Pharmacology

Pharmacology
A Complete Course
By
Dr. Abhaya S. Panwar
Department of Biochemistry
H.N.B. Garhwal University
Srinagar Garhwal
Uttatakahdnd

Pharmacology
• Pharmacology is the science of drugs (Greek: Pharmacon—drug; logos—
discourse in).
• In a broad sense, it deals with interaction of exogenously administered
chemical molecules in a living system.
• Any single chemical substance which can produce a biological response is
a ‘drug’.
• It encompasses all aspects of knowledge about drugs, but most importantly
those that are relevant to effective and safe use for medicinal purposes.

History
• For thousands of years most drugs were crude natural products of unknown
composition and limited efficacy.
• The effects of these substances on the body were and their compositions
were imprecisely known.
• Rudolf Buchheim who founded the first institute of pharmacology in 1847
in Germany.
• In the later part of the 19th century, Oswald Schmiedeberg, regarded as the
‘father of pharmacology’, postulated some of the fundamental concepts in
pharmacology.
• Since then drugs have been purified, chemically characterized and a vast
variety of highly potent and selective new drugs have been developed.
• The mechanism of action including molecular target of many drugs has
been elucidated.

Divisions of pharmacology
Pharmacology
Pharmacodynamics Pharmacokinetics

Pharmacodynamics
Pharmacodynamics (Greek: dynamis—power)
• What the drug does to the body.
• This includes physiological and biochemical effects of drugs and their
mechanism of action at organ system/subcellular/macromolecular levels.
• e.g.—Adrenaline → interaction with adrenoceptors→ G-protein mediated
stimulation of cell membrane bound adenylyl cyclase → increased
intracellular cyclic 3´, 5´AMP → cardiac stimulation, hepatic
glycogenolysis and hyperglycaemia,

Pharmacokinetics
• Pharmacokinetics (Greek: Kinesis—movement)—
• What the body does to the drug. This refers to movement of the drug in and
alteration of the drug by the body; includes absorption, distribution,
binding/localization/storage, biotransformation and excretion of the drug
• e.g. paracetamol is rapidly and almost completely absorbed orally attaining
peak blood levels at 30–60 min; 25% bound to plasma proteins, widely and
almost uniformly distributed in the body(volume of distribution ~ 1L/kg);
extensively metabolized in the liver, primarily by glucuronide and sulfate
conjugation into inactive metabolites which are excreted in urine; has a
plasma half life (t½) of 2–3 hours and a clearance value of 5 ml/kg/min.

Some other important aspects of
pharmacology
Other aspects
of
pharmacology
Pharmacotherapeutics
Clinical pharmacology
Chemotherapy
Pharmacy
Toxicology

Pharmacotherapeutics
• It is the application of pharmacological information together with
knowledge of the disease for its prevention, mitigation or cure.
• Selection of the most appropriate drug, dosage and duration of treatment
and taking into account the specific features of a patient are a part of
pharmacotherapeutics.

Clinical pharmacology
• It is the scientific study of drugs (both old and new) in man.
• It includes pharmacodynamic and pharmacokinetic investigation in healthy
volunteers and in patients.
• Evaluation of efficacy and safety of drugs and comparative trials with other
forms of treatment; surveillance of patterns of drug use, adverse effects, etc
comes under this branch.
• The aim of clinical pharmacology is to generate data for optimum use of
drugs and the practice of ‘evidence based medicine’.

Pharmacy
• It is the art and science of compounding and dispensing drugs or preparing
suitable dosage forms for administration of drugs to man or animals.
• It includes collection, identification, purification, isolation, synthesis,
standardization and quality control of medicinal substances.
• The large scale manufacture of drugs is called Pharmaceutics.
• It is primarily a technological science.

Toxicology
• It is the study of poisonous effect of drugs and other chemicals (household,
environmental pollutant, industrial, agricultural, homicidal) with emphasis
on detection, prevention and treatment of poisonings.
• It also includes the study of adverse effects of drugs, since the same
substance can be a drug or a poison, depending on the dose.

Drug
• Drug (French: Drogue—a dry herb)
• It is the single active chemical entity present in a medicine that is used for
diagnosis, prevention, treatment/ cure of a disease.
• This disease oriented definition of drug does not include contraceptives or
use of drugs for improvement of health.
• The WHO (1966) has given a more comprehensive definition—“Drug is
any substance or product that is used or is intended to be used to
modify or explore physiological systems or pathological states for the
benefit of the recipient.”
• The term ‘drugs’ is being also used to mean addictive/abused/illicit
substances.

DRUG
Pharmacodynamic agents Chemotherapeutic agents
Designed to have
pharmacodynamic
effects in the recipient
Designed
to inhibit/kill invading
parasite/malignant cell
and
have no/minimal
pharmacodynamic
effects in the
recipient

DRUG NOMENCLATURE
• A drug generally has three categories of names:
Chemical name
Non-proprietary name
Proprietary (Brand) name

Chemical name
• It describes the substance chemically.
• e.g. 1-(Isopropylamino)-3-(1-naphthyloxy) propan-2-ol for propranolol.
• This is bulky and not suitable for use in prescribing.
• A code name, e.g. RO 15-1788 (later named flumazenil) may be assigned
by the manufacturer for convenience and simplicity before an approved
name is coined.

Non-proprietary name
• It is the name accepted by a competent scientific body/authority, e.g. the
United States Adopted Name (USAN) by the USAN council.
• Similarly, there is the British Approved name (BAN) of a drug.
• The non-proprietary names of newer drugs are kept uniform by an
agreement to use the Recommended International Nonproprietary Name
(rINN) in all member countries of the WHO.
• Until the drug is included in a pharmacopoeia, the nonproprietary name
may also be called the approved name.
• After its appearance in the official publication, it becomes the official
name.
• In common jargon, the term generic name is used in place of
nonproprietary name.
• This is incorrect: ‘generic’ should be applied to the chemical or
pharmacological group (or genus) of the compound, e.g. phenothiazines,
tricyclic antidepressants, aminoglycoside antibiotics, etc.

Proprietary (Brand) name
• It is the name assigned by the manufacturer(s) and is his property or trade
mark.
• One drug may have multiple proprietary names, e.g. ALTOL, ATCARDIL,
ATECOR, ATEN, BETACARD, LONOL, TENOLOL, TENORMIN for
atenolol from different manufacturers.
• Brand names are designed to be catchy, short, easy to remember and often
suggestive, e.g. LOPRESOR suggesting drug for lowering blood pressure.
• Brand names generally differ in different countries, e.g. timolol maleate eye
drops are marketed as TIMOPTIC in USA but as GLUCOMOL in India.
• Even the same manufacturer may market the same drug under different
brand names in different countries.
• In addition, combined formulations have their own multiple brand names.
• This is responsible for much confusion in drug nomenclature.
• There are many arguments for using the nonproprietary name in
prescribing: uniformity, convenience, economy and better comprehension
(propranolol, sotalol, timolol, pindolol, metoprolol, acebutolol, atenolol are
all β blockers, but their brand names have no such similarity).

Essential Medicines (Drugs) Concept
• The WHO has defined Essential Medicines (drugs) as “those that satisfy
the priority healthcare needs of the population”.
• They are selected with due regard to public health relevance, evidence on
efficacy and safety, and comparative cost effectiveness.
• Essential medicines are projected to be available within the context of
functioning health systems at all times and in adequate amounts, in
appropriate dosage forms, with assured quality and adequate information,
and at a price the individual and the community can afford.
• For optimum utilization of resources, governments (especially in
developing countries) should concentrate on these medicines by identifying
them as Essential medicines.

WHO criteria to guide selection of an essential
medicine
a) Adequate data on its efficacy and safety should be available from clinical
studies.
b) Its quality, bioavailability, and stability on storage be assured.
c) Its choice should depend upon pattern of prevalent diseases; availability of
facilities and trained personnel; financial resources; genetic, demographic
and environmental factors.
d) In case of two or more similar medicines, choice should be made on the
basis of their relative efficacy, safety, quality, price and availability. Cost-
benefit ratio should be a major consideration.
e) Choice may also be influenced by comparative pharmacokinetic properties
and local facilities for manufacture and storage.
f) Most essential medicines should be single compound.
g) Selection of essential medicines should be a continuous process which
should take into account the changing priorities for public health action,
epidemiological conditions as well as availability of better
medicines/formulations and progress in pharmacological knowledge.
h) Recently, it has been emphasized to select essential medicines based on
rationally developed treatment guidelines

Prescription and non-prescription drugs
• Prescribed Drugs- Drugs (all antibiotics) must be sold in retail only
against a prescription issued to a patient by a registered medical
practitioner.
• Non-prescribed drugs / over-the-counter’ (OTC) drugs- Drugs like
simple analgesics (paracetamol aspirin), antacids, laxatives (senna,
lactulose), vitamins, ferrous salts, etc. are considered relatively harmless,
and can be procured without a prescription.
• ‘Non-prescription’ or drugs; can be sold even by grocery stores.
• Orphan Drugs
• These are drugs or biological products for diagnosis/treatment/ prevention
of a rare disease or condition, or a more common disease (endemic only in
resource poor countries) for which there is no reasonable expectation that
the cost of developing and marketing it will be recovered from the sales of
that drug.
• The list includes sodium nitrite, fomepizole, liposomal amphotericin B,
miltefosine, rifabutin, succimer, somatropin, digoxin immune Fab (digoxin
antibody), liothyronine (T3) and many more.
• Though these drugs may be life saving for some patients, they are
commercially difficult to obtain as a medicinal product.

Routes of Drug Administration
• Most drugs can be administered by a variety of routes.
• The choice of appropriate route in a given situation depends both on drug
as well as patient related factors.
• Routes can be broadly divided into
• (a) Local action and (b) Systemic action

Routes of Drug Administration
Local action Systemic action
Topical
Deeper tissues
Arterial supply
Oral
Sublingual (s.l.) or buccal
Rectal
Cutaneous
Inhalation
Nasal
Parenteral

Factors governing choice of route
1. Physical and chemical properties of the drug (solid/liquid/gas; solubility,
stability, pH, irritancy).
2. Site of desired action—localized and approachable or generalized and not
approachable.
3. Rate and extent of absorption of the drug from different routes.
4. Effect of digestive juices on the drug.
5. Speed with which the response is desired (routine treatment or
emergency).
6. Accuracy of dosage required (Intravenous and inhalational can provide
fine modification).
7. Condition of the patient (unconscious, vomiting).

Topical
• This refers to external application of the drug to the surface for localized
action.
• It is often more convenient as well as encouraging to the patient.
• Drugs can be efficiently delivered to the localized injury on skin, nasal
mucosa, eyes, ear canal, anal canal or vagina in the form of lotion,
ointment, cream, powder, rinse, paints, drops, spray, lozengens,
suppositories or pesseries.
• Non-absorbable drugs given orally for action on gestrointestinal mucosa
(sucralfate, vancomycin), inhalation of drugs for action on bronchi
(salbutamol, cromolyn sodium) and irrigating solutions/jellys (povidone
iodine, lidocaine) applied to urethra are other forms of topical medication.

Deeper tissues
Certain deep areas can be approached by using a syringe and needle, but
the drug should be in such a form that complete absorption is slow, e.g.
intra-articular injection (hydrocortisone acetate in knee joint), infiltration
around a nerve or intrathecal injection (lidocaine), retrobulbar injection
(hydrocortisone acetate behind the eyeball).

Arterial supply
Close intra-arterial injection is used for contrast media in angiography;
anticancer drugs can be infused in femoral or brachial artery to localize the
effect for limb malignancies.

1. Oral
• Oral ingestion is the oldest and commonest mode of drug administration. It
is safer, more convenient, does not need assistance, often painless, the
medicine need not be sterile and so is cheaper.
• Both solid dosage forms (powders, tablets, capsules, spansules, dragees,
moulded tablets, gastrointestinal therapeutic systems—GITs) and liquid
dosage forms (elixirs, syrups, emulsions, mixtures) can be given orally

Limitations of oral route of administration
• Action of drugs is slower and thus not suitable for emergencies.
• Unpalatable drugs (chloramphenicol) are difficult to administer; drug may
be filled in capsules to circumvent this may cause nausea and vomiting
(emetine).
• Cannot be used for uncooperative/unconscious/vomiting patient.
• Absorption of drugs may be variable and unpredictable; certain drugs are
not absorbed (streptomycin).
• Others are destroyed by digestive juices (penicillin G, insulin) or in liver
(GTN, testosterone, lidocaine).

2. Sublingual (s.l.) or buccal
• The tablet or pellet containing the drug is placed under the tongue or
crushed in the mouth and spread over the buccal mucosa.
• Only lipid soluble and non-irritating drugs can be so administered.
• Absorption is relatively rapid—action can be produced in minutes.
• Though it is somewhat inconvenient, one can spit the drug after the desired
effect has been obtained.
• The chief advantage is that liver is bypassed and drugs with high first pass
metabolism can be absorbed directly into systemic circulation.
• Drugs given sublingually are—GTN, buprenorphine, desamino-oxytocin.

3. Rectal
• Certain irritant and unpleasant drugs can be put into rectum as
suppositories or retention enema for systemic effect.
• This route can also be used when the patient is having recurrent vomiting
or is unconscious.
• However, it is rather inconvenient and embarrassing; absorption is slower,
irregular and often unpredictable, though diazepam solution and
paracetamol suppository are rapidly and dependably absorbed from the
rectum in children.
• Drug absorbed into external haemorrhoidal veins (about 50%) bypasses
liver, but not that absorbed into internal haemorrhoidal veins.
• Rectal inflammation can result from irritant drugs.
• Diazepam, indomethacin, paracetamol, ergotamine and few other drugs are
some times given rectally.

4. Cutaneous
• Highly lipid soluble drugs can be applied over the skin for slow and
prolonged absorption.
• The liver is also bypassed.
• The drug can be incorporated in an ointment and applied over specified
area of skin.
• Absorption of the drug can be enhanced by rubbing the preparation, by
using an oily base and by an occlusive dressing.

5. Inhalation
• Volatile liquids and gases are given by inhalation for systemic action, e.g.
general anaesthetics.
• Absorption takes place from the vast surface of alveoli—action is very
rapid.
• When administration is discontinued the drug diffuses back and is rapidly
eliminated in expired air.
• Thus, controlled administration is possible with moment to moment
ajustement.
• Irritant vapeurs (ether) cause inflammation of respiratory tract and increase
secretion.

6. Nasal
• The mucous membrane of the nose can readily absorb many drugs;
digestive juices and liver are bypassed.
• However, only certain drugs like GnRH agonists and desmopressin applied
as a spray or nebulized solution have been used by this route.
• This route is being tried for some other peptide drugs like insulin, as well
as to bypass the blood-brain barrier.

7. Parenteral
• Parenteral (Par—beyond, enteral—intestinal)
• Conventionally, parenteral refers to administration by injection which takes
the drug directly into the tissue fluid or blood without having to cross the
enteral mucosa.
• The limitations of oral administration are circumvented.
• Drug action is faster and surer (valuable in emergencies).
• Gastric irritation and vomiting are not provoked.
• Parenteral routes can be employed even in unconscious, uncooperative or
vomiting patient.
• There are no chances of interference by food or digestive juices. Liver is
bypassed.
• Disadvantages of parenteral routes are—
• The preparation has to be sterilized and is costlier, the technique is invasive
and painful, assistance of another person is mostly needed (though self
injection is possible, e.g. insulin by diabetics), there are chances of local
tissue injury and, in general, parenteral route is more risky than oral.

The important parenteral routes are:
1. Subcutaneous (s.c.)
• The drug is deposited in the loose subcutaneous tissue which is richly
supplied by nerves (irritant drugs cannot be injected) but is less vascular
(absorption is slower than intramuscular).
• Only small volumes can be injected s.c. Self-injection is possible because
deep penetration is not needed. This route should be avoided in shock
patients who are vasoconstricted— absorption will be delayed.
• Repository (depot) preparations that are aqueous suspensions can be
injected for prolonged action.
• Some special forms of this route are:
a. Dermojet
In this method needle is not used; a high velocity jet of drug solution is
projected from a microfine orifice using a gun like implement. The solution
passes through the superficial layers and gets deposited in the subcutaneous
tissue. It is essentially painless and suited for mass inoculations.

b. Pellet implantation
The drug in the form of a solid pellet is introduced with a trochar and
cannula. This provides sustained release of the drug over weeks and
months,
e.g. DOCA, testosterone.
c. Sialistic (nonbiodegradable) and biodegradable implants
• Crystalline drug ispacked in tubes or capsules made of suitable materials
and implanted under the skin.
• Slow and uniform leaching of the drug occurs over months providing
constant blood levels.
• The nonbiodegradable implant has to be removed later on but not the
biodegradable one.
• This has been tried for hormones and contraceptives

2. Intramuscular (i.m.)
• The drug is injected in one of the large skeletal muscles—deltoid, triceps,
gluteus maximus, rectus femoris, etc.
• Muscle is less richly supplied with sensory nerves (mild irritants can be
injected) and is more vascular (absorption of drugs in aqueous solution
• is faster).
• It is less painful, but self injection is often impracticable because deep
penetration is needed.
• Depot preparations (oily solutions, aqueous suspensions) can be injected by
this route.
• Intramuscular injections should be avoided in anticoagulant treated
patients, because it can produce local haematoma.

3. Intravenous (i.v.)
• The drug is injected as a bolus (Greek: bolos–lump) or infused slowly over
hours in one of the superficial veins.
• The drug reaches directly into the blood stream and effects are produced
immediately (great value in emergency).
• The intima of veins is insensitive and drug gets diluted with blood,
therefore, even highly irritant drugs can be injected i.v., but hazards are—
thrombophlebitis of the injected vein and necrosis of adjoining tissues if
extravasation occurs.
• These complications can be minimized by diluting the drug or injecting it
into a running i.v. line.
• Only aqueous solutions (not suspensions, because drug particles can cause
embolism) are to be injected i.v. and there are no depot preparations
• for this route.
• Chances of causing air embolism is another risk.
• The dose of the drug required is smallest (bioavailability is 100%) and even
large volumes can be infused.

• One big advantage with this route is—in case response is accurately
measurable (e.g. BP) and the drug short acting (e.g. sodium nitroprusside),
titration of the dose with the response is possible. However, this is the most
risky route—vital organs like heart, brain, etc. get exposed to high
concentrations of the drug

4. Intradermal injection
• The drug is injected into the skin raising a bleb (e.g. BCG vaccine,
sensitivity testing) or scarring/multiple puncture of the epidermis through a
drop of the drug is done.
• This route is employed for specific purposes only.

Adverse Drug Effects
• Adverse effect is ‘any undesirable or unintentional consequence of drug
administration’. It is a broad term, includes all kinds of harmful effect—trivial,
serious or even fatal.
• Adverse drug reaction (ADR) defined as ‘any harmful change which is suspected
to be due to a drug, occurs at doses normally used in man, requires treatment or
decrease in dose or indicates caution in the future use of the same drug’.
• Adverse drug event’ (ADE) ‘any inconvenient medical occurrence that may present
during treatment with a medicine, but which does not necessarily have a causal
relationship with the treatment’.
• All drugs are capable of producing adverse effects, and whenever a drug is given a
risk is taken.
• The magnitude of risk has to be considered along with the magnitude of expected
therapeutic benefit in deciding whether to use or not to use a particular drug in a
given patient.
• Adverse effects may develop quickly or only after prolonged medication or even
after stoppage of the drug.
• Adverse effects are not rare; frequency of 10–25% has been documented in
different clinical settings. They are more common with multiple drug therapy and in
the old ages.

Classification of Adverse Drug effects
• Predictable (Type A or Augmented) reactions (mechanism based adverse
reactions)
Type A are predictable, dose-related toxicities, often identified in
preclinical or clinical trials, and usually occur in overdose settings or with
pre-existing hepatic impairment.
These are based on the pharmacological properties of the drug.
They are more common, dose related and mostly preventable and
reversible.
• Unpredictable (Type B or Bizarre) reactions
These are based on peculiarities of the patient and not on drug’s known
actions; include allergy and idiosyncrasy.
They are less common, often non-dose related, generally more serious and
require withdrawal of the drug.
Some of these reactions can be predicted and prevented if their genetic
basis is known and suitable test to characterize the individual’s phenotype
is performed.

Severity of adverse drug reactions
• Minor: No therapy, antidote or prolongation of hospitalization is required.
• Moderate: Requires change in drug therapy, specific treatment or prolongs
hospital stay by at-least one day.
• Severe: Potentially life-threatening, causes permanent damage or requires
intensive medical treatment.
• Lethal: Directly or indirectly contributes to death of the patient.

Pharmacovigilance
• Pharmacovigilance has been defined by the WHO (2002) as the ‘science and
activities relating to the detection, assessment, understanding and prevention
of adverse effects or any other drug related problems.’
• The information generated by pharmacovigilance is useful in educating
doctors about ADRs and in the official regulation of drug use.
• Its main purpose is to reduce the risk of drug-related harm to the patient.
• It has an important role in the rational use of medicines, as it provides the
base for assessing safety of medicines.
The activities involved in pharmacovigilance are:
a. Post-marketing surveillance and other methods of ADR monitoring such as
voluntary reporting by doctors (e.g. yellow card system of UK), prescription
event monitoring, computerized medical record and case reports by doctors.

b. Spreading of ADR data through ‘drug alerts’, ‘medical letters,’ advisories
sent to doctors by pharmaceuticals and regulatory agencies (such as FDA in
USA, committee on safety of medicines in UK).
b. Changes in the labeling of medicines indicating restrictions in use or
statuary warnings, precautions, or even withdrawal of the drug, by the
regulatory decision making authority.

Causality assessment
• When a patient undergoing drug therapy experiences an adverse event, it may be
due to the drug, or the disease or some other causes. Most of the time, a clear-cut
‘yes/no’ cause and effect relationship between a drug and the adverse event cannot
be well-defined. Causality is assessed on the basis of:
• Temporal relationship: How the time-sequence of the event is related to drug
administration.
• Previous knowledge: Whether the drug is known to produce the event in earlier
recipients with a certain degree of consistency.
• Dechallenge: Whether the event fall down on stopping the drug.
• Rechallenge: Whether the event reappeared when the drug was administered again
after a gap during which the event had subsided. Many times rechallenge is
unethical/dangerous, and is not done.
Assessed on the basis of the above criteria, causality has been graded as:
1. Definite: Causality is proven.
2. Probable: Though not proven, drug is the likely cause of the event.
3. Possible: Drug as well as other causes could be responsible for the event.
4. Doubtful: Drug unlikely to be the cause, but cannot be ruled out.

Prevention of adverse effects to drugs
Adverse drug effects can be minimized but not altogether eliminated by
observing the following practices:
1. Avoid all inappropriate use of drugs in the context of patient’s clinical
condition.
2. Use appropriate dose, route and frequency of drug administration based on
patient’s specific variables.
3. Take into consideration previous history of drug reactions.
4. Bring out the history of allergic diseases (drug allergy is more common in
patients with allergic diseases).
5. Rule out possibility of drug interactions when more than one drug is
prescribed.
6. Adopte correct drug administration technique (e.g. intravenous injection of
vancomycin must be slow).
7. Carry out appropriate laboratory monitoring

Types of Adverse Drug Effect
1. Side effects
2. Secondary effects
3. Toxic effects
4. Intolerance
5. Idiosyncrasy
6. Drug allergy
7. Photosensitivity
8. Drug dependence
9. Drug withdrawal
reactions
10. Teratogenicity
11. Mutagenicity and
Carcinogenicity12. Drug induced
diseases

1. Side effects
• These are unwanted but often unavoidable pharmacodynamic effects that
occur at therapeutic doses.
• Generally, they are not serious, can be predicted from the pharmacological
profile of a drug and are known to occur in a given percentage of drug
recipients.
• Reduction in dose, usually improve the symptoms.
• A side effect may be based on the same action as the therapeutic effect, e.g.
atropine is used in preanaesthetic medication for its antisecretory action.
The same action produces dryness of mouth as a side effect.
• An effect may be therapeutic in one context but side effect in another
context, e.g. codeine used for cough but produces constipation as a side
effect.
• Many drugs have been developed from observation of side effects, e.g.
early sulfonamides used as antibacterial were found to produce
hypoglycaemia and acidosis as side effects which directed research
resulting in the development of hypoglycaemic sulfonylureas and carbonic
anhydrase inhibitor—acetazolamide.

2. Secondary effects
These are indirect consequences of a primary action of the drug
e.g. suppression of bacterial flora by tetracyclines pave the way for
superinfections
corticosteroids weaken host defence mechanisms so that latent tuberculosis
gets activated.

3. Toxic effects
• These are the result of excessive pharmacological action of the drug due to
overdosage or prolonged use.
• Overdosage may be absolute (accidental, homicidal, suicidal) or relative
(i.e. usual dose of gentamicin in presence of renal failure).
• They result from functional alteration or drug induced tissue damage.
• The CNS, CVS, kidney, liver, lung, skin and blood forming organs are most
commonly involved in drug toxicity.
• Toxicity may result from extension of the therapeutic effect itself,
e.g. coma by barbiturates, complete A-V block (Atrioventricular block) by
digoxin, bleeding due to heparin.
• Another action of the drug can also be responsible for toxicity, e.g.—
 Morphine (analgesic) causes respiratory failure in overdosage.
 Imipramine (antidepressant) overdose causes cardiac arrhythmia.
Streptomycin (antitubercular) causes vestibular (body balance) damage
on prolonged use.

Poisoning
• In a broad sense, poisoning implies harmful effects of a
chemical on a biological system.
• It may result from large doses of drugs because ‘it is the dose
which distinguishes a drug from a poison’.
• Poison is a ‘substance which endangers life by severely
affecting one or more vital functions’.
• Specific antidotes such as receptor antagonists, chelating
agents or specific antibodies are available for few poisons.

The general detoxification and supportive measures are:
1. Recovery and maintenance of vital functions
a. Ensure patient airway, adequate ventilation, give artificial
respiration/100% oxygen inhalation as needed.
b. Maintain blood pressure and heart beat by fluid and pressor
agents, cardiac stimulants, pacing, etc, as needed.
c. Maintain body temperature.
d. Maintain blood sugar level by dextrose infusion, especially in
patients with altered perceptions.

2. Termination of exposure (decontamination)
• By removing the patient to fresh air (for inhaled poisons),
washing the skin and eyes (for poisons entering from the
surface), induction of vomiting (Emesis) with syrup ipecac or
gastric lavage (for ingested poisons).
• Emesis should not be attempted in comatose or
haemodynamically unstable patient, as well as for kerosene
poisoning due to risk of aspiration into lungs.
• Emesis/gastric lavage is not recommended if the patient
presents > 2 hours after ingesting the poison; if the poison/its
dose ingested are known to be non life-threatening, or if the
patient has vomited after consuming the poison.

3. Prevention of absorption of ingested poisons
• A suspension of 20–40 g (1g/kg) of activated charcoal, which
has large surface area and can adsorb many chemicals, should
be administered in 200 ml of water.
• However, strong acids and alkalies, metallic salts, iodine,
cyanide, caustics, alcohol, hydrocarbons and other organic
solvents are not adsorbed by charcoal.
• Charcoal should not be administered if there is paralytic ileus
or intestinal obstruction or when the patient reports > 2 hours
after ingesting the poison.

4. Hastening elimination
of the poison by inducing diuresis (furosemide, mannitol) or
altering urinary pH (alkalinization for acidic drugs, e.g.
barbiturates, aspirin).
However, excretion of many poisons is not enhanced by forced
diuresis and this procedure is generally not employed now.
Haemodialysis and haemoperfusion (passage of blood through
a column of charcoal or adsorbant resin) are more efficacious
procedures.

4. Intolerance
• It is the appearance of characteristic toxic effects of a drug in
an individual at therapeutic doses.
• It indicates a low threshold of the individual to the action of a
drug.
• These are individuals who fall on the extreme left side of the
Gaussian frequency distribution curve for sensitivity to the
drug.
• Examples are:
• A single dose of triflupromazine induces muscular dystonia
(repetitive muscle contractions) in some individuals, specially
children.
• Only few doses of carbamazepine may cause ataxia (lack of
voluntary coordination of muscle movements) in some people.
• One tablet of chloroquine may cause vomiting and abdominal
pain in an occasional patient.

5. Idiosyncrasy
• It is genetically determined abnormal reactivity to a chemical.
• The drug interacts with some unique feature of the individual,
not found in majority of subjects, and produces the
uncharacteristic reaction.
• As such, the type of reaction is restricted to individuals with a
particular genotype
Examples
• Barbiturates cause excitement and mental confusion in some
individuals.
• Quinine/quinidine cause cramps, diarrhoea, purpura, asthma
and vascular collapse in some patients.
• Chloramphenicol produces nondose-related serious aplastic
anaemia (body stops producing enough new blood cells) in
rare individuals.

6. Drug allergy / drug hypersensitivity
• It is an immunologically mediated reaction producing symptoms which are
unrelated to the pharmacodynamic profile of the drug.
• Generally occur even with much smaller doses.
• The target organs primarily affected in drug allergy are skin, airways, blood vessels,
blood and gastrointestinal tract.
• Allergic reactions occur only in a small proportion of the population exposed to the
drug and cannot be produced in other individuals at any dose.
• Prior sensitization is needed and a latent period of at least 1–2 weeks is required
after the first exposure.
• The drug or its metabolite acts as antigen (Ag) or more commonly hapten and
induce production of antibody (Ab)/sensitized lymphocytes.
• Presence of Ab to a drug is not necessarily followed by allergy to it.
• One drug can produce different types of allergic reactions in different individuals,
while widely different drugs can produce the same reaction.
• The course of drug allergy is variable; an individual previously sensitive to a drug
may subsequently tolerate it without a reaction and vice versa.

Basic features of drug allergy
• Sign unrelated to the pharmacodynamic actions of the drug.
• Manifestations similar to food/protein allergy, allergic
diseases.
• Severity of reaction poorly correlated with dose of the drug;
even small dose may trigger severe reaction.
• Occur only in few recipients, cannot be produced in other
individuals.
• Prior sensitization is needed.
• Positive dechallenge (on withdrawal of drug) and rechallenge
(even with small dose).

Mechanism and types of allergic reactions
A. Humoral
Type-I (anaphylactic) reactions
• Antibodies (IgE) are produced which get fixed to the mast cells.
• On exposure to the drug, Ag:Ab reaction takes place on the mast cell
surface releasing mediators like histamine, 5-HT, leukotrienes
(especially LT-C4 and D4), prostaglandins, PAF, etc. resulting in
urticaria, itching, angioedema, bronchospasm, rhinitis or
anaphylactic shock.
• Anaphylaxis is usually signed by paresthesia, flushing, swelling of
lips, generalized itching, wheezing, palpitation followed by syncope.
• The manifestations occur quickly after challenge and are called
immediate hypersensitivity.
• Antihistaminic drugs are beneficial in some of these reactions.

Urticaria
Urticaria is also known as ‘nettle rash’ or ‘hives’. This
condition consists of wheals - spots or patches of raised red or
white skin - each of which usually clears away in a few hours
to be replaced by other fresh wheals

Angioedema
Angioedema is an area of swelling of the lower layer of
skin and tissue just under the skin or mucous membranes.

Bronchospasm
or a bronchial spasm is a sudden constriction of the muscles in the
walls of the bronchioles.
It is caused by the release (degranulation) of substances from mast
cells or basophils under the influence of anaphylatoxins.
It causes difficulty in breathing which can be very mild to severe.

Rhinitis
• Also known as coryza, is irritation and inflammation of
the mucous membrane inside the nose.
• Common symptoms are a stuffy nose, runny nose, sneezing,
and post-nasal drip.
• The inflammation is caused by viruses, bacteria, irritants
or allergens.

Anaphylaxis
Is a severe allergic reaction that occurs rapidly and causes a
life-threatening response involving the whole body. This
reaction can lead to difficulty breathing and shock ultimately
leading to death.

Type-II (cytolytic) reactions
Drug + component of a specific tissue cell act as Ag. The resulting
antibodies (IgG, IgM) bind to the target cells; on reexposure Ag:Ab
reaction takes place on the surface of these cells, complement is
activated and cytolysis occurs.
e.g. Thrombocytopenia, agranulocytosis, aplastic anaemia,
haemolysis, organ damage (liver, kidney, muscle), systemic lupus
erythematosus.
Type-III (retarded, Arthus) reactions
These are mediated by circulating antibodies (predominantly IgG).
Ag:Ab complexes bind complement and precipitate on vascular
endothelium giving rise to a destructive inflammatory response.
Manifestations are rashes, serum sickness (fever, arthralgia,
lymphadenopathy), polyarteritis nodosa, Stevens-Johnson syndrome
(erythema multiforme, arthritis, nephritis, myocarditis, mental
symptoms).
The reaction usually subsides in 1–2 weeks.

Systemic lupus erythematosus (SLE)
• Also known simply as lupus, is an autoimmune disease in which the
body's immune system mistakenly attacks healthy tissue in many
parts of the body.
• Symptoms vary between people and may be mild to severe.
• Common symptoms include painful and swollen joints, fever, chest
pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired,
and a red rash which is most commonly on the face.

Serum sickness
Is a reaction to proteins in antiserum derived from a non-
human animal source, occurring 5–10 days after exposure.
It is a type of hypersensitivity, specifically immune complex
hypersensitivity (type III).
The term serum sickness-like reaction (SSLR) is occasionally used to
refer to similar illnesses that arise from the introduction of certain non-
protein substances, such as penicillin.
Arthralgia
(arthro-, joint + -algos, pain) literally means joint pain; it is a symptom
of injury, infection, illnesses (in particular arthritis) or an allergic
reaction to medication
Lymphadenopathy or adenopathy
Is disease of the lymph nodes, in which they are abnormal in size, number,
or consistency. Lymphadenopathy of an inflammatory type (the most
common type) is lymphadenitis, producing swollen or enlarged lymph
nodes.

Polyarteritis nodosa (PAN)
Is a condition that causes swollen arteries. It primarily affects small
and medium arteries, which can become inflamed or damaged. This
is a serious disease of the blood vessels caused by an immune
system malfunction.
Stevens–Johnson syndrome (SJS)
Is a type of severe skin reaction.
Together with toxic epidermal necrolysis(TEN) and Stevens-
Johnson/toxic epidermal necrolysis (SJS/TEN), it forms a spectrum
of disease, with SJS being less severe.
Early symptoms of SJS include fever and flu-like symptoms.
A few days later the skin begins to blister and peel forming painful
raw areas. Mucous membranes, such as the mouth, are also typically
involved.Complications include dehydration, sepsis, pneumonia,
and multiple organ failure.

B. Cell mediated
Type-IV (delayed hypersensitivity) reactions
• These are mediated through production of sensitized T-
lymphocytes carrying receptors for the Ag.
• On contact with the Ag these T cells produce lymphokines
which attract granulocytes and generate an inflammatory
response, e.g. contact dermatitis, some rashes, fever,
photosensitization.
• The reaction generally takes > 12 hours to develop.

Drugs frequently causing allergic reactions
Penicillins Salicylates
Cephalosporins Carbamazepine
Sulfonamides Allopurinol
Tetracyclines ACE inhibitors
Quinolones Methyldopa
Antitubercular drugs Hydralazine
Phenothiazines Local anaesthe

Treatment of drug allergy
• The offending drug must be immediately stopped.
• Most mild reactions (like skin rashes) subside by themselves
and do not require specific treatment.
• Antihistamines (H1) are beneficial in some type I reactions
(urticaria, rhinitis, swelling of lips, etc.) and some skin rashes
• In case of anaphylactic shock or angioedema of larynx the
resuscitation council of UK has recommended the following
measures:

• Put the patient in lie down position, administer oxygen at high flow rate and
perform cardiopulmonary resuscitation if required.
• Inject adrenaline 0.5 mg (0.5 ml of 1 in 1000 solution for adult, 0.3 ml for child 6-
12 years and 0.15 ml for child upto 6 years) i.m.; repeat every 5–10 min in case
patient does not improve or improvement is transient. This is the only life saving
measure. Adrenaline should not be injected i.v. (can itself be fatal) unless shock is
immediately life threatening. If adrenaline is to be injected i.v., it should be diluted
to 1:10,000 or 1:100,000 and infused slowly with constant monitoring.
• Administer a H1 antihistaminic (chlorpheniramine 10–20 mg) i.m./slow i.v. It may
have adjuvant value.
• Intravenous glucocorticoid (hydrocortisone sod. succinate 200 mg) should be added
in severe/recurrent cases.
• It acts slowly, but is specially valuable for prolonged reactions and in asthmatics. It
may be followed by oral prednisolone for 3 days.
• Adrenaline followed by a short course of glucocorticoids is indicated for
bronchospasm attending drug hypersensitivity.
• Glucocorticoids are the only drug effective in type II, type III and type IV reactions.
• Skin tests (intradermal, patch) or intranasal tests may forewarn in case of Type I
hypersensitivity, but not in case of other types. However, these tests are not entirely
reliable—false positive and false negative results are not rare.

7. Photosensitivity
• It is a cutaneous reaction resulting from drug induced
sensitization of the skin to UV radiation.
• The reactions are of two types:
(a) Phototoxic
(b) Photoallergic

Phototoxic
• Drug or its metabolite accumulates in the skin, absorbs light
and undergoes a photochemical reaction followed by a
photobiological reaction resulting in local tissue damage
(sunburn-like), i.e. erythema, edema, blistering which have
fast onset and shorter duration after exposure ends.
• This is followed by hyper-pigmentation.
• The lesions may be more severe with larger doses of the drug.
• The shorter wave lengths (290–320 nm, UV-B) are
responsible.
• Drugs involved in acute phototoxic reactions are tetracyclines
(especially demeclocycline) and tar products.
• Drugs causing chronic and low grade sensitization are
nalidixic acid, fluoroquinolones, dapsone, sulfonamides,
phenothiazines, thiazides, amiodarone.

Photoallergic
• Drug or its metabolite induces a cell mediated immune
response which on exposure to light of longer wave lengths
(320–400 nm, UV-A) produces a papular or eczematous
contact dermatitis like picture that may persist long after
exposure.
• Rarely antibodies mediate photoallergy and the reaction takes
the form of immediate flare (burns), itching on exposure to
sun.
• Even small doses may trigger the reaction and lesions may
extend beyond the exposed area.
• Drugs involved are sulfonamides, sulfonylureas, griseofulvin,
chloroquine, chlorpromazine, carbamazepine.

8. Drug dependence
• Drugs capable of altering mood and feelings are liable to
repetitive use to derive euphoria, recreation, withdrawal from
reality, social adjustment, etc.
• Drug dependence is a state in which use of drugs for personal
satisfaction is accorded a higher priority than other basic
needs, often in the face of known risks to health.
• There is a lot of confusion in terminology and definitions; the
following may serve to describe different aspects of the
problem.

Psychological dependence
• Individual believes that optimal state of wellbeing is achieved
only through the actions of the drug.
• The subject feels emotionally distressed if the drug is not
taken.
• It may start as liking for the drug effects and may progress to
compulsive drug use in some individuals who then lose control
over the use of the drug.
• Obviously, certain degree of psychological dependence go
along with all patterns of self medication.

Reinforcement
• The ability of the drug to produce effects that the user enjoys
and which make him/her wish to take it again or to induce
drug seeking behaviour.
• Certain drugs (opioids, cocaine) are strong reinforcers, while
others (benzodiazepines) are weak reinforcers.
• Faster the drug acts, more reinforcing it is. Thus, inhaled drugs
and those injected i.v. are highly reinforcing—produce an
intense ‘high’ in dependent individuals.

Physical dependence
• It is an altered physiological state produced by repeated
administration of a drug which necessitates the continued
presence of the drug to maintain physiological equilibrium.
• Discontinuation of the drug results in a characteristic
withdrawal (abstinence) syndrome.
• The nervous system get adapted to function normally in the
presence of the drug, it has been called ‘neuroadaptation’.
• Drugs producing physical dependence are—opioids,
barbiturates and other depressants including alcohol and
benzodiazepines.
• Stimulant drugs, e.g. amphetamines, cocaine produce little or
no physical dependence.

Drug abuse
• Refers to use of a drug by self-medication in a manner and amount
that deviates from the approved medical and social patterns in a
given culture at a given time.
• The term conveys social disapproval of the manner and purpose of
drug use.
• For regulatory agencies, drug abuse refers to any use of an illegal
drug.
• The two major patterns of drug abuse are:
a. Continuous use:
The drug is taken regularly, the subject wishes to continuously
remain under the influence of the drug, e.g. opioids, alcohol,
sedatives.
b. Occasional use:
The drug is taken off and on to obtain pleasure or high, recreation
(as in rave parties) or enhancement of sexual experience, e.g.
cocaine, amphetamines, psychedelics, binge drinking (alcohol),
cannabis, solvents (inhalation), etc.

Drug addiction
• It is a pattern of compulsive drug use characterized by vast
involvement with the use of a drug.
• Procuring the drug and using it takes precedence over other
activities.
• Even after withdrawal most addicts tend to relapse.
• Physical dependence, though a strong impetus for continued
drug use, is not an essential feature of addiction.
• Amphetamines, cocaine, cannabis, LSD are drugs which
produce addiction but little/no physical dependence.
• On the other hand, drugs like nalorphine produce physical
dependence without imparting addiction in the sense that there
is little drug seeking behaviour.

Drug habituation
• It denotes less intensive involvement with the drug, so that its
withdrawal produces only mild discomfort.
• Consumption of tea, coffee, tobacco, social drinking are
regarded habituating, physical dependence is absent.
• Basically, habituation and addiction imply different degrees of
psychological dependence and it may be difficult to draw a
clearcut line of distinction between the two.
• Therefore, it is better to avoid using these terms in describing
drug dependence and related conditions.

9. Drug withdrawal reactions
Apart from drugs that are usually recognized as producing
dependence, sudden interruption of therapy with certain other
drugs also results in adverse consequences, mostly in the form
of drug was being used, e.g.:
(i) Acute adrenal insufficiency may be precipitated by abrupt
cessation of corticosteroid therapy.
(ii) Severe hypertension, restlessness and sympathetic
overactivity may occur shortly after discontinuing clonidine.
(iii) Worsening of angina pectoris, precipitation of myocardial
infarction may result from stoppage of β blockers.
(iv) Frequency of seizures may increase on sudden withdrawal
of an antiepileptic.
• These manifestations are also due to adaptive changes and can
be minimized by gradual withdrawal

10. Teratogenicity
• It refers to the capacity of a drug to cause foetal abnormalities
when administered to the pregnant mother.
• The placenta does not constitute a strict barrier, and any drug
can cross it to a greater or lesser extent.
• The embryo is one of the most dynamic biological systems and
in contrast to adults, drug effects are often irreversible.
• The thalidomide disaster (1958–61) resulting in thousands of
babies born with phocomelia (seal like limbs) and other
defects focused attention onto this type of adverse effect.

Drugs can affect the foetus at 3 stages—
(i) Fertilization and implantation—conception to 17 days—
failure of pregnancy which often goes unnoticed.
(ii) Organogenesis—18 to 55 days of gestation—most
vulnerable period, deformities are produced.
(iii) Growth and development—56 days onwards —
developmental and functional abnormalities can occur, e.g.
ACE inhibitors can cause hypoplasia of organs, especially of
lungs and kidneys; NSAIDs may induce premature closure of
ductus arteriosus; androgens and progestins cause
masculanization of female foetus, antithyroid drugs and
lithium cause foetal goiter.

11. Mutagenicity and Carcinogenicity
• It refers to capacity of a drug to cause genetic defects and
cancer respectively.
• Usually oxidation of the drug results in the production of
reactive intermediates which affect genes and may cause
structural changes in the chromosomes.
• Covalent interaction with DNA can modify it to induce
mutations, which may manifest as heritable defects in the next
generation.
• If the modified DNA sequences code for factors that regulate
cell proliferation/growth, i.e. are protooncogenes, or for
proteins that inhibit transcription of protooncogenes, a tumour
(cancer) may be produced.

• Even without interacting directly with DNA, certain chemicals
can promote malignant change in genetically damaged cells,
resulting in carcinogenesis.
• Chemical carcinogenesis generally takes several (10–40) years
to develop.
• Drugs implicated in these adverse effects are—anticancer
drugs, radioisotopes, estrogens, tobacco.
• Generally, drugs which show mutagenic or carcinogenic
potential are not approved for marketing/are withdrawn, unless
they are useful in life-threatening conditions.

12. Drug induced diseases
• These are also called iatrogenic (physician induced) diseases,
and are functional disturbances (disease) caused by drugs
which persist even after the offending drug has been
withdrawn and largely eliminated.
• e.g.: Peptic ulcer by salicylates and corticosteroids.
Parkinsonism by phenothiazines and other antipsychotics.
• Hepatitis by isoniazid.
• DLE by hydralazine.

Bio-Assays
• The determination of the relative strength of a substance (e.g., a drug
or hormone or toxicant) by comparing its effect on a test organism
with that of a standard preparation." is called bioassay.
• Bioassay/ biological assay/ biological assessment/ biological
standardization is a type of scientific experiment which involves the
use of live animal or plant (in vivo) or tissue or cell (in vitro) to
determine the biological activity of a substance, such as a hormone or
drug.
• Bioassays are conducted to measure the effects of a substance on a
living organism and are essential in the development of new drugs
and in monitoring environmental pollutants.
• A bioassay can also be used to determine the concentration of a
particular constitution of a mixture that may cause harmful effects on
organisms or the environment.

Purpose
• Measurement of the pharmacological activity of new or
chemically undefined substances.
• Investigation of the function of endogenous mediators.
• Determination of the side-effect profile, including the degree
of drug toxicity.
• Measurement of the concentration of known substances.
• Assessing the amount of pollutants being released by a
particular source, such as wastewater or urban runoff.
• Determining the specificity of certain enzymes to certain
substrates.

Bioassays may be qualitative or quantitative
Qualitative bioassays are used for assessing the physical effects
of a substance that may not be quantified, such as seeds fail to
germinate or develop abnormally deformity.
Quantitative bioassays involve estimation of the concentration or
potency of a substance by measurement of the biological response
that it produces.
Quantitative bioassays are typically analyzed using the methods of
biostatistics.

Principle of Bioassay
• Active compound to be assayed should show the same measured
response in all animal species.
• Bioassay involves the comparison of the main pharmacological
response of the unknown preparation with that of the standard.
• The method selected should be reliable, sensitive, and reproducible
and should minimize errors due to biological variation and
methodology.
• The degree of pharmacological response produced should be
reproducible under identical conditions.
• The reference standard and test sample should have same
pharmacological effect and mode of action, so that their Dose
Response Curve run parallel and their potency ratio can be
calculated.
• Bioassay might measure a different aspect of the same substance
compared to chemical assay.
• The test solution and standard should be compared for their
established pharmacological effect using a specified
pharmacological technique.

Characteristics of a good assay method
• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Precision
• Accuracy
• Stability

Bioassay can be performed on
• Invivo Intact animals
• Invitro Isolated tissues, Specific cells

Important Terms
• EC50 (ED50)= Drug concentration (dose) that produce 50%
of maximal response.
• Potency= Amount of a drug needed to produce a given
response.
• Maximum effect= the maximum response that could be
caused by a drug after occupying all possible receptors.
• Notes*
 EC increases = potency decreases
 The magnitude of a drug effect depends on the number of
receptors occupied by the drug

Bioassay Methods / Types of
Bioassay
Graded Response Quantal Response
Matching or
bracketing
method
Graphical
method
End point
method

Graded response
• In these response, as the dose increases there is an equivalent
rise in response.
• The potency is estimated by comparing the test sample
responses with the standard response curve.
• Graded dose response assay, relates the size of the response to
the drug in a single biologic unit.
• As the dose administered increased the pharmacological
response also increases and eventually reaches a steady level
called the ceiling effect there will be on further increase in
response even with an increase in dose.

Quantal response
• As the name indicates, the threshold dose of the sample
required to obtain a complete or a particular pharmacological
effect is determined and compared with standard.
• The Quantal dose response curves are useful for determining
dose to which most of the population responds. They have
similar shpae as log dose-response curve

• ED50 is the dose that produce certain pharmacological effect in 50% off the
exposed units.
• LD50 is the dose which produces toxic effect at 50% of exposed units (people) to
the drug
• Therapeutic Index (TI)- Ratio of the dose that produces toxicity in half of the
population (TD50) to the dose that desired therapeutic response (ED50)
• Therapeutic window- The dose range between the minimum effective
concentration (dose) and the minimum toxic concentration or dose. So larger the
therapeutic window or index, the greater the safety range.

Matching point Method:
• In this method a constant dose of the test is bracketed by
varying doses of standard till the exact match is obtained
between test dose and the standard dose.
• Initially, two responses of the standard are taken. The doses
are adjusted such that one is giving response of approximately
20% and other 70% of the maximum.
• The response of unknown which lies between two responses of
standard dose is taken.
• The panel is repeated by increasing or decreasing the doses of
standard till all three equal responses are obtained.
• The dose of test sample is kept constant.

At the end, a response of the double dose of the standard and test
which match each other are taken.
These should give equal responses.
Concentration of the test sample can be determined as follows:

Limitations of Matching point Method
• It occupies a larger area as far as tracings are concerned.
• The match is purely subjective, so chances of error are there
and one cannot determine them.
• It does not give any idea of dose-response relationship.
• However, this method is particularly useful if the sensitivity of
the preparation is not stable. Bioassay of histamine, on guinea
pig ileum is preferably carried out by this method.
Fig.: Bioassay of histamine by the matching method

Graphical Method
This method is based on the assumption of the dose-response
relationship.
Log-dose-response curve is plotted and the dose of standard
producing the same response as produced by the test sample is
directly read from the graph.
In simpler design, 5-6 responses of the graded doses of the
standard are taken and then two equiactive responses of the test
sample are taken.
The height of contraction is measured and plotted against the log-
dose.
The dose of standard producing the same response as produced
by the test is read directly from the graph and the concentration
of test sample is determined by the same formula as mentioned
before.

Fig.: Graphical method of bioassay
The characteristic of log-dose response curve is that it is linear in
the middle (20-80%). Thus, the comparison should be done within
this range only. In other words, the response of test sample must lie
within this range.
Advantage of this method is that, it is a simple method and chances
of errors are less if the sensitivity of the preparation is not changed.
Other methods which are based on the dose-response relationship
include 3 point, 4 point, 5 point and 6 point methods.

In these methods, the responses are repeated several times and
the mean of each is taken. Thus, chances of error are
minimized in these methods. In 3 point assay method 2 doses
of the standard and one dose of the test are used. In 4 point
method 2 doses of standard and 2 doses of the test are used. In
6 point method 3 doses of standard and 3 doses of the test are
used. Similarly one can design 8 point method also.
Fig.: Bioassay of histamine by three point method

The mean responses are calculated and plotted against log-
dose and amount of standard producing the same response as
produced by the test is determined graphically as well as
mathematically:
n1 = Lower standard dose
n2 = Higher standard dose
t = Test dose
S1 = Response of n1
S2 = Response of n2
T = Response of test (t)
Cs = Concentration of standard

Similarly, in 4 point method, amount of standard producing the
same response as produced by the test can be determined by
graphical method. It is determined mathematically as follows:
t1 = lower dose of test
t2 = higher dose of test
T1 = response of t1
T2 = response of t2

End Point Method
• Here the threshold dose producing a positive effect is
measured on each animal and the comparison between the
average results of two groups of animals (one receiving
standard and other the test) is done.
e.g. bioassay of digitalis in cats.
• Here the cat is anaesthetized with chloralose and its blood
pressure is recorded.
• The drug is slowly infused into the animal and the moment the
heart stops beating and blood pressure falls to zero, the volume
of fluid infused is noted down.

• Two series of such experiments-one using standard digitalis
and the other using test preparation of digitalis is done and
then potency is calculated as follows

What is animal experimentation
• Animal experimentation is the term used to explain the use of
animals in experimentation in education, training and research.
• The terms animal testing, animal experimentation, animal
research, in vivo testing and vivisection are often used
interchangeably although they carry different meanings.
• “Vivisection”, a term preferred by those who oppose the use of
animals in research, means cutting into or dissecting a living
animal.
• Researchers prefer to use the term ‘animal experimentation’.

Use of Animals in Research
Animals are used most often in the following cases:
• Disease Treatment
• Prevention
• Treatment of Injuries
• Basic Medical Testing
• Medical Diagnosis
• Vaccinations
• Anesthesia
• Antibiotics
• Numerous medical treatments for various diseases

Contribution of animal experimentation to
therapeutic discoveries
• There is a strong relationship between rapid progress in
experiments on animals and progress in clinical medicine.
• In the 1880s, Behring used horses for production of diphtheria
antitoxin and the development of a vaccine against diphtheria
and tetanus leading to the first Noble prize in physiology or
medicine in 1901.
• Insulin was first isolated from dogs in 1922 and it
revolutionized the treatment of diabetes.
• In the 1970s, antibiotic treatment and vaccines for leprosy
were developed using armadillos.
• Domagk introduced antibacterial activity of prontosil in 1939
by experiments on chicken.

Salient drug discoveries that involved use of animals

Ethics
• Today there exists a wide spectrum of views on this subject,
ranging from those concerned with animal 'rights' to those who
view animals only as a resource to be exploited.
• All of these viewpoints have contributed to the development of
ethical principles of animal use.

Animal Ethics
• Is a term used to describe human-animal relationships and how
animals should to be managed and treated.
• The subject matter includes
 animal rights
 animal welfare
 animal law
 animal cognition
 wildlife conservation
 And the history of animal use

Five Freedom
• The five freedoms were originally developed from a UK
Government report on livestock husbandry in 1965 (Prof.
Roger Brambell) then by Farm Animal Welfare Council
(FAWC) In July 1979

The Three Rs
• The three Rs are encouraged to follow in order to reduce the
impact of research on animals
• The three Rs are:
• Replacement
• Reduction
• Refinement

1. Replacement
• Means replacing 'higher' animals with 'lower‘ animals.
Microorganisms, plants, eggs, reptiles, amphibians, and
invertebrates may be used in some studies to replace warm-
blooded animals.
• Alternately, live animals may be replaced with non-animal
models, such as dummies for an introduction to dissection for
teaching the structure of the animal or the human body,
mechanical or computer models, audiovisual aids, or in vitro
modeling.

Replacing experiments on animals with alternative
techniques such as:
• Experimenting on cell cultures instead of whole animals
• Using computer models
• Studying human volunteers
• Using epidemiological studies

2. Reduction
• It means minimizing the number of animals needed to perform
an experiment or teach a concept.
• By examining these parameters, the IACUC (Institutional
Animal Care and Use Committees) can determine if
thoughtful experimental design was employed to minimize
overall animal use.
• Reducing the number of animals used in experiments by:
• Improving experimental techniques
• Improving techniques of data analysis
• Sharing information with other researchers

• Consulting with a statistician to use only the numbers of
animals required to achieve significance.
• Minimizing variables such as disease, stress, diet, genetics,
etc., that may affect experimental results.
• Performing appropriate literature searches and consulting with
colleagues to ensure that experiments are not duplicated.
• Using the appropriate species of animal so that useful data is
collected.
• Replacement whenever possible.

3. Refinement
• It means refining experimental protocols to minimize pain or
distress Using a Protocol Form. Examples of refinement
include:
• Identifying pain and distress and making plans for preventing
or relieving it.
• Receiving adequate training prior to performing a procedure.
• Using proper handling techniques for animals.
• Refining the experiment or the way the animals are cared for
so as to reduce their suffering by:
• Using less invasive techniques
• Better medical care
• Better living conditions

Ethics and animal use
• The debate surrounding animal use in experiments and teaching
started way back in the 17th century. The animal protection
movement was started in 18th century by a group of people known as
abolitionists in England. Another worldwide initiative was started in
1975 by Societies for Protection and Care of Animals (SPCA) who
opposed all forms of animal research.
• Since years, some researchers have favored animal experimentation
and emphasized that such experiments were necessary for the
advancement of scientific knowledge. Claude Bernard is known as
the “prince of vivisection” and the father of physiology. His wife,
Marie Françoise Martin, established the first anti-vivisection society
in France in 1883. She wrote “the science of life is a superb and
dazzlingly lighted hall which may be reached only by passing
through a long and ghastly kitchen”. Arguing that “experiments on
animals are entirely conclusive for the toxicology and hygiene of
man, the effects of these substances are the same on man as on
animals, save for differences in degree,” Bernard established animal
experimentation as a part of the standard scientific method.

The main concern for animals in experimentation is physical and
mental stress and pain. A “painful procedure” in an animal
study is defined as one that would “reasonably be expected to
cause more than slight or momentary pain or distress in a
human being to which that procedure was applied.” In the
USA (2006) millions of animals were used in procedures that
caused more than momentary pain or pain/distress, while
84,000 were used in studies that would cause pain or distress
that would not be relieved by anesthesia.[36] In the UK,
research projects are classified as mild, moderate, and
substantial in terms of the suffering caused to animals.
Animals that are anesthetized and killed without recovering
consciousness are categorized as “unclassified”.[

Notwithstanding the various regulations, unethical treatment of
animals is being reported worldwide. Some of these ‘so cruel’
episodes attracted worldwide attention. iBritches, a macaque
monkey, was used for an experiment to test sensory substitution
devices for blind people in University of California, Riverside. The
monkeys had their eyes sewn shut and hence attracted a lot of
ctiticism in 1985. The laboratory was raided by Animal Liberation
Front and animals were rescued.[38] The first instance of lab
technicians being fined for animal cruelty in the United Kingdom
happened in 1997, when employees were ordered to pay £250 by
People for the Ethical Treatment of Animals (PETA) for mistreating
dogs.[38] Similarly, PETA fined a contract research organization
after filming their facility in 2004-05. However, these unethical
treatments to animals continued. In 2006, a trial of a monoclonal
antibody in primates triggered a disastrous immune reaction and
widespread organ failure in the six trial animals. This happening in
London generated a lot of media attention.

• On the other hand, animal activists were using extreme measures to
stop animal use. There have been threats to researchers from animal
rights activists. A bomb was placed under the car of a
ophthalmologist experimenting on cats and rhesus monkeys.
Following this and similar incidents, the US government passed the
Animal Enterprise Terrorism Act. The government in U.K. followed
by adding the offense of “Intimidation of persons connected with
animal research organisation” to the Serious Organised Crime and
Police Act 2005.
• It is pertinent here to remember that ethics, whether involving
humans or animals carries varying connotations to different people.
So how do we judge the ethical issues in animal experimentation?
Whenever you consider ethical issues in animal experiments,
critically analyze the following:
• Is the animal the best experimental system for the hypothesis to be
tested?
• Is the problem under review worth solving?
• Can pain and discomfort be minimised for the animal?

International Guidelines of using Animals
in Scientific Procedures
• Animal experiments should be designed only after due consideration of
animal health and the advancement of knowledge on humans or animals
weighed against the potential impacts on the welfare of the animals.
• Researchers should treat animals as sentient and must consider their proper
care and use and the avoidance or minimization of discomfort, distress, or
pain as imperatives. In this field, the 3 ‘R’ principles must be considered at
all animal experiments:
• Replacement of animal experimentation with alternative methods such as
mathematical models, computer simulation and in vitro biological systems,
which replace or complement the use of animals must be considered before
embarking on any procedure involving use of animals.
• Reduction in the number of animals used which means minimum number
of animals required to obtain scientifically valid results. Furthermore,
scientific projects involving the use of animals must not be repeated or
duplicated unnecessarily.
• Refinement of projects and techniques used to minimize impact on animals
which means: (a) Animals chosen must be of an appropriate species and
quality for the scientific projects concerned taking into account their
specific biological properties, including genetic constitution, behavior, and
microbiological, nutritional and general health status.

Introduction
• Insulin was discovered in 1921, which helped millions
suffering from type-1 diabetes.
• It is a hormone made in pancreas, by special cells called “beta
cells.
• Most people now a days use human insulin or insulin analogs.
• Its is also produced by bacteria or by yeast by using genetic
engineering.

Mechanism of action
• Every pancreatic islet contains ~1,000 endocrine cells of which 75% are
insulin-producing beta-cells.
• Insulin is synthesized as pro-insulin and is processed to the biologically
active form inside the secretory granules.
• The beta-cell is electrically excitable and uses changes in membrane
potential to couple variations in blood glucose to trigger insulin secretion.
• The beta-cell contains about 20 different ion channels proteins.
• Two types of ion channels are particularly important for the initiation of
insulin secretion.
• The ATP sensitive potassium ion-channels are active at low glucose
concentrations, because of the high intracellular ADP levels.

Bioassay of insulin
Standard preparation and unit:
It is pure, dry and crystalline insulin. One unit contains
0.04082 mg. This unit is specified by Ministry of Health,
Government of India and is equivalent to international unit.
Preparation of standard solution:
Accurately weigh 20 units of insulin and dissolve it in normal
saline. Acidify it with HCl to pH 2.5.
• Add 0.5% phenol as preservative. Add 1.4% to 1.8% glycerin.
Final volume should contain 20 units/ml.
• Store the solution in a cool place and use it within six months.
Preparation of test sample solution:
The solution of the test sample is prepared in the same way as
the standard solution.

Rabbit Method
• Selection of rabbits: They should be healthy, weighing about
1800-3000 gms. They should then be maintained on uniform
diet but are fasted for 18 hrs. before assay. Water is withdrawn
during the experiment.
• Standard and Sample Dilutions: These are freshly prepared
by diluting with normal NaCl solution so as to contain 1
unit/ml. and 2 units/ml.
• Doses: The dose which can produce suitable fall in blood
sugar level is calculated for the standard.
• Principle: The potency of a test sample is estimated by
comparing the hypoglycemic effect of the sample with that of
the std. preparation of insulin.
• Any other suitable method can also be used.

• Experimental Procedure: Animals are divided into 4 groups
of 3 rabbits each. The rabbits are then put into an animal
holder. They should be handled with care to avoid excitement.
• First part of the Test: A sample of blood is taken from the
marginal ear vein of each rabbit Presence of reducing sugar is
estimated per 100 ml. of blood by a suitable chemical method.
• This concentration is called ‘Initial Blood Sugar Level’.
• The four groups of rabbits are then given subcutaneous
injections of insulin as follows:

• From each rabbit, a sample of blood is withdrawn up to 5 hrs.
at the interval of 1 hr. each. Blood sugar is determined again.
This is known as ‘Final Blood Sugar Level’.
• Second part of the test (Cross over test) : The same animals
are used for the second part.
• The experiment can be carried out after one week.
• Again they are fasted and initial blood sugar is determined.
The grouping is reversed, that is to say, those animals which
received the standard are given the test and those which
received the test are now given the standard.
• Those animals which received the less dose of the standard are
given the higher dose of the test sample and vice-versa. This
test is known as ‘Twin Cross Over Test’. Mean percentage
decrease in blood sugar of the first and second part is
calculated.

Mouse Method
• Mice show characteristic convulsions after subcutaneous
injection of insulin at elevated temperatures. The percentage
convulsions produced by the test and standard preparations are
compared.
• Experimental procedure: Minimum 100 mice weighing
between 18-22 gms. of the same strain are used. They should
be maintained on constant diet. They should be fasted 18 hrs.
prior to the experiment.
• Standard and sample dilutions: Dilutions are prepared with
sterile saline solution, so as to contain 0.064 units/ml. (std
dilution I) and 0.096 untis/ml. (std. dilution II). Similarly, test
sample solutions are also prepared

• Mice are divided into 4 groups each containing 25 mice
and insulin is injected s.c. as follows
• Mice are put in an air incubator at 33°C and observed for
one and a half hr. An air incubator with a glass front
provided with six shelves is used.
• The temperature of is thermostatically controlled. Two
mice are kept in each of the boxes made up of perforated
sheet of metal.

• The mice which convulse or die are taken out of the incubator
and observed.
• These reacting mice usually convulse severely but failure of
the animal to upright itself when placed on its back, should as
well be considered as convulsion.
• Convulsion mice may be saved by an injection of 0.5 ml of
5% dextrose solution.
• Percentage convulsions produced by the test sample are
compared with those of the standard sample .
• Those animals which survive may be used again for another
experiment after an interval of one week.

Log ratio of dose (I) =
Variance of dose (E) = ½ (T2-T1+S2-S1)
Slope = b= tan a =E/I =
Variance of preparation (F) = ½ (T1-T2-S1-S2)
Log potency ratio M=F/b
Dose S1 0.25ml S2 0.25 ml T1 0.25 ml T2 0.25 ml
Convulsions
Percentage
Convulsions

Rat diaphragm method
• Sprague Dawley rats weighing 70–100 g are used. The animals
are sacrificed during anesthesia and the diaphragms still
attached to the rib cages are carefully removed, released from
the rib cages and adhering connective and fat tissues, washed
in Phosphate Buffer Saline, spread out and divided into two
equal pieces.
• For assaying the effects of insulin/compounds/drugs, the
hemidiaphragms are incubated in Krebs-Ringer-HEPES
(KRH) buffer gassed with carbogen (95% O2/5% CO2) in the
presence of 5 mM glucose

Rat Epididymal Fat Method
• Insulin-like activity can be measured by the uptake of glucose
into fat cells.
• Adipose tissue from the epididymal fat pad of rats has been
found to very suitable.
• The difference of glucose concentration in the medium after
incubation of pieces of epididymal rat adipose tissue or
measured oxygen consumption in Warburg vessels,
Radiolabelled 14C glucose, the 14CO2 is trapped and counted.
• The concentration is determined by immuno-assay.

Table of Content
• Introduction on Heparin
– Biological activity of Heparin
– Therapeutical Uses
• Assay of Heparin
– Principle
– Standard Preparations
– Methods
– Results
– Calculations
– Limit of Errors
• Conclusion

Introduction
• Heparin is a highly-sulfated glycosaminoglycan of natural
origin.
• It is also one of the oldest drugs still in widespread use.
• Heparin, along with vitamin K antagonists, have been the main
anticoagulant drugs for more than 70 years, as it has been used
since the 1930s.

Biological Activity
• Heparin can interact and regulate the activities of a wide range
of proteins that are essentials to important biological processes
such as
 Blood clotting
 Pathogen infection
 Cell differentiation
 Cell growth and migration
 Inflammation

Therapeutic Uses
The general medical uses of heparin are the following:
• Acute myocardian infarction
• Curative and prophylactic treatment of arterial and venous thrombo-
embolism.
• Lung thrombo-embolism
• Prevention of deep venous and pulmonary thrombo-embolism during
pregnancy
• Peripheral arterial diseases
• Arterioesclerosis
• Extracorporeal circulation
• Anticoagulant
• Hemodialysis
• Extracorporeal therapies such as heart-lung oxygenation and liver dialysis
• Open heart surgery
• Deep vein thrombosis
• Vitreoretinal surgery
• External use for ulcer treatment
• External use for treatment of varicose veins.

Bioassay of Heparin
Principle
The potency of heparin sodium is determined in
vitro by comparing the concentration necessary to
prevent the clotting of sheep or goat or human
plasma with the concentration of the standard
preparation of heparin sodium.
Its is necessary to give the same effect under the
same conditions of the method of assay.
Requirements of
Heparin Bioassay
Heparin to be
tested
(Synthesized)
Calcium
Chloride
Standard
heparin
solution
Plasma (From
sheep or goats,
human)

Standard Heparin
• Purified freeze-dried heparin sodium salt from bovine/ pork
intestinal mucous.
• The potency of standard heparin has been determined in
relation to the International Standard stated by the World
Health Organization.

Prepared plasma
Collect the blood from sheep or goat or human in vessel
containing 8% w/v of sodium citrate (The ratio of sodium
citrate and blood is 1:19)
Mix gently and centrifuge to pool out plasma
Clean test tube In one ml of pooled plasma, add 0.2 ml of
1% w/v of calcium chloride solution and mix it
The plasma is suitable if clot form within 5 min

Standard Heparin Preparation
The minimum quantity of standard preparation of heparin sodium
which, when added in 0.8 ml of saline solution, maintain fluidity
in 1 ml of prepared plasma for 1 h after addition of 0.2 ml of 1%
w/v calcium chloride.
The potency of standard heparin is determined in relation to the
International Standard stated by the World Health Organization.

Test Solution
Weigh accurately about 25 mg of the test sample
Dissolve in sufficient saline to give the concentration of 1 mg/ml
Dilute the test solution corresponds to that of standard

Method
• In cline test tube, add graded amount of the solution of
standard preparation (the largest dose not exceed 0.8 ml)
• Add sufficient volume of saline to make total volume of 0.8
ml and add 1 ml of prepared plasma to each test tube
• Add 0.2 ml of 1 % w/v solution of calcium chloride, note the
time
• Mix the content properly so entire inner surface of the tube is
wet

• In same manner setup a series of test preparation (complete the
entire process within 20 min after addition of prepared plasma)
• After 1 hr the addition of calcium chloride solution, determine
the extent of clotting in each test tube
• Recognize three grades between zero and full clotting
• Dilution of the test solution which contains same concentration
as that of standard shows same degree of clotting

• If the degree of clotting in dilution of the standard preparation
lies between that observed in 2 of the dilution of test
preparation, the potency of later is estimated
• If there is no correspondence between the degree of clotting by
standard and test, new dilution prepared and assay is repeated
• Calculate the estimated potency of the preparation by
combining the result of assay with standard statistical method

Results
• If the degree of clotting observed in the series of dilutions of
the solution of standard preparation lies between that observed
in two of the series of dilutions of the sample being examined,
the potency of the latter is estimated
• If there is no such correspondence between the degrees of
clotting produced by the solution of standard preparation and
any of the dilutions of the sample being examined, new
dilutions of the latter are prepared and assay is repeated.

Calculations
• The estimated potency of the preparation being examined is
calculated by combining the results of these assays by standard
statistical methods.
• The ratio of a given reference standard dose to the
corresponding unknown dose is designated by R.
• The logarithm of the ratio of potency of the unknown, in
quantities assumed to be equal to those of the reference
standard, is designated by M‘
• Calculations:
M= M’+ log R
Potency(P) = antilog M = antilog M’× R

Conclusion
• Heparin bioassays are performed to monitor and adjust
standard heparin.
• This is done in order to evaluate the concentration of heparin
in blood and helps doctors to monitor therapy.
• If concentrations are within an established therapeutic interval
and the person is doing well clinically i.e. there is no clotting,
excessive bleeding, or other complications – then the dosage is
considered appropriate.

What is histamine?
Histamine is a small ,water soluble molecule or amine
autocoid or locally acting hormone which mediates its effects
by binding to receptors H1, H2, H3, and H4.

Site of synthesis and storage:
Histamine is synthesized and stored in the following sites:
1- Neurons in the brain
2- Entero chromaffin cells in the gastric mucosa
3- Mast cells

Non mast cell sources of histamine
in the body
1- Brain: (functions as neurotransmitter)
2- Entero chromaffin cells (EC) in the stomach
• Function: stimulates HCl secretion by parietal cells of the
stomach

Pharmacological actions of
histamine
• The pharmacological actions of histamine depend on the tissue
and type of receptor present at the area of release.

Histamine Receptors
R subtype Distribution Action Agonist Antagonist
H1 Smooth
Muscle
Contraction of GIT,
Bronchoconstricton.
Hist. Mepyramine
Cyproheptadine
H2 Gastric
mucosa
Acid release Hist. Cimetidine
Ranitidine
H3 Presynaptic Autoregulation of
histamine
release
Hist. Thioperamide
H4 Eosinophils
Neutrophils
CD4 T cells
Modulate the
production of
blood cells &
cytokines
Hist. Thioperamide

Bioassay of histamine can be done by recording
1. Contractions of isolated guinea pig ileum.
2. BP fall in anaesthetised cat or dog .

Bioassay using guinea pig ileum
Bioassay of histamine on isolated guinea pig ileum can be
determined by
• Matching bioassay
• Interpolation bioassay
• Bracketing assay
• Multiple point assays

Ileum
• 3/5 of intestine
• Empties in the large intestine via ileocecal valve
• Bile salts, vitamin B12, water and electrolytes absorption
• Doesn’t have myogenic contraction
• More sensitive to histamine action

Histamine receptors in ileum
• H1 receptors
• Receptor type: G-protein-coupled receptor
• Agonist: Histamine
• Mode of Action: G-protein → ++phspholipase C → splitting
of PIP2 into 1) DAG that increases the opening of calcium
channels 2)IP3 which increases calcium mobilization from
sarcoplasmic stores;
• DAG & IP3 lead to increase in the intracellular concentration
of calcium and smooth muscles contraction.

Requirements
• Instruments:
– thermostatically controlled organ-bath,
– Chymograph (kymograph)
– Aerator
• Physiological solution:
– Tyrode’s solution
• Temperature:32°c
• Animal :
– Guinea pig (Cavy)
• Standard histamine solution (10μg/ml)

Preparing standard
• Take 10 mg of histame + 10 ml of water (1000μg/ml)
• Take 0.1ml and dilute with 10 ml of water(10μg/ml)

3 Point Bioassay
• 2 Standard
• 1 Test
Potency = n1/t1 antilog {[T2-S2) + (T1-S1)/(T2-T1)+(S2-S1)} × log
n2/n1}
n1 = Lower standard dose
n2 = Higher standard dose
t 1 = Lower dose of test
S1 = Response of n1
S2 = Response of n2
T 1 = Response of test (t)
T2 = Response of t2

Bioassay Using Anaesthetised Cat or Dog
• Cat or dog is anaesthetised with chloralose or barbiturate and
prepared for recording of Blood Pressure.
• Sensitivity is determined by injecting standard solution of
0.05,0.1,0.15μg of histamine base per kg bodyweight is given
for 5 min interval.
• A fixed dose of standard producing a fall in BP about 20mm
Hg is injected with changing doses of test at regular intervals
and matching assay is done.

Mechanism of BP Fall
• Histamine binds wth histamine H1 receptor of endothelium
causes release of EDRF (endothelium derived relaxing factor).
• This EDRF diffuses out and reaches the smooth muscle of the
arteriole and causes generation of cyclic-GMP → causing
reduction of Ca++ in smooth muscle relaxation of the smooth
muscle→ arteriolar dilatation.
• Another possibility is that combination of H1 with the
Histamine causes release of PGI2 (prostaglandin I2) which
causes vasodilatation.

Introduction
• Tubocurarine (also known as d-tubocurarine or DTC) is a toxic
alkaloid historically known for its use as an arrow poison.
• Tubocurarine is a naturally occurring mono-
quaternary alkaloid obtained from the bark of the South American
plant Chondrodendron tomentosum.
• In the mid-1900s, it was used in conjunction with an anesthetic to
provide skeletal muscle relaxation during surgery .
• Tubocurarine competes with acetylcholine for the nicotinic
receptors at the neuromuscular junction of skeletal muscles, thereby
inhibiting the action of acetylcholine and blocking the neural
transmission without depolarizing the postsynaptic membrane. This
may lead to skeletal muscle relaxation and paralysis.

Bioassay of d-Tubocurarine
• Can be done by two methods
– Rabbit Head-drop Method
– Frog’s Rectus Abdominis muscle Preparation

Rabbit Head-drop Method
Principle
• d-Tubocurarine hydrochloride is injected into the marginal vain
of a rabbit’s ear till the rabbits neck muscles are relaxed such
that the animal cannot hold its head up.
• The total amount of the test sample required to produce the
endpoint is compared to with the total amount of the standard
sample required to produce similar endpoint.

Selection of Rabbit-
• Rabbits weighting 2 kg are used.
• Animals should free from diseases, obtained from a healthy
colony and should be accustomed to produced similar
endpoint.

Experiment Procedure
• Each rabbit is placed in a holder with its head protruding
outside.
• The head should be freely movable.
• Minimum 8 rabbits are used .
Rabbit head drop method for the bioassay of d-tubocurarine. (i) : i.v. inj. of d-tubocurarine.
(ii) : Head drop after injection.

• They are divided into two groups each containing 4 rabbits.
• First group receive a standard sample and the second group
will receive the sample under test.
• D-Tubocurarine solution is injected in a constant speed by
infusion apparatus through the marginal vein.
• Injection should be given at a rate of 0.4 ml/min and should
take about 10 min. Dose should be 0.012% w/v in saline.
• Infusion is continued till the rabbit will not be in a position to
hold its head erect or there will be no response by focusing
light on the eyes.
• During the experiment there is a possibility of respiratory
embarrassment which is treated by injecting neostigmine,
methyl sulphate (0.05 mg.) and atropine sulphate immediately
through the marginal ear vein.

• Cross-over test is carried out to minimize biological error due
to animal variation.
• Those rabbits which received the standard sample on the first
day will be given test sample on the second day of experiment
and vice versa.
• Mean dose which produces head drop of the test sample is
compared with the mean dose of standard preparation.

Introduction
• Digitalis, drug obtained from the dried leaves of
the common foxglove (Digitalis purpurea) and used
in medicine to strengthen contractions of the heart muscle.
• Belongs to a group of drugs called cardiac glycosides, digitalis
is most commonly used to restore adequate circulation in
patients with congestive heart failure, particularly as caused
by atherosclerosis or hypertension.
• Digitalis can increase blood flow throughout your body and
reduce swelling in your hands and ankles.

Bioassay of Digitalis
Principle:
• Potency of the test sample is compared with that of the
standard preparation by determining the action on the cardiac
muscle.
Standard Preparation and Units:
• The standard preparation is a mixture of dried and powdered
digitalis leaves (1 unit = 76 mg.)
Preparation of Extracts:
• Exact amount of the powder is extracted with dehydrated
alcohol in a continuous extraction apparatus for six hours. The
final extract should contain 10 ml. (5 ml. alcohol + 5 ml.
water) per 10 g. of digitalis powder. It should be stored in
between 5 °C and -5 °C.

Pigeon Method
• Minimum 6 pigeons are used for testing each sample.
• The weight of the heaviest pigeon should not exceed twice the
weight of the lightest pigeon.
• Food is withheld 16-28 hours before the experiment.
• Pigeons are divided on the basis of their sex, weight and breed,
into two groups.
• They are anaesthetized with anesthetic ether.
• One side of the wing is dissected and the alar vein is
cannulated by means of a venous cannula.
• Dilutions are made with normal saline. The test sample and
standard sample is infused through cannula.

• In pigeons, stoppage of heart is associated with a characteristic
vomiting response called ‘emesis’.
• The milk from the crop sac of pigeons is being ejected out.
This may be taken as the end point response of digitalis.
• The lethal dose per kg. of body weight is determined for each
pigeon.
• The potency of the test sample is determined by dividing the
mean lethal dose of standard by the mean lethal dose of the
test sample.

Guinea Pig Method (End Point Method)
• Standard and test samples are diluted with normal saline in
such a way that 1gm of digitalis powder is diluted to 80 ml.
• A guinea pig is anaesthetized with a suitable anesthetic. It is
dissected on the operation table. The jugular vein is dissected
out by removing adhering tissues and cannulated by means of
venous cannula.
• A pin is inserted in the heart, such that it get inserted in the
apex of the heart. In this way, we can observe the heart beats
by up and down movement of the pin.
• The injection is continued through various cannula until the
heart is arrested in systrole.

• The amount of extract required to produce this effect is taken
as the lethal dose of the extract.
• Another sets of 19 animals of same species are used for this
experiment and the average lethal dose is determined.
• It is not necessary to determine the lethal dose of the standard
during each time of the experiment. But it should be
occasionally checked.
• The lethal dose of the test sample is determined in a similar
way using minimum 6 guinea pig of the same strain.
• The potency of the test sample is calculated in relation to that
of the standard preparation by dividing the average lethal
dose of the sample to the test and expressed as units-per gram.

Introduction
• Acetylcholine (ACh) is an organic chemical that functions in the
brain and body of many types of animals, including humans, as
a neurotransmitter—a chemical released by nerve cells to send
signals to other cells.
• Acetylcholine is the neurotransmitter used at the neuromuscular
junction—in other words, it is the chemical that motor neurons of
the nervous system release in order to activate muscles.
• Acetylcholine is also used as a neurotransmitter in the autonomic
nervous system, both as an internal transmitter for the sympathetic
nervous system and as the final product released by
the parasympathetic nervous system
• In the brain, acetylcholine functions as a neurotransmitter and as
a neuromodulator.
• They play an important role in arousal, attention, memory and
motivation.

Principle
• Potency of the test sample is compared with that of the
standard preparation.
• There are several biological methods for its assay

1. Rectus Abdominis Muscle of Frog:
• Dissect the rectus muscle and arrange the assembly as per
assay of d-tubocurarine. Plot log dose-response curve and find
out the potency of the sample of acetylcholine.

2. Cat’s Blood Pressure:
• A cat is anaesthetized with suitable anaesthetic.
• The carotid artery is cannulated for recording BP.
• Femoral vein is cannulated for injecting acetylcholine.
• Trachea is cannulated for giving artificial respiration.
• Acetylcholine produces a fall in BP by dilating peripheral
blood vessels. This principle is utilized for its bioassay.
• The extent to which BP falls due to the test sample is
compared with the fall by the standard preparation.

3. Guinea-pig Ileum:
• Guinea–pig is killed by a blow on the head and bled to death.
• The abdominal wall is dissected out so as to isolate the ileum,
the faecal matter, mesentery and bood vessels are removed
from the piece of ileum.
• It is ligated on both sides and suspended in mammalian organ
bath containing Tyrode solution maintained at 37°C and
oxygenated continuously.
• Acetylcholine contracts the ileum. This principle is utilized
for its bioassay.
• The extent of contraction produced by the test sample is
compared with the standard preparation of acetylcholine.

4. Anaesthetised Rats:
• Compare the extent of fall in BP of the test sample with
that produced by the standard preparation.

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