1 Role of ADME in Drug Discovery (Míriam Zanuy, Biotech App 18 Oct 2021)

Role of ADME in Drug Discovery:
in vitro screening methodology

and in vivo models.
Miriam Zanuy, PhD

Pharmacokinetic and Drug Metabolism Department.
Almirall Research Center
Biotechnological Applications. Master of Biothecnology
Barcelona, 18th October 2021

1
Outline
 Drug discovery and drug development
 Pharmaceutical Industry: Facts & Figures
 R&D efficiency
 The way to a new drug
 Clinical failures
 Overview of the relevance of ADME in Drug Discovery

 General knowledge: What ADME is?
 Absorption
 Distribution
 Metabolism
 Excretion (role of transporters).
2
Outline
 ADME: Methodology and relevance in Drug Discovery

 Screening cascade
 An ADME Department
 ADME objectives in Drug Discovery
 ADME assays in Drug Discovery
 Absorption
 Distribution
 Metabolism
 Excretion
 In vivo
3
Drug Discovery
&
Drug Development
4
Drug Discovery & Drug Development
Pharmaceutical Industry: Facts & Figures
2020 ranking of the global top 10 pharmaceutical and biotech companies based on
revenue (in billion U.S. dollars)
5
Pharmaceutical Industry: Facts & Figures
Top 10 pharmaceutical products by sales worldwide in 2020 (in million U.S. dollars)
6
R&D efficiency
Elliott Med. Chem. Lett. (2012)
7
Once upon a Time
Pharmaceutical and Bioanalysis

The way to a new drug
It is necessary an amount of 700 millions of euros

and more or less 15 years to transform an idea into
a drug with authorization to be sold.
Laufer et al. Angew. Essays (2013)

Clinical failures
1991: Kola I.et al. Nature Review.Drug Discovery (2004)

2000:
11
Overview of the relevance of
ADME
in Drug Discovery
12
Overview of the relevance of ADME in Drug Discovery
What ADME is?
“ADME is the detailed study of all the

processes that a drug could suffer in an
organism”
What ADME is?
 Absorption: process by which drug

molecules move from the site of
administration to the systemic
circulation
 Distribution: movement of drug

molecules from the systemic circulation
to extravascular sites
 Metabolism: enzymatic breakdown

of a drug
 Exrection: passive or active transport

of drug molecules into the urine or bile
ADME -T Journal of Controlled Release 161 (2012) 446–460

Absorption (Release + Absorption)
Active drug
Release Desintegration, disgregation and
Pharmaceutical preparations disolution
http://notass.blogia.com/2008/051003-formas-farmaceuticas-solidas.php
http://www.zambon.es/servicios/atlas/fichas/4002.htm
http://www.forocoches.com/foro/showthread.php?t=2252773
Absorption (Realase + Absorption)
Cellular membrane
Difusion through the membrane

Molecular size
Liposolubility
Ionization
Presence of specific transport mechanisms
http://bioudescucuta.blogspot.com.es/2012/03/organizacion-general-y-
funciones-de-las.html
Absorption (Intestinal absorption)
Despopoulos & Silbernagl Color Atlas of Physiology (2003)
Balimane & Chang Drug Disc. Today (2005)

17
Absorption (Skin absorption)
Penetration
Permeation
Absorption
18
D (Distribution)
 Blood transport
Dissolved drugs travel in the plasma where they can

be bind with proteins like albumin, α-glycoprotein
or lipoproteins or they could be also bind to blood
cells.
 Distribution into the tissues

The drug achieves the tissues thanks to gradient
concentration.
This phase depend on some drug characteristics

like size of the drug, liposolubility, ionization state
and binding to plasmatic proteins, tissue blood flow
and endothelial characteristics.
http://journalmex.wordpress.com/2009/02/21/prevencion-del-tromboembolismo-venoso/
M (Metabolism)
Liver
Phase I metabolism drug
Oxidated
metabolite
Phase II
metabolism
Conjugated Phase I metabolism:
metabolite Oxidation, reduction and hydrolysis
Metabolites Phase II metabolism:

Compounds with Glucuronide conjugation,
better properties to be glutahtion, sulfates, aminoacids…
excreted.
http://www.escuelapedia.com/las-partes-del-cuerpo-humano-en-ingles/
M (Metabolism)
Contribution of the enzymatic systems to the metabolism of different

commercial drugs.
CYP450
(Chem. Res. Toxicol. 2008, 21, 70–83)

M (Metabolism)
Contribution of the enzymatic systems to the metabolism of different

commercial drugs.
Metabolism Enzymes
CYP450
FMO (flavin-containing
monooxygenase )
phase I
XAO (Xanthine oxidase)
Esterases
UGT (UDP-
glucuronosyltransferase )
SULT (sulfotransferase)
phase II
NAT (N-acetyltransferase)
GST (glutathione transferase)

(Chem. Res. Toxicol. 2008, 21, 70–83)
ADME: Methodology and relevance in Drug Discovery
ADME assays in Drug Discovery: METABOLISM
 Phase I metabolism or  Phase II metabolism or conjugative

functionalization reactions reactions
Phase I Phase II
Oxidation (CYP450) Glucuronidation/glucosidation
Oxidation (other) Sulfation
Reduction Methylation
Hydrolysis Acetylation
(Hydration) Amino acid conjugation
Other Glutathione conjugation
Fatty acid conjugation
Condensation
23
M (Metabolism)
Liver
CYP450
Dermic, intestinal, renal, pulmonary, cerebral, …

ttp://es.123rf.com/photo_10750124_concepto-de-rayos-x-del-corazon-humano.html
http://www.escuelapedia.com/las-partes-del-cuerpo-humano-en-ingles/
http://es.dreamstime.com/imagenes-de-archivo-sistema-digestivo-femenino-
image5591554http://blog.masalladelaciencia.es/conferencia-astrologia-hipnosis-y-cerebro/
E (Excretion)
Other fluids
(Salivary glands,
Tear glands..)
Skin
Sweat glands Drug
+
Metabolites
LUNGS
Respiration
KIDNEY
Liver
Urine
Bilis Feces
Elimination way: METABOLISM + EXCRETION

http://didactalia.net/comunidad/materialeducativo/recursos/t
ag/pulmones?rdf
“ADME is the detailed study of all the processes

that a drug could suffer in an organism”
The knowledge of the processes
(absorption, distribution,
metabolism and elimination) and
factors which alter them is essential
for the adequate selection of the
pharmaceutical form, the way of
administration, the dose and the
posology more suitable to achieve
the maximum efficacy with the less
risk in one specific patient.
http://criandoconamor.blogspot.com.es/2011_07_01_archive.html
ADME: Methodology and relevance
in Drug Discovery
27
Screening cascade
TPP (Target Product Profile)
Assay complexity
Enzimatic assay
Cellular assay Citotoxicity
Phase I metabolism Phase II metabolism PAMPA
Selectivity panel PPB Caco-2 PK IT rat
Eficacy in animals models PK IV rat PK PO rat
Output
CYPs inhibition PK IV dog Effect duration in animal models
cardiovasacular security in CNS Tissue distribution Bioactivation
Toxicology 14 days Test AMES Salt screening
Eficacy Security ADME Others

ADME Department
Facilities
Equipment &
Software
Fungible and
biological
goods
Staff
ADME Department
Medical
chemistry
Farmacology
QBCE ADME
Security
Patents Toxicology
Development
Galenic
Development
External
CROs
http://es.123rf.com/photo_2750553_figura-de-la-forma-del-cuerpo-humano-en-3d-aplicable-a-varios-conceptos.html
ADME, some objectives in Drug Discovery
• Compound characterization from research programs to

select the candidates with the major ADME potential for
Clinical Development.
• Animal ADME properties study and human prediction
• Dose and posology in humans based in the relation PK/PD
• Are the administration route the most appropriate to the
new drug?
• There are drug-drug interactions?
• Could compound penetrate to achieve the target?
• Evaluation of the bioactivation potential (reactive
metabolites formation)
• …
ADME objectives in Drug Discovery
ADME
assays
In vitro In vivo
http://es.wikipedia.org/wiki/Diclofenaco
Laufer et al. Angew. Essays (2013)

ADME assays in Drug Discovery:ABSORPTION
Optimised PK profile
DDI identified
ADME issues identified PK/PD in animal models Complete PK profile
Candidate
Feasibility Hit to Lead Lead Optimization
Selection Candidate
► Reference Stds ► Caco-2 ► RBC dist
(ADME profile)
► PAMPA (GIT or Skin)
► Hepatocyte stability ► BBB
► Penetration assay
► Plasma/blood stability ► Metabolite Profile
► PPB
► CYP induction ► Enzyme phenotyping
► Tissue Dist
► Microsomal stability ► Dog PK ► MiniPig or Monkey PK
(PhI, PhII)
► PK/PD studies ► Rat excretion
► Met Id (MS)
► Toxicokinetics
► CYP inhibition
► Bioactivation ► Human PK prediction
(PhI, PhII)
► Rat PK
► Absorption & Distribution

► Metabolism
► Bioactivation & DDI
► In vivo PK studies
► Bioanalysis/PK-PD/Toxicokinetics
Methodology Assay Biological System Parameters to determine
In vitro Oral Absorption Caco-2 Permeability A-B and B-A

PgP substrate
PAMPA Permeability A-B
Derma Skin- PAMPA Permeability
penetration
Derma 3D models Permeability
penetration
Derma Franz Cell assay Penetration
penetration
In vivo Pharmacokinetic Animal: Cmax, tmax
study Rat, mouse, guinea Biodisponibility
minipig, dog,
monkey.
ADME assays in Drug Discovery: DISTRIBUTION
Methodology Assay Biological Parameters to determine

System
In vitro Protein plasma binding (PPBs) Plasma % PPBs or fu (free fraction)
Red Cells Distribution (RBCs) Bood Ratio red blood cell/plasma
Tissue binding Lung, brain & % TBs or fu (free fraction)

skin
Hepatocytes Distribution Hepatocytes Ratio hepatocyte/media
In vivo Blood Brain Barrier (BBB) Animal Ratio brain/plasma
Tissue distribution Animal Ratio tissue/plasma
Pharmacokinetic study Animal Distribution volume


System
In vitro Phase I and phase II Subcellular % Metabolism, metabolites identification
fraction and metabolic clearance; intrinsic clearance
hepatocytes or (mic, hep)
skin
Bioactivation Subcellular Glutathione adducts formation
fraction Acylglucuronides
Reactive metabolites identification
Enzymatic Inhibition Subcellular CYP450 Inhibition , esterases
fraction IC50, Ki (irreversible inhibition )
Enzymatic Induction Hepatocytes Induction CYP3A4 and CYP1A2
Enzymatic isoforms Recombinant CYPs, esterase, UGTs, SULTs, etc.
identification enzymes (Phase I and phase II)

System
In vivo Pharmacokinetic assay Animal Total clearance(Cl), half-life of the drug
Metabolites identification
Toxicokinetics Animal Toxicokinetic profile (exposition (AUC),
security (Cmax)
PK/PD assay Animal Efficacy profile
Human prediction Animal Alometric scale
Vss, Cl, half-life
In silico Meteor Sofware Metabolism prediction
Metasite Sofware Metabolism prediction
ADME assays in Drug Discovery: EXCRETION

System
In vivo Renal excretion Animal Renal clearance (drug+ metabolites)
Biliary excretion Animal Biliary clearance (drug+ metabolites)
GIT PAMPA (Gastrointestinal parallel artificial membrane permeability
assay)
Receiver Passive diffusion

well buffer pH 7.4
4 hours
Donor
well test
compound
in buffer
Peff > 1*10-6 cm/s Good absorption

0.1*10-6 cm/s < Peff < 1*10-6 cm/s Moderate absorption
Peff< 0.1*10-6 cm/s Poor absorption
40
Routinely used in ADME as in vitro model of intestinal absorption
Correlation with gastrointestinal absorption in humans
41
(Chen et al. 2008-Pharmaceutical Research)
Skin PAMPA (Skin parallel artificial membrane permeability assay)
SC: main penetration barrier of the skin

(Hadgraft and Lane, 2005).
42
Caco-2 (human epithelial cells from colon adenocarcinoma):

In vitro intestinal permeability and transport to predict oral absorption
Papp> 10 *10-6cm/s Good absorption

1*10-6 cm/s <Papp< 10*10-6 cm/s Moderate absorption
Papp< 1 *10-6 cm/s Bad absorption
A
Passive diffusion
B
Possible PgP substrate

PgP, BCRP and MRP2 are
expressed.
Highly expressed in the human GI
and brain
Papp A→B / B→A

Compound Ratio F (%) oral rat Comments
(10-6 cm/s)
A 3.2 / 34.3 11 8
B 4.4 / 39.1 8.9 5
Ratio > 2-5, substrate Pgp
C 3.0 / 8.7 3.2 19
D 9.8 / 26.6 2.7 61
F 14.8 / 6.3 0.5 30
G 13.2 / 23.0 1.8 46
H 17.6 / 17.5 1.0 60
I 21.0 / 23.4 1.2 98
A→B > < 1.10-6 cm/s (low)
J 23.6 / 23.4 1.0 95 >5.10-6 cm/s (med-high)
K 23.3 / 22.2 1.0 40
L 31.1 / 20.1 0.7 90
M 32.5 / 19.4 0.6 15 First-pass metabolism
N 35.9 / 25.4 0.7 75

Difussion cells
Procedure
Conditions
 Pig frozen skin

 n= 6 + 1 vehicle sample
 t=24 h
 Acceptance criteria: Mass balance of 100% ± 15%
 Analysis: UPLC-MS/MS
45
Membrane in Franz cell
 Tissue ex vivo (frozen/fresh/cadaver)
 Human:
 Animals: Hairless Rat, Rat, Hairless Mouse, Mouse, Monkeys, Dogs and
Pigs
 Polymeric membranes: usually used for in vitro release testing (IVRT)

cellulose: cellulose acetate/nitrate mixed ester
Polymeric: polysulfone, polyester sulfones, polyamide, polycarbonate,
polydimethylsiloxane
 Human skin equivalents (HSEs): tissues engineered 3D skin constructs.
Adv. Reduce variability
Disadv: higher permeability
46
Human Skin Equivalents (HSEs) Bioengineered substitutes composed
of primary human skin cells
HSEs have two main applications: (keratinocytes, fibroblasts and/or
stem cells) and extracellular
 Clinical skin replacements and grafts components (mainly collagen)
 Models for drug permeability tests and

toxicity screening
47
Robotics allows us to measure levels of compounds

at different time points.
48
49
Skin PAMPA: correlation with Franz diffusion cell assay
 Skin PAMPA (buffer) versus  Skin PAMPA (45%PEG400)

Human Skin versus Human Skin
(Sinkó et al. 2012 European Journal of Pharmaceutical Sciences)
50
The progression
From: *A report of t4 – the transatlantic think tank for toxicology, a collaboration of the toxicologically oriented chairs in Baltimore, Konstanz
and Utrecht sponsored by the Doerenkamp Zbinden Foundation.
Protein Plasma Binding (PPBs) with RED method (rapid

equilibrium dialysis)
PPBs may influence in the dose, efficacy and clearance (PK/PD). Plasma
contain more than 60 proteins (albumin, acidic α-glycoprotein, lipoprotein
and globulins)
BUFFER
SAMPLE
Conc(bufferchamber )
Drug unbound (%) = × 100
Conc( samplechamber )
4h 37ºC PPB (%) = 100 − Drug unbound (%)

STIRRING
Only the unbound fraction (fu) of the drug could cross the cellular
membranes and archive the tissues.
RBC Distribution
Tissue Distribution (in vitro)
The device used is RED also and the principle is the same.
Tissue Distribution (in vivo)

LAS100X1
Tissue/plasma ratio
30
25
20 Blood
Ratio
Liver
15
Brain
10 Lung
Kidney
5
0
0 4 8 12 16 20 24
Time (h)
Important distribution in lung, kidney and liver.

Brain accumulation, may lead to CNS adverse effects
Homogenization
Centrifuge supernatant
400-500 gmax
Centrifuge supernatant
Liver
12,000-13,000 gmax
Homogenate Centrifuge supernatant
Whole cells and

Resuspend pellet
nuclei sediment 104,000-105,000 gmax
S9 fraction Wash and re-pellet

104,000-105,000 gmax
Resuspend pellet (supernatant)
(-80oC)
Mitochondrial fraction
Wash and re-pellet (pellet)
7,500-8,500 gmax
Cytosolic fraction
(supernatant)
SULT
Washed mitochondrial XAO
(-80oC)
fraction GST Microsomal fraction
(pellet) NAT
MT
CYP450 (pellet)
(-80oC)
ALDH FMO (-80oC)
ADH/ALDH
MAO UGT
Esterases
NAT Esterases
GST
55
MT
General
strategy In vitro
In vivo
In silico
Protein precipitation
Metabolite Isolation and SPE
generation purification LLE
Fraction collection
LC MS
(HPLC or Separation NMR
Identification
UPLC) (ICP/MS,
CE radioactivity)
GC
Quantification Synthesis
UV, MS, (need
of standards)
Radioactivity,
ICP/MS
56
General
strategy In vitro
In vivo
In silico
Metabolite
generation
57
Metabolite
generation
 In silico: computational procedures that predict metabolic transformation.

 Invitro
expert knowledge rules in metabolism
 Recombinant
bibliographic information
enzymes
 Subcellular fractions (microsomes, S9 fraction and cytosol)
Complexity
Reliability
 Blood/plasma
Metasite
 Cryopreserved hepatocytes
 Freshly isolated hepatocytes
Meteor
 Freshly isolated liver slices
 InProbable
vivo: plasma, bile, urine, faeces… samples
Plausible
58
General
strategy
Protein precipitation
Isolation and SPE
purification LLE
Fraction collection
59
Sample preparation
 Concentration, isolation and/or purification of generated metabolites
 Protein precipitation
 Precipitate proteins with organic solvent (ACN) and separate supernatant
by centrifugation
 Solid phase extraction

 Separate metabolites according to their physical and chemical properties
 Reverse phase, ion exchange (cation or anion)
 Liquid-liquid extraction
 Separate metabolites on the basis of their relative solubilities in two
different immiscible liquids
 Fraction collection
Valve 2 Valve 1 HPLC
Waste
2 2
4
3
1
6
3
4
1
6
 Preparative or semi-preparative LC
5 5
Fracc. 1
Fracc. 2
60
General
strategy
MS
Identification NMR
(ICP/MS,
radioactivity)
61
Mass spectrometry
Separation Data
MS spectrometer
technique analysis
Ion source Mass analyzer Detector
Atmospheric - Quadrupole (Q) - Electron multiplier

pressure ionization - Triple quadrupole (TQ) - Faraday cup
- Ion trap (IT) - …
- ESI - Linear IT
- APCI - Time of flight (TOF)
-… - Orbitrap
-…
LC-MS is the dominant analytical instrumentation used in the pharmaceutical
industry for the detection, quantification and structure elucidation of drug-
related compounds
62
Mass spectrometry: examples
Case I: hydroxylation of compound A
[M+H+16]+
[M+H]+: 349 365
1.4e6
6.9e5
1.3e6
6.5e5
1.2e6 N
1.1e6
N 6.0e5
5.5e5
1.0e6
5.0e5
9.0e5
4.5e5
HOH2C
8.0e5
H 3C 4.0e5
Intensity, cps
Intensity, cps
7.0e5
3.5e5
6.0e5
3.0e5 HN
5.0e5
HN Metabolite M4 obtained in 2.5e5
4.0e5
vitro (rat liver microsomes) 2.0e5
3.0e5 1.5e5
2.0e5 1.0e5
1.0e5 5.0e4
0.0 0.0
250 260 270 280 290 300 310 320 330 340 350 360 370 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
m/z, amu m/z, amu
63
Mass spectrometry: examples
Case II: glucuronidation of compound B
[M+H]+: 461
461.3
4.0e7
[M+H+176]+: 637 637.3
5.0e7
3.8e7
4.5e7 3.6e7
3.4e7
4.0e7 3.2e7
3.0e7
HOOC
3.5e7 2.8e7 O
HO
3.0e7
HO Glucuronide obtained in 2.6e7
2.4e7
O
Inten sity , cp s
In te nsity, c ps
vivo (rat bile) 2.2e7 HO OH

2.5e7 2.0e7
1.8e7
2.0e7 1.6e7
1.4e7
1.5e7
[M+H-H2O]+: [M+Na]+: 483 1.2e7 [M+Na+176]+:
1.0e7
1.0e7 443 443.4
483.3
8.0e6
659
6.0e6 659.4
5.0e6 4.0e6 461
2.0e6
443
0.0 0.0
100 150 200 250 300 350 400 450 500 550 600 650 700 100 150 200 250 300 350 400 450 500 550 600 650 700
m/z, amu m/z, amu
64
Phenotyping: To determine which is the isoenzyme

responsible of the metabolism
M2
M5
4.0 M6
37oC,
% of metabolite formed
stirring
time 3.0
2.0
rhCYPs: CYP3A4,CYP2D6,CYP2C9,
1.0
Drug CYP2C19 & CYP1A2
COFACTOR: NADPH 0.0
1A2 2C9 2C19 2D6 3A4
Human CYP450
*rhCYPs: recombinants human isoforms

Drug-drug interaction
Inhibition CYP450
Increase plasma levels
Drug A of drug B
Induction CYP450
Drug B
Risk of inefficacy of
is metabolized
drug B
by CYPs http://encontactoconelusuario.blogspot.com.es/2009/03/no-tome-medicamentos-por-su-cuenta.html
CYP450 inhibition. Cisapride (Prepulsid): case study
Clinical interaction study

(CYP3A4 inhibition)
60
Cisapride
50
Plasma conc (ng/ml)
Cisapride+Ketoconazole
40
30
20 THERAPEUTIC RANGE
10
0
0 2 4 6 8 10 12 14
Time (hours)
Cisapride is a CYP3A4 substrate and its metabolism was inhibited with other CYP3A4
substrates/inhibitors. It was withdrawn from the US and European markets due to
QT- related problems (2000).
• Docetaxel is a drug designed

to treat mama, lung, prostate
and gastic cancer.
• It’s eliminated mainly by the

liver (CYP3A4) through hepatic
metabolism.
http://dmaf-biotech-upv.posterous.com/?page=4
The grapefruit juice inhibits CYP3A4, interfiering with

the drug elimination and increasing its toxicity risc.
CYP450 induction. Human hepatocytes (CYP1A2 y CYP3A4)
Hepatocytes isolation (perfusion) Cell culture

(48-well plates, Colagen I)
Cryopreserved Hepatocytes
Liver
(rat, dog, human)
0h 24 h 48 h 72 h 96 h
d1 d2 d3 d4 d5 ANALYSIS
Trawing and culture Test1 Test1 Test1 Substrate compound

(activity, 2 h)
PRE-INCUBATION INCUBATION
1: test compounds include rifampicin (CYP3A4) and omeprazole (CYP1A2), as positive controls
2Substrate compound: testosterone (CYP3A4), 7-etoxyresorufine (CYP1A2)
A compound is considered inductor if the change produced in the CYP activity

is ≥40% from the reference inductor (FDA)
Bioactivation, reactive metabolites
Drug + Subcellular fraction

Trap molecule
Metabolism
37oC, stirring
Instable compound or electrophile. UPLC-MS/MS
analysis
Cofactors
(NADPH, UDPGA)
Interaction with macromolecules disrupting its function
IDIOSINCRATIC REACTIONS
GSH The formation of reactive metabolites for compounds with a

There are some functional groups dose < 10mg/day is irrelevant.
which are susceptible to form
reactive metabolites
Reactive metabolites, Paracetamol & cat

UGTs
SULTs
Conjugated
NO toxics
90% (Renal excretion)
CYP450
5%
Renal
excretion
GSH
Glutathione
5%
conjugated
N-acetyl-p-
benzoquinonemine NO toxics
(NAPQI)
TOXIC http://historiasveterinarias.wordpress.com/tag/toxicidad/
In cats, the poor capacity to do glucoranidation produces an exceed of reactive

metabolite, NAPQI. This compound is capable to bind itself covalently with some
macromolecules of the hepatocyte producing hepatotoxicity and oxidative stress.
ADME assays in Drug Discovery: IN VIVO
Pharmacokinetics
Pharmacokinetics studies the temporal course of the drug and metabolites amount and
concentration in the biological fluids, tissues and excretion fluids. It is also related with the
pharmacological response and constituted optimal models to do data interpretation.
Formulation
preparation
Administration
iv, po, it, sc…
(dose, animal number, times)
Sample extraction
Culex UPLC-MS/MS
Automatic recollection of analysis
samples
(Accusampler)
Pharmakinetics parameters
Cmax (ng/ml): Maxim concentration

Fraction absorbed and velocity
Cmax
AUC (ngxh/ml): area under the curve

Amount absorbed
tmax
tmax (h): time to achieve Cmax

Velocity of absorption
F (%): Biodisponibility, fraction absorbed

Pharmakinetics parameters
Vss (l/kg): Distribution volum
Vss (l/kg)
iv route Physiologic human volumes (70 kg)
Plasma volume 0.04 l/kg
High >5
Plasmatic concentration
Extracellular space 0.21 l/kg

Water body volume 0.6 l/kg Medium 1-5
Low <1
The aqueous volume of the organism in which a
drug could be distributed depending on its
physicochemical properties. It is an apparent
volume and it is not real.
Cl: Clearance %LBF
Time Plasma/blood volume depurated of a compound High <70

by time unit through elimination process. Medium 30-70
Low <30
Cl total = Cl metabolic+ Cl biliar + Cl renal +…
%LBF: Blood Hepatic flux
t 1/2 (h): elminination half-life Therapeutic window

Time of administration
Time to reduce 50% of the concentration  Relation PK/PD
 Human prediction
…and now the quiz to evaluate the knowledge…
https://create.kahoot.it/login
Username:Mzanuy
Password: masterbiotech
Students:
www.kahoot.it
XXXXXXXXXX
Enter a nick name and play ;)

Conclusions
• ADME has an important role in Drug Discovery
• The assays done in ADME Department try to predict which could

happen with the drug when it’s administered to the overall
population
• ADME could perform in vitro and in vivo assays, which represent

a powerful tool to predict the behavior of the drug in human
species always taking into account toxicology and efficacy issues.
Questions
77
79

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Role of ADME in Drug Discovery:

in vitro screening methodology

Miriam Zanuy, PhD

Biotechnological Applications. Master of Biothecnology

Barcelona, 18th October 2021

 Overview of the relevance of ADME in Drug Discovery

 ADME: Methodology and relevance in Drug Discovery

Elliott Med. Chem. Lett. (2012)

Pharmaceutical and Bioanalysis

It is necessary an amount of 700 millions of euros

Laufer et al. Angew. Essays (2013)

1991: Kola I.et al. Nature Review.Drug Discovery (2004)

“ADME is the detailed study of all the

 Absorption: process by which drug

 Distribution: movement of drug

 Metabolism: enzymatic breakdown

 Exrection: passive or active transport

ADME -T Journal of Controlled Release 161 (2012) 446–460

Difusion through the membrane

Despopoulos & Silbernagl Color Atlas of Physiology (2003)

Balimane & Chang Drug Disc. Today (2005)

Dissolved drugs travel in the plasma where they can

 Distribution into the tissues

This phase depend on some drug characteristics

Metabolites Phase II metabolism:

Contribution of the enzymatic systems to the metabolism of different

(Chem. Res. Toxicol. 2008, 21, 70–83)

Contribution of the enzymatic systems to the metabolism of different

GST (glutathione transferase)

 Phase I metabolism or  Phase II metabolism or conjugative

Dermic, intestinal, renal, pulmonary, cerebral, …

Elimination way: METABOLISM + EXCRETION

“ADME is the detailed study of all the processes

Cellular assay Citotoxicity

Phase I metabolism Phase II metabolism PAMPA

Selectivity panel PPB Caco-2 PK IT rat

Eficacy in animals models PK IV rat PK PO rat

cardiovasacular security in CNS Tissue distribution Bioactivation

Toxicology 14 days Test AMES Salt screening

Eficacy Security ADME Others

• Compound characterization from research programs to

Laufer et al. Angew. Essays (2013)

► Absorption & Distribution

Methodology Assay Biological System Parameters to determine

In vitro Oral Absorption Caco-2 Permeability A-B and B-A

Methodology Assay Biological Parameters to determine

Red Cells Distribution (RBCs) Bood Ratio red blood cell/plasma

Tissue binding Lung, brain & % TBs or fu (free fraction)

Hepatocytes Distribution Hepatocytes Ratio hepatocyte/media

In vivo Blood Brain Barrier (BBB) Animal Ratio brain/plasma

Tissue distribution Animal Ratio tissue/plasma

Pharmacokinetic study Animal Distribution volume

Methodology Assay Biological Parameters to determine

Methodology Assay Biological Parameters to determine

Methodology Assay Biological Parameters to determine

Receiver Passive diffusion

Peff > 1*10-6 cm/s Good absorption

Skin PAMPA (Skin parallel artificial membrane permeability assay)

SC: main penetration barrier of the skin

Caco-2 (human epithelial cells from colon adenocarcinoma):

Papp> 10 *10-6cm/s Good absorption

Possible PgP substrate