Upload
audrey-henderson
View
213
Download
1
Tags:
Embed Size (px)
Citation preview
I METODI ALTERNATIVI INTEGRATI
CON LA SPERIMENTAZIONE
ANIMALE: VANTAGGI E
ASPETTI REGOLATORI
Dal Negro G.GlaxoSmithKline S.p.A. Medicines Research CentreVerona, Italy
Copyright 2006 - GlaxoSmithKline SpA
The changing face of R&D: The traditional model
Drug Discovery Clinical Development
Efficacy, metabolism and toxicology
NewMedicinesChemicals Animals People
Copyright 2006 - GlaxoSmithKline SpA
Massive investment in in vitro technologies:
Alternatives
Focused use of animals where no
Alternatives available
Emerging technologies for
validated replacement
Discovery Research
Genetics Research Pre-clinical Development
New Product Development
Drug Discovery
NewMedicinesof ProvenValue
The changing face of R&D: Impact of new technologies on animal use
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO MODELS ARE APPLIED TOALL BIOLOGICAL AND TO SOME NON-BIOLOGICAL DISCIPLINES
PharmacologyMicrobiology Toxicology Pharmacokinetic & MetabolismCosmetic Research Pharmacy
Copyright 2006 - GlaxoSmithKline SpA
RUSSEL & BURCH 3Rs (1954)
• Reduction
• Refinement
• Replacement
Copyright 2006 - GlaxoSmithKline SpA
ECVAM
EUROPEAN CENTRE FOR THE VALIDATION OF ALTERNATIVE METHODS
Copyright 2006 - GlaxoSmithKline SpA
VALIDATION OF ALTERNATIVE METHODS(Balls et al., 1995; Curren et al., 1995)
Scientific purpose definitionDatabaseGLP-compliant protocol Inter-laboratory evaluationData collection and Stat. analysisResults evaluationReportingMethod release
Copyright 2006 - GlaxoSmithKline SpA
MODELLI IN VITRO SCOPI
RIDUZIONE DELL’USO DI ANIMALI ELEVATA PRODUTTIVITA`
RAPIDA RISPOSTA LIMITATA QUANTITA` DI COMPOSTO RICHIESTA
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO ASSAY:THE DREAM
NON TOXIC
TOXIC
TARGET ORGAN TOXICITY?
METABOLISM?
PHARMACOKINETIC?
NCE
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO ASSAY:THE NIGHTMARE
TARGET ORGAN TOXICITY?
METABOLISM?PHARMACOKINETIC?
?CELL LINE?
NCE
Copyright 2006 - GlaxoSmithKline SpA
ALTERNATIVO O COMPLEMENTARE?
Copyright 2006 - GlaxoSmithKline SpA
Pro-clotting enzyme
Divalent cations Endotoxin
Activated clotting enzyme
Coagulogen
Clot protein
LIMULUS AMEBOCYTE LYSATE (LAL) TEST
Copyright 2006 - GlaxoSmithKline SpA
3T3 NEUTRAL RED UPTAKE PHOTOIRRITATION TEST(ECVAM, 01 ottobre 1997)
Preparare due piastre da 96 pozzetti:1x104 cellule/100ml DMEM/pozzetto.
Incubare (37°C/5% CO2/24 ore).
Lavare due volte con 150ml di EBSS.
Trattare con 100ml di composto chimico in esame sciolto in EBSS; (controllo non trattato = EBSS).
Incubare (37°C/5% CO2/1 ora).
Fototossicità: Citotossicità: Esporre una piastra a Mantenere la piastra
radiazioni UVA (5J/cm2) in duplicato per (=1.67mW/cm2) per 50 min. 50 min. al buio a a temperatura ambiente. temperatura ambiente.
Lavare due volte con 150ml di EBSS.
Sostituire EBSS con il medio di coltura.Incubare (37°C/5% CO2/24 ore).
Controllare le alterazioni morfologiche.
Lavare con 150ml di EBSS.Aggiungere 100ml di soluzione di Rosso Neutro.
Incubare (37°C/5% CO2/3 ore).
Rimuovere il colorante Rosso Neutro e lavare una volta con 150ml di EBSS.Aggiungere 150ml di soluzione di estrazione (Etanolo/Acido Acetico).
Agitare la piastra per 10 minuti.
Leggere allo spettrofotometro a 540nm.
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO BONE MARROW PROGENITOR CELL CULTURES IN THE ASSESSMENT OFHAEMATOTOXIC POTENTIAL OF NEW DRUGS: ENHANCEMENT OF THE CURRENT MODEL
Copyright 2006 - GlaxoSmithKline SpA
CFU-GM EXPERIMENTAL PROTOCOL
BMC suspension
Mouse Femur Bome Marrow
Flush with IMDM + antibiotics
Centrifuge at 400Xgthen, resuspend in IMDM +
30%FBS
Adjust the suspension at 1.5x106
Mix diluted BMC+ compound
+ MCM
Distribute in Petri dishes
Incubate for 1 week Score the colonies
Copyright 2006 - GlaxoSmithKline SpA
Human Cord Blood or
CIPROFLOXACIN
0
20
40
60
80
100
120
0 100 200 300 400 500
CO NCENTRATION (mMol/L)
Per
cent
of g
row
th o
n co
ntro
l
OFLOXACIN
020
4060
80100
120
0 200 400 600 800 1000
CO NCENTRATIO N (mMol/L)Pe
rcen
t o
f gr
owth
on
cont
rol
CFU-GM CLONOGENIC ASSAY
Copyright 2006 - GlaxoSmithKline SpA
CFU-GM CLONOGENIC ASSAY
CEFAZOLIN
0
20
40
60
80
100
120
0 500 1000 1500
CO NCENTRATIO N (mMol/L)
Per
cent
age
of g
row
th o
n co
ntro
l
CEFOTAXIME
0
20
40
60
80
100
120
0 1000 2000 3000 4000
CO NCENTRATIO N (mMol/L)
Per
cen
tag
e o
f g
row
th o
n
con
tro
l
Copyright 2006 - GlaxoSmithKline SpA
STAVUDINE
0
20
40
60
80
100
120
0 2 4 6 8
CO NCENTRATIO N (mMol/L)
Per
cent
age
of g
row
th o
n co
ntro
l
ZIDOVUDINE
0
20
40
60
80
100
120
0 1 2 3 4 5 6
CO NCENTRATIO N (mMol/L)P
erce
ntag
e of
gro
wth
on
cont
rol
CFU-GM CLONOGENIC ASSAY
Copyright 2006 - GlaxoSmithKline SpA
PHENYLBUTAZONE
0
20
40
60
80
100
120
140
0 500 1000 1500
CO NCENTRATIO N (mMol/L)
Per
cen
tag
e o
f g
row
th o
n
con
tro
l
INDOMETHACIN
0
20
40
60
80
100
120
0 500 1000 1500
CO NCENTRATIO N (mMol/L)
Per
cent
age
of g
row
th o
n co
ntro
l
CFU-GM CLONOGENIC ASSAY
Copyright 2006 - GlaxoSmithKline SpA
CFU-GM CLONOGENICMECHANISM-NAIVE ASSAY
NCE
CFU-GM CFU-GM + Met.Act.
MYELOTOXIC POTENTIAL
Copyright 2006 - GlaxoSmithKline SpA
METABOLIC ACTIVATIONdifferent metabolizing systems
• co-culture with stromal cells (Dexter, 1977)
• primary hepatocytes
• intestinal/liver microsomes
• intestinal/liver S9
Copyright 2006 - GlaxoSmithKline SpA
MODELLI IN VITRO PER LA CARATTERIZZAZIONE DEL POTENZIALE EPATOTOSSICO DI XENOBIOTICI
Copyright 2006 - GlaxoSmithKline SpA
EPATOTOSSICITA` IN VITROCOLTURE PRIMARIE
PRO CONDIZIONI
SPERIMENTALI STANDARDIZZATE
ECONOMICITA` RAPIDITA` DI
RISPOSTA
CONTRO VITALITA` E
FUNZIONALITA` LIMITATE NEL TEMPO
RELAZIONI INTERCELLULARI
METAB. ENERGETICO BINDING PROTEICO PERDITA DI ALCUNE
CARATTERISTICHE WILDTYPE
ESTRAPOLAZIONE VITRO-VIVO?
Copyright 2006 - GlaxoSmithKline SpA
in vitro hepatotoxici
ty
PRINCIPLE
Collagenase perfusion
Liver
Leave to attach overnight
Hepatocytes
Treat with compound (different timepoints)
MTTEnzyme Leakage
Molecular Markers
Copyright 2006 - GlaxoSmithKline SpA
VALUTAZIONE QUANTITATIVA
AST*
*
0
20
40
60
80
100
controllonegativo
100 200
Concentrazione (mM)
Ril
asc
io e
nzim
ati
co
(%
)
MTT
0,00
0,10
0,20
0,30
0,40
controllonegativo
10 50 100 150 200
Concentrazione (μM)
Ass
orb
anza
Composto X
n=4
Copyright 2006 - GlaxoSmithKline SpA
CONTROLLO
COMPOSTO Y(20mM)
APPROCCIOMECCANICISTICO:MORFOLOGIA
Copyright 2006 - GlaxoSmithKline SpA
1
2
1 2 3
1
2
1 2 3
1
2
1 2 3
0
1
2
3
4
5
6
7
8
1 2 3
0
2
4
6
8
10
12
1 2 3
0
0.5
1
1.5
2
2.5
3
1 2 3
0
1
2
3
4
5
6
1 2 3
0
1
2
3
4
5
6
7
8
1 2 3
0.8
1.8
1 2 3
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 2 3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1 2 3
0
1
2
3
4
5
6
7
8
9
1 2 3
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
0.5
1.5
1 2 3
1
2
1 2 3
D30666, Acyl CoAsynthetase II
D37920, Squaleneepoxidase
rc_AA859980,unknown
X55286,HMG CoA reductase
J05210, ATPcitrate lyase
CholesterolBiosynthesis
CholesterolBiosynthesis
TCA Cycle
Fatty AcidMetabolism
0/5 3/5 3/3 0/5 2/5 5/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
ChloroquinMaprotiline
AmiodaroneAcetaminophen
Dexamethasone
Transcripts Correlating with Hepatic Phospholipidosis
Genes Pathways
Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
L M H L M H L M H L M H L M H
Copyright 2006 - GlaxoSmithKline SpA
EPATOTOSSICITA` IN VITRO: CYP450ESTRAPOLAZIONI INTER-SPECIE
2E1, 3A:+++
1A1, 1A2, 17A (1B1 e 4A?): ++
2D: +
2A, 2B, 2C: ?
(Guernerich, 1997)
Copyright 2006 - GlaxoSmithKline SpA
Source: Human livers procured but not used for transplantation
Two-step collagenase digestion Viability: >70% Yield: 10-30 billion viable cells per
isolation
Isolation of Human Hepatocytes*
* Li, A. P., Roque, M. A., Beck, D. J., Kaminski, D. L. (1992): J. Tissue Culture Methods 14:139-146.
Copyright 2006 - GlaxoSmithKline SpA
CELLULE UMANESi`, pero`.....
DIFFICOLTA` DI REPERIMENTO DIFFICOLTA` NELLA PIANIFICAZIONE SPERIMENTALE.
STATO DI CONSERVAZIONE DEL PEZZO ANATOMICO.
CONDIZIONI PATOLOGICHE DEL PEZZO ANATOMICO.
POLIMORFISMO GENICO SCARSA RIPETIBILITA`.
Copyright 2006 - GlaxoSmithKline SpA
Individual Differences in CYP3A Activity in Human Hepatocytes
0
20
40
60
80
100
120
140
160
180
200
CYP3A4
CYP2D6
Donors
Acti
vit
y(p
mol/
min
/million
cells)
Copyright 2006 - GlaxoSmithKline SpA
Individual Variations in CYP3A Induction by Rifampin
0
200
400
600
800
1000
1200
1400
1600
% Induction
Donors
Copyright 2006 - GlaxoSmithKline SpA
CELLULE UMANE CRIO-PRESERVATEPRO......
PIANIFICAZIONE SPERIMENTALE.
CELLULE SANE.
USO DELLO STESSO LOTTO DI CELLULE IN DIVERSI ESPERIMENTI RIPETIBILITA`.
CARATTERIZZAZIONE GENICA DI ALCUNE IMPORTANTI “ATTITUDINI” DEL DONATORE (PER ESEMPIO, METABOLISMO).
Copyright 2006 - GlaxoSmithKline SpA
POLIMORFISMO GENICO POOL DI CELLULE
PROVENIENTI DA DONATORI CON “ATTITUDINI”
METABOLICHE DIVERSE (“BUONI” E “CATTIVI” METABOLIZZATORI).
POSSIBILITA` DI ESPLORARE L’EFFETTO TOSSICO
IN CELLULE CON CARATTERISTICHE BORDERLINE PER UNA SPECIFICA VIA METABOLICA.
POSSIBILITA` DI ESPLORARE MECCANISMI SPECIE-SPECIFICI.
CELLULE UMANE CRIO-PRESERVATE......PRO
Copyright 2006 - GlaxoSmithKline SpA
AN IN VITRO MODEL TO ASSESS THE (PHOSPHO)LIPIDOGENIC POTENTIAL
OF NEW DRUGS
Copyright 2006 - GlaxoSmithKline SpA
Cationic Amphiphilic DrugsCommon features
Hydrophobic ring structure Hydrophilic side chain with a charged
cationic amine group
NCl
NHNEt2
hydrophobic
hydrophilic
Chloroquine
Copyright 2006 - GlaxoSmithKline SpA
CATIONIC AMPHIPHILIC DRUGS
Class /Drug
AnoreticChlorpentermineCloforexFenfluramine
AntiarrhythmicAmiodaronePerhexiline
Antidepressant1-Choloramitriptyline1-Chloro-10,11-
dehydroamitriptylineImipramineIprindoleMaprotilineZimelidineChlomipramine
AntimalarialChloroquineMepacrine
Antianginal4,4’-Diethylamino-
ethoxyhexestrol
AntibioticGentamicinErythromycin
AntiestrogenicTamoxifen
AntithromboticRMI 10.393
AntihistaminicChlorcyclizineMeclizineCyclizineNorchlorcyclizineHomochlorcyclizineHydroxyzine
AntianxietyEthylfluclozepateHaloperidol
Cholesterol synthesisinhibitor
AY-9944BoxidineTriparanol
Interferon inducerTiloroneS3458-0
NeurolepticClozapine
SchistomicidalIA-3
SecretolyticAmbroxolBromhexine
Serotonin uptake inhibitorFluoxetineCitalopram
TranquilizerAC-3579
Reasor M. 1989, Toxicology and Applied Pharmacology 97: 47-56
Copyright 2006 - GlaxoSmithKline SpA
Mechanisms which may lead to phospholipid accumulation
CADs bind to phospholipids
pHCADs
bind to phospholipases
lysosome
protein kinase C inhibited - exocytosis reduced
X
Copyright 2006 - GlaxoSmithKline SpA
Macrophage - mesenteric lymph node.
Copyright 2006 - GlaxoSmithKline SpA
PHOSPHOLIPIDOSIS: MULTI-LAMELLAR BODIES
Copyright 2006 - GlaxoSmithKline SpA
Target organs
Range of organs affected - varies with different agents
Primarily tissues with high cell membrane turnover (eg macrophages in lung or immune system) or organs with high lipid or phospholipid biosynthesis (eg adrenals and retina)
Organs affected can be different for the same compound in different species (Tafenoquine - lung in rat and dog, cornea in man)
Copyright 2006 - GlaxoSmithKline SpA
Target organs in vivo for phospholipid inducing agents
Drugs liver lungs retina cornea endocrine kidney brain
Amiodarone
Chloroquine ?Chlorpromazine
Triparanol ?
Clomipramine ?
Copyright 2006 - GlaxoSmithKline SpA
Phospholipids in toxicology studies
Primary detection is by light microscopy
This is not definitive as various forms of vacuolation may occur which appear identical to phospholipid accumulation at light microscopy
Diagnosis is via electron microscopy in which multilamellar bodies are definitive
Other staining techniques are under evaluation
Copyright 2006 - GlaxoSmithKline SpA
Phospholipid risk assessment
Recent experience with the FDA Neurosciences division (SB271046)
“The accumulation of phospholipid in several organs and tissues which has been observed in several animal studies is of lesser concern, since such a finding has been noted with a number of other drugs and is not believed to have any pathological consequences”
Copyright 2006 - GlaxoSmithKline SpA
Phospholipid risk assessment
However,
“ If the foamy macrophages identified in the preclinical studies are determined to be secondary to phospholipidosis, this will have implications in the follow-up required in future clinical trials. We would appreciate additional information concerning the identification of these lesions”
Copyright 2006 - GlaxoSmithKline SpA
ControlImipramine 65mMProcaine 100mMCompound aCompound bCompound c
Phospholipidogenic potential assessmentU-937 cell line model
Copyright 2006 - GlaxoSmithKline SpA
ControlImipramine 65mMProcaine 100mMCompound aCompound bCompound c
Phospholipidogenic potential assessmentU-937 cell line model
Copyright 2006 - GlaxoSmithKline SpA
ControlImipramine 65mMProcaine 100mMCompound aCompound bCompound c
Phospholipidogenic potential assessmentU-937 cell line model
Copyright 2006 - GlaxoSmithKline SpA
Y/N TESTEDIn vivo
Y IMIPRAMINE PhospholipidosisDrew et al., 1981; Hansson et al., 1997; Xia et al.,1997 and 2000
Y AMIODARONE Phospholipidosis Honegger et al., 1993; Schneider et al., 1997
Y FLUOXETINE Phospholipidosis Reasor, 1989; Wladyslawa et al., 1996
Y QUINIDINE Phospholipidosis Jagel et al., 1984
Y PROCAINENO phospholipidosis
(in vitro)Jagel et al., 1984
Y CHLOROQUINE Phospholipidosis Colombo et al., 1988; Schneider et al., 1997
Y AZITHROMYCIN Phospholipidosis Bambeke et al., 1996
Y TELITROMYCIN Phospholipidosis Non published reports on in vivo studies
Y ERYTHROMYCIN Phospholipidosis Reasor, 1989; Montenez et al., 1999
Y CITALOPRAM Phospholipidosis Lullmann-Rauch et al., 1983
Y MAPROTILINE Phospholipidosis Tanaka et al., 1975; Staubli et al., 1978
N VALPROIC ACIDNO phospholipidosis
(lipid deposition)Tang et al., 1995
N ZIDOVUDINENO phospholipidosis
(lipid deposition)Agarwal et al., 1997; Benbrik et al., 1997;Pessayre et al., 1999
N ACETAMINOPHENNO phospholipidosis
(lipid deposition)Jaeschke et al., 2002; Waters et al., 2001
N2,3-DIDEHYDRO-3-
DEOXYTHYMIDINENO phospholipidosis
(lipid deposition)Gerschenson et al., 2001; Johnson et al., 2001;Moyle, 2000
N2,3-
DIDEOXYCYTIDINE(DDC)
NO phospholipidosis(lipid deposition)
Benbrik et al., 1997; Kakuda, 2000; Moyle, 2000
N2,3-DIDEOXYINOSINE
(DDI)NO phospholipidosis
(lipid deposition)Benbrik et al., 1997; Kakuda, 2000; Moyle, 2000
N FENOFIBRATE NO lipid deposition Milosavljevic et al., 2001
N TETRACYCLINENO phospholipidosis
(lipid deposition)JW Horn et al., 1996
Casartelli et al. (2003) Cell Biology and Toxicology 19(3): 161-176
>80% PREDICTIVITY
Copyright 2006 - GlaxoSmithKline SpA
RESULTS
Copyright 2006 - GlaxoSmithKline SpA
P-glycoprotein is 1280 amino acid (170kDa) long with 12 transmembrane domains consisting of two homologous parts. Each half contains a hydrophylic domain for ATP binding. (member of the ABC active transporter family)
P-gp is located: luminal surface of hepatocytes and duct cells kidney proximal cells, enterocytes, capillary endothelial cells of brain and testes
BACKGROUND
Copyright 2006 - GlaxoSmithKline SpA
+Blood
Brain
Endothelium
Pgp
Transcellular Paracellular Transport Proteins
Receptor-Mediated EndocytosisEffluxEfflux
AdsorptiveEndocytosis
+ ++
P-glycoprotein and the BBB
Copyright 2006 - GlaxoSmithKline SpA
potentially low and extremely variable oral bioavailability
potentially low brain penetration
potentially high hepatic or renal excretion
interactions with other substrates or inhibitors
possible changes in kinetics with multiple dosing
POTENTIAL CONSEQUENCES ONSUBSTRATE DISPOSITION
Copyright 2006 - GlaxoSmithKline SpA
Distribution of 14C-XXX 2 hours after single oral 50mg/kg dose of 14C-XXX in male FVB mdr1a/1b (-/-) knockout or mdr1a/1b (+/+) control mice
A
BB r a i n C S F
Copyright 2006 - GlaxoSmithKline SpA
Distribution of 14C-XXX 2 hours after single oral 50mg/kg dose of 14C-XXX in male CD1 mice pretreated with the P-gp inhibitor GF120918
A
BBrain CSF
Copyright 2006 - GlaxoSmithKline SpA
apical side
basolateral side
Caco-2 Cells
Use of transfected cell lines with MDR1MDR1 construct for:
•1st HTS 1st HTS fluorescent probe substrate for P-gp (such as Rhodamine 123) against test compounds (as inhibitors).
•2nd Screening2nd Screening Transport experiments to show the directional transport (HPLC-MS based)
Copyright 2006 - GlaxoSmithKline SpA
PHARMACYPHARMACY
Intravenous Precipitation Intravenous Precipitation
ModelsModels:
available approaches and applications to screen different formulations
Copyright 2006 - GlaxoSmithKline SpA
Why?
To avoid:
– Pain
– Phlebitis
– Erratic bioavailability
Because many drugs must be administered at concentrations higher than their aqueous solubility
Copyright 2006 - GlaxoSmithKline SpA
How study precipitation?
In silico modelsIn Vitro models
– Static serial dilution Dropwise with stirring
– Dynamic dynamic injection dropwise without stirring
In Vivo models
Copyright 2006 - GlaxoSmithKline SpA
SCHEMES OF THE TWO MODELS
1:1 1:2 1:4 1:8 1:1024
Waste
Water BathT= 37 °C
Peristaltic Pump
Flow cellT= 37 °C
UV/VIS source Detector
Cir
cula
ting
Flu
id
distance (d)
Static Dynamic
Copyright 2006 - GlaxoSmithKline SpA
selected where the drug does not absorb.Every absorption is due to the passage of a precipitate (or to mixing effects)
0
0.5
1
1.5
2
2.5
0 50 100 150 200Time (sec)
Ab
so
rban
ce
420nm 600 nm 800 nm
Variation of the absorbance as function of wavelength for phenytoin (Zentropil) injected @1ml/min into ISPB7.4 @8ml/min
DYNAMIC INJECTION MODELWavelengh
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO SAFETY EVALUATION OF
POTENTIAL HAEMOLYTIC SURFACE ACTIVE
INJECTABLE DRUGS
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO ASSESS THE SURFACE-ACTIVE POTENTIAL OF A DRUG
De Nuoy Type Ring Tensiometry (DNT): measurement of the decrease in surface tension at increasing concentrations.
Nuclear Magnetic Resonance (NMR): evaluation of the chemical shift in proton position association of molecules.
Photon Correlation Spectroscopy (PCS): evaluation of presence and size of aggregates.
Critical Micellar Concentration
Copyright 2006 - GlaxoSmithKline SpA
IN VITRO SAFETY EVALUATION OF POTENTIAL HAEMOLYTIC SURFACE ACTIVE INJECTABLE DRUGSPARAMETERS TO BE TAKEN INTO ACCOUNT
Temperature pH Drug concentration Injection/infusion rate Formulation Animal species
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO EVALUATE THE HAEMOLYTIC POTENTIAL OF A DRUG IN VIVO:
Species: dog
Endpoints: haematology (RBC, MCV, HCT , Ret., RDW, Morphology), clinical chemistry (LDH, plasma HGB), clinical signs (anaemia, haemoglobinuria, vomiting, cardiovascular signs).
IN VITRO:
Species: man
Endpoints: haemolysis (by mean of HGB leaked % and K leaked %)
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO EVALUATE THE HAEMOLYTIC POTENTIAL OF A DRUG
In Vivo
PLASMA HAEMOGLOBIN CONCENTRATION
0
100
200
300
400
500
600
Pre. I.A.D. +30' +4h +8h +24h
Timepoint
mg
/dL
51 M GP152 M GP253 M GP354 M GP455 M GP5
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO EVALUATE THE HAEMOLYTIC POTENTIAL OF A DRUG
In Vitro
pump 1blood
pump 2test drug
HGB Leakage %K Leakage %
haemolysis occurs in the immediate vicinityof the injection site (where the drug concentration is the highest)
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO EVALUATE THE HAEMOLYTIC POTENTIAL OF A DRUG
In Vitro
R
v
(mL/min)inj.
(mL/min)= mL solut. /mLblood
Q
injection rate
blood flow rate
Copyright 2006 - GlaxoSmithKline SpA
HAEMOLYSIS AND SURFACE AGENTSHOW TO EVALUATE THE HAEMOLYTIC POTENTIAL OF A DRUG
In Vitro
0
20
40
60
80
100
HGB LEAKAGE %
M 3mL/min.
M 1mL/min.
F 3mL/min.
F 1mL/min.
Copyright 2006 - GlaxoSmithKline SpA
Genetics Research
Roles
Identify Disease Genes
Validate Disease Targets
“Right Medicine for Right Patient"
Predictive Toxicology
Copyright 2006 - GlaxoSmithKline SpA
EXAMPLE OF REDUCTION AND OPTIMIZATIONPharmacology
YESTERDAY
Isolated organs
TODAY
Immortalized transfected cell lines (human or animal)
Cell membranes obtained from cell lines
transgenic
normal
ad hoc species
Copyright 2006 - GlaxoSmithKline SpA