Experiment NO.

General introduction of pharmacology and experimental pharmacology.

Pharmacology:

The word pharmacology is derived from Greek words “Pharmacon” which means
drug and “logia” study of. Pharmacology is the science of drugs. It deals with the
interaction of exogenously administered chemical molecules (drug) with the living
system.

Drug:

It is an active chemical entity present in a medicine that is used for diagnosis,

prevention, treatment and cure of a disease.

Clinical pharmacology:

Clinical pharmacology is a branch of biomedical science. Clinical pharmacology is

the study of drugs in humans. It includes drug discovery, the study of the effects of
drugs on their targets in living systems and their clinical use, as well as the study of
biological function related to these chemicals. Clinical pharmacology includes
application of pharmacological principles, such as pharmacodynamics and
pharmacokinetics.

Pharmacokinetics:

Pharmacokinetics refers to what the body does to the drug. It includes absorption
distribution, metabolism and elimination.

Absorption:

Absorption is the movement of a drug from the site of administration towards the
systemic circulation. The rate and extent of absorption depends on the environment
from where the drug is absorbed, chemical characteristics of the drug and the route
of administration.
General Principles:

 Nonpolar Lipid soluble drugs traverse the cell membrane more easily than
ionic or polar water soluble drugs.

 Low molecular weight drugs diffuse across cell membrane more easily than
do high molecular weight drugs.

 Drugs bound to proteins and macromolecules do not easily cross the cell
membrane.

Distribution:

Drug distribution is the process by which a drug reversibly leaves the bloodstream
and enters the interstitium (extracellular fluid) and the tissues.

Metabolism:

It is the conversion of drug into a form which can be excreted. It is needed to

convert the nonpolar (lipid soluble) compounds in to polar (lipid insoluble)
compounds so that they are not reabsorbed in the renal tubules and are excreted.

This mechanism of metabolizing the drug protects the body from toxicity.

Excretion:

It is the removal of drug from the body. Drug and their metabolites are excreted
in:

 Urine

 Feces

 Exhaled air

 Saliva

 Skin (sweating)
Pharmacodynamics:

The action of drug on the body and the influence of drug concentration on the
magnitude of the response.

Bioavailability:

The fraction of unchanged drug reaching the systemic circulation following

administration by any route is known as bioavailability.

Receptor:

Any target molecule with which a drug molecule combines in order to elicit
its specific effect.

OR

Any biological molecule to which a drug binds and produces a measurable

response is known as a receptor.

Ligand:

Any molecule which attaches to a particular receptor or site is known as

ligand.

Agonists, partial agonist and antagonists are examples of ligand.

Agonist:

An agent which activates a receptor to produce an effect similar to that of

the physiological signal molecule is known as an agonist.

Antagonist:

An agent which prevent the action of an agonist on a receptor, but does not
have any effect of its own is termed as an antagonist.

Affinity:

The ability to bind with the receptor is designated as affinity. Affinity

describes the strength of interaction (binding) between a ligand and its receptor.
Efficacy:

Efficacy is the magnitude of response that a drug cause when it interact with
a receptor. It is the maximum effect of a drug.

Maximum efficacy of a drug assumes (E max) assumes that all the receptors are
occupied by the drug and no increase in response is observed if higher
concentration of drug is given.

Potency:

Potency is a measure of the amount of drug necessary to produce an effect

of a given magnitude.

OR

The quantity of a drug required to produce a desired response is potency of drug.

The lower the dose required for given response the more potent is the drug.

Posology:

The word posology is derived from Greek word 'posos' means how much
and 'logos' means science. Posology is the branch of medical science that deals
with dose or quantity of drugs which can be administered to the patient to get
desired pharmacological action.

Toxic effects:

These are the harmful effects of the drug which is related to over dose.

Therapeutic Index:
Therapeutic index is the ratio of lethal dose to effective dose. It an indication
of safety of a drug

TI = LD50/ED50, (higher is better).

Half-life:
Time taken for plasma concentration of a drug to drop/decrease to 50% of
initial level is called as half-life.
Experimental Pharmacology:

It is the branch of pharmacology which deals with the study of effects of

drugs on the experimental animals for the determination of various affects related
to mechanism of action.

Objectives of Experimental Pharmacology:

1. To screen drug substance for their biological activities.

2. To test the toxicity of drugs.
3. To study the mechanism of actions and as well as site of action of drugs.

Tolerance:
A state in which an individual no longer respond to a drug at same dose. A
higher dose is required to achieve the same effect.
Dependence:
Dependence develops when the body adapts to the repeated drug exposure
and only functions normal in the presence of the drug.
When the drug is withdrawn severe physical and psychological reactions occur.
 Experiment NO.2

Study of animals used for experimental pharmacology.

Why do we use animals for research?

The function of cells and organs are basically the same in animal and human. What
we learn from animals is useful in humans and animal medicine. It helps us in
preventing drug-drug and drug-food interactions, toxic and adverse effects of the
drugs which otherwise would have been damaging. All drugs, devices and medical
procedures have been developed with some animal research. Studies in sheep led
to the treatment of respiratory distress syndrome, a major cause of death in
premature infants.

Animals used for experiment:

List of some animals used in experimental pharmacology is given below:

 Frog
 Rats
 Rabbits
 Mice
 Guinea Pigs
 Dogs
 Cats

1) Frog:

Characteristics:

i. It is a cold blooded animal.

ii. It has three chambers in its heart, 2 atria, 1 ventricle.
iii. It is used for the study of heart, abdomen and central nervous system.
 2) Rats:
i. Small in size
ii. It is a warm blooded animal.
iii. Gall bladder and tonsils are absent.
iv. Vomiting center is also absent so oral administration of drug is easy.

Uses in experimental pharmacology

1. Evaluation of psychopharmacological agents.

2. Toxicity studies: Hepatotoxicity, nephrotoxicity, teratogenic and
carcinogenic activity.
3. Study of hepatoprotective and nephroprotective activity of drug.
4. Rats are used for the study of antiulcer and gastro protective activity of a
drug.
5. It is used for the study of antihypertensive effects of a pharmacological
agent.
6. Rats are also used for the study of antidibetic activity.
7. It is also used for the study of analgesic activity of drug, bioassay of various
hormones such as insulin and oxytocin.

3) Rabbit:
i. Docile animal.
ii. New Zealand white strains are
iii. Enzyme atropine esterase is present in rabbit liver so can tolerate large doses
of belladonna (atropine).

Uses in experimental pharmacology

1. Pyrogen testing.
2. Anti-diabetic studies
3. Bioassay of insulin
4. Embryo toxicity studies.
5. For the study of drugs used in glaucoma.

4) Dogs:
 i. Supportive in nature for the experiments.
ii. They can easily be trained for behavioral studies.

Uses in experimental pharmacology

1. Antihypertensive studies.
2. Vaccination
3. Diabetes and antiulcer experiments.

5) Guinea Pig

It is susceptible to anaphylaxis and is highly sensitive to histamine and penicillin.

Uses in experimental pharmacology

1. Evaluation of bronchodilators.
2. Study of histamine and anti-histamine drugs.
3. Evaluation of local anesthesia.

Experiment No.3
 Handling of Lab animals

Why do we use animals for research?

The function of cells and organs are basically the same in animals and humans.
What we learn from animals is useful in humans and animal medicines. Virtually
all the drugs, devices and medical procedures have been developed with some
animal research.

Example:

The anti-AIDS treatment developed from experiments and researches in animals

have greatly extended life expectancy and quality of life for Aids victims.

Precautions to be taken before handling animals:

1) Before handling first tap or sooth the animal by slow deliberate movement
on their body.
2) Overcrowding near the animal cage should be avoided.
3) Noise should be kept to a minimum as much as possible.
4) Do not hold the animal too hard, it may face difficulty in breathing and may
die.
5) Never agitate the animal it may become violent for self-protection.

Handling of rats:

The animal should be grasped by the tail, preferably the proximal third and lifted
clear of its cage. It should then be placed on a surface such as a cage top. The
scruff can be grasped between the thumb and forefinger while maintaining a grip
on the tail. The animal is then secure and can be examined or injected safely.

Handling of rabbits:

Rabbits are especially susceptible to the effects of stress and should always be
approached in a calm and confident manner. The handler restrains the rabbit firmly
by the scruff with one hand and with the other hand supports the animal’s
hindquarters.

The rabbit should be held by its head tucked under the handler’s arm and with the
back and hindquarters supported by the handler’s forearms.
Rabbits may also be restrained for injection by wrapping the animal in a towel. If
the rabbit is securely wrapped, it will not struggle. Intravenous injections into the
rabbit’s marginal ear vein can be readily made if the rabbit is restrained with a
towel.

Frog:

Frog can be held in one hand and stunned with other hand by a sudden sharp blow
of wooden hammer or passing along pithing needle into the spinal cord while
making the rotatory movement and placed over a wooden block.

TO STUDY THE PYROGEN TEST OF GIVEN SAMPLE

REQUIREMENT:

Animal: Rabbits (of either sex weighing 1500g or more.)

Apparatus: Rabbit holder, Automatic temperature recording device calibrate to

0.10C with applicator, 24 Gauge needle and 30 ml syringe.

PRINCIPLE:

Rabbits and human being are equally responsive to the threshold level of pyrogen
given intravenously, on a dose per kg basis. If a sample containing pyrogen is
injected it produce rise in temperature Within 3 hr. according to official methods
given in B.P. and U.S.P. etc. If maximum rise to temperature within 3 hours after
injection of sample exceeds 0.60C. the sample is considered to be pyrogenic and is
discarded. This is rabbit fever response test.

THEORY:

1) Chemically pyrogens are phospholipids attached to the polysaccharide

carrier molecules.
2) They are synthesized in the gram negative bacteria and gram positive
bacteria.
3) Pyrogens include increase in the body temperature if they are administered
to human being or animals by parenteral route. To check the presence of
pyrogens in the injectable formulation, different biological assay are used.

PROCEDURE:
Pyrogen test by rabbit methods:

1) In this methods rise in the body temperature of the rabbits after intravenous
administration of the test solution is studied.
2) The rabbit used for pyrogen test:
3) They have body weight more than 1.5 kg.
4) It can be of either sex.
5) They are maintained in quite place with constant room temperature and
humidity.
6) They are not used for the pyrogen testing more frequently than once every
48 hours.
7) The rabbit which has been used in testing of a pyrogen and has shown more
than 0.60C rise in body temperature is not used for other pyrogen testing
before two weeks.
8) The rabbit are kept in the holders one hour before the actual commencement
of test. They are given diet overnight before the test and are withheld from
water during the testing.

A) Sham Test:

It is performed to select the rabbits for actual testing.

1) The animals satisfying above state are selected at random and are
administered with 10ml/kg pyrogen free saline solution (warmed at 38.0C)
through the marginal ear vein.
2) The body temperature recording is started 90 minutes before this dosing and
is continued for further 3 hrs at the interval of 30 minutes.
3) The rabbit showing more than 0.60C rise in the body temperature are not
used for main test.
B) Main test:

1) The test substance is dissolved in pyrogen free saline solution and warmed at
38.0C. Initially, three rabbits are used for testing. The recording of body
temperature is start 90 minutes before the injection of test materials.
2) Initial body temperatures are recorded at the interval of 30 minutes, not
preceding 40 minutes of the injection.
3) The initial body temperature in individual rabbits should not be less than
380C or greater than 39.80C.
4) Each rabbit selected for the test is injected with the test solution (<0.5 ml/kg
body weight and not > 10 ml/kg body weight) through marginal ear vain
slowly and steadily within 4 minutes.
5) The height body temperature reached in each rabbit after injection is
considered to be the response. (Readings are taken for at least 3 hrs at the
interval of 30 minutes).

RESULTS:

The drugs said to pass the test if:

Sum of rise in the body temperature of three rabbits is not more than 1.4 oC and in
any individual rabbit the rise in the body temperature does not exceed 0.60C.

The test sample fails if:

Rise in body temperature in individual rabbits is more than or equal to 0.60C and
sum of the responses in the three rabbits is more than or equal to 1.40C.

If the sample fails this initial test, the test is carried out in FIVE more rabbits and
pooled observation from all eight rabbits is considered.
The sample passes the pyrogen tests if:

Not more than three rabbits shows individual rise by more than or equal to 0.60C
and sum of rise in body temperature in all eight rabbits does not exceed 3.70C.

Experiment No.5

To study the anticoagulant effect of drugs on clotting time.

Theory

Anticoagulants prolong the clotting time of blood by different mechanisms.

Warfarin an anticoagulant prevents clotting of blood by blocking vitamin K
synthesis.

Vitamin K is necessary for the formation of “prothrombin” in the liver which is

essential for blood clotting. It is synthesized by bacteria in large intestine and is
widely distributed in green vegetables.

Sodium citrate and EDTA are chelating agents that bind with calcium ions making
it inaccessible for blood coagulation.

Apparatus:

10 mL syringe, Alcohol swab, test tube, capillary tube.

Subject:

Healthy Rabbit

Drug used:

Sodium citrate 3mg/mL, EDTA 0.2mg/mL and warfarin.

Procedure:

1. Wash and dry the apparatus as per standard operating procedure (S.O.P).
2. Take 4 test tubes and mark them as A, B, C and D. Test tube A serve as
control while test tube B, C and D are experimental tubes.
3. Then add sodium citrate 3mg/mL in the test tube B, EDTA 0.2mg/mL in the
test tube C and Warfarin in test tube D respectively.
4. Collect about 8 ml of blood from marginal vein of rabbit ear and transfer
2mL of blood in each test tube.
5. Test tube B, C and D are gently rotated with hand to allow mixing of blood
with anticoagulants.
6. Tilt test tube A, B, C and D then dip capillary tubes in each of it. The
capillary tubes are allowed to fill.
7. The filled capillary tubes are broken at regular intervals to check thread
formation.
Precautions:

 Measure the required concentration of anti-coagulant carefully.

 Handle the test animal gently.
 Avoid hematoma formation from the site of blood with drawl.

Result:

Normal clotting time of rabbit was 5 minutes. It was around about 15 minutes with
sodium citrate and was found to be 25 minutes with EDTA and above 30 minutes
with warfarin. Hence warfarin is more effective anti-coagulant drug.

Experiment (write on blank page)

To study the anticoagulant effect on clotting time of rabbit.

Apparatus:

10 mL syringe, Alcohol swab, test tube, capillary tube.

Subject:

Healthy Rabbit

Drug used:

Sodium citrate 3mg/mL, EDTA 0.2mg/mL and warfarin.

Calculation:

Normal Sodium Citrate EDTA Warfarin

Clotting time 5 minute 15 minute 25 minute 30 minutes
Result:

Normal clotting time of rabbit was 5 minutes. It was around about 15 minutes with
sodium citrate and was found to be 25 minutes with EDTA and 30 minutes with
warfarin. Hence warfarin is more effective anti-coagulant drug.
Hemostasis:

The cessation of blood loss from a damaged blood vessel.

Thrombus:

A clot that adheres to the vessel wall is called.

Embolus:

A detached thrombus becomes an embolus.

1. Drugs for the Treatment of Bleeding OR Hemostatic Agents

a) Protamine Sulfate

b) Vitamin K

c) Aminocaproic acid

d) Tranexamic acid

2. Platelet Aggregation Inhibitors

a) Aspirin

b) Clopidogrel (inhibit binding of ADP to its receptors).

c) Tirofiban (binds to GP IIb/IIIa receptors and blocks the binding of

fibrinogen).

3. Anticoagulants (drugs which prevent the formation of clot)

a) Heparin

b) Warfarin

c) Rivaroxaban

d) Enoxaprin

4. Thrombolytic Drugs

a) Alteplase
 b) Streptokinase

c) Urokinase

Antidotes

Drugs Antidotes

Heparin Protamine Sulfate

Warfarin Vitamin K

Fibrinolytic state Tranexamic acid

1) Vitamin K can stop bleeding problems due to warfarin.

2) Protamine Sulfate antagonizes the anticoagulant effects of heparin.

Handling Drug label information

Drug Label

 Label means a display of written or printed material on a container of a drug

package.
Need of a drug label

 Label provides drug information for the use of medical practitioner,

pharmacists or nurses and consumer.

Legal requirements of drug label

 Name of the preparation

 Strength and dosage form

 Quantity

 Instructions for the use

 Precautions and warnings

 Registration number

 Batch number

 Manufacturing and expiry date

 Price

 The name and address of the pharmaceutical industry

Name of the preparation

 It must include generic and brand name of the drug.

 Example: Brufen (ibuprofen)

 Ibuprofen is the generic name whereas brufen is the brand name.

Strength

 It is the amount of drug per unit dose.

 Example:
  Amoxicillin 250 mg capsules and Amoxicillin 500 mg capsules

Dosage form

 Dosage form of the medicine should be mention on the label.

 Example: different dosage forms of Amoxicillin i.e. capsules and oral

suspensions.
Quantity:

The quantity and volume is indicated as per packing units.

Instructions:

Important and necessary instructions should be mentioned on the label.

Example:

 Keep in refrigerator.

 Shake well before use.

 Protect from light.

Precautions and warning:

 Keep out of the reach of children.

 For external use only.

 Inflammable (alcoholic preparations).

Registration number:

 A number given to a specific drug when it is registered according to specific

rules by registration board set by the federal government.

Batch number:
  A number printed on the label of a drug that indicates the batch and
production history of the drug.

Manufacturing and expiry date

 The manufacturing and expiry of the drug should be clearly mentioned on

the label of the drug.

 Expiry date (date stated on the label after which a drug is not expected to
retain its claimed efficacy and safety).

Manufacturing information:
 The name and address of the pharmaceutical industry should the mentioned
on the label of the drug.
 Classification of Antibiotics

1. Macrolides (bind to the 50s subunit of the bacterial ribosome)

a) Erythromycin

b) Azithromycin

c) Clarithromycin

d) Roxitthromycin

2. Aminoglycosides (Binds 30s subunit resulting in misreading of genetic code)

a) Amikacin

b) Gentamicin

c) Neomycin

d) Kanamycin

e) Tobramycin

f) Streptomycin

3. Tetracycline’s (binds to 30S subunit of bacterial ribosome and prevents the

binding of tRNA to the mRNA)

a) Doxycycline

b) Minocycline

c) Tetracycline
 4. QUINOLONES & FLUOROQUINOLONES (Target DNA Gyrase enzyme)

Quinolones:

a) Nalidixic acid
b) Cinoxacin

Fluoroquinolones:

a) Norfloxacin

b) Ofloxacin

c) Ciprofloxacin

d) Moxifloxacin

e) Gatifloxacin

f) Levofloxacin

g) Sparfloxacin

h) Pefloxacin

i) Lomefloxacin

5. Cell wall Synthesis Inhibitors

a) Vancomycin

b) Fosfomycin (inhibits the enzyme enolpyruvate transferase and blocks

the addition of Phosphoenolpyruvate to UDP-N-acetylglucosamine)

c) Bacitracin

d) Cycloserine (inhibits Racemase enzyme which converts L-alanine to D-

alanine)

e) Beta Lactam Antibiotics

 6. Beta Lactam Antibiotics

a) Penicillins (inhibits transpeptidase so that cross linking does not take place)

b) Cephalosporins and Cephamycins

c) Carbapenems (imipenem)

d) Monobactams (Aztreonam)

7. β-lactamase Inhibitors

The β-lactamase inhibitors are:

a) Clavulanic acid
b) Sulbactam
c) iii.Tazobactam

Example:

Augmentin = Amoxicillin + Clavulanic acid

8. Classification of PENICILLIN antibiotics

i. Natural Penicillin antibiotics

a) Penicillin G

b) Penicillin V

ii. Anti-staphylococcal Penicillin antibiotics

a) Nafcillin

b) Cloxacillin

c) Dicloxacillin

d) Oxacillin
iii. Broad Spectrum Penicillin antibiotics

a) Amoxicillin

b) Ampicillin

iv. Antipseudomonal Penicillins

a) Piperacillin
b) Ticarcillin
c) Carbenicillin
d) Mezlocillin

Nonsteroidal Anti-Inflammatory Drugs and Antipyretic-Analgesics

Classification:

A) Nonselective COX inhibitors:

1. Aspirin

2. Ibuprofen

3. Naproxen

4. Ketoprofen

5. Flurbiprofen

6. Mephenamic acid

7. Diclofenac

8. Aceclofenac

9. Indomethacin

10.Piroxicam
 B) Selective Cyclooxygenase 2 (COX-2) inhibitors:

1. Celecoxib

2. Etoricoxib

3. Parecoxib

Difference between COX-1 & COX-2:

Cyclooxygenase 1 (COX-1) is responsible for physiological production of

prostaglandins, whereas COX-2 occur at the site of inflammation.

Adverse effects of NSAIDs:

 a) Gastrointestinal effects (Ulcer and GI bleeding)
b) Increased risk of bleeding

Gastric Drugs

1. Classification of Proton Pump inhibitors (PPIs):

a) Omeprazole
b) Esomeprazoe
c) Lansoprazole
d) Dexlansoprazole
e) Rabeprazole
f) Pantoprazole

Prostaglandin E
Prostaglandin E stimulates the secretion of mucus which forms a protective
layer over the walls of the stomach.

Prostaglandin Drug name:

Misoprostol

Mechanism of antacids
Antacids are weak bases that react with gastric acid and neutralize it by
forming salt and water.

Examples of antacids: Aluminum hydroxide, Magnesium Hydroxide, Sodium

bicarbonate, Calcium carbonate.

2. Classification of H2 Receptor Blockers

a) Cimetidine
b) Famotidine
c) Nizatidine
d) Ranitidine
 CLASSIFICATION OF ANTIHYPERTENSIVE DRUGS

1. Angiotensin Converting Enzyme Inhibitors (ACE INHIBITORS):

a) Captopril
b) Enalapril
c) Ramipril
d) Lisinopril
2. Angiotensin II Receptor Blockers (ARBs):
a) Losartan
b) Valsartan
c) Candesartan
d) Olmesartan
e) Irbesartan
3. Calcium Channel Blockers:
a) Diltiazem
b) Verapamil
c) Amlodipine
d) Nicardipine
e) Nifedipine
4. Non-Selective Beta blockers: ( Act at both Beta 1 and Beta 2 receptors)
a) Propranolol
b) Nadolol
c) Pindolol
d) Timolol
5. Selective Beta 1- blockers:
a) Metoprolol
b) Atenolol
c) Esmolol
d) Bisoprolol
e) Nebivolol
6. Alpha- Blockers:
a) Prazosin
b) Terazosin
 c) Doxazosin

Classification of Diuretics

Diuretics are the drugs that increase the volume of urine excreted.

1. Thiazide Diuretics:
a) Chlorothiazide
b) Hydrochlorothiazide
2. Loop Diuretics:
a) Furosemide
b) Torsemide
c) Bumetanide
d) Ethacrynic acid
3. Potassium sparing Diuretics:
a) Spironolactone
b) Amiloride
c) Triamterene
4. Carbonic Anhydrase Inhibitors:
a. Acetazolamide