Drug Developement
DRUG DEVELOPEMENT 2 Summary Acronyms used in preclinical development Life cycle of medicine Clinical trials for Beginners The Funnel Road map Example of drug development: Chemical level Pharmaceutical level Preclinical pharmacology Preclinical Toxicology Preclinical ADME 3
Preclinical development acronyms (1)
ADME Absorption, distribution, metabolism, and excretion API Active pharmaceutical ingredient: any component intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease. CFR Code of Federal Regulations CGMP, GMP (Current) good manufacturing practice CMC Chemistry manufacturing and controls CoA Certificate of analysis CRO Contract research organization CTM Clinical trial material
Cmax Maximum plasma concentration 4 Preclinical development acronyms (2) DP Drug product: finished dosage form (for example, tablet, capsule, solution) that contains an active drug ingredient, generally in association with inactive ingredients DS Drug substance: any substance that is represented for use in a drug and that, when used in manufacturing, processing, or packaging of a drug, becomes an active ingredient or a finished drug form FDA US Food and Drug Administration FIH First in human FRS Foreign related substances GLP Good laboratory practice HPLC High performance liquid chromatography ICH International Conference on Harmonization IND Investigational New Drug application MTD Maximum tolerated dose NCE New chemical entity 5 Preclinical development acronyms (3)
NDA New drug application NCI National Cancer Institute NIA National Institute on Aging NOAEL No observed adverse effect level PIB Powder in bottle PK Pharmacokinetics RAID Rapid Access to Intervention Development (preclinical program) SBIR Small Business Innovative Research (grant) STTR Small Business Technology Transfer (grant) TI Therapeutic Index TK Toxicokinetic Tmax Time of maximum plasma concentration after dose administration TTP Target product profile 6 Drug development for beginners 7 8 9 The Funnel Translating a Discovery into a Product 10 Typical problems 11 'begin with the end in mind
This adage is particularly appropriate for preclinical development, as the resulting IND must support the planned clinical trial design. For example, a clinical trial involving daily chronic administration requires repeat-dose toxicity studies in preclinical animal models. 12 Road map The drug development process is typically divided into three major steps: Discovery, Preclinical development, and Clinical trial. Preclinical development Research and Development Product development Preclinical studies Toxicology studies (PK, ADME) GLP toxicology studies IND package 13
Ethical Principles in Preclinical Study 14 Requirement to publish
Experiments should be conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and should have the approval of the Ethics Committee for Animal Experiments. 15 Ethics for experimental studies AMERICAN UNIVERSITY OF Public and private research in France BEIRUT: INSTITUTIONAL has established ethics committees ANIMAL CARE AND USE that analyze experimental protocols COMMITTEE submitted to them. REQUEST FORM FOR RESEARCH The committees may ask researchers PROTOCOL APPROVAL to develop protocols to reduce or Search for Alternatives: eliminate, if possible, any animal FRAME Guide to Searching suffering. for Alternatives to the Use of Laboratory Animals This is an additional guarantee for the civil society about the scientific justification of experiences and animal welfare Search for alternatives is encouraged! 16 Binding study Cell test
Animal models Toxicology study
Preclinical evaluation
Investigational New Drug application summarizes the results of the above activities for submission to the US FDA 17 IND Table of Contents
1. Form FDA 1571 [21 CFR 312.23(a)(1)] 2. Table of contents [21 CFR 312.23(a)(2)] 3. Introductory statement [21 CFR 312.23(a)(3)] 4. General investigational plan [21 CFR 312.23(a)(3)] 5. Investigator's brochure [21 CFR 312.23(a)(5)] 6. Protocol(s) [21 CFR 312.23(a)(6)] a. Study protocols [21 CFR 312.23(a)(6)] b. Investigator data [21 CFR 312.23(a)(6)(iii)(b)]a c. Institutional review board data [21 CFR 312.23(a)(6)(iii)(b)]a 7. Chemistry, manufacturing, and control data[21 CFR 312.23(a)(7)] 8. Pharmacology and toxicology data [21 CFR 312.23(a)(8)] 9. Previous human experience [21 CFR 312.23(a)(8)] 10. Additional information [21 CFR 312.23(a)(10)] 18 Discoveries during last years
Anti-TNF in auto-immune disease (PR)
AIDS: 5 mechanism of action
Cardiology : prevention – NOAC (Dabigatran/Rivaroxaban/Apixaban)
Anti-Alzheimer: Bapineuzumab
New anticancer drugs: Tinibs 19 2003 Blockbusters at the Drugstore (US/Worldwide; out of ~$500b)
Lipitor (Pfizer) cholesterol $6.8/10.3 billion (66%) Zocor (Merck) cholesterol $4.4/6.1 billion (72%) Zyprexa (Eli Lilly) antipsychotic $3.2/4.8 billion (72%) Norvasc (Pfizer) blood pressure $2.2/4.5 billion (40%) Procrit (J&J) anemia $3.3/4.0 billion (83%) Prevacid (TAP) ulcers $4.0/4.0 billion (100%) Nexium (AstraZeneca) ulcers $3.1/3.8 billion (82%) Plavix (BMS-Sanofi) blood thinner $2.2/3.7 billion (59%) Seretide (GSK) asthma $2.3/3.7 billion (62%) Zoloft (Pfizer) depression $2.9/3.4 billion (85%) Epogen (Amgen) anemia $3.1/? billion (?%) Celebrex (Pfizer) arthritis $2.6/? billion (?%) Source: IMS Health, March 2004 20 Me too drugs: antidepressants
1986 Fluvoxamine (Luvox; Solvay) SSRI
1987 Fluoxetine (Prozac; Lilly) SSRI
1992 Sertraline* (Zoloft; Pfizer) SSRI/NRI
1993 Venlafaxine (Effexor; Wyeth) SSRI/NRI
1996 Buproprion (Wellbutrin; Wyeth) SNRI/DRI
2002 Escitalopram (Lexapro; Forrest) SSRI
2004 Duloxetine (Cymbalta; Lilly) SSRI/NRI 21 “Big Pharma” Drug Discovery in the 21st Century
The pharmaceutical industry is short of new drugs. In the 2nd part of the 20th century, about 50-60 new drugs (NCEs) were approved by the FDA every year. In contrast, in 2002, a historical low of 18 NCEs were approved (in 2001, 24 NCEs, in 2000, 27 NCEs, in 2003, 21 NCEs). Conversely, research costs for a new drug are estimated to be in the $1-1.5 Bi. range. 22 Clinical Trial: Phase I
In Phase 1, we seek to understand what the organism does to the new molecule, which is also known as 1. pharmacokinetics: Healthy volunteers, Bioavailability and the best route of administration. 2. Research for the right dose. 3. In fact, the primary objective is to ascertain safety. Thirty percent of synthesized molecules is lost in Phase 1. 23 Clinical Trial: Phase II
It is called the therapeutic exploratory phase. In Phase 2, we seek to establish the efficacy of a drug (in subjects) with a specific illness. We also compare how the illness responds to established standard drugs or to a placebo. Phase 2 studies also allow us to determine the level of short-term safety and range of appropriate dosage. Test in a relatively small population of about 300 to 500 patients. That is where the wisdom of clinical researchers is found. 24 Clinical Trial: Phase III
This phase is also known as the therapeutic confirmatory phase. It consists of large-scale studies designed to find the efficacy and safety of the new drug relative to already accepted drugs. Thousands of subjects are involved at this point, including people from different populations. These studies are usually controlled with a placebo or with drugs that are universally accepted for treating that specific condition. Phase 3 studies allow us to build a good profile of how the drug behaves when it is used in large populations. 25 Clinical Trial: Phase IV The drug is submitted to health authorities and if it is approved, the phase 4 begins. It consists of studies that differentiate the investigational drug from other drugs in its class; studies that compare the drug’s efficacy and demonstrate the drug’s benefits in terms of pharmaco-economics. Phase 4 is never-ending. 26 27 Remarks Transition from discovery to preclinical development is a continuum, and results of preliminary pharmacology and toxicology testing often contribute to lead drug candidate selection. Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Investigational New Drug application 28 Preclinical drug development stages.
Following identification of a drug target and candidate compounds, several early activities, such as pharmacology, in vivo efficacy, and experimental toxicology, can contribute to the selection of a lead candidate for preclinical development. These preclinical activities provide the basis for an Investigational New Drug (IND) application to the FDA for permission to initiate clinical testing in humans. ADME, absorption, distribution, metabolism, and excretion; API, active pharmaceutical ingredient; PK, pharmacokinetics; Prep, preparation; Tox, toxicity. 29 Lead product Once a lead candidate is identified, a typical preclinical development program consists of six major efforts: Manufacture of drug substance (DS)/active pharmaceutical ingredient (API); Pre-formulation and formulation (dosage design); Analytical and bio-analytical methods development and validation; Metabolism and pharmacokinetics; Toxicology, both safety and genetic toxicology and possibly safety pharmacology; And good manufacturing practice (GMP) manufacture and documentation of drug product for use in clinical trials. 30 Time is money 31 Dépenses en R&D 32
DRUG DISCOVERY 33 Medicinal Chemistry The science that deals with the discovery or design of new therapeutic agents and their development into useful medicines. It involves: Synthesis Structure-Activity Relationships (SAR) Receptor interactions 34 2003 Blockbusters at the Drugstore
HO O O (US/Worldwide; out of ~$500b) CH3 O N N O H N OH H OH N O N H H O Zyprexa CH3
OH N H C Lipitor H S CH3 3 F Zocor O H N OH 2 S O O N Ph H HO (H2C)4 N N S N O CF3 HN O CF3 HO N CH3 (CH2)6 OH Prevacid Seretide CO2H H C 3 Celebrex H N NH O 2 NHCH3 HCl N MeO C CO Et 2 2 Cl CO2Me MeO Cl S N 2+ )2 Mg N Zoloft N O S - HSO4 Nexium Norvasc Cl Plavix Cl 35 Why Is It So Difficult to Make Drugs? 40 Estimates of the number of possible drug molecules average 10 . In contrast, the number of seconds since the Big Bang is only 1017.
If 10,000 chemists were available to to prepare 1 compound each per second, it would take 10,000,000,000,000,000,000,000,000,000 years to finish the job. 36 New drugs from old poisons
CH3 O HO CH3O O O O O N CH3 N CH3 H N CH3 H O H HO HO O CH3 morphine codeine heroin The reductionist approach to medicine began with the isolation of opium alkaloids 37 At least a quarter of all prescriptions dispensed in the US and UK contain, as active compounds, molecules derived from flowering plants. Other data show that 12 out of the top 25 highest earning drugs in 1995 were derived from natural products. The importance of plants as medicines in the developing world should also be acknowledged. Here, they are estimated to comprise 80% of the medication used in primary healthcare (Source: Houghton, P. J., "Roots of remedies: Plants, people and pharmaceuticals." Chem. Ind. 1999, 15). • Ethnographies show that humans are great botanical experimentors • Perhaps human brains, drugs and spices evolved together! 38 Examples of Natural Products as Leads & Drugs Cardiac glycosides, morphine, quinine, salicylic acid, taxol, camptothecin, penicillin, cyclosporin A, warfarin, artemisine…. O HO OH OH N HO OMe H H H OH O N H H H H HO O O HO HO O RO O O OMe 17-ethynylestradiol norethindrone R = H: Morphine R = Me: Codeine O OH (the "Pill"; contraceptive) (pain killer) Clarithromycin (antibacterial)
CO2H HO O CO2H O N O N OH O S O 3 H H O OH N N NH2 N 11 N 10 1 N 2 4 N O H O O Ampicillin Clavulanic acid O O HN (antibiotic) -lactamase inhibitor) O H 7 9 8 N 6 N 5 O N N H O O Augmentin (antibiotic) Cyclosporine A 39 Current use of natural product extracts
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DCM & MeOH High-Pressure Accelerated Extracts Solvent Extractor System
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Multiwell Plates QuickTime™ and a TIFF (Uncompressed) decompressor For HTS are needed to see this picture. Parallel HPLC
HT Solvent Concentrator 40 Drug Discovery One way to ―discover‖ drugs 41 Serendipitous Drug Discovery The use of nitrous oxide and ether as narcotic gases in surgery resulted from the observation that people who inhaled these chemicals [in parties] did not experience any pain after injury. The vasodilatory activity of amyl nitrite and nitroglycerin was discovered by chemists who developed strong headaches after inhaling or ingesting minor amounts. A wrong working hypothesis on chloral hydrate, which was supposed to degrade metabolically to narcotic chloroform, led to its application as a strong sedative (in reality, the metabolite trichloroethanol is the active form). Similarly, urethane was supposed to release ethanol but is a hypnotic by itself. Acetylsalicylic acid was thought to be just a better tolerable prodrug of salicylic acid, but turned out to have a unique mechanism. Warfarin was used a rat poison. 42 Serendipitous Discovery of Librium
In 1955 Roche set out to prepare a series of benzheptoxdiazines as potential new tranquilizer drugs, but the actual structure was found to be that of a quinazoline 3-oxide. N R1
X N+ O- Y R2 2.5
No active compounds were found, so the project was abandoned 43 In 1957, during a lab cleanup, a vial containing what was thought to be 1 2 the latter compound (X = 7-Cl, R = CH2NHCH3, R = C6H5) was sent for testing, and it was highly active. Further analysis showed that the actual structure of the compound was the benzodiazepine 4-oxide, Librium, presumably produced in an unexpected reaction of the corresponding chloromethyl quinazoline 3-oxide with methylamine. H NHCH3 N CH2Cl N.. CH NH CH Cl + 3 2 2 N - N - Cl O Cl + O
2.6
CH3NH2 . NHCH3 HCl NHCH3 N N CH2NHCH3 N + CH2 Cl N+ N - .. Cl - Cl O Cl N O OH
chlordiazepoxide HCl 2.3 Librium 44 Rational Drug Discovery Nearly every modification of neurotransmitters dopamine, serotonin, histamine, or acetylcholine by classical medicinal chemistry led to a compound with modified activity and selectivity. Steroid hormone modifications led to similar success stories. Many enzyme inhibitors were developed from leads that mimic the transition state of the corresponding enzyme. Protease inhibitors started from cleavage-site peptides by converting the critical amide bond into another functionality. For example, aspartyl protease inhibitors should contain the amino acids at both sides of the cleavable peptide bond, and the latter bond needs to be replaced by a stable isostere that resembles the transition state. In the 1980’s and 1990’s, computer modeling of enzyme-substrate complexes became a major driving force for rational drug discovery and the interpretation of SAR results. 45 Structure-Activity Relationships (SARs)
1868 - Crum-Brown and Fraser: Examined neuromuscular blocking effects of a variety of simple quaternary ammonium salts to determine if the quaternary amine in curare was the cause for its muscle paralytic properties. Conclusion: the physiological action is a function of chemical constitution Most drugs act at specific sites (receptor or enzyme), Activity/potency susceptible to small changes in structure, 46 Rational Drug Discovery - Piroxicam
It took Pfizer about 18 years to develop the anti-inflammatory drug piroxicam, which was launched in 1980 during the ―golden age of rational drug discovery‖. The starting point for the development was chemistry-driven, ie. to identify acidic, but not carboxylic acid-containing (salicylic acid) structurally novel compounds. Measurement of a physical property (pKa) as well as serum half-life in dogs was the guide for the synthesis program. Several generations of leads were refined and ultimately led to a successful structure with an acceptable safety and activity profile: O NH2 O O O Ar Ar N S R O O O O
OH O O NHR
NHR O N R N R O O 47 Bioisosterism
Substituents or groups with chemical or physical similarities that produce similar biological properties. Can attenuate toxicity, modify activity of lead, and/or alter pharmacokinetics of lead. 48 Drug Discovery Paradigm
Currently: Biology-driven drug discovery
Chemistry-driven drug discovery - back to the future? 49
MODERN CHEMISTRY Combinatorial Chemistry Drug design in silico 50 Usual organic synthesis: classic chemistry:
A B C
Aren’t enough to find New Chemical Entity active as Extraction of natural pharmaceutic compounds product 51
Concept of combinatorial chemistry:
Combinatorial Chemistry is a new method to reduce the time and cost of producing effective, marketable and competitive new drugs. Scientists use Combinatorial Chemistry to create large numbers of molecules that can be detected efficiently. 52 Classic synthesis, Parallel Synthesis and combinatorial Synthesis:
Combinatorial Chemistry
Parallel Organic Synthesis
Organic Synthesis Classic way 53 High Throughput Screening 54 High Throughput Screening (HTS)
This new technological requires Allows testing thousands of special HTS program: products per week overlooked Instrumentation and automation one or more biological targets (automated operating platform) simultaneously. Compound library Biochemical screening assays and / or cellular Software to record data Multidisciplinary program involving biology, biochemistry, analytical chemistry, synthetic chemistry, engineering of automated systems, and computer sciences. 55 Computer-Aided Drug Design
The method "in silico" has permeated all search paths to the discovery of new drug molecules, including the identification of "Leader" optimizing "Leader", the ADMET prediction and reorientation of a molecule data. Drug design based on the structure resulted in fruitful success of drug discovery obeying in the study of protein-ligand interactions to the law of protein-protein interactions. 56 3D Protein structures Collection of three-dimensional structures of biological macromolecules: Protein DataBank (PDB). It contains nearly 92,000 structures (June 2013) of proteins, nucleic acids (DNA and RNA) and ribonucleo-protein complexes. Techniques used to determine the protein structure: Crystallography X-ray diffraction: over 70,000 structures NMR: over 9,000 structures Cryo-electron microscopy (most recently used technique): more than 450 structures
57 Protein modeling Protein 58 Drug design in silico Molecular docking Fragment based drug design: FBDD aims to discover novel chemical leads with expected pharmacological properties, namely tight binder, from small ―efficient‖ binder. FBDD approach has been successfully utilized to guide pharmaceutical design inhibitors against a variety of targets, such as CDK4, estrogen receptor, factor Xa or HIV protease. Focused library design 59
PHARMACOLOGICAL TEST Efficacy/Toxicity/ADME 60 OBJECTIVES Found :
The mechanism of action The efficacy The safety Choose the pharmaceutical form
12
10 TOXIC RANGE
8
6 THERAPEUTIC RANGE
4
2 SUB-THERAPEUTIC Plasma Concentration Plasma 0 0 1 2 3 4 5 6 7 8 9 Dose 61 Pharmacological study: Pharmacodynamic Justify the investigation in humans if the molecule is not dangerous and effective in a relevant animal model (predictive value). Determination of the target (receptor) Selection of the ligand (x) / receptor(s) Experimental pharmacology studies (Enzymes, isolated organs, cells) Pharmacokinetic: ADME 62 Identify the impact point: The effects of most drugs result Target from their interaction with macromolecular components of the organism. Receptor: the component of the organism with which the drug is presumed to interact. The drug selectively binds to its receptor and initiates its pharmacological effect. Membranous Receptor Nuclear receptor Ionic channel Cell Process Etc… Cell specificity 63 DRUG–RECEPTOR INTERACTIONS
Intracellular Receptor Ligand Effector Effect
Extracellular Cell effect, Organ effect Message Pharmacological effect In vitro, In vivo effect
Therapeutic effect Clinical studies Clinical trials Binding study Pharmacodynamic study Pre clinic study 64 PROTEIN RECEPTORS
Quantitatively, proteins Proteins involved in form the most important transport processes class of drug receptors. + + Examples (Na ,K -ATPase); receptors for hormones, Structural proteins Receptors for (tubulin). neurotransmitters the enzymes of crucial Nucleic acids are metabolic or regulatory important drug receptors, pathways particularly anticancer (dihydrofolate reductase, acetylcholinesterase); agents. Pharmacology Introduction 65 CYTOPLASMIC SECOND MESSENGERS
Binding of an agonist to a receptor provides the first message in receptor signal transduction to effector to affect cell physiology. The first messenger promotes the cellular production or mobilization of a second messenger, which initiates cellular signaling through a specific biochemical pathway. Synthesis or release and degradation or excretion of these second messengers reflects the activities of many pathways
66 67 Nuclear receptors
The family of nuclear includes nearly 100 members, many of which are still classified as orphan receptors because their ligands, if they exist, remain to be identified. Most nuclear receptors are classified based on the nature of their ligands. 68 Which target ?
The target should be validated already in human disease « Evidence Based Medicine» Clinical Pharmacology Pathophysiology in human
Reality Genetic Genetically modified animals Animal Pharmacology
Clinical Cell Pharmacology Genomics - 69 Objectives These studies should: Provide non-clinical proof of mechanism of action and effectiveness Understanding the pharmacodynamic properties Schedule the remaining work and establish a road map Schedule various doses Provide information about the selection of the species to test Determine the starting dose Selection of the biomarker for the next investigations Identifying the pathways involved in the mechanism of action is also essential for the selection of biomarkers, it can be used clinically to optimize dosages and treatment regimens. Justify eventually the pharmaceutical combinations 70
PHARMACOLOGICAL TEST Example of anticancer drug 71 Cancer cell lines
In vitro studies performed in cell lines, cell-free systems Often form the basis for screening and optimization during discovery Animal screening is too expensive for routine use Cellular uptake and membrane transport MDR, MRP, etc Predictions of bioavailability and distribution In vitro drug metabolism: P450 isoenzyme inhibition or induction Effects on hERG channels (prolonged QT interval risk) Preliminary protein binding studies 72 The NCI-60: Assessing drug effectiveness NCI's In Vitro Cell Line Screening Project, better known as the NCI-60 analyzes the anti-cancer properties of a compound in human tumor samples from 60 different cell cultures, sometimes referred to as lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The NCI-60 project, which has been testing lines since 1990 in the Developmental Therapeutics Program of NCI's Division of Cancer Treatment and Diagnosis, screened 17,200 compounds in 2011, roughly evenly divided between natural and synthetic agents. The most promising—the "hits"—move on to further testing. Since 1990, more than 100,000 natural products have gone through the NCI screening process, driving the number of drugs in NCI's repository that have had some kind of screening process to over 400,000. There is no cost to the researcher for NCI-60 screening. 73 Comparison of variant counts across tumor types (NCI-60) 74 Animal models for human prostate cancer
Spontaneous tumors: Idiopathic Carcinogen- induced Transgenic/gene knockout animals: p53, RB, etc 75 Murine Xenograft Sites
Subcutaneous tumor (NCI method of choice) with IP drug administration: Intraperitoneal Intracranial Intrasplenic Renal subcapsule Site-specific (orthotopic) organ inoculation 76 Xenograft Advantages Disadvantages Many different human tumor cell Brain tumors difficult to model lines transplantable Metastases rare Wide representation of most Survival not an ideal endpoint: death human solid tumors from bulk of tumor, not invasion Allows for evaluation of therapeutic index Shorter doubling times than original growth in human Good correlation with drug regimens active in human lung, Less necrosis, better blood supply colon, breast, and melanoma Difficult to maintain animals due to cancers infection risks Several decades of experience Ability to mimic the human tumor microenvironment is limited 77
ANIMAL MODELS 78 Animal models
Efficacy demonstrated in disease specific animal models: Proof of therapeutic principle Groundwork for clinical development planning Evaluation of therapeutic index Toxicity versus efficacy 79 Animal models (2)
Ideal Animal Model Validity Selectivity Predictability Reproducibility 80 Animal models Analysis of 10,000 open access publications shows that the mouse is overwhelmingly the preferred laboratory animal. Rats and mice are the most widely used C57BL / 6 BALB / c Sprague-Dawley rats and Wistar rats The majority of these animals are supplied by four major suppliers, The Jackson Laboratory laboratories, Charles River Laboratories, Taconic Farms and Harlan Laboratories. 81 animal lignée num fournisseurs num souris C57BL/6 116 The Jackson 68 Laboratory Charles River 18 Taconic Farms 9 Harlan Laboratories 9 souris BALB/C 26 The Jackson 10 Laboratory Charles River 6 Taconic Farms 5 Harlan Laboratories 2 souris CD-1 8 souris SCID 8 souris A/J 4 rat Sprague-Dawley 9 rat Wistar 6 82 Mice and Rats Animal models contribute to the better understanding the function of individual genes, and the mechanisms of various diseases, as well as the efficacy and toxicity of various medicines and chemicals. The inbred lines, congenic and transgenic mice, are commonly used. An inbred line is defined as a line which was obtained by crossing siblings of 20 generations and the animals of the same inbred strain are considered genetically identical. The congenic lines are obtained by repeated backcrossing with inbred line to select a single marker on a minimum of 10 generations. 83 C57BL/6 La lignée C57BL/6, également appelée "C57Black 6" ou simplement "Black 6", a l'avantage d’être une lignée stable et à reproduction facile. C'est aussi la première lignée de souris dont le génome a été entièrement séquencé en 2005, devancée seulement par le génome humain. L’International Mouse Phenotyping Consortium (CGIP), lancé le 29 Septembre 2011, vise à cataloguer la fonction de chaque gène murin dans cette lignée à l’aide de technologies de « knock out ». Les souris C57BL/6 sont utilisées dans trois domaines principaux. Le plus commun est de servir de modèles physiologiques ou pathologiques pour des expériences in vivo. elles sont souvent utilisées pour construire des modèles de souris transgéniques. Enfin, les souris C57BL/6 sont utilisées comme lignée de référence pour la génération de lignées congéniques avec des mutations spontanées ou induites 84 Les souris BALB/c
BALB/c est une lignée consanguine albinos, immunodéficiente. Les caractéristiques des souris BALB/c sont une reproduction facile et des variations de poids minimes entre les mâles et les femelles. Un autre point important est la faible incidence des tumeurs mammaires chez les souris BALB / c, mais elles sont très sensibles à des agents cancérigènes, et peuvent développer des tumeurs du poumon, des tumeurs réticulaires, tumeurs rénales et autres. En outre, l'injection d'huile minérale peut facilement induire plasmacytomes dans la lignée BALB/c, et cette lignée a été largement utilisée pour la création d’hybridome et la production d'anticorps monoclonaux. Des souris BALB/c sont utiles pour la recherche en thérapie contre le cancer et l'immunologie 85 Les souris CB17 SCID
Les souris CB17 SCID (SCID réfère à immunodéficience combinée sévère) est une lignée albinos avec mutation SCID spontanée. La mutation empêche le développement et la maturation des cellules T et B. Cependant, les souris SCID ont des cellules NK normales, des macrophages et des granulocytes. Ils partagent la même apparence que les souris normales. En raison de la mutation SCID, le taux de réussite de la greffe de tumeur humaine est très élevé (encore plus élevé que les souris Nude; déficientes en cellules T), ce qui fait de ces souris un modèle animal immunodéprimé précieux pour tester de nouveaux traitements contre le cancer et comme hôtes pour les tissus du système immunitaire humain 86 Rat Sprague-Dawley Wistar Par rapport aux souris, les rats sont plus Le rat Wistar est une autre lignée albinos gros, plus féroces et plus résistants contre hybride. divers maux. Il a l'honneur d'être la première lignée de Les rats Sprague-Dawley et Wistar sont rat développé pour servir de modèle deux lignées non consanguines de rats animal. albinos les plus fréquemment utilisées. Les La lignée de rats Sprague-Dawley est rats de laboratoire sont de l'espèce Rattus dérivée de cette lignée. norvegicus. Le rat Sprague-Dawley est une lignée albinos hybride avec des têtes longues et étroites. Il a un taux de reproduction élevé et une faible incidence de tumeurs spontanées. Son tempérament calme et sa maniabilité en font un modèle de choix pour les scientifiques et techniciens de laboratoire. 87 Animal models for Arterial Hypertension 88 Spontaneous Hypertensive Rats SHR DOCA-sel Undoubtedly is the best known strain among hypertensive rats. It is a Japanese strain SHR (Spontaneous Hypertensive Rats) isolated in Administration of the sixties by Okamoto and Aoki. These rats desoxycorticostérone + High- have a primary hypertension, salt diet. The researchers measured the blood pressure of hundreds of Wistar rats breeding Kyoto University, a male and a female hypertensive being retained to initiate the colony. In six generations by selection of progenitors was obtained a hypertensive strain really inbred to the twentieth generation. With the same method, they created a strain Wistar Kyoto (WKY) normotensive rats control ; elementary to any experimentation with SHR. 89 Rats Zucker
These rats are characterized by obesity, insulin resistance and hyperinsulinemia. They develop a blood hypertension and hyperlipidemia. Kidney damage is a focal segmental glomerulosclerosis proteinuria. This model was used to assess the effect of drugs (antihypertensive, lipid- lowering, etc.) on the progression of kidney damage. 90 Animal Pharmacokinetic models Animal pharmacokinetics can guide dose and schedule selection ADME data can be generated in parallel with clinical development Preliminary evaluation of candidate biomarkers 91
TOXICOLOGY TESTS 92 General recommendations
At least two species, one belonging to rodents. Animal sex: For at least one of the species, there must be animals of both sexes. Number of animals: In the case of rodents, each group must include at least five animals per sex. For other species, each group must include at least two males and two females. Route of Administration: Typically, oral as it is the normal route of administration. However, some regulators also call the parenteral route. In cases where the parenteral route was chosen for the man, it is sufficient to use this channel for testing in animals. 93 Toxicology Tests Acute Toxicity tests: which describe the adverse effects of a substance that result either from a single exposure or from multiple exposures in a short space of time (usually less than 24 hours). Accumulation Toxicity The accumulation of drugs and their metabolic byproducts in organs can be toxic, leading to organ damage. Subchronic Toxicity Studies that continue for 90 days or for up to 10% of a test subject's life span are considered subchronic. Chronic Toxicity Chronic toxicity is a property of a substance that has toxic effects on a living organism, when that organism is exposed to the substance continuously or repeatedly. 94 Acute toxicity and chronic toxicity
Determine DL50 Therapeutic index Organs targeted (predictable toxicity) Methodology: 3 unique dose 10 animal per dose Observation during at least 2 weeks Sub-chronic toxicity: informations about dose accumulation 3 doses 1 high dose > therapeutic dose 1 low dose 1 high dose without lethality Duration: 28 or 90 days Animals rodents and primates 95 Duration of Toxicology Studies
Recommended duration of repeated-dose toxicity studies to support the conduct of clinical trials Rodents Non-Rodents Up to 2 weeks 2 weeks 2 weeks Between 2 weeks Same as clinical trial Same as clinical trial and 6 month > 6 month 6 month 9 month 96 Duration of Toxicology Studies (2)
Recommended duration of repeated-dose toxicity studies to support marketing Duration of indicated Rodent Non-Rodent treatment Up to 2 weeks 1 month 1 month > 2 weeks to 1 month 3 month 3 month > 1 month to 3 month 6 month 6 month > 3 month 6 month 9 month 97 Long term toxicity studies
Repeated dose Duration Dose EU USA Many doses 6 month 12 month Low Mean High
No toxic effect in animal doesn’t assure the safety use in human. In general, no correlation between acute and subchronic or chronic toxicity. 98 Genotoxicity Genotoxicity is defined as a destructive effect on a cell's genetic material (DNA, RNA). Genotoxins are mutagens; they can cause mutations. A genotoxic substance is known as a genotoxin. There are three primary effects that genotoxins can have on organisms by affecting their genetic information. Genotoxins can be carcinogens, or cancer-causing agents, mutagens, or mutation- causing agents, or teratogens, birth defect-causing agents. Usually not required if biologically irrelevant Test contaminants Use relevant assay 99 Carcinogenicity The term "carcinogen" denotes a chemical substance or a mixture of chemical substances which induce cancer or increase its incidence. Substances which have induced benign and malignant tumours in well performed experimental studies on animals are considered also to be presumed or suspected human carcinogens unless there is strong evidence that the mechanism of tumour formation is not relevant for humans. In rat and mouse; 2 year in length. Often difficult route of exposure for 2-year study, e.g., IV, ICV. Not generally required unless scientifically justified Assays can include p53 or Tg.AC transgenic models, SHE cell assay (Syrian hamster embryo), in vitro assays (e.g., 3T3 transformation assay), nude mouse xenograft models, mice transfected with human protein 100 Toxicity Studies
ReproTox Segment I – fertility (testis histopath,, sperm mobility, menstrual cycling) Segment II – teratology (developmental tox) – organogenesis, placental transfer) Segment III – prenatal/postnatal (secretion of drug in milk) Immunotoxicology Many biologic therapeutics are immunomodulators – that is their MOA Potential effects on:innate immunity (macrophage, neutrophil, NK cells, acquired immunity - cellular (T cell cytotoxicity) and humoral (antibody response), host resistance (bacterial, viral, tumor)