Design and Conduct Safety Pharmacology Toxicology Study

Design and Conduct Safety Pharmacology & Toxicology Study for Pharmaceuticals Dr. Basavaraj K. Nanjwade M. Pharm. , Ph. D Professor of Pharmaceutics Department of Pharmaceutics, KLE University, BELGAUM – 590010, Karnataka, India E-mail: bknanjwade@yahoo. co. in Cell No: +919742431000

R & D Pharmacology and Toxicology PHARMACOLOGY • Oncology • Pain & Inflammation • Metabolic Disorders • Respiratory Diseases • Psychopharmacology models IN-VIVO TOXICOLOGY • General toxicology • In-vivo Genotoxicity studies • Carcinogenicity • Development and reproductive studies • Special toxicity studies 30/12/2009 IN-VITRO TOXICOLOGY • In-vitro toxicity assays • In-vitro genotoxicity assays SAFETY PHARMACOLOGY • CNS studies • CVS studies • Respiratory evaluations/ system Nepal Research Foundation. , Birgunj, Nepal. DRUG METABOLISM & PHARMACOKINET ICS STUDIES • In-Vivo DMPK studies • In-vitro DMPK studies • Plasma Protein Binding studies • Drug – Drug interaction studies • Bioanalysis • Special Analytical studies HISTOPATHOLOG ICAL AND BIOCHEMISTRY STUDIES 2

PHARMACOLOGY 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 3

Oncology i. Xenograft model n n n This model provides consistent and reproducible cell growth and permits easy access to the tumor for treatment and calliper measurement Cell implant subcutaneously into immuno-compromised animals Tumors measured during growth phase Animals treated with vehicle, test article and positive control Various protocol-specified parameters/markers assessed ex vivo or in vivo assays Report preparation 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 4

Oncology ii. In vivo targeted inhibition model n In PK/PD studies the tumors are from the test article treated mice group along with respective control groups. n Then the effect of test article at molecular level is investigated in different cell based assays n The MTD of the compound is also analysed 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 5

Pain and Inflammation 1. Chemical Induced n Capsaicin induced Hyperalgesia in rats n FCA induced Hyperalgesia n Carrageenan induced paw edema n Acetic acid/Formalin induced pain in mice n Mouse Ear Edema n Adjuvant-Induced Arthritis (AIA) n Collagen-Induced Arthritis (CIA) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 6

Pain and Inflammation 2. Thermal & Mechanical Induced Hyperalgesia n Tail flick model of Hyperalgesia n Hot plate model of Hyperalgesia n Neuropathic Pain (PSL and CCI induced PAIN) n Diabetic neuropathic pain model (DNP) n Post operative pain model 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 7

Pain and Inflammation 3. Miscellaneous n Novel technique to quantitatively assess Inflammatory mediators-In Vitro assay measuring cytokine production/inhibition in rat/mice n Mouse LPS model-LPS-stimulated inflammation in mice 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 8

Metabolic Disorder 1. Acute Model n OGITT/IPGTT Model 2. Chronic Model n STZ induced Diabetes in mice/rats n STZ induced Diabetes in neonatal rats n Sucrose Fed Diet induced Diabetes n High-Fat/carbohydrate Fed Diet and STZ treated Mice model for Diabetes n Glucose and Insulin estimation n ob/ob mouse n db/db mouse n ZDF rats 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 9

Respiratory Diseases n n n Antigen-induced Airway Hyper responsiveness in mice/rats Antigent-induced Pulmonary eosinophilia in mice/rats Passive Cutaneous Anaphylaxis Active Cutaneous Anaphylaxis LPS-induced Meutrophilia in rats/mice 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 10

Psychopharmacology Models 1. Muscle Relaxation n Rota-rod test n Inclined screen test n Grip Strength test 2. Behavioral test n Irwin test 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 11

IN-VIVO TOXICOLOGY 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 12

General Toxicology n n n Single dose studies Repeated dose sub-acute and sub-chronic studies (14, 28 & 90 days) Chronic toxicology studies (6, 9 & 12 months) Carcinogenicity studies Toxicokinetics Pathology 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 13

In-Vivo Genotoxicity Studies n n n Micronucleus test in mouse or rat bone marrow (OECD 474) Mammalian bone marrow chromosome aberration test in rats or mice (OECD) Unscheduled DNA synthesis (UDS) with rat hepatocytes (OECD 486) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 14

Development & Reproduction toxicology n n n n n Fertility (Segment I) Embryo-fetal development (Segment II) Perinatal and postnatal development, including maternal function (Segment III) Multigenerational studies Endocrine disruptors Selected neurobehavioral tests Juvenile dosing studies (rodent, dog) Sample collection (TK/PK and absorption analysis, maternal and fetal blood, amniotic fluid, milk) Spermatogenesis evaluations of cellular endpoint (morphology, motility, spermatid head count) via the IVOS system Vaginal cytology evaluations 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 15

Special Toxicity Studies n n n The potential, adverse effects of human pharmaceuticals and biotech products on the immune system is acknowledged as an important issue. According to the FDA, evaluation of potential immunotoxic effects should be incorporated into standard drug development It incorporates immunotoxicology assays using standard rodent species (Wistar rat and CD-1 mouse) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 16

IN – VITRO TOXICOLOGY 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 17

In – Vitro Toxicology n n n Human skin corrosion assay (OECD 431) Human skin irritation assay (draft proposal for a new guideline OECD) Hen’ Egg Test-Chorioallantoic Membrane (HET-CAM) test 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 18

In-Vitro Genotoxicity Studies n n n Bacterial reverse mutation (Ames) test with salmonella typhimurium and Escherichia coli (OECD 471) Chromosome aberration test (CAT) with human lymphocytes (OECD 473) Mammalian gene mutation test (MLA) with mouse lymphoma cells (TK-locus) (OECD 476) Unscheduled DNA synthesis (UDS) with isolated rat hepatocytes (OECD 482) Micronucleus test with human lymphocytes (draft OECD 487) Single cell gel electrophoresis (COMET) Assay 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 19

SAFETY PHARMACOLOGY 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 20

Scope and Principle n Design and conduct safety pharmacology study can be applied to marketed pharmaceuticals when appropriate e. g. 1. When adverse clinical events 2. A new patient population 3. A new route of administration raises concerns not previously addressed. n Some safety pharmacology endpoint can be incorporated in the design of toxicology, kinetic and clinical studies 30/12/2009 29/12/2009 Nepal Research Foundation. , Birgunj, Nepal Pharmaceuticals Ltd. , 21

Definition of Safety Pharmacology n Pharmacology studies can be divided into three categories: 1. Primary pharmacodynamic 2. Secondary pharmacodynamic 3. Safety Pharmacology studies 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 22

Objectives of Studies n To identify undesirable pharmacodynamic properties of a substance that may have relevance to its human safety n To evaluate adverse pharmacodynamic and/or pathophysiological effects of a substance observed in toxicology and /or clinical studies n To investigate the mechanism of the adverse pharmacodynamic effects observed and/or suspected 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 23

General Considerations in Selection and Design of Safety Pharmacology n Effects related to therapeutic class of the test substance, since the mechanism of action may suggest specific adverse effects n Adverse effects associated with members of the chemical or therapeutic class, but independent of the primary pharmacodynamics effects 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 24

General Considerations in Selection and Design of Safety Pharmacology n Ligand binding or enzyme assay data suggesting a potential for adverse effects n Results from previous safety pharmacology studies, from secondary pharmacodynamic studies, from toxicology studies, or from human use that warrant further investigation to establish and characterize the relevance of these findings to potential adverse effects in humans. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 25

Test Systems 1. General Considerations on Test Systems 2. Use of In Vivo and In Vitro Studies 3. Experimental Design a. Sample Size and Use of Controls b. Route of Administration 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 26

General Considerations on Test Systems n Consideration should be given to the selection of relevant animal model or other test systems so that scientifically valid information can be derived. n Selection factors can include the pharmacodynamic responsiveness of the model, pharmacokinetic profile, species, strain, gender and age of the experimental animals, the susceptibility, sensitivity, and reproducibility of the test system and available background data on the substance. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 27

Use of In Vivo and in Vitro Studies n Animal models as well as ex vivo and in citro preparations can be used as test systems. n Ex vivo and vitro systems can include, but are not limited to: isolated organs and tissues, cell cultures, cellular fragments, subcellular organelles, receptors, ion channels, transporters and enzymes. n In vitro systems can be used in supportive studies n In conducting in vivo studies, it is preferable to use anaesthetized 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 28

Sample Size and Use of Controls n The sample size should take into consideration the size of the biological effect that is of concern for humans n Appropriate negative and positive control group should be included in the experimental design n In well-characterized in vivo test systems, positive controls may not be necessary. n The exclusion of control from studies should be justified 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 29

Route of Administration n In general, the expected clinical route of administration should be used when feasible. n Regardless of the route of administration, exposure to the parent substance and its major metabolites should be similar to or greater than that achieved in humans hen such information is available. n Assessment of effects by more than one route may be appropriate if the test substance is intended for clinical use by more than one route of administration 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 30

Dose Levels or Concentrations of test Substance n In Vivo Studies n In Vitro Studies 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 31

In Vivo Studies n In vivo safety pharmacology studies should be designed to define the dose-response relationship of the adverse effect observed. n The time course of the adverse effect should be investigated, when feasible. n Generally, the doses eliciting the adverse effect should be compared to the doses eliciting the primary pharmacodynamic effect in the test species or the proposed therapeutic effect in humans, if feasible. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 32

In Vitro Studies n In vitro studies should be designed to establish a concentration-effect relationship. n The range of concentrations used should be selected to increase the likelihood of detecting an effect on the test system. n The upper limit of this range may be influenced by physico-chemical properties of the test substance and other assay specific factors. n In the absence of an effect, the range of concentrations selected should be justified. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 33

Duration of Studies n Safety pharmacology studies are generally performed by single-dose administration n When pharmacodynamic effects occur only after a certain duration of treatment, or when results from repeat dose non-clinical studies or results from use in humans give rise to concerns about safety pharmacological effects, the duration of the safety pharmacology studies to address these effects should be rationally based. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 34

Studies on Metabolites, Isomers and Finished Products n Metabolites from humans are known to substantially contribute to the pharmacological actions of therapeutic agent, it could be important to test such active metabolites. n When the in vivo studies on the parent compound have not adequately assessed metabolites, as discussed above, the tests of metabolites can use in vitro systems based on practical considerations. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 35

Studies on Metabolites, Isomers and Finished Products n In vitro or in vivo testing of the individual isomers should also be considered when the product contains an isomeric mixture n Finished product formulations should be conducted only formulations that substantially alter the pharmacokinetics and/or pharmacodynamics of the active substance in comparison to formulations previously tested 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 36

Safety Pharmacology Core Battery 1. Central Nervous System 2. Cardiovascular System 3. Respiratory System 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 37

Central Nervous System n Effects of the test substance on the central nervous system should be assessed appropriately. n Motor activity, behavioral changes, coordination, sensory/motor reflex responses and body temperature should be evaluated. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 38

Cardiovascular System n Effect of the test substance on the cardiovascular system should be assessed appropriately. n Blood pressure, heart rate and the electrocardiogram should be evaluated n In vivo, in vitro and /or ex vivo evaluations, including methods for repolarzation and conductance abnormalities, should also be considered. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 39

Respiratory System n Effects of the test substance on the respiratory system should be assessed appropriately n Respiratory rate and other measures of respiratory function should be evaluated. n Clinical observation of animals is generally not adequate to assess respiratory function, and thus these parameters should be quantified by using appropriate methodologies. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 40

Follow-up and Supplemental Safety Pharmacology Studies 1. Follow-up Studies For Safety Pharmacology Core Battery a. Central Nervous System b. Cardiovascular System c. Respiratory System 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 41

Central Nervous System, Cardiovascular System & Respiratory System n Behavioral pharmacology, learning and memory, ligandspecific binding, neurochemistry, visual, auditory, and/or electrophysiology examination n Cardiac output, ventricular contractility, vascular resistant, the effects of endogenous and/or exogenous substances on the cardiovascular responses n Airway resistance, compliance, pulmonary arterial pressure, blood gases, blood p. H 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 42

Follow-up and Supplemental Safety Pharmacology Studies 2. Supplemental Safety Pharmacology Studies a. Renal/Urinary System b. Autonomic Nervous System c. Gastrointestinal System d. Other Organ Systems 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 43

Renal/Urinary System n Effect of the test substance on renal parameters should be assessed. e. g Urinary volume, specific gravity, osmolality, p. H, fluid/electrolyte balance, protein, cytology, and blood chemistry determinations such as blood urea nitrogen, creatinine, and plasma proteins can be used 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 44

Autonomic Nervous System n Effects of the test substance on the autonomic nervous system should be assessed. e. g Binding to receptors relevant for the autonomic nervous system, functional responses to agonists or antagonists in vivo or in vitro, direct stimulation of autonomic nerves and measurement of cardiovascular responses, baroreflex testing, and heart rate variability can be used. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 45

Gastrointestinal System n Effects of the test substance on the gastrointestinal system should be assessed. e. g Gastric secretion, gastrointestinal injury potential, bile secretion, transit time in vivo, ileal contraction in vitro, gastric p. H measurement and pooling can be used 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 46

Other Organ Systems n Effects of the test substance on organ systems not investigated elsewhere should be assessed when there is a reason for concern. e. g. Dependency potential or skeletal muscle, immune and endocrine functions can be investigated. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 47

Conditions Under Which Studies are Not Necessary n Safety pharmacology studies may not be needed for locally applied n Safety pharmacology studies prior to the first administration in human may not be needed for cytotoxic agents for treatment of end-stage cancer patients. There may be additional exceptions where safety pharmacology testing is not needed e. g n Case of a new salt having similar pharmacokinetics and pharmacodynamics 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 48

Timing of Safety Pharmacology Studies in Relation to Clinical Development 1. Studies Prior to First Administration in Humans 2. Studies During Clinical Development 3. Studies Before Approval 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 49

Studies prior to First Administration in Human n The effects of a test substance on the functions listed in the safety pharmacology core battery should be investigated prior to first administration in humans n Any follow-up or supplemental studies identified as appropriate, based on a cause for concern, should also be conducted. n Information from toxicology studies adequately designed and conducted to address safety pharmacology endpoint can result in reduction or elimination of separate safety pharmacology studies. 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 50

Studies During Clinical Development n Additional studies may be warranted to clarify observed or suspected adverse effects in animals and humans during clinical development 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 51

Studies Before Approval n Safety pharmacology effects should be assessed prior to product approval, unless not warranted, in which case this should be justified. n Available information from toxicology studies adequately designed and conducted to address safety pharmacology endpoint, or information from clinical studies, can support this assessment and replace safety pharmacology studies 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 52

Application of Good Laboratory Practice (GLP) n This is normally accomplished through the conduct of the studies in compliance with GLP. n Due to the unique design of and practical considerations for, some safety pharmacology studies, it may not be feasible to conduct these in compliance with GLP n The safety pharmacology core battery should ordinarily be conducted in compliance with GLP 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 53

Application of Good Laboratory Practice (GLP) n Follow-up and supplemental studies should be conducted in compliance with GLP to the greatest extent feasible. n Safety pharmacology investigations can be part of toxicology studies; in such cases, these studies would be conducted in compliance with GLP n Primary pharmacodynamic studies do not need to be conducted in compliance with GLP 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 54

Application of Good Laboratory Practice (GLP) n Generally, secondary pharmacodynamic studies do not need to be conduct in compliance with GLP n In some circumstances, results of secondary pharmacodynamics studies may make a pivotal contribution to the safety evaluation for potential adverse effects in humans, and these are normally conducted in compliance with GLP 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 55

DRUG METABOLISM & PHARMACOKINETIC STUDIES 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 56

In-vivo DMPK Studies n n n In vivo preclinical ADME studies are routinely performed in rat and dog with radiolabeled compounds usually using carbon-14 or tritium isotopes However, all other animal species or isotopes can be considered These studies are performed using all common routes of administration This includes mass balance, tissue distribution, bile cannulation and dermal absorption studies Pharmacokinetic evaluation using validated Win. Non. Lin software is performed in house 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 57

In-vitro DMPK Studies n n Cytochrome P 450 studies Metabolite Profiling metabolite Identification & Metabolic Stability Studies Blood Distribution Studies Drug Transport Using Caco-2 cell and skin Absortion, transport and Metabolism studies 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 58

Plasma Protein Binding n n n The plasma protein binding of a drug is determined in plasma of different species (including human) using either equilibrium dialysis or ultrafiltration In addition, the stability of the drug in plasma is investigated These studies are performed using radiolabeled and non-labeled compounds 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 59

Drug –Drug Interaction Studies n n Using pooled human liver microsomes or other in vitro metabolism models The interaction of a drug on the metabolism of another drug is determined 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 60

Special Analytical Studies Metabolite Identification/Elucidation n This provides metabolite identification in sample available from in vitro and in vivo studies, as well as from environmental fate studies n The method is then adapted for the specific compound n Generally method development is finished within three days n Data are generated using LC-PDA-MS analysis with data-dependent MS-MS n Additional MS analysis is possible, as well as accurate mass determination 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 61

HISTOPATHOLOGICAL & BIOCHEMISTRY STUDIES 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 62

Hematology n n Complete Blood Count (CBC) with no Differential (WBC, RBC, HCT, HGB, MCV, MCH, MCHC, MPV, RDW, CHCM) Platelet (automated count), Reticulocytes Differential Count Complete Blood Count (CBC) with Differential 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 63

Clinical Chemistry n n n Alanine Aminotransferase Albumin Alkaline Phosphatase Aspartate Aminotrasferase Calcium Chloride Cholesterol Creatinine Glucose Prealbumin A/G/ Ratio 30/12/2009 n n n Globulin, Phosphorous Potassium Sodium Total Bilirubin Total Protein Triglycerides Urea Nitrogen Osmolality Anion Gap Nepal Research Foundation. , Birgunj, Nepal. 64

Biomarkers Advanced Lipid biomarkers n n n n HDL-C LDL-C VLDL Cardiac risk ratio Apo A 1 Apo B FFA Cardiac biomarkers n n n n 30/12/2009 BNP Troponin I Myoglobin CK CK-MB Homocysteine Hs-CRP D-Dimer Nepal Research Foundation. , Birgunj, Nepal. 65

LIST OF EQUIPMENTS 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 66

General Pharmacology Equipments n n n n n Auto analyzer (semi-automated) Blood cell counter UV Spectrophotometer HPLC CO 2 incubator with air jacket ELISA microplate reader Inverted microscopes Non Invasive BP Measurements (Tail cuff) Physiograph Polygraph 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 67

General Pharmacology Equipments n n n n n Deep freezer (420 L) Electrophoresis Cooling microcentrifuge CNS Pharmacology related instruments Student kymographs Respiratory pumps Langendorff’s apparatus UGO Basile plethysmometer UGO Basile analgesiometer UGO Basile ECT 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 68

List of Critical Equipment for Pharmacology n n n n IVC units Boyle’s apparatus Hot plate Plethysmometer Automated cell counter (Mythic 18) Plate reader (Biotech) Plantar Aesthesiometer 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 69

List of Critical Equipments for ADME and PK n n n n HPLCs (Water, Agilent) LC/MS/MS (API 3000, Applied Biosciences) Spectramax-Quartz plate reader (Molecular Devices) Zymark Evaporator (Caliper) Vacuum manifield (Whatman) Apricot Centrifuge (Hettich) Shaker incubator (Jeotech) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 70

List of Critical Equipment for Assay Development n n n FLIPR (Molecular Devices) Janus Liquid Handaling system (Perkin. Elmer) Novostar-single channel robotic plate reader (BMG Labtech) Fluostar-single channel robotic plate reader (BMG Labtech) Victor (Perkin. Elmer) Cell Harvesters (Tomtec, Skatron) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 71

List of Critical Equipment for Assay Development n n n n Wallac Beta and Gamma counter (Perkin. Elmer) Inverted fluorescence microscope (Nikon TE 200 U) CO 2 incubator (Thermo) Ultracentrifuge (Beckman coulter) Cold centrifuge (Beckman coulter) Cryopreservation Equipment (Thermo) Hydra (Matrix) Lyophilizer 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 72

List of Critical Equipments for Molecular Biology and Gene Expression n n n Gradient PCR machines (Biometra, Applied Biosciences) Gel Imaging System Bio-Rad) Shaker incubators (VWR, Zhicheng) DNA Electrophoresis system (Owl) UV-Vis Spectrophotometer (Beckman Coulter) Electroporator (Bio-Rad) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 73

List of Critical Equipments for Molecular Biology and Gene Expression n n n FPLC (Bio-Rad) Gel dryer (Bio-Rad) Southern/Northern blot chamber (Amersham Biosciences) Western transfer apparatus (Bio-Rad) Table-top refrigerated centrifuges (Beckman Coulter, Eppendroff) Protein Electrophoresis (Bio-Rad) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 74

PHARMACOLOGY LAB LAY OUT (ANIMAL HOUSE) Sterile room for exchange of clothes, belongings etc Library Animal house for RATS Animal House for MICE Entrance Quarantine room Restricted Entry Senior and Junior Research Officers Dosing Room Restricted Entrance Dosing HOD’s Room Office Well equipped Sample’s Analysis Room Discussion/Meeting Room Operation Room for invasive procedures Animal House for Guinea Animal House for Mating Room for Rats and Mice Rabbits Pigs 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 75

Rodent Facility n n Preclinical research facility encompasses a state-of-art building with a total built up area of 33, 000 sq. ft. with 32 Rodent experimental rooms (200 sq. ft) and separate provision of rooms for quarantine of animals and 5 procedure rooms wherein different pharmacological studies are carried out as on need basis The facility is registered with the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 76

International Guidelines n ICH www. ich. og International Conference on Harmonization FDA www. fda. org Food and Drug Administration n n EPA www. epa. org Environmental Protection Agency n OECD www. oecd. org Organization for Economic Co-operation & Development n CPCSEA www. cpcsea. com Committee for the Purpose of Control and Supervision of Experiments on Animals 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 77

Thanking You 30/12/2009 Nepal Research Foundation. , Birgunj, Nepal. 78