DATE : / /

EXPERIMENT NO. 1

OBJECT: ​To study dose calculation in pharmacological experiments.

REFERENCES:

1. Medhi B., Prakash A., Practical manual of Experimental and Clinical Pharmacology,
First edition, Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, 2010, 23.
2. Badyal D., Practical Manual of Pharmacology, First edition (Reprint), Jaypee
Brothers Medical Publishers (P) Ltd., New Delhi, 2010, 9.
3. Kulkarni S. K., “Handbook of Experimental Pharmacology”, 4​th Edition (Reprint),
New Delhi: Vallabh Prakashan; 2016, 20.
4. https://www.researchgate.net/publication/287995503_Guidelines_on_dosage_calculat
ion_and_stock_solution_preparation_in_experimental_animals'_studies

REQUIREMENTS:

Animals: Frog/Rat/Mice

Chemicals: Acetylcholine or any other drug

THEORY:

Dose :

It is an appropriate amount of the drug required to produce a certain degree of

response in any patient given at a time and which can be repeated at an appropriate interval to
produce a desired therapeutic effect on its quantity of drug which is sufficient to diagnose,
prevent or cure a disease.

Posology :

It is the study of drug dosage, dosage quantity and prescription.

Types of doses :

1. Effective dose : It is a dose or concentration of a drug that produces a biological

response.

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 ED​50 is dose effective in producing certain expected response in 50% of the animal
group.
2. Lethal dose (LD) : It is an indication of lethal toxicity of a given drug.
LD​50​ is the dose lethal to 50% of the animal group.
3. Maintenance dose : It is the maintenance rate [mg/h] of drug administration equal to
the rate of elimination at steady state.
4. Loading dose : It is an initial higher dose of a drug that may be given at the beginning
of a course of treatment before dropping down to a lower maintenance dose.
5. Defined daily dose : ​It is a statistical measure of ​drug​ consumption, defined by
the ​World Health Organization​ (WHO). It is used to standardize the comparison of
drug usage between different drugs or between different health care environments.

The doses of drugs are expressed as mg/kg in case of animals. The most common
route of drug administration is intraperitoneal route.

OECD Guidelines :

OECD’s full form is Organization for Economic Co-operation and Development. It is

established on 30 September 1961. It is an inter-governmental economic organization with 35
member countries in North America, Europe and the Pacific, as well as the European
Commission, meet to coordinate and harmonise policies, discuss issues of mutual concern,
and work together to respond to international problems. Most of the OECD’s work carried
out by more than 200 specialized Committees and subsidiary groups. The OECD Secretariat,
located in Paris, France, its work is conducted by various committees and subsidiary groups.
The work of the OECD related to chemical safety is carried out in the Environment, Health
and Safety Programme. As part of its work on chemical testing, the OECD has issued the
OECD Test Guidelines, a collection of methods used to assess the hazards of chemicals and
of the chemical preparations such as pesticides. These methods cover tests for physical and
chemical properties, effects on human health and wild life, and accumulation and degradation
in the environment. The OECD test guidelines are recognized world-wide as the standard
reference tool for chemical testing. The mission of OECD is to promote the policies that will
improve the economic and social well being of around the world. It works with government
to understand what drives economic, social and environmental change. It sets international
standards on a wide range of thing, from agriculture and tax to the safety of chemicals. India
is one of many non-member economies with which OECD has working in addition to its

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member countries. The OECD has co-operating with India since 1995. The OECD council at
ministerial level adopted a resolution on 16 may 2007 to strengthen the co-operation with
India, as with Brazil, China, Indonesia, and South Africa through a program of enhanced
engagement.

Dose Calculation :

General methods for drug calculation are following as:

1. Proportional calculation: Involves mixing of two similar preparation of different

strength to produce a preparation of intermediate strength.
Proportions are equation containing ratios of equal value.
H:V=D:X
Where,
H = Drug on hand,
V = Vehicle drug from tablet/liquid/capsule,
D = Desired dose as ordered,
X = Known amount to give,
2. Calculation by conversion factor: Sometimes, doses of a drug are not known for the
animals, then it may be converted to the respective animal drug dose by the help of
conversion factor developed according to the body surface area.
There are some conversion factors of animals-
From human drug dose to animal drug dose -

ANIMALS BODY WEIGHT (in gram) CONVERSION FACTORS

Mice 20 0.0026
Rat 200 0.018
Guinea Pig 400 0.031
Rabbit 1500 0.07

From animal to animal drug dose-

MALS Y WEIGHT (in gram) VERSION FACTORS

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 a Pig

3. Calculation by basic formula: This formula is applicable in most of the cases and
used very commonly,
C​1​ × V​1​ = C​2​ × V​2

Where,

C​1 is
​ the concentration of solution 1,

C​1 is
​ the concentration of solution 2,

V​1 is
​ the volume of solution 1,

V​2​ is the volume of solution 2.

Note: The unit of the solution concentration must be same such as if it is in mg/ml then both
solution concentrations should be in mg/ml

4. Dosage calculation and preparation of stock solution of crude plant extract for
experimental animals : Stock solutions and doses of a plant extract (With selected
doses, 200 mg/kg and 400 mg/kg) for a rat weighing 120 g can be calculated by
following formula :

Body W eight × Dose (in mg)

Required dose for animal (in mg) = 1000 gm

5. Dosage calculation and preparation of stock solution of a reference drug for

experimental animals :
Body W eight of animal (in gm)×Standard dose (in mg)
Required dose for animal = 1000 gm

The calculated dose is then dissolved in 10ml/kg body weight according to Organization for
Economic Co-operation and Development (OECD) guidelines
[Standard dose = dose for an animal (mg/kg)]
6. Calculation according to body surface area :
Body Surface Area (BSA) of animal = Body Weight (kg) × Height (cm) × 0.007184
Required dose for animal = Drug dose × BSA

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METHOD/INSTRUCTION:

1. Calculation by conversion factor:-

(A) Human drug dose to animal drug dose:-
1. Convert given human drug dose into absolute dose (absolute dose = total dose
required for human).
2. Multiply absolute dose by conversion factor.
3. Convert into per kg dose according to animal.
4. Now, convert the dose in gram according to animal weight.
5. If this dose is given in X ml, then weigh the required dose of drug and dissolve
in required quantity of appropriate solvent and make up the volume of the
drug solution.

For example,
Suppose, human dose of drug A =100 mg/kg/day [ for the calculation, dose will be
calculated either for 20 gm mice or 200 gm rat (as per animal selection)]
Step I: Dose given = 100 mg/kg
Step II: Convert into absolute dose*
Human dose given =100 mg/kg = 70 × 100 = 7000 mg/70 kg
(Absolute dose is converted for 70 kg of adult).
*If, a adult dose is taken, then avoid step II. For example, drug dose is 50mg/day, then
it is assumed that, it is for 50 mg/70 kg of adult.
Step III: Multiply by conversion factor
For
Mice (20gm) = 7000 × 0.0026 = 18.2 mg/20 gm of mice
Rat (200gm) = 7000 × 0.018 = 126 mg/200gm of rat
Step IV: Then, convert into per kg dose according to animal
Mice: 18.2 mg/20 gm = 910 mg/kg/day
Rat: 126 mg/200 gm = 630 mg/kg/day
(Reference: Medhi B., Prakash A.)
(B) Animal to animal drug dose:-
1. Convert given animal drug dose into absolute dose (absolute dose = total dose
required for animal).

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 2. Multiply absolute dose by conversion factor.
3. Convert into per kg dose according to animal.

For example,
Mice dose of drug A is 5 mg/kg, then, calculate the drug dose for rat
Step I: Dose given = 5 mg/kg
Step II: Convert into absolute dose
For mice (20gm) = 5 mg/kg = 0.1 mg/20gm
Step III: Multiply by conversion factor
For
Rat (200 gm) = 0.1 × 7.0 = 0.7 mg/200 gm
Step IV: Then, convert into per kg dose according to animal
From Step III
Rat: 0.7mg/200gm = 3.5mg/kg/day
(Reference: Medhi B., Prakash A.)
2. Dosage calculation and preparation of stock solution of crude plant extract for
experimental animals :
1. Calculate the required dosage for animal.
2. Dissolve the required quantity in normal saline.
3. Make up the volume and administer via oral route.

For example,
With reference to table 1 above, stock solutions and doses of a plant extract (With
selected doses, 200 mg/kg and 400 mg/kg) for a rat weighing 120 g be calculated
as follows;
Step1: Calculation of required dose of drug for animal
Body weight of animal = 120 gm
Body W eight × Dose (in mg)
Required dose of animal in mg = 1000 gm
120 gm × 200 mg
Required dose of animal in mg = 1000 gm =​ 24 mg
Step 2​: ​Dissolution of dose in a suitable vehicle for oral administration
From the OECD’s ​guidelines,

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120 g rat requires 24 mg of the crude plant extract which should be constituted in not
more than 1.2 ml of normal saline (see table 1 above) according to the OECD
guideline.
In a nut shell, 120 gm= 24 mg = 1.2 ml of normal saline.
Bulk volume of the stock solution required for large number of animals can be calculated
by​ ​multiplying both side by a constant value as follow;
24 mg = 1.2 ml
40 × 24 mg = 40 × 1.2 ml
960 mg
96​0 mg of crude plant extract will be dissolved in 48 ml of normal saline = 48 ml

= 20 mg/ml
3. Dosage calculation and preparation of stock solution of a reference drug for
experimental animals :
1. Calculate the require dose of the drug for animal
2. Dissolve the required dose in appropriate solvent
3. Make up the volume of the drug according to 10 ml/kg dose of the drug.

For example
The required dose of silymarin (70 mg/tablet) for a rat weighing 130 gm at a standard
dose 25 mg/kg can be calculated as follows;
Step I: Dosage calculation
Body W eight of animal (in gm)×Standard dose (in mg)
Required dose for animal = 1000 gm
130 gm × 25 mg
= 1000 gm ​= 3.25 mg
Step 2​: ​Dissolution of sylimarin in a suitable volume of vehicle for oral administration
From the above calculation, 130 g rat requires 3.25 mg of sylimarin and this dosage (3.25
mg) should be constituted in not more than 1.3 ml of normal saline according to
the​ ​OECD guideline.
In a nut shell, ​130 g ​≡ 3.25 mg ≡ 1.3 ml ​of normal saline. If ​3.25 mg would be constituted
in ​1.3 ml​ of normal saline,
Then, one tablet of sylimarin (​70 mg​) would be constituted in
1.3 ml ×70 mg 70 mg
= 3.25 mg = ​28 ml of normal saline. That is 28 ml = ​2.5 mg/ml
From this stock solution, dosages can be administered to animals of varying body weights
based on the OECD’s 10 ml/kg standard volume rationale.

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 4. Calculation by basic formula:-
Suppose, volume of solution B and concentrations of solution A & B are known, but volume
of solution A is unknown, then-
1. Calculate required volume of solution A.
2. Take calculated volume of solution A in a test tube.
3. Make up its volume equal to the volume of solution B by appropriate solvent.

For example
Suppose you are provided with the 50 ml solution of a 5 mg/ml concentration and the
instructor wants you to make it, 10 ml of 3.5 mg/ml.
So, you have
C​1​ = 5 mg/ml V​1​ = ? = A
C​2​ = 3.5 mg/ml V​2​ = 10 ml
Apply the formula
C​1​ × V​1​ = C​2​ × V​2
So, 5 mg/ml × A = 3.5 mg/ml × 10 ml
3.5 mg/ml × 10 ml
Hence, A = 5 mg/ml

= 3.5 ×10 ml
5 =​ 7 ml
Therefore, take 7 ml of the solution 1 in a test tube and make it volume to the 10 ml by
the solvent. The resultant solution will be 10 ml of 3.5 mg/ml.

PROCEDURE:

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RESULT:

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INFERENCE:

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FREQUENTLY ASKED QUESTIONS:

1. What do you understand by proportional calculation ?

2. What is the basic formula of dose calculation ?
3. What is the conversion factor of mice to convert human dose to animal dose ?
4. What is lethal dose ?
5. What is maintenance dose ?
6. What is the significance of dose calculation ?
7. What are ED​50​ and LD​50​ ?

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