LOGO The requirements for the conduct of preclinical studies and clinical trials of drug products. The requirements of Good Laboratory Practice and Good Clinical Practice LECTURE 4 N.I. Gudz, Associate Professor (Drugs Technology and Biopharmaceutics Department), Ph.D.

Animals in scientific investigation The use of animals in scientific investigation has been traced back to several centuries BC. For instance, the writings of Aristotle (384–322 BC) and Erasitratus (304–258 BC) indicated that they had studied the anatomy of various animals. Tests in intact animals are necessary to understand how a drug will work in the context of the myriad metabolic and homeostatic mechanisms that are active in vivo. Screening tests are commonly conducted with in vitro systems and isolated tissues or organs to identify and, in some cases, to act as a bioassay to help purify pharmacologically active agents.

THE PURPOSE OF ANIMAL STUDIES In vivo studies must be conducted to identify unexpected adverse effects and to estimate the dosages that may be pharmacologically active without producing unwanted effects. Thus, the purpose of the animal studies is to verify the pharmacologic activity of the new drug, identify any unexpected pharmacologic activity, to identify any adverse effects and develop an initial data base on the action of the drug in vivo

SPECIES DIFFERENCES IN SENSITIVITY TO SOME DRUGS For most substances, the mechanism of action will be the same in humans and other mammals. Therefore, quantitative rather than qualitative differences in response are most common. Humans may be more sensitive to some drugs than certain laboratory animals are, but in many cases some animal species are more sensitive than humans are. For example, the mouse is most sensitive to atropine, the cat is less sensitive, and the dog and the rabbit tolerate atropine at doses 100 times higher than does the human. Species differences in sensitivity can often be explained by differences in metabolism, including quantitative and qualitative differences in the ability to detoxify drugs and also differences in the rates of absorption, transport, distribution, and elimination of chemicals.

PHARMACOLOGIC STUDIES In vivo pharmacology screening programs and pharmacologic studies include models for detection of pharmacologic activity of the major therapeutic classes of compounds. For example, spontaneously hypertensive rats can be used to screen compounds for antihypertensive effects and for effects on heart rate. The animals are dosed for one or a few days. Blood pressure and heart rate are measured by means of an inflatable cuff around the tail. Most classes of antihypertensives will be detected. Agents such as beta-adrenergic antagonists will be detected by decreased heart rate. Other rat models include deoxycorticosterone acetate (DOCA)-induced hypertensive, renal hypertensive (one or both renal arteries clamped), and stroke-prone spontaneously hypertensive rats. Hypertensive dogs produced by clamping one or both renal arteries may also be used to test or verify antihypertensive activity in a second species.

ANIMALS IN DRUG SAFETY TESTING Before a new drug can be given to people, it must be tested in animals to determine its side effects and at what dosage those side effects will appear. This testing must be done in vivo because the effects of the processes of absorption, distribution, metabolism, excretion, the interactions among these processes, and the interactions among the various organs and neuroendocrine systems within the whole animal cannot be duplicated in vitro. To characterize the nature of the side effects to be expected from a new drug, it is usually necessary to give much higher dosages than would be given clinically and sometimes to give the drug over prolonged periods of time. Acute toxicology testing, repeat-dosing toxicity studies, reproductive toxicology studies, mutagenicity studies are referred to laboratory toxicology studies.

ACUTE TOXICOLOGY STUDIES In acute toxicology testing, animals are given single doses of a drug. The most common study design is to give groups of rats or mice (such as 5 per sex) single treatments over a wide range of dosages and then observe them over a period of 7–14 days for survival and for physical or behavioral signs of toxicity. In most cases a LD50 (the dosage at which 50% of the animals die) will be determined; however, this is not always feasible or necessary.

ACUTE TOXICOLOGY STUDIES (CONTINUE) The reasons of conducting the acute toxicity test are following: This is the first toxicity test that will be done for a new drug, and it provides the basis for choosing dosages that will be used in subsequent toxicity studies involving repeat dosing. This test provides information regarding the effects of single large doses of a new drug. This information may be used to predict the effects of overdosing (either accidental or purposeful) in humans. The acute toxicity test is a convenient safety check on new batches of drug. Even though extensive chemical analyses are conducted on new batches of any drug, a quick test in live animals to assure that there have been no dramatic changes in acute toxicity provides a desirable level of comfort before making the new drug batch available for human use. The acute toxicity test is also a convenient method to determine any interaction between two drugs that would cause unexpected toxicity in clinical use.

Reproductive toxicology studies cover the entire process of reproduction from mating to pregnancy, birth, weaning and, sometimes, the reproductive function of subsequent generations. The species most commonly used in these studies are rats and rabbits, although in special circumstances, other species such as mice, dogs, or monkeys may be used. A series of studies is usually conducted so as to cover all the phases reproduction. Within each study, groups of animals corresponding to untreated controls and two or three drug-treated groups are used. One type of typical study, usually conducted in rats, provides information on fertility and general reproductive performance, particularly on gonadal function, estrous cycles, mating behavior, conception rates, and early stages of gestation. This study is usually referred to as a fertility study.

MUTAGENICITY STUDIES (GENOTOXITY TESTING) Genotoxity tests can be defined as in vitro and in vivo tests desighed to detect compounds that induce genetic damage directly or indirectly by varios mechanisms. New drugs are routinely screened for their potential to cause gene mutations and/or chromosome aberrations by in vitro tests. An in vivo test is also required by most regulatory agencies.

MUTAGENICITY STUDIES (GENOTOXITY TESTING) A new drug showing a strong positive response in an in vitro mutagenicity test probably would not be developed. In vivo tests serve to determine whether a drug has potential to cause genetic alterations under conditions closer to those that one would obtain under clinical use. The significance of these studies to the safety of a drug is twofold. First, a drug that causes genetic damage could produce such damage in the sperm or ovaries of the patient and thus potentially cause genetic abnormalities in the children of that patient. Second, it is widely accepted that any agent capable of causing genetic damage is also likely to be a carcinogen. The most common in vivo tests for genetic damage are chromosome damage.

Carcinogenicity studies Carcinogenicity bioassays are long-term studies in mice and rats conducted according to standard guidelines established by the National Cancer institute and the International Conference on Harmonization. In these studies the animals are treated repeatedly for periods of 18–24 months for mice and of 24 months for rats. Treatment is by a route consistent with the intended clinical route. The study design usually includes vehicle control and two or three drug-treated groups. The highest dosage is selected as the maximum dosage that will be tolerated by the animals.

Carcinogenicity studies This dosage may cause minimal signs of toxicity but should cause no more than a 10% decrement in body weight gain compared with control groups and should not cause toxicity, other than that related to a neoplastic response, that would be predicted to shorten the animals natural life span. This high dosage is necessary to give a maximum test of the potential of a new drug to cause cancer. The animals are examined frequently for palpable masses. At the end of the treatment period, the animals are necropsied and histologic examinations of the tissues are conducted with particular attention given to the detection and identification of tumors.

The GLP regulations The GLP regulations set out the rules for good practice and help researchers perform their work in compliance with their own pre-established plans and standardized procedures. The regulations are not concerned with the scientific or technical content of the research programmes. Nor do they aim to evaluate the scientific value of the studies. All GLP texts, irrespective of their origin, stress the importance on the following five points: 1. Resources: organization, personnel, facilities and equipment 2. Characterization: test items and test systems 3. Rules: study plans (or protocols) and written procedures 4. Results: raw data, final report and archives 5. Quality Assurance.

Clinical drug development is generally divided into four phases: preapproval segments (Phases 1 through 3) and a postapproval segment (Phase 4). For each study conducted within a particular phase, specific information is collected according to the requirements for individual drugs being developed. Collection of safety, efficacy, and pharmacokinetic data is the focus of most clinical trials. CLINICAL TRIALS. THE PURPOSE, BASIC PRINCIPLES AND REQUIREMENTS OF GCP

The primary goal of Phase 1 studies is to demonstrate safety in humans to collect sufficient pharmacokinetic and pharmacological information to permit the determination of the dose strength and regimen for Phase 2 studies. Phase 1 studies are closely monitored, are typically conducted in healthy adult subjects, and are designed to meet the primary goal (i.e., to obtain information on the safety, pharmacokinetics, and pharmacologic effects of the drug). In addition, the metabolic profile, adverse events associated with increasing dosages, and evidence of efficacy may be obtained. The dose range and route of administration should be established during Phase 1 studies. PHASE 1

Generally, the first study in humans is a rising, single- dose tolerance study. The initial dose may be based on animal pharmacology or toxicology data, such as 10 % of the no-effect dose. Doses are increased gradually until an adverse event is observed that satisfies the predetermined criteria of a maximum tolerated dose (MTD). Although the primary objective is the determination of acute safety in humans, the studies are designed to collect meaningful pharmacokinetic information. SUBDIVISION OF PHASE 1. The features of the first study

The first study of Phase 1 Generally, healthy male volunteers are recruited, although patients sometimes are used (e.g., when testing a potential anticancer drug that may be too toxic to administer to healthy volunteers). Participants in the first study are usually hospitalized or enrolled in a clinic so that clinical measurements can be performed under controlled conditions and any medical emergency can be handled in the most expeditious manner. The first study in humans is usually not considered successfully completed until an MTD has been reached. An MTD must be reached because the relationship between a clinical event (e.g., emesis) and a particular dose level observed under controlled conditions can provide information that will be extremely useful when designing future trials.

A multiple-dose safety study typically is initiated once the first study in humans is completed. The primary goal of the second study is to define an MTD with multiple dosing before to initiating well controlled efficacy testing. The study design of the multiple-dose safety study should simulate actual clinical conditions in as many ways as possible. Typically, dosing in the second study lasts for 2 weeks. The length of the study may be increased depending on the pharmacokinetics of the drug so that both drug and metabolite concentrations reach steady state. Also, if the drug is to be used to treat a chronic condition, a 4-week study duration may be appropriate. To obtain information for six dose levels with six subjects receiving active drug and two receiving placebo for each of three cohorts, a minimum enrollment of 24 subjects should be anticipated. Similar to the first study in humans, these subjects would be hospitalized for the duration of the study. The second study of Phase 1

2 The focus of these Phase 2 studies is on efficacy, while the pharmacokinetic information obtained in Phase 1 studies is used to optimize the dosage regimen. Phase 2 studies are not as closely monitored as Phase 1 studies and are conducted in patients. These studies are designed to obtain information on the efficacy and pharmacologic effects of the drug, in addition to the pharmacokinetics. Additional pharmacokinetic and pharmacologic information collected in Phase 2 studies may help to optimize the dose strength and regimen and may provide additional information on the drugs safety profile (e.g., determine potential drug–drug interactions). On completion of the efficacy trial, a therapeutic window for plasma drug concentrations can be defined by reviewing the correlation between plasma drug concentrations and key safety and efficacy parameters. The goal is to improve efficacy and safety of the drug by individualizing the dosage based upon previous plasma drug concentration profiles in the same patient. The features of Phase 2

2 The goals of Phase 3 studies are to confirm the therapeutic effect, establish dosage range and interval, and assess long-term safety and toxicity. Less common side effects and AEs (adverse experiences) that develop latently may be identified. In addition, studies targeted to evaluate and quantify specific effects of the drug, such as drowsiness or impaired coordination, are conducted during this phase. Phase 3 studies are also used to identify the most appropriate population or subpopulation for the study drug and to establish a place for the drug in its therapeutic class. A drug may be developed in a therapeutic class that already has effective alternatives, but the investigative compound may have a better safety profile than its established competitors. A Phase 3 clinical study can be designed to assess relative safety profiles. The features of Phase 3

The large numbers of patients in Phase 4 studies make it easier for researchers to determine rare AEs and can help identify patient populations that are at particular risk for certain AEs. For example, demographic trends toward side effects involving geographic locus, gender, or race may be determined from postmarketing surveillance data.