Assessing oral drug absorption and metabolism in human intestinal tissues
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Transcript of Assessing oral drug absorption and metabolism in human intestinal tissues
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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log M [phenylephrine]
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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
so
rp
tio
n (
%)
© 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: [email protected]
Tel: +44 141 330 3831
www.biopta.com