UNIT I : INTRODUCTION TO DRUG DISCOVERY AND DEVELOPMENT LEARNING OBJECTIVES: ‘At the end of the lesson, the student will be able to: ‘a. Understand the background and development of product research. b._Demonstrate knowledge in understanding basic biology of diseases. INTRODUCTION Drug Discovery and Development (DDD) Is a significant approach to the overall advancement of the healthcare community. Yet, it is broad and complex. From the pre-discovery phase up to the Post Marketing ‘Surveillance involves a lot of procedures and protocols that must be followed throughout the process and may even take more than 10 years. In this unit, we will discuss the overview of DDD, ethnopharmacological characteristics of natural product research and understanding the biology of disease. Overview of Drug Discovery and Development. Medical and pharmaceutical research provides a basis for the development of new therapeutic approaches to human and animal disease. This process of drug discovery research can be basic (seeking an understanding of biological phenomena that are unknown) or applied (using principles that are known to produce a desired new product or effect). In either case, drug discovery research results from an unmet clinical need, a recognized deficit in treatment options. Drug discovery is the process by which drugs are discovered or designed. In the past most drugs were discovered either by identification of the active Ingredient from traditional remedies or by serendipitous discovery. Today we know how disease and infection are controlled at the molecular and physiological level. Drug discovery involves the identification of candidates, synthesis, characterization, screening, and assays for therapeutic efficacy. Drug discovery és still a lengthy, ‘expensive, difficult, and inefficient process with a low rate of new therapeutic discovery. The outcome of a successful drug discovery program is the generation of a therapeutic entity where none previously existed or the replacement of established therapies in favor of a newer modiality that Is safer and more efficacious. ‘The main function of the pharmaceutical Industry is to create products (i.e., drugs that have an impact on health care). Drug development is the process of bringing a new drug to the market once a lead compound has been Identified through drug discovery. Drug development includes preclinical research (microorganisms/animal) and clinical trials (human). Products of this type can be foreseen to some extent through knowledge and study and thus are amenable to planned research and development (R&D). For example, if the cause of a disease has been identified as an infection by a microorganism, a search can be undertaken for an agent that will prevent ‘or cure the infection. However, in some instances, the etiology of a disease is unknown despite intensive Investigation. In this situation, the pathway to a satisfactory cure or method of prevention cannot be foreseen or forecast. In such cases, products may only be developed after application of careful investigations, from a revolutionary new approach, or perhaps from a serendipitous finding. The major objective of research in the pharmaceutical industry is to produce safe drugs that prevent, cure, or ameliorate disease. Interim research goals that lead to this major objective are to: 1. Understand the molecular basis of biological mechanisms in health and disease. 2. Develop new biological testing procedures relevant to human and veterinary medicine. 3. Develop a quantitative understanding of the interaction of drugs with key biological systems, leading to the more rational design of drugs. 4. Understand the absorption, transport, and mode of action of drugs. Develop drugs of low toxicity, reproducible delivery, and high specificity for a given pathological state or target organ.Tis the mission of pharmaceutical research companies to take the path from understanding a disease to bringing a safe and effective new treatment to patients. Scientists work to piece tagether the basic causes of disease at the level of genes, proteins and cells. Out of this understanding emerge “targets,” which potential new drugs might be able to affect. Putting everything into perspective takes about an average of 10-15 years just to develop one new medicine from the earliest stages of discovery to the time it Is available for treating Patients. Researchers work to: ‘A. validate these targets, B. discover the right molecule (potential drug) to interact with the target chosen, C. test the new compound in the lab and clinic for safety and efficacy and D. gain approval and get the new drug into the hands of doctors and patients. For the first time in history, scientists are beginning to understand the inner workings of human disease at the molecular level. Recent advances in genomics, proteomics and computational power present new ways to understand illness. The task of discovering and developing safe and effective drugs is even more promising as ‘our knowledge of disease increases. AAs scientists work to harness this knowledge, It Is becoming an increasingly challenging undertaking. ‘The average cost to research and develop each successful drug is estimated to be $800 million fo $1 billion. This number includes the cost of the thousands of fallures: For every 5,000- 10,000 compounds that enter the research and development (R&D) pipeline, ultimately only one receives approval. ‘These numbers defy imagination, but a deeper understanding of the R&D process can explain why so many compounds don't make it and why It takes such a large, lengthy effort to get one medicine to patients. Success requires immense resources — the best scientific minds, highly sophisticated technology and complex project management. It also takes persistence and, sometimes, luck. Ultimately, though, the process of drug discovery brings hope and relief to millions of patients. ‘The discovery process includes all early research to identify a new drug candidate and testing it in the lab. ‘The process takes approximately 3-6 years. By the end, researchers hope to have a promising candidate drug to test in people. Modern drug discovery Is the product of cooperation. Many sectors contribute, particularly in building the basic science foundations. Both public and private organizations play unique but increasingly interdependent roles in translating basic research into medicine. Major biopharmaceutical companies are the primary source of R&D funding for new medicines, both for projects in thelr own laboratories as well as for research licensed from other sectors. Smaller companies also crive Innovation, conducting basic research, drug discovery, preclinical experiments and, in some cases, dinical trials. The National Institutes of Health (NIH) provides leadership and funding support to universities, medical schools, research centers and other nonprofit institutions, and stimulates basic research and early- stage development of technologies that enable further targeted drug discovery and development. ‘Typically, researchers discaver new drugs through:= New insights into @ disease process that allow researchers to design @ product ta stop or reverse the effects of the disease. ‘+ Many tests of molecular compounds to find possible beneficial effects against any of a large number of diseases. ‘= Existing treatments that have unanticipated effects. '* New technologies, such as those that provide new ways to target medical products to specific sites within the body or to manipulate genetic material. Ethnopharmacology Drug discovery from Ethnopharmacology and Ethnomedicine literally begins with an ethnopharmacologist ‘or plant botanist. This approach is based on botany, chemistry and pharmacology and ather various disciplines. Based on these considerations, Ethnopharmacology is defined as ‘the interdisciplinary scientific exploration of biologically active agents traditionally employed or observed by man’. Ethnopharmacology is the cross-cultural study of how people derive medicines from plants, animals, fungi, ‘or other naturally occurring resources. Up to now, the field has focused mostly on developing drugs based on ‘the medicinal use of plants by indigenous people. The discipline researches human interactions with biologically active plants (and other living things) as medicines, poisons, and intoxicants with a primary focus on indigenous and non-Western cultures. Ethnopharmacology seeks to document plants and animals used by various cultures, land describe their use and preparation. These plants and their preparations are then studied to identify, isolate, and characterize the active compounds responsible for the plant actions on people. Due to the potential for profit, some drug companies have teamed up with botanists, anthropologists, biochemists, conservation organizations, and governments of less-developed countries to protect biologically diverse areas and search for new drugs and this has renewed the interest of younger indigenous people in preserving their culture, and led to grass-roots, indigenous-based, entrepreneurial businesses targeting alternative medicine markets. ‘An example of ethnopharmacological study is the discovery of reserpine from a traditional medicinal plant (Rauvolfia serpentina). This plant which is called Cheta-chand in Hindi, has been used by local people of ‘alayan Mountains for snakebite. A local legend claims that in ancient times mongooses were observed to feed on the plant before engaging in combat with cobra. Copying the reputed activity of the mongoose, local people found that the shrub could serve as a potent antidote to snakebite. In Bihar Province of India people use the plant to treat insanity, epilepsy and insomnia. Reserpine isolated from Rauvolfia is a potent drug for hypertension. Interestingly, there is a similar legend among Turkmens of Iran concerning a different plant, Verbascum, called Segher-ghureg in Turkmeni. Verbascum species are also used to treat snakebites. Despite the Rauvoifia which finally captured the attention of scientists, Verbascum sp. are still waiting for scientists to have a closer and detailed look on them. Ethnophar-macology as a science bridges the gap between natural sciences and anthropology, should look at ‘symbolic and cognitive aspects. People may select plants because of their specific pharmacological properties, but also because of the symbolic power they may believe is in a plant. Understanding these aspects requires cognitive and symbolic analysis of field data Natural Product Research Natural products are the sources of pharmacologically active agents and recent progress in discovering NCEs has resulted in new drugs that are useful in treating cancer, viral, bacterial and immunosuppressive diseases (Butler, 2005). Discovering new chemical entities (NCEs) from plant sources has been the first mind stuck for the plant chemists in both academics and industry. Compounds of natural origin still play a major role in development of new drugs. About 40% of the world’s best selling drugs are derived directly or indirectly from ‘compounds of plant origin. Paclitaxel, world’s largest selling anticancer drug is the synthetic derivative of Taxol, isolated from Taxus brevifolia (Newman & Cragg, 2007).Opportunities in the field of natural product research: 1, Vast literature in the field of plant studies were reported in the traditional Ayurvedic medical scriptures like Rig veda, Charaka sambita etc. but these literature was not used by natural chemists in this modern ‘era. Now there is a need to use these reference data to understand pharmacology and hence describing the phytochemistry behind the pharmacological aspects of plant materials. 2. In the Ayurvedic system of medicine, traditional people used plants in their surroundings for preventing them from various diseases. In short, the Ayurvedic system is the only system of medicine which claims direct health benefits of plant material on human beings, 3. Lack of standardization report on medicinal plants. 4, Lack of Chemical fingerprints of medicinal plants. Challenges in the field of natural product research: 1. Lack of funds and interest by the government of the developing countries for discovery of natural products. 2. Itis very difficult to isolate and identify new compounds from the plant extracts and also itis very time ‘consuming. 3. Multi Component Analysis (MCA) of plant extracts. Testing and identifying biologically active compounds from plant extracts should be accomplished with defining the structure of that particular compound which is performed by using modern phytochemical and analytical techniques. Liquid Chromatography (LC) and Gas Chromatography (GC), Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (NMR) are methods that are very helpful in Multi Component Analysis (MCA) in the herbal extracts. These techniques are sensitive, selective and fast where simultaneous separations as well as identification of separated components in a mixture are possible. Globally, there is an increasing trend towards natural product research due to large number of opportunities, modernization of analytical tools and availability of validated methods of High Performance Liquid Chromatography (HPLC), GC, GC/MS, LC/MS, LC/MS/NMR, LC/MS/MS. These analytical tools are the important milestones in the natural product characterization, identification and quantification. Ever increasing cost and increasing failure at the end of discovery make medicine unaffordable to the developing countries. The natural products are the new approaches which are more attractive and also with advancement in the analytical methodologies make the research more innovative for drug discovery. It is known to all of us that the drug discovery pipeline in modern drug discovery is getting dry and this modern world is, looking again towards natural products with great expectations.Understanding the Disease ‘Before any potential new medicine can be discovered, scientists work to understand the disease to be treated ‘as well as possible, and to unravel the underlying cause of the condition. They try to understand how the genes are altered, how that affects the proteins they encode and haw those proteins interact with each other in living cells, how those affected cells change the specific tissue they are in and finally how the disease affects the ‘entire patient. This knowledge is the basis for treating the problem. Researchers from government, academia and industry all contribute to this knowledge base. However, even with new tools and insights, this research takes many years of work and, too often, leads to frustrating dead ends. And even if the research is successful, it will take many more years ‘of work to turn this basic understanding of what causes a disease into a new treatment. Recent advances in molecular medicine and powerful tools to enhance computational capacity are enabling researchers to better understand the inner workings of human disease at the molecular level. As our knowledge ‘of disease increases, so does the potential of discovering and developing innovative medicines. ‘Biopharmaceutical companies perform basic research independently and in partnership with researchers and ‘others from across the biomedical research ecosystem, including disease foundations and patient groups, venture capital, and pre-competitive consortia. ‘Models for Studying Diseases The following tools help researchers gain insights into how disease develops. 1. Cell cultures: By growing both diseased and healthy cells in cell cultures, researchers can study differences in cellular processes and protein expression. 2. Cross-species studies: Genes and proteins found in humans may also be found in other species. The functions of many human genes have been revealed by studying parallel genes in other organisms. 3. Bioinformatics: The scientific community generates huge volumes of biological data daily. Bioinformatics helps organize that data to form a clearer picture of the activity of normal and diseased cells. 4, Biomarkers: These are substances, often proteins, that can be used for measuring a biological function, identifying a disease process, or determining responses to a therapy. Thay also can be used for diagnosis, for prognosis, and for guiding treatment. 5. Proteomics: Proteomics is the study of protein activity within a given cell, tissue or organism. Changes in protein activity can shed light on the disease process and the impact of medicines under study.UNIT Ii: DRUG DISCOVERY AND DEVELOPMENT AS A PROCESS: LEARNING OBJECTIVES: ‘To demonstrate knowledge on discovery of lead compounds. b. To understand the significance of early safety tests and technology in developing lead compounds. c._ To identify the different characteristics to consider in conducting preformulation studies. INTRODUCTION This chapter tackles on the different processes and principles that are involved in Drug Discovery and Development specifically the discovery, optimization, validation, early safety test of the lead compound and reformulation studies for the lead compound. ‘Target Identification and Validation Armed with an idea, researchers work to identify biological targets for a potential medicine. A drug target is a ‘molecular structure in the body that, when it interacts with a potential drug compound, produces a clinical effect (lreatment of prevention of a disease, for example). Once they have enough understanding of the underlying cause of a disease, pharmaceutical researchers select a “target” for a potential new medicine. A target Is generally a single molecule, such as a gene or protein, which Is Involved in a particular disease. Even at this early stage in drug discovery it Is critical that researchers pick a target that is “druggable,” |.¢., one that can potentially interact with and be affected by 2 drug molecule. The investigators conduct studies in cells, tissues and animal models to determine whether the target can be influenced by a medicine. After choosing @ potential target, scientists must show that it actually is involved in the disease and can be acted upon by a drug. Researchers demonstrate that a particular target Is relevant to the disease being studied through complicated experiments in both living cells and in animal models of disease. Target validation is crucial to help scientists identify the most promising approaches before going into the laboratory ta develop potential drug candidates, increasing the efficiency and effectiveness of the RED process. Once identified, the target then needs to be fully prosecuted. Validation techniques range from i vitro tools through the use of whole animal models, to modulation of a desired target in disease patients. While each approach Is valid In its own right, Confidence in the observed outcome is significantly increased by a multi-validation approach. Lead Identification Armed with their understanding of the disease, scientists are ready to begin looking for a drug. They search for a molecule, or “lead compound,” that may act on thelr target to alter the disease course. If successful ‘over long odds and years of testing, the lead compound can ultimataly become a new medicine. ‘These are a few ways to find a lead compound: 1. Nature: Until recently, scientists usually turned to nature to find interesting compounds for fighting disease. Bacteria found in soil and moldy plants both led to important new treatments, for example. Nature still offers many useful substances, but now there are other ways to approach drug discovery. 2. Be nevo: The word De novo means “scratch”. It does mean that scientists start from the beginning. ‘They can use sophisticated computer modeling to predict what type of molecule may work. Tt is done by utilizing the 3D structure of the receptor to come up with new molecules that can bind or suit the receptor and involves structural determination of the lead target complexes using molecular modeling tools,(Principle of De nove Drug Desian) 3. High-throughput Screening(HTS): This process is the most common way that leads are usually Found. Advances in rabotics and computational power allow researchers to test hundreds of thousands of compounds against the target to identify any that might be promising. Based on the results, several lead compounds are usually selected for further study. This is @ key process used in drug discovery to- identify hits from compound libraries that may become leads for medicinal chemistry optimization. High-Throughput Screening Speeding Up CF Drug, (Principle of HTS being used in Cystic Fibrosis Transmembrane Conductance Regulater (CFTR) modulator drug discovery by Vertex Pharmaceuticals) 4. Biotechnology: Scientists can also genetically engineer living systems to produce disease-fighting biological molecules. Blotechnological methods have became an important tool in pharmaceutical drug research and development. Today approximately 15 % of drug revenues are derived from biopharmaceuticals. The most relevant indications are oncology and metabolic disorders, Early Safety Test for the Lead Compound Lead compounds go through a series of tests to provide an early assessment of the safety of the lead compound. Scientists test Absorption, Distribution, Metabolism, Excretion and Toxicological (ADME/Tox) properties, or “pharmacokinetics,” of each lead. Successful drugs must be: A. Absorbed into the bloodstream 8. Distributed to the proper site of action in the body C. Metabolized efficiently and effectively D. Successfully excreted from the body E. Demonstrated to be not toxic. These studies help researchers prioritize lead compounds early in the discovery process. ADME/Toxicity studies are performed in living cells, in animals and via computational models. Lead Optimization: Alter the structure of lead candidates to improve properties Lead compounds that survive the initial screening are then “optimized,” or altered to make them more effective and safer. By changing the structure of a compound, scientists can give it different properties. They can also modify the therapeutic index of the drug in order for it to be eligible for clinical trials. For example, they can make it less likely to interact with other chemical pathways in the body, thus reducing the potential for side effects. Hundreds of different variations or “analogues” of the initial leads are made and tested. Teams of bioloaists and chemists work toaether closely: The bioloaists test the effects of analoaues onBiological systems while the chemists take this information to make additional alterations that are then retested bby the biclagists. The resulting compound is the candidate drug. Even at this early stage, researchers begin to think about how the drug will be made, considering formulation (the recipe for making a drug, including inactive ingredients used to hold it together and allow it to dissolve at the right time), delivery mechanism (the way the drug is taken by mouth, injection, inhaler) and large-scale manufacturing (how you make the drug in large quantities). ‘Computer-aided Drug Design (CADD) Drug Design is the inventive process of finding new medications based on the knowledge of a biological target. It involves the design of molecules that are complementary in shape and charge to the biomolecular target with Which they interact and therefore will bind to it. Drug design Frequently but not necessarily relies on computer ‘modeling techniques. This type of modeling is sometimes referred to as Computer-Aided Drug Design. When the disease process is understood at the molecular level and the target molecule(s) are defined, drugs can be designed specifically to interact with the target molecule in such a way as to disrupt the disease, ‘New techniques have revolutionized the ability of researchers to optimize potential drug molecules. Thanks to technologies such as magnetic resonance imaging and X-ray crystallography, along with powerful computer ‘modeling capabilities, chemists can actually “see” the target in three dimensions and design potential drugs to more powerfully bind to the parts of the target where they can be most effective. In addition, new chemistry techniques help scientists to synthesize the new compounds quickly. Computer aided drug design (CADD) is an evolving cascade of research areas encompassing many Facets. ‘Computer-aided drug design (CADD) is an exciting and diverse discipline where various aspects of applied and basic research merge and stimulate each other. The theoretical basis of CADD involves quantum mechanics and ‘molecular modeling studies like structure based drug design; ligand-based drug design; database searching and binding affinity based on the knowledge of a biological target. CADD represents computational methods and resources that are used to facilitate the design and discovery of now tharanautie enlitinns Drug design with the help of computers may be used at any of the following stages of drug discovery: A. Hit identification using virtual screening (structure- or ligand-based design) Hit-to-lead optimization of affinity and selectivity (structure-based design, QSAR, ete.) Lead optimization: optimization of other pharmaceutical properties while maintaining affinity. =) = =a zz (Basic CAD workflow In drug discovery. Wet-lab, SBDD and LADD CADD techniques are outlined in solid lines, dashed lines or dotted lines, respectively. Double headed arrows indicate the two techniques can be used interactively In several iterative rounds of ligand desian.) Goals of CADD: 2. Te shift from: ‘2. Random screening against disease assays. b. Natural products, synthetic chemicals a. Rational drug design and testing b._Speed-up screening process‘¢. Efficient screening (focused, target directed) d. De novo design (target directed) ‘e. Integration of testing into design process. Fail drugs fast (remove hopeless ones as early as possible) Toward the design of new drugs, computer-aided drug design (CADD) can be combined with wet-lab techniques to elucidate the mechanism of drug resistance, to search for new antibiotic targets and to design navel antibiotics for both known and new targets. Notably CADD methods can produce an atomic level structure activity relationship (SAR) used to facilitate the drug design process thereby minimizing time and costs. ‘Two Types of Drug Design 4. Structure-based ‘2. relies on knowledge of the three dimensional structure of the biological target obtained through i. Xray crystallography i, Nuclear Magnetic Resonance (NMR) spectroscopy. 1b. fan experimental structure of a target is not available, it may be possible to create a homology model of the target based on the experimental structure of a related protein. Homology modeling, also known as comparative modeling of protein, refers to constructing an atomic resolution model of the “target” and an experimental three-dimensional structure of a related homologous protein (the “template” ‘<_ Using the structure of the biological target, candidate drugs that are predicted to bind with high affinity and selectivity to the target may be designed using: i. Interactive graphics Intelligence of a medicinal chemist. i. Various automated computational procedures may be used to suggest new drug candidates. 2. Ligand-basea 18. Relies on knowledge of other molecules that bind to the biological target of interest. used to derive @ pharmacophore model that defines the minimum necessary structural characteristics: ‘a molecule must possess in order to bind to the target. b. A model of the biological target may be built based on the knowledge of what binds to it, and {this model in turn may be used to dasign new molecular entities that interact with the target. © Alternatively, a quantitative structure-activity relationship (QSAR), in which 2 correlation between calculated properties of molecules and thelr experimentally determined biological activity, may be derived. These QSAR relationships in turn may be used to predict the activity ‘of new analogs. Pre-formulation Studies Preformulation studies were evolved in 1950 & early 1860. Preformulation testing is the first step in the rational development of dosage forms of a drug substance. It can be defined as.an investigation of physical and chemical Properties of 2 drug substance alone and when combined with excipients. The overall objective of reformulation testing Is to generate information useful to the formulator in developing stable and bioavailable dosage forms that can be mass produced. Preformulation investigations are designed to deliver all necessary, data especially physicochemical, physico- mechanical and bio pharmaceutical properties of drug substances, excipients and packaging materials. Preformulation studies help in assessing the “drugability’ of 2 molecule. Prefermulation can thus be considered 13: a critical decision-making tool during both — drug discovery and development phase. A comprehensive understanding of physicochemical properties and its effect on biological performance, allows selection of Potential lead molecules and in identification of drug delivery challenges.‘To establish the physical characteristics ‘To establish the kinetic rate profile. ‘To establish the compatibility with the common excipient. ‘To. choose the correct form of a drug substance. mone 1. Physical Characteristics: Organoleptic properties Bulk characteristics Solid state characteristics Flow properties b. a. e £ 9. Crystalline he i i k. lL rn. °. UNIT ITT: PRECLINICAL PHASE Objectives: a. To identify the steps involved in the preclinical phase of drug discovery and development. b. To demonstrate a complete understanding on the importance of toxicity screening in DDD. c. To demonstrate proper ethical protocols and principles in conducting human and animal testing, INTRODUCTION Tn this chapter, the concept of preclinical phase will be discussed. This includes the biologic activity test, chemical synthesis, formulation studies, stability testing and safety test on animal and human subjects.Preclinical Phase Many of the lead compound's critical properties. This team might continue to work with the compound throughout the entire development process or the development responsibilities right be transferred to another Group of scientists during the clinical testing phase. Preclinical testing includes: 1. Discovery testing to ensure biological activity in vivo 2. Chemical synthesis and scale-up to ensure adequate quantities of high purity can be made 3. Formulation development and stability testing to characterize various chemical properties, develop the initial drug delivery system, and determine the stability of the compound 4. Animal safety testing to ensure limited toxicities of the lead agent. At this stage of the development process, good laboratory practices (GLPs) are followed and Qualifications of personnel and requirements for standard operating procedures are specified. During discavery testing, the specifics of the compound's properties, such as the mechanism of action in animal models, compound specificity, duration of action, and structure-activity relationships, are determined. Adequate quantities of the new chemical ‘compound must be produced at a high level of purity. Impurities present at concentrations greater than 0.1% must be characterized and tested for toxicity. The physicochemical properties of the active compound are determined, and development of the drug delivery system to be used in human testing begins. Animal testing provides initial data regarding the absorption, distribution, metabolism, and excretion (ADME) in a living system. Possible side effects and toxicities are noted. Toxicity studies of at least the same duration as the proposed human testing and @ minimum of two weeks must be completed. Active and inactive metabolites must be characterized. Often, the most appropriate animal model to predict human response is not known, thus toxicity studies are conducted in at least 2 animal species, one rodent and one nonrodent, to obtain a comprehensive view of the potential toxicity. Early absorption, distribution, metabolism, and excretion or toxicity problems may be corrected by slight modifications in the chemical structure of the new entity. Animals should be given the new drug product by the same route intended for humans. Certain dosage forms, such as aerosol, nasal, or buccal delivery systems, might be difficult to administer to animals. In these circumstances, alternative drug delivery routes may be used, and the selected route of administration should ensure sufficient exposure to the new chemical entity. During animal safety testing, dosing studies are conducted, and the highest no-effect dose is determined. In addition to dose, plasma concentrations of the Grug are followed, and noted toxicities are correlated to dose andjor plasma concentration. Generally, once discovery testing shows therapeutic promise, the chemical synthesis, formulation development, and animal safety testing occur concurrently (see Figure below). Although resources may be wasted on earlier failures, successful candidates will be ready for human testing more quickly. The administration of drugs in humans at the earliest time possible ultimately saves valuable resources, as highly toxic compounds can be eliminated and alternative lead compounds can be developed. Additional preclinical studies may be conducted during clinical testing to support larger trials and, eventually, the marketing of the drug product. Formulation development continues throughout the process, and the data gained from both animal and human testing allow for optimization of the drug delivery system. It is imperative to identify and resolve formulation problems early in the development process, as unresolved problems will surely reemerge later, costing the company both time and money as clinical testing is delayed. More chronic animal exposure experiments are conducted to support further clinical testing.‘Drug Omcovey fond Lead Compound "Soloction Phase ‘Clinical Tras FDA Conditionat FDA ‘Approval Post-Approval Safety Ropers te FDA Moke Drug Safety Evaluation With one or more optimized compounds in hand, researchers turn their attention to testing them extensively to determine if they should move on to testing in humans. Scientists carry out in vitro and in vivo tests. In vitro tests are experiments conducted in the lab, usually carried out in test tubes and beakers (“vitro” is “glass” in Latin) and in vivo studies are thase in living cell cultures and animal models (“vivo" is “life” in Latin). Scientists try to understand how the drug works and what its safety pprofile looks like. The U.S. Food and Drug Administration (FDA) requires extremely thorough testing before the candidate drug can be studied in humans. During this stage researchers also must work out how to make large enough quantities of the drug for dlinical trials. Techniques for making a drug in the lab on a small scale do not translate easily to larger production. This is the first scale up. The drug will need to be scaled up even more if it is approved for use in the general patient population. At the end of several years of intensive work, the discovery phase concludes. After starting with approximately 5,000 to 10,000 compounds, scientists now have winnowed the group down to between one and five molecules, “candidate drugs,” which will be studied in clinical trials. Toxicity testing or studies of new compounds is essential for the drug development process. The preclinical toxicity testing on various biological systems reveals the species-, organ- and dose- specific toxic effects of an investigational product. The toxicity of substances can be observed by (a) studying the accidental exposures to a substance (6) in vitro studies using cells/ cell lines (¢) in vivo exposure on experimental animals. The preclinical toxicity testing helps to calculate "No Observed Adverse Effect Level” which is needed to initiate the clinical evaluation of investigational products. Usually toxicants are classified based on their chemical nature, mode of action, or class (exposure class and use class). The exposure class classifies toxicants as occurring in food, air, water, or soil. The use class dlassifies drugs as drugs of abuse, therapeutic drugs, agriculture chemicals, food additives, pesticides, plant toxins (phytotoxins), and cosmetics. Usually toxicants are classified based on their chemical nature, mode of action, or class (exposure class and use class). The exposure class classifies toxicants as occurring in food, air, water, or soll. The use class dassifiesdrugs a5 drugs of abuse, therapeutic drugs, agriculture chemicals, food additives, pestades, plant toxins (phytotoxins), and cosmetics. In vive methods In witro toxicity testing shouid build upon test madels that are relevant for the species to be protected. Proper test development requires well defined test compounds with high quality 47 vivo data (gold standard) and cell systems that mimic Jp vitro the key events that are known to.eccur Jn vive, This type of screening ie carried out in the laboratory ware in the absence of living animals. The onimals widely used for this method are rodents (mice, rats, rabbits, hamsters), most of which are raised commercially to offer the pharmacologists uniformity of genetic, nutritional, sex and health standards. Toxicity Studies 1 Acute toxicity testing A. Acute toxicity testing Is carried out to determine the effect of a single dose on 2 particular ‘animal species. In general, it is recommended that acute toxicity testing be carried out with two different animal species (one rodent and one nonrodent). In acute toxicological testing, the investigational product is administered at different dese levels, and the effect is observed for 14 days. All mortalities caused by the investigational product during the experimental period are recorded and morphological, biochemical, pathological, and histological changes in the dead animals are investigated. I. Acute toxicity testing for inhalation A. Acute inhalation toxicity testing is performed for aerosol-like preparations. Rats are the most preferred animal species. The animals are acclimatized to Inboratory conditions (temperature preferably 22°C + 2°C). They are maintained in an air flow of 12-15 air changes per hour with adequate oxygen (1994/h). The animal is exposed to the test substance for 2 minimum of 4h, and then It is monitored for 14 days. Food Is withheld during the exposure period, and water may be withheld under certain conditions. During the observation period, the animal is observed for tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Mortality during the exposure and observation periad is noted. Dead animals are exemined for histological and Pathological changes. At the end of the study, the animals are sacrificed, and pathological changes are evaluated III. Acute toxicity testing for topical preparations A. The eye inntation test and skin imtation test are very Important for topical preparations. Dermal ‘and ophthalmic preparations can be tested using Draize tests. The Draize eye inritancy test and the Draize skin inritancy test are used to measure the harmfulness of chemicals and pharmaceutical substances in rabbits and guinea pigs. In the eye irritation test, 0.5 mil of a tast_ substance is administered to an animal's eyes, and the animal is restrained for 4 h. Redness, swelling, discharge, ulceration, hemorrhage, and blindness are assessed and monitored for 14 days. B. In the skin irritation test, 0.5 g of a test substance is applied to the surface of an animal's skin. During te observation period (14 days), signs such as erythema and edema are assessed. ‘Some alternative Jin vitro testing methods are available that can be used in place of the Draize eye irritancy test. At the end of the study, the animals are sacrificed and pathological changes ‘are evaluated. IV. Skin sensitization tests ‘A. Skin sensitization tests are carried out using the guinea pig as a model. Skin sensitization is ‘assessed using the Draize test. open epicutaneous test, optimization test, split adjuvant test, ‘guinea pig maximization test (GPMT), Buehler test, and murine local lymph node assay (LLNA). The LLNA method Is used 25 an alternative to the guinea pig Draize test, and It Is widely ‘accepted that this method meets regulatory requirements. In the LLNA test, the test substance is applied on the surface of the ears of a mouse for three consecutive days, and the proliferation ‘of lymphocytes in the draining lymph node is measured at the end, V. Mutagenicity testing A. Mutagenicity testing is used to assess submicroscopic changes in the base sequence of DNA, ‘chromosomal aberrations, and structural aberrations in DNA including duplications, insertions, inversions, and trenslocations. Certain types of mutations result in carcinogenesis (alteration inproto-oncogenes of tumor suppressor gene mutation), and so the determination of the mutagenicity is essential in the drug development process. 17 wire testing Is carried out in two or three different bacteria and mammalian cells to cover the end points of gene mutations, dlastogenicity, and aneuploidy. The test generally indudes a bacterial reverse mutation assay. “The choice: of an additional test depends on the chemical structure/class of the substance. 2 vivo mutagenicity which is dese dependent ic used to determine the cace-by-cace basis risk assessment of the test substances. Mutagenicity studies with transgenic animals are more appropriate assay techniques to determine the toxicity of a test substance. VI. Carcinogenicity testing ‘A. Both rodents and non rodent animal species may be used in carcinogenicity testing. The tests. are carried out over the greater portion of an anima''s lifespan. During and after exposure to test substances, the experimental animals are observed for signs of toxicity and development of tumors. If these are not found, a test may be terminated after 18 months in the case of mice ‘and hamsters and after 24 months with rats. IF the animals are healthy, hematological analysis |S performed after the 12 months ang the 18 months, respectively, and the study is terminated. The animals are sacrificed, and grass pathological changes are noted and histopathological studies are carried out on all the tissues. Ethical Considerations in Human and Animal Experimentation Before using animals, it is mandatory for researchers to clearly clarify their scientific purpose. There should be a reasonable expectation that the research will result in increasing scientific knowledge in different aspects of biomedicine and also will increase understanding of the species under study or provide results that could improve the quality of health or welfare of humans or ather animals. The scientific purpose of the research should he of sufficient potential significance to justify the use of animals. The species. chosen for study shauld bbe the best suited to answer the question(s) posed. Moreover, it is noteworthy that good experimental design hhelps reduce the number of animals used in research since they allow scientists to collect data using the minimum number of animals required. However, a sufficient number must be used to enable precise statistical analysis and results, preventing the repetition of experiments and the consequent need to use more animals. All procedures on animals are to be reviewed by 2 local animal care committee to ensure that the procedures are appropriate and humane. In the event that it is not possible to constitute an appropriate local animal care ‘committee, scientists are encouraged to seek advice from a corresponding committee of 2 cooperative Institution, Responsibilities for the conditions under which animals are kept, beth within and outside of the ‘context of active experimentation or teaching, rests with the researcher under the supervision of the animal ‘care committee and with individuals appointed by the institution to oversee animal care. Ethical Requirements For Human Testing “When people are invited to participate in research, there is a strong belief that it should be their choice based on their understanding of what the study is about, and what the risks and benefits of the study are,” said Dr. Christine Grady, chief of the NIH Clinical Center Department of Bioethics, to Clinical Center Radio in a podcast. Clinical research advances the understanding of science and promotes human health. However, it is important to remember the individuals who volunteer to participate in research. There are precautions researchers can take ~ in the planning, implementation and follow-up of studies ~ to protect these participants in research. Ethical guidelines are established for clinical research to protect patient volunteers and to preserve the integrity of the science. The National Ethical Guidelines is a distinct manifestation of the country's commitment to the protection of the rights, welfare, and well-being of human participants in research, and to research integrity. Ethical assessment of health and health-related research requires a framework that may consist of principles, values,Guidelines for Ethical Conduct in the Care of Human subjects (Clinical Phase) in Drug Testing ‘Social Value ‘The participation of human beings in research can only be justified if the study has social value. Social value refers to the relevance of the study to an existing social or health problem such that the results are expected to bring about a better understanding of related issues, or contribute to the promotion of well-being of individuals, their families, and communities. ‘The significance of the study shall be clearly described in a separate section of the protocol with an ‘accurate and updated description of the status of the social or health problem, and how the study will help arrive at a solution. ‘The study design, methodology, and data collection, overall, should be able to generate information ‘supportive of the objectives of the study. Social value can only be realized if the study is scientifically valid. ‘A dissemination plan for the study results shall be included in the protocol. Dissemination is essential to achieving social value. ‘The REC shall determine the appropriateness and the practicability of the dissemination plan, as well as the suitability of the recipient(s) of the information, ‘Informed Consent 10, Ll. Informed consent is a decision of a competent potential participant to be involved in research after receiving and understanding relevant information, without having been subjected to coercion, undue influence, or inducement. Obtaining informed consent is a process that is begun when initial contact is made with a potential participant and continues throughout the course of the study. By informing the potential participants, by repetition and explanation, by answering their questions as they arise, by ensuring that they understand each procedure, and by obtaining agreement from them, researchers elicit their informed ‘consent, and in so doing manifest respect for their dignity and autonomy. For all research involving humans, the researcher shall obtain the voluntary informed consent of the prospective research participant. In the case of an individual who is incapable of giving or who has. diminished capacity to give informed consent, the researcher must exert effort to obtain his or her ‘assent and the consent of a legally authorized representative (LAR), in accordance with applicable laws. In obtaining informed consent, sponsors and researchers shall have the duty to avoid deception, undue influence, or intimidation. Informing the potential participant shall not be simply a ritual recitation of the contents of a written document. Rather, the researcher shall convey the information, whether orally, in writing, or other modes of communication, in a language and manner that suit the individual's capacity and level of understanding. ‘The researcher shall ensure that the prospective participant has adequately understood the information. The researcher shall give each one the full opportunity to ask questions, and should ‘answer them honestly, promptly, and completely.Essential information for participants 12. The researcher shall provide the following information to the potential research participant, whether orally or in writing, in a language that suits the participant's level of understanding: 121. 12. ‘That the individual is invited to participate in the research which is being undertaken by the researcher (name of researcher) from the institution (name of institution); ‘The reasons for considering the individual suitable for the study, and that participation is voluntary; 126. 127. ae. 12.9. 12.10. aa. 12.12. 12.13. 1.34. 12.15. 12.16. 12.47. 12.18. 12.19. “Any Toresceable Wicks, pain oF discomfort, or inconvenience to the individual (or others) associated with participation in the research (in both the control and experimental group), Including risks to the health or well-being of the individual's spouse or partner; “The direct benefits, if any, expected to manifest to Individuals from participating in the research; ‘Whether money or other forms of material goods will be provided in return for the individual's Participation and, if so, the kind and amount; "Tne expected benents of the research {0 the community OF to society at large, or contripution to scientific knowledge: Whether, when, and how, any intervention proven by the research to be safe and beneficial will be made available to the individuals after they have completed their participation in the research, and whether they will be expected to pay for them; ‘The provisions to ensure respect for the privacy of research participants and the confidentiality of records in which they are identified, Including documentation through taking of pictures and recording of the interview and that these might be displayed in publications and conferences or fora; Legal of other limits to the researcher's ability to safeguard confidentiality, and the possible consequences of breaches of confidentiality; “The participants are free to withdraw from the research at any time without having to give any reason, and without penalty or loss of benefits to which he or she is entitled; ‘The sponsors or funders of the research, the institutional affiliation of the researchers, and the ature and sources of funding for the research: ‘The possible research uses, direct or secondary, of the individual's medical or health records, land the possible future use and final disposition of biological specimens; If the speamens collected will not be destroyed, then where, how, and for how long they are going to be stored; ‘That the research participants have the right to decide about future use, continued storage, or destruction of collected specimens and/or personal information; Whether commercial products may be developed from biological specimens, and whether the research participant shall receive monetary or other benefits from the development of such products; ‘The extent of the researcher's responsibility to ensure needed services to the research participant; “That treatment and rehabilitation will be provided free of charge for specified types of research= related injury or for complications associated with the research, the nature and duration of such care, the name of the medical service or organization that will provide the treatment and whether there is any uncertainty regarding funding of such treatment; “That a PHREB-accredited REC has approved or cleared the research protocol; “The contact information of persons designated to respond to the following: 12.221. Queries on the details of the protocol; 12.22.2. Issues relating to the human rights of participants; 12.22.3. Related concerns and grievances; and 2.22.4. Management of research-related Injuries. Documentation of consent 13. As @ general rule, documentation of informed consent Includes an actual signature or thumb mark of the prospective participant on the informed consent form. 114, When the use of an informed consent form is not feasible or unacceptable to the prospective participant, {2 description of the process, attested by a witness, may be an alternative that needs prior approval of the REC.‘Waiver of the informed consent 15. 16. 17. Waiver of individual informed consent is to be regarded as exceptional, and must be approved by an REC. ‘The informed consent process may be waived in specific research contexts, such as: 16.1. Archival research involving publicly available documents; 16.2. Research that uses the method of naturalistic observation (often described as “covert” method) in data collection provided that all of following requirements are complied with: Thorough iustification for the use of naturalistic abservation: 16.3, Plan for now the data collected will be used; 16.4. Assurance that risks to participants are unlikely; and 16.5. Mechanism to ensure confidentiality and anonymity of observed individuals and their data (e.9., observations are recorded in such a way that the Individuals involved are net identifiable). ‘Some or all of the elements in the informed consent may be waived or altered (with prior approval of the REC) if all these conditions are met: 17.1. ‘The research presents no more than minimal risk; 17.2. The waiver or alteration will not adversely affect the rights and welfare of the participants; 473. The research cannat be practicably carried aut without the waiver ar alteration; 17.4. The participants will be provided with additional pertinent information after thelr participation (whenever appropriate). Renewing consent 18. ‘The informed consent of each research participant shall be renewed under the following conditions: 18.1. If there are any significant changes in the circumstances or procedures of the research; 18.2. If new information becomes available that could affect the willingness of research participants, to continue to participate; or 18.3. _ Th long-term studies at predetermined intervals even if there are no changes in the design or objectives of the research, Vulnerability of Research Participants 19. 2. Vulnerable participants shall require special protection because of certain characteristics or situations that render them as such. Vulnerable participants are these who are relatively or absolutely incapable of deciding for themselves whether or not to participate in a study for reasons such as physical and mental disabilities, poverty, asymmetric power relations, and marginalization, among others and who are at greater risk for some harms. Vulnerable groupe chall not be included in research unless such research: 20.1.1. Ts necessary to promote the welfare of the population represented: 20.1.2. Cannot be performed on non-vulnerable persons or groups. Researchers, sponsors, or RECs shall not arbitrarily exude women of reproductive age from biomedical research. The potential for becoming pregnant during a study shall net, in itself, be used as a reason for precluding or limiting women’s participation in research. Competent advice and assistance shall be provided to participants who, by virtue of social, economic, political or medical disadvantages, are liable to give consent under duress or without the benefit of adequate information. Caution shall be exercised in obtaining informed consent for a research project It the research participant Is in a dependent relationship with the researcher (e.g., as a research participant) to ensure that the consent is net given under duress or undue influence.Risks, Benerits, ana Sarety 23. Research Is justified if there Is 9 reasonable likelihood that the population from which the participants are derived stand to benefit from the research. 24. Ailresearch involving human participants shall be preceded by @ careful assessment of predictable risks, burdens, and foresaeable benems to the research partiopant or to others. 25. Every precaution shall be taken to minimize the negative Impact of the study on the research 26, Research shail be conducted only if there is an acceptable positive benefit-risk ratio. 39. The researcher/funder/sponcor shall endeavar to encure the rearonable availability and accocsibilty of favorable research outcomes to the communtty- 28. When there is ethical and sclentin Justification to conduct research with Individuals capable of olving: IGemed consent, the nak from fescarch interventions that do nat held out the prospect af direct bene for the individual participant shall ba ne more likely ahd no greater than the risk attached to FOUUNe medical or peychological examination OF such person=. Slight or minor increases above such risk may the REC has approved therm. Privacy and Confidentiality of Information 29. Researchers shall adhere to the principles of transparency, legitimate purpose, and proportionality in the collection, retention, and processing of personal Information (Data Privacy Act of 2012). 30. "Researchers must respect participants’ right to privacy. Unless required by law, the confidentiality OF Information shall at all times be observed. Records that link individuals to SPeciNc information shall not be released. This requirement shall be included In the informed consent form. 31. Researchers shall refrain from identifying individuals or groups when release of information about them can expose them to possible harm of social stigma unless required by law. 32. Where there Is. some likelihood or opportunity for the researcher to observe the occurrence of illegal or harmful behaviors (e.g., child abuse, substance use, self-harm, of suicide ideation), the researcher ‘shall: 32.1. Explicitly indicate the limits of confidentiality in the Informed consent process, such as when tne researcher Is ethically and legally obligated t disclose the identity of the respondent £O forestall imminent harm to self or others; 32.2. Emphasize the right of the respondent to withdraw from the study or withdraw his or her data, and to refuse to answer any question; and 32.3. Prepare a concrete: and realistic protocol for reporting and referral in the event that imminent harm and/or a criminal act is disclosed or discovered in the process of data collection. 33. Researchers shall recognize that collecting data using group methods (e.g., FGDs) has implications for the privacy and confidentiality of individuals. As It might not be possible for researchers to ensure the confidentiality of Information or the anonymity of research participants, the researcher shall ensure that the nature of the study and the questions would cause minimal harm should confidentiality or anonymity The researcher shall describe his or her data protection plan in the protocol, including the steps to be taken so that all who have access to the data and the identities of the respondents can safeguard privacy and confidentiality. For example, the researcher shall provide adequate and clear Instructions to research assistants, transcribers of audio recordings, or translators of transcriptions.2 analyses shall be clearly described. 3. All procedures, whether Invasive or not, shall be satisfactorily described in detall. 4. The research protocol shall adequately address the elements of research ethics as part of the Ethical Considerations section. 5. The protocol shall provide information on how the safety and welfare ef research partidpants shail be protected. UNIT IV: CLINICAL TRIALS. LEARNING OBJECTIVES: 3. define clinical trials BL describe the design and types of clinical trials: €: discuss the importance of conducting clinical trials in drug discovery According to WHO, Clinical tials ore a type of research that studies new tests and treatments and evaluates their effects on human health outcomes. People volunteer tO take part in clinical trials. to test inedical interventions including drugs. cells and other biological products, surgical procedures, radiological Procedures, devices, behavioural treatments and preventive care. Clinical trials are carcfully designed, reviewed and complated, and need to be approved before they can start. People of all ages can take part in clinical trials, including children. While precinical research answers basic pout a orug’s safety, it Is not a substitute ror ‘stuches of ways the Urug will Intoract with the human body. +Ciinical research” refers tr stiches, OF tii, a "ine re cheniie tite: clinical stuns, tivey. wall Consicier whist they “went es1. Controlled clinical trial = Subjects are divided into the treatment group (those given the investigational drug) and 2 control group = Depending on the purpose of the trial, patients in the control group get no treatment, a placebo (an inactive product resembling the investigational drug). another drug known to be effective or a different dose of the drug under study = Treatment and control group must have the disease = Treatment and control should also be similar in age. weight, general health 2. Randomization = Subjects are randomly assigned to treatment or control 3. Blinding = In Sinale blind study, patients do not know whether they are receiving the investigational drug or placebo In double blind study, patients, investigators and data analysts do not know which patients received the investigational drug. Only when the assignment code is broken, usually at the end of the trial, is it possible to identify treatment and control patients. Preparing a promising new drug to start a clinical trial is a significant stage in the drug development. process. There has been a significant investment in identifying an active pharmacologic compound , developing a stable formulation and performing pharmacologic and toxicological tests. Safety in animal models has been established to a degree that the pharmaceutical firm feels that there are sufficient data to being testing in humans. Links: Clinical Trials — What you need to know 5 //vwws. 1tch?v=6B5S3-nTdD4 Giinical Trial Players: http://www youtube.com/watch?v=0uPdioONiJa,