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Principles of Pharmacological and Pre-clinical Evaluation of DrugsDepartment of Pharmaceutical Sciences, Nagpur01/02/20131Introduction:-Pharmacology Science of drugs Categories of PharmacologyPharmacognosy study of characteristics of natural drugs and their sourcesPharmacodynamics study of what drugs do to the bodyPharmacokinetics what the body does to drugsPharmacotherapeutics study of how drugs are used to treat diseaseToxicology study of poisonous effects of drug PharmacodynamicsStudy of what a drug does to the body

Includes interaction between the drug and target cells or tissues and the bodys response to that interaction01/02/20132 PharmacokineticsThe study of what the body does to a drug includes:Absorbs converts a drug into a form the body can useMetabolizes drug molecules are transformed into simpler products Distributes transporting a drug from its site of administration to its site of action Excretes manner in which a drug is eliminated from the body

Principles of PharmacologyMedication safety Medication legislation Naming Classifications Schedules PharmacokineticsStorage and security Autonomic pharmacology 01/02/20133Metabolism and excretion Mechanism of action Phases of medication activity Medication response relationshipsMedication interactions Toxicity Principles of Pharmacology (contd) Pharmacotherapeutics- Safety and efficacySafety is how many and what kinds of adverse effects are associated with the drug.An adverse reaction may require immediate action.Be alert to complaints from patient after starting new drug.

Efficacy:- Drug working as excepted

01/02/201344ToxicologyStudy of poisonous effect or toxicity of drugs Toxic effects includes: Adverse effect on a fetus or infant Adverse reactions reported in clinical trials Adverse effect in pediatric or elderly patients

01/02/20135Pre-clinical Evaluation of Drugs Introduction Before a pharmaceutical company can initiate the testing in humans, it must conduct extensive preclinical or laboratory research

This research typically involve years of experiment in animals and human cells Definition Research using animals to find if a drug, procedure or a treatment is likely to be useful

Preclinical study take place before any testing in humans is done Objective of preclinical study Support for human pharmacology

Support for human toxicology

Prediction of human pharmacokinetics

Screening of new dosage form and formulation

01/02/20136Preclinical Study DesignAssess pharmacology fate in relevant animal model of disease/injury, as feasible Assess safety/toxicology in healthy animals pharmacology-toxicology study designApply the 3 Rs Reduce, Refine, Replace

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Commonly used Laboratory animal in pharmacological research

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Selection of Animal Species 01/02/20139

01/02/201310Standard testing used in laboratory animal Animal test are conducted all over the world

Majority of animals are used for testing are rats and mice. Other animals used are hamster, rabbit, guinea pig, dogs, cats, nonhuman primates, sheep and cattle Millions of the animals listed above are locked inside cold cages in laboratory, waiting in fear to be experimented with next painful procedure

Every year more than 100 million animals suffer and die from animal testing

Before a product is claimed as human friendly hundreds of animals are poisoned, blinded and kill every year01/02/201311Types of animal testingIn vivo studies Human-basedSkin irritationPyrogenicityModular immune in vitro study In vitro studies Cell culture and tissue engineering Molecular targets01/02/201312Preclinical Development in vitro Studies Pharmacology:

Molecular mechanisms of action Mechanisms of drug resistance Determinant of response Intracellular pharmacodynamics Molecular pharmacology Eg: In vitro studies of anticancer agentsPreclinical Development in vivo Studies-Animal Models:

Proof of therapeutics principle Animal pharmacokinetic and pharmacodynamics Toxicology Starting dose and schedule for clinical trial01/02/201313Euthanasia refers to the practice of intentionally ending a life in order to relieve pain and suffering.

Classification of euthanasia

Voluntary euthanasia Euthanasia conducted with the consent of the patient is termed voluntary euthanasia

Non-voluntary euthanasia Euthanasia conducted where the consent of the patient is unavailable is termed non-voluntary euthanasia. Eg: Child euthanasia

Involuntary euthanasia Euthanasia conducted against the will of the patient is termed involuntary euthanasia.Euthanasia01/02/201314 Passive and active euthanasia Voluntary, non-voluntary and involuntary euthanasia can all be further divided into passive or active variants.Passive euthanasia implies the withholding of common treatments, such as antibiotics, necessary for the continuance of life. Active euthanasia implies the use of lethal substances, such as administering a lethal injection, to kill. The choice of a method will depend on the nature of study, the species of animal to be killed. The method should in all cases meet the following requirements: Death, without causing anxiety, pain or distress with minimum time lag phase. 2. Minimum physiological and psychological disturbances. 3. Compatibility with the purpose of study and minimum emotional effect on the operator. 4. Location should be separate from animal rooms and free from environmental contaminants. 01/02/201315Euthanasia Of Laboratory Animals

01/02/20131601/02/20131701/02/201317Precautions:-Method should be reliable, reproducible and irreversible.-Not in the housing environment.-It should not produce distress vocalizations, pheromones in anxiety.-Confirmation by observing the cessation of the heart best, respiration and absence of reflexes.-Can be guaranteed by exsanguinations or decapitations.

-Physical method: Concussion- Blow on the head of the animals.

RECENT DEVELOPMENTS

-Microwave irradiation: Use of microwave beam-Neuropharmacological studies-Useful for fixation of brain metabolites-Without destroying the anatomical areas of brain.01/02/201318Regulations For The Laboratory Animal Care And Ethical Requirements CPCSEA Guidelines For Laboratory Animal Facility (committee for the purpose of control and supervision of experiments on animals, Ministry of Environment and Forests, Government of India)Good Laboratory Practices (GLP) for animal facilities is proposed to assure quality maintenance and safety of animals used in laboratory studies while performing behavioral research and testing of productsGoal The goal of these Guidelines is to promote the humane care of animals used in behavioral research And testing with the basic objective of providing specifications that will enhance animal well being, quality in the purpose of advancement of biological knowledge that is relevant to humans and animals.GLP addresses: Regulatory requirements Occupational Health and Safety Housing and Routine Care Animal Identification Sex Determination Clinical Signs of Illness Euthanasia1801/02/201319Regulatory Requirements

To ensure the humane treatment of laboratory animals, animal research is regulated by two federal agencies:

The United States Department of Agriculture (USDA) / Animal Care enforcing the Animal Welfare Act Regulations

The National Institutes of HealthHousing and Routine Care:Animal Crowding Animal crowding in a cage affects environmental quality (the accumulation of urine, for example, leads to excess ammonia and moisture).

Crowding can also cause newborn pups to be injured or killed. Crowding is a special concern for multiple litters in a cage since pups grow very quickly and rapidly increase their output of excreta.

In particular, if the mother is about to give birth to a second litter, the first litter should be weaned and removed to new cages to prevent smothering and trauma of the newborns.01/02/201320Routine Sanitation

Routine sanitation and environmental controls are necessary for protecting animal health and for minimizing the introduction for non-experimental variables which could undermine the quality of research data.

Sanitation schedules vary according to the type of mouse caging.

Based on the types of caging and bedding in use at your facility, your institution will have a standard operating procedure (SOP) on the sanitation schedule for mouse cages.PHYSICAL FACILITIESBUILDING MATERIALS should be selected to facilitate efficient and hygienic operation of animal facilities. CORRIDOR(S) should be wide enough to facilitate the movement of personnel as well as equipments and should be kept cleanUTILITIES such as water lines, drain pipes, and electrical connections should preferably be accessible through service panels or shafts in corridors outside the animal roomsANIMAL ROOM DOORS should be rust, vermin and dust proof01/02/201321EXTERIOR WINDOWS they may be necessary to provide alternate source of light and ventilationFLOORS should be smooth, moisture proof, nonabsorbent, skid-proof, resistant to wear, acid, solvents, adverse effects of detergents and disinfectantsDRAINS To prevent high humidity, drainage must be adequate to allow rapid removal of water and drying of surfaces

WALLS & CEILINGS Walls should be free of cracks, unsealed utility penetrations, or imperfect junctions with doors, ceilings, floors and cornersSTORAGE AREAS Separate storage areas should be designed for feed, bedding, cages and materials not in useFACILITIES FOR SANITIZING EQUIPMENT AND SUPPLIES An area for sanitizing cages and ancillary equipment is essential with adequate water supply EXPERIMENTAL AREA All experimental procedures should be carried out in a separate area away from the place where animals are housed

01/02/201322Environmental Controls The Guide for the Care and Use of Laboratory Animals establishes standards for laboratory animal environments in regard to:

Room temperature 18 to 29C (64.4 to 84.2F) Humidity 30% to 70% Ventilation Illumination and light schedule Noise abatement

The details of these standards are generally not a concern to researchers who house their animals in an institutional animal facility, because it is the animal care staff who have the responsibility of operating the facility in compliance with these environmental standards

Researchers should be aware of the lighting schedules used in the rodent housing rooms (commonly 12 hr light:12 hr dark or 14 hr light:10 hr dark)

If researchers working late turn on the lights in the animal rooms during the dark period, the disruption of the light schedule may cause the animals to be perturbed, which may have effects on breeding performance and on circadian rhythms.

01/02/201323 It's important to remember the following:

As a small animal, mice can be easily injured if handled roughly. You should learn how to handle them firmly but gently and with confidence to avoid injuring these delicate animals

Mice are inclined to become aggressive and bite. Although their teeth seldom break through your skin, a bite can hurt! Develop your confidence in handling mice by learning from an experienced mentor and practice hand restraint first on anesthetized mice

Animal Handling and Restraint

01/02/201324Hand Restraint There are two common hand methods for restraining mice. Mice are picked up the following way:

If your mice are in barrier or containment housing, please open their cages in an appropriate hood or laminar-flow workbench

Remove the cage top if they are housed in a filter-top cage

Place the wire lid top sideways on top of the cage

Pick up a mouse by the tail (away from the tail tip) and lift the mouse directly to the wire lid. You will find that the wire lid is a useful area to which the mouse will want to hang on with its front feet, allowing you the opportunity to reposition your grasp

01/02/201325Tail Wrap MethodIn the tail wrap method, you transfer the tail from your thumb and forefinger to the 4th and 5th fingers of the same hand. These fingers press into your palm the mouse's tail, close to the animal's rump

Then, your thumb and forefinger grasp a scruff of skin at the occiput of the mouse

In this method, the mouse is held by two points: the occiput and the base of the tailTo further immobilize the hindquarters, you may gently entrap one leg along with the tail beneath your 5th finger. It is important to provide back support to the mouse and avoid hyperextension of its back01/02/201326Points to remember: When picking up a mouse

Pick up the tail at the middle, not the tip. A mouse does not need to be picked up at the base of the tail like a rat does, because the mouse is light and its weight will not damage its tail

Never dangle a mouse by its tail, but instead lift it directly to the cage wirelid or some other support. When dangled, mice appear anxious as seen by the their behavior in hyper extending their feet

If you need a place to briefly sort and hold your mice, say while you are rapidly administering injections to a cage of mice, each mouse can be placed on the wirelid after its injection01/02/201327

Sex and Age

01/02/201328Reproductive Data Of MiceStage

Sexual maturity

Estrous cycle

Gestation

Weaning age

Reproductive life

Life span

Pups begin eating dry foodAge

40-60 days

4 5 days

21 days

21-30 days

8 months

1 - 3 years

12 14 daysProestrus (12 h)Estrus (9-15 h)Metestrus (20 h)Diestrus (60-70 h)01/02/201329Types of Observations A common approach to assessing animal appearance and behavior is through observation of the following:

Activity Level e.g., hypoactivity (hunched, huddled, lethargic), hyperactivity, restlessness, lack of inquisitiveness

Attitude e.g., arousal, depression, awareness of surroundings

Behavior, Spontaneous e.g., vocalization, self-trauma, isolation from cage mates. These observations are made without disturbing the animal

Behavior, Provoked e.g., vocalization, hiding, aggressiveness, minimal response. These observations are made when the animal is disturbed or even prodded

Body Condition e.g., emaciation, missing anatomy

Food and Fluid Intake e.g., elimination of feces and urine

01/02/201330 Fur and Skin e.g., unkempt or greasy or dull fur; porphyrin staining around eyes and nostrils; cyanotic, pale, or congested mucous membranes or skin (ears, feet, tail); skin lesions; soiled anogenital area

Eyes e.g., clarity/condition of lens, cornea; position of globe (e.g., sunken in orbit or protruding); condition of eyelids, encrustation

Posture e.g., hunched back, tucked abdomen; prostrate; head tucked down Locomotion e.g., gait, ataxia, lameness, action of each limb, position of tail when ambulating

Neurological e.g., tremor, convulsion, circling, paralysis, head tilt, coma

Vital Signs e.g., respiratory distress (open mouth breathing, pronounced chest movement)

Other clinical parameters that are relevant to your study e.g., presence and status of tumors, infection, or surgical wounds

Types of Observations 01/02/201331Physical Examination After assessing the animals appearance and behavior, conduct a physical exam using methods that are appropriate to the species and experimental model

You may evaluate:

Behavior

Body weight

Surface lesions (wounds, masses)

Hydration status

Body temperature

Blood parameters (Blood collection can be difficult/stressful in mice; may be used to confirm disease or failed treatment.)3101/02/201332Acclimation Upon arrival to your facility, your mice should have an acclimation period before they are used in research studies

This period of time allows animals to adapt to a new environment

Effects of transportation stress include alterations in various blood parameters, immune cell function, food intake, and animal behavior.

The period of time necessary for biological stabilization will depend on the parameters to be studied

Typically, acclimation periods can range from days to over a week, depending on the studies involved

01/02/201333Ethical treatment of laboratory animals Justification of ResearchResearch should be undertaken with a clear scientific purpose.There should be a reasonable explanation that the research will:Increase knowledge of the processes being studiedProvide results that benefit the health and welfare of humans and other animals01/02/201334Justification of ResearchThe scientific purpose of the research should be of sufficient potential significance to justify the use of animals. The species chosen for study should be best suited to answer the question(s) posed. Research on animals may not be conducted until the protocol has been reviewed by an appropriate animal care committee, to ensure that the procedures are appropriate and humane. 01/02/201335PersonnelAnimal use procedures must conform with federal regulations regarding personnel, supervision, record keeping, and veterinary care. Ensure that all individuals who use animals under their supervision receive instruction in experimental methods and in the care, maintenance, and handling of the species being studied. 01/02/201336Care and housing of animalsAnimals are to be provided with humane care and healthful conditions during their stay in the facility. Researchers are encouraged to consider enriching the environments of their laboratory animals and should keep current with literature on well-being and enrichment for the species with which they work.

01/02/201337Acquisition of animalsAnimals not bred in the research facility are to be acquired lawfully. Researchers should make every effort to ensure that those responsible for transporting the animals to the facility provide adequate food, water, ventilation, space, and impose no unnecessary stress on the animals. Endangered species should be used only with full attention to required permits and ethical concerns.

01/02/201338Experimental proceduresProcedures in which the animal is anesthetized and insensitive to pain throughout the procedure and is euthanized before regaining consciousness are generally acceptable. Behavioral studies that involve no aversive stimulation to, or overt sign of distress from, the animal are acceptable. 01/02/201339Experimental proceduresProcedures involving more than momentary or slight aversive stimulation, which is not relieved by medication or other acceptable methods, should be undertaken only when the objectives of the research cannot be achieved by other methods. Procedures involving the use of paralytic agents without reduction in pain sensation require particular prudence and humane concern. 01/02/201340Experimental proceduresExperimental procedures that require prolonged aversive conditions or produce tissue damage or metabolic disturbances require greater justification and surveillance. An animal observed to be in a state of severe distress or chronic pain that cannot be alleviated and is not essential to the purposes of the research should be euthanized immediately.In surgical procedures animals should be maintained under anesthesia until the procedure is ended.

01/02/201341Experimental proceduresAnimals reared in the laboratory should not be released because, in most cases, they cannot survive or they may survive by disrupting the natural ecology. Euthanasia shall be accomplished in a humane manner, disposal of euthanized animals should be accomplished in a manner that is in accord with all relevant legislation. 01/02/201342LABORATORY ANIMAL ETHICS All scientists working with laboratory animals must have a deep ethical consideration for the animals they are dealing with. From the ethical point of view it is important that such considerations are taken care at the individual level, at institutional level

TRANSGENIC ANIMALS Transgenic animals are those animals, into whose germ line foreign gene have been engineered, whereas knockout animals are those whose specific gene have been disrupted leading to loss of function These animals can be bred to establish transgenic animal strains. Transgenic animals are used to study the biological functions of specific genes, to develop animal models for diseases of humans or animals, to produce therapeutic products, vaccines and for biological screening, etc. MAINTENANCE Housing, feeding, ventilation, lighting, sanitation and routine management practices for such animals are similar to those for the other animals of the species as given in guidelines DISPOSAL The transgenic and knockout animals should be first enthanized and then disposed off as prescribed in the guidelines BREEDING AND GENETICS For initiating a colony, the breeding stock must be procured from CPCSEA registered breeders or suppliers ensuring that genetic makeup and health status of animal is knownThe health status should indicate their origin, e.g. conventional, specific pathogen free 01/02/201343

01/02/201344Limitations of animal paradigms No site specific claimParticular mechanism may not be targetedNon-selective, partial agonist-antagonist creates the problemSurgical intervention develops other complicationsPredicting therapeutic efficacy is far from perfectTissue culture: Inability to replicate the in-vivo environment (cell-cell-tissue interaction)01/02/201345Limitations of animal paradigmsPhenotype: no counterpart in animals for mania, delusion or migraine attack

Common cause: some common or complex diseases like Alzheimers, osteoarthritis and Parkinsons diseases.

Similarity of pathophysiology: depression and anxiety; psychosis and reward

Treatment response: drug treatment as test of validity for models and miss the other potential mechanisms.

Species differences01/02/201346

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