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Dr. Pranesh V. PawaskarFi rs t Year Res identDepartment o f Pharmaco logyL.T.M.M.C . S ion , Mumbai – 400022Date: 30/01/2017

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STUDY OF ANTI-INFLAMMATORY EFFECT OF

SIMVASTATIN IN RATS

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ARTICLEAUTHORS :• Ranga Satya Venkatesh1, Lakshmi Deepika Patchva2,

Singamma Muppa2

JOURNAL :• International Journal of Basic and Clinical

Pharmacology.

YEAR : July – August 2016VOLUME : 5ISSUE : 4PAGE : 1520

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ARTICLE

RECEIVED : 26 MAY 2016

REVISED : 04 JULY 2016

ACCEPTED : 08 JULY 2016

CORROSPONDENCE : Dr. Ranga Satya Venkatesh

EMAIL : drrsvenkatesh25@ gmail.com

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ABSTRACTBACKGROUND:-• 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.

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ABSTRACTMETHODS:- • 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.

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ABSTRACTRESULTS: • 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

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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.

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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.

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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).

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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.

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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.

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

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

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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.

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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.

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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.

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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

edema

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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.

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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).

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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).

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DISCUSSION

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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.

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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.

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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

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REFERENCES1. 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.

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REFERENCES5. 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.

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CRITICISM

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GUIDELINESARRIVE GUIDELINES (Animal Research Reporting Of An In Vivo Experiment)

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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.

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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

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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.

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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.

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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.

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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

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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)

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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.

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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.

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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.

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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)

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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 ???

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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.

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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)

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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.

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RESULTS• Statistical tools have not been applied to Figure no. 1

and results are interpreted as significant.

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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.

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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.

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FUNDING & CONFLICT OF INTEREST

[List all funding sources (including grant number) and the role of the funder(s) in the study.]

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REFERENCE STYLE

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