Screening Methods for Antipyretic Agents

SCREENING METHODS FOR ANTIPYRETIC AGENTS

Treatment with antipyretics has been very important in the preantibiotic era. Nevertheless, for treatment of acute viral diseases and for treatment of protozoal infections like malaria, reduction of elevated body temperature by anti-pyretics is still necessary. For antiinflammatory compounds, an antipyretic activity is regarded as a positive side effect. To evaluate these properties, fever is induced in rabbits or rats by injection of lipopolysaccharides or Brewer’s yeast.

Antipyretic Testing in Rats

The subcutaneous injection of Brewer’s yeast suspension is known to produce fever in rats. A decrease in temperature can be achieved by administration of compounds with antipyretic activity.

Procedure

A 15% suspension of Brewer’s yeast in 0.9% saline is pre-pared. Groups of six male or female wistar rats with a body weight of 150 g are used. By insertion of a thermocouple to a depth of 2 cm into the rectum the initial rectal tempera-tures are recorded. The animals are fevered by injection of 10 ml/kg of Brewer’s yeast suspension subcutaneously in the back below the nape of the neck. The site of injection is massaged in order to spread the suspension beneath the skin. The room temperature is kept at 22–24°C. Immedi-ately after yeast administration, food is withdrawn. 18 h post challenge, the rise in rectal temperature is recorded. The measurement is repeated after 30 min. Only animals with a body temperature of at least 38°C are taken into the test. The animals receive the test compound or the standard drug by oral administration. Rectal temperatures are recorded again 30, 60, 120, and 180 min postdosing.

Evaluation

The differences between the actual values and the starting values are registered for each time interval. The maximum reduction in rectal temperature in comparison to the control group is calculated. The results are compared with the effect of standard drugs, for example, aminophenazone 100 mg/ kg p.o. or phenacetin 100 mg/kg p.o.

Modifications of the method

Stitt and Shimada (1991) and Shimada et al. (1994) induced fever in rats by microinjecting 20 ng PGE1 directly into one of the brain’s circumventricular organs of the rat known as the organum vasculosum laminae terminalis.

Luheshi et al. (1996) induced fever by intraperitoneal injection of 100 μg/kg lipopolysaccharide into rats and measured the inhibition of fever by interleukin-1 receptor antagonist.

Telemetry has been used to record body temperature in animals (Riley et al. 1978; Gallaher et al. 1985; Clement et al. 1989; Guillet et al. 1990; Kluger et al. 1990; Bejanian 1991; Watkinson et al. 1996; Miller et al. 1997).

Antipyretic Testing in Rabbits

Lipopolysaccharides from Gram-negative bacteria, for example, E. coli, induce fever in rabbits after intravenous injection. Only lipopolysaccharide fractions are suitable, which cause an increase of body temperature of 1°C or more at a dose between 0.1 and 0.2 μg/kg after 60 min. In the rabbit, two maxima of temperature increases are observed. The first maximum occurs after 70 min and the second after 3 h.

Procedure

Rabbits of both sexes and of various strains with a body weight between 3 and 5 kg can be used. The animals are placed into suitable cages and thermocouples connected with an automatic recorder are introduced into the rectum. The animals are allowed to adapt to the cages for 60 min. Then 0.2 ml/kg containing 0.2 μg lipopolysaccharide are injected intravenously into the rabbit ear. After 60 min, the test compound is administered either subcutaneously or orally. Body temperature is monitored for at least 3 h.

Evaluation

A decrease of body temperature for at least 0.5°C for more than 30 min as compared with the temperature value before administration of the test compound is regarded as positive effect. This result has been found after 45 mg/kg phenylbutazone s.c. or 2.5 mg/kg indomethacin s.c.

Modifications of the method

Cashin and Heading (1968) described a simple and reliable assay for antipyretic drugs in mice, using intracerebral injec-tion of pyrogens. Davidson et al. (1991) tested the effect of human recombinant lipocortin on the pyrogenic action of the synthetic polyribonucleotide polyinosini:polycytidylic acid in rabbits. Yeast-induced pyrexia in rats has been used for antipyretic efficacy testing by Loux et al. (1982) and Cashin et al. (1977). van Miert et al. (1977) studied the effects of antipyretic agents on fever and ruminal stasis induced by endotoxins in conscious goats. Petrova et al. (1978) used turpentine-induced fever in rabbits to study antipyretic effects of dipyrone and acetylsalicylic acid. Lee et al. (1985) studied the antipyretic effect of dipyrone on endotoxin fever of macaque monkeys. Loza Garcia et al. (1993) studied the potentiation of chlorpromazine-induced hypothermia by the antipyretic drug dipyrone in anesthe-tized rats. Shimada et al. (1994) studied the mechanism of action of the mild analgesic dipyrone preventing fever induced by injection of prostaglandin E1 or interleukin-1β into the organum vasculosum terminalis of rat brain.