Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito.

Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.

Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes.

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria "chemical vaccine", combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian (Anopheles stephensi) or African (Anopheles gambiae) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field.

Evidence of Microbiome-Drug Interaction between the Antimalarial Lumefantrine and Gut Microbiota in Mice.

The antimalarial drug lumefantrine exhibits erratic pharmacokinetics. Intersubject variability might be attributed, in part, to differences in gut microbiome-mediated drug metabolism. We assessed lumefantrine disposition in healthy mice stratified by enterotype to explore associations between the gut microbiota and lumefantrine pharmacokinetics. Gut microbiota enterotypes were classified according to abundance and diversity indices from 16S rRNA sequencing. Pharmacokinetic parameters were computed using noncompartmental analysis. Two distinct enterotypes were identified. Maximal concentration (C max) and total drug exposure measured as the area under the drug concentration-time curve (AUC0-24) differed significantly between the groups. The mean and standard deviation of C max were 660 ± 220 ng/mL versus 390 ± 59 ng/mL (P = 0.02), and AUC0-24 was 9,600 ± 2,800 versus 5,800 ± 810 ng × h/mL (P = 0.01). In healthy mice intragastrically dosed with the antimalarial drug lumefantrine in combination with artemether, lumefantrine exposure was associated with gut bacterial community structure. Studies of xenobiotic-microbiota interactions can inform drug posology and elucidate mechanisms of drug disposition.

Kinetic Driver of Antibacterial Drugs against Plasmodium falciparum and Implications for Clinical Dosing.

Antibacterial drugs are an important component of malaria therapy. We studied the interactions of clindamycin, tetracycline, chloramphenicol, and ciprofloxacin against Plasmodium falciparum under static and dynamic conditions. In microtiter plate assays (static conditions), and as expected, parasites displayed the delayed death response characteristic for apicoplast-targeting drugs. However, rescue by isopentenyl pyrophosphate was variable, ranging from 2,700-fold for clindamycin to just 1.7-fold for ciprofloxacin, suggesting that ciprofloxacin has targets other than the apicoplast. We also examined the pharmacokinetic-pharmacodynamic relationships of these antibacterials in an in vitro glass hollow-fiber system that exposes parasites to dynamically changing drug concentrations. The same total dose and area under the concentration-time curve (AUC) of the drug was deployed either as a single short-lived high peak (bolus) or as a constant low concentration (infusion). All four antibacterials were unambiguously time-driven against malaria parasites: infusions had twice the efficacy of bolus regimens, for the same AUC. The time-dependent efficacy of ciprofloxacin against malaria is in contrast to its concentration-driven action against bacteria. In silico simulations of clinical dosing regimens and resulting pharmacokinetics revealed that current regimens do not maximize time above the MICs of these drugs. Our findings suggest that simple and rational changes to dosing may improve the efficacy of antibacterials against falciparum malaria.

Long-acting injectable atovaquone nanomedicines for malaria prophylaxis.

Chemoprophylaxis is currently the best available prevention from malaria, but its efficacy is compromised by non-adherence to medication. Here we develop a long-acting injectable formulation of atovaquone solid drug nanoparticles that confers long-lived prophylaxis against Plasmodium berghei ANKA malaria in C57BL/6 mice. Protection is obtained at plasma concentrations above 200 ng ml-1 and is causal, attributable to drug activity against liver stage parasites. Parasites that appear after subtherapeutic doses remain atovaquone-sensitive. Pharmacokinetic-pharmacodynamic analysis indicates protection can translate to humans at clinically achievable and safe drug concentrations, potentially offering protection for at least 1 month after a single administration. These findings support the use of long-acting injectable formulations as a new approach for malaria prophylaxis in travellers and for malaria control in the field.

Hollow-Fiber Methodology for Pharmacokinetic/Pharmacodynamic Studies of Antimalarial Compounds.

Knowledge of pharmacokinetic/pharmacodynamic (PK/PD) relationships can enhance the speed and economy of drug development by enabling informed and rational decisions at every step, from lead selection to clinical dosing. For anti-infective agents in particular, dynamic in vitro hollow-fiber cartridge experiments permit exquisite control of kinetic parameters and the study of their consequent impact on pharmacodynamic efficacy. Such information is of great interest for the cost-restricted but much-needed development of new antimalarial drugs, especially since the major human pathogen Plasmodium falciparum can be cultivated in vitro but is not readily available in animal models. This protocol describes the materials and procedures for determining the PK/PD relationships of antimalarial compounds.

Targeting the gyrase of Plasmodium falciparum with topoisomerase poisons.

Drug-resistant malaria poses a major public health problem throughout the world and the need for new antimalarial drugs is growing. The apicoplast, a chloroplast-like organelle essential for malaria parasite survival and with no counterpart in humans, offers an attractive target for selectively toxic new therapies. The apicoplast genome (plDNA) is a 35 kb circular DNA that is served by gyrase, a prokaryotic type II topoisomerase. Gyrase is poisoned by fluoroquinolone antibacterials that stabilize a catalytically inert ternary complex of enzyme, its plDNA substrate, and inhibitor. We used fluoroquinolones to study the gyrase and plDNA of Plasmodium falciparum. New methods for isolating and separating plDNA reveal four topologically different forms and permit a quantitative exam of perturbations that result from gyrase poisoning. In keeping with its role in DNA replication, gyrase is most abundant in late stages of the parasite lifecycle, but several lines of evidence indicate that even in these cells the enzyme is present in relatively low abundance: about 1 enzyme for every two plDNAs or a ratio of 1 gyrase: 70 kb DNA. For a spectrum of quinolones, correlation was generally good between antimalarial activity and gyrase poisoning, the putative molecular mechanism of drug action. However, in P. falciparum there is evidence for off-target toxicity, particularly for ciprofloxacin. These studies highlight the utility of the new methods and of fluoroquinolones as a tool for studying the in situ workings of gyrase and its plDNA substrate.

Model system to define pharmacokinetic requirements for antimalarial drug efficacy.

Malaria presents a tremendous public health burden, and new therapies are needed. Massive compound libraries screened against Plasmodium falciparum have yielded thousands of lead compounds, resulting in an acute need for rational criteria to select the best candidates for development. We reasoned that, akin to antibacterials, antimalarials might have an essential pharmacokinetic requirement for efficacy: action governed either by total exposure or peak concentration (AUC/CMAX), or by duration above a defined minimum concentration [time above minimum inhibitory concentration (TMIC)]. We devised an in vitro system for P. falciparum, capable of mimicking the dynamic fluctuations of a drug in vivo. Using this apparatus, we find that chloroquine is TMIC-dependent, whereas the efficacy of artemisinin is driven by CMAX. The latter was confirmed in a mouse model of malaria. These characteristics can explain the clinical success of two antimalarial drugs with widely different kinetics in humans. Chloroquine, which persists for weeks, is ideally suited for its TMIC mechanism, whereas great efficacy despite short exposure (t1/2 in blood 3 hours or less) is attained by CMAX-driven artemisinins. This validated preclinical model system can be used to select those antimalarial lead compounds whose CMAX or TMIC requirement for efficacy matches pharmacokinetics obtained in vivo. The apparatus can also be used to explore the kinetic dependence of other pharmacodynamic endpoints in parasites.

Membrane active chelators as novel anti-African trypanosome and anti-malarial drugs.

Malaria (Plasmodium spp.) and human African trypanosomiasis (Trypanosoma brucei spp.) are vector borne, deadly parasitic diseases. While chemotherapeutic agents for both diseases are available, difficulty in disease eradication and development of drug resistance require that new therapies targeting unexplored pathways or exploiting novel modes of action be developed. Intracellular Plasmodium and extracellular Trypanosoma brucei may have unique and essential requirements for divalent metal ions, beyond that deemed physiological for the host. Membrane Active Chelators (MACs), biologically active only in a hydrophobic lipid environment, are able to bind metal ions at elevated non-physiological concentrations in the vicinity of cell membranes. A dose-response relationship study using validated viability assays revealed that two MAC drugs, DP-b99 and DP-460, were cytotoxic for these parasites in vitro. The 50% effective concentration (EC50) values for DP-b99 and DP-460 were 87 µM and 39 µM for Trypanosoma brucei brucei and 21 µM and 28 µM for erythrocytic Plasmodium falciparum, respectively. Furthermore, drug potency was maintained for at least 24h in serum containing medium at 37°C. While the exact mechanism of action of MACs against intracellular malaria and extracellular African trypanosome parasites has yet to be determined, their potential as antiparasitic agents warrants further investigation.

Malaria-infected mice are completely cured by one 6 mg/kg oral dose of a new monomeric trioxane sulfide combined with mefloquine.

Sixteen new anilide derivatives of the natural trioxane artemisinin were prepared and evaluated for antimalarial efficacy in Plasmodium berghei infected mice. Of these 16 new anilides administered orally as one 6 mg/kg dose combined with 18 mg/kg mefloquine hydrochloride, only sulfide 3-arteSanilide 12d was completely curative: on day 30 after infection, all mice in this group had no detectable parasitemia, gained as much weight as the uninfected control mice, and behaved normally.

Integrating cognitive and affective dimensions of pain experience into health professions education.

Pain is prevalent in clinical settings, and yet it is relatively under-represented in the education of most students in the health professions. Because pain includes both sensory-discriminative and affective features, teaching students about pain presents unique challenges and opportunities. The present article describes the evolution of a new blueprint for clinical excellence that, among other competencies, incorporates a need for the emotional development of clinical trainees. The framework has been applied to the development and implementation of two new courses in pain. The first course is designed to provide a comprehensive foundation of medical knowledge regarding pain, while integratively introducing students to the affective dimensions of pain. The second course is designed to enhance students' appreciation for the protean effects of pain through use of the humanities to represent medical experience. It is concluded that, to be most effective, fostering the emotional development of trainees in the health professions necessitates the incorporation of affect-focused learning objectives, educational tasks, and assessment methods.

A new program in pain medicine for medical students: integrating core curriculum knowledge with emotional and reflective development.

OBJECTIVE: Improvements in clinical pain care have not matched advances in scientific knowledge, and innovations in medical education are needed. Several streams of evidence indicate that pain education needs to address both the affective and cognitive dimensions of pain. Our aim was to design and deliver a new course in pain establishing foundation-level knowledge while comprehensively addressing the emotional development needs in this area. SETTING: One hundred eighteen first-year medical students at Johns Hopkins School of Medicine. OUTCOME MEASURES: Performance was measured by multiple-choice tests of pain knowledge, attendance, reflective pain portfolios, and satisfaction measures. RESULTS: Domains of competence in pain knowledge included central and peripheral pain signalling, pharmacological management of pain with standard analgesic medications, neuromodulating agents, and opioids; cancer pain, musculoskeletal pain, nociceptive, inflammatory, neuropathic, geriatric, and pediatric pain. Socio-emotional development (portfolio) work focused on increasing awareness of pain affect in self and others, and on enhancing the commitment to excellence in pain care. Reflections included observations on a brief pain experience (cold pressor test), the multidimensionality of pain, the role of empathy and compassion in medical care, the positive characteristics of pain-care role models, the complex feelings engendered by pain and addiction including frustration and disappointment, and aspirations and commitments in clinical medicine. The students completing feedback expressed high levels of interest in pain medicine as a result of the course. DISCUSSION: We conclude that a 4-day pain course incorporating sessions with pain specialists, pain medicine knowledge, and design-built elements to strengthen emotional skills is an effective educational approach. SUMMARY: Innovations in medical education about pain are needed. Our aim was to design and deliver a new course for medical students addressing both the affective and cognitive dimensions of pain. Combining small-group sessions with pain specialists, active-learning approaches to pain knowledge, and design-built elements to strengthen emotional skills was highly effective.

Antitrypanosomal activities of fluoroquinolones with pyrrolidinyl substitutions.

Fluoroquinolones with pyrrolidinyl substitutions were tested against Trypanosoma brucei and mammalian cells. Bulky substituents at C-7 or a 1-2-bridging thiazolidine ring increased antitrypanosomal activity and selective toxicity. These compounds trap protein-DNA complexes and inhibit nucleic acid biosynthesis in trypanosomes, characteristics of topoisomerase II inhibition.

Antitrypanosomal activities of tryptanthrins.

New drugs and molecular targets are needed against Trypanosoma brucei, the protozoan that causes African sleeping sickness. Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione), a traditional antifungal agent, and 11 analogs were tested against T. brucei in vitro. The greatest activity was conferred by electron-withdrawing groups in the 8 position of the tryptanthrin ring system; the most potent compound had a 50% effective concentration of 0.40 microM.