NON CLINICAL DEVELOPMENT OF NON CLINICAL DEVELOPMENT OF DRUG SPECIALITIESDRUG SPECIALITIES

________________________________________________

1. 1. Preclinical studies Preclinical studies

Bernard MARCHANDBernard MARCHAND

ExploExploExplo

ProjectProjectProject

Preclinical Stage A

Preclinical Preclinical Stage AStage A Phase IPhase IPhase I Phase IIPhase IIPhase II

Phase IIIPhase IIIPhase III NDANDANDA

Post NDAPost Post NDANDA

Preclinical Stage B

Preclinical Preclinical Stage BStage B

BioPharmaceutical

ResearchTOXICOADME

Salt Selection

Phase IFormulation

PK Interactions

PB/PK

PK/PD

Up scaled Formulation

Toxicological and kineticsExpertises

PopulationKinetic

Interspecies metabolismcomparison

Pharmaceutical File

NewFormulation

PharmaceuticalSupport

RegulatoryAffairs

Pharmacopoeia

Copy Analysis

PharmacokineticPharmacokineticToxicologyToxicology

PhysicochemistryPhysicochemistryPreclinicalPreclinicalDevelopmentDevelopment

New targets New tools comingfrom development

RESEARCHPHARMACOLOGY

CHEMISTRY

StructureActivity

Relationships

BIOPHARMACEUTICALSCREENING

DEPARTMENT

Hits Identification

Lead Optimisation

Candidat Selection

Analytical methods, Absorption

Metabolic stabilitySolubility,Specific

questions...

HTS SDS Preclinical Development

ClinicalTrial

1 drug1- 3 drugs30 - 3 drugs300 - 30 drugs

Lead Optimisation Back-up

Intestinal absorption P450 Isoenzymes Metabolic Stability InhibitionMetabolic pathway InductionOther parameters BBB permeation/Cell toxicity

DISCOVERY PROCESS & BIOPHARMACY DESCRIPTORS

MAJOR TECHNICAL EVOLUTIONS IN BIOPHARMACY

* ** ** * * * * * * * * * * * * * * * **HPLC

LC/MS/MS

ANALYTICAL

DETECTION

SAMPLE PREPARATION

CELLLULAR MODELS(Caco2, hepatocytes)

GENETIC TOOLS(Human DNA)

Automation (96 wells)

SUBCELLULARMODELS

HepaticMicrosomes

(animal + man)

BIO INFORMATIC

Data AnalysisModelisation

Cassette Dosing

ABSORBED FRACTION

SOLUBILITY

Fraction of the dosesolubilised

in the intestin

SOLUBILITY

Fraction of the dosesolubilised

in the intestin

ABSORBEDFRACTION

PERMEABILITY SOLUBILITY

LIPOPHILY

PERMEABILITY

Molecular WeightNitrogenOxygene

Hydrogen BondsIonisation

PERMEABILITY

Molecular WeightNitrogenOxygene

Hydrogen BondsIonisation

Caco2 PERMEABILITY MODEL

Different TransportMechanisms

- Transcellular (passive)(lipophilic)

- Paracellular (passive)(hydrophilic)

- Transcellular (active)(transportors)

- Efflux Process(PGP)

HUMAN ENTEROCYTE CELLS

Intestin

Liver

GeneralCirculation

HEPATIC BARRIER

Metabolism RateComponent of theterminal half time

Metabolic Bioavailability(first pass effect)Metabolism

rapidity

LIPOPHILY

PREDICTION OF IN VIVO METABOLIC BIOAVAILABILITY

Km vitro Vm vitro

Km vivoVm vivo

g prot/g liver and g liver/animal

MetabolicBiovailability

Q*fu*Vm*S/(Km+S) BloodFlow

Q+fu*Vm*S/(Km+S)

PlasmaProteins

ConcentrationIN

ConcentrationOUT

MetabolitesConcentration

Ka Dose

SIMULATION IN RELATION TO DOSE

predicted clinical doses

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0,01

0,08

0,64

5,12

40,9

6

327,

68

2621

,44

SimLin

Dose (mg/kg)

DOG

+++

MAN

0%

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0,01

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5,12

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327,

68

2621

,44

SimLin

Dose (mg/kg)

+

RATRAT

0%

10%

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50%

60%

70%

80%

90%

100%

0,01

0,08

0,64

5,12

40,9

6

327,

68

2621

,44

2097

1,52

SimLin

Dose (mg/kg)

+ ++

Met

abol

ic B

ioav

aila

bilit

y

Bio

disp

onib

ilité

mét

abol

ique

Bio

disp

onib

ilité

mét

abol

ique

In vivo/In vitrocorrelation in one species :Mixture of products (cassette dosing 5/Rat - 50/Dog

1A2

6%2A6

2%2C910%

2C194%

2D630%

2E1

5%

3A443%

NATURE AND NUMBER OF INVOLVED P450

Interest in screening :- Evaluate inhibition- Avoid metabolism by only one P450

MetabolicStability

± specific inhibitors

Enterocyte humain : transport et métabolisme

UGTUGT1A61A63A43A4UGTUGT2B272B273A43A4

hOATP-B(?)

MRP-3

MRP-1

MRP-(5?)

BCRP

ISBT

LRP

MRP-1(?)

MRP-2

P-gp (MDR-1)

PepT1

ASBT

Na+/SLGT1

SPNT1

DipeptideTripeptideTransporters

MCT(drug/H+co-transporter)

SANG

VEINE

PORTE

I

N

T

E

S

T

I

N

Noyau 1A11A1

3A43A4

1A21A2

Jonctions serréesJonctions serrées

Jonctions serréesJonctions serrées

BLOOD BRAIN BARRIER MODEL

Transport Study(filter +/- cells)

Astrocytes(confluent in 3

weeks)Ringer HEPES

Drug

6 wales plates

BBCE (confluent and differenciated

in 1 week and ready to use for 5 days)

Basolateral

ApicalLC-MS-MS

Quantitation(10, 15, 20, 30, 45

min)

Co-culture of Bovin Brain Capillary Endothelial Cellswith rat astrocytes (Pr Ceccheli - Lille)

CORRELATION Caco2/BHE

-13

-12

-11

-10

-9

-8

-7

-6

-10 -9 -8 -7 -6 -5 -4

Log Papp BBB

Log

Papp

Cac

o2

Mannitol

Terbutaline Pipenzepine

Sucrose Urée

DopamineAc Acetylsalicylique

NicotineDexamethazonePindolol

HydrocortisonePropanolol

CafeinePhenytoinDiazepamR = 0.74

SCREENING IN TOXICOLOGY

MutagenesisMutagenesisMutagenesisAmes II

Automatised Micronucleus ?

Morphology, Viability, Glutathion levelCellular Toxicology with cryopreserved hepatocytes ? Cellular Cellular Toxicology with cryopreserved hepatocytes Toxicology with cryopreserved hepatocytes

In vitro model answering in vivo issues In vitro model In vitro model answering answering in vivo issues in vivo issues Ex : vacuolisation on cultured fibroblastes

Toxicogenomics ?Toxicogenomics Toxicogenomics ??

? ?

RetrotranscriptionRetrotranscription, , amplification, amplification, labelinglabeling

of of messagermessager RNARNAComplementary Complementary RNARNA

CTP-Cy3CTP-Cy5

Extraction of RNAExtraction of RNA

Treated cells or tissues

Control cells or tissues

Total RNATotal RNA

Pool of treated and Pool of treated and control control cRNAscRNAs

HybridizationHybridization of of cRNAcRNA with with

complementary complementary probesprobes

Scan : ratio of Scan : ratio of fluorescencesfluorescences

Data analysisData analysis

TOXICOGENOMICS APPROACH

maxmin

Control Phenobarbital

CYP2BCYP3A

RESULTS

Gene Category Selected Genes

•Apoptosis

•Cell cycle

•DNA damage/Repair

•Inflammation

•Oncogene

•Stress response

•Peroxisome Proliferators

•Transcription factors, growth factors

•Plasma transport

•Phase I

•Phase II

•Phase 0/III

•CYPs regulating nuclear factors

•Bax, Bcl-2, Bcl-X, c-myc, c-fos, caspase 7-8,CD 27, TNF, Smp30

•Cyclin A-B1- D1/2/3-E1, cdK 2-4-6,JNK-1, Telomerase

•GADD45, GADD153, MGMT, p16, poly(ADP-ribose) synthetase

•IL 1, IL-6 ,IL11, IL-15, cyclooxygenase-2

•c-jun, c-myc, elk-1

•Oxidative stress genes, ApoJ, Hsp70, Heme oxygenase 2, SOD

•Enoyl coA hydratase, PPAR α, Acyl coA oxidase

•C/EBPα, IκB-α, NFκB, erk-1, p38, HGF, TGFB RII•albumin, transferrin

•CYP P450s (22), FMO, EH, MAO

•GST (4), UGT(10), SULT(4)

•MDR1, MRP (6), BSEP, OATP (4), OAT (2), OCT (2)

•CAR, PXR, RXR, GR

175 human genes involved in drug metabolism at the hepatic level

From gene expression … to phenotypic outcome

Hub of metabolism

Predose rat10

Time2.00 4.00 6.00 8.00 10.00

%

0

100179003003 1: TOF MS ES+

BPI2.91e4

0.69212.10

1.64154.05

10.50531.41

5.14679.51

3.58194.08

1.69217.12 2.74

190.055.09

220.14

10.22391.28

9.95611.367.93

326.387.31304.30

5.94307.20 6.74

343.30

9.37282.28

8.74254.25

10.65208.04

Metabonomics : structural tools

Separation of intended compounds(lipids, amino-acids, peptides…)

UPLC-TOF-MSanalysis

Fingerprint of biological medium

1H- NMR analysis

Loading plot

Control Treated

One markerm/z, Rt

Biomarkers per subject(≈ 20 000 compounds) to be reduced to a couple of hundreds

Omics : From Fingerprints to Biomarkers

Analysis of unchanged

Structuralanalysis

Biomarkers

Kinetic parameters(enzymologie and PK)

Metabolic pathways

adapt the designs of the study

Biochemical pathways

MetabonomicsMetabonomics0 20 40 60 80 100 120 140

Time (min)

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PK /PD

Metabonomics Approach

HANDLING THE DATA

Databases for correct data use, the new challenge for tomorrow ?

Tools for rapid assessment of metabolism are available but how we handle the data has not yet been completely mastered

Data base

Log K ’CalculatedLogP, Rate of metabolism

Solubility, LogP Caco-2 Papp MicrosomalKm/Vm IC50 inhibition n-in-one dosing CYP450 Km, Vm Ki

inhibition constant induction potential

n-in-one dosingin Man ?

Sorting molecules with Warningsand Metabolic SAR Sorting molecules with

in vivo scaled up data and Metabolic QSAR

Sorting molecules with partial or total

rebuilding of theentire population

Theoreticalapproach

HTS SDSPreclinical

development1st administration

to Man

Back up

PRECLINICAL STUDYPRECLINICAL STUDY PROGRAMMEPROGRAMME

Stage B6 6 monthsmonths

Stage A4 4 monthsmonths

-Dose Ranging (3-7d) Rat + Non-Rodt- Ames test- Mouse Lymph.

-Drug Subst. Analyt. Chem.- Degradation

- 4 wk Tox Rat + Non-Rodt- Acute studies

Rat + Mouse PO & IP or IV- Rat Bone Marrow

micronucleus

- Choice of Salt- Tablet Formul. + Stability

- Assay Validation- Plasma Stability- TK 4 wk Rat- TK 4 wk Non-Rodt- TK Micronucleus- Def. PK Non-Rodt- Induct. Potential Rat + Non-Rdt*

* if Rat positive - Enzymes identif. (human)- Intersp. Comp. 14C- Σ Label. Cpd

- Assay developt- TK DoseRanging Rat+ Non-Rodt- PK Rat - Prel. PK Non-Rodt- Blood/Pl.Ratio- Prel. Metab. in vivo Rat- Prel. Prot. Bind. - Inhib. Potential- Intersp. Comp.- Feasibility label. cpd

200 g200 g 2.5 kg2.5 kg

Regulatory Toxicology – Early Programme #

p Phase I requirements *Single dose toxicityRepeat dose toxicity studiesGenotoxicity studiesReproductive toxicity studies

* Other requirementsPharmacology (actions relevant to the proposed route)Safety pharmacologyPharmacokinetics (preliminary studies on absorption, distribution, metabolism and excretion) and in vitrometabolism studies

## ICH M3 : ICH M3 : NonclinicalNonclinical Safety StudiesSafety Studies

Regulatory Toxicology – Later Programme #

p Phase II, III marketing application requirements *Chronic dose toxicity studiesCarcinogenicity studiesReproductive toxicity studiesAppropriate toxicity/genotoxicity studies on metabolites,impurities and/or excipient

* Other requirementsAdditional safety pharmacology (if necessary)Additional genotoxicity studies (if necessary)Phamacokinetics (studies on absorption, distribution, metabolism and excretion)

## ICH M3 : ICH M3 : NonclinicalNonclinical Safety StudiesSafety Studies

Duration of Toxicity Studies #

* NOTE in US and EU, as an alternative to repeat dose studies, single dose toxicity studies with extended examinations may support single dose human trials

Minimum duration of toxicity

Duration of clinical trials Rodent Non –rodent

Single dose 2 weeks* 2 weeks

Up to 2 weeks 2-4 weeks* 2 weeks

Up to one month one month one month

Up to 3 months 3 months 3 months

Up to 6 months 6 months 6 months - 1year

> 6 months 6 months 1 year

To support phase I and II trials in EU and phase I, II and III trials

## ICH M3 : ICH M3 : NonclinicalNonclinical Safety StudiesSafety Studies

The battery can be completed with additional test(s) when necessary.Should permit to discard at the beginning of development potential genotoxic carcinogen compounds

AMES TEST: detection of reverse mutation onS. typhimurium and E. coli (= procaryotes)

MOUSE LYMPHOMA : detection of forwardmutation on cell lineage (= eucaryotes)can also detect clastogenic effects

IN V

ITRO

IN V

ITRO

IN V

IVO

IN V

IVO

MICRONUCLEUS on rat bone marrow: detection of chromosome breaks = clastogenicity

PRECLINICAL STUDIES

GENOTOXICITY : Standard Test Battery

- ACUTE :Route: intended for human -If oral route for human :

ORAL + PARENTERAL (IV or IP)Species: MOUSE and RATExaminations :

MORTALITYCLINICAL SIGNS/ BEHAVIORGROSS OBSERVATION AT NECROPSY(Histopathology for gross lesions)

Acute toxicology profileMNLD = Maximal non-lethal doseMLD = Minimal lethal dose

PRECLINICAL STUDIES

GENERAL TOXICOLOGY

- SUBCHRONIC and CHRONICRoute : intended for human Species : Rodent = RAT

Non-Rodent = DOG or MONKEYDosing : daily (or twice daily), 3 doses + controlDuration : up to 6 months (rodents)

9 to 12 months (non-rodents)

Investigations : pluridisciplinary contributions

Define NOEL : No Effect Level or

NOAEL: No Adverse Effect Level

TARGET ORGANS - BIOMARKERS

PRECLINICAL STUDIESGENERAL TOXICOLOGY

Clinical observationsbehavior

Toxicokinetic/Metabolism (enzyme induction/inhibition)

BodyweightFood/Water intakes

Necropsy Gross observations

≈ 40 organs/tissue samplesHistology process

Histopathology Electronmicroscopy

Urinalysis

BloodHematologyred, white cells andplatelet countsBiochemistry20 to 25 parameters

STANDARD TOXICOLOGY EVALUATIONS

- EMBRYOFETAL TOXICOLOGY :Hysterectomy

- uterus content: implantations, resorptions…- external- visceral examinations of fetus- skeletal

Teratogenic effect?

- FERTILITY : Reproductive performanceMale : sperm analysis

+ histopathology of gonadsand accessory glands

Female : oestrus cycle

PRECLINICAL STUDIES

REPRODUCTIVE TOXICOLOGY

- PERI-and POSTNATAL TOXICOLOGYParturitionLactationPhysical, sensory and behavioral development of pupsSecond generation study

Species : Rodent = RAT+ Non Rodent = RABBIT (Lagomorph)for embryofetal studies

PRECLINICAL STUDIES

REPRODUCTIVE TOXICOLOGY (contld)

These studies remain necessary to detect non-genotoxiccarcinogens.Two species: RAT and MOUSETwo-year duration: LIFE SPAN for these speciesInvestigations :. Clinical observations and mortality. Feed and water intakes. Palpations: for detection of masses (subcutaneous, mammary glands,…)

. Necropsygross observationsorgan weightshistomorphologic evaluations≈ 40 tissues or organs + masses

. Statistical analysis Conclusion about carcinogenic potential

PRECLINICAL STUDIESCARCINOGENESIS

PlasmaConcentration

1000

100

10

0 1 2 3 45

Time (h)

Toxicity treshold

Peak effect

Toxic effectsToxicokinetics

Pharmacologiceffects

Pharmacokinetics

TOXICOLOGY

PRECLINICAL STUDYPRECLINICAL STUDY PROGRAMMEPROGRAMME

PrePre-- ProjectProject

Stage BStage A

4 4 monthsmonths 6 6 monthsmonths 2 2 monthsmonths1 1 monthmonth

Decision PointPreclinical Research

- Dose Ranging(3-7d)Rat + Non-Rodt

- Ames test- Mouse Lymph.

- 4 wk Tox Rat + Non-Rodt- Acute studies

Rat + Mouse PO & IP or IV- Rat Bone Marrow

micronucleus

- Drug Subst.Analyt. Chem.

- Degradation

- Choice of Salt- Tablet Formul.

+ Stability

- Assay Validation- Plasma Stability- TK 4 wk Rat- TK 4 wk Non-Rodt- TK Micronucleus- Def. PK Non-Rodt- Induct. Potential

Rat + Non-Rdt* * if Rat positive

- Enzymes identif. (human)

- Intersp. Comp. 14C- Σ Label. Cpd

Check List

Preclinical SummaryBoard Committee

Development Decision

- Production clinical batchPhase I (capsule)

Investigator brochure

- Clinical Assay- Plasma Stab. (man)- TK assay (transfer to CRO)- TK Dose Ranging

ReproTox - WBA Rat- Mass Bal. Rat & in vivo Met. - Def. Prot. Bind. (label. cpd)

- Dose Ranging ReproTox

- Assay developt- TK DoseRanging Rat+

Non-Rodt- PK Rat - Prel. PK Non-Rodt- Blood/Pl.Ratio- Prel. Metab. in vivo Rat- Prel. Prot. Bind. - Inhib. Potential- Intersp. Comp.- Feasibility label. cpd

16 kg16 kg200 g200 g 2.5 kg2.5 kg

IMPD