Evaluation of parasitemia in malaria cultures

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DISCUSSION

In the current study I have observed that clofazimine, primarily an anti-mycobacterial agent, does not possess anti-plasmodial activity at therapeutically relevant concentrations in vitro. This observation is in agreement with a previous report (Sheagren, 1968). Interestingly, however, substitution of the isopropyl substituent on the imino functional group at position 2 of the phenazine nucleus of clofazimine with a IMP group resulted in acquisition of activity against P. jalciparum. Of the seven IMP-substituted phenazines, all of which varied according to the number and type of substituents on the aniline and phenyl rings attached at positions 3 and 10 of the phenazine nucleus respectively, two, B4119 and B4158, exhibited anti-plasmodial activity which was comparable with that of chloroquine. B4119, the most potent of the two agents, is halogenated whereas B4158 is isopropyiated at their respective aniline and phenyl rings.
These agents were investigated for activity against drug-resistant strains ofP. jalciparum. Both agents, at sub-microgram concentrations, were found to be active against all the drug-resistant strains ofthe malaria parasite, the level of susceptibility in the case of chloroquine-resistant strains being similar to that of chloroquine-sensitive strains. Ihese observations suggest that the sensitivity ofP. jalciparum to the IMP-substituted phenazines is unaffected by the mechanisms which confer resistance to conventional antimalarial agents. Chloroquine and quinine are lysosomotropic weak bases which accumulate within food vacuoles, killing the parasite by mechanisms related to interference with haem polymerization (Ridley, 1997).
Sulfadoxine/pyrimetharnine is an antimetabolite which inhibits plasmodial folate metabolism, resulting in parasite death (peterson et.al, 1990).
Considerable interest remains in the identification of compounds which potentiate the activity of classical antimalarials to decrease toxicity and counter development of drug resistance without compromising clinical efficacy (Winter et.al, 1997; Yeo et.al, 1997). Both B4119 and B4158 potentiated the activity of mefloquine as well as that ofchloroquine, suggesting that combinations of the conventional anti-plasmodial agents with the IMP-substituted phenazines may have therapeutic potential. Ihe change in chloroquine sensitivity (from 0.074 to 0.38!!M) that occurred in the absence ofdrug pressure during continued culturing of the PtUP 1 0 strain ofP. jalciparum indicates that changes in culturing conditions may affected the sensitivity patterns that emerge due to selection ofchloroquine-resistant clones (Freese et.ai, 1991).

Stringently synchronised cultures were used to investigate the stages of growth at which the parasite is most vulnerable to the TMP-substituted phenazines. In these experiments B4119 and B4158 were added to ring and late trophozoite cultures of parasites. The test agents were found to interfere with parasite development at both stages of the life cycle, with the late phase of parasite development (the last 24 hours) being most sensitive. These observation suggest that B4119 and B4158 affect metabolic events which are essential for parasite survival throughout the life cycle.
Because the prototype riminophenazine, clofazimine, has been reported to be a membrane active agent (Van Rensburg et.ai, 1993; De Bruyn et.ai, 1996), the ability ofB4119 and B4158 to alter erythrocyte membranes and inhibit parasite invasion and maturation was also investigated. Schizont-infected red cell suspensions were allowed to rupture and release merozoites in vitro. Neither agent inhibited erythrocyte invasion by merozoites. However, the resulting ring forms failed to mature into trophozoites at 4 and 811M of both compounds. This observation suggests that the TMP-substituted phenazines do not alter the structure of the erythrocyte receptor for parasite invasion (glycophorin-A) (Gratzer and Dluzewski, 1993) and that the parasite adherence structures on the red cell surface are insensitive to B4119 and B4158. The compounds therefore act subsequently on the development and maturation of rings into trophozoites. These results imply that the target sites for the TMP-substituted phenazines are located intracellularly and may be indispensable for parasite maturation. Laboratory work conducted in Chapter 4 will serve to identifY the antimalarial mode of action of these two compounds.

PARASITES

Plasmodium berghei parasites were obtained from Dr. Ian Havlik; Department of Experimental and Clinical Pharmacology, University of the Witswatersrand. The parasites were kept in liquid nitrogen as a 1 : 2 dilution of infected blood in 28% glycerol. The parasite was passaged once in Balb/C mice before use in each experiment. Briefly, a frozen vial was thawed and three mice received a single inoculum of 0.2m1 of the parasite suspension intraperitoneally. Parasitemia was allowed to develop to about 20% at which time blood was collected to prepare an infective inoculum for subsequent administration to experimental mice. For preparation of the inoculum, the mice were anaesthetized using halothane (Rhone-Poulenc-Rhorer, Lyon, France) and the thoraxes were opened. Blood was collected in citrate from the dorsal vena carva at the hepatic junction.
The blood was centrifuged at 200g for Smin and the buffY coat of leukocytes as well as excess citrate were removed. The blood from the three mice was then pooled. Part of the suspension was diluted in washing medium to approximately 1 to 2.S X106 parasitized red blood cells per 0.2m! of the suspension. The recipient mice were injected with a single inoculum of 0.2ml intraperitoneally. The other part of the parasite suspension was mixed with freezing medium (28% glycerol) and stored in liquid nitrogen (cryopreservation). The course of clinical infection was monitored by weighing the mice daily and evaluating parasitemia microscopically. Briefly, the tails were pricked with a thin needle and thin smears were prepared on microscope slides and airdried. After fixing with ethanol, the smears were stained with Giemsa solution (0.2ml Giemsa I 2ml PBS) for 5min. After staining, the slides were washed under running tap water, dried and investigated under a light microscope at a magnification of 787.5. The level of parasitemia was determined by counting 1000 red blood cells per mouse.

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Effects of pretreatment of mice with B4119 on the growth of P. berghei.

Female Balb/C mice (inbred), 6 – 8 weeks old, were divided into groups of ten mice. Mouse food with 0.025% (30mglkglday) B4119 was given three weeks prior to infection (pre and postinfection treatment group). On the day of infection another group of mice was given mouse food with 0.025% (30mglkglday) B4119 (postinfection treatment group) and treatment was continued in the latter group. All mice were inoculated intraperitoneally with 2.5 X 106 infected red cells per 0.2ml suspension ofP. berghei parasites. The course of infection and drug effects were evaluated every second day starting from day 4 as detailed in section 6.3.2 and treatment was terminated on day 10. Results are expressed as percentage parasitemia, survival rate and weight.

Effects of chloroquine on the growth P. berghei-infected Balb/C mice.

Female, 6-8 week old, Balb/C mice (inbred) were divided into groups of five mice. The mice were injected intraperitoneally with 2 X 106 infected red cells per 0.2ml suspension ofP. berghei parasites. On the same day the mice were injected with varying doses of chloroquine (1.25 – 25)lglkglday in normal saline). Control mice were injected with 0.2ml normal saline only. The course of infection and drug effects were evaluated every second day from day 4 as detailed in section 6.3.2 and treatment was terminated on day 10. Results are expressed as percentage parasitemia and survival rate.

CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
1.1 THE MALARIA PARASITE
1.2 ERYTHROCYTE MEMBRANE STRUCTURE AND FUNCTION
1.3 THE ERYTHROCYTE AND MALARIA PARASITE INVASION
1.4 THE MALARIA-INFECTED ERYTHROCYTE
1.5. TREA TMENT AND PREVENTION OF MALARIA INFECTIONS
1.6. RIMINOPHENAZINES
1.7 AIMS AND OBJECTIVES OF THE STUDY
CHAPTER 2
EVALUATION OF PARASITEMIA IN MALARIA CULTURES
2.1 INTRODUCTION
2.2 AIMS AND OBJECTIVES
2.3 MA TERIALS AND METHODS
2.4 RESULTS
2.5 DISCUSSION
CHAPTER 3
IN VITRO ANTIMALARIAL ACTIVITIES OF TMP-SUBSTITUTED PHENAZINES, B4119 AND B4158
3.1 INTRODUCTION
3.2 AIMS AND OBJECTIVES
3.3 MA TERIALS AND METHODS
3.4 RESULTS
3.5 DISCUSSION
CHAPTER 4
HEME POLYMERIZATION INHIBITORY ACTIVITY (HPIA) OF B4119 AND B4158: AN INFRARED SPECTROMETRIC STUDY
4.1 INTRODUCTION
4.2 AIMS AND OBJECTIVES
4.3 MATERIALS AND METHODS
4.4 RESULTS
4.5 DISCUSSION
CHAPTER 5
CYTOTOXIC ACTIVITY OF B4119 AND B4158 AGAINST NORMAL HUMAN ERYTHROCYTES
5.1 AIMS AND OBJECTIVES
5.2 MA TERIALS AND METHODS
5.3 RESULTS
5.4 DISCUSSION
CHAPTER 6
PLASMODIUM BERGHEI MOUSE MODEL: ANTIPLASMODIAL ACTIVITY OF B4119
6.1 INTRODUCTION
6.2 AIMS AND OBJECTIVES
6.3 MATERIALS AND METHODS
6.4 RESULTS
6.5 DISCUSSION
CHAPTER 7
CONCLUDING DISCUSSION
REFERENCES

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