EVALUATION OF QUINAZOLINONE DERIVATIVES AS POTENTIAL ANTI-INFLAMMATORY AND ANTIARTHRITIC

AGENTS

Protocol of Dissertation Submitted

By

Mr. RAMESH. B. NIDAVANI

To

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGLORE,KARNATAKA

Under the guidance of

Mr. CHANDRASHEKHAR V. M Asso. Professor

(2009-2010)

0

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FORDISSERTATION

1. Name of the Candidate and Address RAMESH. B. NIDAVANI

DEPARTMENT OF PHARMACOLOGY,

H.S.K.COLLEGE OF PHARMACY,

B.V.V.S.CAMPUS,

BAGALKOT-587101, KARNATAKA

2. Name of the Institution H.S.K.COLLEGE OF PHARMACY,

B.V.V.S.CAMPUS,

BAGALKOT-587101, KARNATAKA

3. Course of Study and Subject MASTER OF PHARMACY IN

PHARMACOLOGY

4. Date of Admission of Course 18-06-2009

5. Title of the topic:

Evaluation of Quinazolinone Derivatives as potential Anti-inflammatory and

Antiarthritic agents.

1

6. Brief resume of the intended work

6.1 Need for the study: Inflammation is a response of a tissue injury and it has two faces, one which is good side

to protect the body and second is bad side, which have created many types of allergies and

autoimmune diseases including rheumatoid arthritis (RA) and others. Consequently anti-

inflammatory drugs are extensively employed in virtually all branches of medicine1. Design

of agents for fast and effective relief from inflammation and pain in the human being is a

major challenge for medicinal fields. Search continues to develop new drugs that have potent

anti-inflammatory activity with minimum side effects2.

RA is the most common systemic inflammatory disease and is characterized by symmetrical

joint involvement. RA is estimated to have prevalence of 1% to 2% and doesn’t have any

racial predilections. It can occur at any age, with increasing prevalence up to the seventh

decade of life3. The prevalence rate is higher in women (25.4%) than in men (17.6%), and in

the elderly (50.0% of those ≥ 65 years of age versus 29.3% of those between 45 and 65 years

of age)4. In people aged 15 to 45 years, women predominate by a ratio of 6:1; the sex ratio is

equal among patients in first decade of life and in more than 60 years old. Vasculitis is seen

in patient with long-standing rheumatoid arthritis, which may also cause the breakdown of

skin especially in lower extremities. Pulmonary complications like Pulmonary fibrosis may

develop as a result of rheumatoid involvement. Other complications such as

lymphadenopathy, amyloidosis may occur with rheumatoid arthritis. This arthritis causes

other complications such as cardiac complications, myocarditis which may occur resulting in

the accumulation of fluid and may risk the cardiovascular mortality associated with

arthritis5,6. The importance of anti-inflammatory agents cannot be exaggerated because of

their utility, often as life-saving drugs in many diseases such as arthritis, rheumatoid fever,

2

lupus erythromatosus and pemphigus7. This disease is challenging for the international

community and requires significant modified therapies.

The present available therapies for the treatment of rheumatoid arthritis have serious side

effects and adverse effects like renal toxicity, myelo-suppression, ulcers, severe bleeding in

intestine, anemia, hepatotoxicity8, 9.

The present work is an extension and identification of new molecules for anti-

inflammatory and further selected compound for antiarthritic activity with minimal

gastrointestinal ulceration side effects. With this background in this present study was design

to screening of new Quinazolinone derivatives for anti-inflammatory and antiarthritic

activities.

6.2 Review of literature:

Non-steroidal anti-inflammatory drugs (NSAID’s) are commonly for the treatment of

acute and chronic inflammation, pain and fever. However long term clinical usage of

NSAID’s are associated with significant side effects like GIT bleeding and nephrotoxicity.

Therefore the discovery of new safer anti-inflammatory drug represents a challenging goal

for such a research area. On our going research programme we found that quinazolines and

condensed quinazolines exhibit potent CNS activities like analgesic and anti-inflammatory.

Quinazolinone is a compound made up of two fused six-membered simple aromatic rings,

a benzene ring and a pyrimidine ring. Its chemical formula is C8H6N2. The 4-(3H)-

quinazolinones bearing a heterocyclic nucleous at N-3 are associated with a range of

pharmacological properties such as antiparkinson, anti-depressant and other central nervous

system activities10. Quinazolin-4(3H)-ones with 2,3-disubstituion is reported to posses

significant analgesic, anti-inflammatory11, antimicrobial, antidepressant12, hypoglycemic and

antitumor activity already reported scientifically. Interests in quinazolines as anticancer agent

3

7.

proved active against a panel of human cancer cell lines. Also 4-anilinoquinazolinones

represent as a new class of antitumor drugs13. With this background, in the department of

inorganic chemistry, Karnataka University, Dharwad were synthesized various derivatives

of Quinazolinones and these synthesized compounds were subjected for their anti-

inflammatory and antiarthritic activity.

6.3 Objectives of the study :

Scientifically well known chemical compound Quinazolinone derivatives, as anti-

inflammatory and antiarthritic will be used for the present study. The objectives of the

present study are to evaluate:

1. Anti-inflammatory activities of Quinazolinones.

2. Anti-arthritic activity of the Quinazolinones.

3. Effect of drug on FCA induced arthritic rat’s behavioral studies.

4. Effect of drug on morphological alterations in joints

5. Effect of drug on physiological parameters associated with arthritis.

6. Effect of drug on histopathological changes.

Material and methods :

7.1 Source of data :

In order to improve the above pharmacological properties and gain more insight in the

structure activity relationships of Quinazolinones. Synthesis of new derivatives in the

department of chemistry, Karnataka University, Dharwad were carried out by Schiff base14

and these synthesized pure compounds were subjected evaluation of anti-inflammatory and

antiarthritic activity in preclinical animal model in our department of pharmacology.

All the data will be collected from the experimental animal model and the animals will

be subdivided in to twenty four groups containing eight animals in each group, and doses

4

were selected as per acute toxicity:

I) Normal group with vehicle as a positive control.

II) Effect of vehicle on carrageenan induced changes in rat paw edema.

III) Effect of Standard drug on carrageenan induced changes in rat paw edema.

IV) Effect of lower dose on carrageenan induced changes in rat paw edema.

V) Effect of higher dose on carrageenan induced changes in rat paw edema.

VI) Effect of vehicle on FCA induced changes in rat edema.

VII) Effect of Standard drug on FCA induced changes in rat paw edema.

VIII) Effect of lower dose on FCA induced changes in rat paw edema.

IX) Effect of higher dose on FCA induced changes in rat paw edema. X) Effect of lower and higher doses on ulcerogenic index.

7.2 Materials

a) Animals : Female Sprague-Dawley rats.

b) Chemicals : All the chemicals to be used in the present study will be of AR grade.

c) Instruments : Digital Plethysmometer (UGO BASILE 7140 Plethysmometer),

Refrigerate centrifuge, Tissue homogenizer, UV-Spectrophotometer.

7.3 Methods:

Anti-inflammatory activity:

Carrageenan-induced paw oedema in rats: Male Sprague-dawley rats (150-200g) were used.

The animals were provided with standard diet and water ad libitum, divided into five groups,

each containing eight animals. Two groups served as a standard (Diclofen; 10mg/kg), and

negative control; and another group as a control (1%CMC) and remaining groups for test drugs.

Test compounds and Ibuprofen were suspended in 1%CMC, as vehicle. The drugs were given

orally. After 1 hour, a sub-plantar injection of 0.05ml of 1% Carrageenan is to be administered.

5

The volume of the injected paw is measure with plethysmograph immediately. Again measured

after three hours. The average paw volume in a group of drug treated rats is compared with that

of the control, and percentage inhibition of oedema is to be calculated15.

Antiarthritic activity:

Induction of arthritis: Following anesthesia (45mg/kg of Ketamine chloride i.p.), 0.1 ml of

Freund’s adjuvant [Sigma Chemical Co., USA] was injected in the sub-plantar tissue of right

posterior paw of rat. Every day animals were carefully and thoroughly inspected, by examining

the affected paw and the animal’s general status. Evaluation of anti-inflammatory effects of

quinazolinones was performed by monitoring the oedema in the right paw. In control animals,

sub-plantar injection of Freund’s adjuvant produced a pronounced local oedema after few hours

with progressive increase, reaches to maximum the 8th day after inoculation or in the 28th day

after induction of arthritis.

Acute treatment with Quinazolinone derivatives:

On day 0, all animals were subjected to behavioral test, assessment of body weight and

measurements of right paw (Test-I). Subsequently, Freund’s adjuvant was injected in the right

paw. Five days after administration of Freund’s adjuvant, animals were again subjected to the

tests (Test-II) and were randomly assigned to one of four groups. Control which receives

distilled water (1mg/kg); and standard group (Diclofenac; 10 mg/kg)16, Experimental group-1

(lower dose) and Experimental Group-2 (higher dose) of followed by the test. On the 8th day

administration of test solution, test III, IV, and V were repeated respectively.

Chronic treatment with Quinazolinone derivatives:

On day 0, the animals were subjected to behavioral test and assessment of body weight and right

paws measurement, followed by the injection of Freund’s adjuvant in the right paw (Test I). On

the 10th day after adjuvant administration, all tests are repeated (Test-II). On 20th day, animals

6

were treated with distilled water (1ml/kg of body weight), and drug (lower & higher dose) and

again subjected to the tests the next day (Test-III). The test solution was administered daily and

testing application was done on 28th day after administration of Freund’s adjuvant tests IV, V,

VI and VII respectively17.

Histopathological changes:

All the rats will be sacrificed by anesthetic ether necrosis. Hind limbs will be removed and

fixed in 10% buffered formalin. The limbs will be decalcified in 5 % formic acid, processed for

paraffin embedding sectioned at 5µm, thickness, and subsequently stained with haemotoxylin-

eosin for examination under a light microscope. Sections will be examined for the presence of

hyperplasia of the synovium, pannus and destruction of the joint spaces18.

Morphological alterations in joints:

Radiographic evaluation will be performed on the basis of radiographs and coned down

views of lower limbs. Radiographs will be taken with Siemens, Multiphos 10 (version1.0) X-ray

machine19.

Evaluation of ulcerogenic index:

Sprague-Dawley rats (150-200g) of either sex were divided into various groups each of

eight animals. Control group were administered only with 10% v/v tween 80 suspension (i.p.).

One group is administered with Aspirin (20mg/kg; i.p.) once daily for three days. Remaining

groups are treated with test drugs (20mg/kg; i.p.). On fourth day, pylorus is ligated (Shay

method). Animals were fasted for 36 hours before the pylorus ligation procedure. 4 hours after

ligation, animals were sacrificed. The stomach is removed and opened along with the greater

curvature. Then ulcer index is calculated20.

Statistical analysis:

The data collected from the present study will be analyzed by one way ANOVA followed

7

by the Dunnett’s multiple comparison test method and will be confirmed by the student’s t-test

for intergroup differences.

7.4 Does the study require any investigations or to be conducted on patients or other

animals? If so please describe briefly.

Yes, the above study requires to be carried out in rats. The effects of preparation of arthritis

will be studied and evaluated by considering physiological, pathological and biochemical

parameters in Freund’s complete adjuvant induced arthritis animal model.

7.5 Has ethical clearance been obtained from your institution for performing various tests

on animals?

Yes, the study is cleared from Institutional Animal Ethics Committee (IAEC). The copy is

enclosed with this protocol.

8

8. References:

1. Rang H. P., Dale M. M., Ritter J. M., Flower R. J., Rang and Dale’s Pharmacology, 6 th ed.

Churchil Livingstone Elsevier 2007:226-247.

2. Sachin S. L., Sudhir G. W., Sharad K. M, Studies on some biologically active 4-(3H-)-

quinazolinones part 1. Synthesis, characterization and anti-inflammatory activity of 6,8-

disubstituted 2-phenyl-3-(substituted-benzothiazol-2-yl)-4(3H)quinazolinones. ARKIVOC

2006:1-6.

3. Joseph T. D., Robert L. T., Gary C. Y., Gary R. M., Barbara G. W., Michael P. L.,

Pharmacotherapy, A Pathophysiologic Approach, 6th ed. Mc Graw Hill Medical publishing

division 1999:1671-1674.

4. Marry Anne K. K., Lloyd Yee Y., Brian K. A., Robbin L.C., Joseph G. B., Wayne A. K.,

Bradley R.W., Applied Therapeutics, The Clinical use of Drugs, 9th ed. The

PointLippincott Williams & Wilkins 2009: 43-1.

5. Choi H. K., Hernan M. A., Seeger J. D. Methotrexate and mortality in patients with

Rheumatoid arthritis: A prospective study. Lancet 2002; 359: 1173- 1177.

6. Wallberg J. S., Johansson H., Ohman M.L., Rantappa D. S, Extent of inflammation

predicts cardiovascular disease and overall mortality in seropositive rheumatoid arthritis,

A retrospective cohart study from disease onset. J. Rheumatology 1999; 26: 2562-2571.

7. Robert. A. Turner. Screening Methods In Pharmacology, Academic Press – An imprint of

Elsevier 2009;Vol-I;152-163.

8. Walker R., Edwards C. Clinical Pharmacy and Therapeutics. 5 th ed. New York: Churchill

Livingston 2003:791-793.

9

9. American college of Rheumatology. Committee on clinical guidelines. Guidelines for the

management of rheumatoid arthritis. Arthritis Rheum 1996; 32: 713-22.

10. Daidone G., Maggio B., Raffa D., Plescia S., Bajardi M. L., Curuso A., Catuli V.M.C.,

Amico-roxas M, Synthesis and pharmacological study of ethyl 2-methyl-5-[2-substituted-

4-oxo 3(4H)-quinazolinyl]-1H-pyrazole-4-acetates, Eur J Med Chem 1994; 29:707-711.

11. Veerachamy A, Sankarnarayan M. Synthesis and pharmacological evaluation of some 3-

(4-Methoxyphenyl)-2-substituted amino quinazolin-4(3H)-ones as analgesic and anti-

inflammatory agents. Chem. Pharm Bull 2007; 55(1):76-80.

12. Varsha J., Pradeep M., Sushil K., Stables J. P. CNS depressant and anti-convulsant

activities of some novel 3-[5-substituted1,3,4-thiadiazole-2-yl]-2-styryl-quinazoline-

4(3H)-ones. Eur J Med Chem 2008; 43:1945-1954.

13. Sushil K. K., Varsha K, Pradeep M., Jain N. K., Stables J. P. Synthesis, anticonvulsant

and CNS depressant activity of some new Bioactive 1-(4-substituted-phenyl)-3-(4-oxo-2-

phenyl/ethyl 4-H-quinozolin-3-yl)-urea. Eur J Med Chem 2009; 44:4335-4343.

14. Fulop F., Simeonov M., Pinhlaja K., Formation of 1,2-dihydroquinazolin-4(3H)-ones–

Reinvestigation of a recently reported 1,3,4-binzotriazepine synthesis. Tetrahedron 1992;

48(3):531

15. Kulkarni S.K. Hand book of Experimental Pharmacology. 3rd ed. Vallabh Prakashan,

Delhi, India 2007:193.

16. Suthakaran R., Kavimani S., Venkappayya D., Farhat S., Suganthi K. Inflammation:

Synthesis and pharmacological investigation of some new 4(3H)- Quinazolinone analogs

as antioxidant, antihistaminic, anti-inflammatory and antitumor agents, Rasayan J. Chem

2008; 1:2:263-275.

10

17. Monica L. A., Eduardo H., Santos R., Ana A. B.de silva., Seirgo T., Maria de L., Sara V.

Evaluation of acute and chronic treatments with Harpagophytum procumbens on Freund’s

adjuvant induced arthritis in rats. J. Ethnopharmacol 2004; 91: 325-330.

18. Campo M., Avenosa, Campo S., Ferlazzo A. M., Altavilla D., Calatroni A. Efficacy of

treatment with glycosaminoglycans on experimental collagen induced arthritis in rats.

Arths Res Therp 2003; 5: 122-129.

19. Carlson R.P., Datko L.J., Lynn O.N., Frank D., Beidman R., Lewis A.J. Comparison of

changes in established Type-II collagen and adjuvant induced arthritis using outbred

Wister rat. Int. J. Immunopharmacol 1985; 7 (6): 811-826.

20. Goyal R. K., Chakrabarthi A., Sanyl A. K., Planta media 1985; 29:85-88.

11

9. SIGNATURE OF CANDIDATE (R. B. NIDAVANI)

10. REMARKS OF THE GUIDE The quinazolinones are reported for potential CNS activity like analgesic, anti-inflammatory and aticonvulsant extensively. The new molecule modification of quinazolinones explored for anti-inflammatory and antiarthritic action with less ulcerogenic index.

11. NAME AND DESIGNATION OF THE GUIDE

Mr. CHANDRASHEKHAR V.M Asso. Professor

12. SIGNATURE

13. CO-GUIDE

14. SIGNATURE

15. HEAD OF THE DEPARTMENT Dr. I.S.MUCHANDIH.O.D. , Department of Pharmacology

H.S.K.College of Pharmacy,B.V.V.S. Campus, Bagalkot-587101.

16. SIGNATURE

17. REMARKS OF THE PRINCIPAL The above mentioned information is correct and I recommended the same for approval.

18. NAME OF THE PRINCIPAL Dr. I.S.MUCHANDIH.O.D. , Department of Pharmacology

H.S.K.College of Pharmacy, B.V.V.S. Campus, Bagalkot-587101.

19. SIGNATURE

12

OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA

REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE “BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision)

RULES 1998”

Ref: HSKCP/IAEC, Clear / 2009-10 / 1-8

CERTIFICATE

This is to certify that Mr. RAMESH B. NIDAVANI, a student of FIRST M.Pharm is

permitted to carry out experiments on animals for the dissertation / thesis work entitled

“Evaluation of Quinazolinone derivatives as potential Anti-inflammatory and

Antiarthritic agents” as per details mentioned and after observing the usual formalities laid

down by IAEC as per provision made by CPCSEA.

Animal House In charge CHAIRMAN

13

Form B

See rule [6 (a) and 8(a)]

PART A

(1) Name and address of the Establishment: H.S.K. COLLEGE OF PHARMACYBAGALKOT, KARNATAKA.

(2) Date and Registration Number of the Establishment: 821/01/a CPCSEA

(3) Name, address and Registration No. of the breeder from whom acquired and the date ofacquisition:

OFFICE OF CPCSEA,MINISTRY OF ENVIRONMENT AND FOREST, 3rd SEAWARD ROAD, VALMIKINAGAR,THRIRUVANMIYUR,CHENNAI-600041.

(4) Place where the animals are presently kept:ANIMAL HOUSEH.S.K. COLLEGE OF PHARMACYBAGALKOT, KARNATAKA.

(5) Place where the experiment is to be performed:

DEPARTMENT OF PHARMACOLOGYH.S.K. COLLEGE OF PHARMACYBAGALKOT, KARNATAKA.

(6) The date on which the experiment is to commence and the duration of the experiment:

01 JUNE 2010

The protocol form for the research proposal - PART B in the case of experiments using other than non-human primate animals for ongoing/new projects, PART C for use of non-human primates for new projects and PART D for use of non-human primate for extension of ongoing projects – should be duly filled, singed and annexed with this form.

Signature

Dated: (Name and Designation)Place: Bagalkot

14

PART – B

Protocol form for Research Proposal to be submitted to the Committee on use of small animals / Animals other than non human Primate in Biomedical Research for ONGOING / NEW PROJECTS

1. Project Title : “Evaluation of Quinazolinone derivatives as potential anti-inflammatory and antiarthritic agents”

2. Investigation (s) : Designation

CHANDRASHEKHAR V.M Associate Professor

3. Department (s) :DEPARTMENT OF PHARMACOLOGYH.S.K. COLLEGE OF PHARMACYBAGALKOT, KARNATAKA.

4. (a) Funding Source (s): if any ----

(b) Are sufficient funds available for purchase and maintenance of the animals yes

(c) Duration of present project :

(1) Number of months : 6 months

(2) Date of start of the Project : (Experiment) 01 JUNE 2010

(3) Date of termination of the project : 01 NOVEMBER 2010

5. Date by which approval is needed in case the project is to be funded by outside agency (If less than six weeks from the date of admission, please justify below).

-----

15

6. Summary of project briefly summarize in laymen’s term the background, the objective and the experiment approach.

(a) Background: Enclosed

(b) Objectives Enclosed

(c) Experimental procedure: Enclosed

7. (a) Name of species Sprague-Dawley rats

Age Sex Weight

4-6 Months Female 150-200 g

(b)Rationale for selection

Approximate number of animals required during the

First 12 months. 192

Justification of number (define treatment group and

number per group)Twenty four groups, Each group containing eight animals.

Number of animals housed per weeks 40 Animal

8. List all invasive Non Surgical Animal Procedures and Potentially Stressful Noninvasive procedures to be used (Example IM injection, foot pad injection, venapunctures).

Invasive non-surgical animal procedures.

Procedure and Approximate Frequency:----

16

9.

10.

11.

Anesthetic and/or Analgesic and Dosage:

Ketamine chloride, 45mg/kg, given i.p. route.

Test substance injected and/or applied:

Test substance will administer post orally.

Does the protocol prohibit the use of anesthetic and analgesic for the conduct of painful procedures?

No.

With surgical procedure/Experimental procedure is performed?

No.

(a) Will the animal be sacrificed after surgery?

No.

(b) Give anticipated post operative survival time:

----

Will hazardous agent such as radioisotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?

No

INVESTIGATOR SIGNATURE

DATE: _________________

17