DEVELOPMENT & REGULATION OF DRUGS

Table: The development and testing process required to bring about a drug to market in the USA.

In-vitro studies

Animal testing

Clinical testing

Marketing

Biological products

Phase I (Is it safe? pharmacokinetics)

Lead compound

Phase II (Does it work in patients?) Efficacy selectivity mechanism Phase III (Does it work, double blind?) Phase IV (Postmarketing surveillance)

Generics become available

Chemical synthesis Drug metabolism, safety assessment

4 IND

20

NDA (Patent expires 20 years after filing of application)

Years (average)

* Some of the requirements may be different for drugs used in life threatening diseases.

Clinical Trials
ICH Guideline for Good Clinical Practice defines Clinical Trial/Study as any investigation in human subjects intended to discover or verify the clinical, pharmacological and/or other pharmacodynamic effects of an investigational product(s), and/or to identify any adverse reactions to an investigational product(s), and/or to study absorption, distribution, metabolism, and excretion of an investigational product(s) with the objective of ascertaining its safety and/or efficacy. US National Institute of Health organizes Clinical Trials into five types: y Preventive Trials. To look for better ways to prevent disease in people who have never had the disease or to prevent disease from returning. Diagnostic Trials. To find new tests or procedures for diagnosing a particular disease or condition. Treatment Trials. To test experimental treatments, new combination of drugs or new approaches to surgery or radiation therapy. Quality of Life Trials. To minimize sufferings and improve the quality of life in patients with chronic disease. Compassionate Use Trials. To allow for use of drugs which have not as yet passed all the phases of clinical trials in patients suffering from that disease on compassionate basis after prior permission sought from FDA.

y y

Phases of Clinical Trials

Phase O (Exploratory Investigational New Drug Trials/Micro-dosing Trials): - Phase O trials are held to assess the safety of the lead compound in 10-15 healthy human volunteers. Drug is given in a subtherapeutic dose so that actual safety and efficacy in therapeutic doses cannot be assessed; however, go or no go decision for actual clinical trials can be facilitated. These trials give a basic idea about the preliminary pharmacokinetic profile of the drug in question and the volunteers are adequately paid. Phase I (Dose Escalation Trials): - Phase I trials give an estimate of the drug safety and tolerability in 20-100 healthy human volunteers. However in case severe toxicities are likely, as in case of drugs intended for HIV or cancer, real patients suffering from that disease are used. These trials give a basic idea about the preliminary pharmacokinetic profile of the drug, and provide a safe clinical dose range and therapeutic window of the drug. The subject is usually observed until several half-lives of the drug have passed. There are three different kinds of phase I trials: Single ascending Dose Trials In this case,a single dose of a drug is given to one group of volunteers who are then closely monitored for the safety and efficacy of the drug. Subsequently, higher dose of the same drug is given to another group of volunteers who are again monitored closely. This method evades any likelihood of toxicity due to multiple dosing in the same individual. Single Ascending Dose studies are those in which groups of three or six patients are given a small dose of the drug and observed for a specific period of time. If they do not exhibit any adverse effect, a new group of patients is then given a higher dose. This is continued until intolerable adverse effects start showing up, at which point the drug is said to have reached the Maximum tolerated dose (MTD). ii. Multiple ascending dose trials In this case, different doses of a drug in the increasing order are given to the same group of volunteers with adequate wash period in between and observed for various pharmacological and toxic effects. Multiple Ascending Dose trials are conducted to better understand the pharmacokinetics/pharmacodynamics of the drug. iii. Food drug trials Drug is given before meals first followed by after meals so as to observe the effect of food on drug absorption. Phase I trials are non-blind or open, i.e., both the investigator as well as the volunteer knows what is being given. Phase I trials are usually conducted in research centers by specially trained clinical pharmacologists. Phase II: - Phase II trials focus on determining the efficacy of a drug. These trials are performed on a modest number of patients, 100-300. These trials are conducted in special clinical centres, e.g., university hospitals.The development process of an investigational new drug commonly fails during Phase II trials due to the discovery of poor efficacy or toxic effects. Phase II trials are sometimes divided into Phase II-A and Phase II-B. y In Phase II-A, actual dose to be used is determined. y In Phase II-B, efficacy of that particular dose is determined. In phase II trials, case controlled studies, cohort studies, or randomized placebo controlled studies are undertaken. A single-blind design is often used, with an inert placebo medication and an established active drug (positive control) in addition to the investigational agent. Phase III: - In Phase III trials, the drug is tested in a large number of patients, 200-3000, to further establish the safety and efficacy. Using the information obtained from Phase I and II, Phase III trials are designed to minimize the errors caused by placeboeffects, variable course of the disease, etc. Therefore, double-blind and crossover techniques are frequently used. Group I II III Week 1 Aspirin Novent Placebo Week 2 Novent Placebo Aspirin Week 3 Placebo Aspirin Novent i.

Phase III trials are usually performed in settings similar to those anticipated for the ultimate use of the drug. Phase III trials can be difficult to design and execute and are usually expensive and time consuming because of the large number of patients involved and the masses of data that must be collected and analyzed. The investigators are usually specialists in the disease being treated. Certain toxic effects, especially those caused by immunologic processes, may first become apparent in phase III. Phase IV: - Phase IV Trials are also known as Post Marketing Surveillance trials. Phase IV trials involve the safety surveillance (pharmacovigilance) and ongoing technical support of a drug after it receives permission to be sold. Phase IV trials may be required by regulatory authorities or may be undertaken by the sponsoring company for competitive or other reasons (for example, the drug may not have been tested for interactions with other drugs or on certain population groups such as pregnant women, who are unlikely to subject themselves to trials). The safety surveillance is designed to detect any rare or long term adverse effects over a much larger patient population that was not possible during the phase I-III clinical trials. Harmful effects discovered by Phase IV trials may result in a drug being no longer sold, or restricted to certain uses.However, several hundred thousand patients may have to be exposed before the first case of toxicity is observed that occurs with an average incidence of 1 in 10,000. Recent examples of drugs withdrawn from market include Cerivastatin, Troglitazone and Rofecoxib.

Ethical & Legal Considerations

In each of the three formal phases of clinical trials, volunteers or patients must be informed of the investigational status of the drug as well as the possible risks; the researchers must obtain the full and informed consent of participating human subjects, and they must be allowed to decline at any amount of time during the trials. These regulations are based on the ethical principles set forth in the Declaration of Helsinki. In addition to the approval of sponsoring organization and the FDA, an interdisciplinary institutional review board at the facility where the clinical trials will be conducted must review and approve the plans for testing in humans. In India, Institutional Ethics committee supervises the conduct of clinical trials; as proposed by ICMR, Institutional Ethics Committee has the following constitution: Chairperson, who should be from outside the institution; 1-2 medical scientists; 1-2 clinicians from outside the institution; Legal advisor who should be preferably a retired judge or an advocate; 1 social scientist preferably from a reputed NGO; 1 philosopher/theologist or an ethics activist; A layman from the society; Member secretary from the institution who shall run the business of the IEC and call for various periodic meetings. The total number of members in IEC may range from 7-9, but should not exceed 15; the minimum number of members required for a periodic meeting should be 5-7. Representation has to be given to all persons, i.e., with respect to age, gender, and qualification. (i) (ii) (iii) (iv) (v) (vi) (vii) (viii)

IND Application (INDA)

Prior to the conduct of a clinical trial, an IND Application must be filled with the Drug Regulatory Authority. An Investigational New Drug (IND) application should carry the following information: i. ii. iii. iv. v. Source and chemical composition of the drug. Manufacturing information including formulation, stability and shelf life of the drug. Animal pharmacology and toxicology data. Design and protocol of the clinical trials. Name and credentials of the clinicians who will conduct the clinical trials.

Preclinical Studies
Acute Toxicity Studies: These studies are used to determine ED50, i.e., dose which produces effect in 50% test animals; this is observed when there is a graded response, i.e., the response increases with increase in dose.Otherwise, LD50, i.e., dose which causes death in 50% test animals, is determined; this is observed when there is a quantal response, i.e., all or none response. From LD50& ED50, therapeutic index can be calculated as:

Therapeutic Index = LD50 / ED50

Therapeutic index should be greater than 1 for a drug to be safe. These studies are also known as LD50 studies. A single large dose of test drug is givento one species and toa particular strain. For acute toxicity studies, inbred strains should not be used. It is advisable to use hybrid strains so that the toxicities can be easily predicted. There are three methods for the LD50 calculation: (1) Arithmetic, (2) Graphical, and (3) Statistical. For this study, two doses are selected: the one of which is the highest tolerated maximum dose (0% mortality) and the lowest is the lowest tolerated minimum dose (100% mortality). In between these two doses, five doses are selected by one of the following methods: 1. 2. 3. 4. Krabers Method (1931). Read & Muench Method (1938). Miller & Tainter Method (1944). Lichfield & Wilcoxon Method(1948). Thus total of seven doses are employed. For this study, two species: one rodent (mice, rats) and one non-rodent (Dogs, Cats), are used, and two routes of administration: oral/parenteral and the other is the one that is intended to be used as the therapeutic route of drug administration in humans, are employed. After dose selection, 10 animals for each of the seven groups are to be used. Following the drug administration, the animals are observed for mortality for the next 2 hours and it may be continued up to 12 hours. For other toxicities, the animals are observed for 72 hours. The percentage mortality is determined after 2, 4, 8, 16 hours; then the percentage mortality is converted into probit scale and plotted against log dose. The probit scale value of 5 corresponds to LD50 value on x-axis as shown below:

Humans are more sensitive to drug toxicities: 6 times more than dogs and 10 times more than rats. The smaller the animal, the greater is the surface area to body weight ratio; thus, there will be more loss of heat, therefore body temperature decreases faster. So, metabolic rate will be higher, dose is be high and effects are eliminated rapidly. First LD50 is determined in rodents and then taking into consideration the surface area of bigger animals, the dose of LD50 is calculated and given to 3-4 bigger animals. If no difference occurs, LD50 remains same but if it differs, LD50 is again calculated in bigger animals. Subacute Toxicity Studies: These studies require to species: one rodent and one non-rodent, and two routes of administration are employed. In these studies, maximum tolerated dose is determined and doses are given to same species with increments. This study is continued up to maximum tolerated dose, i.e., when maximum toxicities appear and some animals even get killed. The animals have to be maintained at this dose for 2-3 weeks. The increments in dose can be given on daily basis. Cage side monitoring of animals is to be done on daily basis for changes in their body weight, food intake water intake, renal function, hepatic function, hematological parameters, BP, heart rate and levels of drug in the blood. After monitoring the animals at the maximal tolerated dose, the animals are sacrificed and observed for various parameters including RBC and WBC count, blood urea, blood glucose, serum cholesterol, serum creatinine, SOGT, SGPT, ALP, Hb, ESR, albumin and total proteins. The various vital organs like kidneys, lungs, adrenals, pancreas, and uterus are removed, weighed followed by their gross examination and histopathological examination by a veterinarian. These studies last for 4 weeks and maximally up to 2 months. Chronic Toxicity Studies: For this, two species (one rodent and one non-rodent) are selected and two routes of administration (one oral and one parenteral) are employed. Drug is given for 90 days which may extend over two years in case the drug is intended for chronic use in humans. A minimum of three dose levels are chosen; the lowest being two times the maximum clinical dose; the highest being ten times the maximum clinical dose; and the third lies in between the highest and lowest doses. The animals are closely monitored throughout the study; after every 14 days the animals are observed for various morphological, physiological, biochemical, and pathological parameters and any change in the following parameters are recorded: o o o o o o o o o o o o o o o o o o o o o o o o o o Decrease in weight. Increase in weight. Salivary secretion (watery/viscid) Piloerection. Exopthalmus. Catatonia. Catalepsy. Stupor. Spasticity. Fecal pellets. Urinary output. Tremors. Clonic convulsions. Tonic extensions. Straubes reaction (flipping of tail). Anesthesia. Hyperesthesia. Loss of righting reflex. Sedation. Ataxia. Hypnosis. Muscle relaxation. Arching & rolling. Ptosis. Lachrymation, Diarrhea.

o o o o

Writhing. Vasodilation. Skin color. Rate of respiration.

In addition, chronic toxicity studies involve carcinogenic and mutagenic potential of drugs. If the drug is intended to be used in pregnant women, the teratogenic studies have to be undertaken. In case the drug is intended for use in infants, it has to be tested in rats born not less than 24 hours ago, and after every 14 days blood tests are to be conducted. Mutagenicity Mutagenicity is the capability of a substance to cause alterations in the base sequence of DNA. Clastogenicity Clastogenicity is the capability of a drug to cause alterations in the gross structure and content of chromosomes. Mutagen Any agent (i.e., physical, chemical, or biological) that can induce a genetic mutation or increase the rate of mutation is known as mutagen. Mutagenicity Testing (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x) (xi) (xii) (xiii) (xiv) (xv) (xvi) (xvii) (xviii) (xix) (xx) Salmonella mutation assay (Ames test). E. coli test system. Recombinant assay on Bacillus Subtilis. Neurospora crassa test system. Transdescentia test system. Drosophila test system. Barley test system. Soyabean test system. Specific locus mutation induction system in yeast. Specific locus mutation induction system in mice. Specific locus mutation induction system in mammalian cell culture. Yeast mitotic recombination, gene conversion and non-disjunction. Mammalian spot test. Mammalian inheritable translocation test. Mammalian dominant lethal assay. Unscheduled DNA synthesis. Vicia faba and Alium cepa test system. Mammalian sister chromatid exchange. Mammalian micronucleus test. Mammalian chromatid aberration test. Ames Test Ames test is based on the principle of reversion of mutation in the mutant strains of salmonella typhi upon exposure to a mutagen. Two strains are chosen, one wild and other mutant. Mutant strains are made such that they cant synthesize one amino acid, histidine; while the wild strains are able to synthesize all the amino acids. Both wild and mutant strains are supplied with basic carbon source; however, the mutant strainsare supplemented with histidine. Both of the strains grow and multiply fairly. But as the histidine supplied exhausts, mutagens, if any, induce reversion of mutation in the mutant strains, and now they are able to synthesize the histidine amino acid from the basic carbon source upon exposure to the mutagen drug.

Carcinogenicity & Carcinogens Carcinogenicity is the capability of a substance to cause cancer. Alteration of DNA is the first step in the complex, multistage process of carcinogenesis. Carcinogens are chemical substances that cause cancer and can directly interact with DNA or act at a later stage to increase the likelihood that mutation will result in a tumor. Carcinogens are divided into two groups: 1. Genotoxic Carcinogens (i.e., agents that are themselves responsible for causing genetic damage, and thereby cancer) They are further divided into: y Primary carcinogens that act directly on DNA. y Secondary carcinogens, which must be converted to a reactive metabolite before they affect DNA, most clinically important carcinogens are secondary. Epigenetic Carcinogens (i.e., agents that do not themselves cause genetic damage but increase the likelihood that such damage will cause cancer). The most important types are as follows: y Promoters, they produce cancers when given after a genotoxic agent; examples include phorbol esters and cigarette smoke. y Cocarcinogens, these enhance the effect of genotoxic agents when given simultaneously; examples include phorbol esters and various aliphatic and aromatic hydrocarbons. y Hormones, some tumor are hormone dependent; for example, estrogen dependent breast and uterine cancer and androgen dependent prostate cancer. Primary Carcinogen Secondary Carcinogen Cocarcinogens Promoters

2.

Metabolizing Enzymes

Reactive Metabolite

Alteration of DNA (Mutation)

Oncogene Expression

Malignant Transformation

USFDA Classification of Carcinogens

1. 2.

3. 4. 5. 6.

Category A Human carcinogens with convincing evidences of human carcinogenicity (11 chemicals). Category B y Category B1: probable human carcinogens with limited evidences (6 chemicals). y Category B2: probable human carcinogens with sufficient evidences (64 chemicals). Category C Possible human carcinogens with inadequate animal data for stronger classification (40 chemicals). Category D Unclassifiable human carcinogens with inadequate animal data for stronger classification (53 chemicals). Category E Unclassifiable carcinogens with no animal/ human data (58 chemicals). Category F Non-carcinogenic for humans with sufficient evidence of non-carcinogenicity in animals.

IARC Classification of Carcinogens 1. 2. 3. 4. 5. Human carcinogens (88 chemicals). Probable human carcinogens (64 chemicals). Possible human carcinogens (237 chemicals). Unclassifiable human carcinogens (495 chemicals). Probable human non-carcinogens (1).

Carcinogenicity Testing

Teratogen Teratogen is a physical (as in, radiation), chemical (as in, the drug thalidomide) or biological(as in, rubella virus) factorcausing birth defects and reproductive abnormalities. US FDA Classification of Teratogenic Drugs

1. Category A: No demonstrated risk; possibility of fetal harm is removed. 2. Category B: Either no evidence of fetal risk in extrapolation from animal data plus no information from
human studies or evidence of fetal risk in animals that could not be confirmed from human studies.

3. Category C: Either evidence of fetal risk in animal studies combined with no studies in humans or animal
and human studies are not available. Drugs are given to pregnant women only when possible benefits outweigh risk involved. 4. Category D: Positive evidence of fetal risk; drugs may be given to pregnant women when benefits are acceptable. 5. Category E: Not known to affect animal reproduction but no human data is available. 6. Category X: Animal/human studies or human experience has revealed fetal abnormalities; risk of use in pregnant women outweighs any possible benefit. Teratogenicity Teratogenicity is the capability of a substance to interfere with developmental and reproductive processes. Teratogenic Insult Extent and rate of teratogenic insult depends on the stage of pregnancy; the various stages of pregnancy are as follows: y y Blastocyst formation: conception to 17 days; this stage is a state of active cell division exposure leads to failure of pregnancy which often goes unnoticed. Organogenesis: 18 to 55 days of gestation; in this stage, actual differentiation, migration, apoptosis, etc. takes place this is the most vulnerable stage, deformities are produced.

Growth & development: 56 days onwards; this is the development stage Exposure may lead to development & functional abnormalities.

Teratogenicity Testing Safety Evaluation of Drugs for Development & Reproductive Toxicity for human Use (1960):1) Segment I (Evaluation of reproductive toxicity): The drug is administered to both sexes before mating, during mating, and after mating, and monitored for potential risks on fertility. The various parameters evaluated during this evaluation phase include: Female (1) litter size, (2) fetal viability. Male (1) fertility, (2) sperm count, (3) sperm motility, and (4) weight of sex organs. 2) Segment II (Evaluation of development toxicity): The drug is administered right from the day of implantation and stopped one day before delivery, and closely monitored for teratogenic potentials; during the study, 50% animals are sacrificed before delivery, and 50% are sacrificed after delivery. 3) Segment III (Postnatal evaluation): The drug is administered throughout the last trimester of pregnancy and continued until lactation and weaning; even the studies are continued till third generation. Note: Mating should be done between male & female of the same species and same strains; mating of siblings should be avoided.