Reversal of Chloroquine Resistance of Plasmodium vivax in Aotus Monkeys.

Chloroquine-resistant (CQR) vivax malaria has emerged as a threat to the malaria elimination agenda. The objective of this study was to assess if a combination of chloroquine (CQ) and prochlorperazine was able to reverse CQ resistance of the Plasmodium vivax AMRU-1 strain from Papua New Guinea in infected Aotus monkeys. For this purpose, in two independent experimental drug efficacy trials, a total of 18 Aotus monkeys infected with blood obtained from donor animals were randomly assigned to treatment and control groups and orally administered CQ at 10 mg/kg or prochlorperazine at 20 mg/kg, alone or in combination, for five consecutive days. Reversal of CQR was achieved in animals that received the drug combination, whereas neither drug alone produced cures. This same drug combination reverses CQR in P. falciparum and could be an alternative for treatment in humans with chloroquine-resistant P. vivax infections.

Spectrophotometric detection of susceptibility to anti-malarial drugs.

BACKGROUND: With malaria drug resistance increasing in prevalence and severity, new technologies are needed to aid and improve the accuracy and clinical relevance of laboratory or field testing for malaria drug resistance. This study presents a method based on simple and reagentless spectroscopic measurements coupled with comprehensive spectral interpretation analysis that provides valuable quantitative information on the morphological and compositional responses of Plasmodium falciparum and infected red blood cells (IRBCs) to anti-malarial treatment. METHODS: The changes in the size, internal structure, nucleotide and haemozoin composition of the parasites as well as the morphology (size and shape) and haemoglobin composition of the IRBCs treated with dihydroartemisinin (DHA) and mefloquine (MFQ) were investigated using a spectral interpretation analysis. RESULTS: DHA treatment reduced the sizes of the parasites and their structural organelles. The haemoglobin composition of the host IRBCs determined from spectroscopic analysis changed negligibly following DHA treatment. MFQ treated parasites grew to the same size as those from parallel non-treated cultures but lacked haemozoin. Lesser deformation of the cell shape and no haemoglobin depletion were detected for the IRBCs of MFQ treated cultures. CONCLUSIONS: The spectroscopic analysis method proved to be sensitive for recognition of the effects of anti-malarial treatment on the structure and composition of the parasites and IRBCs. The method can have significant potential for research and clinical applications such as evaluating patient specimens for drug action, drug effects or for therapeutic monitoring.

Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin.

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

Radical curative efficacy of tafenoquine combination regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca mulatta).

BACKGROUND: Tafenoquine is an 8-aminoquinoline being developed for radical cure (blood and liver stage elimination) of Plasmodium vivax. During monotherapy treatment, the compound exhibits slow parasite and fever clearance times, and toxicity in glucose-6-phosphate dehydrogenase (G6PD) deficiency is a concern. Combination with other antimalarials may mitigate these concerns. METHODS: In 2005, the radical curative efficacy of tafenoquine combinations was investigated in Plasmodium cynomolgi-infected naïve Indian-origin Rhesus monkeys. In the first cohort, groups of two monkeys were treated with a three-day regimen of tafenoquine at different doses alone and in combination with a three-day chloroquine regimen to determine the minimum curative dose (MCD). In the second cohort, the radical curative efficacy of a single-day regimen of tafenoquine-mefloquine was compared to that of two three-day regimens comprising tafenoquine at its MCD with chloroquine or artemether-lumefantrine in groups of six monkeys. In a final cohort, the efficacy of the MCD of tafenoquine against hypnozoites alone and in combination with chloroquine was investigated in groups of six monkeys after quinine pre-treatment to eliminate asexual parasites. Plasma tafenoquine, chloroquine and desethylchloroquine concentrations were determined by LC-MS in order to compare doses of the drugs to those used clinically in humans. RESULTS: The total MCD of tafenoquine required in combination regimens for radical cure was ten-fold lower (1.8 mg/kg versus 18 mg/kg) than for monotherapy. This regimen (1.8 mg/kg) was equally efficacious as monotherapy or in combination with chloroquine after quinine pre-treatment to eliminate asexual stages. The same dose of (1.8 mg/kg) was radically curative in combination with artemether-lumefantrine. Tafenoquine was also radically curative when combined with mefloquine. The MCD of tafenoquine monotherapy for radical cure (18 mg/kg) appears to be biologically equivalent to a 600-1200 mg dose in humans. At its MCD in combination with blood schizonticidal drugs (1.8 mg/kg), the maximum observed plasma concentrations were substantially lower than (20-84 versus 550-1,100 ng/ml) after administration of 1, 200 mg in clinical studies. CONCLUSIONS: Ten-fold lower clinical doses of tafenoquine than used in prior studies may be effective against P. vivax hypnozoites if the drug is deployed in combination with effective blood-schizonticidal drugs.

Synthesis, structure-activity relationship, and antimalarial activity of ureas and thioureas of 15-membered azalides.

Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class. Ureas and thioureas of 15-membered azalides, N''-substituted 9a-(N'-carbamoyl-ß-aminoethyl), 9a-(N'-thiocarbamoyl-ß-aminoethyl), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-ß-aminoethyl)], 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-ß-aminoethyl)], 9a-{N'-[ß-(ethoxycarbonyl)ethyl]-N'(carbamoyl-ß-aminoethyl)}, and 9a-[N'-(ß-amidoethyl)-N'-(carbamoyl-ß-aminoethyl)] of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin), particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a-position. The improved in vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of molecules represents a good starting platform for the design of novel antimalarial azalides.

An automated, polymer-assisted strategy for the preparation of urea and thiourea derivatives of 15-membered azalides as potential antimalarial chemotherapeutics.

A series of 15-membered azalide urea and thiourea derivatives has been synthesized and evaluated for their in vitro antimalarial activity against chloroquine-sensitive (D6), chloroquine/pyremethamine resistant (W2) and multidrug resistant (TM91C235) strains of Plasmodium falciparum. We have developed an effective automated synthetic strategy for the rapid synthesis of urea/thiourea libraries of a macrolide scaffold. Compounds have been synthesized using a solution phase strategy with overall yields of 50-80%. Most of the synthesized compounds had inhibitory effects. The top 10 compounds were 30-65 times more potent than azithromycin, an azalide with antimalarial activity, against all three strains.

In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones.

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.

Establishment of an in vitro assay for assessing the effects of drugs on the liver stages of Plasmodium vivax malaria.

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.

An outbreak of Plasmodium falciparum malaria in U.S. Marines deployed to Liberia.

In 2003, 44 U.S. Marines were evacuated from Liberia with either confirmed or presumed Plasmodium falciparum malaria. An outbreak investigation showed that only 19 (45%) used insect repellent, 5 (12%) used permethrin-treated clothing, and none used bed netting. Adherence with weekly mefloquine (MQ) was reported by 23 (55%). However, only 4 (10%) had serum MQ levels high enough to correlate with protection (> 794 ng/mL), and 9 (22%) had evidence of steady-state kinetics (MQ carboxy metabolite/MQ > 3.79). Tablets collected from Marines met USP identity and dissolution specifications for MQ. Testing failed to identify P. falciparum isolates with MQ resistance. This outbreak resulted from under use of personal protective measures and inadequate adherence with chemophrophylaxis. It is essential that all international travelers make malaria prevention measures a priority, especially when embarking to regions of the world with high transmission intensity such as west Africa..

Quantitative analysis of morphological alterations in Plasmodium falciparum infected red blood cells through theoretical interpretation of spectral measurements.

Spectroscopic analysis can provide valuable insights into morphological and biochemical cellular transformations caused by diseases. However, traditional spectroscopic methods and the corresponding spectral interpretation approaches have been challenged by the complexities of the cell shape, orientation, and internal structure. Here we present an elegant spectral interpretation model that enables accurate quantitative analysis of the UV-visible spectra of red blood cells (RBCs) parasitized by the lethal human malaria parasite, Plasmodium falciparum. The model is based on the modified Mie theory (MMT) approach that incorporates the effects of the nonsphericity and orientation and multilayered cell structure to account for complex composition of the infected RBCs (IRBCs). We determine the structure and composition of the IRBCs and address unresolved matters over the alterations induced by the intraerythrocytic development of P. falciparum. The results indicate deformation and swelling of the IRBCs during the trophozoite stage of P. falciparum that is followed by substantial shrinkage during the schizont stages. We determine that up to 90% depletion of hemoglobin from the RBC cytosol does not lead to a net loss of iron from the infected cells. We quantitatively follow the morphological changes in the parasites during the intraerythrocytic development by applying the interpretation model to the UV-visible spectroscopic measurements of the IRBCs. We expect this method of quantitative spectroscopic characterization of the diseased cells to have practical clinical utility for rapid diagnosis, therapeutic monitoring, and drug susceptibility testing.

Adaptation of a Thai multidrug-resistant C2A clone of Plasmodium falciparum to Aotus monkeys and its preliminary in vivo antimalarial drug efficacy-resistance profile.

A multidrug-resistant (MDR) clone of Plasmodium falciparum (C2A) from Thailand was adapted through serial passage to Aotus monkeys. During adaptation, the parasite showed resistance to a single 20 or 40 mg/kg oral dose of mefloquine (MQ). Infection was only cured when MQ was administered orally at 40 mg/kg once in combination with intravenous artesunic acid at 20 mg/kg for 3 days. Similarly, the parasite clone was found to be resistant to quinine, failing at 20 mg/kg orally for 5 days in combination with an experimental dihydrofolate reductase (DHFR) inhibitor (WR297608) at 10, 20, or 40 mg/kg orally for 3 days, and with atovaquone/proguanil at 25 mg/kg for 3 days. This new model will allow in vivo testing of new antimalarial compounds or their combinations against a currently circulating MDR P. falciparum strain.

New chimeric antimalarials with 4-aminoquinoline moiety linked to a tetraoxane skeleton.

The synthesis of the chimeric molecules consisting of two pharmacophores, tetraoxane and 7-chloro-4-aminoquinoline, is reported. The tetraoxanes 2, 4, and 8 show relatively potent in vitro antimalarial activities, with IC90 values for the Plasmodium falciparum strain W2 of 2.26, 12.44, and 10.74 nM, respectively. In addition, two compounds, 2 and 4, cured mice in a modified Thompson test for antimalarial blood stage activity, with a minimum curative dose of 80 mg/kg, a minimum active dose of 20 mg/kg/day, and a maximum tolerated dose of >960 mg/kg.

Novel 4-aminoquinolines active against chloroquine-resistant and sensitive P. falciparum strains that also inhibit botulinum serotype A.

We report on the initial result of the coupling of 4-amino-7-chloroquinoline with steroidal and adamantane constituents to provide small molecules with excellent in vitro antimalarial activities (IC90 (W2) down to 6.74 nM). The same entities also inhibit the botulinum neurotoxin serotype A light chain metalloprotease at low micromolar levels (7-31 microM). Interestingly, structural features imparting increased antimalarial activity also provide increased metalloprotease inhibition, thus allowing for simultaneous compound optimizations against distinct targets.

Chemical stability of the peroxide bond enables diversified synthesis of potent tetraoxane antimalarials.

Of 17 prepared 1,2,4,5-tetraoxacyclohexanes stable to reductive and acidic conditions, 3 of them were more active than artemisinin against CQ and MFQ resistant strain TM91C235 and all compounds were more active in vitro against W2 than against D6 strain. In vivo, amines 10 and 11a cured all mice at higher doses with MCD < or = 37.5 (mg/kg)/day. Triol 13 was exceptionally active against melanoma (LOX IMVI) and ovarian cancer (IGROV1), both with LC 50 = 60 nM.

Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo.

BACKGROUND AND METHODOLOGY: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. PRINCIPAL FINDINGS AND CONCLUSIONS: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. SIGNIFICANCE: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

Malaria causal prophylactic activity of imidazolidinedione derivatives.

A series of acid-stable carboxamide derivatives of 2-guanidinoimidazolidinedione (5a-c and 6a-c) were prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate to good causal prophylactic activity in mice infected with Plasmodium yoelii sporozoites. Three compounds were further tested for causal prophylactic activity in Rhesus monkeys infected with Plasmodium cynomolgi sporozoites, and all showed a delay in patency from 13 to 40 days at 30 mg/kg/day x 3 days by IM dosing. Two out of four compounds tested for radical curative activity in Rhesus showed cure at 30 mg/kg/day x 3 days. The other two compounds showed delay in relapse from 16 to 68 days. Conversion of new carboxamides (5 and 6) to s-triazine derivatives (7) was demonstrated in mouse and human microsomal preparations and in rat plasma. The results suggest the metabolites, s-triazine derivatives 7, may be the active species of the new carboxamides 5a-c and 6a-c prepared in this study.

Deoxycholic acid-derived tetraoxane antimalarials and antiproliferatives(1).

The synthesis of deoxycholic acid (DCA)- and cholic acid (CA)-derived mixed tetraoxanes revealed that N-(2-dimethylamino)ethyl derivatives are potent antimalarials in vitro and in vivo. The tetraoxanes presented in this paper are dual inhibitors: besides curing mice in vivo without observed toxic effects, they kill cancer cell lines at very low concentrations. For example, DCA and CA derivatives 16 and 25 cured 3/5 (160 mg/kg/day) and 2/5 (40 mg/kg/day, MTD >960 mg/kg), respectively, and they were extremely active against melanoma LOX IMVI cancer, LC50 = 22 nM and 69 nM, respectively.