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.

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.

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.

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.

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.

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.

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.

Tetraoxane antimalarials and their reaction with Fe(II).

Mixed tetraoxanes 5a and 13 synthesized from cholic acid and 4-oxocyclohexanecarboxylic acid were as active as artemisinin against chloroquine-susceptible, chloroquine-resistant, and multidrug-resistant Plasmodium falciparum strains (IC50, IC90). Most active 13 is metabolically stable in in vitro metabolism studies. In vivo studies on tetraoxanes with a C(4' ') methyl group afforded compound 15, which cured 4/5 mice at 600 and 200 mg.kg-1.day-1, and 2/5 mice at 50 mg.kg-1.day-1, showing no toxic effects. Tetraoxane 19 was an extremely active antiproliferative with LC50 of 17 nM and maximum tolerated dose of 400 mg/kg. In Fe(II)-induced scission of tetraoxane antimalarials only RO* radicals were detected by EPR experiments. This finding and the indication of Fe(IV)=O species led us to propose that RO* radicals are probably capable of inducing the parasite's death. Our results suggest that C radicals are possibly not the only lethal species derived from peroxide prodrug antimalarials, as currently believed.

Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa.

BACKGROUND: In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. METHODS: The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. RESULTS: 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. CONCLUSION: No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.

In vitro metabolism of phenoxypropoxybiguanide analogues in human liver microsomes to potent antimalarial dihydrotriazines.

Phenoxypropoxybiguanides, such as 1 (PS-15), are prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, 1a (WR99210), the active metabolite of 1, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Unfortunately, manufacturing processes and gastrointestinal intolerance have prevented the clinical development of 1. In vitro antimalarial activity and in vitro metabolism studies have been performed on newly synthesized phenoxypropoxybiguanide analogues. All of the active dihydrotriazine metabolites exhibited potent antimalarial activity with in vitro IC(50) values less than 0.04 ng/mL. In vitro metabolism studies in human liver microsomes identified the production of not only the active dihydrotriazine metabolite, but also a desalkylation on the carbonyl chain, and multiple hydroxylated metabolites. The V(max) for production of the active metabolites ranged from 10.8 to 27.7 pmol/min/mg protein with the K(m) ranging from 44.8 to 221 microM. The results of these studies will be used to guide the selection of a lead candidate.

Mixed steroidal 1,2,4,5-tetraoxanes: antimalarial and antimycobacterial activity.

Mixed 1,2,4,5-tetraoxanes possessing simple spirocycloalkane and spirocholic acid-derived substituents were prepared and shown to have significantly higher in vitro antimalarial activity than bis-substituted tetraoxanes. Out of 41 synthesized tetraoxanes, 12 were in vitro more potent against Plasmodium falciparum chloroquine-resistant W2 clone than artemisinin, and the most potent one was 2.4 times as active as arteether. In addition, 9 compounds exhibit higher activity than chloroquine against P. falciparum chloroquine-susceptible D6 clone. Cytotoxicity was assessed for most active compounds against the Vero cell line, showing a cytotoxicity/antimalarial potency ratio of 1/(1400-9500). For the first time, tetraoxanes were screened against Mycobacterium tuberculosis with MICs as low as 4.73 microM against H37Rv strain. Mixed tetraoxanes were synthesized in a simple procedure from cholic acid methyl esters by direct coupling of steroidal gem-dihydroperoxide to simple ketones and further transformed into corresponding acids and amides.

Design, synthesis, and evaluation of new chemosensitizers in multi-drug-resistant Plasmodium falciparum.

A series of new chemosensitizers (modulators) against chloroquine-resistant Plasmodium falciparum were designed and synthesized in an attempt to fabricate modulators with enhancing drug-resistant reversing efficacy and minimal side effects. Four aromatic amine ring systems-phenothiazine, iminodibenzyl, iminostilbene, and diphenylamine-were examined. Various tertiary amino groups including either noncyclic or cyclic aliphatic amines were introduced to explore the steric tolerance at the end of the side chain. The new compounds showed better drug-resistant reversing activity in chloroquine-resistant than in mefloquine-resistant cell lines and were generally more effective against chloroquine-resistant P. falciparum isolates from Southeast Asian (W2 and TM91C235) than those from South America (PC49 and RCS). Structure-activity relationship studies revealed that elongation of the alkyl side chain of the molecule retained the chemosensitizing activity, and analogues with four-carbon side chains showed superior activity. Furthermore, new modulators with phenothiazine ring exhibited the best chemosensitizing activity among the four different ring systems examined. Terminal amino function has limited steric tolerance as evidenced by the dramatic lose of the modulating activity, when the size of substituent at the amino group increases. The best new modulator synthesized in this study possesses all three optimized structural features, which consist of a phenothiazine ring and a pyrrolidinyl group joined by a four-carbon alkyl bridge. The fractional inhibitory concentration (FIC) index of the best compound is 0.21, which is superior to that of verapamil (0.51), one of the best-known multi-drug-resistant reversing agents. Some of the analogues displayed moderate intrinsic in vitro antimalarial activity against a W-2 clone of P. falciparum.

Pharmacokinetic investigation on the therapeutic potential of artemotil (beta-arteether) in Thai patients with severe Plasmodium falciparum malaria.

Pharmacokinetic data were obtained to evaluate the therapeutic potential of Artemotil (beta-arteether) in 56 Thai patients with severe Plasmodium falciparum malaria. Intramuscular administration was given at 1) a low dose of 3.2 mg/kg on day 0 and 1.6 mg/kg/day on days 1-4 and 2) a high dose of 4.8 mg/kg on day 0 at 0 hours, 1.6 mg/kg at 6 hours, and 1.6 mg/kg/day on days 1-4. Cmax values of 63.7 ng/mL at 6.1 hours and 140.8 ng/mL at 5.7 hours were reached in low-dose and high-dose patients, respectively. Drug concentrations decreased slowly with half-lives of 12.5-22.4 hours on day 0 and 31.6-40.7 hours on day 4 for both dosage regimens. Although the maintaining dosage on the last day was much lower than the loading dose on day 0, the area under the curve (AUC) and Cmax on day 4 were significantly increased (2.85-4.55 fold), suggesting drug accumulation in the blood. Dihydroartemisinin (DHA), an active metabolite of Artemotil, was detected in most patients. The mean ratios of DHA and Artemotil were 0.16-0.19 in both dosage regimens for the entire study period. Similar to previous reports, all patients showed a slow response to treatment with mean values of 77.2 hours for the fever clearance time (FCT) and 75.8 hours for the parasite clearance time (PCT) (low dose) and 70.1 hours for the FCT and 64.4 hours for the PCT (high dose). Interestingly, a very rapid response to the treatment was exhibited in patient 151, with an FCT of 4 hours and a PCT of 36 hours, with different pharmacokinetic data from others on day 0. The patient had a very high Cmax (2,407 ng/mL) and AUC (12,259 ng.hr/mL) values without an intramuscular absorption phase on the first day. These values were approximately 21.9 (Cmax) and 2.6 (AUC) times higher than in other patients; this patient may have been to be injected through a vessel at first dosing. In conclusion, the patients treated with the high dosage regimen had higher AUC values and higher antimalarial efficiency (cure rate = 48%) than the low-dose subjects (cure rate = 23%). Despite the high accumulation and longer exposure time (9-11 days) when compared with other artemisinin agents, due to the slow prolonged absorption of Artemotil from injection sites, the two dosage regimens did not show a better therapeutic effects than other artemisinin drugs, including alpha/beta-arteether dissolved in peanut oil used in Indian patients.

Structure-activity relationship study of antimalarial indolo [2,1-b]quinazoline-6,12-diones (tryptanthrins). Three dimensional pharmacophore modeling and identification of new antimalarial candidates.

A widely applicable three-dimensional QSAR pharmacophore model for antimalarial activity was developed from a set of 17 substituted antimalarial indolo[2,1-b]quinazoline-6,12-diones (tryptanthrins) that exhibited remarkable in vitro activity (below 100 ng/mL) against sensitive and multidrug-resistant Plasmodium falciparum malaria. The pharmacophore, which contains two hydrogen bond acceptors (lipid) and two hydrophobic (aromatic) features, was found to map well onto many well-known antimalarial drug classes including quinolines, chalcones, rhodamine dyes, Pfmrk cyclin dependent kinase inhibitors, malarial FabH inhibitors, and plasmepsin inhibitors. The phamacophore allowed searches for new antimalarial candidates from multiconformer 3D databases and enabled custom designed synthesis of new potent analogues.

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.

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 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..

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.