Assessing oral drug permeability and

absorption using ex vivo human tissues

© Biopta Ltd 2014

© Biopta Ltd 2014

Biopta- Leaders in Fresh

Human Tissue Research

Beltsville,

Maryland

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

Constriction

↑ blood pressure

Relaxation

↓ blood pressure

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Biopta – How we work

Fresh Human

Tissue

Pharmacology

Techniques

Scientific

Experience

Scientific

Question

Biopta Client

Protocol design

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Decision-making data

Relaxation to

Acetylcholine

Constriction to

Phenylephrine

Functional data Analysed data Interim and final reports

© Biopta Ltd 2014

• Biopta possess all the necessary specialist equipment and expertise for ex vivo pharmacology studies

Fresh Functional Tissues:

Wide Range of Endpoints

Wire Myographs

(Small smooth / cardiac

muscle contractility)

Ussing Chambers

(Mucosal membrane

transport)

Tissue baths

• Smooth muscle and

cardiac muscle

contractility

• Nerve-muscle

interaction

Ussing

chambers

• GI drug absorption,

transporters and

metabolism

• Ion channel

function

Wire myographs

• Smooth muscle and

cardiac muscle

contractility

• Nerve-muscle

interaction

Perfusion

myographs

• Pressure and

flow studies in

tubular tissues

• Vascular

permeability

Ex vivo

cultures

• Mediator release

assays (e.g.

cytokines)

© Biopta Ltd 2014

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

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

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

Species differences in oral bioavailability

Figure taken from Grass & Sinko, (2002). Adv. Drug Delivery

Reviews, 54, 433-451.

• 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

R2 = 0.97

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human oral absorption (%)

rat

oral

ab

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%)

© Biopta Ltd 2014

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

Ussing Chamber Set-Up

Tissue forms a barrier

between right and left

chambers

Voltage and current

electrodes: electrical

parameters can be

measured to monitor ion

flow, cell integrity etc

Samples can be collected

from each chamber at

various time-points

Addition of test substance to

apical or basolateral surface

of membrane

Bi-directional

membrane transport

can be measured

Heated, gassed physiological

saline solution

© Biopta Ltd 2014

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

Permeability

Human Duodenum Reference Compound Papp

Mean values + SD

n=number of patients, Papp is low for non-permeable compounds and high for highly

permeable compounds

© Biopta Ltd 2014

Biopta Fresh Human Ileum

Ussing Chamber Validation

Drug Biopta Ileum

Papps (x10-6 cm/s)

Published human gut

Papps (x10-6 cm/s)

Pharma CACO

Papps (x10-6 cm/s)

Propranolol 12 22 (ileum) 32

Cimetidine 10 3.7 (jejunum) 1.5

Antipyrine 47 50 (jejunum 52

© Biopta Ltd 2014

1.00E-09

1.00E-08

1.00E-07

1.00E-06

1.00E-05

1.00E-04

1.00E-03

1.00E-02

1.00E-01

1.00E+00

0 10 20 30 40 50 60 70 80 90 100

Pap

p c

m/s

ec

Human Clinical Fraction Absorbed (%)

Biopta Human Papp

Caco-2

PAMPA

Rabbit Colon

Rat Jejunum

PEG-4000 Mannitol Methotrexate Clonidine Verapamil

Human tissue data is the best predictor

of clinical absorption

© Biopta Ltd 2014

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

Phase 1 Metabolic Profile of Human Duodenum

Mucosa in Ussing Chamber

2C9

3A4

1A2

2D6

2C19

OH- Midazolam

OH- Tacrine

OH- Diclofenac

OH- Bufuralol

OH- Mephenytoin

© Biopta Ltd 2014

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

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: davidbunton@biopta.com

Tel: +44 141 330 3831

www.biopta.com