USE OF EXPERIMENTAL PHARMACOLOGY IN DRUG DISCOVERY AND DEVELOPMENT

The discovery and development of new drugs to provide medicines for treating diseases is the main role of the pharmaceutical industry. It is a challenging and expensive activity of pharmaceutical industry. Biological organisms and especially human beings are extraordinarily complex, and our understanding of how they function at the molecular level remains rudimentary, although considerable advances in knowledge have been made in recent decades. Whilst an advanced industrial society was able to plan and deliver a man to the moon following a 10-year program, almost 50 years on we are still only able to treat about 60% of cancer patients effectively, and do not understand how to correct most mental diseases. Development of new medicines is complex, time consuming and very expensive. The average cost of developing a new drug is estimated to be about US $ 1-1.2 billion, including expenditures on failed projects. This amount is about four times the price of an Airbus A380 at US $ 270 million, or five times that of a Boeing B - 787 Dreamliner at US $ 200 million.

Total drug development time grew from an average of 8.1 years in the 1960s to 11.6 years in the 1970s, to 14.2 in the 1980s, to 15.3 years for drugs approved from 1990 through 1995. Pharmaceutical companies and regulatory authorities are working together to reduce this time span. With the advent of technologies in biological screening procedures of new chemical entities the time involved in drug discovery has gone down in recent years but the cost of drug discovery has touched a new high. Success rate in getting from an initial compound to an approved and commercially available product is very low. Typically, tens of thousands of compounds are screened and tested, and only a handful makes it onto the market as drug products. The statistics are such that, out of the 10,000 compounds that show initial promise, only 0.3% will reach the testing stage for sub-acute study, five will go into human clinical trials, and only one will become an approved drug.

                    Table 1: Key stages of drug discovery and developments

Drug Discovery	Drug Developments
1.  Program selection (choosing a disease to                 work on)         2. Identification and validation a drug target         3.     Assay development         4.     Identification of a “lead compound”         5.     Lead optimization         6.     Identification of a drug candidate	1.     Preclinical study         2.     Clinical trials         3.     Release of the drug         4.     Follow-up monitoring

The process involves finding out the target that causes the disease. Next, chemical or biological compounds are screened and tested against these targets or assays, which are representative of these targets, to find leading drug candidates for further development. Many new scientific approaches are now used to determine targets (most targets are receptors or enzymes) and obtain the lead compounds; including the use of genomic technology, synthetic chemistry, recombinant DNA (rDNA) technology, laboratory automation and bioinformatics. Tests are performed on the lead compounds in test tubes (in vitro) and on animals (in vivo) to check how they affect the biological systems. The tests, often called preclinical research activities, include toxicology, pharmacodynamics and pharmacokinetics, as well as optimization of drug delivery systems. The leading compounds are modified and synthesized to improve their interactions with the targets, or to reduce the toxicity or improve pharmacokinetics performance. At the end of this process, an optimized compound is found and this becomes a potential drug ready for clinical trial in human. The development work has to follow Good Laboratory Practice (GLP) to ensure that proper quality system and ethical considerations are established. Only compounds that satisfy certain performance and safety criteria will proceed to the next stage of clinical trial. Then release of the drug occurs and follow up monitoring is required for confirming the effectiveness of the drug.           

Figure 1: Drug discovery and development processes.

After completion of preclinical studies successfully, the investigators files an ‘Investigational New Drug’ application (IND) to the government bodies such as Central Drugs Standard Control Organization (CDSCO), for allowance of initial testing in human beings. After the successful clinical trials and laboratory work, the investigators may file a New Drug Application (NDA). Permission to market a drug product will be given by the drug control authority after confirming the drug’s safety and effectiveness.

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.

Maintenance of Laboratory Animals as per CPCSEA Guidelines

All establishments engaged in research and education involving animals in India are required to comply with the various guidelines, norms and stipulations set out by CPCSEA. The aim of these guidelines is to ensure humane and ethical treatment of animals, while facilitating legitimate scientific research involving experiments on animals and to make judicious use of animals for experimental purposes.

Committee for Purpose of Control andSupervision of Experiments on Animals (CPCSEA):

The Prevention of Cruelty to Animals Act 1960 as amended in 1982, is to prevent the unnecessary pain or suffering on animals. The Central Government has constituted a Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) which ensures that animals are not subjected to unnecessary pain or suffering before, during or after the performance of experiments on them. For this purpose, the Government has made "Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998" as amended during 2001 and 2006, to regulate the experimentation on animals.

Mandate of CPCSEA:

1. Registration of establishments conducting experiments on animals.
2. Registration of establishments engaged in breeding of laboratory animals.
3. Constitution of IAECs in the establishments registered with CPCSEA.
4. Approval of animal house facilities for small and large animals.
5. Permission for conducting experiments on large animals.
6. Recommendation for import of animals for experimentations and breeding.

CPCSEA has issued the following guidelines:

1. Guidelines for the Reuse and Rehabilitation of dogs.
2. Guidelines for Constitution/Reconstitutions of IAECs.
3. Care and Management of Equines used in the product of biologicals.
4. Standard operating procedures for IAEC.

Maintenance of laboratory animals as per CPCSEA guidelines:

CPCSEA has set guidelines for laboratory animal facility. The goal of these guidelines is to promote the humane care of animals used in biomedical and behavioral research and testing with the basic objective of providing specifications that will enhance animal well being, quality in the pursuit of advancement of biological knowledge that is relevant to humans and animals. The CPCSEA provides the guidelines to be followed by all research institutions in the country.

1. Adequate veterinary care must be provided by a veterinarian or a person who has training or experience in laboratory animal sciences and medicine. Institutions should employ people trained in laboratory animal science or provide for both formal and on-the-job training for the care of animals. It is essential that the animal care staff maintain a high standard of personal cleanliness.
2. All animals must be acquired lawfully as per the CPCSEA guidelines. The transport of animals from one place to another is very important and must be undertaken with care. The main considerations for transport of animals are the mode of transport, the containers, the animal density in cages, food and water during transit, protection from transit infections, injuries and stress. Quarantine is the separation of newly received animals from those already in the facility. An effective quarantine minimizes the chance for introduction of pathogens into an established colony.
3. All animals should be observed for signs of illness, injury, or abnormal behavior. Health and nutritional status of the animals should be properly maintained.
4. Animals should be fed palatable, non-contaminated and nutritionally adequate food daily unless the experimental protocol requires otherwise. Ordinarily animals should have continuous access to fresh, potable, uncontaminated drinking water, according to their particular requirements. 
5. Laboratory animals are very sensitive to their living conditions. It is important that they shall be housed in an isolated building located as far away from human habitations as possible and not exposed to dust, smoke, noise, wild rodents, insects and birds. The location, building, cages, material and environment of animal rooms are the major factors, which affect the quality of animals.
6. Housing, care, breeding and maintenance of experimental animals is necessary to keep them in physical comfort, good health and behave normally. Bedding should be removed and replaced with fresh materials as often as necessary to keep the animals clean and dry.
7. Sanitation is essential in an animal facility. Animal rooms, corridors, storage spaces, and other areas should be cleaned with appropriate detergents and disinfectants as often as necessary.
8. The Institute shall maintain SOPs describing procedures/methods adapted with regard to animal husbandry, maintenance, breeding, animal house microbial analysis and experimentation records. The animal house should maintain the different records of animals as instructed by CPCSEA.
9. Acceptable experimental techniques and procedures for anaesthesia and euthanasia are applied during the study.
10. Transgenic animals are used to study the biological functions of specific genes, to develop animal models for diseases of humans or animals, to produce therapeutic products, vaccines and for biological screening. Housing, feeding, ventilation, lighting, sanitation and routine management practices for such animals are similar to those for the other animals of the species as given in guidelines. However, special care has to be taken with transgenic/gene knockout animals where the animals can become susceptible to diseases.
11. All scientists working with laboratory animals must have a deep ethical consideration for the animals they are dealing with. From the ethical point of view it is important that such considerations are taken care at the individual level, at institutional level and finally at the national level.

Alternatives to animal screening procedures: 3 Rs concept

British researchers William Russell and Rex Burch in 1959 formulated this concept of the 3 Rs in their book ‘The Principles of Humane Experimental Technique’, which argues that humane science is the best science. Alternative methods fall into three broad categories. These are called the 3 Rs: Replacement, Reduction, and Refinement. Since the introduction of these principles, they have become widely accepted internationally as the basic principles guiding animal use in research, teaching and testing. Ron Banks added the 4^th R i.e. Rehabilitation and Reuse of animals.

Institutional Animal Ethics Committee (IAEC):

As defined in “Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998”: Institutional Animals Ethics Committee means a body comprising of a group of persons recognized and registered by the CPCSEA performed in an establishment which is constituted and operated in accordance with procedures specified for the purpose by the committee. The primary responsibility of a person who has been nominated to represent CPCSEA on IAEC is the wellbeing and welfare of the animals housed or kept for experiments/breeding.

Composition and Constitution of IAEC

IAEC includes 8 members as described below:

A. Institutional Members (5 members from the institute)

1. A Scientist In Charge of Animals Facility
2. A Veterinarian Involved In the Care of Animals
3. A Biological Scientist
4. Two Scientists from Different Biological Disciplines

B. CPCSEA Nominated members (3+1 members from outside institute)

1. A CPCSEA Main Nominee
2. A CPCSEA Link Nominee
3. A Scientist From Outside the Institute
4. A Socially Aware Nominee

The Chairperson and member secretary of the committee are nominated by the institution and should take care of the functioning of IAEC in accordance with procedures specified for the purpose by the committee.

Objectives of IAEC:

1. Experiments to be avoided wherever it is possible to do so.
2. Experiments on larger animals are avoided when same results can be achieved in smaller animals.
3. Rationalize usage of animals- 3 Rs.
4. Animals to be properly looked after before and after experiments.

The main functions of IAEC are:

1. IAEC will review of research proposals and prevent infliction of unnecessary pain and sufferings before, during and after experiments on animals, to follow the CPCSEA guidelines.
2. IAEC reviews and approves all research proposals involving small laboratory animal experiments with a view to assure quality maintenance and welfare of animals used in pre-clinical research.
3. For experiments on large animals, the IAEC will forward its recommendation to the CPCSEA, for its approval process.
4. IAEC ensures that experiments shall be performed in every case by or under the supervision of a qualified person and under the responsibility of the Principle Investigator.
5. IAEC reviews the proposals before start of the study as well as monitor the research throughout the study and after completion of the study through annual reports, final report.
6. IAEC team will visit the laboratory in the animal house/respective department where the experiments are conducted. The committee also ensures compliance with all regulatory requirements, applicable guidelines and laws.
7. Monitor and inspect the housing of animals and ensure that it is as per specified standards.

Institutional Biosafety Committee (IBSC):

The Institutional Biosafety Committee shall be the point for interaction within institution for implementation of the guidelines. Any research project which is likely to have biohazard potential (as envisaged by the guidelines) during the execution stage or which involve the production of either micro-organisms or biologically active molecules that might cause biohazard should be notified to IBSC. IBSC will allow genetic engineering activity on classified organisms only at places where such work should be performed as per guidelines. Provision of suitable safe storage facility of donor, vectors, recipients and other materials involved in experimental work should be made and may be subjected to inspection on accountability.

The Institutional Biosafety Committee is engaged in hazardous chemical use, genetic engineering research and production activities. This committee shall also examine the proposal on animal experiments involving hazardous agents in addition to its existing functions. IBSC whose members are knowledgeable about hazardous agents, are in place in most of the higher level education, research institutes and in many pharmaceutical industries for safety issues. Institutional Biosafety Committee (IBSC) is to be constituted in all centres engaged in genetic engineering research and production activities. The committee will constitute the following.

1. Head of the institute or his nominee

2. Three or more scientist engaged in DNA work or molecular biology with an outside expert in the relevant discipline.

3. A member with medical qualification- Biosafety officer (in case of work with pathogenic agents/ large scale used).

4. One member nominated by Department of Biotechnology (DBT), Government of India.

The main functions of IBSC

The functions and activities of IBSE include the following:

1. Registration of Biosafety Committee membership composition with Review Committee on Genetic Manipulation (RCGM) and submission of report. ISBC will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any. A computerized Central Registry for collation of periodic reports on approved projects will be setup with RCGM to monitor compliance on safeguards as stipulated in the guidelines.

2. Review and clearance of project proposals falling under restricted category that meets the requirements under the guidelines. IBSC would make efforts to issue clearance certificates quickly on receiving the research proposals from investigators.

3. Tailoring biosafety program to the level of risk assessment.

4. Training of personnel on biosafety.

5. Instituting health monitoring program for laboratory personnel, complete medical check-up of personnel working in projects involving work with potentially dangerous microorganism should be done prior to starting such projects. Follow up medical check-up including pathological test should be done periodically, annually for scientific workers involved in such projects. Their medical record should be accessible to the RCGM. It will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any.

6. Adopting emergency plans.

Note: Above mentioned mandate and guidelines are documented in the CPCSEA websites i.e. http://cpcsea.nic.in.

Other guidelines for maintenance of laboratory animals

The Organization for Economic Cooperation and Development (OECD) and the International Conference on Harmonization (ICH) are the different organizations that have issued certain guidelines regarding toxicity and safety studies in animals.

The OECD has issued a guideline for toxicity testing of various chemicals. OECD is an intergovernmental organization in which representatives of 29 industrialized countries in North America, Europe and the Pacific. The OECD Secretariat located in Paris, France and 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 program. The OECD has issued the OECD test guidelines for chemical testing i.e. collection of methods used to assess the hazards of chemicals and of chemical preparations such as pesticides. These methods cover tests for physical and chemical properties, effects on human health and wild life, accumulation and degradation in the environment. The OECD provides the guidelines on selection of animal species, housing and feeding conditions and preparation of animals for toxicity study. OECD test Guideline 420, 423 and 425 are used for acute toxicity study and 452 for chronic toxicity study. The OECD test guidelines are recognized worldwide as the standard reference tool for chemical testing.

The ICH is a sole project that unites the regulatory authorities of Europe, Japan and the United States.  The experts from pharmaceutical industries from the above three regions should discuss scientific and technical aspects of product registration. The object of such harmonization is a more economical use of human, animal and material resources and the elimination of unnecessary delay in the global development and availability of new medicines by maintaining the quality, safety and efficacy and regulatory obligations to protect public health. The ICH guidelines are divided into four major categories i.e. a) Quality: relating to chemical and pharmaceutical quality assurance, b) Safety- relating to in vitro and in vivo preclinical studies, c) Efficacy- relating to clinical studies, and d) Multidisciplinary- topics that do not fit uniquely into one of the above categories.

Note: Certain guidelines regarding toxicity and safety studies in animals for new chemical or pharmaceutical products are placed in both OECD and ICH. These guidelines are documented in the websites of WWW.oecd.org and WWW.ich.org.

References:

Committee for Purpose of Control and Supervision of Experiments on Animals, 2017. General Guidelines for Nominees of CPCSEA. Government of India, Ministry of  Environment, Forest and Climate Change, Animal welfare Division., CPCSEA, New Delhi, India.

Committee for the Purpose of Control and Supervision on Experiments on Animals, 2003. CPCSEA Guidelines for Laboratory Animal Facility. Indian Journal of Pharmacology; 35, 257-274.

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.