Download parenteral controlled drug delivery system

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: [email protected]
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
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Nepal Research Foundation., Birgunj, Nepal.
3
Oncology
i. Xenograft model
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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
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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
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5
Pain and Inflammation
1. Chemical Induced

Capsaicin induced Hyperalgesia in rats
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FCA induced Hyperalgesia
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Carrageenan induced paw edema
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Acetic acid/Formalin induced pain in mice
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Mouse Ear Edema
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Adjuvant-Induced Arthritis (AIA)
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Collagen-Induced Arthritis (CIA)
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Nepal Research Foundation., Birgunj, Nepal.
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Pain 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
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Nepal Research Foundation., Birgunj, Nepal.
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Pain 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
30/12/2009
Nepal Research Foundation., Birgunj, Nepal.
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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
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Nepal Research Foundation., Birgunj, Nepal.
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Respiratory Diseases
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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.
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Psychopharmacology Models
1. Muscle Relaxation
 Rota-rod test
 Inclined screen test
 Grip Strength test
2. Behavioral test
 Irwin test
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IN-VIVO TOXICOLOGY
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General Toxicology
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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
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13
In-Vivo Genotoxicity Studies
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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)
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Nepal Research Foundation., Birgunj, Nepal.
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Development & Reproduction toxicology
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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
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Special Toxicity Studies
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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)
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IN – VITRO TOXICOLOGY
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In – Vitro Toxicology
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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
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In-Vitro Genotoxicity Studies
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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
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SAFETY PHARMACOLOGY
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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
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30/12/2009
Nepal
Nepal Research
Pharmaceuticals
Foundation.,
Ltd., Birgunj,
Birgunj, Nepal.
Nepal.
21
Definition of Safety Pharmacology

Pharmacology studies can be divided into
three categories:
1. Primary pharmacodynamic
2. Secondary pharmacodynamic
3. Safety Pharmacology studies
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Objectives of Studies
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To identify undesirable pharmacodynamic
properties of a substance that may have
relevance to its human safety
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To evaluate adverse pharmacodynamic and/or
pathophysiological effects of a substance
observed in toxicology and /or clinical studies
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To investigate the mechanism of the adverse
pharmacodynamic effects observed and/or
suspected
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General Considerations in Selection and Design of
Safety Pharmacology
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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
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General Considerations in Selection and Design of
Safety Pharmacology
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Ligand binding or enzyme assay data
suggesting a potential for adverse effects
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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.
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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
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General Considerations on Test Systems
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Consideration should be given to the selection of
relevant animal model or other test systems so that
scientifically valid information can be derived.
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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.
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Use of In Vivo and in Vitro Studies
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Animal models as well as ex vivo and in citro
preparations can be used as test systems.
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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.
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In vitro systems can be used in supportive studies
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In conducting in vivo studies, it is preferable to use
anaesthetized
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Sample Size and Use of Controls
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The sample size should take into consideration the size
of the biological effect that is of concern for humans
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Appropriate negative and positive control group should
be included in the experimental design
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In well-characterized in vivo test systems, positive
controls may not be necessary.
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The exclusion of control from studies should be justified
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Route of Administration
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In general, the expected clinical route of administration
should be used when feasible.
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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.
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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
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Dose Levels or Concentrations of test Substance
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In Vivo Studies
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In Vitro Studies
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In Vivo Studies
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In vivo safety pharmacology studies should be designed
to define the dose-response relationship of the adverse
effect observed.
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The time course of the adverse effect should be
investigated, when feasible.
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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.
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In Vitro Studies
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In vitro studies should be designed to establish a
concentration-effect relationship.
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The range of concentrations used should be selected to
increase the likelihood of detecting an effect on the test
system.
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The upper limit of this range may be influenced by
physico-chemical properties of the test substance and
other assay specific factors.
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In the absence of an effect, the range of concentrations
selected should be justified.
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Duration of Studies
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Safety pharmacology studies are generally performed by
single-dose administration
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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.
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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.
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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
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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
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Safety Pharmacology Core Battery
1. Central Nervous System
2. Cardiovascular System
3. Respiratory System
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Central Nervous System
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Effects of the test substance on the central
nervous system should be assessed
appropriately.
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Motor activity, behavioral changes, coordination,
sensory/motor reflex responses and body
temperature should be evaluated.
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Cardiovascular System
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Effect of the test substance on the cardiovascular
system should be assessed appropriately.
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Blood pressure, heart rate and the
electrocardiogram should be evaluated
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In vivo, in vitro and /or ex vivo evaluations, including
methods for repolarzation and conductance
abnormalities, should also be considered.
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Respiratory System
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Effects of the test substance on the respiratory system
should be assessed appropriately
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Respiratory rate and other measures of respiratory
function should be evaluated.
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Clinical observation of animals is generally not adequate
to assess respiratory function, and thus these
parameters should be quantified by using appropriate
methodologies.
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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
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Central Nervous System, Cardiovascular System
& Respiratory System
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Behavioral pharmacology, learning and memory, ligandspecific binding, neurochemistry, visual, auditory, and/or
electrophysiology examination
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Cardiac output, ventricular contractility, vascular
resistant, the effects of endogenous and/or exogenous
substances on the cardiovascular responses
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Airway resistance, compliance, pulmonary arterial
pressure, blood gases, blood pH
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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
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Renal/Urinary System
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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
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Autonomic Nervous System
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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.
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Gastrointestinal System
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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
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Other Organ Systems
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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.
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Conditions Under Which Studies are Not
Necessary
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Safety pharmacology studies may not be needed for
locally applied
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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
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Case of a new salt having similar pharmacokinetics and pharmacodynamics
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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
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Studies prior to First Administration in Human
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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.
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Information from toxicology studies adequately designed
and conducted to address safety pharmacology endpoint
can result in reduction or elimination of separate safety
pharmacology studies.
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Studies During Clinical Development
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Additional studies may be warranted to clarify observed
or suspected adverse effects in animals and humans
during clinical development
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Studies Before Approval
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Safety pharmacology effects should be assessed prior to
product approval, unless not warranted, in which case
this should be justified.
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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
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Application of Good Laboratory Practice (GLP)
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This is normally accomplished through the conduct of the
studies in compliance with GLP.
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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
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The safety pharmacology core battery should ordinarily
be conducted in compliance with GLP
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Application of Good Laboratory Practice (GLP)
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Follow-up and supplemental studies should be
conducted in compliance with GLP to the greatest extent
feasible.
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Safety pharmacology investigations can be part of
toxicology studies; in such cases, these studies would be
conducted in compliance with GLP
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Primary pharmacodynamic studies do not need to be
conducted in compliance with GLP
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Application of Good Laboratory Practice (GLP)
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Generally, secondary pharmacodynamic studies do not
need to be conduct in compliance with GLP
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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
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DRUG METABOLISM &
PHARMACOKINETIC STUDIES
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In-vivo DMPK Studies
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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
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In-vitro DMPK Studies
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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
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Nepal Research Foundation., Birgunj, Nepal.
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Plasma Protein Binding
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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
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Drug –Drug Interaction Studies
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Using pooled human liver microsomes or
other in vitro metabolism models
The interaction of a drug on the metabolism
of another drug is determined
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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
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HISTOPATHOLOGICAL &
BIOCHEMISTRY STUDIES
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Hematology
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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
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Clinical Chemistry
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Alanine Aminotransferase
Albumin
Alkaline Phosphatase
Aspartate Aminotrasferase
Calcium
Chloride
Cholesterol
Creatinine
Glucose
Prealbumin
A/G/ Ratio
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Globulin,
Phosphorous
Potassium
Sodium
Total Bilirubin
Total Protein
Triglycerides
Urea
Nitrogen
Osmolality
Anion Gap
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Biomarkers
Advanced Lipid biomarkers
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HDL-C
LDL-C
VLDL
Cardiac risk ratio
Apo A1
Apo B
FFA
Cardiac biomarkers
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BNP
Troponin I
Myoglobin
CK
CK-MB
Homocysteine
Hs-CRP
D-Dimer
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LIST OF EQUIPMENTS
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General Pharmacology Equipments
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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
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General Pharmacology Equipments
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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
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List of Critical Equipment for Pharmacology
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IVC units
Boyle’s apparatus
Hot plate
Plethysmometer
Automated cell counter (Mythic 18)
Plate reader (Biotech)
Plantar Aesthesiometer
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List of Critical Equipments for ADME and PK
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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)
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List of Critical Equipment for Assay
Development
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

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
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
Nepal Research Foundation., Birgunj, Nepal.
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List 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
30/12/2009
Nepal Research Foundation., Birgunj, Nepal.
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List of Critical Equipments for Molecular
Biology and Gene Expression
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


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.
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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)
30/12/2009
Nepal Research Foundation., Birgunj, Nepal.
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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.
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Rodent Facility
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
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)
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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
30/12/2009
Nepal Research Foundation., Birgunj, Nepal.
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Thanking You
30/12/2009
Nepal Research Foundation., Birgunj, Nepal.
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