Essentials of Experimental Pharmacology, General Concepts

Bibliography

Chapter 1

Introduction

Pharmacology is a branch of biology which deals with study of Drug. There are several branches viz- Pharmacokinetics dealing with study of Absorption, Distribution, Metabolism and excretion of drugs (What body does to Drug), Pharmacodynamics dealing with study of actions and effects of drug (What drug does to body), Toxicology deals with study of toxic effects of drugs and treatment of poisons/toxicities. Experimental Pharmacology deals with study of various models and parameters to test the hypotheses related to either Pharmacokinetic /Pharmacodynamic /toxicologic characteristics of the given agent. The therapeutics (Pharmacotherapeutics) is also one branch dealing with study of applications of medicinal agents in therapeutic (treatment of diseases). Clinical Pharmacology is application of Pharmacology in Clinical Practice i.e. during treatment of patients (in Clinic).

This brief introduction of some important branches of Pharmacology underlines importance of experimental pharmacology, which forms base for study of various hypotheses and bring out some answers to contribute other branches of Pharmacology. The experimental Pharmacology can be broadly divided in two major types of study-

A. Preclinical studies and B. Clinical studies

Thus preclinical studies involve a very broad field which utilizes biological sources other than human beings for acquiring information on many aspects like-

¹ Animal Model- Inducing the human disease like situation (simulation) by using suitable method (may be induced by either one or more techniques like chemically, alteration of housekeeping, surgically or genetically altered physiology simulating the disease status in humans)

² End Points- The product studied in the animal model should demonstrate a beneficial effect analogous to the intended outcome in humans. Primary shidy endpoints, which should be specifically discussed with the review division, generally are the enhancement of survival or prevention of major morbidity in most of the studies. The dose response for these endpoints should be explored fully and established. Although secondary endpoints can provide useful information about the animal model and the activity of the product as studied in the animal model, ordinarily, only primary endpoints can serve as the basis of approval.

³Parameters- The data required to substantiate tlie effects of given agent e.g. blood sugar level, blood pressure, agonistic/antagonistic effects produced etc.

E.g., Diabetes can be induced in animal by various ways (Chemically using streptozotocin/ alloxan, surgically- by pancreactomy and diet induced) accordingly called as alloxan model of diabetes. End points can be Primary-survival with and without treatment and impact on complications. Secondary endpoints / Parameters can be blood glucose level, glycosylated haemoglobin level, insulin level, ketone bodies and other which can predict mechanism of action e.g. estimation of Glucose Transporters or potassium channel activity etc.

Clinical Studies: The studies performed using human subjects / volunteers are known as Clinical Studies. Clinical Studies involve clinical research and clinical trials.

At the core of the drug development process is a fundamental understanding of the clinical pharmacology of the drug substance. Clinical pharmacology can be thought of as a translational science in which basic information about the relationship between a drug's dose, local or systemic exposure and response (related to either efficacy or safety) is applied in the context of patient care. Knowledge of this relationship, which is a key to successful therapeutics, and how it is altered by the intrinsic (age, gender, renal function, etc.) and extrinsic (diet, drugs, life-style) factors of an individual patient is one of the major contributions of clinical pharmacology to drug development and regulatory decision-making.

⁴ NCE- New Chemical Entities: New Chemical Entity- According to the U.S. Food and Drug Administration, a new chemical entity (NCE) (also known as new molecular entity (NME)) is a drug that contains no active moiety that has been approved by FDA in any other application (Molecule not recognized as Drug), is a chemical molecule developed by the innovator company in the early drug discovery stage, which after undergoing clinical trials could translate into a drug

⁵ Bioavailability: Rate (per unit time) (quantity) by which the drug reaches systemic circulation after administering it extra-vascularly (other than directly in blood vessel). It gives both - Rates applied for ADME and quantity (concentration) changing with respect to time.

successful therapeutics, and how it is altered by the intrinsic (age, gender, renal function, etc.) and extrinsic (diet, drugs, life-style) factors of an individual patient is one of the major contributions of clinical pharmacology to drug development and regulatory decision-making.

Once a lead compound with the intended pharmacological action is identified, the step-wise process to characterize and potentially optimize its pharmacokinetic (PK) properties (i.e., absorption, distribution, metabolism, and excretion), as well as to minimize its pharmacokinetic limitations (e.g., poor absorption), begins in humans as part of phase I human clinical trials. Soon after, other principles of clinical pharmacology [e.g., pharmacokinetic-pharmacodynamic (PD) relationships] become critical to the evaluation and selection of the most appropriate dosing regimen of the drug in a carefully selected target population enrolled in phase II clinical trials. These trials form the scientific rationale for subsequent dose selection in large-scale phase III clinical trials where the primary goal is to provide adequate evidence of efficacy and relative safety of the drug. Phase III trials are the most expensive and time-consuming component of the overall drug development process and many believe that paying careful attention to doing clinical pharmacology homework has the greatest potential to reduce the failure rate of new drugs at this near final stage of development.

Often, in parallel with phase III clinical trials, a group of clinical pharmacology studies, such as those in special populations, are conducted in human volunteers to develop a knowledge database of factors influencing drug exposure. These data are crucial for an understanding of when, and how much, to adjust dosage regimens. Because these studies typically focus on changes in systemic exposure, as a surrogate marker for either efficacy or toxicity, the availability and the intelligent use of exposure (e.g., dose, PK measurementsj-response (e.g., biomarkers, surrogate clinical endpoints, clinical outcomes, PD) relationships to interpret the results of these studies become critical to information for various sections of the product label.

These studies can be broadly classified into two broad categories: 1. those dealing with patient-intrinsic factors that include gender, age, race, diseases states (primarily renal and/or hepatic impairment), and genetic (e.g., activity of cytochrome P450 enzymes) factors, and 2. those dealing with patient extrinsic factors that include drug-, herbal- and nutrient-drug interactions, environmental variables (e.g., smoking, diet), and lifestyle factors.

Clinical Research: The NIH definition of clinical research is based on the 1997 Report of the NIH Director's Panel on Clinical Research that defines clinical research in the following three parts: 1. Patient-oriented research. Research conducted with human subjects (or on material of human origin such as tissues, specimens and cognitive phenomena) for which an investigator (or colleague) directly interacts with human subjects. Excluded from this definition are in vitro studies that utilize human tissues that cannot be linked to a living individual. Patient-oriented research includes: (a) mechanisms of human disease, (b) therapeutic interventions, (c) clinical trials, or (d) development of new technologies. 2. Epidemiologic and behavioural studies, 3. Outcomes research and health sendees research.

Clinical Trial: a clinical trial is operationally defined as a prospective biomedical or behavioural research study of human subjects that is designed to answer specific questions about biomedical or behavioural interventions (drugs, treatments, devices, or new ways of using known drugs, treatments, or devices).

As per the revised Schedule Ύ’ of the Drugs & Cosmetic Act (2005), a clinical trial is a systematic study of new drug(s) in human subject to generate data for discovering and/or verifying the clinical, pharmacological (including pharmacodynamic, and pharmacokinetics), and/or adverse effects with the objective of determining the safety and/or efficacy of the new drugs. Clinical trial of drugs is a randomised single or double blind controlled study in human participants, designed to evaluate prospectively the safety and effectiveness of new drugs/ new formulations. The new drug as defined under the Drugs and Cosmetic Rules 1945 (DCR), and subsequent amendments include:

Clinical trials are used to determine whether new biomedical or behavioural interventions are safe, efficacious and effective. Clinical trials of experimental drug, treatment, device or behavioural intervention may proceed through four phases:

Phase I Clinical Trial: Human Pharmacology- These are done to test a new biomedical or behavioural intervention in a small group of people (e.g. 20-80) for the first time to explore activities, PK and evaluate safety (e.g. To determine a safe dosage range, and identify side effects). These trails are conducted to- Assess tolerance, Define/describe PK and PD, Explore drug metabolism and drug interactions, Estimate activity. E.g. Dose-tolerance studies, Single and multiple dose PK and/or PD studies, Drug interaction studies.

Phase II Clinical Trial: Therapeutic Exploratory- These are done to study the biomedical or behavioural intervention in a larger group of people (several hundred) to detennine efficacy and to further evaluate its safety. These trails are conducted so as to explore use for the targeted indication, estimate dosage for subsequent studies, provide basis for confirmatory study design, endpoints, and methodologies. E.g. Earliest trials of relatively short duration in well- defined narrow patient populations, using surrogate or pharmacological endpoints or clinical measures, Dose-response exploration studies.

Phase III Clinical Trial: Therapeutic Confirmatory- These are done to study the efficacy of the biomedical or behavioural intervention in large groups of human subjects (from several hundred to several thousand) by comparing the intervention to other standard or experimental interventions as well as to monitor adverse effects, and to collect information that will allow the intervention to be used safely. These trails are conducted to demonstrate /confirm efficacy. Establish safety profile, Provide an adequate basis for assessing the benefit/risk relationship to support licensing, establish doseresponse relationship. E.g. Adequate and well controlled studies to establish efficacy, randomised parallel dose response studies, clinical safety studies, studies of mortality/ morbidity outcomes, large sample trials, and comparative studies.

NIH-Defined Phase III Clinical Trial: Therapeutic Use- For the purpose of the Guidelines on the Inclusion of Women and Minorities, an NIH-defined Phase III clinical trial is a broadly based prospective NIH-defined Phase III clinical investigation, usually involving several hundred or more human subjects, for the purpose of evaluating an experimental intervention in comparison with a standard or control intervention or comparing two or more existing treatments. Often the aim of such investigation is to provide evidence leading to a scientific basis for consideration of a change in health policy or standard of care. The definition includes pharmacologic, non-pharmacologic, and behavioural interventions given for disease prevention, prophylaxis, diagnosis, or therapy. Community trials and other populationbased intervention trials are also included.

Phase IV Clinical Trial: (POST-MARKETING SURVEILLANCE): Therapeutic Use- studies are done after the intervention has been marketed. These studies are designed to monitor effectiveness of the approved intervention in the general population and to collect information about any adverse effects associated with widespread use. Because of genetic diversity among humans, it is possible that a new drug will cause adverse effects in only a small group of genetically similar people, which may not have been apparent during clinical trials. As the new drug is given to more and more people, careful monitoring is necessary to avoid this possibility. Drugs are taken off the market if post-marketing surveillance reveals previously undetected side effects. This phase of clinical trails is conducted to refine understanding of benefit/risk relationship in general or special populations and/or environments, identify less common adverse reactions, and refine dosing recommendation. E.g. Comparative effectiveness studies, studies of mortality/morbidity Outcomes, studies of additional endpoints, large sample trials, pharmacoeconomic studies

Clinical testing is complex and time-consuming, averaging 14 years to complete Phase I through III testing to gain FDA approval. Sometimes, drugs will fail in clinical tests because the animal tests did not accurately predict their effects in humans. Often people wonder why it takes so long to develop a new drug and why sometimes a drug treatment is not found to be dangerous until after it is sold to the public.

Chapter 2

Understanding Basics

Before starting experiments it is important to know various basics covering Pharmacology with respect to basic phannacology, drug-receptor interactions, quantitative and qualitative aspects of drug actions. Further, it is also important to learn basics, terminology and methods for making solutions of various concentrations.

Receptor: A cellular macromolecule, or an assembly of macromolecules, that is concerned directly and specifically in chemical signalling between and within cells. Combination of a hormone, neurotransmitter, drug, or intracellular messenger with its receptor(s) initiates a change in cell function. Several types of receptors may be identified: peripheral membrane proteins, many hormone and neurotransmitter receptors are transmembrane proteins i.e. transmembrane receptors are embedded in the phospholipid bilayer of cell membranes which allow the activation of signal transduction pathways in response to the activation by the binding molecule called as ligand and Metabotropic receptors are coupled to G proteins and affect the cell indirectly through enzymes which control ion channels.

Ionotropic receptors (also known as ligand-gated ion channels) contain a central pore which opens in response to the binding of ligand.

Another major class of receptors is intracellular proteins such as those for steroid and intracrine peptide hormone receptors. These receptors often can enter the cell nucleus and modulate gene expression in response to the activation by the ligand.

Ligand: A molecule which binds to a receptor is called a ligand, and may be a peptide (such as a neurotransmitter), a hormone, a pharmaceutical drug, or a toxin, and when such binding occurs, the receptor goes into a conformational change which ordinarily initiates a cellular response. Ligand-induced changes in receptors result in physiological changes which constitute the biological activity of the ligands. Ligand may be agonist or antagonist.

Receptor reserve: There is said to be a receptor reserve (also known as spare receptors) for that particular agonist in that particular tissue. This was speculated after it was found that despite of blocking receptors with irreversible antagonist (making no receptor available for agonist), the agonist could exhibit the action. It is believed that there is no receptor reserve for a drug which acts as a partial agonist in the tissue. The receptor reserve may vary between tissues, depending on the number of receptors in the particular tissue and the efficiency of coupling between them and their effector mechanism. Consequently, a partial agonist in one tissue may appear to act as a full agonist in a tissue with a higher receptor reserve

2-State Receptor Concept: It is also believed that many receptors exist in 2 states -Active and inactive state. Full agonist can convert maximum receptors in active state while partial agonist does it partially. The agonist prevents activation of receptor by agonist.

Orphan Receptor: The receptors which are identified and the binding of ligand is also known but their Effector Mechanism (see below second messenger) / effects are not elucidated.

Species homologue (or species variant): A receptor for a particular neurotransmitter which mediates the same physiological function in two species and is formd in similar tissue locations. The two receptors differ in amino-acid sequence to a small degree (approx. 10% or less), giving rise to differences in the affinity of some antagonists or the relative potencies of agonists

Subtype: Subty pes of receptor are those which, in a single species, are activated by the same family of endogenous ligands but exhibit sufficient differences in their pharmacological properties or molecular structure to justify being classified separately. Traditionally, subtypes have been identified using drugs which can selectively activate them or antagonize the effects of agonists with markedly different