Chronotherapy of malaria: An approach to malaria chemotherapy

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Chronotherapy of Malaria: An Approach to Malaria Chemotherapy I. Landau, A. Chabaud, G. Cambie and H. Ginsburg Chronotherapy is the science of the timing of drug application so as to achieve optimal therapeutic success for the treatment of disease. Here, Irene Landau, Alain Chabaud, Gilles Cambie and Hagai Ginsburg show how a suitable animal model can be chosen, how the stage of parasite development most susceptible to the drug can be identified, and how this can eventually be used for the improvement of drug treatment.

Chronotherapy is based on the universality of biochemical and physiological rhythmicity in living organisms that cause temporal variations in the disposition of drugs and the response of the pharmacological target to them. Given the circadian rhythms of malarial parasites and their vertebrate hosts, chronotherapy of malaria seems, a priori, an attractive approach for the treatment of the disease.

cological effect of a potion. Chronotoxicity and chronotolerance indicate rhythmic changes in toxic effects and in resistance to a given drug, respectively. Similarly, chronopharmacokinetics signifies time-related changes in the pharmacokinetics of a drug and chronoesthesy designates the sensitivity of the drug's target. Thus, chronotherapy deals with the augmentation of a desired pharmacological effect and/or t',,~ diminution of unwanted effects by ascertaining the most auspicious biological time for drug administration. Chronopharmacological investigations, therefore, entail the manipulation of both the time of dosing and the dose itself (see Box 2). Circadian changes in dose-response relationships are a common phenomenon. Malaria is a typical circadian

Box I. Terminology of Circadian Rhythms

Biological R h y t h m s and M e d i c i n e

Most living organisms, from the unicellular eukaryote to the mammal, display some type of biological rhythmicity. The characterization of biological phenomena by measurable chemical or physical parameters reveals that these measures are not constant with time but oscillate with a uniform and predictable periodicity t. Biological rhythms in different organisms are governed by endogenous oscillators (biological clocks) with a complex hierarchy that matches the complexity of the organism. Biological clocks are set by external signals, or synchronizers, that are inherent properties of the organism's natural environment. Synchronizers are able to shift the phase of the period, or the acrophase (see Box I), but do not create the rhythm itself. Biological rhythms are genetically inherited and the modern view holds that they have evolved to allow the organism to adapt to predictable changes in various environmental factors. Since rhythmicity is expressed at the biochemical and physiological levels, toxic and pharmacological effects of drugs can also vary with biological rhythms. Thus, chronopharmacology is the rhythmic alteration in the pharma-

phenomenon. The life cycle of the parasite is a multiple of 24-h duration. Human malarias are infradian (eg. tertian or quartan) and exhibit an amplitude characterized by the malaria fever. The synchronizer of this cycle may be either the circadian rhythm of the host or the circadian habits of the mosquito vector. Except for the pioneering work of Hawking and colleagues 2, these aspects of the host-Plasmodium relationship have not been thoroughly investigated. Such studies would be very complex because of the interactions between the biological clocks of the parasite, vector and host, particularly as the latter two are synchronized by external signals. Clock-clock inter-relationships could have important consequences for the optimization of anti-malarial chemotherapy.

Biological rhythms are portrayed and measured by the following parameters: = Period. The time span between two habitual repeating events, such as maxima or minima of a measurable circumstance. Periods can be circadian (24 h), circannual ( I y) and ultradian ( < 2 0 h). • Acrophase. The circadian peak-time location with respect to a reference time (eg. noon or midnight). • Amplitude. One-half the maximal alteration in a measurable parameter, between trough and peak. • Mesor. The rhythm-adjusted average, ie. the arithmetic mean of data collected at fixed intervals.

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schizogony always appears 24h postinoculation, irrespective of the host's rhythm, and subsequent stages appear regularly at six-hourly intervals 13. The chronotherapeutic approach has therefore been tested, in vivo, using the P. v. petteri-laboratory mouse (ie. Mus musculus) model and chloroquine, which is known to have a relatively short duration of active concentration in the blood, and was used at a concentration known to have sub-maximal activity in this model. Two different tests were used. In the first, a single dose of the drug (5mgkg -I body weight) was injected subcutaneously during the first 24 h after infection at six-hourly intervals, ie. when different developmental stages of the parasite were present in the blood; the duration of the prepatent period was determined by microscopic inspection. In the second test, the drug was injected on the fourth day post-infection, when the parasitaemia was close to I%. This

Box 2. Pharmacokinetics Traces in the figure display the simulated pharmacokinetics of a drug injected subcutaneously or intramuscularly. This classical kinetic behaviour describes the increase in blood drug concentration following drug injection, a rapid decrease due to partitioning of the drug in the tissues (the spike) and a slower decline as a result of excretion. The DS and DR lines depict the levels of drug needed to exterminate drugsensitive and drug-resistant parasites, respectively. Obviously, drug-resistant parasites will be affected only if the duration of the spike, say at half-maximal height (designated by tl), is sufficient to cause irreversible inhibition of parasite viability. If t~ of the first injection is too short, i,t can be prolonged by extended infusion or by consecutive injections given at intervals equal to t=, resulting in t2 for two injections and t3 for three injections. For drug-sens, itive parasites, the given dose is in excess and could exacerbate unwarranted side effect.,; and select for drug-resistant strains. Hence, for these parasites a much smaller dose should suffice.

The development of a chronotherapeutic approach would entail a precise understanding of the chronopharmacokinetics of anti-malarial drugs and the ways in which these could be influenced by the course and gravity of the disease and of ckronoesthesy in relation to the parasite's cycle. Since a great deal has been recently learned about the pharmacokinetics of antimalarial drugs 3'4 and some information concerning stage-dependent susceptibility of the parasite to drug is available s-8, it seems that the time is ripe for a more earnest consideration of chronotherapy in the treatment of malaria.

chabaudi infections are synchronous and depend on the host's circadian rhythm and, for P. vinckei petteri, synchronous infection is independent of the host's rhythm ~~.~2.While all three species can be used as models of human malarias, P. v. petteri is the most useful since

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37.8 Cultures of malaria parasites, in vitro, are experimentally convenient for the identification of stages sensitive to drug treatment. However, the parasite in culture, while possessing:an endogenous biological clock, does not experience physiological and biochemical alterations arising from the circadian rhythm of the host that could serve as synchronizers. This, and the fact that parasites in culture are particularly susceptible to drug treatment 9, casts some doubt on the extrapolation of results thus obtained to conditions, in vivo. Hence, although animal models are less amenable to experimentation, results obtained with them may indicate what happens in the human host. Variations between circadian rhythms of species are best interpreted by the ability of merozoites to survive in the circulation and invade red blood cells at different times of the day ~°. Consequently, of the three species that infect the rodent Thamnomys rutilans in Central Africa, P. yoelii yoelii infections are highly asynchronous, P. chabaudi

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permitted the determination of the percentage of each particular stage present in the blood 13. The drug was also injected at six-hourly intervals before the onset of high parasitaemia and uncontrolled parasite multiplication (this characteristic has little in common with the disease in wild rodents). Both tests yielded identical results. A single drug injection almost exclusively affected mid-term trophozoites, At the doses of chloroquine used (5mgkg i body weight), the development of other stages was not affected. In the second test, as the parasites reached the sensitive stage, ie. mid-term trophozoite, they disappeared from the circulation, No difference in drug efficacy was detected when similar experiments were performed with mice maintained in a reverse day-night regimen (which did not affect the parasite's cycle), suggesting that drug disposal does not depend on the circadian rhythm of the host 13, Following a single injection of chloroquine, the population of sensitive midterm trophozoites is eliminated but, since schizogony is not absolutely synchronous, other stages that did not respond to the submaximal drug dose continued to develop. When they reached the sensitive stage they were affected, indicating that the drug concentration up to 12h past the first treatment was still high enough to kill the parasites. As a result of the selective elimination of the sensitive stage, the pattern of parasitaemia was considerably changed and the circadian rhythm of the parasite markedly altered. When this was taken into consideration and a second injection timed to coincide with the reappearance of mid-term trophozoites, 18h after the first injection, maximal therapeutic effect was obtained, Other 'two-hits' schedules, ie, the first injection at the time of the presence of other stages or shorter or longer time intervals between consecutive injections, were less efficient 13. This finding clearly suggests that chronotherapy might be of importance in the treatment of malaria.

Chloroquine in Falciparum Malaria It is widely accepted that the paroxysmal periodicity in all types of human malarias corresponds to the end of the schizogonic cycle, when the merozoites together with residual bodies are discharged from the ruptured host red blood ceils into the bloodstream ~4. While in P. vivax and P. malariae infec-

tions febrile peaks are high and narrow, P. falciparum infections show an elevated temperature plateau interrupted by declining peaks prior to the rupture of schizonts (Fig. I). In falciparum infections, the fever may be initially quotidian (daily) and irregular in about half the patients, subsequently becoming tertian. Hence, the febrile curve can be used as a guide for the chronotherapeutic administration of chloroquine only if parasitaemia is tertian, However, P. falciparurn-infected erythrocytes are sequestered in the deep vasculature when the parasite matures to the trophozoite stage, Hence, periodic microscopical inspection could be used to determine the parasitic cycle and the timing of drug treatment. Examination of Fig. I suggests that drug treatment during the first 24h after merozoite release would have little efficacy (if extrapolation from the mouse model is made) since only relatively insensitive stages would be present during this period. If, or when, the chronotherapeutic approach is applied to the chemotherapy of malaria, great care must be taken in discriminating the effects of clearance of bloodstream forms and the sequestration of the mature forms in the deep organs ~s. However, that such an approach is warranted has been underscored by Chinese research on qinghaosu (artemisinine) and related compounds which has identified ring stage as the most desirable time for treatment ~6.

Chronotherapy and Drug Resistance In principle, it seems that a chronotherapeutic approach may resolve some of the problems of drug resistance. Drug resistance is never absolute; increasing drug concentrations eventually lead to therapeutic resolution. Studies on the pharmacokinetics of chloroquine show that drug injection results in a spike of drug concentration in the blood with peak levels some tenfold higher than those achieved by oral administration and deemed sufficient for curative purposes w. The duration of the spike at half-maximal level is approximately I h, ie, shorter than the time needed to accomplish the full drug effect. If this duration could be prolonged to 3-4h, either by infusion or by repetitive injections of smaller doses at short time intervals, one should be able to achieve blood concentrations that would be effective even against drug resistant parasites. Temporal

elevation of drug levels might cause some acute side effects t7 but could possibly avoid chronic effects and the development of resistant parasites. Obviously, chloroquine treatment should be synchronized with the presence of the mid-term trophozoite stage most susceptible to the drug, ie. shortly after the disappearance of the ring forms from the circulation. The patient's blood should then be observed for the appearance of young ring stages of those parasites that were not cleared by the first treatment and the next injection should be timed to the reappearance of mid-term trophozoites.

Acknowledgements Thanks are extended to Wilfred Stein for critical reading of the manuscript. The authors' work cited was financially supported by the UNDP/World Special Programme for Research and Training in Tropical Diseases, and the EEC.

References

I Reinberg, A. and Smolensky, M. (1983) Biological Rhythms and Medicine, SpringerVerlag 2 Hawking, F. , Worms, M.J.and Gammage,K. (1968) Trans. R. Sac. Trap. Med. Hyg. 62, 731-760 3 Aderounmu, A.F. , Lindstrom, S. and Ekman, L. (I 987)J. Pharm. Pharrnacol 39, 234-235 4 Frisk-Holmberg, M. et al. (1984) Eur. J. Clin. Pharrnacot. 26, 521-530 5 Yayon, A. et al. (1983)J. Protozool. 30, 642-647 6 Zhang,Y., Asante, K.S.O.and Jung,A. (I 986) J. Parasitol. 92, 830-836 7 Dieckmann, A. and jung, A. (1986) Z. Parasitenkd. 72, 591-594 8 Geary,T.G., Divo, A.A. andJensen,J.B.(I 989) Am. J. Trap. Med. Hyg. 40, 240-244 9 Peters, W. (1987) Chemotherapy and Drug Resistance in Malaria (Vol. 2), Academic Press I 0 Cambie,G., Landau,I. and Chabaud,A. (1990) C. R. Acad. Sci. Pans 3t0, 183-188 II Montalvo-Alvares, A.M. et al. (1988) C. R. Acad. Sci. Paris 307, 5-10 12 Landau, I., Cambie, G. and Chabaud, A. (1990) Ann. Parasitol. Hum. Camp. 65, 101-103 13 Cambie,G. et at. ( 1991) Ann. Parasitol. Hum. Camp. 66, 14-2I 14 Garnham,P.C.C.(1968)Malaria Parasites and Other Haernosporidia, Blackwell Scientific 15 White, N.J.and Krishna,S.(I 989) Trans. R. Sac. Trap. Med. Hyg. 83, 767-777 16 Jiang,J.B.et at. (I 982) Lancet ii, 285-288 17 White, N.J. eta/. (1987)J. Infect. Drs. 155, 192-201

Irene Landau, Alain Chabaud and Gilles Carnbie are at the Laboratoire de ZoolagieVers, associ~ au CNRS, Museum National d'Histaire Naturelle, 61 rue Buffon, 75231 Paris Cedex 05, France and Hagai Ginsburg is at the Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel.