i metodi alternativi integrati con la sperimentazione animale: vantaggi e aspetti regolatori dal...

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


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


IN VITRO MODELS ARE APPLIED TOALL BIOLOGICAL AND TO SOME NON-BIOLOGICAL DISCIPLINES

PharmacologyMicrobiology Toxicology Pharmacokinetic & MetabolismCosmetic Research Pharmacy


RUSSEL & BURCH 3Rs (1954)

• Reduction

• Refinement

• Replacement


ECVAM

EUROPEAN CENTRE FOR THE VALIDATION OF ALTERNATIVE METHODS


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


MODELLI IN VITRO SCOPI

RIDUZIONE DELL’USO DI ANIMALI ELEVATA PRODUTTIVITA`

RAPIDA RISPOSTA LIMITATA QUANTITA` DI COMPOSTO RICHIESTA


IN VITRO ASSAY:THE DREAM

NON TOXIC

TOXIC

TARGET ORGAN TOXICITY?

METABOLISM?

PHARMACOKINETIC?

NCE


IN VITRO ASSAY:THE NIGHTMARE

TARGET ORGAN TOXICITY?

METABOLISM?PHARMACOKINETIC?

?CELL LINE?

NCE


ALTERNATIVO O COMPLEMENTARE?


Pro-clotting enzyme

Divalent cations Endotoxin

Activated clotting enzyme

Coagulogen

Clot protein

LIMULUS AMEBOCYTE LYSATE (LAL) TEST


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.


IN VITRO BONE MARROW PROGENITOR CELL CULTURES IN THE ASSESSMENT OFHAEMATOTOXIC POTENTIAL OF NEW DRUGS: ENHANCEMENT OF THE CURRENT MODEL


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


Human Cord Blood or

CIPROFLOXACIN

0

20

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80

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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



CEFAZOLIN

0

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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

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0 1000 2000 3000 4000


Per

cen

tag

e o

f g

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th o

n

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tro

l


STAVUDINE

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0 2 4 6 8


Per

cent

age

of g

row

th o

n co

ntro

l

ZIDOVUDINE

0

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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



PHENYLBUTAZONE

0

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Per

cen

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th o

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INDOMETHACIN

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0 500 1000 1500


Per

cent

age

of g

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th o

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l



CFU-GM CLONOGENICMECHANISM-NAIVE ASSAY

NCE

CFU-GM CFU-GM + Met.Act.

MYELOTOXIC POTENTIAL


METABOLIC ACTIVATIONdifferent metabolizing systems

• co-culture with stromal cells (Dexter, 1977)

• primary hepatocytes

• intestinal/liver microsomes

• intestinal/liver S9


MODELLI IN VITRO PER LA CARATTERIZZAZIONE DEL POTENZIALE EPATOTOSSICO DI XENOBIOTICI


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?


in vitro hepatotoxici

ty

PRINCIPLE

Collagenase perfusion

Liver

Leave to attach overnight

Hepatocytes

Treat with compound (different timepoints)

MTTEnzyme Leakage

Molecular Markers


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


CONTROLLO

COMPOSTO Y(20mM)

APPROCCIOMECCANICISTICO:MORFOLOGIA


1

2

1 2 3

1

2

1 2 3

1

2

1 2 3

0

1

2

3

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5

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7

8

1 2 3

0

2

4

6

8

10

12

1 2 3

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1 2 3

0

1

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5

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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

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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

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2.5

3

3.5

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4.5

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0

1

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7

8

9

1 2 3

0

0.5

1

1.5

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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


EPATOTOSSICITA` IN VITRO: CYP450ESTRAPOLAZIONI INTER-SPECIE

2E1, 3A:+++

1A1, 1A2, 17A (1B1 e 4A?): ++

2D: +

2A, 2B, 2C: ?

(Guernerich, 1997)


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.


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`.


Individual Differences in CYP3A Activity in Human Hepatocytes

0

20

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60

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100

120

140

160

180

200

CYP3A4

CYP2D6

Donors

Acti

vit

y(p

mol/

min

/million

cells)


Individual Variations in CYP3A Induction by Rifampin

0

200

400

600

800

1000

1200

1400

1600

% Induction

Donors


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).


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


AN IN VITRO MODEL TO ASSESS THE (PHOSPHO)LIPIDOGENIC POTENTIAL

OF NEW DRUGS


Cationic Amphiphilic DrugsCommon features

Hydrophobic ring structure Hydrophilic side chain with a charged

cationic amine group

NCl

NHNEt2

hydrophobic

hydrophilic

Chloroquine


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


Mechanisms which may lead to phospholipid accumulation

CADs bind to phospholipids

pHCADs

bind to phospholipases

lysosome

protein kinase C inhibited - exocytosis reduced

X


Macrophage - mesenteric lymph node.


PHOSPHOLIPIDOSIS: MULTI-LAMELLAR BODIES


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)


Target organs in vivo for phospholipid inducing agents

Drugs liver lungs retina cornea endocrine kidney brain

Amiodarone

Chloroquine ?Chlorpromazine

Triparanol ?

Clomipramine ?


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


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”


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”


ControlImipramine 65mMProcaine 100mMCompound aCompound bCompound c

Phospholipidogenic potential assessmentU-937 cell line model


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


RESULTS


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


+Blood

Brain

Endothelium

Pgp

Transcellular Paracellular Transport Proteins

Receptor-Mediated EndocytosisEffluxEfflux

AdsorptiveEndocytosis

+ ++

P-glycoprotein and the BBB


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


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


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


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)


PHARMACYPHARMACY

Intravenous Precipitation Intravenous Precipitation

ModelsModels:

available approaches and applications to screen different formulations


Why?

To avoid:

– Pain

– Phlebitis

– Erratic bioavailability

Because many drugs must be administered at concentrations higher than their aqueous solubility


How study precipitation?

In silico modelsIn Vitro models

– Static serial dilution Dropwise with stirring

– Dynamic dynamic injection dropwise without stirring

In Vivo models


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


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


IN VITRO SAFETY EVALUATION OF

POTENTIAL HAEMOLYTIC SURFACE ACTIVE

INJECTABLE DRUGS


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


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


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 %)


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



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)



In Vitro

R

v

(mL/min)inj.

(mL/min)= mL solut. /mLblood

Q

injection rate

blood flow rate



In Vitro

0

20

40

60

80

100

HGB LEAKAGE %

M 3mL/min.

M 1mL/min.

F 3mL/min.

F 1mL/min.


Genetics Research

Roles

Identify Disease Genes

Validate Disease Targets

“Right Medicine for Right Patient"

Predictive Toxicology


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


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