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Steps of drug development (cont'd...)

2. Preclinical development

In this step the ability of the new molecule to satisfy the requirement as a drug is checked. It includes four types of evaluation:

i. Pharmacological testing

This is done to check whether the molecule has serious unwanted effects or potential hazards (eg. ability to change the vital signs like blood pressure, body temperature, respiratory rate, heart rate or severe adverse drug reaction like bronchoconstriction, ataxia etc.). The study is done in the laboratory on animals (usually on rat, mice, rabbit, and guineapig) [Rang MP et. al., 2007].

ii. Preliminary toxicological assessment

The objective of this step is to determine the safety margin of the incoming molecule which is done by calculating median lethal dose, LD50 (acute toxicity study) and the maximum non toxic therapeutic dose (median effective therapeutic dose, ED50). The safety margin of the drug is evaluated by measuring therapeutic index (TI), calculating LD50 and ED50. Here we should define the median lethal dose (LD50) and median effective therapeutic dose (ED50) and TI [Rang MP et. al., 2007].

Median lethal dose (LD50)

This is the dose of a drug required to produce lethal action on 50% of the population, drug applied on. This is also called the maximum non toxic dose. It is determined to calculate the TI of the drug.

Determination of Acute toxicity

Acute toxicity (LD50)

The globally harmonized system (GHS) defined acute toxicity as follows:
the adverse drug reactions, occurred due to administration of single dose of a substance by oral or dermal route, or due to administration of a second dose of the drug within twenty four hours by oral route and 4 hours by inhalation, is called the acute toxicity. There are five popularly accepted methods to determine acute toxicity: fixed dose method, acute toxicity class method, up and down method, acute dermal toxicity and acute inhalation toxicity [Bogdan T]. Here, one of the methods is explained in detail.

Fixed dose method to determine LD50

Animals are divided into four groups (each containing at least 6 animals), after acclimatization in the laboratory for at least 24 hours with adequate access to water and food. Different doses (5, 50, 300 and 2000 mg/kg body weight) of the test molecule are given to different group of animals. Dose can be fixed on the basis of literature survey, if any data available. After 24 hours, each roup of animals are checked for toxicity. Further group of animals are tested for higher or lower doses of the molecule, depending upon the result and the dose for the toxicity is calculated [Bogdan T].
The primary step to calculate the LD50 is to select the route of drug administration. It is usually selected on the basis of future use of the lead. The dose is dependant on the route and dose increases gradually in the following manner: intravenous (iv), intraperitoneal (ip), subcutaneous (sc) and oral.

We represent the flow diagram to give an illustration, how the experiment is done in practical:

Step 1. Divide the animals into four following groups: Group A (5 mg/kg); Group B (50 mg/kg); Group C (300 mg/kg); Group D (2000 mg/kg). Observation after 24 hours: no death is found case of group A, B and C. All animals died in group D.

Step 2. Animals are further divided into four groups: Group A (500 mg/kg); Group B (1000 mg/kg); Group C (1250 mg/kg); Group D (1500 mg/kg). Observation after 24 hours: no death is found case of group A and B. 2 animals died in group C and 7 animals are dead in group D.

Step 3. Animals are further divided into four groups: Group A (1300 mg/kg); Group B (1350 mg/kg); Group C (1400 mg/kg);Group D (1450 mg/kg). Observation after 24 hours: 3 animals died in group A, 5 animals are dead in group B, 7 are dead in group C and D.

Step 4. Animals are further divided into four groups: Group A (1350 mg/kg);Group B (1365 mg/kg); Group C (1380 mg/kg); Group D (1400 mg/kg). Observation after 24 hours: 5 animals died in group A and B, 6 animals are dead in group C and D.

Step 5. Animals are further divided into four groups: Group A (1365 mg/kg); Group B (1370 mg/kg); Group C (1375 mg/kg); Group D (1380 mg/kg) Observation after 24 hours: 5 animals died in group A, 6 animals are dead in group B, C and D.

Step 6. Animals are further divided into four groups: Group A (1365 mg/kg); Group B (1367 mg/kg); Group C (1368 mg/kg); Group D (1370 mg/kg); Observation after 24 hours: 5 animals died in group A, 6 animals are dead in group B, C and D.

Therefore, the conclusion from the interpretation of the result is that the LD50 for the selected molecule is considered as 1365 mg/kg of the body weight.

The primary step to calculate the $LD_{50}$ is to select the route of drug administration. It is usually selected on the basis of future use of the lead. The dose is dependant on the route and dose increases gradually in the following manner: intravenous (iv), intraperitoneal (ip), subcutaneous (sc) and oral.

Reference

[Bogdan T] Bogdan T, George F, Leon M, Radu I, Chapter 15: Determination of drug toxicity in animals, Experimental model in rodents, 231-260.
Rang MP et. al., 2007] Rang HP, Dale MM, Ritter J M, Moore P K, Pharmacology, 5th Edition, Elsevier, 2-752, 2007.

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