Screening of Antiinflammatory Drugs

Diana Holidah

Outline
Describe the inflammatory response Anti-inflammatory drugs Explain methods for screening of Antiinflammatory drugs Discuss the lab work

Inflammation
Response of immune system to foreign organism or antigenic substance Characterized by edema, erythema, swelling, and pain or tenderness

Inflammatory Mediators
Phospholipids

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Phases of inflammation
Acute: vasodilation & increase capillary permeability
Delayed: infiltration of leukocytes and phagocyte cells Chronic proliferative: tissue degeneration and fibrosis

Anti-inflammatory Drugs
Steroidal: ex. Corticosteroids (inhibit gene transcription ) Non-steroidal Anti-inflammatory Drugs (NSAID) (inhibit COX) Ex: ASA, indomethacin, ibuprofen Selective COX-2 inhibitors

Screening methods For Antiinflammatory Drugs

In vitro screening In vivo screening

In vitro screening
The screening methods to be selected have to be oriented towards the predominant & fundamental pathophysiological processes involved in inflammatory & allergic reaction

Sites for drug intervention:

Arachidonic acid metabolism Phagocytic & other cell functioned in inflammatory process Complement factors Autoimmune processes

Methods
Cyclooxygenase test (prostaglandin synthetase) 5-lipoxygenase test Complement test Granulocyte-chemoluminescence test Histamine release test from leucocytes T-lymphocyte transformation test Chemotaxis

In vivo screening
Acute and subacute inflammation:
UV-erythema in guinea pigs Vascular permeability Croton oil ear edema Paw edema Pleurisy test

Chronic proliferative inflammation:

Cotton wool granuloma Glass rod granuloma PVC sponge granuloma

Granuloma Pouch edema method

Croton oil Control vs. treated Calculate % inhibition

14-03-07

Gamra Hamad

Procedure:
For tests in mice the irritant is composed as follows (v/v): 1 part Croton oil, 10 parts ethanol, 20 parts pyridine, 69 parts ethyl ether. For tests in rats the following mixture is prepared (v/v): 4 parts Croton oil, 10 parts ethanol, 20 parts pyridine, 66 parts ethyl ether. The standards and the test compounds are dissolved in this solution.

 Ten animals are used for controls and each test group. The test compounds are dissolved in a concentration of 0.03 mg/ml to 1 mg/ml for mice and in a 3 to 10 time's higher concentration for rats in the irritant solution. On both sides of the right ear 0.01 ml in mice or 0.02 ml in rats are applied. Controls receive only the irritant solvent. The left ear remains untreated. The irritant is applied under ether anaesthesia.

 Four hours after application the animals are sacrificed under anaesthesia. Both ears are removed and discs of 8 mm diameter are punched. The discs are weighed immediately and the weight difference between the treated and untreated ear is recorded indicating the degree of inflammatory edema. In the originally described method the ears are removed by sharp, straight scissors 6 h after application and weighed as total. The animals were sacrificed 48 h after topical ad-ministration and the thymus glands were removed, weighed and expressed as mg thymus/100 g body weight.

Evaluation:
The antiphlogistic effect can be determined by expressing the increase in weight of the treated ear as percentage of the weight of the contra lateral control ear The difference of both weights is divided by the weight of the contra lateral ear times 100. Otherwise, the difference between either ears or excised discs is calculated as the average values for treated and control groups and the effect is evaluated by statistical methods

Rat-paw edema
Edema induction done on the animal feet (intraplanar injection of carrageenan) The sample is given orally an hour before the carrageenan injection The size of the feet oedema measured using plestimometer (an equipment that works based on Archimedes theory)

White rat Wistar strain with 180-200 g in weight 10 rats for each group Divided into 2 groups:
Control group Test group with several doses

Procedure
1. 2. The rats are fasted + 18 hours before the test On the testing date, weight the rats and divided into groups: a. control group: carrageenan induction b. test group: carrageenan induction & sample with several doses 3. Control group 1 ml/10 g BW NaCl p.o. Test group suspension of sample 1 ml/100 g BW p.o. several concentration

Procedure
4. An hour after giving the sample suspension or control, the left feet of each rat are injected with 0,05 ml carrageenan suspension intraplanarry The left feet volume are measured using the pletismometer every 15 for 3-4 hours after the giving of carrageenan All of the data are tabulated and the average feet volume of each groups are calculated

5.

6.

Evaluation
Statistical point of view the feet volume of test group and control are compared significancy ????? If it is significantly difference, calculate the % of inflammatory reduction on the test group:
a b % reduction 100 % a

Note: a = average edema volume on control group b = average edema volume on test group

 Cotton Pellet-Induced Granuloma in Rats

This model is based on the foreign body granuloma which is provoked in rats by subcutaneous implantation of pellets of compressed cotton After several days, histologiclly gaint cells and undiffentiated connective tissue can be observed beside the fluid infiltration The amount of newly formed connective tissue can be measured by weighing the dried pellets after removal More intensive granuloma formation has been observed if the cotton pellets have been impregnated with carrageenan

Procedure:
The animals used in this method are rats divided into five groups (n=6), fasted overnight and allowed free access to water. The animals are administered with vehicle, standard drug and test drugs. One hour after the first dosing, the animals are anesthetized with anesthetic ether and 20 mg of the sterile cotton pellet is inserted one in each axilla and groin of rats by making small subcutaneous incision. The incisions are sutured by sterile catgut (Crunkhon et al., 1971).

 The animals are sacrificed by excess anesthesia on the 8th day and cotton pellets are removed surgically. Pellets are separated from extraneous tissue and dried at 60C until weight become constant. The net dry weight, i.e. after subtracting the initial weight of the cotton pellet will be determined. The average weight of the pellet of the control group as well as of the test groups is calculated. The percent change of the granuloma weight relatively with vehicle control is determined and statistically evaluated.

 The percentage inhibition increase in the weight of the cotton pellet is calculated.
% Inhibition = Wc - Wd / Wc C 100
Wd = difference in pellet weight of the drug treated group. Wc = difference in pellet weight of the control group.

References
Dey, P.M. and Harborne, J.B., 1991. Methods in Plant Biochemistry. Volume 6. Assays for Bioactivity. Edited by Hostettmann, K., Academic Press., Inc., San Diego, USA.
Kelompok Kerja Ilmiah Phyto Medica, 1993. Penapisan Farmakologi, Pengujian Fitokimia dan Pengujian Klinik. Yayasan Pengembangan Obat Bahan Alam Phyto Medica, Jakarta

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