compound Papp Mean values + SD © Biopta Ltd 2014 n=number of patients, Papp is low for non-permeable compounds and high for highly permeable compounds
  13. 13. Biopta Fresh Human Ileum Ussing Chamber Validation Drug Published human gut Papps (x10-6 cm/s) Pharma CACO Papps (x10-6 cm/s) Propranolol 12 22 (ileum) 32 Cimetidine © Biopta Ltd 2014 Biopta Ileum Papps (x10-6 cm/s) 10 3.7 (jejunum) 1.5 Antipyrine 47 50 (jejunum 52
  14. 14. Human tissue data is the best predictor of clinical absorption PEG-4000 1.00E+00 Mannitol Methotrexate Clonidine Verapamil 1.00E-01 1.00E-02 Papp cm/sec 1.00E-03 Biopta Human Papp Caco-2 1.00E-04 PAMPA 1.00E-05 Rabbit Colon Rat Jejunum 1.00E-06 1.00E-07 1.00E-08 1.00E-09 0 © Biopta Ltd 2014 10 20 30 40 50 60 70 Human Clinical Fraction Absorbed (%) 80 90 100
  15. 15. Human Intestinal Metabolism Modelled in the Ussing Chamber • Ussing chambers offer the opportunity to model human absorption taking into account intestinal metabolism. F = Fa x Fg x Fh • Human duodenal mucosa mounted in the Ussing chamber shows time-dependent Phase 1 and Phase 2 metabolism of model enzyme substrates. • Ussing chambers also allow the opportunity to highlight differences in intestinal absorption and metabolism between preclinical species © Biopta Ltd 2014
  16. 16. Phase 1 Metabolic Profile of Human Duodenum Mucosa in Ussing Chamber 3A4 1A2 2C9 2D6 2C19 OH- Midazolam OH- Tacrine OH- Diclofenac OH- Bufuralol OH- Mephenytoin © Biopta Ltd 2014
  17. 17. Phase 2 Metabolic Profile of Human Duodenum Mucosa in Ussing Chamber 7-OH- Coumarin Sulphate 7-OH Coumarin Glucuronide Diclofenac glucuronide Hydroxy diclofenac glucuronide 1 © Biopta Ltd 2014
  18. 18. Why Biopta? • Add commercial value and reduce risk of clinical failure • Efficacy, absorption and safety data generated in target species eliminating species differences • Conduct experiments to directly compare animal and human data • Biopta is the world leading GLP-compliant laboratory for testing in fresh, functional human tissues Email: Tel: +44 141 330 3831 © Biopta Ltd 2014