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Preclinical Pharmacological Experiments.pptx

  1. WELCOME Priclinical Pharmacological Experiments Mr. Akash Anil kale (B. Pharm, VII Sem) Department of pharmaceutical sciences Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440033 2020 – 2021 Presented By 1
  2. In drug development preclinical Experimental studies , is a stage of research that begins before clinical trials (testing in humans) can begin, and during which important feasibility, iterative testing and drug safety data are collected, typically in laboratory animal. The main goals of preclinical studies are to determine a starting, safe dose for first-in-human study and assess potential toxicity of the product, which typically include new medical devices, prescription drugs, and diagnostics. 2 Introduction
  3. Objective of the Preclinical studies 3 • Preclinical studies aim at providing information about safety and efficacy of a drug candidate before testing it in humans. • The purpose of pre-clinical study is to develop adequate data to decide that it is reasonably safe to proceed with human trials of the drug • Means, a laboratory test of a new drug or a new medical device, usually done on animal subjects, to see if the treatment really works and if it is safe to test on humans. • However, the main objective is to collect the data to submit to the FDA for IND filing. • Screening of new dosage form and formulation.
  4. 4 Basic Terminologies • IN VIVO :- Studies that are in vivo are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism. • IN VITRO :- means studies or experiments conducted on microorganisms and cells outside of their normal biological environment. • Ex vivo:- In this type of experiment the living tissue are not created artificially but directly taken from living organism. • In situ:-. In this the experiment is carried out in their natural habit or environment. • In silico:- In silico technique the experiment is carried out bythe use of computational methods.
  5. Animals used 5 NAME OF Animals USE OF Animals 1. RAT AND MICES. • Used For toxic study , bioassay of drug , testing of analgesics etc. • Tissue Used for the study are colon , uterus , ceacum , vas deference , abdominal stomach muscle etc. 2. Guine Pig (Cavia procellus) • They are very sensitive to Tuberculosis and anaphylaxis. • The commonly used tissue is ileum for study. 3. Rabbits (Oryctolagus Cuniculus) • Used for pyrogen testing, Bioassay of anti-diabetics, curare derivatives and sex hormones. • Used to study of meiotic and mydriatics.
  6. 6 Name of Animals Use OF Animals 4. Frog (RanaTigrina) • Isolated Heart, rectus abdominal muscles used to study of drugs. • Used for evolution of local anaesthetic. • Use as research model form many years. 5.Transgenic Animals A transgenic animal is one whose genome has been altered by the transfer of a gene or genes from another species or breed. • Transgenic rodents play a number of critical roles in drug discovery and development. • Importantly, they enable scientists to study the function of specific genes at the level of the whole organism which has enhanced the study of physiology and disease biology and facilitated the identification of new drug targets.
  7. 1. Behavioural Models of Animals 7 • Animal model’s:- The animal model is living organism in which normative biology or behaviour can be studied , or in which a spontaneous or induced pathological processes can be investigated. • Types of behavioural animal models:- A. Elevated plus maze B. Eddy’s hot plate C. Rota rod D. Elecroconvulsiometer A. Elevated plus maze. :- • The elevated plus maze task is a simple method to assess anxiety-like behaviors in rodents.
  8. 8 B. Eddy’s hot plate • The hot plate test is used in basic pain research and in testing the effectiveness of analgesics by observing the reaction to pain caused by heat. C. Rota rod :- • Rotarod test is widely used to evaluate drug effects on motor coordination, balance and motor learning in rodents. D. Elecroconvulsiometer :- • This instrument is used to study the anti- convulsant activity of phenytoin against maximal electro-shock induced convulsions in rat or mice.
  9. 2. Irwin test (Battery Test) 9 • The Irwin test is an observational screening that is comprised of a battery of tests used to assess a mouse or rat’s neurobiological and physiological state. • Parameters that are evaluated include autonomic and sensorimotor functions, convulsive behaviour, and other activities produced a drug after administration. • The Irwin Test is use to evaluate the qualitative effects of the New chemical entity on behaviour and physiological function, from the first dose that has observable effects up to doses that induce clear behavioural toxicity or even death.
  10. 10 3. Biochemical assays • Biochemical assays are analytical methods used to measure and quantify metabolic reactions and cellular processes such as cell signalling and apoptosis. Biochemical assays helps to analyse the biomolecules and their functions during drug discovery and development. • There are so many techniques like ELISA and Western blotting techniques which are used to quantify metabolic activities and measuring functional behaviour of biomolecules such as proteins, enzyme, small molecules, cofactors. A. WESTERN Blotting technique. B. Enzyme-linked immunosorbent assay (ELISA) C. ENZYMATIC ASSAY A. WESTERN Blotting techniques :- In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein. B. ELISA :- In ELISA, various antigen-antibody combinations are used, always including an enzyme-labeled antigen or antibody, and enzyme activity is measured colorimetrically.
  11. I. Continuous assays :- 11 C. ENZYMATIC ASSAY The enzymes or proteins present in the human body exhibit catalytic activity, the ability to perform tasks resulting in reactions. Hence, it is necessary for enzymes synthesized industrially to exhibit the correct functions. There are two main types of enzymatic assays based on sampling methods. I. Continuous assays. II. Discontinuous assays. This is the type of enzymatic assay which give the continuous readings of activity. Methods Used :- 1. Spectrophotometric. 2. Fluorimetric. II. Discontinuous assays:- In this type of enzymatic assay the samples is taken after the specific time interval from the enzyme reaction at intervals and the product production and substrate consumption is measured in the sample by different chemicals methods. • Methods used :- 1. Chromatographic 2. Radiometric
  12. 4.Protein Estimation :- 12 Proteins estimations or protein assays are done to estimate the concentration or amount of protein present in product. A. Biuret method:- Principle :- Protein reacts with this alkaline copper complex and colour changes to violet. The protein can then be estimated by reading the absorption at 540nm. This method takes 20-30 minutes to complete. Principle :- The principle behind the Lowry method of determining protein Concentrations lies in the reactivity of the peptide nitrogen[s] with the copper[II] ions under alkaline conditions. B. Lowry Method :-
  13. D. Bradford assay(Dye Binding method):- 13 C. BCA assay(Bicinchonic acid Method):- • This method is highly sensitive and detects proteins at a low concentration of 1 µg. In this method, Copper ions bind to Nitrogens in protein and the complex is then bound to bicinchoninic acid resulting in the change of colour to purple depending on protein concentration. • This is a very sensitive method and simple dye binding assay. This method uses Coomassie brilliant blue-250 dye that binds with negatively charged protein molecules. • The dye colour changes based on protein concentrations and the absorption is measured at 595nm E. UV absorption:- In this method, no reagents are required, the liquid protein sample is monitored under UV absorption at OD 280nm.
  14. 5.Nucleic acid estimation 14 • In nucleic acid estimation blotting techniques are very widely used as analytical tools for the specific identification of desire DNA and RNA fragments from thousands of molecule. • Blotting refers to process of immobilization of sample nucleic acid on solid support. • The blotted nucleic acid then used as target in the hybridization experiments for their specific detection. • Types of blotting:- A) Northern blotting ( FOR RNA) B)Dot Blotting ( for RNA) C) Southern blotting ( for DNA)
  15. • Toxicology and safety studies identify potential target organs for adverse effects and define the therapeutic Index to set the initial starting doses in clinical trials. • Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up, select the best formulation, determine the route, frequency, and duration of exposure, and ultimately support the intended clinical trial design. • It plays an important role in development of new drug or chemical entity. IMPORTANCE OF PRECLINICAL EXPERIMENTS IN DRUG DISCOVERY PROCESS
  16. References :- • • Granchelli, J.A., Pollina, C. and Hudecki, M.S., 2000. Pre-clinical screening of drugs using the mdx mouse. Neuromuscular Disorders, 10(4-5), pp.235-239 • Badyal, Dinesh K., and Chetna Desai. "Animal use in pharmacology education and research:The changing scenario." Indian journal of pharmacology 46, no. 3 (2014): 257. • • • •
  17. THANK YOU 17