Br. J. clin. Pharmac. (1992), 33, 575-581
Pharmacology and parasitology: integrating experimental methods and approaches to falciparum malaria P. A. WINSTANLEY & W. M. WATKINS Kenya Medical Research Institute, and Department oj Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3BX
Introduction
Falciparum malaria is a major global health problem responsible for about 1 million deaths annually, and is expected to remain a menace despite the enormous efforts made to curb it. Because most cases of malaria occur in the community, remote from hospitals it could be argued that expenditure on hospital-based treatment is wasteful and that money should be spent on primary health care including malaria prevention. However, whatever progress is made on that front, treatment of acute episodes will continue to be the mainstay of control in many areas for some years to come and attempts to reduce malaria mortality and morbidity must continue to include hospital-based interventions (WHO, 1991). Better hospital staffing levels, drug supply and organisation are needed in many parts of the tropics, but advances in the basic and clinical sciences remain essential if improvements in the management of malaria are to continue.
malaria (White et al., 1983a, 1987) and, though it would be difficult to prove, clinical benefit has almost certainly followed. This research effort must continue since the parasite is adept at developing drug resistance, and much is still to be learned about the optimal use of antimalarial drugs both old and new.
Development of supportive therapies Most work on the treatment of severe malaria has focused on antimalarial drugs with relatively less attention being paid to interventions in specific pathological processes, such as lactic acidosis, seizures and intracranial hypertension. While this topic has little to do with parasitology, it promises to be an area of particular interest to tropical clinical pharmacology in the near future. These areas are now receiving attention: the use of dichloracetate, an inducer of pyruvate dihydrogenase, in lactic acidosis is being explored in S.E. Asia; phenobarbitone for seizure prophylaxis is being studied in Thailand (White et al., 1988) and Kenya (Winstanley et al., 1992a); and the recent demonstration that young children with cerebral malaria have intracranial hypertension (Newton et al., 1991) will lead to drug intervention studies (though not with dexamethasone; Warrell et al., 1982).
The priority areas A replacement for chloroquine for non-severe malaria
Numerically and economically, the treatment of nonsevere malaria is a much larger problem than the treatment of severe forms of the disease. In the past, reliance has been placed on chloroquine but now that there is widespread chloroquine resistance (Centres for Disease Control, 1985), its usefulness has been seriously eroded. There is an urgent need to identify alternative first line drugs but mefloquine and halofantrine are too expensive for routine use in Africa, and artemisinin derivatives are not available. The need here is the identification of an effective, safe and cheap replacement for chloroquine, preferably one which is rapidly eliminated which might be expected to reduce the risk of resistant strains
Parasitology and pharmacology When considering antimalarial drugs, the parasitologist has traditionally been concerned with the effects of the drug on the parasite in vitro. In particular, parasitologists have tended to concentrate on such questions as the degree of variability in parasite chemosensitivity within and between locations, and optimal drugs/combinations against given strains. Clinical pharmacology has only recently begun to address the tropical pharmacopoeia (Breckenridge et al., 1987). Pharmacologists tend to be interested more in the host than the parasite; in particular they are interested in intersubject variability in drug response, assessment of risk:benefit relationships and rationalisation of drug usage. Parasitological and pharmacological expertise will both be essential in future studies of malaria chemotherapy, and we believe that a combined approach will yield the best results. The main aim of the present review is to
emerging. Optimisation of drugs for severe disease Cerebral malaria remains the commonest severe manifestation of falciparum malaria, and even with intensive therapy its mortality rate is 10-40% (Warrell et al., 1990). Much work has been undertaken in recent years in an effort to improve the chemotherapy of severe
Correspondence: Dr P. A. Winstanley, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 2BX
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define areas in which these two disciplines have worked, or could usefully work, together; these areas include the definition of therapeutic ranges, adaptation of analytical methods, clinical pharmacokinetics and comparisons of drug efficacy.
Delineation of therapeutic ranges Most drugs produce their effects by combining with receptors, either native or foreign. There are no drugs with absolute receptor specificity and, partly as a result of this, all drugs have adverse effects. By the term 'therapeutic range' we usually mean the difference between the lowest therapeutic and toxic drug concentrations in blood or plasma. Such ranges can only ever be approximate, because of variation in host response (Figure 1; Bourne & Roberts, 1982) and, in the case of antimalarial drugs, because of strain-specific differences in chemosensitivity. Tests to measure the in vitro chemosensitivity of Plasmodium falciparum became important with the advent of chloroquine resistance in the early 1960s. The first of these, the 'macro test', measured inhibition of the maturation of the parasite as a function of drug concentration (Rieckmann et al., 1968). It was reliable in assessing chemosensitivity in the field, and produced a minimum inhibitory concentration (MIC) which correlated with in vivo sensitivity (Peters & Seaton, 1971; Rieckmann & Lopez-Antunano, 1971). The 'macro' test was succeeded by the 'micro' test (Rieckmann et al., 1978) where susceptibility to a number of drugs could be determined from a single finger-stick blood sample. This is now the standard test for sensitivity to 4-aminoquinolines, quinine, quinidine and mefloquine, and has been modified for antifolate sensitivity (although consensus on a suitable test for this purpose is still lacking) (Payne & Wernsdorfer, 1989). In vitro chemosensitivity testing, although having little impact on drug policy (WHO, 1990), remains an essential tool for assessing temporal and geographical trends. 100 03) C
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Increased reliance is now quite rightly given to in vivo assessment of drug efficacy, especially treatment failures. However, the impossibility of distinguishing between parasite recrudescence and reinfection, together with the difficulty of determining the type and concentrations of antimalarial drugs in the patient together place fundamental limitations on in vivo data alone. Ideally, a composite picture of the complex host/parasite/drug relationship requires in vitro and in vivo test data collected over a time scale calibrated by pharmacokinetic measurements. The conditions under which the parasite is exposed to drug varies considerably between the in vitro and in vivo situations. For example, in all in vitro tests the patient's blood is diluted, which lowers haematocrit. This is likely to affect the test endpoint for drugs which concentrate in blood cells (e.g. chloroquine; Bergqvist & Domeij-Nyberg, 1983). Furthermore, where potent ligands are present in blood (e.g. acute phase proteins like a1-acid-glycoprotein; AAG) diluted blood will present a changed ratio of free/bound drug to the parasitised erythrocyte, and in the case of quinine this can be shown to affect test endpoints (Elford, personal communication). Finally, the presence of both humoral and cellular immune factors in the test system represents a further complication; blood from a semi-immune host is capable of artificially lowering test endpoints (Carlin et al., 1984). Despite these differences, in vitro chemosensitivity has been shown to correlate with clinical results in the case of chloroquine (Sixsmith et al., 1983), pyrimethamine (Spencer et al., 1986) and pyrimethaminesulphadoxine. Clinical dose-response relationships are much harder to define. The recommended dose regimens of antimalarial drugs are usually derived from percentage cure and adverse effect rates of semi-empirical dose schedules (Warhurst & Schofield, 1989). Such an approach is most useful in designing regimens for prophylaxis and uncomplicated malaria. Severe malaria, on the other hand, is a medical emergency where the fast achievement of therapeutic drug concentrations is a priority (Warrell et al., 1990; White et al., 1983). It is in this setting that therapeutic ranges of antimalarial drugs become clinically important. Although it remains impossible to define clinical dose-response relationships precisely, one can show that they exist. In a recent trial of quinine in severe malaria, relationships were sought between achieved quinine concentrations and the rates of parasite clearance. Parasitaemia was measured 6 hourly, and the slope of the log-linear phase of parasite clearance (see White & Krishna, 1989) was calculated. These values were then plotted against the area under the plasma concentration vs time curve of quinine for the first dose interval (AUC(0,12 h)), and the resulting correlation was significant (Pasvol et al., 1991). Addition of further data since the end of the original study has improved the correlation (Figure 2). Since the main effects of quinine are against sequestered stages of the parasite (Geary et al., 1989), this observation may seem surprising. However, on reflection it becomes evident that the parasite 'clearance' slope represents a hybrid rate constant, the components of which are disappearance of ring forms from the blood, and the
Falciparum malaria
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-1 are not unusual (Winstanley et al., unpublished observation), and this may be a cause for concern. Temporary blindness could easily be missed in unconscious children (permanent blindness is a recognised sequel of cerebral malaria; Molyneux et al., 1989) and ocular adverse effects appear to occur at lower concentrations than cardiotoxicity (Dyson et al., 1985), which has received more attention in the setting of severe malaria (White et al., 1983b). It remains true that effective quinine concentrations need to be achieved rapidly in patients with severe malaria, and that a loading dose is required to achieve this (White et al., 1983a). However, it is also important to avoid serious drug toxicity which may be possible by optimising dose regimens, particularly in young children with cerebral malaria, where detailed kinetic data are less complete than in adults.
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of new rings from the sequestered mass (White & Krishna, 1989). The observed quinine concentration-response relationship probably indicates effects of the drug on rate of emergence rather than rate of disappearance, and this view is supported by work on parasite viability described below. The positive y axis intercept seen in Figure 2 is interesting, since untreated synchronous infections probably show a rising sine wave pattern of increase, exhibiting a fall in peripheral parasitaemia every 24 h (White & Krishna, 1989); this intercept may represent the negative slopes of such waves. The concentration-dependent toxicity of antimalarial drugs varies, from low (e.g. proguanil) to potentially very high (e.g. chloroquine; Riou et al., 1988; Stead & Moffat, 1983). As above, toxic drug concentrations are of most importance in the setting of severe malaria where parenteral administration is the rule. In the case of quinine, assessment of toxic drug concentrations is complicated by disease-induced changes in plasma protein binding. In attempted suicides, plasma quinine concentrations > 10 mg 1-1 invariably cause temporary or permanent blindness (Dyson et al., 1985), whereas in cerebral malaria concentrations exceeding 20 mg 1-1 are not unusual, and have been thought to be benign (White et al., 1983a). This disparity is explained by the smaller fraction of unbound drug in patients with cerebral malaria (Silamut et al., 1985), which results from higher concentrations of AAG (Mansor et al., 1990, 1991; Silamut et al., 1991). Thus the delineation of toxic quinine concentrations would be best made using unbound quinine concentrations. Since the fraction unbound in health is about 20% (Mihaly et al., 1987), the demonstrably oculotoxic plasma concentration of emergence
> 10 mg 1-1 would result in an unbound concentration of > 2 mg 1-1. In children with cerebral malaria on quinine therapy, unbound concentrations of over 2 mg
Adaptation of analytical methods The most widely employed technique of drug analysis is currently high performance liquid chromatography (h.p.l.c.), which requires expensive equipment and skilled personnel. The investigator in the tropics has the choice between setting up methods locally (successfully done in many centres) or sending stored specimens to collaborators-usually overseas. This latter approach has been widely used and has the advantage that equipment need not be maintained in the tropics, where facilities are often inadequate. The disadvantages include difficulty in sample transportation, delayed availability of data, minimal interaction between laboratory and clinical personnel, missed opportunities for local staff training and insensitivity to local scientific priorities. In many African countries h.p.l.c. grade solvents are often unobtainable or prohibitively expensive and must be imported; those solvents which are bought locally are often found to be of inadequate purity and require redistillation. Consequently, where possible mobile phase mixtures with a high water content are preferable, and recirculation of mobile phase (for as long as chromatography is satisfactory) further reduces costs. Likewise, because helium is expensive, isocratic delivery systems which can be de-gassed using ultrasound are preferable. Since voltages may fluctuate greatly, all equipment containing solid-state circuitry should be protected by a voltage stabiliser (servomotor-controlled rheostats available at moderate cost). In addition, power supplies of many tropical cities are often prone to occasional millisecond voltage surges, with which a stabiliser would not adequately cope and which would ruin solid circuitry. Ensuring that each piece of equipment is supplied through an (inexpensive) spike protector (basically a large capacitor) reduces this risk. As well as measures designed to protect equipment and contain costs, analytical methods must be made compatible with clinical and practical requirements. For example, work in young anaemic children (the characteristic African population with severe malaria) must minimise blood sample volume. Where the blood:plasma concentration coefficient of a drug is about 1, and analytical recovery and reproducibility from blood are
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satisfactory, sample volume can be reduced by using blood rather than plasma (furthermore, blood is, arguably, a better choice of biological fluid for pharmacokinetic analysis). Secondly, even with local analytical facilities, the transportation of frozen samples is complicated by the lack of dry ice, lack of freezers in provincial airports (in the event of flight cancellations) and bad roads (especially when it rains). The adaptation of h.p.l.c. techniques for filter paper absorbed samples-first used by Patchen et al. (1983) for chloroquine-reduces these problems and has been used by our group for quinine (Mberu et al., 1991), phenobarbitone (Winstanley et al., 1992a) and pyrimethamine/sulphadoxine (Winstanley et al., 1992b). Furthermore, since the viability of human immunodeficiency virus is reduced markedly by drying (Evengard etal., 1988) laboratory safety is probably improved. Thus it is possible to undertake h.p.l.c. to a high standard in the tropics so long as attention is paid to practical details. Furthermore, h.p.l.c. will adequately measure free drug when total plasma drug concentrations are in the mg 1-1 range (e.g. quinine). Ultrafiltration (Lindup, 1986) using commercially available plastic kits fitted with disposable dialysis membranes can be set up relatively easily in the tropics. The selective incorporation by viable P. falciparum of a radioisotope is a convenient, accurate and reproducible method of assessing parasite chemosensitivity in vitro (Desjardins et al., 1979). The method can be inverted to measure drug concentrations in biological fluids, provided that the test organism is of known drug sensitivity. Such bioassays have been employed by Watkins et al. (1987), where radioisotope uptake was used to measure triazine metabolite concentrations, and Scott etal. (1988), where cycloguanil concentrations were measured (using microscopy rather than radioisotope uptake). Bioassays have been found to be of comparable accuracy and sensitivity to h.p.l.c. and, though they lack specificity, could be used to advantage in concert with h.p.l.c. in the search for pharmacologically active, but unidentified, drug metabolites. Since little sophisticated equipment is required, the method may prove valuable for groups in the tropics who wish to conduct pharmacokinetic studies, but who have been discouraged by the cost and technical demands of establishing h.p.l.c. Each of the above methods is time-consuming and labour-intensive. For drug measurement in large numbers of samples ELISA is an obvious choice, and antibodies to a large number of antimalarial drugs are now available. In the clinical work at Kilifi, screening of patient's plasma for chloroquine is now routinely done by ELISA, a technique well adapted for use in the field (see Churchill (1989) for a review of field-adapted methods).
Clinical pharmacokinetics
Detailed description of drug disposition, and recommendations for improved use are the final fruits of the considerations discussed in the two previous sections; a discussion of the role of clinical pharmacokinetics in improving the use of antimalarial drugs will be found elsewhere in this series of articles (White, 1992).
Comparisons of drug efficacy The first part of this review has dealt with the means of optimising drug use, but of equal importance to tropical clinical pharmacology are the means of comparing drug treatments. The ideal method remains the controlled trial (Pocock, 1988). In non-severe malaria relevant comparisons between groups can be made relatively easily, using criteria like parasite and fever clearance rates, but in severe malaria comparisons are more difficult. Small differences in mortality, rate of neurological sequelae and rate of clinical recovery are of most importance, and the first two of these require large numbers of patients to achieve adequate power. For example, in most units the mortality associated with strictly defined cerebral malaria is about 20% (Warrell et al., 1990); the detection of a 25% drop in mortality, clinically a very impressive result, would need about 1000 patients in each arm. Few centres are able to recruit such numbers within an acceptable study period, and the answers to such fundamentally important questions require a multicentre approach (Marsh et al., 1991). Since multicentre studies rely on the recruitment of comparable patients, close agreement between centres on interpretation of physical signs and their prognostic importance is essential. Cerebral malaria is defined as 'unrousable coma not attributable to any other cause in a patient with falciparum malaria' (Warrell et al., 1990). Other causes of coma may include recent sedative drugs, recent seizure, unsuspected head injury or concomitant CNS infections, and as in the assessment of any sick patient careful attention to detail in the history and physical examination is essential, both for clinical and scientific purposes. An exact definition of 'unrousable coma' is also needed between centres; many coma scoring systems exist, some meant for adults others for children (Molyneux et al., 1989; Reilly et al., 1988) and agreement between centres on which system to use is an obvious essential in trial design. However, even if the same system is used there can be great interobserver variation in scoring; this area requires urgent investigation, particularly in paediatric practice, to allow more rational study design. Comparisons between treatments in terms of clinical recovery rate in severe malaria can also be very difficult. Time taken to localise a painful stimulus, to drink, to sit unsupported and to clear fever lend themselves best to study. For these to function as valid endpoints in a clinical trial they must be defined prospectively and unambiguously (e.g. the first of two consecutive observations 6 h apart), assessments must be made frequently (probably no less than 6 hourly), regularly (including overnight) and in a standard manner between observers. Furthermore, the study design should anticipate deterioration in some patients after they have reached an endpoint (e.g. 'secondary' lapse into coma after initial recovery of consciousness); directions for the handling of such deterioration data also need to be established prospectively and unambiguously. Are there any meaningful ways to compare drug treatments, in the setting of severe disease, using parasitological endpoints? Quantitative change in asexual parasitaemia has been used for many years to assess the action of drugs in vivo. However, stains such as Giemsa
Falciparum malaria do not differentiate between live and dead parasites and, although 'damage' can be assessed from morphological changes, the blood slide essentially provides a measure of total parasite count, the usefulness of which is often uncertain (see White & Krishna, 1989). The ability of single, viable bacteria to grow to macroscopic colonies represents a technique for measuring viability which is widely and routinely used, but an equivalent method for assessing the viability of P. falciparum is yet to be devised. However, since all erythrocytic stages of P. falciparum can be cultured in vitro, while only the early erythrocytic forms are usually found in vivo, a potential method of viable counting is apparent, which is being studied in Kilifi. Briefly, only those rings which develop to mature stage parasites (pigmented trophozoites and schizonts), with the ability to reinvade RBCs, are functionally viable. For a true 'viability count' it would be necessary to remove or inactivate all inhibitory processes in contact with the parasite at the time of sampling. This is impossible at present; repeated washing before in vitro culture, the only practicable method of inactivation, will remove native serum and WBCs, extracellular drug, and probably most of the unbound intracellular drug. Washing will not remove 'inhibitors' bound to active centres within the parasite. Using such an experimental system, we find that when small samples of parasitised RBCs are taken from malaria patients, before treatment, washed several times and cultured in vitro, a fraction of the original population of ring-form parasites is able to mature and reinvade fresh RBCs, demonstrating functional viability, while a fraction does not develop. Changes in the percentage viability of parasites can then be followed during the course of drug treatment. We have observed drug-dependent effects on the viability of parasites ex vivo, and work in this area is in progress. An important application of this technique will be in the assessment of drug action on the ring-forms which remain in the circulation during antimalarial treatment. The sequestration of large numbers of mature trophozoites in the cerebral vasculature is probably a critical step leading to severe disease (MacPherson et al., 1985). Thus a prime objective in studies of severe malaria is the identification of drugs which will arrest the development of the early ring-forms and thus prevent further cerebral sequestration; drugs exhibiting this characteristic have a most important potential role in reducing mortality (though it should not be forgotten that such drugs might have quite the reverse effect on mortality/morbidity if a
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Herxheimer-type reaction occurs). There is considerable scope for an expansion of this technique into other areas of malaria research, especially immunology and pathophysiology.
Conclusions Among the medical sciences, clinical pharmacology is performing an important role in the global attempt to reduce the mortality and morbidity associated with falciparum malaria. The multidisciplinary approach to research bringing together scientists of differing backgrounds, skills and outlooks is essential for groups working in the tropics. In malaria this is proving to be particularly fruitful for tropical clinical pharmacology and parasitology. So what next? Tropical clinical pharmacology has tended to concentrate heavily on the severe manifestations of falciparum malaria, and has been tied to hospital and laboratory aspects of the disease as a result. In Kenya, and many other African countries, most childhood malaria is treated in the community, often by drugs of inadequate efficacy and/or suboptimal dosage. In the past chloroquine, still the first-line drug, was a cheap and effective treatment, but there is now an urgent need to identify alternative drugs to treat the massive burden of malaria in the community. This challenge will involve close collaboration between clinical pharmacology/parasitology and field-based epidemiology. Similarly, collaboration is essential between the diminishing number of tropical centres able to undertake large-scale interventions such as the forthcoming trials of artemether vs quinine (funded by WHO). A discernible problem for British tropical clinical pharmacology is its distance from the tropics, both physical and psychological. This gap can only be bridged effectively by the periodic practice of clinical tropical medicine, and by receptiveness to ideas generated from the field, often by sister disciplines. We would like to thank the director of the Kenya Medical Research Institute for his continuing support and help. The unpublished data quoted in this review were produced with the help of our colleagues in the KEMRI/Wellcome Trust Collaborative Research Programme at Kilifi, funded in part by the Wellcome Trust of Great Britain. WMW is grateful to the Wellcome Trust for personal support; PAW is a Medical Research Council Special Training Fellow and has support from the World Health Organisation and the Royal Society.
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Churchill, F. C. (1989). Field-adapted assays for chloroquine and its metaboites in urine and blood. Parasitol. Today, 5, 116-126. Colwell, E. J., Phintuyothin, P., Sadudee, N., Berijapong, W. & Neopatimanondh, S. (1972). Evaluation of an in vitro technique for detecting chloroquine-resistant falciparum malaria in Thailand. Am. J. trop. Med. Hyg., 21, 6-12. Desjardins, R. E., Canfield, C. J., Haynes, J. D. & Chulay, J. D. (1979). Qualitative asessment of antimalarial activity in vitro by semi-automated microdilution technique. Antimicrob. Agents Chemother., 16, 710-718. Dyson, E. H., Proudfoot, A. T., Prescott, L. F. & Heyworth, R. (1985). Death and blindness due to overdose of quinine. Br. med. J., 291, 31-33. Evengard, B., von Sydow, M., Ehrnst, A., Pehrson, P. O., Lunbergh, P. & Linder, E. (1988). Filter paper sampling of blood infected with HIV: effect of heat on antibody activity and viral infectivity. Br. med. J., 297, 1178. Geary, T. G., Divo, A. A. & Jensen, J. (1989). Stage specific actions of antimalarial drugs on Plasmodium falciparum in culture. Am. J. trop. Med. Hyg., 40, 240-244. Lindup, W. E. (1987). Plasma protein binding of drugs-some basic and clinical aspects. In Progress in Drug Metabolism, eds Bridges, J. W., Chasseaud, L. & Gibson, C. G. London, Taylor and Francis Ltd. MacPherson, G. G., Warrell, M. J., White, N. J., Looareesuwan, S. & Warrell, D. A. (1985). Human cerebral malaria: a quantitative ultrastructural analysis of parasitised erythrocyte sequestration. Am. J. Path., 119, 385-401. Mansor, S. M., Molyneux, M. E., Taylor, T. E., Ward, S. A., Wirima, J. J. & Edwards, G. (1991). Effect of Plasmodium falciparum infection on the plasma concentration of alpha1 acid-glycoprotein and the binding of quinine in Malawian children. Br. J. clin. Pharmac., 32, 317-323. Mansor, S. M., Taylor, T. E., McGraith, C. S., Edwards, G., Ward, S. A., Wirima, J. J. & Molyneux, M. E. (1990). The safety and kinetics of intramuscular quinine in Malawian children with moderately severe falciparum malaria. Trans. Roy. Soc. trop. Med. Hyg., 84, 482-488. Marsh, K., Newton, C. J. R. C., Winstanley, P. A. & Kirkham, F. J. (1991). Clinical malaria: new problems in patient management. In Malaria, waiting for the vaccine. Chichester: Wiley. Mberu, E. K., Ward, S. A., Winstanley, P. A. & Watkins, W. M. (1991). Measurement of quinine in filter-paper absorbed blood by high performance liquid chromatography. J. Chromatogr., 570, 180-184. Mihaly, G. W., Ching, M. S., Kleijn, M. B., Paull, J. & Smallwood, R. A. (1987). Differences in the binding of quinine and quinidine to plasma proteins. Br. J. clin. Pharmac., 24, 769-774. Molyneaux, M. E., Taylor, T. E., Wirima, J. J. & Borgstein, J. (1989). Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Quart. J. Med., 71, 441-459. Newton, C. R. J. C., Kirkham, F. K., Winstanley, P. A., Pasvol, G., Peshu, N., Warrell, D. A. & Marsh, K. (1991). Intracranial pressure in African children with cerebral malaria. Lancet, 337, 573-576. Pasvol, G., Newton, C. R. J. C., Winstanley, P. A., Watkins, W. M., Peshu, N. M., Were, J. B. O., Marsh, K. & Warrell, D. A. (1991). Quinine treatment of severe falciparum malaria in African children; a randomised comparison of three regimens. Am. J. trop. Med. Hyg., 45, 702-713. Patchen, L. C., Mount, D. L., Schwartz, I. K. & Churchill, F. C. (1983). Analysis of filter-paper absorbed finger-stick blood samples for chloroquine and its major metabolite using high performance liquid chromatography with fluorescence detection. I. Chromatogr., 278, 81-89. Payne, D. & Wernsdorfer, W. H. (1989). Development of
blood culture medium and a standard in vitro microtest for field testing the response of Plasmodium falciparum to antifolate antimalarials. Bull. WHO, 67, 59-64. Peters, W. & Seaton, D. R. (1971). Sensitivity of Plasmodium falciparum to chloroquine in Africa. Ann. trop. med. Parasitol., 65, 267-269. Pocock, S. J. (1988). Clinical trials: a practical approach. Chichester: John Wiley and Sons. Reilly, P. L., Simpson, D. A., Sprod, R. & Thomas, L. (1988). Assessing the conscious level of infants and young children: a paediatric version of the Glasgow coma score. Child. Nerv. System, 4, 30-33. Rieckmann, K. H., McNamara, J. V., Frischer, H., Stockert, T. A., Carson, P. E. & Powell, R. D. (1968). Effects of chloroquine, quinine and cycloguanil upon the maturation of asexual erythrocytic forms of two strains of Plasmodium falciparum in vitro. Am. J. trop. Med. Hyg., 17, 661-671. Rieckmann, K. H. & Lopez-Antunano, F. J. (1971). Chloroquine resistance of Plasmodiumfalciparum in Brazil detected by a simple in vitro method. Bull. WHO, 45, 157-167. Rieckmann, K. H., Sax, L. J., Campbell, G. H. & Mrema, J. E. (1978). Drug sensitivity of Plasmodiumfalciparum: an in vitro microtechnique. Lancet, i, 22-23. Riou, B., Barriot, P., Rimailho, A. & Baud, F. J. (1988). Treatment of severe chloroquine poisoning. New Engl. J. Med., 318, 1-6. Scott, H. V., Edstein, M. D., Veenedaal, J. R. & Rieckmann, K. H. (1988). A sensitive bioassay for serum cycloguanil using Plasmodiumfalciparum in vitro. Int. J. Pharmac., 18,
605-609. Silamut, K., Molunto, P., Ho, M., Davies, T. M. E. & White, N. J. (1991). Alpha1 acid-glycoprotein (orosomucoid) and plasma protein binding of quinine in falciparum malaria. Br. J. clin. Pharmac., 32, 311-317. Silamut, K., White, N. J., Looareesuwan, S. & Warrell, D. A. (1985). Binding of quinine to plasma proteins in falciparum malaria. Am. J. trop. Med. Hyg., 34, 681-686. Sixsmith, D. G., Spencer, H. C., Watkins, W. M., Koech, D. K. & Chulay, J. D. (1983). Changing in vitro response to chloroquine of Plasmodium falciparum in Kenya. Lancet, ii, 1022. Spencer, H. C., Watkins, W. M., Sixsmith, D. G. & Koech, D. K. (1986). Response of Plasmodium falciparum to dihydrofolate reductase inhibitors. Trans. Roy. Soc. trop. Med. Hyg., 80, 291-203. Stead, A. H. & Moffat, A. C. (1983). A collection of therapeutic, toxic and fatal blood drug concentrations in man. Hum. Toxicol., 3, 437-464. Warhurst, D. C. & Shofield, C. J. (1989). Halofantrine in the treatment of multidrug resistant malaria. Cambridge, UK: Elsevier.
Warrell, D. A., Looareesuwan, S., Warrell, M. J., Kasemsarn, P., Intaraprasert, R., Bunnag, D. & Harinasuta, T. (1982). Dexamethasone proves deleterious in cerebral malaria. A double blind trial in 100 comatose patients. New Engl. J. Med., 306, 313-319. Warrell, D. A., Molyneux, M. & Beales, P. F. (1990). Severe and complicated malaria. Trans. Roy. Soc. trop. Med. Hyg., 84, suppl. 2. Watkins, W. M., Chulay, J. D., Sixsmith, D. G., Spencer, H. C. & Howells, R. E. (1987). A preliminary pharmacokinetic study of the antimalarial drugs proguanil and chlorproguanil. J. Pharm. Pharmac., 39, 261-265. White, N. J. (1992). Antimalarial pharmacokinetics and treatment regimens. Br. I. dlin. Pharmac., 34, (in press). White, N. J., Looareesuwan, S., Warrell, D. A., Warrell, M. J., Chanthavanich, P., Bunnag, D. & Harinasuta, T. (1983a). Quinine loading dose in cerebral malaria. Am. J. trop. Med. Hyg., 32, 1-5. White, N. J., Looareesuwan, S. & Warrell, D. A. (1983b).
Falciparum malaria Quinine and quinidine: a comparison of EKG effects during the treatment of malaria. J. cardiovasc. Pharmac., 5, 173-175. White, N. J., Watt, G., Bergquist, Y. & Njelesani, E. (1987). Parenteral chloroquine for treating falciparum malaria. J. infect. Dis., 155, 192-201. White, N. J., Looareesuwan, S., Phillips, R. E., Chanthavanich, P. & Warrell, D. A. (1988). Single dose phenobarbitone prevents convulsions in cerebral malaria. Lancet, ii, 64-66. White, N. J. & Krishna, S. (1989). Treatment of malaria: some considerations and limitations of the current methods of assessment. Trans. Roy. Soc. trop. Med. Hyg., 83, 767-777. WHO (1990). Practical chemotherapy of malaria. Technical report series 805, World Heath Organisation. WHO (1991). Tropical diseases: progress in research 1989-
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1990. World Health Organisation, tenth programme report. Winstanley, P. A., Newton, C. R. J. C., Pasvol, G., Kirkham, F. J., Mberu, E., Peshu, N., Ward, S. A., Were, J. B. O., Warrell, D. A. & Marsh, K. (1992a). Prophylactic phenobarbitone in young children with severe falciparum malaria: pharmacokinetics and clinical effects. Br. J. clin. Pharmac., 33, 149-154. Winstanley, P. A., Watkins, W. M., Newton, C. R. J. C., Nevill, C., Mberu, E., Warn, P. A., Waruiru, C. M., Mwangi, I. N., Warrell, D. A. & Marsh, K. (1992b). The disposition of oral and intramuscular pyrimethamine/ sulfadoxine in Kenyan children with high parasitaemia but clinically non-severe falciparum malaria. Br. J. clin. Pharmac., 33, 143-148.
Pharmacology and parasitology: integrating experimental methods and approaches to falciparum malaria P. A. WINSTANLEY & W. M. WATKINS Kenya Medical Research Institute, and Department oj Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3BX
Introduction
Falciparum malaria is a major global health problem responsible for about 1 million deaths annually, and is expected to remain a menace despite the enormous efforts made to curb it. Because most cases of malaria occur in the community, remote from hospitals it could be argued that expenditure on hospital-based treatment is wasteful and that money should be spent on primary health care including malaria prevention. However, whatever progress is made on that front, treatment of acute episodes will continue to be the mainstay of control in many areas for some years to come and attempts to reduce malaria mortality and morbidity must continue to include hospital-based interventions (WHO, 1991). Better hospital staffing levels, drug supply and organisation are needed in many parts of the tropics, but advances in the basic and clinical sciences remain essential if improvements in the management of malaria are to continue.
malaria (White et al., 1983a, 1987) and, though it would be difficult to prove, clinical benefit has almost certainly followed. This research effort must continue since the parasite is adept at developing drug resistance, and much is still to be learned about the optimal use of antimalarial drugs both old and new.
Development of supportive therapies Most work on the treatment of severe malaria has focused on antimalarial drugs with relatively less attention being paid to interventions in specific pathological processes, such as lactic acidosis, seizures and intracranial hypertension. While this topic has little to do with parasitology, it promises to be an area of particular interest to tropical clinical pharmacology in the near future. These areas are now receiving attention: the use of dichloracetate, an inducer of pyruvate dihydrogenase, in lactic acidosis is being explored in S.E. Asia; phenobarbitone for seizure prophylaxis is being studied in Thailand (White et al., 1988) and Kenya (Winstanley et al., 1992a); and the recent demonstration that young children with cerebral malaria have intracranial hypertension (Newton et al., 1991) will lead to drug intervention studies (though not with dexamethasone; Warrell et al., 1982).
The priority areas A replacement for chloroquine for non-severe malaria
Numerically and economically, the treatment of nonsevere malaria is a much larger problem than the treatment of severe forms of the disease. In the past, reliance has been placed on chloroquine but now that there is widespread chloroquine resistance (Centres for Disease Control, 1985), its usefulness has been seriously eroded. There is an urgent need to identify alternative first line drugs but mefloquine and halofantrine are too expensive for routine use in Africa, and artemisinin derivatives are not available. The need here is the identification of an effective, safe and cheap replacement for chloroquine, preferably one which is rapidly eliminated which might be expected to reduce the risk of resistant strains
Parasitology and pharmacology When considering antimalarial drugs, the parasitologist has traditionally been concerned with the effects of the drug on the parasite in vitro. In particular, parasitologists have tended to concentrate on such questions as the degree of variability in parasite chemosensitivity within and between locations, and optimal drugs/combinations against given strains. Clinical pharmacology has only recently begun to address the tropical pharmacopoeia (Breckenridge et al., 1987). Pharmacologists tend to be interested more in the host than the parasite; in particular they are interested in intersubject variability in drug response, assessment of risk:benefit relationships and rationalisation of drug usage. Parasitological and pharmacological expertise will both be essential in future studies of malaria chemotherapy, and we believe that a combined approach will yield the best results. The main aim of the present review is to
emerging. Optimisation of drugs for severe disease Cerebral malaria remains the commonest severe manifestation of falciparum malaria, and even with intensive therapy its mortality rate is 10-40% (Warrell et al., 1990). Much work has been undertaken in recent years in an effort to improve the chemotherapy of severe
Correspondence: Dr P. A. Winstanley, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 2BX
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define areas in which these two disciplines have worked, or could usefully work, together; these areas include the definition of therapeutic ranges, adaptation of analytical methods, clinical pharmacokinetics and comparisons of drug efficacy.
Delineation of therapeutic ranges Most drugs produce their effects by combining with receptors, either native or foreign. There are no drugs with absolute receptor specificity and, partly as a result of this, all drugs have adverse effects. By the term 'therapeutic range' we usually mean the difference between the lowest therapeutic and toxic drug concentrations in blood or plasma. Such ranges can only ever be approximate, because of variation in host response (Figure 1; Bourne & Roberts, 1982) and, in the case of antimalarial drugs, because of strain-specific differences in chemosensitivity. Tests to measure the in vitro chemosensitivity of Plasmodium falciparum became important with the advent of chloroquine resistance in the early 1960s. The first of these, the 'macro test', measured inhibition of the maturation of the parasite as a function of drug concentration (Rieckmann et al., 1968). It was reliable in assessing chemosensitivity in the field, and produced a minimum inhibitory concentration (MIC) which correlated with in vivo sensitivity (Peters & Seaton, 1971; Rieckmann & Lopez-Antunano, 1971). The 'macro' test was succeeded by the 'micro' test (Rieckmann et al., 1978) where susceptibility to a number of drugs could be determined from a single finger-stick blood sample. This is now the standard test for sensitivity to 4-aminoquinolines, quinine, quinidine and mefloquine, and has been modified for antifolate sensitivity (although consensus on a suitable test for this purpose is still lacking) (Payne & Wernsdorfer, 1989). In vitro chemosensitivity testing, although having little impact on drug policy (WHO, 1990), remains an essential tool for assessing temporal and geographical trends. 100 03) C
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Increased reliance is now quite rightly given to in vivo assessment of drug efficacy, especially treatment failures. However, the impossibility of distinguishing between parasite recrudescence and reinfection, together with the difficulty of determining the type and concentrations of antimalarial drugs in the patient together place fundamental limitations on in vivo data alone. Ideally, a composite picture of the complex host/parasite/drug relationship requires in vitro and in vivo test data collected over a time scale calibrated by pharmacokinetic measurements. The conditions under which the parasite is exposed to drug varies considerably between the in vitro and in vivo situations. For example, in all in vitro tests the patient's blood is diluted, which lowers haematocrit. This is likely to affect the test endpoint for drugs which concentrate in blood cells (e.g. chloroquine; Bergqvist & Domeij-Nyberg, 1983). Furthermore, where potent ligands are present in blood (e.g. acute phase proteins like a1-acid-glycoprotein; AAG) diluted blood will present a changed ratio of free/bound drug to the parasitised erythrocyte, and in the case of quinine this can be shown to affect test endpoints (Elford, personal communication). Finally, the presence of both humoral and cellular immune factors in the test system represents a further complication; blood from a semi-immune host is capable of artificially lowering test endpoints (Carlin et al., 1984). Despite these differences, in vitro chemosensitivity has been shown to correlate with clinical results in the case of chloroquine (Sixsmith et al., 1983), pyrimethamine (Spencer et al., 1986) and pyrimethaminesulphadoxine. Clinical dose-response relationships are much harder to define. The recommended dose regimens of antimalarial drugs are usually derived from percentage cure and adverse effect rates of semi-empirical dose schedules (Warhurst & Schofield, 1989). Such an approach is most useful in designing regimens for prophylaxis and uncomplicated malaria. Severe malaria, on the other hand, is a medical emergency where the fast achievement of therapeutic drug concentrations is a priority (Warrell et al., 1990; White et al., 1983). It is in this setting that therapeutic ranges of antimalarial drugs become clinically important. Although it remains impossible to define clinical dose-response relationships precisely, one can show that they exist. In a recent trial of quinine in severe malaria, relationships were sought between achieved quinine concentrations and the rates of parasite clearance. Parasitaemia was measured 6 hourly, and the slope of the log-linear phase of parasite clearance (see White & Krishna, 1989) was calculated. These values were then plotted against the area under the plasma concentration vs time curve of quinine for the first dose interval (AUC(0,12 h)), and the resulting correlation was significant (Pasvol et al., 1991). Addition of further data since the end of the original study has improved the correlation (Figure 2). Since the main effects of quinine are against sequestered stages of the parasite (Geary et al., 1989), this observation may seem surprising. However, on reflection it becomes evident that the parasite 'clearance' slope represents a hybrid rate constant, the components of which are disappearance of ring forms from the blood, and the
Falciparum malaria
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-1 are not unusual (Winstanley et al., unpublished observation), and this may be a cause for concern. Temporary blindness could easily be missed in unconscious children (permanent blindness is a recognised sequel of cerebral malaria; Molyneux et al., 1989) and ocular adverse effects appear to occur at lower concentrations than cardiotoxicity (Dyson et al., 1985), which has received more attention in the setting of severe malaria (White et al., 1983b). It remains true that effective quinine concentrations need to be achieved rapidly in patients with severe malaria, and that a loading dose is required to achieve this (White et al., 1983a). However, it is also important to avoid serious drug toxicity which may be possible by optimising dose regimens, particularly in young children with cerebral malaria, where detailed kinetic data are less complete than in adults.
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of new rings from the sequestered mass (White & Krishna, 1989). The observed quinine concentration-response relationship probably indicates effects of the drug on rate of emergence rather than rate of disappearance, and this view is supported by work on parasite viability described below. The positive y axis intercept seen in Figure 2 is interesting, since untreated synchronous infections probably show a rising sine wave pattern of increase, exhibiting a fall in peripheral parasitaemia every 24 h (White & Krishna, 1989); this intercept may represent the negative slopes of such waves. The concentration-dependent toxicity of antimalarial drugs varies, from low (e.g. proguanil) to potentially very high (e.g. chloroquine; Riou et al., 1988; Stead & Moffat, 1983). As above, toxic drug concentrations are of most importance in the setting of severe malaria where parenteral administration is the rule. In the case of quinine, assessment of toxic drug concentrations is complicated by disease-induced changes in plasma protein binding. In attempted suicides, plasma quinine concentrations > 10 mg 1-1 invariably cause temporary or permanent blindness (Dyson et al., 1985), whereas in cerebral malaria concentrations exceeding 20 mg 1-1 are not unusual, and have been thought to be benign (White et al., 1983a). This disparity is explained by the smaller fraction of unbound drug in patients with cerebral malaria (Silamut et al., 1985), which results from higher concentrations of AAG (Mansor et al., 1990, 1991; Silamut et al., 1991). Thus the delineation of toxic quinine concentrations would be best made using unbound quinine concentrations. Since the fraction unbound in health is about 20% (Mihaly et al., 1987), the demonstrably oculotoxic plasma concentration of emergence
> 10 mg 1-1 would result in an unbound concentration of > 2 mg 1-1. In children with cerebral malaria on quinine therapy, unbound concentrations of over 2 mg
Adaptation of analytical methods The most widely employed technique of drug analysis is currently high performance liquid chromatography (h.p.l.c.), which requires expensive equipment and skilled personnel. The investigator in the tropics has the choice between setting up methods locally (successfully done in many centres) or sending stored specimens to collaborators-usually overseas. This latter approach has been widely used and has the advantage that equipment need not be maintained in the tropics, where facilities are often inadequate. The disadvantages include difficulty in sample transportation, delayed availability of data, minimal interaction between laboratory and clinical personnel, missed opportunities for local staff training and insensitivity to local scientific priorities. In many African countries h.p.l.c. grade solvents are often unobtainable or prohibitively expensive and must be imported; those solvents which are bought locally are often found to be of inadequate purity and require redistillation. Consequently, where possible mobile phase mixtures with a high water content are preferable, and recirculation of mobile phase (for as long as chromatography is satisfactory) further reduces costs. Likewise, because helium is expensive, isocratic delivery systems which can be de-gassed using ultrasound are preferable. Since voltages may fluctuate greatly, all equipment containing solid-state circuitry should be protected by a voltage stabiliser (servomotor-controlled rheostats available at moderate cost). In addition, power supplies of many tropical cities are often prone to occasional millisecond voltage surges, with which a stabiliser would not adequately cope and which would ruin solid circuitry. Ensuring that each piece of equipment is supplied through an (inexpensive) spike protector (basically a large capacitor) reduces this risk. As well as measures designed to protect equipment and contain costs, analytical methods must be made compatible with clinical and practical requirements. For example, work in young anaemic children (the characteristic African population with severe malaria) must minimise blood sample volume. Where the blood:plasma concentration coefficient of a drug is about 1, and analytical recovery and reproducibility from blood are
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satisfactory, sample volume can be reduced by using blood rather than plasma (furthermore, blood is, arguably, a better choice of biological fluid for pharmacokinetic analysis). Secondly, even with local analytical facilities, the transportation of frozen samples is complicated by the lack of dry ice, lack of freezers in provincial airports (in the event of flight cancellations) and bad roads (especially when it rains). The adaptation of h.p.l.c. techniques for filter paper absorbed samples-first used by Patchen et al. (1983) for chloroquine-reduces these problems and has been used by our group for quinine (Mberu et al., 1991), phenobarbitone (Winstanley et al., 1992a) and pyrimethamine/sulphadoxine (Winstanley et al., 1992b). Furthermore, since the viability of human immunodeficiency virus is reduced markedly by drying (Evengard etal., 1988) laboratory safety is probably improved. Thus it is possible to undertake h.p.l.c. to a high standard in the tropics so long as attention is paid to practical details. Furthermore, h.p.l.c. will adequately measure free drug when total plasma drug concentrations are in the mg 1-1 range (e.g. quinine). Ultrafiltration (Lindup, 1986) using commercially available plastic kits fitted with disposable dialysis membranes can be set up relatively easily in the tropics. The selective incorporation by viable P. falciparum of a radioisotope is a convenient, accurate and reproducible method of assessing parasite chemosensitivity in vitro (Desjardins et al., 1979). The method can be inverted to measure drug concentrations in biological fluids, provided that the test organism is of known drug sensitivity. Such bioassays have been employed by Watkins et al. (1987), where radioisotope uptake was used to measure triazine metabolite concentrations, and Scott etal. (1988), where cycloguanil concentrations were measured (using microscopy rather than radioisotope uptake). Bioassays have been found to be of comparable accuracy and sensitivity to h.p.l.c. and, though they lack specificity, could be used to advantage in concert with h.p.l.c. in the search for pharmacologically active, but unidentified, drug metabolites. Since little sophisticated equipment is required, the method may prove valuable for groups in the tropics who wish to conduct pharmacokinetic studies, but who have been discouraged by the cost and technical demands of establishing h.p.l.c. Each of the above methods is time-consuming and labour-intensive. For drug measurement in large numbers of samples ELISA is an obvious choice, and antibodies to a large number of antimalarial drugs are now available. In the clinical work at Kilifi, screening of patient's plasma for chloroquine is now routinely done by ELISA, a technique well adapted for use in the field (see Churchill (1989) for a review of field-adapted methods).
Clinical pharmacokinetics
Detailed description of drug disposition, and recommendations for improved use are the final fruits of the considerations discussed in the two previous sections; a discussion of the role of clinical pharmacokinetics in improving the use of antimalarial drugs will be found elsewhere in this series of articles (White, 1992).
Comparisons of drug efficacy The first part of this review has dealt with the means of optimising drug use, but of equal importance to tropical clinical pharmacology are the means of comparing drug treatments. The ideal method remains the controlled trial (Pocock, 1988). In non-severe malaria relevant comparisons between groups can be made relatively easily, using criteria like parasite and fever clearance rates, but in severe malaria comparisons are more difficult. Small differences in mortality, rate of neurological sequelae and rate of clinical recovery are of most importance, and the first two of these require large numbers of patients to achieve adequate power. For example, in most units the mortality associated with strictly defined cerebral malaria is about 20% (Warrell et al., 1990); the detection of a 25% drop in mortality, clinically a very impressive result, would need about 1000 patients in each arm. Few centres are able to recruit such numbers within an acceptable study period, and the answers to such fundamentally important questions require a multicentre approach (Marsh et al., 1991). Since multicentre studies rely on the recruitment of comparable patients, close agreement between centres on interpretation of physical signs and their prognostic importance is essential. Cerebral malaria is defined as 'unrousable coma not attributable to any other cause in a patient with falciparum malaria' (Warrell et al., 1990). Other causes of coma may include recent sedative drugs, recent seizure, unsuspected head injury or concomitant CNS infections, and as in the assessment of any sick patient careful attention to detail in the history and physical examination is essential, both for clinical and scientific purposes. An exact definition of 'unrousable coma' is also needed between centres; many coma scoring systems exist, some meant for adults others for children (Molyneux et al., 1989; Reilly et al., 1988) and agreement between centres on which system to use is an obvious essential in trial design. However, even if the same system is used there can be great interobserver variation in scoring; this area requires urgent investigation, particularly in paediatric practice, to allow more rational study design. Comparisons between treatments in terms of clinical recovery rate in severe malaria can also be very difficult. Time taken to localise a painful stimulus, to drink, to sit unsupported and to clear fever lend themselves best to study. For these to function as valid endpoints in a clinical trial they must be defined prospectively and unambiguously (e.g. the first of two consecutive observations 6 h apart), assessments must be made frequently (probably no less than 6 hourly), regularly (including overnight) and in a standard manner between observers. Furthermore, the study design should anticipate deterioration in some patients after they have reached an endpoint (e.g. 'secondary' lapse into coma after initial recovery of consciousness); directions for the handling of such deterioration data also need to be established prospectively and unambiguously. Are there any meaningful ways to compare drug treatments, in the setting of severe disease, using parasitological endpoints? Quantitative change in asexual parasitaemia has been used for many years to assess the action of drugs in vivo. However, stains such as Giemsa
Falciparum malaria do not differentiate between live and dead parasites and, although 'damage' can be assessed from morphological changes, the blood slide essentially provides a measure of total parasite count, the usefulness of which is often uncertain (see White & Krishna, 1989). The ability of single, viable bacteria to grow to macroscopic colonies represents a technique for measuring viability which is widely and routinely used, but an equivalent method for assessing the viability of P. falciparum is yet to be devised. However, since all erythrocytic stages of P. falciparum can be cultured in vitro, while only the early erythrocytic forms are usually found in vivo, a potential method of viable counting is apparent, which is being studied in Kilifi. Briefly, only those rings which develop to mature stage parasites (pigmented trophozoites and schizonts), with the ability to reinvade RBCs, are functionally viable. For a true 'viability count' it would be necessary to remove or inactivate all inhibitory processes in contact with the parasite at the time of sampling. This is impossible at present; repeated washing before in vitro culture, the only practicable method of inactivation, will remove native serum and WBCs, extracellular drug, and probably most of the unbound intracellular drug. Washing will not remove 'inhibitors' bound to active centres within the parasite. Using such an experimental system, we find that when small samples of parasitised RBCs are taken from malaria patients, before treatment, washed several times and cultured in vitro, a fraction of the original population of ring-form parasites is able to mature and reinvade fresh RBCs, demonstrating functional viability, while a fraction does not develop. Changes in the percentage viability of parasites can then be followed during the course of drug treatment. We have observed drug-dependent effects on the viability of parasites ex vivo, and work in this area is in progress. An important application of this technique will be in the assessment of drug action on the ring-forms which remain in the circulation during antimalarial treatment. The sequestration of large numbers of mature trophozoites in the cerebral vasculature is probably a critical step leading to severe disease (MacPherson et al., 1985). Thus a prime objective in studies of severe malaria is the identification of drugs which will arrest the development of the early ring-forms and thus prevent further cerebral sequestration; drugs exhibiting this characteristic have a most important potential role in reducing mortality (though it should not be forgotten that such drugs might have quite the reverse effect on mortality/morbidity if a
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Herxheimer-type reaction occurs). There is considerable scope for an expansion of this technique into other areas of malaria research, especially immunology and pathophysiology.
Conclusions Among the medical sciences, clinical pharmacology is performing an important role in the global attempt to reduce the mortality and morbidity associated with falciparum malaria. The multidisciplinary approach to research bringing together scientists of differing backgrounds, skills and outlooks is essential for groups working in the tropics. In malaria this is proving to be particularly fruitful for tropical clinical pharmacology and parasitology. So what next? Tropical clinical pharmacology has tended to concentrate heavily on the severe manifestations of falciparum malaria, and has been tied to hospital and laboratory aspects of the disease as a result. In Kenya, and many other African countries, most childhood malaria is treated in the community, often by drugs of inadequate efficacy and/or suboptimal dosage. In the past chloroquine, still the first-line drug, was a cheap and effective treatment, but there is now an urgent need to identify alternative drugs to treat the massive burden of malaria in the community. This challenge will involve close collaboration between clinical pharmacology/parasitology and field-based epidemiology. Similarly, collaboration is essential between the diminishing number of tropical centres able to undertake large-scale interventions such as the forthcoming trials of artemether vs quinine (funded by WHO). A discernible problem for British tropical clinical pharmacology is its distance from the tropics, both physical and psychological. This gap can only be bridged effectively by the periodic practice of clinical tropical medicine, and by receptiveness to ideas generated from the field, often by sister disciplines. We would like to thank the director of the Kenya Medical Research Institute for his continuing support and help. The unpublished data quoted in this review were produced with the help of our colleagues in the KEMRI/Wellcome Trust Collaborative Research Programme at Kilifi, funded in part by the Wellcome Trust of Great Britain. WMW is grateful to the Wellcome Trust for personal support; PAW is a Medical Research Council Special Training Fellow and has support from the World Health Organisation and the Royal Society.
References Bergquist, Y. & Domeij-Nyberg, B. (1983). Distribution of chloroquine and its metabolite desethylchloroquine in human blood cells and its implication for the quantitative determination of these compounds in serum and plasma. J. Chromatogr., 272, 137-148. Bourne, H. R. & Roberts, J. M. (1982). Drug receptors and pharmacodynamics. In Basic and Clinical Pharmacology, ed. Katzung. B. G. Los Altos, USA: Lange Medical Publications. Breckenridge, A. M., Orme, M. L'E. & Edwards, G. (1987).
Clinical pharmacology looks at tropical medicine. Trans. Roy Soc. trop. Med. Hyg., 81, 529-533. Carlin, J. M., Van de Waa, J. A., Jensen, J. B. & Akwood, M. A. S. (1984). African serum interference in the determination of chloroquine sensitivity in Plasmodium falciparum. Zeitschrift fPr Parazitenkunde, 70, 589-597. Centres for Disease Control (1985). Revised recommendations for preventing malaria in travellers to areas with chloroquineresistant Plasmodium falciparum. Mord. Mort. Weekly Rep., 34, 185-195.
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