Title: Design and Conduct Safety Pharmacology & Toxicology Study for Pharmaceuticals
1Design 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_at_yahoo.co.in
- Cell No 919742431000
2R D Pharmacology and Toxicology
- PHARMACOLOGY
- Oncology
- Pain Inflammation
- Metabolic Disorders
- Respiratory Diseases
- Psychopharmacology
- models
- IN-VITRO TOXICOLOGY
- In-vitro toxicity assays
- In-vitro genotoxicity
- assays
- 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
- SAFETY PHARMACOLOGY
- CNS studies
- CVS studies
- Respiratory evaluations/
- system
- IN-VIVO TOXICOLOGY
- General toxicology
- In-vivo Genotoxicity studies
- Carcinogenicity
- Development and reproductive
- studies
- Special toxicity studies
3 4Oncology
- i. Xenograft model
- 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
5Oncology
- 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
6Pain 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)
7Pain and Inflammation
- 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
8Pain and Inflammation
- 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
9Metabolic 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
10Respiratory 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
11Psychopharmacology Models
- 1. Muscle Relaxation
- Rota-rod test
- Inclined screen test
- Grip Strength test
- 2. Behavioral test
- Irwin test
12 13General 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
14In-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)
15Development 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
16Special 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)
17 18In 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
19In-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
20 21Scope 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
29/12/2009
21
Nepal Pharmaceuticals Ltd., Birgunj, Nepal.
22Definition of Safety Pharmacology
- Pharmacology studies can be divided into three
categories - 1. Primary pharmacodynamic
- 2. Secondary pharmacodynamic
- 3. Safety Pharmacology studies
23Objectives 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
24General 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
25General 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.
26Test 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
27General 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.
28Use 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
29Sample 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
30Route 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
31Dose Levels or Concentrations of test Substance
- In Vivo Studies
- In Vitro Studies
32In 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.
33In 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.
34Duration 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.
35Studies 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.
36Studies 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
37Safety Pharmacology Core Battery
- 1. Central Nervous System
- 2. Cardiovascular System
- 3. Respiratory System
38Central 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.
39Cardiovascular 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.
40Respiratory 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.
41Follow-up and Supplemental Safety Pharmacology
Studies
- Follow-up Studies For Safety Pharmacology Core
Battery - Central Nervous System
- Cardiovascular System
- Respiratory System
42Central 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
43Follow-up and Supplemental Safety Pharmacology
Studies
- 2. Supplemental Safety Pharmacology Studies
- Renal/Urinary System
- Autonomic Nervous System
- Gastrointestinal System
- Other Organ Systems
44Renal/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
45Autonomic 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.
46Gastrointestinal 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
47Other 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.
48Conditions 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.g
- Case of a new salt having similar
pharmacokinetics and pharmacodynamics
49Timing of Safety Pharmacology Studies in Relation
to Clinical Development
- Studies Prior to First Administration in Humans
- Studies During Clinical Development
- Studies Before Approval
50Studies 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.
51Studies During Clinical Development
- Additional studies may be warranted to clarify
observed or suspected adverse effects in animals
and humans during clinical development
52Studies 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
53Application 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
54Application of Good Laboratory Practice (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
55Application of Good Laboratory Practice (GLP)
- 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
56- DRUG METABOLISM PHARMACOKINETIC STUDIES
57In-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
58In-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
59Plasma 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
60Drug 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
61Special 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
62- HISTOPATHOLOGICAL BIOCHEMISTRY STUDIES
63Hematology
- 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
64Clinical 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
65Biomarkers
- Advanced 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
66 67General 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
68General Pharmacology Equipments
- Deep freezer (420 L)
- Electrophoresis
- Cooling microcentrifuge
- CNS Pharmacology related instruments
- Student kymographs
- Respiratory pumps
- Langendorffs apparatus
- UGO Basile plethysmometer
- UGO Basile analgesiometer
- UGO Basile ECT
69List of Critical Equipment for Pharmacology
- IVC units
- Boyles apparatus
- Hot plate
- Plethysmometer
- Automated cell counter (Mythic 18)
- Plate reader (Biotech)
- Plantar Aesthesiometer
70List 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)
71List 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)
72List of Critical Equipment for Assay Development
- 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
73List 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)
74List 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)
75PHARMACOLOGY LAB LAY OUT (ANIMAL HOUSE)
Sterile room for exchange of clothes, belongings
etc
Library
Quarantine room
Animal House for MICE
Animal house for RATS
Entrance
Dosing Room
Senior and Junior Research Officers
Restricted Entry
Restricted Entrance
HODs Office
Well equipped Samples Analysis Room
Operation Room for invasive procedures
Animal House for Rabbits
Dosing Room
Mating Room for Rats and Mice
Animal House for Guinea Pigs
Discussion/Meeting Room
76Rodent 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)
77International 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
78Thanking You