THE TESTING OF DRUGS AGAINST EXOERYTHROCYTIC FORMS OF P. GALLINACEUM IN TISSUE CULTURE

Abstract

In 1938 James and Tate described a tissue phase of P. gallinaceum in chicks which they called the exoerythrocytic phase of the parasite, and in 1945 Hawking succeeded in growing these forms in tissue culture. At the time of writing, similar forms have not been demonstrated in mammalian malaria, but it is likely that they do exist, and that the relapses common in benign malaria are due to their presence.The tissue culture technique has been used as an in vitro method for testing the effect of drugs against the tissue phase of P. gallinaceum. It is hoped that this test can be adapted so that the effect of drugs against the tissue phase of all types of Plasmodia can be investigated. A drug which was active against the tissue phase of the malaria parasite in this test would probably be a causal prophylactic and have a beneficial effect on the relapse rate of benign tertian malaria. METHOD Ten-day-old chicks were injected with sporozoites of P. gallinaceurm and killed [7][8] days later, when they were heavily infected with tissue forms of the parasite. In a typical experiment the spleen from such an infected chick was removed aseptically, minced finely in Tyrode's solution, and set up in Carrel flasks, each of which contained 3-4 coverslips, using the method described by Hawking (1945). The fluid phase consisted of Tyrode's solution containing 20 per cent (v/v) chick serum and 3-4 per cent (v/v) chick embryo extract; 0.05 per cent phenol red was added to indicate changes in the pH of the medium. Penicillin was also added to make a final concentration of 3 units per ml. as an additional safeguard against possible contamination by bacteria. (Later 0.5 unit/ml. was used.) Drugs to be tested were dissolved in Ringer's solution, sterilized by boiling, and appropriate concentrations made by serial dilution. 2.5. ml. volumes of the fluid phase were then run into the flasks containing the infected spleen explants, followed by 0.5 ml. volumes of the drug solutions-the control flasks receiving 0.5 ml. volumes of sterile Ringer solution in place of drug. In this way a series of flasks was set up in which the same infected material was in contact with varying concentrations of several different drugs.Under these conditions the final concentration of the drug in the flasks is less than the nominal concentration owing to the absorptive powers of the plasma and spleen explants used in the course of the experiments; the ratio of the nominal concentration to the final concentration may also vary because the amounts of spleen and plasma used were not

Activation of Paludrine

“…The simplest explanation for these facts is that paludrine undergoes some chemical modification which converts it into an active compound. This hypothesis would explain the contradictory findings of Tonkin (1946) that paludrine had no antimalarial action when added to tissue cultures of exo-erythrocytic forms of P. gallinaceum, and of Black (1946), who reported that serum from a patient treated with paludrine arrested the development in vitro of trophozoites of P. falciparum. Marshall (1947) has reported that paludrine solutions are active in inhibiting the uptake of oxygen by P. gallinaceum in vitro; but the concentrations of paludrine required to produce this effect were 10' to 10' times as great as those used in our experiments (1 in 3,000 to 1 in 6,000 as compared with 1 in 1,000,000 to 1 in 10,000,000).…”

“…The tubes were incubated at 370 C. for 4 hours and then centrifuged, and the supernatants removed. These provided the "liver extracts" and " liver extract plus' paludrine," the use of which is illustrated in the typical protocol reproduced in Table II up, without allowing a preliminary period for growth (Tonkin, 1946). Serum A was obtained from a normal fowl and it was used as serum for all the cultures unless stated otherwise.…”

“…The exo-erythrocytic forms of P. galfinaceum were grown in tissue culture at 370 C. by the technique described by Hawking (1945) and used by Tonkin (1946) to study the action of antimalarial 'compounds. The implants were obtained from the spleen of chickens, at the stage of infection when exo-erythrocytic parasites were present.…”

“…ClK< 3NH.C(=NH) NH.C(= NH) NH.CH(CH3), Tonkin (1946), in this laboratory, investigated the effect of adding paludrine to tssue cultures containing exo-erythrocytic forms of Plasmodium gallinaceum and found that the parasites developed in the highest concentrations (2-5 mg. per litre) tolerated by the macrophages. Since this concentration is many times greater than that which is believed to be present in the plasma during human therapy (probably about 0.3 mg. per litre), and since Davey (1946) had reported that paludrine was highly effective in curing the exo-erythrocytic infections of P. gallinaceum in chickens, as well as the endo-erythrocytic ones, Tonkin's finding was surprising and led to further investigations, which were undertaken with both P. gallinaceum and P. cynomolgi.…”

Proguanil—the Isolation of a Metabolite With High Antimalarial Activity

“…Hawking and Perry (1948) showed that whilst the serum from animals dosed with proguanil was active in vitro against Plasmodium gallinaceum, the drug itself had no demonstrable effect on the exo-erythrocytic forms of the parasite grown in tissue cultures or on the blood forms of P. cynomolgi (see also Hawking, 1947, andTonkin, 1946). Incubation of proguanil with minced liver was said to produce in vitro activity.…”