Webinar on research methodologies and pharmacological screening ppt dr bhanu final 20 06-2020
1. Department of Pharmacy, IEC Group of Institutions
Organizing a Webinar on
“Research Methodologies and Pharmacological
Screening of Anti-hepatotoxic & Anti-Diabetic Drugs”
Date : June 20, 2020 (Saturday)
Time : 2-3 pm
by
Prof. (Dr.) Bhanu P. S. Sagar
Professor & Director
DOP, IECGI, Greater Noida
)
1
3. Research :
The research is defined as a
logical, scientific and systematic
search for new and useful
information on a specific topic.
Introduction
3
4. Original addition to
the available
knowledge
Solve the scientific
and non-scientific
problems
Discover new
things & tests
existing facts
Systematic
Analysis to Gain
New Knowledge
Scientific
Investigation to
Achieve Innovation
Logical, Scientific &
Systematic Search
for new & useful
information
Research
Introduction..
4
5. 1.
Aim, Objectives,
Research Envisaged,
Methodology,
Standardization and
Validation of Scientific
Protocols
2.
Experimental
studies collection
of data,
observations,
analysis of data,
and comparison of
data with
Standard
3.
Results &
Discussions,
Summary and
final
Conclusions
Research Work
5
6. Objectives of Research
One Aim with multiple objectives.
The main aim of research is to find out the truth which is hidden
and which has not been discovered at yet .
To get research degree for better employment or promotion.
To develop new scientific method, concept and theories to solve
the scientific and non-scientific problems.
To discovered new things and tests the existing facts.
To gain familiarity with new insights into a phenomenon.
To analyse the frequency with which something occurs or its
association with something else.
6
7. Research Methods Vs. Methodology
Research Methodology Research Methods
Way in which research problems are solved
systematically.
Methods which are adopted for conducting
research studies.
Science of studying how research is to
designed Scientifically.
Science of studying how research is to be
conducted Scientifically.
Study of various steps involved in research
protocols.
Complete detail of procedures to be used in
different methods.
It includes all techniques used like screenings,
chromatographic & spectral, and
pharmacological screening techniques,
formulation development etc.
Refers to the methods which are used in
research work eg : experimental procedures,
collection of data , analysis of data etc.
7
8. Significance of Research
“
All progress is born of inquiry.
Doubt is often better than over-
confidence, as it leads to inquiry,
and inquiry leads to invention” —
Hudson Maxim
Research inculcates innovative and
scientific thinking which promote logical
thinking, implementations of scientific
thoughts for systematic and organized
experimental research work.
8
9. Importance of Research
Helps to study the application of existing theories &
concepts.
Important tool in all Pharmaceutical industries for higher
growth & to improve the quality of products / discover new
products. Research gives the solution for problems of large
and small scale industries.
Isolation , identification, characterization and discovery
of new APIs / Develop new formulation.
Research is one of the important parameter for National
/ International University / College Ranking.
9
12. 7
Types of Research
Descriptive Research :
Means description of the state of affairs as it exist at present.
Researcher only reports only what has happened or what is
happening.
Applied research :
Aims at finding solution of an immediate problem face by society
or industry / business organization. Most of research, case
studies, and inter – disciplinary research are essentially applied
research .
Qualitative Research :
Concerned with qualitative phenomena , i.e. phenomena relating
to or involving quality or nature / kind / type. The detection of
compound by Physical or chemical tests is Qualitative analysis.
12
13. 7
Types of Research
Quantitative Research :
This is done for structured or predetermined methodology after
qualitative analysis (based on measurement of quantity). It can
be expressed in terms of quantity like determination of amount
by volumetric , calorimetric, or instrumental methods.
Analytical Research:
Researcher has to use facts on information already available and
analyze these to make a critical evaluation of the material .
Fundamental Research / basic research :
It is also called theoretical research and not concerned with
solving problem of immediate interest. Study of some natural
phenomena or research related to pure science termed as basic
research.
13
16. 7
Procedural Steps In Research
1. Formulating the Research Problem :
It should be from your area of interest (having good knowledge
of basic fundamentals).
The best way to select it by discussion it with colleagues or
with those having expertise in specific field.
In an academic institution the researcher can take help from
guide who is expert in research area.
Researcher can search what are currently published in
scientific journals and what are the new possibilities / new
research can be added in those areas.
Resources required and Resources available (parameters).
16
17. 7
Procedural Steps In Research
2. Literature Survey / Literature Review:
Once the problem is formulated, researcher should undertake
extensive literature review connected with the problem.
It is a collection of research publication, books and other
documents related to the defined problem.
Why Literature Review?
i. Assist in defining statement of the problem
ii. Strengthening the argument of selection of a
research topic (Justification )
iii. It helps to get familiar with various types of
methodology that might be used in the study
(Design)
17
18. 7
Procedural Steps In Research
3. Formulation of Hypothesis :
Hypothesis are scientifically reasonable prediction.
The expected solution based on available data and tentative
explanations constitute the hypothesis.
It should be very specific and its role is to guide the
researcher ad keep him on right track.
4. Research Design :
It should indicate various approaches to be used in solving
the research problem, sources and information related to
problem, the measurement procedures, the frame of analysis,
time schedule and cost budget.
18
19. What will be
the sample
design?
What periods
of time will the
study include?
What techniques
of data collection
will be used?
How will
the data be
analysed?
What is the
study
about?
Why is the
study being
made?
Where will
the study be
carried out?
Where can the
required data be
found?
Research Design
19
20. 7
Procedural Steps In Research
5. Experimental Research, Data analysis & interpretation :
Qualitative, Quantitative, Analytical, Interpretation and
appropriate statistical tools are used for data analysis.
The research outcome must be tested by Chi-square test, t-
test, ANOVA Variance analysis etc.
6. Research report :
It is an oral or written presentation of important and useful
aspects / anticipated outcome. It helps to get feedback from
other researcher.
It is important to publish the research outcome in appropriate
journal or conference. 20
21. Tips for a Good Researcher
Follows Research Guide: Guidance of Research Guide / Supervisor is
essential & indispensable (to achieve targets).
Patience : Researcher needs to cool and calm with patience to avoid /
prevent repetition of experiment (disposable)
Right Information : Researcher must what is right source of
informations while compiling experimental design.
Validation of Research Protocols: Right approach should be followed to
develop standardized and validated protocols.
Keep Day to Day Record: It is always essential to keep records of
experimental observations on daily basis. Maintain secrecy.
Good Experimental Hand : Researcher must have good
knowledge of Instruments handling and research techniques.
21
22. Tips to Prevent Plagiarism
Present the information / data in your own language. Squeeze the
sentences by merging them in your own one sentence.
All informations taken from literature should be properly acknowledged
with references.
Don’t use more than 1% information from any research publication /
source (otherwise high-lighted).
Try to present the data in tabular forms / in flow charts / graphs /
schematic diagrams (data can presented in better way)
Write scientific content in the Thesis by accommodating volume of data
/ information (keep plagiarism less then 20%). Avoid header –Footer.
Avoid repetition of sentences (even 7-10 words) otherwise high-lighted.
22
23. Pharmacological Screening
According to FDA 92% of drugs that passes preclinical testing, highly found to
be fail in clinical trials.
In vivo assays are not usually performed after achieving the lead optimization
stage.
When animal models are employed in the study of human disease, they are
frequently selected because of their similarity to humans in terms of genetics,
anatomy, and physiology. Rodents are the most common type of mammal
employed in experimental studies, and extensive research has been
conducted using rats, mice, guinea pigs, and hamsters.
All Animals studies need to be approve by CPCSEA through IAEC.
Animals are sacrificing their life for human, but we have nothing to sacrifice for
them.
23
25. Methods of Screening
• In-vitro :
▫ Experimental process in a given procedure which is mainly done
outside the body in a controlled condition
Activity assays (screen the activity)
Bioassays (define the molecular mechanism)
Toxicity assays (Toxicity of chemicals)
Types: Biological assay using isolated tissues/organs
(skeletal/smooth muscles, aorta, heart etc.,)
Antioxidant assays
Xanthine oxidase activity
Antiglycation activity
DNA, protein, RNA level
assays
Immunological assays
Chemical Assay using regents
Cell culture studies
Toxicity(cyto) assays
Immunological assays Cancer cell
line studies
25
26. Ex-vivo:
▫ Experimental process which is performed outside the
living body in an ‘artificial invivo environment’
▫ This usually lasting up to 24 hrs
In vivo
▫ Experimental process which is performed in the living
body using laboratory animals
Insilico
▫ Process which is performed on computer or via
computer simulator
26
27. STEP -I
◦ Toxicological assessment of chemicals
◦ LD50 estimation
◦ Using Acute/sub-acute, chronic etc., studies
STEP - II
◦ Evaluation of Pharmacological activity
◦ Animal models of induced disease and injury
STEP - III
◦ ADME Studies
Absorption studies
Tissue/organ/fluid conc. estimation
Histopathological studies
Serum estimation of biological indicators for drug &
metabolites
Factors:
Species
Strains
Sex Age
Disease
Induction
Environmental
27
28. Pharmacological Screening of Anti-
hepatotoxic and Anti-Diabetic Drugs
The liver is a master chemist of human body.
It is cone shaped, dark reddish-brown, one of the largest solid glandular organs in
the human body (weighing about 1.5 Kg / about 3 pounds in adults; representing
approximately 2.5% of adult body weight) and essential for life.
It is situated in the right upper quadrant of the abdomen (upper right hand portion
of the abdominal cavity, under the diaphragm and on top of the stomach, right
kidney and the intestines; in the protection of rib cage).
It is covered by Glisson's capsule, a visceral continuation of the peritoneum.
The liver holds about 13 percent of the body's total blood supply at any given
moment.
It divided into two lobar segments (right and left), and two accessory lobes.
Further subdivided into eight (Couinaud) segments based upon vascular supply
and bile duct distribution.
28
29. Figure : Human Liver.
Figure : Anatomy of Human Liver
Figure : Segmental anatomy of Liver
34. Screening Models for Anti-hepatotoxic Drugs.
i. Carbon tetrachloride (CCl4) : Cause lipid peroxidation.
ii. Galactosamine: Induce decrease the bile flow and its content,
reduces rate of oxygen consumption, simulate viral hepatitis.
ii. Thioacetamide : Its metabolite (s-oxide) interferes with
movement of RNA from the nucleus to cytoplasm.
iv. Paracetamol: hepatic necrosis, nuclear pyknosis.
v. Anti-tubercular (Isoniazid - rifampicin) cause hepatoxicity by
multiple drug regimens.
vi Ethanol : Necrosis, steatosis (fatty changes), FFA overload.
34
35. Biomarkers of Hepatotoxicity
Biochemical parameter Histopathological lesion Reason of abnormality
Alanine aminotransferase
(ALT/SGPT)
Hepatocellular necrosis Leakage from damaged
tissue
Albumin Hepatic dysfunction Decreased synthesis
Alkaline phosphatase Hepatobilary injury
Choleastasis
Overproduction,
releases in blood
Arginase Hepatocellular Release from injured
hepatocytes
(AST/SGOT) Aspaertate
aminotransferase
Hepatocellular necrosis Damaged tissue leakage
Bile acids Hepatobiliary disease Regurgitation into blood
Glumate dehydrogenase Hepatic necrosis Leakage from damaged
tissue
Glutathione S-transferase Necrosis of hepatocytes Rapidly leaks from
hepatocytes
Lactate dehydrogenase Hepatocytes’s necrosis Leaking damaged tissue
Malate dehydrogenase Necrosis of liver cells Damaged tissue which
results in its leakage
Sorbitol dehydrogenase Liver cell necrosis Damaged tissue leaking
Total bilirubin Hepatobiliary injury as
well as cholestasis
Hepatic clearance is
decreased
Total protein Hepatic dysfunction Reduced synthetic
36. Hepatoprotection is achieved by following
two categories of drugs :
Hepato-protective Drugs: These drugs preserve the normal
anatomical architecture and physiological functions of the liver
and prevent toxic action of hepatotoxins (prophylactic action).
Anti-hepatotoxic Drugs : Drugs which reduce injurious effect
caused by hepatotoxic agents, significant alleviation of the
serum enzyme activities (Biochemical parameters like SGPT,
SGOT etc.), increase glutathione store, membrane stabilization
through increased protein synthesis (Therapeutic action;
antagonise the effects of any hepatotoxin).
36
37. Evaluation of Liver Function Test (LFTs) / Liver Damage Assessment
Non-invasive functional methods:
• Ascorbic acid content in urine
• Pentobarbitone induced sleeping time (TBARS)
• Bromosulphthaline clearance test (a dye cleared by normal liver)
Biochemical analysis of blood for the following:
a. SGPT & SGOT
c. Alkaline phosphatase
d. Serum bilirubin
e. Total proteins
• Morphological test-weight of liver/100 g body weight
• Histopathology of Liver
Free radical scavengers
a. Glutathione
b. Lipid peroxidation
c. Superoxide dismutase
d. Catalase 37
38. Screening Models for Anti-diabetic Drugs.
Screening methods
IDDM Screening methods
Chemically induced diabetes
Surgically induced diabetes
NIDDM Screening methods
Chemically induced diabetes
Normoglycemic animal model
Isolated pancreas of rat [in vitro]
In vitro assay of insulin on adipocyte
Glucose uptake by isolated diaphragm from mice/rat
Insulin receptor binding assay
38
39. Diabetes mellitus (DM)
It is a group of metabolic
disorder characterized by
high blood sugar levels over a
prolonged period.
Diabetes is due to either
the pancreas not producing
enough insulin or the cells of
the body not responding
properly to the insulin
produced.
39
40. Type-I: IDDM
Type-1 Diabetes Mellitus
• Absolute lack of insulin
• β cell mass reduction
Mechanisms for
destruction of islet cells
• Genetic susceptibility
• Acute autoimmunity
• Environmental insult
40
41. Type-II: NIDDM
Type 2 Diabetes Mellitus
• Insulin resistance
• Relative impairment
in insulin secretion
Mechanisms for development of
NIDDM
•Defect at Insulin receptor level
•Defect at β-cell function
•Combination of both
41
42. Screening Methods
IDDM
Chemically Induced DM
Surgically Induced DM
Miscellaneous
Genetic model
Hormone induced DM
Insulin antibody induced DM
DM induced by viral agent
NIDDM
• Chemically Induced DM
Normoglycemic Animal model
Miscellaneous
• Genetic model
• Isolated pancreas of Rat [in-vitro]
• In-vitro assay of insulin on
adipocyte
• Glucose uptake by isolated
• diaphragm from mice/rat
• Insulin receptor binding assay
43. Alloxan induced DM (150 / 160/170 mg/kg by i.p. / i.v. in 5% saline )
Disadvantage
• High mortality
• Ketosis in animals
• Diabetes induced- reversible
• Guinea pig are resistant to its effect
Principle :
Alloxan have capacity to produce reversible diabetes.
It is a toxic cyclic urea analogue which destroys β-cells of the islets of
Langerhans in pancreas.
This compound causes severe necrosis of pancreatic β-cells .
It has been suggested that alloxan induces the production of H2O2 and
of some free radicals such as O2 and OH- that produce first damage and
later the death of β-cells.
44. Alloxan Induced DM
Mechanism of
action
Free oxygen
radical
Free radical β-
cell death
Reaction with -
SH group of
protein
Cell necrosis
44
45. Streptozotocin (STZ) induced DM
Broad
spectrum
antibiotic
produced
from
streptomyces
acromogens
Induce DM in
almost all
species
Cyclosporine-
A enhance
STZ-
diabetogenic
efficacy
45