Evaluation of antimalarial compounds in mosquito test systems

358 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 65. No. 3. 1971.

EVALUATION OF ANTIMALARIAL COMPOUNDS TEST SYSTEMS

IN MOSQUITO

EUGENE J. GERBERG Insect Control and Research Inc., Baltimore, Maryland 21228, U.S.A. During the last decade, decreased susceptibility of malaria parasites to the more commonly used antimalarial drugs has resulted in a resurgence of interest in the search for new antimalarial compounds. The meetings of the W H O Scientific Group on Resistance of Malaria Parasites to Drugs held in 1964 underlined the world wide interest in this serious problem. PETERS (1967) in an excellent review of rational methods in the search for antimalarial drugs, evaluated the various screening systems and noted that drug screening in the mosquito vector can give useful leads to compounds having sporontocidal action against Plasmodia. Such a method is relatively cheap and easy. The testing of antimalarial compounds on the sporogonous stages of plasmodia in mosquitoes may be carried on by administering the candidate compound to the Plasmodium-infected vertebrate host after which the mosquitioes may be allowed to feed on the blood of the vertebrate host. Conversely the candidate compound may be fed to the mosquito in an acceptable solution or suspension in sugar-water either before or after the vector insect is given an infective bloodmeal upon a vertebrate host infected by Plasmodium. Many Of the earlier workers (KRITSCHEWSKIand PINES, 1934, LUMSDEN and BERTRAM, 1940, RAMAKRISHNANet al., 1952, SINGH, et al., 1953, SHUTE and MARYON, 1954) used the former technique with avian malarias. TERZlAN (1947) was the first to feed antiplasmodial compounds directly to the mosquito vector in Order to observe any effect of such compounds on the sporogonous cycle of Plasmodium gallinaceum in the insect. The mosquitoes received the test compounds by feeding on cotton balls soaked in a solution of test compound and 4% sucrose. Results were evaluated by microscopic examinations of the sporogonous stages of the Plasmodium in the dissected mosquitoes or by allowing the compound-treated infected insects to feed upon chicks which were in turn followed for development or failure of parasitaemia. Terzian also injected ground compound-treated infected mosquitoes into chicks to test for presence of viable sporozoites. A number of investigators (JOHNSON and AKINS, 1948, GEIGY and RAHlVi, 1949, TERZIAN, et al., 1949, TERZIANand WEATHERSBY, 1949, SINGH et al., 1953, NARAYANDAS and RAy, 1954) used similar techniques, feeding drugs directly to infected mosquitoes. The criteria for activity of a compound consisted of o6cyst counts in experimental versus control mosquitoes or of observations of the presence or absence of viable infective sporozoites in the salivary gland. These earlier test systems were primarily designed for purposes of basic research or for use in small scale screening of antimalarials. This study was supported by the U.S. Army Medical Research and Development Command Department of the Army, under Contract DA-49-193-MD-2771, under the auspices of Walter Reed Army Institute of Research. This paper is contribution number 717 from the Army Research Program on Malaria. I greatfully acknowledge the assistance of J. W. Gentry, J. V. Gerberg, T. M. Hopkins and F. W. Kutz.

EUGENE J. GERBERG

359

To meet the requirements of a mass screening programme desired by the Division of Medicinal Chemistry of the Walter Reed Army Institute of Research, a primary screen, using mass production techniques was developed by GERBERG et al., (1966). This system can now screen 1,000 compounds a week for antiplasmodial activity. The advantages of the system are that it is economical in cost, and only requires a few mgs. of candidate compound for statistically valid testing. The method is quick (results can be obtained in 7-14 days) and reliable. The main disadvantage is that the system will select only those compounds that inhibit sporogony. Drugs such as quinine, chloroquine and mepacrine are not active, or show slight activity in this system. Materials and m e t h o d s

Avian malaria system Aedes aegypti mosquitoes are mass-reared under carefully controlled environmental conditions in order to provide uniform test insects in large numbers. 40 female pupae are placed in a clear plastic container 10.7 cm. in diameter and 7.8 cm. high, containing water to a depth of 1.5 cm. The screw top lid of the plastic vessel has an 8 cm. opening covered with 18 mesh plastic screening. The mosquitoes eclose from the pupae within 48 hours. After eclosion is complete the plastic cages are drained by inverting them and allowing the water to pass through the screen. The plastic cages are placed on aluminium trays which in turn are arranged on wheeled galvanized-iron racks. Both pupae and adults are held in a room maintained at 27°C (80°F) and 80% relative humidity. A specially designed aluminium feeding rack is placed over each tray of 22 cages. By means of the feeding rack individual disposable plastic pipettes, each containing a different candidate compound or diverse concentrations of a compound are placed in contact with the respective cages. Each tray of test compounds is provided with a known drug control (Proguanil) and a sucrose control. The feeding solution consists of 10% sucrose, 0-1% to 0.00001% candidate compound and 0.5% suspending agent. (either a hydrocolloid of high molecular weight or sodium carboxymethyl guar gum) in water. The test compounds are prepared by weighing out 15 mg. of the candidate compound, placing it in a vial with 15 c.c. of sucrose solution containing also the suspending agent. The compound is broken into fine particles and dispersed by means of a sonic probe. In our system all drugs, regardless of solubility, are considered to be insoluble in water. 3-day old mosquitoes are allowed to feed on the compound containing suspension; for 2 days, and are then starved for 2 days. The mosquitoes are starved merely to increase their avidity for blood. The insects are then offered a blood-meal by placing the plastic mosquito cage in contact with each side of a restrained infected chick. 10 day old hybrid (white Leghorn) chicks are infected by intramuscular inoculation with 1/20 c.c. of blood infected with Plasmodium gallinaceum (Brumpt 8 A strain). The parasitaemia on day 7 post-inoculation varies from 5-25%. The chick room is kept at a temperature of 27 ° ± I°C and 80% 4- 5% RH. The mosquitoes regularly engorge within 10 minutes and 30-40 cages can be fed in succession on a single bird. The percentage of engorged insects is about 90%. Using these techniques, nearly 100% of the engorged mosquitoes become infected. These figures are based on thousands of feedings. After the infective blood-meal, the drug feeding racks are replaced and the mosquitoes are again offered the sucrose/drug mixture. After 9 days, the cages are removed and a sample of 4 mosquitoes from each cage is dissected. The mosquito ovaries are checked for egg development to validate blood feeding. The gut is examined for the presence of o/3cysts and the salivary glands are examined for the presence of sporozoites. All data are recorded and transferred to computer cards.

360

EVALUATION OF ANTIMALARIAL COMPOUNDS I N MOSQUITO TEST SYSTEMS

TABLE. Comparison of 3 antimalarial screening systems: lowest confirmed sporontocidal concentration. Compound

WRAIR No.

Avian

Simian

Human

%

%

%

Quinine

2976

Chloroquine phosphate

1544

0.1 ±

Sontoquine

7429

n.t.

Hydroxychloroquine

1545

Mepacrine

1543

--

0-1

Primaquine

2975

0.1

0-1

0.1 ±

Pentaquine

6021

0-01+

0'1 ±

0-1 ±

Pamaquine

4234

Isopentaquine

6020

Metachloridine

6010

0"1

0-1

0"01

Sulphadiazine

7557

0"1

0"1

0"I

DDS

0448

Proguanil

3091

0"01

0"01

0-01

Pyrimethamine

2978

0-0001

0"0001

0.0001

Lap±none

26041

Cycloguanil

5473

±

0-1

n.t.

0'01

-0" 00001

0'1 0' 00001

0.001

indicates no sporontocidal activity. ± indicates variable or inconsistent sporontocidal activity.

--

n.t. indicates not tested.

Simian malaria system Anopheles stephensi mosquitoes are mass reared according to the schedule and methods of GERBERG et al. (1968). Cages for holding test mosquitoes are made from one half gallon paper cartons with a rubber-dam closed opening in the side. Before adding pupae, each cage is filled with water to a depth of 2 inches (5 cm.). Approximately 500 female pupae are put in each container. The carton is covered with nylon netting held in place by a paper rim made from the cover of the carton. The cover is then taped firmly to the carton. After eclosion of the adult mosquitoes, water is poured out by inverting the screened cartons. The cages are then placed On wheeled racks and transferred to the holding room for subsequent blood feeds on infected monkeys. Adult mosquitoes are maintained on 10% sucrose solution. Humidity in the holding area is held at approximately 80% and the temperature is kept at 27°C.

EUGENEJ. GERBERG

361

Rhesus monkeys (Macaca mulatta) of both sexes, are infected with Plasmodium cynomolgi~ by intravenous inoculation of parasitized-blood, or by the "bites" of sporozoite-bearing mosquitoes. Blood-induced infections are aroused by the injection of approximately 100,000 parasites. Sporozoite-induced infections are aroused by the "bites" of five or more mosquitoes having heavily infected salivary glands (1,000 plus sporozoites per gland, evaluated postprandially). Blood films (Giemsa stained) are prepared, and examined daily, starting 3 days after trophozoite inoculation and 9 days after sporozoite introduction. At first both thick and thin blood films are made but when the infections become well established only thin films are made. The timing of infective bloodmeals for mosquitoes is determined by the parasitaemia of the vertebrate host. Mosquitoes are offered a blood-meal when the parasitaemia of the monkey reaches approximately 0.5-1% accompanied by the presence of mature gametocytes. Feeding of test mosquitoes is practised daily until the parasitaemia peaks and starts to decline. (Parasitaemia surges in P. cynomolgiinfection tend to resolve by crisis). Monkeys are subsequently splenectomized and again used for infecting mosquitoes when the parasitaemia' again begins to mount. After allowing the test mosquitoes to feed on an infected host, samples of 40 fullyengorged females are gently aspirated from the feeding cage into similarly constructed test cages made from 1 pint cartons. The aspiration of engorged mosquitoes is accomplished by a mechanical pump with adjustable vacuum. The pump is attached to a plastic cover which fits snugly over the opening of the carton. The operator places one end of a suitable length of glass-tipped rubber tubing into the rubber-dam opening of the one pint test cage, and suctions off the required number of fully engorged mosquitoes. The test cages are set up on aluminium trays, 22 per tray. A second bloodmeal from an uninfected monkey is provided for the infected mosquitoes on the 3rd or 4th day after the infective blood-meal. A 3rd blood-meal from an uninfected monkey is provided on day 6 or 7 following the infective blood-meal. Compounds to be tested are prepared in the manner described above as for the avian malaria test system. After their preparation, compounds are applied to the cages of test mosquitoes by individual plastic pipettes. The drugs are available to the mosquitoes immediately after the infective blood-meal and continue available until day 14, except for such brief times as are necessary to give the supplementary blood-meals. On day 14, following the infective blood-meal a sample of 5 mosquitoes is taken from each test cage. The insects are dissected arid examined for the presence of oScysts and sporozoites. The main criteria are the presence or absence of sporozoites, as many of the oScysts have burst by day 14.

Human malaria system Anopheles stephensi is used as the vector species and test insect. Various strains of Plasmodium falciparum, depending upon availability were used. The following strains were included in tests--Vietnam (Smith), Malaya (Taylor) and Malaya (Pooley), (see CLYDE et al., 1969). Methods of rearing and handling mosquitoes are similar to those described for the simian system. The vertebrate hosts are prisoner volunteers at the Maryland House of Correction and the medical and parasitological aspects of the study are carried on by the University of Maryland School of Medicine. The criterion for *The Plasmodium cynomolgi strain was obtained from Dr. Leon Schmidt of Southern Research Institute. It is known as the PYS or RO/S strain.

362

EVALUATION OF ANTIMALARIAL COMPOUNDS IN MOSQUITO TEST SYSTEMS

feeding of mosquitoes on the human host is a gametocytaemia of 200 gametocytes/mm3. over 3 days. Immediately upon their return to our laboratory, the cartons of fed mosquitoes are enclosed in a glove box and 25 engorged females are removed and placed in a pint-sized cardboard ice cream carton. The mosquitoes are then fed the candidate compound in the same manner as in the avian and simian systems. After 14 days, the mosquitoes are anaesthetized and dissected to determine the degree of o6cyst and sporozoite development. As in the other systems the presence or absence of sporozoites is considered the main criterion. Discussion

The results of earlier workers have shown that a parallelism of activity exists between some drugs that are active against human malaria and those that are active in a bird malaria system. The criterion for ascertaining activity was the presence or absence of sporozoites. As this was a high output system, a quick, simple criterion was required. The mass screening system described here is primarily searching for causal prophylactic drugs. If some of the commonly used antimalarial drugs are examined (see Table), it may be observed that, proguanil, pyrimethamine and cycloguanil are active in all three screening systems. Quinine, chloroquine, and mepacrine are not active in the avian system, and show variable activity in the simian and human systems. The variable activity in the simian and human systems may be due to the increased time for drug feeding. It is of interest that these three drugs (quinine, cMoroquine, mepacrine) are neither active in most strains of falciparum malaria against the pre-erythrocytic or early tissue stages in the liver, nor do they show good sporonticidal action in the mosquitoes. On the other hand, proguanil and pyrimethamine are active against the pre,erythrocytic or early tissue phase of the parasite and are also sporontocidal. In our system these two drugs (proguanil and pyrimethamine) provide complete suppression of orcysts when fed to mosquitoes at the 0,01% level. Pyrimethamine will completely suppress development of sporozoites in the mosquito at the 0-0001% level in both avian and simian systems. TERZAKIS, SPRINZ and WARD (1967) point out that in avian malaria, sporozoite formation is strikingly similar to the process of merozoite formation. It might then be surmised that a substance that interferes with sporozoite formation might likewise interfere with merozoite formation, or for that matter with any phase that involves nuclear division of the parasite. The mechanism of this action is not fully understood, but POWELL (1966) believes that it may involve interference with the folic acid metabolism of the parasite and consequently with DNA synthesis. This theory is supported by the present test system since folic acid antagonists (pyrimethamine, etc.) are found to suppress the development of sporozoites in the mosquito. Results

Over 80,000 compounds have been tested during the 3 year period from August 1966 to July 1969 in the test systems we have described. From August 1968 to July 1969, 42,792 tests were conducted of which 2,257 or 5% showed some antimalarial activity on the part of the compounds involved. Comparisons of the three screening systems indicate that the cynomolgi-stephensi system is slightly more sensitive than the gallinaceum-aegypti system. The cynomolgistephensi and falciparum-stephensi systems appear equal in sensitivity. To compare the sensitivity of the gallinaceum system with the cynomolgi system, 111 compounds were selected and the tests reconfirmed.

EUGENEJ. GERBERG

363

were active in the gallinaceum system only were active in the cynomolgi system only were more active in the gallinaceum system were more active in the cynomolgi system showed the same inactivity or activity in both systems of which 22.5% showed the same activity in both systems 43.3% were inactive in both systems 10-8% 7-2% 0.9% 15.3% 65.8%

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