Design and Conduct Safety Pharmacology & Toxicology Study for Pharmaceuticals Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D Professor of Pharmaceutics Department

Design and Conduct Safety Pharmacology &

Toxicology Study for PharmaceuticalsDr. Basavaraj K. Nanjwade M.Pharm., Ph.D

Professor of PharmaceuticsDepartment of Pharmaceutics, KLE University,

BELGAUM – 590010, Karnataka, IndiaE-mail: [email protected]

Cell No: +919742431000

R & D Pharmacology and ToxicologyR & 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

IN-VITRO TOXICOLOGY

• In-vitro toxicity assays•In-vitro genotoxicity assays

SAFETY PHARMACOLOGY

• CNS studies• CVS studies• Respiratory evaluations/ system

DRUG METABOLISM & PHARMACOKINETICS STUDIES

• In-Vivo DMPK studies• In-vitro DMPK studies• Plasma Protein Binding studies• Drug – Drug interaction studies• Bioanalysis• Special Analytical studies

HISTOPATHOLOGICAL AND BIOCHEMISTRY STUDIES

30/12/2009 2Nepal Research Foundation.,

Birgunj, Nepal.

PHARMACOLOGY


Birgunj, Nepal.

Oncology

i. Xenograft modelThis 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


Birgunj, Nepal.

Oncology

ii. In vivo targeted inhibition model In PK/PD studies the tumors are from the test

article treated mice group along with respective control groups.

Then the effect of test article at molecular level is investigated in different cell based assays

The MTD of the compound is also analysed


Birgunj, Nepal.

Pain and Inflammation

1. Chemical Induced Capsaicin induced Hyperalgesia in rats FCA induced Hyperalgesia Carrageenan induced paw edema Acetic acid/Formalin induced pain in mice Mouse Ear Edema Adjuvant-Induced Arthritis (AIA) Collagen-Induced Arthritis (CIA)


Birgunj, Nepal.


2. Thermal & Mechanical Induced Hyperalgesia Tail flick model of Hyperalgesia Hot plate model of Hyperalgesia Neuropathic Pain (PSL and CCI induced

PAIN) Diabetic neuropathic pain model (DNP) Post operative pain model


Birgunj, Nepal.


3. Miscellaneous Novel technique to quantitatively assess

Inflammatory mediators-In Vitro assay measuring cytokine production/inhibition in rat/mice

Mouse LPS model-LPS-stimulated inflammation in mice


Birgunj, Nepal.

Metabolic Disorder

1. Acute Model OGITT/IPGTT Model

2. Chronic Model STZ induced Diabetes in mice/rats STZ induced Diabetes in neonatal rats Sucrose Fed Diet induced Diabetes High-Fat/carbohydrate Fed Diet and STZ treated Mice

model for Diabetes Glucose and Insulin estimation ob/ob mouse db/db mouse ZDF rats


Birgunj, Nepal.

Respiratory Diseases

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


Birgunj, Nepal.

Psychopharmacology Models

1. Muscle Relaxation Rota-rod test Inclined screen test Grip Strength test

2. Behavioral test Irwin test


Birgunj, Nepal.

IN-VIVO TOXICOLOGY


Birgunj, Nepal.

General Toxicology

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


Birgunj, Nepal.

In-Vivo Genotoxicity Studies

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)


Birgunj, Nepal.

Development & Reproduction toxicology

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


Birgunj, Nepal.

Special Toxicity Studies

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)


Birgunj, Nepal.

IN – VITRO TOXICOLOGY


Birgunj, Nepal.

In – Vitro Toxicology

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


Birgunj, Nepal.

In-Vitro Genotoxicity Studies

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


Birgunj, Nepal.

SAFETY PHARMACOLOGY


Birgunj, Nepal.

29/12/2009Nepal Pharmaceuticals Ltd.,

Birgunj, Nepal. 21

Scope and Principle 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.

Some safety pharmacology endpoint can be incorporated in the design of toxicology, kinetic and clinical studies


Birgunj, Nepal.

30/12/2009 22

Definition of Safety Pharmacology

Pharmacology studies can be divided into three categories:

1. Primary pharmacodynamic

2. Secondary pharmacodynamic

3. Safety Pharmacology studies

Nepal Research Foundation., Birgunj, Nepal.

30/12/2009 23

Objectives of Studies To identify undesirable pharmacodynamic

properties of a substance that may have relevance to its human safety

To evaluate adverse pharmacodynamic and/or pathophysiological effects of a substance observed in toxicology and /or clinical studies

To investigate the mechanism of the adverse pharmacodynamic effects observed and/or suspected


30/12/2009 24

General Considerations in Selection and Design of Safety Pharmacology

Effects related to the therapeutic class of the test substance, since the mechanism of action may suggest specific adverse effects

Adverse effects associated with members of the chemical or therapeutic class, but independent of the primary pharmacodynamics effects


30/12/2009 25

General Considerations in Selection and Design of Safety Pharmacology

Ligand binding or enzyme assay data suggesting a potential for adverse effects

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 26

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 27

General Considerations on Test Systems

Consideration should be given to the selection of relevant animal model or other test systems so that scientifically valid information can be derived.

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 28

Use of In Vivo and in Vitro Studies

Animal models as well as ex vivo and in citro preparations can be used as test systems.

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.

In vitro systems can be used in supportive studies

In conducting in vivo studies, it is preferable to use anaesthetized


30/12/2009 29

Sample Size and Use of Controls

The sample size should take into consideration the size of the biological effect that is of concern for humans

Appropriate negative and positive control group should be included in the experimental design

In well-characterized in vivo test systems, positive controls may not be necessary.

The exclusion of control from studies should be justified


30/12/2009 30

Route of Administration

In general, the expected clinical route of administration should be used when feasible.

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.

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 31

Dose Levels or Concentrations of test Substance

In Vivo Studies

In Vitro Studies


30/12/2009 32

In Vivo Studies

In vivo safety pharmacology studies should be designed to define the dose-response relationship of the adverse effect observed.

The time course of the adverse effect should be investigated, when feasible.

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 33

In Vitro Studies In vitro studies should be designed to establish a

concentration-effect relationship.

The range of concentrations used should be selected to increase the likelihood of detecting an effect on the test system.

The upper limit of this range may be influenced by physico-chemical properties of the test substance and other assay specific factors.

In the absence of an effect, the range of concentrations selected should be justified.


30/12/2009 34

Duration of Studies

Safety pharmacology studies are generally performed by single-dose administration

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 35

Studies on Metabolites, Isomers and Finished Products

Metabolites from humans are known to substantially contribute to the pharmacological actions of the therapeutic agent, it could be important to test such active metabolites.

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.


Studies on Metabolites, Isomers and Finished Products

In vitro or in vivo testing of the individual isomers should also be considered when the product contains an isomeric mixture

Finished product formulations should be conducted only for formulations that substantially alter the pharmacokinetics and/or pharmacodynamics of the active substance in comparison to formulations previously tested


Birgunj, Nepal.

30/12/2009 37

Safety Pharmacology Core Battery

1. Central Nervous System

2. Cardiovascular System

3. Respiratory System


30/12/2009 38

Central Nervous System

Effects of the test substance on the central nervous system should be assessed appropriately.

Motor activity, behavioral changes, coordination, sensory/motor reflex responses and body temperature should be evaluated.


30/12/2009 39

Cardiovascular System

Effect of the test substance on the cardiovascular system should be assessed appropriately.

Blood pressure, heart rate and the electrocardiogram should be evaluated

In vivo, in vitro and /or ex vivo evaluations, including methods for repolarzation and conductance abnormalities, should also be considered.


30/12/2009 40

Respiratory System

Effects of the test substance on the respiratory system should be assessed appropriately

Respiratory rate and other measures of respiratory function should be evaluated.

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 41

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


Central Nervous System, Cardiovascular System& Respiratory System

Behavioral pharmacology, learning and memory, ligand-specific binding, neurochemistry, visual, auditory, and/or electrophysiology examination

Cardiac output, ventricular contractility, vascular resistant, the effects of endogenous and/or exogenous substances on the cardiovascular responses

Airway resistance, compliance, pulmonary arterial pressure, blood gases, blood pH


Birgunj, Nepal.

30/12/2009 43

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 44

Renal/Urinary System

Effect of the test substance on renal parameters should be assessed.

e.g

Urinary volume, specific gravity, osmolality, pH, fluid/electrolyte balance, protein, cytology, and blood chemistry determinations such as blood urea nitrogen, creatinine, and plasma proteins can be used


30/12/2009 45

Autonomic Nervous System

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 46

Gastrointestinal System

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 pH measurement and pooling can be used


30/12/2009 47

Other Organ Systems

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 48

Conditions Under Which Studies are Not Necessary

Safety pharmacology studies may not be needed for locally applied

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.gCase of a new salt having similar pharmacokinetics and pharmacodynamics


30/12/2009 49

Timing of Safety Pharmacology Studies in Relation to Clinical

Development1. Studies Prior to First Administration in Humans

2. Studies During Clinical Development

3. Studies Before Approval


Studies prior to First Administration in Human

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

Any follow-up or supplemental studies identified as appropriate, based on a cause for concern, should also be conducted.

Information from toxicology studies adequately designed and conducted to address safety pharmacology endpoint can result in reduction or elimination of separate safety pharmacology studies.


Birgunj, Nepal.

Studies During Clinical Development

Additional studies may be warranted to clarify observed or suspected adverse effects in animals and humans during clinical development


Birgunj, Nepal.

Studies Before Approval

Safety pharmacology effects should be assessed prior to product approval, unless not warranted, in which case this should be justified.

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


Birgunj, Nepal.

30/12/2009 53

Application of Good Laboratory Practice (GLP)

This is normally accomplished through the conduct of the studies in compliance with GLP.

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

The safety pharmacology core battery should ordinarily be conducted in compliance with GLP



Follow-up and supplemental studies should be conducted in compliance with GLP to the greatest extent feasible.

Safety pharmacology investigations can be part of toxicology studies; in such cases, these studies would be conducted in compliance with GLP

Primary pharmacodynamic studies do not need to be conducted in compliance with GLP


Birgunj, Nepal.


Generally, secondary pharmacodynamic studies do not need to be conduct in compliance with GLP

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


Birgunj, Nepal.

DRUG METABOLISM & PHARMACOKINETIC STUDIES


Birgunj, Nepal.

In-vivo DMPK Studies

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 WinNonLin software is performed in house


Birgunj, Nepal.

In-vitro DMPK Studies

Cytochrome P450 studies Metabolite Profiling metabolite Identification

& Metabolic Stability Studies Blood Distribution Studies Drug Transport Using Caco-2 cell and skin

Absortion, transport and Metabolism studies


Birgunj, Nepal.

Plasma Protein Binding

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


Birgunj, Nepal.

Drug –Drug Interaction Studies

Using pooled human liver microsomes or other in vitro metabolism models

The interaction of a drug on the metabolism of another drug is determined


Birgunj, Nepal.

Special Analytical Studies

Metabolite Identification/Elucidation This provides metabolite identification in sample

available from in vitro and in vivo studies, as well as from environmental fate studies

The method is then adapted for the specific compound

Generally method development is finished within three days

Data are generated using LC-PDA-MS analysis with data-dependent MS-MS

Additional MS analysis is possible, as well as accurate mass determination


Birgunj, Nepal.

HISTOPATHOLOGICAL & BIOCHEMISTRY STUDIES


Birgunj, Nepal.

Hematology

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


Birgunj, Nepal.

Clinical Chemistry Alanine Aminotransferase Albumin Alkaline Phosphatase Aspartate Aminotrasferase Calcium Chloride Cholesterol Creatinine Glucose Prealbumin A/G/ Ratio

Globulin, Phosphorous Potassium Sodium Total Bilirubin Total Protein Triglycerides Urea Nitrogen Osmolality Anion Gap


Birgunj, Nepal.

BiomarkersAdvanced Lipid biomarkers

HDL-C LDL-C VLDL Cardiac risk ratio Apo A1 Apo B FFA

Cardiac biomarkers

BNP Troponin I Myoglobin CK CK-MB Homocysteine Hs-CRP D-Dimer


Birgunj, Nepal.

LIST OF EQUIPMENTS


Birgunj, Nepal.

General Pharmacology Equipments

Auto analyzer (semi-automated) Blood cell counter UV Spectrophotometer HPLC CO2 incubator with air jacket ELISA microplate reader Inverted microscopes Non Invasive BP Measurements (Tail cuff) Physiograph Polygraph


Birgunj, Nepal.

General Pharmacology Equipments

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


Birgunj, Nepal.

List of Critical Equipment for Pharmacology

IVC units Boyle’s apparatus Hot plate Plethysmometer Automated cell counter (Mythic 18) Plate reader (Biotech) Plantar Aesthesiometer


Birgunj, Nepal.

List of Critical Equipments for ADME and PK

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)


Birgunj, Nepal.

List of Critical Equipment for Assay Development

FLIPR (Molecular Devices) Janus Liquid Handaling system

(PerkinElmer) Novostar-single channel robotic plate reader

(BMG Labtech) Fluostar-single channel robotic plate reader

(BMG Labtech) Victor (PerkinElmer) Cell Harvesters (Tomtec, Skatron)30/12/2009 71


Wallac Beta and Gamma counter (PerkinElmer) Inverted fluorescence microscope (Nikon TE 200U) CO2 incubator (Thermo) Ultracentrifuge (Beckman coulter) Cold centrifuge (Beckman coulter) Cryopreservation Equipment (Thermo) Hydra (Matrix) Lyophilizer

List of Critical Equipment for Assay Development


Birgunj, Nepal.

List of Critical Equipments for Molecular Biology and Gene

Expression 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)


Birgunj, Nepal.

List of Critical Equipments for Molecular Biology and Gene

Expression 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)


Birgunj, Nepal.

PHARMACOLOGY LAB LAY OUT (ANIMAL PHARMACOLOGY LAB LAY OUT (ANIMAL

HOUSE)HOUSE)

Entra

nce

Sterile room for exchange of clothes,

belongings etc

Restricted Entry

HOD’s

Office

Discussion/Meeting Room

Library

Senior and Junior

Research Officers

Well equipped Sample’s Analysis Room

Restricted Entrance

Quarantine room

Dosing Room

Animal House for MICEAnimal house for RATS

Operation Room for invasive

procedures Animal House for

Rabbits

Animal House

for Guinea

Pigs

Dosing

Room

Mating Room for

Rats and Mice


Birgunj, Nepal.

Rodent Facility

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)


Birgunj, Nepal.

International Guidelines

ICH www.ich.og International Conference on Harmonization

FDA www.fda.org

Food and Drug Administration

EPA www.epa.org

Environmental Protection Agency

OECD www.oecd.org Organization for Economic Co-operation & Development

CPCSEA www.cpcsea.com Committee for the Purpose of Control and Supervision of Experiments on Animals


Birgunj, Nepal.

Thanking You


Birgunj, Nepal.

Documents

Design and Conduct Safety Pharmacology & Toxicology Study for Pharmaceuticals Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D Professor of Pharmaceutics Department