Pharmacodynamics, ADRs & Preclinical drug testing

Manipal College of Pharmaceutical Sciences
14-Sep-18
Pharmacodynamics
Dr Yogendra Nayak
yogendra.nayak@manipal.edu; yogendranayak@gmail.com
Mobile: 9448154003

Manipal College of Pharmaceutical Sciences - MAHE Manipal 2
Pharmacodynamics - Definition
• Study of the biochemical & physiological effects
of drugs & their mechanisms of action at
– Organism level
– system
– organ
– Tissue
– Sub-cellular
– Macromolecular
– Molecular level

Principles of Drug Action
1. Stimulation
2. Depression
3. Irritation
4. Replacement
5. Cytotoxic action

Principles of Drug Action - Stimulation
• Selective enhancement of activity
– Adrenaline stimulates heart
– Pilocarpine stimulates salivary glands
• Excessive stimulation  opposite action
– Picrotoxin  CNS stimulant  Coma,
Respiratory depression
Pilocarpus
Anamirta cocculus

Principles of Drug Action - Depression
• Selective diminution of activity
– Quinidine depresses heart
– Barbiturates depress CNS
– ACh  Stimulates intestinal smooth muscle
– ACh  Depresses SA-node in heart

Principles of Drug Action - Irritation
• Nonselective, noxious effect
• Mild irritation may stimulate associated
functions
• Bitters increase salivary & gastric secretion
• Counter irritants  ↑ blood flow to the site
• Strong irritation  inflammation, corrosion,
necrosis & morphological damage  loss of
function

Principles of Drug Action - Replacement
– Parkinsonism ( Levodopa)
– Diabetes (Insulin)
• Use of natural metabolites, hormones or their
congeners in deficiency states

Principles of Drug Action - Cytotoxic Action
• Selective toxicity
– Invading parasites
– Cancer cells
– Antimicrobials (Penicillin)
– Anticancer drugs (Cyclophosphamide)

Mechanism of Drug Action
1. Physical
2. Chemical
3. Enzymes
4. Ion channels
5. Receptors

Targets

Targets
Receptors
Ion channels
Enzymes
Carrier molecules (transporters)

Physical Actions
• Adsorptive Property : Charcoal,
Kaolin
• Mass: Bulk laxatives, Protectives
• Osmosis: Mannitol, Magnesium
sulphate
• Radioactivity: I131
• Radio Opacity: Barium sulphate

Chemical Actions
• Antacids  neutralization of gastric HCl
• Potassium Permanganate  oxidizing
property
• Chelating agents (EDTA, dimercaprol) 
Chelation of heavy metals
• Cholestyramine  sequestration of
cholesterol in the gut
• Mesna  Scavenging reactive metabolites of
cyclophosphamide/ cisplatin

Actions through Enzyme

• Enzyme Inhibitors
• Nonspecific
• Specific
– Competitive
• Equilibrium type
• Nonequilibrium type
– Noncompetitive

• Nonspecific Inhibition
– Heavy metal salts
– Strong acids & alkalies
– Alcohol, Formaldehyde, Phenol
– Denaturing the proteins

• Competitive Inhibitors
– Equilibrium & Nonequilibrium type

• Noncompetitive Inhibitors
• Drug reacts with an adjacent site & not with the catalytic
site, but alters enzyme & loses its catalytic property
– Acetazolamide - Carbonic anhydrase
– Disulfiram –Aldehyde dehydrogenase
– Omeprazole- H-K-ATPase
– Theophylline- Phosphodiesterase
– Sildenafil- Phosphodiesterase-5

Actions through Receptors
• Receptor
– Component of a cell or organism
– Interacts with a drug
– Initiates the chain of biochemical events
– Leading to the drug's observed effects

• A macromolecule or binding site
• Surface or inside the cell
• Recognize the drug/ signal
molecule – High specificity
• Initiate the response to the drug
• Itself has no other function

Anatomical Receptors

Nicotinic Receptor

• Ligand
• Agonist
• Inverse agonist
• Antagonist
• Partial agonist

Actions through Receptors - Agonist
• Affinity
– Ability of the drug to combine with the receptor
– A molecule which can bind to receptor is called
ligand (drug)
• Intrinsic activity ( Efficacy)
– Ability of the drug to activate the receptor
(conformational change) consequent to receptor
occupation

• Based on affinity & efficacy 
Ligands are classified
– Agonist
– Inverse agonist
– Antagonist
– Partial agonist

• Agonism
• Antagonism

• High affinity
• High intrinsic activity
• ACh  Nicotinic receptor
• Adr  Beta receptor

Actions through Receptors - Antagonist
• High affinity
• No intrinsic activity
• d-Tubocurarine  Nicotinic
receptor
• Propranolol  Beta receptor

Actions through Receptors - Partial Agonist
• High affinity
• Sub maximal intrinsic activity
• Nalorphine  partial agonist of
opioid receptor

Actions through Receptors - Inverse agonist
• High affinity
• Inverse efficacy (intrinsic
activity)  opposite action
• Beta carbolines are inverse
agonists of benzodiazepine
receptors

Actions through Receptors – Benzodiazepine Receptors
• Benzodiazepine is agonist
(sedation)
• Beta carboline, inverse agonist
(excitation)
• Flumezanil is antagonist - Antidote

(sedation)
(excitation)

How Drugs binds Receptors?
• Ionic bond
• Covalent bond
• Dipole interaction
• Hydrogen bond
• Hydrophobic interaction
• Van der Waals interaction

Drug Receptor Interaction - Theories
• Occupation theory
• Rate theory
• Induced fit theory
• Occupation activation theory -
“Two State” receptor model

– Non-activated (Ri)
– Activated (Ra)
Ri Ra

Agonist
Ri Ra Response
Ri Ra
Antagonist
No Response
Inverse agonist
Ri Ra Opposite Response

Actions through Receptors - Types

Actions through Receptors -Types
• Nicotinic acetylcholine receptor (nAChR; N)
• GABAA receptor
• Glutamate receptors (NMDA, AMPA &
kainate types)

• Muscarinic acetylcholine receptor
(mAChR; M)
• Adrenoceptors
• Chemokine receptors

• Cytokines & growth factors
• Atrial natriuretic factor (ANF) - Guanylyl
cyclase type

• Gene transcription
• Steroid hormones
• Thyroid hormone
• Retinoic acid & vitamin D

Receptor Types, Sub-types – Naming
• Nicotinic  NN, NM
• Muscarinic  M1, M2, M3, M4, M5
• Adrenergic
– α1, α2
– β1, β2, β3
• Dopamine
– D1, D2, D3, D4…

Receptor Types, Sub-types – Naming
• International Union of Pharmacological
Sciences (IUPHAR)

Basic & Clinical Pharmacology
Translational
Pharmacology

Basic & Clinical Pharmacology
Translational Pharmacology
Experience Based Medicine Evidence based Medicine

Ligand Gated Ion Channels
Ion Channel Receptors
GABAA, Benzodiazepine, Kainate,
5HT3, NMDA & AMPA, Glycine, etc

GPCR: α – Adrenergic Receptors
7 - α-helical structure
3 - Intracellular loops
3 - Extracellular loops
N-terminus
C-terminus

Structure of GPCR: β – Adrenergic Receptors
7 - α-helical structure
3 - Intracellular loops
3 - Extracellular loops
N-terminus
C-terminus

Water-soluble
hormone
Receptor
G protein
Blood capillary
Binding of hormone (first messenger)
to its receptor activates G protein,
which activates adenylate cyclase
Adenylate cyclase
1
Water-soluble
hormone
Receptor
G protein
cAMP
Second messenger
Activated adenylate
cyclase converts
ATP to cAMP
Blood capillary
Adenylate cyclase
ATP
1
2
Water-soluble
hormone
Receptor
cAMP serves as a
second messenger
to activate protein
kinases
G protein
Protein kinases
cAMP
Second messenger
Activated adenylate
cyclase converts
ATP to cAMP
Blood capillary
Adenylate cyclase
ATP
1
2
3 Activated
protein
kinases
Water-soluble
hormone
Receptor
cAMP serves as a
second messenger
to activate protein
kinases
G protein
Protein kinases
cAMP
Activated
protein
kinases
Second messenger
Activated adenylate
cyclase converts
ATP to cAMP
Activated protein
kinases
phosphorylate
cellular proteins
Blood capillary
Adenylate cyclase
ATP
1
2
4
3
Protein— P
ADP
Protein
ATP
Water-soluble
hormone
Receptor
cAMP serves as a
second messenger
to activate protein
kinases
G protein
Protein kinases
cAMP
Activated
protein
kinases
Protein—
Second messenger
Activated adenylate
cyclase converts
ATP to cAMP
Activated protein
kinases
phosphorylate
cellular proteins
Millions of phosphorylated
proteins cause reactions that
produce physiological responses
Blood capillary
Adenylate cyclase
P
ADP
Protein
ATP
ATP
1
2
4
3
5
Water-soluble
hormone
Receptor
cAMP serves as a
second messenger
to activate protein
kinases
G protein
Protein kinases
cAMP
Activated
protein
kinases
Protein—
Second messenger
Phosphodiesterase
inactivates cAMP
Activated adenylate
cyclase converts
ATP to cAMP
Activated protein
kinases
phosphorylate
cellular proteins
Millions of phosphorylated
proteins cause reactions that
produce physiological responses
Blood capillary
Adenylate cyclase
P
ADP
Protein
ATP
ATP
1
2
6
4
3
5
Mechanism of Action
of Water-soluble Hormones
Signal Transduction
Transducer Mechanism

G-Proteins
• GPCRs linked to G-proteins
• For signal transduction or
response effectuation
• GTP – Binding proteins
– α, β & γ – subunits

* RGS: Regulators of G-protein Signaling

* GRKs: G protein receptor kinases
↑ : Stimulating
↓ : Inhibiting

G-Proteins - Functions
• α – subunit binding proteins
– Gs  Stimulate Adenylyl cyclase, Ca2+ - Channel opening
– Gi  Inhibit Adenylyl cyclase, K+ - Channel opening
– Go  Ca2+ - Channel opening
– Gq  Stimulates Phospholipase C

• β-γ – subunit binding proteins
• Activate K+ channels
• Inhibit voltage-gated Ca2+ channels
• Activate GPCR kinases
• Activate mitogen-activated protein
kinase cascade (MAPKinase Cascade)

GPCRs GP-Coupler
Muscarinic M2 Gi, Go
Muscarinic M1, M3 Gq
Dopamine D2 Gi, Go
β-adrenergic Gs
α1-adrenergic Gq
α2-adrenergic Gi, Go
GABAB Gi, Go
Serotonin 5-HT1 Gi, Go
Serotonin 5-HT2 Gq
Prostanoid Gs, Gi, Gq

β-Adrenergic & M2-Muscarinic Receptors

Signal Transducer Mechanism
• Adenylyl cyclase: cAMP pathway
• Phospholipase C: IP3-DAG pathway
• Channel regulation

GPCR Transduction

Kinase-linked & Related Receptors
• Receptor tyrosine kinases (RTKs)
– Insulin, EGF, NGF, PDGF, VEGF,
Herceptin, imatinib
• Serine/threonine kinases
– Transforming growth factor (TGF)
• Cytokine receptors
– Interleukins & other cytokines
• Toll-like receptors
– LPS, bacterial products
• Membrane-bound GC
– Natriuretic peptides, Nesiritide

Central Role of Kinase Cascades in Signal Transduction

Nuclear Receptors
Intracellular Glucocorticoid
Receptor Mechanism
Glucocorticoids, mineralocorticoids,
androgens, estrogens, progesterone,
thyroxine, Vit D & Vit A

Receptors Regulation
• Exist in dynamic state
• Density & efficacy to elicit
response
– Subject to regulation by the
level of on-going activity
– Feedback from their own
signal output
– Other physiopathological
influences

• Estrogens increase the density of oxytocin
receptors on the myometrium
– Sensitivity of uterus to oxytocin increases during third
trimester of pregnancy/near term
• Denervation / continued use of antagonist /
drug
– Super sensitivity of receptor as well as effector
system to agonist
• Clonidine/ CNS-depressant/ opioid withdrawal
syndromes, sudden discontinuation of
propranolol in angina pectoris, etc.

• Continued/ intense receptor stimulation
causes desensitization or refractoriness
– Receptor becomes less efficient in transducing
response to the agonist
• Bronchial asthma patients treated
continuously with β adrenergic agonists
• Parkinsonian patients treated with high
doses of levodopa

Receptors Regulation - Mechanisms
• Down regulation
– Decreased synthesis/ increased destruction
of receptor
• Homologous & heterologous
desensitization

Drug Dose & Dose Response
• Potency
• Efficacy
• Dose response curve (DRC)
– Intensity of response

DRC – In Vitro Experiments

DRC of ACh on Chick Ileum
1 μg 2 μg 4 μg 8 μg 16 μg
Log dose (μg)
%Response(mm)
Dose Response Plot DRC: Dose Response Curve
30%
70%

DRC – Median effective dose - ED50
Log dose
PainRelief
DRC: Dose Response Curve
ED50: Dose at which 50% of
maximum response50%
ED50

DRC – TD50
Log dose
Toxicity
TD50: Dose at which 50% of
maximum toxicity is seen50%
TD50

DRC – Lethal Dose, LD50
Log dose
Lethality
LD50: Dose at which 50% of
subjects were lethal50%
LD50

DRC – Potency & Efficacy
Log dose
%Response
DRC: Dose Response Curves
50%
ED50 TD50 LD50

DRC – Therapeutic Index
Log dose
%Response
50%
ED50 LD50
Therapeutic Index
= LD50 / ED50

DRC – Potency
• Potency
– Amount of drug required to produce a certain
response
• Eg: morphine >10 times pethidine
– 10 mg of morphine = 100 mg of pethidine
• Position of DRC on the dose axis is an
index of drug potency

DRC – Potency
Potency: Drug A > Drug B
Log dose
%Response
50%
Drug A
Drug B

DRC – Efficacy
• Efficacy: Maximal response elicited by
drug
• Upper limit of DRC is the index of
efficacy
• Eg: Degree of analgesia by morphine is
not obtainable with any dose of aspirin
– Efficaciousness: Morphine>Aspirin
• Efficacy is a more decisive factor in the
choice of a drug

DRC – Efficacy
Log dose
%Response
50%
Drug A
Drug B
Efficacy: Drug A = Drug B

DRC – Potency
Potency: Morphine > Pethidine
Efficacy: Morphine = Pethidine
Log dose
%Response
50%
10 100
Morphine Pethidine

DRC – Efficacy
Log dose
%Response
50%
Morphine
Aspirin
Efficacy: Morphine > Aspirin
Potency: Morphine > Aspirin

Log dose
%Response Salbutamol
Bronchodilator Salbutamol
Cardio stimulation

Log dose
%Response Isoprenaline
Bronchodilator
Isoprenaline
Cardio stimulation

Log dose
%Response
Salbutamol Bronchodilator
Salbutamol Cardio stimulation
Isoprenaline Bronchodilator
Isoprenaline Cardio stimulation

DRC – Slope
• Steep slope  a moderate ↑
dose will markedly ↑ response
• Flat  little ↑ in response occur
over a wide dose range

Exercise
Compare the Potency & Efficacy of Drug A, B, C, & D

Combined Effects of Drugs
• Synergism
• Antagonism
• Synergism : Action of one drug is
facilitated or increased by other
– Additive
– Supra additive (Potentiation)

Combined Effects of Drugs - Additive
• Effect of combination is equal to sum
of the individual components
• Drugs A + B = Drug A + Drug B
• Eg: Aspirin + Paracetamol 
Analgesic/ Antipyretic

Combined Effects of Drugs - Additive
Log dose
%Response
A BA + B

Combined Effects of Drugs - Potentiation
• Effect of combination is greater than the
individual effects
• Drugs A + B > Drug A + Drug B
• Eg: Acetylcholine + Physostigmine
Levodopa + carbidopa

Combined Effects of Drugs - Potentiation
Log dose
%Response
A
B
AB

Combined Effects of Drugs - Antagonism
• One drug decreases or inhibits action of
another, they are said to be antagonists
• Drug A+B < effect of A + effect of B
• Antagonism classified depending on
mechanism involved
– Physical
– Chemical
– Physiological
– Receptor

Combined Effects of Drugs – Antagonism
• Physical: Based on physical property of
drugs
– Charcoal prevents absorption of alkaloids
• Chemical antagonism: Drugs react
chemically & form an inactive product
– Eg: chelating agents
• Some drugs may react when taken in the
same syringe
– Penicillin + succinylcholine
– Heparin + penicillin / tetracycline

Combined Effects of Drugs – Antagonism
• Physiological / Functional antagonism
– Two drugs act on different receptors or by
different mechanisms, but have opposite
effects on the same physiological system
– Eg. Histamine & adrenaline on the
bronchial muscles
– Glucagon & insulin on blood sugar level

Receptor Antagonism
• Antagonist interferes with binding of the
agonist to receptor
– Atropine antagonises M-receptors
– Propranolol antagonises β-receptors
• Competitive
• Noncompetitive

Receptor Antagonism
What is A, B & C?

Factors Modifying Drug Actions
• Quantitative
– Dose related
– Pentobarbintone 25 mg/kg, i.p. produces
anesthesia in rats  CNS depression
– Pentobarbitone 50 mg/kg, i.p. produces
Respiratory arrest  CNS depression
• Qualitative
– Response related
– MgSO4 orally produces purgation
whereas, CNS depression by i.p.

2. Age
– Child dose = {Age (in years) X adult dose} ÷ { age + 12 }
--- (Young’s formula)
– Child dose = {Age (in years X adult dose} ÷ 20
---- (Dilling’s formula)
– BSA can also be employed to calculate child dose

4. Species & race
– Rabbits are resistant to atropine
– Rats & mice are resistant to digitalis
– Racial differences are observed in
humans
– Indians tolerate thiacetazone better than
white
– Blacks require higher & Mongols require
lower dose of atropine

6. Route of Administration
• Governs speed & intensity of drug response
• Pentobarbitone sodium rapid onset of action by parenteral
but slow onset by oral
• Magnesium sulfate causes purgation when given orally &
CNS depression & respiratory arrest when given
intraperitonially in rats

9. Pathological status
• GI diseases
– Alters absorption
– In coeliac diseases absorption of amoxycillin is increased but
that of cotrimoxazole is decreased
• Liver diseases – can influence drug disposition
– bioavailability of drugs with high first pass metabolism
– Serum albumin is reduced
– Metabolism of morphine, phenobaritone is reduced
– Prednisone, sulindac are less active  Prodrugs

Drug Toxicity – Adverse Drug Reactions (ADRs)
• Adverse effect is ‘any undesirable or
unintended consequence of drug
administration’
• Predictable (Type A or Augmented)
reactions
– mechanism based adverse reactions
• Unpredictable (Type B or Bizarre) reactions
– not on drug’s known actions

ADRs Contd..
• Minor
• Moderate
• Severe
• Lethal

ADRs - Pharmacovigilance
• Pharmacovigilance has been defined by WHO (2002)
• Science & activities relating to detection, assessment,
understanding & prevention of ADRs or any other
drug related problems
• Post marketing surveillance

ADRs – 1. Side Effect
• Promethazine  Antiallergic  Sedation
• Codeine  Cough  produces constipation
• Digoxin  Atrial fibrillation  A-V block

ADRs – 2. Secondary Effect
• Suppression of bacterial flora by tetracyclines paves
the way for superinfections
• Corticosteroids weaken host defence mechanisms so
that latent tuberculosis gets activated

ADRs – 3. Toxic Effect
• Excessive Pharmacological actions due to prolonged
use or over-dosage
• Coma by barbiturates
• Respiratory depression by Morphine
• Streptomycin (antitubercular)  vestibular damage
on prolonged

ADRs – 4. Intolerance
• Few doses of carbamazepine causes ataxia in some
people
• Single tablet of chloroquine causes vomiting &
abdominal pain in an occasional patient
• Triflupromazine induces muscular dystonias in some
individuals, specially children

ADRs – 5. Idiosyncrasy
• Abnormal reactivity  genetically determined
• Barbiturates cause excitement & mental confusion in
some individuals
• Quinine/quinidine cause cramps, diarrhoea, purpura,
asthma & vascular collapse in some patients
• Chloramphenicol produces non-dose-related serious
aplastic anaemia in rare individuals

ADRs – 6. Drug Allergy
• Immunologically mediated reaction
• Only in small proportion of population
– Humoral
• Type II & Type III allergic reactions
• Phenytoin  Stevens-Johnson syndrome
– Cell mediated
• Type IV reactions
• contact dermatitis, rashes, fever, photosensitization

ADRs – 7. Photosensitivity
• Cutaneous reaction  drug induced sensitization
• Phototoxic
– Tetracycline  Erythema, edema, blistering
• Photoallergic
– Sulfonamides, sulfonylureas, griseofulvin, chloroquine,
chlorpromazine, carbamazepine  immediate flare, itching
& wheal on exposure to sun

ADRs – 8. Drug Dependence
• Psychological dependence
– Induce drug seeking behaviour
– Opioids, cocaine, Benzodiazepines
• Physical dependence
– Withdrawal (abstinence) syndrome  neuroadaptation
– Opioids, barbiturates, alcohol & benzodiazepines

ADRs – 8. Drug Dependence contd…
• Drug abuse
– Self-medication
– Regulatory body  Use of ilicit drugs
– Continuous & occasional uses
• Drug Addiction
– Drug seeking behaviours
• Drug habituation
– Consumption of tea, coffee, tobacco, social drinking are
regarded habituating, physical dependence is absent

ADRs – 9. Drug Withdrawal Reactions
• Acute adrenal insufficiency by abrupt cessation of
corticosteroid
• Severe hypertension, restlessness & sympathetic
over-activity  clonidine discontinuation
• Angina, myocardial infarction  from stoppage of β-
blockers in hypertension
• Seizures increase  sudden withdrawal of an
antiepileptic

ADRs – 10. Teratogenicity
• Effect of drugs on growing embryo in pregnant
women
• Fertilization & implantation—conception to 17 days 
failure of pregnancy which often goes unnoticed
• Organogenesis 18 to 55 days of gestation  most
vulnerable period, deformities are produced
– Thalidomide tragedy
• Growth & development  56 days onwards
– ACE inhibitors, NSAIDs, Androgens & progestins,
antithyroid drugs, lithium

ADRs – 11. Mutagenicity & Carcinogenicity
• Anticancer drugs
• Radioisotopes
• Estrogens
• Tobacco

ADRs – 12. Drug Induced Diseases
• Iatrogenic/ iatrogenicity  Physician induced
• Peptic ulcer by salicylates & corticosteroids
• Parkinsonism by phenothiazines & other
antipsychotics
• Hepatitis by isoniazid
• Discoid lupus erythematosus (DLE) a chronic
skin condition  Hydralazine

Rational Use of Drugs
• Correct medicine for correct
disease/ problem
• Dose
• Time
• Duration
• Monitoring/ Prognosis
• Therapeutic Management

• Components
– Supply-use chain of drugs
– Selection
– Procurement
– Storage
– Prescribing
– Dispensing
– Monitoring
– Feedback

• Rational Prescriptions
• Irrational prescriptions
• Irrational combinations of
drugs
• Polypharmacy

New Drug Discovery – Preclinical Pharmacology

Preclinical Pharmacology
• Blind/ Random Screening
• Tests on isolated organs, bacterial
cultures
• Tests on animal models of human disease
• Focussed trials to systemic pharmacology
• Quantitative tests  DRC, potency,
efficacy, comparative studies
• Pharmacokinetics
• Toxicokinetics

• Acute toxicity
– 1 - 3 days  toxicity, lethality, LD50
• Subacute toxicity
– Repeated doses  2–12 weeks
– Cumulative toxicity, generally 28-days toxicity
in rats
• Chronic toxicity
– Long term toxicity, 6–12 months
– 91 days toxicity in rats

• Special toxicities
– Mutagenicity
– Carcinogenicity
– Reproduction & teratogenicity
– Developmental toxicity
• GLP  Good Laboratory
Practices  animal studies

Manipal College of Pharmaceutical Sciences 155
Thank you
Ph: 9448154003
yogendra.nayak@manipal.edu; yogendranayak@gmail.com

Pharmacodynamics, ADRs & Preclinical drug testing

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