hypolipidaemic agents, anti inflammatory effects

1. First Year Resident
Department of Pharmacology
L.T.M.M.C. Sion, Mumbai –
400022
Date: 30/01/2017
1

2. STUDY OF ANTI-INFLAMMATORY EFFECT
OF SIMVASTATIN IN RATS
2

3. ARTICLE
AUTHORS :
• Ranga Satya Venkatesh1, Lakshmi Deepika Patchva2,
Singamma Muppa2
JOURNAL :
• International Journal of Basic and Clinical Pharmacology.
YEAR : July – August 2016
VOLUME : 5
ISSUE : 4
PAGE : 1520
3

4. ARTICLE
RECEIVED : 26 MAY 2016
REVISED : 04 JULY 2016
ACCEPTED : 08 JULY 2016
CORROSPONDENCE : Dr. Ranga Satya Venkatesh
EMAIL : drrsvenkatesh25@ gmail.com
4

5. ABSTRACT
BACKGROUND:-
• Inflammation is a complex reaction in tissues that consists
mainly of response of blood vessels and leukocytes.
• Simvastatin is a hypolipidemic drug belonging to the class
of statins.
• Statins are competitive Inhibitors Of 3-hydroxy-3-
methylglutaryl-coenzyme a (HMG-CoA) Reductase, the
rate limiting enzyme in cholesterol synthesis, are widely
prescribed for the treatment of Hyperlipidaemia.
• This study was conducted to evaluate the anti-
inflammatory activity of simvastatin in albino rats using
digital Plethysmometer.
5

6. ABSTRACT
METHODS:-
• Male Wistar Albino Rats weighing between 200-250 gm
were selected for the study and rats were randomly
divided into groups (control, standard, and test).
• The anti-inflammatory activity was evaluated by using
carrageenan induced paw edema volume by using digital
Plethysmometer.
• Control group rats were administered 0.2 ml of normal
saline, whereas, test group rats were administered
Simvastatin 40 mg and standard group Diclofenac 50mg
as single dose half an hour before injecting 0.1 ml of 1%
Carrageenan to the sub-plantar region of hind paw of rat.
• The paw edema of each rat was measured at 0 hour and
after 3 hours.
6

7. ABSTRACT
RESULTS:
• At a dose of 40 mg Simvastatin showed anti-inflammatory
effect which is statically highly significant.
CONCLUSIONS:
• However, the above preclinical experiments only give us an
idea about the anti-inflammatory activity, but large scale
clinical trials are necessary for final assessment.
KEYWORDS:
• Simvastatin, Diclofenac sodium, Normal saline,
Carrageenan, Plethysmometer
7

8. INTRODUCTION
• Inflammation is a dynamic process by which living tissue
reacts to injury, particularly vascular and connective
tissue injury.
• It is a complex reaction in tissues that consists mainly
response of blood vessels and leukocytes.
• The word inflammation is taken from the Latin word
"inflammare" meaning burning.
• It is defined as; “a process which follows sub lethal injury
to tissue and ends with complete healing” as proposed by
Ebert.
8

9. INTRODUCTION
• Inflammation has multifactorial causes.
• Almost anything that injures living tissue can cause
inflammation.
• The vascular and cellular reactions of inflammation are
triggered by soluble factors that are produced by various
cells or derived from plasma proteins and generated or
activated in response to the inflammatory stimulus.
• Mediators of inflammation are Prostaglandins,
Leukotrienes, Histamine, Bradykinin, Cytokines Growth
Factors; Lysosomal Contents Of Neutrophils, Reactive
Oxygen Species etc. 9

10. INTRODUCTION
• Simvastatin is a Hypolipedemic drug belonging to the
class of Statins.
• It is a Lipid-lowering Agent, and is derived synthetically
from Lovastatin (formerly known as Mevinclin) which was
isolated from Aspergillus Terreus.
• Simvastin is chemically modified derivative of lovastatin.
• It reduces very low density lipoprotein (VLDL),
triglycerides (TG) and increases high density lipoprotein
cholesterol (HDL-C).
10

11. INTRODUCTION
• Statins exert beneficial effects beyond cholesterol
reduction.
• They are improvement in Endothelial Function,
Decreasing Vascular Inflammation, Inhibiting Smooth-
muscle Proliferation And Immunomodulation.
• In the present study, Anti-inflammatory effect of
simvastatin was evaluated and it was compared with
diclofenac sodium by using Digital Plethysmometer.
11

12. METHODS
• The animals used for the study were Male Albino Rats
(200-250 g).
• Animals are housed at central animal house of Dr.
Pinnamenani Siddhartha Institute of Medical Sciences
and Research foundation which is maintained under
standard conditions.
• The rats are divided into 3 groups, and each group
contains 6 rats.
• A mark is made at the ankle joint (tibio-tarsal joint) of
each rat.
• Initial paw edema of each rat was measured before giving
drug by using Digital Plethysmometer (0 Hour Reading).
And paw edema of each rat was measured at 3 hours after
administration of drug.
12

13. CHEMICAL
• Carrageenan,
• Diclofenac Sodium,
• Simvastatin,
• Double Distilled Water,
• Normal Saline,
• DMSO.
13

14. EQUIPMENT
• Digital Plethysmometer,
• Insulin Syringes,
• Tuberculin Syringes,
• Measuring Jar,
• Glass Beakers,
• Animal Weighing Balance,
• Animal Cages,
• Cotton.
14

15. CARRAGEENAN INDUCED PAW EDEMA
MODEL
• To study the acute and sub-acute phases of inflammation
in rats. Carrageenan is a widely used irritant or
inflammogen or a Phlogistic Agent.
• Chemically, it is a sulphated polysaccharide obtained from
Sea Weed (Rhodophyceae).
• The experimental tissue injury caused by this irritant
initiates a cascade of inflammatory events leading to
formation of exudates.
• The inflammation induced by it is biphasic in nature. 15

16. CARRAGEENAN INDUCED PAW EDEMA
MODEL
• The First Phase is attributed to the release Of Histamine,
5-hydroxy Tryptamine (Serotonin) And Kinin.
• The second phase is related to the release Prostaglandins.
• 1% w/v suspension of Carrageenan was prepared freshly in
normal saline and injected into sub planter region of left
hind paw (usually 0.1 ml in rats).
• In Control Groups animal 0.2 ml Normal Saline, Standard
Group 50 mg of Diclofenac Sodium and in Test Group 40
mg Simvastatin were injected Intraperitonally half an hour
before injecting 0.1 ml of 1% freshly prepared Carrageenan
to the sub-plantar region of left hind paw and the paw
edema of each rat is measured after 3 hours.
16

17. CARRAGEENAN INDUCED PAW EDEMA
MODEL
• Difference in Rat Paw Volume and % Reduction In Paw
Edema was calculated using the following formula:
% Reduction in Edema= [(Mean edema in control group -
Mean edema in drug treated group)/Mean edema in control
group)]×100.
• The readings were recorded by using a Digital
Plethysmometer. Results are tabulated and “Unpaired t-
test” is used to find out the statistical difference in between
the standard and test group animals.
17

18. RESULTS
• In the given table statistical tools have not been applied.
• Un-paired student’s t-test used for evaluation of the
results.
• After comparing the results there is a difference in the rat
paw volume in test versus control and standard versus
control.
• Simvastatin shows significant reduction in rat paw edema18

19. RESULTS
Figure 1: Comparison of Paw Edema in Simvastatin group
and Diclofenac group at 0 and 3.
• Control group (0.2 ml of NS) no decrease in rat paw edema.
• In standard group (50mg of Diclofenac) showed significant
inhibition of rat paw edema compared to normal saline
• In test group (40mg of Simvastatin) showed significant
inhibition of rat paw edema compared to standard
Diclofenac. 19

20. RESULTS
• When the two drugs Simvastatin 40 mg and Diclofenac 50
mg were compared and the results are evaluated.
• Simvastatin was found to have anti-inflammatory activity
which was highly significant (p<0.01).
20

21. DISCUSSION
• This study comprises HMG-COA reductase inhibitor -
Simvastatin tested and compared for its anti-
inflammatory activity with a standard drug - Diclofenac
(non-selective COX inhibitor) by one of the acute method
i.e., rat paw edema method.
• The current study demonstrates significant Anti-
inflammatory Activity, by reduction in Carrageenan
induced paw edema method.
• The percentages of rat paw inhibition of standard and
test drug at 0 and 3 hours are 0.70% and 21.52% and
13.99% and 43.05% respectively (Figure 2).
21

22. DISCUSSION
22

23. DISCUSSION
• The possible mechanism of anti-inflammatory activity of
simvastatin is probably related to Inhibition Of The
Production Pro-inflammatory Mediators.
• It is difficult to define in vivo the specific molecular
mechanism by which statins affect the Cell migration,
because of complexity of the Cholesterol Synthesis.
• Statins Interrupt The Proinflammatory Signalling by
down-regulation of Rho related protein activation.
• Simvastatin requires peroxisome proliferator-activated
receptor (PPAR) α expression to exert its anti-
inflammatory effects in in-vivo models of acute local
inflammation and in-vitro in macrophages and
neutrophils.
23

24. CONCLUSION
• This study was carried out to evaluate the anti-
inflammatory of Simvastatin on rat hind paw edema
using digital plethysmometer.
• At a dose of 40 mg Simvastatin showed anti-inflammatory
effect which is statically highly significant.
• However, the above preclinical experiments only give us
an idea about the anti-inflammatory activity, but large
scale clinical trials are necessary for final assessment.
24

25. ACKNOWLEDGEMENTS
• Authors are very grateful to Dr. M. Singamma, Professor
and HOD, Dept. of Pharmacology, Dr. PSIMS and RF for
her valuable guidance for this study.
• Funding: No funding sources.
• Conflict of interest: None declared.
• Ethical approval: The study was approved by the
Institutional Ethics Committee, Dr. PSIMS and RF
25

26. REFERENCES
1. Kumar V, Abbas AK, Nelson F, Richard NM. Acute and
chronic inflammation. Robbins basic pathology, 8th
edition; WB Saunders Co; 2007:31-58.
2. Goodman and Gillman, Anti-inflammatory, Antipyretic
and Analgesic agents; Pharmacotherapy of Gout, chapter
34, The Pharmacological basis of Therapeutics, 12th
edition, The Pharmacological basis of therapeutics,12th
edition, The Mac Graw Hill companies inc; 2011:986-987
3. Tripathi KD. Hypolipidemic drugs and plasma
expanders, Chapter-45. Essential of Medical
Pharmacology, 7th edition. Jaypee brother publishers;
2013:638.
4. Liao JK, Laufs U. Pleiotropic effects of statins. Annu Rev
Pharmacol Toxicol. 2005;45:89-118. 26

27. REFERENCES
5. Evans M, Roberts A, Davies S, Rees A. Medical lipid-
regulating therapy: current evidence, ongoing trials and
future developments. Drugs. 2004;64:1181- 96.
6. Srivastava K. Drugs for dyslipidaemia, section 5, a
complete textbook for medical pharmacology volume 1,
1st edition, Avichal Publishing company; 2012:327.
7. Anderson WAD. Inflammation and healing. pathology,
9th Edition, C.V. Mosby co. 1990;1:67.
8. Di Rosa M, Sorrenttino L. Biological properties of
carrageenan. J Pharma Pharmacol. 1972;89:102.
9. Gosh MN. Experimental pharmacology.5th edition.
Hilton and company. Gosh S.K publisher; 2007:120- 123.
27

28. CRITICISM
28

29. GUIDELINES
ARRIVE GUIDELINES (Animal Research Reporting
Of An In Vivo Experiment)
29

30. TITLE
[ Provide a Title as accurate and concise a description of the content of
the article as possible. ]
ORIGINAL TITLE :
Study of anti-inflammatory effect of simvastatin in rats.
PROPOSED TITLE :-
Study of Anti-Inflammatory effect of Simvastatin by
Carageenan induced Paw Edema in Male Wistar Albino
Rats.
30

31. ABSTRACT
[ Provide an accurate summary of the background, research objectives,
including details of the species or strain of animal used, key method,
principal findings and conclusions of the study. ]
• Summary of the background given, Research objectives
mentioned.
• Details of the species or strain of animals is given.
• Key methods elaborated.
• Principal findings and Conclusions of the study is
provided.
• Keywords : Simvastatin, Diclofenac Sodium,
Carrageenan, Normal Saline, Plethysmometer, Anti-
Inflammatory, Hypolipidaemic Agent, HMG-CoA
reductase inhibitor 31

32. BACKGROUND
[Include sufficient scientific background (including relevant references to
previous work) to understand the motivation and context for the study,
and explain the experimental approach and rationale.]
• Scientific background is provided.
• No relevant references regarding anti-inflammatory
activity of Simvastatin is given neither similar previous
studies have been mentioned.
• Motivation behind conducting study is provided.
• Experimental approach is given.
32

33. BACKGROUND
[Explain how and why the animal species and model being used can
address the scientific objectives and, where appropriate, the study’s
relevance to human biology.]
• Study gives the details about the animal models used for
this study. Why the particular model used is not
explained.
• Given study model is appropriate to achieve scientific
objectives in this study.
• Study is relevant to the human biology.
33

34. OBJECTIVE
[Clearly describe the primary and any secondary objectives of the study,
or specific hypotheses being tested.]
• Primary objective of study is to evaluate Anti-
Inflammatory effect of Simvastatin and is given clearly.
• Study do not describe any secondary objective.
34

35. ETHICAL STATEMENT
[Indicate the nature of the ethical review permissions, relevant licences
(e.g. Animal [Scientific Procedures] Act 1986), and national or
institutional guidelines for the care and use of animals, that cover the
research.]
• The study was approved by the Institutional Ethics
Committee, Dr. PSIMS and RF
35

36. STUDY DESIGN
[For each experiment, give brief details of the study design including:
a. The number of experimental and control groups.
b. Any steps taken to minimise the effects of subjective bias when
allocating animals to treatment (e.g. randomisation procedure) and
when assessing results (e.g. if done, describe who was blinded and
when).
c. The experimental unit (e.g. a single animal, group or cage of
animals).
A time-line diagram or flow chart can be useful to illustrate how
complex study designs were carried out.]
• No. of experimental and control groups is given.
• No mention about allocation of animals into groups
(blinding) 36

37. STUDY DESIGN
• No. of animals in each group is mentioned but total no. of
animals is not mentioned.
• No timeline diagram or flowchart has been provided.
37

38. EXPERIMENTAL PROCEDURES
[ For each experiment and each experimental group, including controls,
provide precise details of all procedures carried out. For example:
a. How (e.g. drug formulation and dose, site and route of administration,
anaesthesia and analgesia used [including monitoring], surgical
procedure, method of euthanasia). Provide details of any specialist
equipment used, including supplier(s).
b. When (e.g. time of day).
c. Where (e.g. home cage, laboratory, water maze).
d. Why (e.g. rationale for choice of specific anaesthetic, route of
administration, drug dose used). ]
• Dose, Site, Route of drug administration is given.
• Rationale behind drug dose and route of administration is
not given.
38

39. EXPERIMENTAL PROCEDURES
• Source of Drugs and chemicals also the Suppliers of
Instruments are not mentioned.
• No mention about role of DMSO in procedure.
• Vehicle used for i.p. injection preparation of Simvastatin is
not mentioned.
• Time of drug administration is given.
39

40. EXPERIMENTAL ANIMALS
[a. Provide details of the animals used, including species, strain, sex,
developmental stage (e.g. mean or median age plus age range) weight
(e.g. mean or median weight plus weight range).
b. Provide further relevant information such as the source of animals,
international strain nomenclature, genetic modification status (e.g.
knock-out or transgenic), genotype, health/immune status, drug or test
naïve, previous procedures, etc.]
• Animal species and strain is given.
(Why Wister albino instead of Sprague dawley?)
• Animal weight is provided.
(Std S/D 100-150gm)
40

41. HOUSING AND HUSBANDRY
[a. Housing (type of facility e.g. specific pathogen free [SPF]; type of cage
or housing; bedding material; number of cage companions; tank shape
and material etc. for fish).
b. Husbandry conditions (e.g. breeding programme, light/dark cycle,
temperature, quality of water etc for fish, type of food, access to food and
water, environmental enrichment).
c. Welfare-related assessments and interventions that were carried out
prior to, during, or after the experiment.]
• Housing of animals is mentioned as under standard
condition but no specific details about bedding material,
light dark cycle, temperature, food and water given to
animal is mentioned.
• Overnight fasting ???
41

42. SAMPLE SIZE AND ANIMAL ALLOCATION
[a. Specify the total number of animals used in each experiment, and the
number of animals in each experimental group.
b. Explain how the number of animals was arrived at. Provide details of
any sample size calculation used.
c. Indicate the number of independent replications of each experiment, if
relevant.
d. Give full details of how animals were allocated to experimental
groups, including randomisation or matching if done.]
• Sample size of animals is not mentioned.
• n Value not given.
• No. of animal in each group is given
• How they derived no. of animals in each group to be placed
is not given.
• How the animals allocated in each group is also not given.
42

43. STATISTICAL METHOD
[a. Provide details of the statistical methods used for each analysis.
b. Specify the unit of analysis for each dataset (e.g. single animal, group
of animals, single neuron).
c. Describe any methods used to assess whether the data met the
assumptions of the statistical approach]
• Statistical method used is mentioned as unpaired t test.
• Statistical difference has been studied between standard
and test drug.
• p value is considered significant when (p<0.01) (p<0.05)
43

44. RESULTS
[Report the results for each analysis carried out, with a measure of
precision (e.g. standard error or confidence interval).]
• Statistical tools have not been applied to table no. 1 and
results are interpreted as significant.
44

45. RESULTS
• Statistical tools have not been applied to Figure no. 1 and
results are interpreted as significant.
45

46. ADVERSE EVENTS
[a. Give details of all important adverse events in each experimental
group.
b. Describe any modifications to the experimental protocols made to
reduce adverse events.]
• No mention of any adverse event.
• No mention about any mortality in the animals used.
46

47. INTERPRETATIONS
[a. Interpret the results, taking into account the study objectives and
hypotheses, current theory and other relevant studies in the literature.
b. Comment on the study limitations including any potential sources of
bias, any limitations of the animal model, and the imprecision associated
with the results2.]
• Results have been interpreted taking into account the
study objectives and hypothesis.
• No references of relevant studies done in past have been
mentioned.
• Possible mechanism of anti-inflammatory effect of
Simvastatin is mentioned.
• Study limitation has been mentioned.
47

48. FUNDING & CONFLICT OF INTEREST
[List all funding sources (including grant number) and the
role of the funder(s) in the study.]
48

49. REFERENCE STYLE
49

50. 50