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.

1. Assessing oral drug permeability and
absorption using ex vivo human tissues
© Biopta Ltd 2014

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
© Biopta Ltd 2014