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NON CLINICAL DEVELOPMENT OF DRUG SPECIALITIES ________________________ Bernard MARCHAND

NON CLINICAL DEVELOPMENT OF DRUG SPECIALITIES ________________________ Bernard MARCHAND. Post NDA. NDA. Phase III. Preclinical Stage A. Phase II. Phase I. Explo. Preclinical Stage B. Project. Toxicological and kinetics Expertises. New Formulation. Bio Pharmaceutical Research.

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NON CLINICAL DEVELOPMENT OF DRUG SPECIALITIES ________________________ Bernard MARCHAND

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  1. NON CLINICAL DEVELOPMENT OF DRUG SPECIALITIES ________________________ Bernard MARCHAND

  2. Post NDA NDA Phase III Preclinical Stage A Phase II Phase I Explo Preclinical Stage B Project Toxicological and kinetics Expertises New Formulation Bio Pharmaceutical Research PK/PD Population Kinetic Pharmaceutical Support RegulatoryAffairs TOXICO ADME PK Interactions Up scaled Formulation Interspecies metabolism comparison Salt Selection PB/PK Pharmacopoeia Pharmaceutical File Copy Analysis Phase I Formulation

  3. RESEARCH PHARMACOLOGY CHEMISTRY BIOPHARMACEUTICAL SCREENING DEPARTMENT New targets New tools coming from development Hits Identification Analytical methods, Absorption Metabolic stability Solubility,Specific questions... Structure Activity Relationships Lead Optimisation Candidat Selection Pharmacokinetic Toxicology Physicochemistry Preclinical Development

  4. DISCOVERY PROCESS & BIOPHARMACY DESCRIPTORS Lead Optimisation Back-up Preclinical Development Clinical Trial HTS SDS 1- 3 drugs 1 drug 300 - 30 drugs 30 - 3 drugs  Intestinal absorption  P450 Isoenzymes  Metabolic Stability  Inhibition  Metabolic pathway  Induction  Other parameters  BBB permeation/Cell toxicity

  5. HPLC ** * * ** ** * * * * * * * * * * * * * * MAJOR TECHNICAL EVOLUTIONS IN BIOPHARMACY CassetteDosing LC/MS/MS DETECTION ANALYTICAL SAMPLE PREPARATION Automation (96 wells) CELLLULAR MODELS (Caco2, hepatocytes) GENETIC TOOLS (Human DNA) BIO INFORMATIC SUBCELLULAR MODELS Hepatic Microsomes (animal + man) Data Analysis Modelisation

  6. ABSORBED FRACTION PERMEABILITY Molecular Weight Nitrogen Oxygene Hydrogen Bonds Ionisation ABSORBED FRACTION SOLUBILITY Fraction of the dose solubilised in the intestin PERMEABILITY SOLUBILITY LIPOPHILY

  7. Caco2 PERMEABILITY MODEL HUMAN ENTEROCYTE CELLS Different Transport Mechanisms - Transcellular (passive) (lipophilic) - Paracellular (passive) (hydrophilic) - Transcellular (active) (transportors) - Efflux Process (PGP)

  8. HEPATIC BARRIER Metabolism Rate Component of the terminal half time I n t e s t i n Liver General Circulation Metabolic Bioavailability (first pass effect) Metabolism rapidity LIPOPHILY

  9. PREDICTION OF IN VIVO METABOLIC BIOAVAILABILITY Kmvitro Vmvitro Km vivo Vm vivo g prot/g liver and g liver/animal Metabolic Biovailability Blood Flow Q*fu*Vm*S/(Km+S) Q+fu*Vm*S/(Km+S) Concentration OUT Plasma Proteins Ka Dose Concentration IN Metabolites Concentration

  10. RAT RAT SimLin 100% 90% 80% 70% 60% 50% 40% 30% 20% + + 10% + 0% 5,12 0,01 0,64 0,08 40,96 327,68 2621,44 20971,52 Dose (mg/kg) SIMULATION IN RELATION TO DOSE DOG MAN SimLin SimLin 100% 100% 90% 90% 80% 80% 70% 70% 60% 60% Metabolic Bioavailability Biodisponibilité métabolique Biodisponibilité métabolique 50% 50% + 40% 40% + + 30% 30% + 20% 20% 10% 10% 0% 0% 0,01 5,12 0,08 0,64 0,01 0,08 0,64 5,12 40,96 40,96 327,68 2621,44 327,68 2621,44 Dose (mg/kg) Dose (mg/kg) predicted clinical doses In vivo/In vitrocorrelation in one species : Mixture of products (cassette dosing 5/Rat - 50/Dog

  11. NATURE AND NUMBER OF INVOLVED P450 1A2 3A4 2A6 6% 43% 2% 2C9 10% 2C19 4% Metabolic Stability ± specific inhibitors 2E1 2D6 5% 30% Interest in screening : - Avoid one polymorphic enzyme - Avoid metabolism by only one P450

  12. Enterocyte humain : transport et métabolisme Jonctions serrées S A N G V E I N E P O R T E BCRP I N T E S T I N hOATP-B(?) ISBT LRP MRP-3 MRP-1(?) MRP-2 UGT 1A6 3A4 UGT 2B27 3A4 Noyau P-gp (MDR-1) 1A1 3A4 1A2 PepT1 ASBT MRP-1 MCT (drug/H+co-transporter) Na+/SLGT1 MRP-(5?) SPNT1 Dipeptide Tripeptide Transporters Jonctions serrées

  13. BLOODBRAIN BARRIER MODELE Co-culture of Bovin Brain Capillary Endothelial Cells with rat astrocytes (Pr Ceccheli - Lille) 6 wales plates Transport Study (filter +/- cells) Drug BBCE (confluent and differenciated in 1 week and ready to use for 5 days) Apical LC-MS-MS Quantitation (10, 15, 20, 30, 45 min) Basolateral Astrocytes (confluent in 3 weeks) Ringer HEPES

  14. CORRELATION Caco2/BHE R = 0.74

  15. SCREENING IN TOXICOLOGY Mutagenesis Ames II Automatised Micronucleus ? Cellular Toxicology with cryopreserved hepatocytes ? Morphology, Viability, Glutathion level In vitro model answering in vivo issues Ex : vacuolisation on cultured fibroblastes Toxicogenomics ?

  16. TOXICOGENOMICS Global approach Cells / Tissues RNA Hybridization Image analysis Significant gene induction or repression Database mining and mecanistic interpretation

  17. min max RESULTS CYP2B CYP3A Control Phenobarbital

  18. 175 human genes involved in drug metabolism at the hepatic level 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 , Acyl coA oxidase • C/EBP, IB-, NFB, 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

  19. HANDLING THE DATA Tools for rapid assessment of metabolism are available but how we handle the data has not yet been completely mastered Back up Preclinical development 1st administration to Man HTS SDS Theoretical approach Log K ’Calculated LogP, Rate of metabolism Solubility, LogP Caco-2 Papp Microsomal Km/Vm IC50 inhibition n-in-one dosing CYP450 Km, Vm Ki inhibition constant induction potential Data base n-in-one dosing in Man ? Sorting molecules with Warnings and Metabolic SAR Sorting molecules with in vivo scaled up data and Metabolic QSAR Sorting molecules with partial or total rebuilding of the entire population Databases for correct data use, the new challenge for tomorrow ?

  20. PRECLINICAL STUDYPROGRAMME Stage A Stage B 4 months 6 months - Dose Ranging (3-7d) Rat + Non-Rodt - 4 wk Tox Rat + Non-Rodt - Acute studies Rat + Mouse PO & IP or IV - Rat Bone Marrow micronucleus - Ames test - Mouse Lymph. - 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 -  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 2.5 kg 200 g

  21. Regulatory Toxicology – Early Programme # • Phase I requirements * Single dose toxicity Repeat dose toxicity studies Genotoxicity studies Reproductive toxicity studies *Other requirements Pharmacology (actions relevant to the proposed route) Safety pharmacology Pharmacokinetics (preliminary studies on absorption, distribution, metabolism and excretion) and in vitro metabolism studies # ICH M3 : Nonclinical Safety Studies

  22. Regulatory Toxicology – Later Programme # • Phase II, III marketing application requirements * Chronic dose toxicity studies Carcinogenicity studies Reproductive toxicity studies Appropriate toxicity/genotoxicity studies on metabolites, impurities and/or excipient * Other requirements Additional safety pharmacology (if necessary) Additional genotoxicity studies (if necessary) Phamacokinetics (studies on absorption, distribution, metabolism and excretion) # ICH M3 : Nonclinical Safety Studies

  23. 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 To support phase I and II trials in EU and phase I, II and III 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 # ICH M3 : Nonclinical Safety Studies

  24. PRECLINICAL STUDIES GENOTOXICITY : Standard TestBattery • AMES TEST: detection of reversemutation on S. typhimurium and E. coli (= procaryotes) • MOUSE LYMPHOMA : detection of forwardmutation on cell lineage (= eucaryotes) can also detect clastogenic effects IN VITRO • MICRONUCLEUS on rat bone marrow: detection of chromosome breaks =clastogenicity IN VIVO The battery can be completed with additional test(s) when necessary. Should permit to discard at the beginning of development potential genotoxic carcinogen compounds

  25. PRECLINICAL STUDIES GENERAL TOXICOLOGY - ACUTE : Route: intended for human -If oral route for human :ORAL + PARENTERAL(IV or IP) Species:MOUSEandRAT Examinations : MORTALITY CLINICAL SIGNS/ BEHAVIOR GROSS OBSERVATION AT NECROPSY (Histopathology for gross lesions) Acute toxicology profile MNLD = Maximal non-lethal dose MLD = Minimal lethal dose

  26. PRECLINICAL STUDIES GENERALTOXICOLOGY - SUBCHRONIC and CHRONIC Route :intended for human Species : Rodent =RAT Non-Rodent = DOG or MONKEY Dosing : daily (or twice daily), 3 doses + control Duration :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

  27. STANDARD TOXICOLOGY EVALUATIONS Blood Hematology red, white cells and platelet counts Biochemistry 20 to 25 parameters Clinical observations behavior Bodyweight Food/Water intakes Urinalysis Toxicokinetic/Metabolism (enzyme induction/inhibition) Necropsy Gross observations  40 organs/tissue samples Histology process Histopathology Electron microscopy

  28. - EMBRYOFETAL TOXICOLOGY : Hysterectomy - uterus content: implantations, resorptions… - external - visceral examinations of fetus - skeletal Teratogenic effect? PRECLINICAL STUDIES REPRODUCTIVE TOXICOLOGY - FERTILITY : Reproductive performance Male :sperm analysis + histopathology of gonads and accessory glands Female :oestrus cycle

  29. PRECLINICAL STUDIES REPRODUCTIVE TOXICOLOGY (contld) - PERI-and POSTNATAL TOXICOLOGY Parturition Lactation Physical, sensory and behavioral development of pups Second generation study Species :Rodent =RAT + Non Rodent = RABBIT (Lagomorph) for embryofetal studies

  30. PRECLINICAL STUDIES CARCINOGENESIS These studies remain necessary to detectnon-genotoxic carcinogens. Two species: RATandMOUSETwo-year duration:LIFE SPANfor these species Investigations : . Clinical observations and mortality . Feed and water intakes . Palpations: for detection of masses (subcutaneous, mammary glands,…) . Necropsy gross observations organ weights histomorphologic evaluations  40 tissues or organs + masses . Statistical analysis  Conclusion about carcinogenic potential

  31. Peak effect Toxicokinetics Toxic effects Toxicity treshold TOXICOLOGY Plasma Concentration 1000 100 10 Pharmacologic effects Pharmacokinetics 0 1 2 3 4 5 Time (h)

  32. Toxicological Requirement • Depending on toxicity, a frequently used “safety margin” for volunteer studies is 10 fold between dose which produces toxicity in animals and dose given to humans • First dose can be 1/100 of NOAEL • Final dose can be 1/10 of NOAEL • Plasma data on drug will give levels at which toxic effects may be expected and so must not be exceeded • Awareness that animal data not necessarily predict clinical toxicity

  33. PRECLINICAL STUDYPROGRAMME 4 months 6 months 1 month 2 months Preclinical Research Decision Point Pre- Project Stage B Stage A Check List - 4 wk Tox Rat + Non-Rodt - Acute studies Rat + Mouse PO & IP or IV - Rat Bone Marrow micronucleus - Dose Ranging ReproTox - Dose Ranging (3-7d) Rat + Non-Rodt - Ames test - Mouse Lymph. Preclinical Summary - Choice of Salt - Tablet Formul. + Stability - Production clinical batch Phase I (capsule) - Drug Subst. Analyt. Chem. - Degradation Board Committee - 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) - 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 - 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 -  Label. Cpd Development Decision IMPD Investigator brochure 200 g 2.5 kg 16 kg

  34. PHASE 1 STUDIES Dosing 600-900 samples in real time

  35. ABSORPTION SINGLE DOSE PK GASTRO INTESTINAL TRACT ORAL ROUTE Administration Bile Portal vein First pass effect LIVER TARGET ORGANS (TISSUES) 1000 Cmax 100 BLOOD CIRCULATION DISTRIBUTION + ELIMINATION Concentration 10 AUC Exposure 1 EXCRETION tmax 0 4 8 12 16 20 24 Time (h)

  36. 1000 100 Concentration 10 1 0 4 8 12 16 20 24 1000 Time (h) 100 Concentration 10 1 0 4 8 12 16 20 24 Time (h) Integration of physiological parameters and in vitro measurements Qheart Kplung Kpheart Qtissues Kptissues Qmuscles Kpmuscles Q hepatic. a Kp liver Q portal v. Vmax Km fabs

  37. DMPK ISSUES IN DRUG DEVELOPMENT Inter/intra subject variability Evaluation of simple drug metabolism parameters Bioavailability Drug-drug interaction Intestinal absorption Metabolic stability Metabolic pathways P450 Isoenzymes Inhibition potential Induction potential Prediction of the main drug characteristics with respect to the entire population Rebuild (predict) of the in vivo situation

  38. PREDICTIONS IN VIVO Interindividual variability CYP1A1 CYP1A2 CYP2C9 Extreme subjects of the simulation Concentration (µM) Time (h)

  39. + inhibitor + + inductor + IN VIVO PREDICTIONS Drug-drug interactions 1000 100 Concentration 10 1 0 4 8 12 16 20 24 Time (h)

  40. PHASE II PK/PD

  41. Why a modelling approach? A clinical study per formulation Pharmacodynamic objectives Sustained release formulation PK/PD model : Relationship between plasma concentrations and effects PK model : Absorption, distribution, elimination Dissolution model : Relationship between In vitro and In vivo dissolution Simulations

  42. FORMULATION RESEARCH Oral Route Tablets and Capsules Fast Dissolving Forms Slow Release Formulation Delayed Formulation Transmucosal Route Buccal Nasal Pulmonary Transdermal Route Patches Iontophoresis devices Injectable Route Bolus and Infusion Slow Release Formulation

  43. ORAL ROUTE/PROLONGED RELEASEHYDROPHILIC MATRIX  Gastro intestinal fluids penetrate the polymer layer which, consequently, swellsand forms a gel which controls the release kinetics of the drug substance HPMC H2O

  44. ORALROUTE / HYDROPHILIC MATRIX In vitro dissolution profile % 100 50 Prolonged Release Immediate Release 0 H 0 4 8 12 16

  45. ORAL ROUTE / HYDROPHILIC MATRIX In Vivo [ng/ml] 80 Immediate Release 60 Prolonged Release 40 20 H 0 0 24 48 72

  46. ORAL ROUTE / PROLONGED RELEASESEMI SOLID LIPOPHILIC MATRIX • Control of the release kinetics is obtained by the choice of the excipient and by its hydrophilic-lipophilic balance (HLB). • Manufacturing process, excipient melting, drug substance dispersion in the molten mass and, pouring into hard gelatin capsules

  47. ORAL ROUTESEMI SOLID MATRIX STABILISATION

  48. ORAL ROUTE / PROLONGED RELEASEMICROPARTICLES • Multiparticulate dosage formas small spherical reservoir beads (0.5 à1.5 mm diameter) • Dissolution ratecontrolled by a semi-permeable membrane

  49. PROLONGED RELEASE MICROPARTICLES Filters Semi permeable membrane Drug substance + Excipients Coating solution Hot air

  50. ORAL ROUTESUSTAINED RELEASE MICROPARTICLES IR 3 x day PR 1 x day IR PR

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