Assessing the utility of an anti-malarial pharmacokinetic-pharmacodynamic model for aiding drug clinical development

ABSTRACT: BACKGROUND: Mechanistic within-host models relating blood anti-malarial drug concentrations with the parasite-time profile help in assessing dosing schedules and partner drugs for new antimalarial treatments. A comprehensive simulation study to assess the utility of a stage-specific pharmacokinetic-pharmacodynamic (PK-PD) model for predicting within-host parasite response was performed. METHODS: Three anti-malarial combination therapies were selected: artesunate-mefloquine, dihydroartemisinin-piperaquine, and artemether-lumefantrine. The PK-PD model included parameters to represent the concentration-time profiles of both drugs, the initial parasite burden and distribution across the parasite life cycle, and the parasite multiplication factor due to asexual reproduction. The model also included the maximal killing rate of each drug, and the blood drug concentration associated with half of that killing effect (in vivo EC50), derived from the in vitro IC50, the extent of binding to 0.5% Albumax present in the in vitro testing media, and the drugs plasma protein binding and whole blood to plasma partitioning ratio. All stochastic simulations were performed using a Latin-Hypercube-Sampling approach. RESULTS: The simulations demonstrated that the proportion of patients cured was highly sensitive to the in vivo EC50 and the maximal killing rate of the partner drug co-administered with the artemisinin derivative. The in vivo EC50 values that corresponded to on average 95% of patients cured were much higher than the adjusted values derived from the in vitro IC50. The proportion clinically cured was not strongly influenced by changes in the parameters defining the age distribution of the initial parasite burden (mean age of 4 to 16 hours) and the parasite multiplication factor every life cycle (ranging from 8 to 12 fold/cycle). The median parasite clearance times, however, lengthened as the standard deviation of the initial parasite burden increased (i.e. the infection became more asynchronous). CONCLUSIONS: This simulation study demonstrates that the PD effect predicted from in vitro growth inhibition assays does not accord well with the PD effect of the anti-malarials observed within the patient. This simulation-based PK-PD modelling approach should not be considered as a replacement to conducting clinical trials but instead as a decision tool to improve the design of a clinical trial during drug development.     

The influence of host hormones and cytokines on Echinococcus multilocularis signalling and development

Parasitic helminths display highly complex life-cycles in which the establishment of adults or larvae within host target organs as well as the transition of one developmental stage to the following is influenced by host-derived factors. Due to its approachability concerning in vitro cultivation, the larval stage of the fox-tapeworm Echinococcus multilocularis has recently emerged as a model system to study the molecular nature of such host-derived stimuli and their influence on developmental control in the parasite. Data obtained so far indicate that cytokines which are used by the host for cell-cell communication can also be exploited by the parasite as clues to find suitable target organs. This involves direct interactions of evolutionary conserved signalling systems of the receptor tyrosine--and the receptor serine/threonine-kinase pathways of the parasite with corresponding host cytokines of the insulin-, the epidermal growth factor-, and the transforming growth factor-beta-families. In the present article, we will briefly review in vitro cultivation approaches undertaken so far for E. multilocularis larvae as well as our current knowledge on the parasite's signalling systems and their interaction with host cytokines.     

Impact of continuous axenic cultivation in Leishmania infantum virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species.     

Immunopathology of Entamoeba histolytica infections

Entamoeba histolytica infects almost 10% of the world's population and results in about 100 000 deaths annually(1). Relatively little information is available concerning the immune response and the immunopathology elicited by this parasite, probably due in part to the lack of a truly appropriate animal model(2-4). However, there has been some progress - particularly concerning the interaction of this parasite with cells of the immune system(5,6). This review summarizes the salient features of the cellular immune response and immunopathology, largely from in vitro studies and studies using the gerbil model for invasive amoebiasis(7,8). Overall, the results suggest that invasive amoebtasis induces profound immune dysfunction both at the effector level of macrophages and on their accessory cell potential.     

Ultrastructural study of asexual development of Cryptosporidium parvum in a human intestinal cell line.

The lack of a well-defined in vitro model of Cryptosporidium infection has severely hampered research on the biology of parasitic invasion of the host cell and on intracellular development of the parasite. In vitro infection of the differentiated human enterocyte cell line HT29.74 was studied by electron microscopy to detect changes in parasite and host cell morphology. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned differentiated HT29.74 cells. Parasites and infected cells were evaluated by transmission electron microscopy at 20 min, 1 h, 6 h, 24 h and 7 days. Sporozoite invagination within the epithelial cell microvilli and subsequent penetration was evident at 1 h. At 6 h, the development of a dense band and feeder layer was visible. Development of the trophozoite into a schizont occurred over 24 h. Micronemes and dense granules were clearly visible within sporozoites and merozoites. Organization of vacuoles within the cytoplasm of the host cell was evident below the dense band. A sexual Cryptosporidium development in vitro was morphologically no different from initial development in vivo. In vitro infection of HT29.74 cells provides an excellent model to study parasite-host cell interaction and asexual parasite development.     

Cryopreserved Plasmodium vivax and cord blood reticulocytes can be used for invasion and short term culture

The establishment of a Plasmodium vivaxin vitro culture system is critical for the development of new vaccine, drugs and diagnostic tests. Although short-term cultures have been successfully set up, their reproducibility in laboratories without direct access to P. vivax-infected patients has been limited by the need for fresh parasite isolates. We explored the possibility of using parasite isolates and reticulocytes, both cryopreserved, to perform invasion and initiate short-term culture. Invasion results obtained with both cryopreserved isolates and reticulocytes were similar to those obtained with fresh samples. This method should be easily replicated in laboratories outside endemic areas and will substantially contribute to the development of a continuous P. vivax culture. In addition, this model could be used for testing vaccine candidates as well as for studying invasion-specific molecular mechanisms.     

Novel diaryl ureas with efficacy in a mouse model of malaria

Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cultures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum. Compound 18 demonstrated EC₅₀ values of 37 and 55 nM versus in vitro cultured parasite strains and promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at day 31 post-treatment when administered subcutaneously at 256 mg/kg. This series of compounds provides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue antimalarial drug discovery efforts.     

A quantitative in vitro cultivation technique to determine cell number and growth rates in strains of Crithidia bombi (Trypanosomatidae), a parasite of bumblebees

The protozoan parasite Crithidia bombi and its host, the bumblebee Bombus terrestris, are used as a model system for the study of the evolutionary ecology of host-parasite interactions. In order to study these interactions we established a method for in vitro cultivation of single parasite strains. Additionally, a high-throughput method is developed for the determination of cell numbers in cultures by means of optical density (OD) measurements. The protocol for in vitro cultivation allowed for growing different strains on agar plates as well as in culture medium. A calibration curve for the relationship between cell number and OD has been developed. Subsequently, growth rates for different genotypes of C. bombi have been recorded. Significant differences in the growth rates and generation times between these genotypes were demonstrated. As this might be related to the virulence of the parasite, this relationship may be confirmed by in vivo growth rate determination. In comparison with conventional cell counting, the application of OD measurements allows for high-throughput experiments as the time taken to record each sample is reduced by a factor of 30. The in vitro cultivation method allows for controlled infection experiments in order to study host-parasite interactions.     

An alternative model for heme biosynthesis in the malarial parasite

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.     

Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens

The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high-throughput screening of inhibitory compounds.     

Induction of Toxoplasma gondii cystogenesis and multiplication arrest by treatments with a phosphatidylcholine-specific phospholipase C inhibitor.

A model of tissue cyst formation was developed using D609, a specific inhibitor of phosphatidylcholine specific-phospholipase C. The phospholipase inhibitor induced a decrease in Toxoplasma gondii multiplication and several successive treatments could lead to an arrest in parasite multiplication and full encystment of the parasites. This could be a first step towards an in vitro model of T. gondii reactivation.     

Giardia lamblia -- a model organism for eukaryotic cell differentiation

Giardia lamblia is a binucleated, flagellated protozoan parasite that inhabits the upper small intestine of its vertebrate hosts. The entire life cycle, which can be completed in vitro, is simple with cycling between a vegetative trophozoite and a highly resistant cystic form. The parasite is one of the earliest diverging eukaryotes known and more than 95% of the genome is sequenced. This makes Giardia an excellent model system for studies of basic eukaryotic processes like cell differentiation. In this review we will discuss recent data concerning Giardia differentiation with a focus on DNA replication and cytokinesis.     

Blood platelets and schistosome egg excretion

The eggs of helminths of the Schistosoma genus require to be extravasated in order to continue the life cycle of the parasite. The possible mode by which this takes place was investigated in a mouse model. Suppression of platelet activity in Schistosoma mansoni-infected mice by administering rabbit anti-mouse platelet serum or a selection of "antiplatelet drugs" resulted in a significant reduction of parasite egg excretion. This reduction was best achieved when antiplatelet agents were administered just before the onset of parasite egg excretion. The association between parasite eggs and platelets was illustrated in vivo and in vitro where platelet aggregates on egg surfaces were seen in both light and electron microscopy. In addition, eggs that had been isolated from infected mouse tissues induced platelet aggregation in whole mouse blood, and this was inhibited by preincubation with the beta-lactam antibiotic, ticarcillin. Isolated eggs were also capable of inducing ex vivo platelet aggregation in mice, which was dependent on presensitization with eggs. These data suggest a role for platelets in the extravasation and excretion of parasite eggs in schistosomiasis.     

Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR

Control of human leishmaniases relies on appropriate diagnosis and reliable methods for monitoring chemotherapy. The current method used for estimation of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type of Leishmania DNA detection-based method is extremely sensitive, but could fail in distinguishing viable Leishmania from slowly degenerating ones. We have used an in vitro model to monitor the duration of Leishmania DNA persistence in mouse macrophages following exposure to l-leucine ester, a molecule otherwise known to rapidly kill intracellular Leishmania amazonensis amastigotes. At 1h of post l-leucine ester exposure, more than 98% of amastigote-loaded macrophages harbored killed parasites and parasite remnants, as assessed by microscopy. This dramatic decrease in parasite load and the microscopic parasite follow-up over the 120 h time period studied were correlated with Leishmania DNA as quantified by real-time PCR. Our results indicate that kinetoplast and nuclear parasite DNA degradation occurs very rapidly after amastigote death. These data add further weight to the argument that PCR assays represent not only a robust method for diagnosis but can also be reliable for monitoring parasite size reduction rate post any intervention (Leishmania-targeting molecules, immunomodulators...).     

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture.

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.     

Glycolipids and other constituents from Desmodium gangeticum with antileishmanial and immunomodulatory activities

Nineteen compounds of various classes, such as flavonoid glycosides, pterocarpanoids, lipids, glycolipids, and alkaloids, were isolated and identified from the Desmodium gangeticum whole plant. Aminoglucosyl glycerolipid (8) is reported here for the first time. Its structure has been elucidated by spectroscopic and degradation studies. This novel compound exhibited in vitro antileishmanial and immunomodulatory activities, as it enhanced nitric oxide (NO) production and provided resistance against infection established in peritoneal macrophages by the protozoan parasite Leishmania donovani. Another known compound, glycosphingolipid (cerebroside) (7) was found to possess significant in vitro antileishmanial and immunomodulatory activities against the same parasite. Other compounds were found to be inactive.     

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.     

A model for estimating total parasite load in falciparum malaria patients

We describe an age-structured mathematical model of the malaria parasite life cycle that uses clinical observations of peripheral parasitaemia to estimate population dynamics of sequestered parasites, which are hidden from the clinical investigator. First, the model was tested on parasite populations cultured in vitro, and was found to account for approximately 72% of the variation in that sub-population of parasites that would have been sequestered in vivo. Next, the model was applied to patients undergoing antimalarial therapy. Using individual data sets we found that although the model fitted the peripheral parasite curves very well, unique solutions for the fit could not be obtained; therefore, robust estimates of sequestered parasite dynamics remained unavailable. We conclude that even given detailed data on individual parasitaemia, estimates of sequestered numbers may be difficult to obtain. However, if data on individuals undergoing similar therapy are collected at equal time intervals, some of these problems may be overcome by estimating specific parameters over groups of patients. In this manner we estimated sequestered parasite density in a group of patients sampled at identical time points following antimalarial treatment. Using this approach we found significant relationships between changes in parasite density, age structure and temperature that were not apparent from the analysis of peripheral parasitaemia only.     

HeLa cell nucleus, a source of thymidine for Trichomonas vaginalis growing in vitro

Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.