W.M.C. Eling

Measurement by membrane feeding of reduction in Plasmodium falciparum transmission induced by endemic sera.

The standard laboratory test for reduction in malaria transmission is based on the measurement of oocyst numbers in mosquitoes fed on blood meals containing test and control sera. Interpretation of the results, however, is often hampered by the large variation in numbers of infected mosquitoes and oocysts. The objective of this study was to compare 3 measures for the assessment of transmission reduction (so-called R values) and to define the experimental criteria that allow interpretation of the results. To determine variability in R values of control sera, a replicate experiment was performed with 10 non-endemic sera of Dutch blood donors. Furthermore, 2 measures for calculation of transmission reduction were compared in a triplicate experiment using Plasmodium falciparum, Anopheles gambiae and malaria endemic sera. Calculations using the geometric mean of Williams are currently used to identify blocking and non-blocking sera. However, calculations using log-transformed data could distinguish more gradual levels of transmission reduction activity by endemic sera--i.e. blocking, reducing and non-blocking activity. Grading of transmission reduction activity is important for epidemiological studies on transmission immunity and for validation of future transmission-blocking vaccines.

Can Babesia Infections be used as a Model for Cerebral Malaria

Infections with certain species of Plasmodium and Babesia induce, among other symptoms, cerebral pathology. The finding of heavily parasitized cerebral capillaries upon postmortem examination has led to the assumption that blockage of capillaries with infected red blood cells caused the cerebral symptoms and subsequent death. As this type of cerebrovascular pathology is found both in humans dying from malaria and in cattle dying from babesiosis, the latter could possibly be used as an animal model for the study of human cerebral malaria. However, before such a model system is adopted, the experimental data concerning cerebral pathology of babesiosis needs critical evaluation. Here, Theo Schetters and Wijnand Eling review the pathological mechanisms in cerebral babesiosis and relate these to cerebral malaria. Finally, they discuss the use of animal model systems for specific aspects of the pathological picture.

The Parenteral Controlled Release of Liposome Encapsulated Chloroquine in Mice

Abstract— Free (0.6 mg), and liposome encapsulated chloroquine (0.6, 3 mg), were injected intraperitoneally, intramuscularly and subcutaneously in mice. Intraperitoneal administration of liposome‐encapsulated chloroquine resulted in high and long lasting concentrations of chloroquine in the blood compared with intraperitoneal administration of free chloroquine. After administration of the liposome‐encapsulated chloroquine the concentrations in the spleen were also higher, indicating that chloroquine liposomes reached the blood compartment intact after intraperitoneal administration. After intramuscular and subcutaneous administration the chloroquine liposomes acted as a local depot, giving a slower release from the subcutaneous fat layer than from the muscle depot. After the 0.6 mg dose a burst effect was found at about 7 h in most of the animals; this was not found after the 3 mg dose. This finding and the slower release after the 3 mg dose than after the 0.6 mg dose could be explained by the formation of aggregates after the injection.

Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

ABSTRACT During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.

Pharmacokinetic and Pharmacodynamic Aspects of Artelinic Acid in Rodents

Abstract— The efficacy of artelinic acid and artemisinin, orally administered at 10 and 50 mg kg−1 day−1, was compared in Plasmodium berghei infected mice. Subsequently, the pharmacokinetics of artelinic acid after intravenous, intramuscular, oral and rectal administration of a 20 mg kg−1 aqueous solution to rabbits were studied in a four‐way randomized cross‐over experiment. After intravenous administration, artelinic acid concentrations in blood plasma were high (C0: 76 ± 15 mg L−1), and the drug was rapidly eliminated from the central compartment, showing linear elimination kinetics with an elimination half‐life of 15 ± 3 min. A large inter‐subject variation appeared in the absorption rate and the extent of absorption (2–92%) over the 120 min interval after intramuscular administration. Also, a large inter‐subject variation in individual rectal bioavailability (17–100%) was shown, which was dependent on the site of absorption in the rectum. The estimated oral bioavailability was low (4·6 ± 1·7%), probably due to a high first‐pass effect and possible decomposition in the acidic gastric environment.

Liposomes and immunoliposomes for controlled release or site specific delivery of anti-parasitic drugs and cytostatics

Abstract The potential of liposomes for the intramuscular (i.m.) or subcutaneous (s.c.) delivery of drugs is discussed. Protection against a malaria infection after an i.m. or s.c. injection of chloroquine (CQ) containing liposomes was studied in mice. At predetermined time intervals after administration, the mice were challenged with a dose of Plasmodium berghei parasites, which was lethal in untreated mice. CQ liposomes (3 mg CQ/mouse) protected the animals for at least ten days after injection. Preliminary pharmacokinetic data suggested that protection is due to a sustained release of effective concentrations of CQ from a liposome encapsulated CQ depot. The second part of this paper focuses on cytostatic containing (immuno) liposomes in the treatment of ovarian carcinomas. The removal of liposomes from the peritoneal cavity of rats was monitored. Intraperitoneally administered protein coated, cisplatin (cDDP)-containing liposomes were removed much more slowly than ‘free’ cDDP. In vitro a specific and strong interaction of immunoliposomes (OV-TL 3 Fab fragments covalently bound to liposomes) to human ovarian carcinoma cells was observed. Potential pathways to induce therapeutic action in vivo by these adhering immunoliposomes are indicated: (triggered) release, endocytosis and fusion.

Kinetics and prophylactic efficacy of increasing dosages of liposome-encapsulated chloroquine after intramuscular injection into mice

Abstract Chloroquine (CQ) levels in blood, heart, liver, spleen and kidney as a function of time were determined after intramuscular (i.m.) injection of increasing amounts (0.64,1.32,1.98,2.63 and 3.30 mg) of liposome-encapsulated chloroquine (lip-CQ) to mice. Maximum CQ concentration in blood (C max ), area under the curve ( AUC ), mean residence time (MRT) and the absorption rate constant ( k a ) were determined. The prophylactic efficacy of lip-CQ was studied in Plasmudium berghei -infected mice. Parasitaemia was determined after single infection and repeated infection of the mice. CQ concentrations in blood could be monitored during at least 8 days (0.64 mg lip-CQ) up to 15 days ( 3.30 mg lip-CQ ). AUC and MRT values initially increased with dose and finally reached a plateau value, whereas k a values initially decreased and finally reached a plateau value at higher dosages. Mean C max values decreased with the dose. The prophylactic efficacy of lip-CQ increased with the dose. After intramuscular administration of 0.64 mg lip-CQ and infection one week later, all mice developed infection. When 3.30 mg was administered no mice developed infection when parasites were injected 7 days later and 83% and 33% of mice remained negative when parasites were injected 14 and 21 days respectively, after injection of the lip-CQ depot. CQ concentrations in heart were low in all cases which is favorable regarding cardiotoxicity of the drug. The CQ content of spleen, liver or kidney either 15 h or 24 h after injection of lip-CQ was independent of dosage. No dose dependency in chloroquine content, neither 24 h nor 15 days after injection was observed in spleen, liver or kidney. Within the range tested, injection of higher doses of lip-CQ exhibit a prolonged and a comparatively slower release of CQ. This is probably caused by mechanical compaction of the liposomal depot by the surrounding tissue. Prophylactic efficacy increased with the lip-CQ dose.

Influence of injection volume on the release kinetics of liposomal chloroquine administered subcutaneously or intramuscularly to mice

Abstract Encapsulation of chloroquine in liposomes has proven to reduce toxicity and to improve therapeutic efficacy against malaria parasites in mice. In this study the influence of the injection volume on the kinetics of chloroquine containing ‘gel’ state liposomes after intramuscular (im) and subcutaneous (sc) injection was investigated. Mice were given 0.6 mg liposome-encapsulated chloroquine dispersed in different injection volumes (20,40, 60, 80 and 100μl) either im or sc. Blood samples were taken at regular time intervals and chloroquine concentrations were determined with HPLC. Chloroquine concentrations were also measured in heart, liver, spleen and kidney at several time intervals after injection (24 h, 8 days or 22 days). Chloroquine-containing liposomes act as a local depot and provide prolonged release. In most cases chloroquine concentrations in blood were measurable up to 8 days after injection depending on the injection volume. In the case of sc injection of 80 or 100μl, detectable chloroquine concentrations could be found for a period of 4 days. Mean absorption rate constants ( k a ) after the injection of 20,40,60, 80 or 100 μl were 0.10,0.17,0.23,0.31 and 0.66 h −1 respectively after im administration and 0.06, 0.05, 0.10, 0.18 and 0.35 h −1 respectively after sc injection. A positive correlation (Kendalls rank order test, α = 0.05) was found between k a , and injection volume. This might be related to differences in spreading behaviour of the vehicle and differences in caking of the remaining depot by the muscle tonus. Mean peak chloroquine concentrations in blood were higher after im injection than after subcutaneous injection and increased with larger injection volumes. No correlation was found between AUC 0–1 (area under the curve from start to the time of last measurement above detection limit) and injection volume within each administration route. In all cases mean AUC value after im injection was significantly higher than after sc injection. Chloroquine concentrations in spleen increased in time. This was ascribed to the tendency of chloroquine to accumulate in red blood cells and the subsequent clearance of these cells by the spleen.

Human Plasmodium liver stages in SCID mice: A feasible model?

In a recent issue of Parasitology Today, Stanley and Virgin have stressed the potential of B- and T-cell deficient mice, among which severe combined immunodeficiency (SCID) mice are most frequently used, as models for the study of parasites. One of the most tantalizing prospects has been in the development of liver stages (LS) of human Plasmodium.

Targeting with IgG and Immunoliposomes to Circulating Cells: The ‘Target Cell Dragging’ Concept

In conventional drug targeting approaches attempts are made to accumulate a drug-carrier-homing device combination at the target site. Three important issues should be dealt with appropriately to make this approach successful: (1) access of the combination to the target site, (2) timing of the delivery of the drug and (3) retention of the drug at the target site (Tomlinson, 1987). Most target cells or tissues are localized in a fixed position in the body. The drug-carrier-homing device combination needs to gain access to the target sites, adhere to them and release its load, either inside or in the close proximity of the target. Unfortunately, accumulation of relatively large fractions of the drug at target sites outside the circulation (e.g. solid tumors) after intravenous administration has been found to be more the exception than the rule. This applies both to macromolecular carriers and to particulate carrier systems. Access to target sites present in the blood circulation (e.g. certain endothelial cells and macrophages, and blood clots) is (in principle) less restricted. Successful delivery of drugs to macrophages with particulate systems, such as liposomes laden with immunomodulating agents, antibiotics, antiparasitic or antiviral agents, has been described extensively in reviews (e.g. Emmen and Storm, 1987; Alving, 1988; Nassander et al., 1990; Storm et al., 1991). Huang and co-workers demonstrated that immunoliposomes bearing antibodies with the proper specificity for mouse lung endothelial cells indeed accumulate to a high degree in mouse lungs upon intravenous injection (Hughes et al., 1989).