DOSE CALCULATION IN PHARMACOLOGICAL EXPERIMENTS

Experiments on animals are necessary for drug discovery and development as well as to advance pharmaceutical, medical and biomedical research. The best means to extrapolate from animal dose to human dose or human dose to animal dose has been an area of interest in experimental pharmacology for a number of years. Dosage calculation and stock solution preparation based on dosage rationale formula are prerequisites to drug administration in experimental animals also have the utmost importance.

1) Calculate the dose for experimental animals

The human or animal dose that is given to other animals is on the basis of weight or relative surface area. It has been argued that body surface area (BSA) provides a more accurate basis for dose calculation, because total body water, extracellular fluid volume, and metabolic activity are better paralleled by BSA. If the dose of a drug for an animal is unknown then it may be converted from human doses or other animal doses with the help of appropriate conversion factors developed according to the body surface area (as per table 1 given by Paget and Barnes, 1964).

Table 1: Converting factors as per surface area ratios of human and some common laboratory animals (Paget and Barnes, 1964)

Absolute Dose	70 kg Human	12 kg Dog	4 kg Monkey	2 kg Cat	1.5 kg Rabbit	400 g G. pig	200 g Rat	20 g Mouse
70 kg Human	1.0	0.32	0.16	0.076	0.07	0.031	0.018	0.0026
12 kg Dog	3.1	1.0	0.52	0.24	0.22	0.1	0.06	0.008
4 kg Monkey	6.1	1.9	1	0.45	0.42	0.19	0.11	0.016
2 kg Cat	13	4.1	2.2	1.0	0.92	0.41	0.23	0.03
1.5 kg Rabbit	14.2	4.5	2.4	1.08	1.0	0.44	0.25	0.04
400 g G. pig	31.5	10.2	5.2	2.4	2.25	1.0	0.57	0.08
200 g Rat	56.0	17.8	9.2	4.2	3.9	1.74	1.0	0.14
20 g Mouse	387.9	124.2	64.1	29.7	27.8	12.25	7.0	1.0

The dose to be given to a particular species on the basis of surface area can be extrapolated by referring to the above table 1.1. To determine the absolute dose for a species in the column, the absolute dose given to the species in a row is multiplied by the factor given at the intersection of the relevant row or column.

A) From human drug dose to animal dose:

All adult doses for humans are available in the absolute dose form i.e. for 70 kg body weight. If the human dose of a drug is available in mg/kg/day then first convert the dose into an absolute dose. The absolute human dose is converted for 70 kg adult. Conversion of an absolute dose is important because it keeps the dose as per the bodyweight of the animal. Then the 70 kg human dose is converted for 20 g mice or 200 g rat or 400 g guinea pig or 1500 g rabbit or 2 kg cat or 4 kg monkey or 12 kg dog, by multiplying with an appropriate conversion factor. Then convert into per kg dose.

B) From animal drug dose to human or another animal dose:

The animal dose of a drug is mostly available in mg/kg/day. Hence, at first, convert the dose into absolute dose i.e. for 20 g mice or 200 g rat or 400 g guinea pig or 1500 g rabbit or 2 kg cat or 4 kg monkey or 12 kg dog. Then the absolute dose is converted for human or other animal’s dose by multiplying with conversion factor and further convert into per kg dose.

2) Calculate the volume of the vehicle for dissolving the drug for administering to experimental animals.

A solvent or vehicle is a medium in which a drug is dissolved or suspended and administered to the experimental animals. It should be biologically inert, have no toxic effects on the animals, and not influence directly or indirectly to the results obtained for the compound under investigation. Suitable vehicles for animal research include; distilled water or normal saline (0.9% sodium chloride). If the drug is not soluble in the vehicle there may be added polyethylene glycol (50%), Tween 80 (5 to 10%), methylcellulose, or carboxymethylcellulose (0.25%). Water for injection or sterile water may be used for dilution of injectable preparations. In most research involving experimental animals, dosages are usually calculated from the stock solution of the test drugs dissolved in the appropriate volume of solvent (vehicle).

According to the Organization of Economic Corporation and Development’s (OECD’s) guidelines:

Dosage of the drug (mg) should be constituted in an appropriate volume not usually exceeding 10 ml/kg (1 ml/100 g) body weight of experimental animals (mice and rats) for non-aqueous solvent in oral route of administration. However, in the case of aqueous solvents, 20 ml/kg (2 ml/100 g) body weight can be considered (OECD, 2000).

Large dose volumes (40 ml/kg body weight) can cause unnecessary stress to animals and can also overload the stomach capacity and pass immediately into the small bowel or can result in passive reflux in the stomach, aspiration pneumonia, pharyngeal, esophageal, and gastric irritation or injury with stricture formation, esophageal and gastric rupture and stress.

Lower volume (5 ml/kg) can be considered to dissolve highly soluble solute drugs.  Such low volume would ease the administration of drugs in solution.

Highly viscous drug solution should be diluted, whenever possible, for ease of administration. However, the final dilution volume should not exceed 20 ml/kg.

For parenteral administration, an appropriate volume of vehicle ranging between 2 ml/kg to 5 ml/kg in rodents is recommended.

Based on 10 ml/kg volume selection, the required dose volume (x) for a 100 g rat can be calculated as :

X= 100g ×10ml/1000g = 1 ml

Table 2: Calculation of volume based on animal’s body weight

For animals	Animal’s  body weight (g)	Calculated volume (ml) based on 10 ml/kg, oral administration	Calculated volume (ml) based on 2 ml/kg, i.p. administration
For Rat	100 g	1 ml	0.2 ml
	150 g	1.5 ml	0.3 ml
	200 g	2 ml	0.4 ml
For Mice	20 g	0.2 ml	0.04 ml
	25 g	0.25 ml	0.05 ml
	30 g	0.3 ml	0.06 ml

 3) Dosage calculation and dissolution of dose in a suitable vehicle for oral administration.

        Dosage calculation and preparation of a stock solution of a drug for administering the accurate dose to the experimental animals is an important aspect of the pharmacological experiment. Stock solutions of a selected dose (50 mg/kg) for a rat weighing 150 g can be calculated as follows:

Step 1: Dosage calculation

Bodyweight of animal =150 g

Dosage (mg) = 150g × 50mg/ 1000g

Step 2: Dissolution of dose in a suitable vehicle for oral administration

150 g rat requires 7.5 mg of the drug which should be constituted in not more than 1.5 ml of normal saline (see table 1.2 above).

Therefore, 150 g ≈ 7.5 mg of the drug ≈ dissolved in 1.5 ml of normal saline.

For bulk volume of the stock solution required for a large number of animals can be calculated by multiplying both sides by a constant value.

References

Panigrahi G., Patra A., 2020. Experimental Pharmacology- III: bridges the gap between animal models and computer simulation models. 1^st edition, Nirali Prakashan, Pune, India, .

Ghosh, M.N., 2011. Fundamentals of Experimental Pharmacology. 3^rd edition, Hliton & Company, Kolkata.

Goyal, R.K., 2007. Practicals in Pharmacology. 8^th edition, B.S. Shah Prakashan, Ahmedabad.

Kulkarni, S.K., 2007. Practical pharmacology and clinical pharmacy. 1^st edition, Vallabh Publications, Delhi.

Medhi, B., Prakash, A., 2010. Practical Manual of Experimental and Clinical Pharmacology. 1^st edition, Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.