Experiment No.

: 1

Aim: To study the introduction of the experimental Pharmacology.

Reference: Mude G., Wakodkar S, Usman R., Sheikh N. ‘’A Practical Manual of
Pharmacology’’,PV Books, Pageno. 1 to 3.

Theory :

Pharmacology: The pharmacology term comes from two Greek words: Pharmakon-drug or
medicine and logos- the truth about or a rational discussion. It is a science of drugs. It deals with
exogenously administered chemical molecules (drug) interaction with the living system. It covers
information about the history, source, physicochemical properties, physiological actions,
mechanism of drug action, absorption of drug, distribution, metabolism, and excretion of the
drug.

Pharmacodynamics: The pharmacodynamic term comes from the Greek word: dynamics-
Power. It is the study of drug effects, which includes the physiological and biochemical effects
of drugs and their mechanisms of action in any organ/system.

Pharmacokinetics: The pharmacokinetics term comes from the Greek word: kinesis-movement.
Pharmacokinetics is the movement of drugs in and alteration of drugs by the body. It is the
qualitative study of drug movement including absorption, distribution, metabolism and excretion
(ADME) of the drug. E.g. Digoxin is 70% absorbed orally, 25% bound to plasma proteins,
localized in the heart, skeletal muscle, liver, and kidney.

Pharmacotherapeutics: It is the overall knowledge of pharmacological information for the

prevention of disease, mitigation, or cure is known as Pharmacotherapeutics. It also includes the
selection of the most appropriate drug, dosage, and duration of the treatment for the specific
features of a patient.

Toxicology is the study of the poisonous effect of drugs and other chemicals with emphasis on
the detection, prevention, and treatment of poisonings. It also includes the study of the adverse of
drugs.

Chemotherapy: It is the treatment of systemic infection or malignancy with specific drugs that
have selective toxicity for the infecting organism or malignant cell having effect on host cells.

Clinical pharmacology: It is the scientific study of drugs in human. It includes

pharmacokinetics and pharmacodynamics investigation in healthy volunteers and in patients, for
evaluation of efficacy and safety of drugs.
Pre-clinical Pharmacology: It is a scientific study which involves laboratory animals like rats
and mice using wide-ranging doses of the study drug to obtain preliminary efficacy (action that
is done before or in preparation for the event), toxicity, and pharmacokinetic information.

Sr.no. Name of Scientist Achievement

1. Oswald Schmiedeberg Father of Pharmacology
2. Rudolf Bucheim Pioneer of Experimental Pharmacology
3. John Jacob Abel Father of American Pharmacology
4. Louis Lasagna Father of clinical pharmacology
5. Francois Mangendie Pioneer of Experimental Physiology
6. Claude Bernard Father of physiology
7. Paul Ehrlich Father of Chemotherapy
8. Sir Ram Nath Chopra Father of Indian Pharmacology

Pharmacokinetics: It is the branch of science deals with study of genetic sequence and genetic
variation that change drug metabolism and drug response. Pharmacogenetics mainly focused on
hereditary disease or drug reaction.

Pharmacoepidemiology: It is the study of drug effects at the population level. It is deals with
the variability of drug effects between individuals in a population, and between populations.

Pharmacovigilance: This is a science and activities related to the detection, assessment,

understanding and prevention of adverse effects or any other related problems. This mainly
focused on adverse drug reactions.

Types of experimental pharmacology:

The sequential testing is involved in pharmacological screening of new chemical compound or

extracts from biological materials from isolated organs by means of tests in whole animals,
mostly rats and mice but also used higher animals if needed.

The main aim of experimental pharmacology is the development of new drugs, the discovery of
new drugs, the study of the mechanism and site of action of different drugs, and the study of the
toxicity and effects of the drugs.

Experimental pharmacology involves Pre-clinical study and clinical study.

Pre-clinical study: Pre-clinical study contains in-vivo (derived from the Latin words within the
living) the study performed within the living being for example study performed on cattle) and
in-vitro experiments which basically means within the glass or the experiment performed is done
outside the living organism for example in a test tube or lab) using a variable range of doses of
the sample drug to obtain preliminary efficacy, toxicity, and pharmacokinetics information. In-
vivo (Animal testing) study is used to measure the drug absorption into the blood, its breakdown
chemically in the body, the toxicity of the drug and its breakdown products or metabolic
products, and how its metabolic products are excreted from the body. The duration of
experiments consists of short-term testing which is from 2 weeks to 3 months and long-term
testing which is from a few weeks to several years. In-vitro (Test-tube) experiments, instead of
using animals in the test they are using a cell and the tissue culture as product ingredients. The
development of in-vitro methods based on biological materials like skin or human body cells will
be suitable for reliably verifying the safety and compatibility of product ingredients. The
advantages of in-vitro tests are fast and cheap, and human cells and tissues can be used.

Clinical Trials: It is the scientific study of drugs in humans. It includes pharmacokinetic and
pharmacodynamic investigation in healthy volunteers and in patients. A clinical trial is a research
study that tests a new medical treatment or a new way of using an existing treatment to see the
prevention and screen for diagnosis or treatment of a disease. Clinical trials for new drugs are
commonly classified into four phases. The drug development process will normally proceed
through all four phases over many years. If the drug successfully passes through Phases I, II, and
III, it will be approved by the national regulatory authority for use in the general population.
Phase IV is post-approval (Post-marketing surveillance) studies. Phase 0 or micro-dosing studies
are performed to know the action of drug candidates in human beings at an early stage.

RESULT:

Experiment No.: 2

Aim: To study common laboratory animals used in experimental pharmacology.

Reference:
1. https://www.slideserve.com/19402/common-laboratory-animals-experimental-
pharmacology
2. A practical book of pharmacology, Mandeep kumar arora, Nirali Prakashan, pg.
no. 13-19, 2019
3. A practical manual of Pharmacology, Gaurav munde et al., PV books, 2022
4. https://www.britannica.com/animal/hamster
5. https://mypetguineapig.com/guinea-pig-vs-hamster/

Theory: Laboratory animals are those animals that can be bred and reared in laboratory under
suitable conditions for conducting various experiments. Experimental animals have been a part
of biomedical and behavioral research for several millennia; initial experiments with animals
were conducted in Greece over 2000 years ago. All experiments on animals should be conducted
according to CPCSEA (C) guidelines.
Various species of laboratory animals are used in experimental pharmacology for different
studies such as:
1. To investigate dose –biological response relationship (Bioassays),
2. For estimation of the toxicity of various drug substances, and
3. To investigate the pharmacokinetic and pharmacodynamic parameters of drugs molecules.

Common laboratory animals:

Rodents: Mouse, Rats, Guinea Pig, and Hamster
Non- Rodents: Rabbits, Dog, Cat, Monkey, and Pigs.
Miscellaneous: Frog, Pigeon, Zebra fish, etc.

Specific animal is selected on the basis of specific feature/ characteristic required and the type/
nature of the study. Animal which is phylogenetically closer to human will be considered for the
study. Common specific features of the animals which are considered for the study are:

1. Size/ weight: Smaller size of the animal is preferred because they are easy to handle and less
quantity of drug is required.
2. Availability: Animals which are commonly available should be selected eg : Frog, rats,
Rabbits, etc.
3. Sensitivity: Animals should be selected according to their sensitivity to particular test drug.
eg.: Guinea pig is sensitive to histamine.
4. Sex: Males and females have difference in hormonal secretions which may affect the fate of
drug. Hence, gender should be selected wisely according to the requirement.
5. Age: Age of the animal can affect the study results. Hence, age should be selected prior to
the experiments.
6. Condition of the animals can also affect the study. For eg: Pregnancy, Infections, diseased, or
any abnormality.

Description of the animals:

1. Mouse: (Mus musculus)

Various strains: Balb/c, Swiss mice, C57BL/6, DB/DB, etc.
Adult weight: 20-25 g
Suitable Age: 6-8 weeks
Life span: 2-3 yrs.
Smallest, cheap, and easy to handle.
No vomiting Center
Normal Temp.: 37.4 o C
Gestation Period: 19-21 days
Easy and Short breeding and Weaning time (19-21 days)
Nocturnal animal, and need small amount of dose to be administered.
 Commonly used: Swiss albino and Balb/c mice

Appplications:
1. Used in teratogenic & toxicity studies, bioassays (insulin).
2. Used in screening analgesics and CNS active drugs.
3. Immuno-deficient mice are used for cancer and AIDS research.
4. Used in screening of hepato-protective, hepatotoxic, anti-diabetic, anti-ulcer agents, etc.

2. Rats: (Rattus norvegicus)

Various strains: Albino, Wistar kyoto, Sprague-Dawley (SD rats), Long evans
Adult weight: 200-250 g
Suitable Age: 10-12 weeks
Life span: 3-4 yrs.
Small, cheap, and easy to handle.
No vomiting Center, it becomes easy to give oral drug
There is no Gall bladder (continuous flow of bile into intestine) and tonsils.
Pancreas is diffused, hence it becomes difficult to perform pancreactomy.
Nocturnal animal, caprophagy, and need small amount of Drug
Commonly used: Wistar rats (wide head and short tail) and SD rats (long and narrow head
with long tail)
Appplications:
1. Used in screening analgesics and CNS active drugs (anti-convulsant drug)
2. Used in studying the oestrus cycle,
3. Screening of Hepatotoxicity studies, toxicology studies, bioassays of hormones like
Oxytocin, insulin, vasopressin, etc.
4. Chronic studies on blood Pressure
5. Gastric acid secretion studies

3. Guinea Pig: (Cavia porcellus)

Adult weight: 500-800 g
Size: 20-40 cm
Suitable Age: 10-12 weeks
Body Temp.: 38.5o C
Life span: 3-5 yrs. (sombetimes, till 7-8 yrs)
Small, sensitive and docile animal without tail
Easy to raise and it is herbivorous
It is highly sensitive to histamine

Appplications:
1. Vaccines for diphtheria and tuberculosis
2. Hypersensitivity (histamine and penicillin) and anaphylactic shock studies
 3. Evaluation of bronchodialators and local anaesthetics
4. Screening of spasmodic and antispasmodic agents
5. Nutritional Studies (for eg.: Ascorbic Acid metabolism)
6. Biassay of Digitalis
7. Experimental procedures: Subcutaneous, Intracutaneous, and Intraperitoneal inoculation,
blood collection

4. Hamster: (Mesocricetus auratus and Cricetulus griseus)

Various strains: Syrian/golden Hamsters (Mesocricetus auratus), Chinese Hamsters
(Cricetulus griseus), Roborovski Hamster, European hamster, Campbell’s Hamster, Winter
White Hamster, etc.
Adult weight: 80-100 g
Size: 5-15 cm
Suitable Age for experiments: 4-8 weeks
Life span: 2-4 yrs.
It has short legs with diminutive fluffy tail and cheek pouches extending up to shoulders.

Appplications:
1. Chinese hamsters have a low number of chromosomes which makes it useful for
cytological genetic tissue culture and radiation research.
2. Bioassay of Prostaglandins
3. Research related to virology, immunology, and implantation studies.
4. Chronic studies on blood Pressure
5. Gastric acid secretion studies
5. Rabbit (Oryctolagus cuniculus)
Strain: New Zealand white rabbits are used
Adult weight: 1.5-2 kg
Size: 40-90 cm
Life span: 5-6 yrs
Body Temp.: 38.5o C
Life span: 3-5 yrs. (sometimes, till 7-8 yrs)
It is also a docile animal with large ears
Easy to raise and it is herbivorous
It has huge caecum and large appendix.
One peculiar thing about rabbits is that they are resistant to the actions of atropine as they
contain atropine-esterase enzyme, the presence of which is genetically determined.

Appplications:
1. Pyrogen testing
2. Tissue and organ study (Heart, aorta, duodenum, and ileum)
3. Bioassay of anti-diabetic, insulin, curare mimetic drugs.
 4. Studies related to anti- fertility drugs
5. Study of miotics and mydriatics
6. Testing topical agents as skin is sensitive, skin and eye irritation studies.
7. To understand the various human diseases (Cardiovascular disease, cancer & AIDS)
8. Also used as bioreactors for production of pharmaceutical proteins.
Used for toxicity and safety testing of drugs, chemicals

EXPERIMENT NO. 3

Aim: To study commonly used instruments in experimental pharmacology.

(A) To study the basic instruments used for in-vitro/ ex-vivo experiments

Reference : Kulkarni S.K. ,”Handbook of experimental pharmacology”, Published by Vallabh

Prakashan , 4th Edition 2012, Reprint Edition.

Background:

Isolated tissue preparations are commonly used to study the effects of drugs on specific type of
receptors. These preparations are also used:

 For bioassay of drugs,

 For characterization of specific receptor or its subtypes,
 To determine concentration response curve of an agonist,
 To study antagonism of drug and in new drug discovery.

The in-vitro/Ex-vivo isolated preparation represents an isolated organ or a piece of tissue from
freshly killed animal.
 Optimum ionic environment
 Adequate supply of nutrition and oxygen
 And a stable temperature
 These basic requirements should be provided if one has to maintain the isolated tissue in
living state.

Student's Organ Bath (Isolated Organ Bath):

Besides tremendous development in electronic devices, gazetteries and recording system,

conventional equipments (e.g., "Student's organ bath") are routinely used in the experimental
pharmacology. The complete assembly of "Student's organ bath" consists of many parts,
including organ bath, aerator, levers, and Sherrington rotating drum. "Organ bath" (isolated
organ bath) is used to study the effects of drugs on isolated tissues. It was first designed by
Rudolph Magnus in 1904. This equipment essentially consists of the following parts:
1. WATER BATH OR OUTER JACKET:

The water bath is made up of steel, glass or 'perspex' (transparent thermoplastic resin). It holds
water and other parts of organ bath. It consists of a glass vessel called 'organ tube' surrounded by
a perspex or glass tank filled with water maintained at a fixed temperature by an electric heater
and thermostat. The organ tube is connected through polythene or rubber tube to the reservoir
which contains physiological saline solution (Ringer's or Tyrode's solution). In between the
reservoir and organ tube, there is a glass preheating coil. The tissue is suspended in organ tube
by means of a tissue holder that also serves the purpose of aeration (hence known as 'aeration
tube cum tissue holder'). One end of the tissue is tied to this and the other end is connected to
lever, the recording device (kymograph or physiograph).

2. RECORDING DEVICE:

The commonly used recording devices are 'levers'. For recording of isometric contraction, 'spring
lever' is used; while for recording of isotonic contraction, 'isotonic frontal writing lever' is
commonly used. Isotonic contractions indicate the change in length at uniform tension. Isometric
recordings are records of change in tension (force) developed irrespective of the length. There is
another type of recording called 'auxotonic' in which the change in force of contraction is
recorded with respect to change in length.

3. LEVER:

It is the device by virtue of which response of isolated tissue can be recorded and magnified.
Principle:

I. Fulcrum: The point around which the lever moves on the lever holder is the fulcrum.
II. Stylus: Is the writing point which records the tracing on the smoked paper of the drum. It is
either made of celluloid parchments paper, aluminium foil or photographic or X-ray film.

Magnification: The fulcrum (F) should be so placed that there is some magnification of the
actual contraction (response). In order to achieve this, the distance between the writing point and
the fulcrum (F) should always be greater than the distance between fulcrum and point of
attachment of tissue (T).

Adjustment for the load or tension:

The muscle preparation has to be properly relaxed without affecting the normal tone and
rhythmic activity so that eificient contractions are achieved when stimulated and it also relaxes to
its full length after wards. This is achieved by the following way:

 Select the proper length of longer and shorter arm after fixing magnification for the
particular tissue and fix the fulcrum.
 Balance the lever by putting the weight (plasticin) at the end or shorter arm and mark the
point of tissue attachment.
At equidistance i.e. the distance between the F and T from the (F) on the longer arm of
lever the desired load required for particular tissue, The tension (load) prescribed for various
commonly used tissues is as follows:
1. Guinea pig ileum 1 gm
2. Guinea pig trachea 0.2 gm
The writing levers are light in weight, rigid and are generally made up of wood (straw), light
aluminium or stainless steel. The levers are of two types.

(i) Isotonic type: i.e. change in length due to contraction is recorded while the tension on the
muscle remains the same e.g. simple lever frontal writing lever.

(ii) Isometric type: These levers are used under special circumstances for instance, when at
witch is produced by stimulating a muscle suspended between two rigid points, one being a
strong spring, the muscle does not shorten but only creates a force or tension which is recorded;
the twitch is also much faster in action. e.g. Paton's auxotonic lever will serve purpose well.

Different types of lever:

(i) Simple Lever (sideway writing): It is simplest type of lever made up of wood (straw),
stainless steel or aluminium. A celluloid writing tip (stylus) is attached at the end of the longer
arm. The contractions are recorded as curved lines.

(ii) Frontal writing Lever (writes frontally): This lever is designed in such a way that the
writing point rotates freely about its axle. This helps in reducing the tension between the smoked
paper and the recording tip. The contraction is recorded as straight line.

(iii) Starling's or Heart Lever: This lever is used to record the contraction of heart. The
differences between this and other isotonic levers is that fulcrum lies at one end beyond the point
of attachment. It consists of a frame carrying a light lever arm with holes and notches supported
by a fine adjustable nickel silver spring attached to adjustable hook.

(iv) Universal Lever (Brodie's): It is a lever of versatile utility there is an adjustable spring
support which counters the pull on the writing arm. The spring helps bringing the pulled writing
arm back to its original position. This lever is mainly use for recording sudden repetitive
contraction of muscles/movements of a part of th body e.g. contraction of a gastrocnemius
muscle in response to sciatic nerve stimulation.

Preparation of Different Materials:

1. Adjustment of Magnification:
In order to achieve 10 times magnification, the distance from the fulcrum to the point of
recording (frontal point) should be 10 times more than the distance from fulcrum to the point of
tissue attachment. Therefore,
Magnification = Distance from fulcrum to the writing point
Distance from fulcrum to the point of attachment of tissue
2. Adjustment of Tension:
The lever is made horizontal by applying the plasticin (modeling wax) at the shorter end.A small
amount of plasticin is also placed on the point of attachment of tissue.

3. Smoking of Drum:
Tissues responses are recorded on 'smoked drum' which is prepared as under-Glazed paper is laid
down on the table, keeping glazed surface downwards. One end of the paper is gummed. Drum
cylinder is placed in the middle of the paper. Proximal ungummed end is rolled around the drum
and held tightly between the thumbs. Other end is also rolled on the other side and the gummed
end is pasted on the proximal ungummed end. Cylinder with paper is passed over a rod fixed in a
smoking rack. A flame is obtained by passing the gas through benzene or using a mixture of
benzene and kerosene in the ratio of 1: 9. Burner is brought nearer to the drum that is rolled
uniformly at the maximum possible speed. Outer orange zone of flame should touch the paper.
Uniform rolling of drum should be continued so as to set the uniform deposit of soot.

4.Fixing of Graph (Varnishing of Graph):

The paper is cut after obtaining the recordings and then it is dipped in a solution of resin
in methylated spirit. This solution is prepared by dissolving 150 g of resin in 2 L of spirit.
After passing the paper through the solution, it is drained and then allowed to dry.

5. Physiological Salt Solution or Ringer's Solution:

Ionic requirement and nutritional supply can be provided by 'physiological salt solution' or
"Ringer's solution". Composition of Ringer provides artificial media, resembling the inorganic
composition of blood plasma together with buffer mechanism to maintain optimal pH at about
7.0 to 7.2 and glucose to facilitate the tissue metabolism.

6. Aeration:

Physiological salt solution in organ bath should be bubbled with air with sufficient
oxygen or carbogen (95% Oz + 5% CO2). This is important for regular supply of oxygen,
at the maintained pH. Bubbling of air gives the uniform distribution of drug.

Useful Hints for Students:

 Step by step setup the assembly first.
 Assembly should be on right hand side and drum should be on left hand side.
 The drum should move from right to left or away from the writing point.
 The speed of drum should be minimum.
 The writing lever should be horizontal and the writing point should touch the drum.
 All tracing should begin after the overlap of the paper.
 Always start with the minimum concentration of drug.
 Write the name and amount of drug.

Result: