Antimalarial activity of tigecycline, a novel glycylcycline antibiotic.

Tigecycline is a novel glycylcycline antibiotic with a broad antibacterial spectrum. Tigecycline was tested with 66 clinical isolates of Plasmodium falciparum from Bangladesh using the histidine-rich protein 2 in vitro drug susceptibility assay. The 50% and 90% inhibitory concentrations of tigecycline were 699 (95% confidence interval, 496 to 986) and 5,905 nM (4,344 to 8,028). Tigecycline shows no activity correlation with traditional antimalarials and has substantial antimalarial activity on its own.

Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand.

To determine differences in the distribution of drug resistance mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genes of P. falciparum isolates in Thailand, a study was conducted using polymerase chain reaction-restriction fragment length polymorphism to detect mutations in P. falciparum isolates obtained from three areas with different levels of in vivo mefloquine (MQ) resistance. All isolates carried mutant allele T76 of the pfcrt gene and wild-type allele D1246 of the pfmdr1 gene except for one isolate, which showed the wild-type K76 allele. This isolate was obtained from Chanthaburi Province, an area with high MQ resistance. Relatively low rates of the mutant alleles D1042 and Y86 of the pfmdr1 gene were found among Thai isolates of P. falciparum. However, a statistically significant difference in the distribution was noted. Most of the mutant isolates were found among isolates from areas with moderate or low MQ resistance. Only one isolate with mixed mutant and wild-type N1042 and D1042 and two mutants of Y86 were found among the isolates from areas with high MQ resistance. The findings provide limited support for the hypothesis that mutant alleles of pfmdr1 may be associated with increased sensitivity to MQ.

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma.

A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at lambda(ex)=267 nm and lambda(em)=443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 microg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.

Pharmacodynamic interactions of amodiaquine and its major metabolite desethylamodiaquine with artemisinin, quinine and atovaquone in Plasmodium falciparum in vitro.

The antimalarial in vitro activities of amodiaquine and desethylamodiaquine in combination with atovaquone, quinine and artemisinin against Plasmodium falciparum were investigated in strains F-32, FCR-3 and K-1. These parasitic strains have different sensitivity profiles to the standard antimalarial chloroquine, but all can be considered sensitive to the test drugs, representing the recommended situation for introduction of two partner drugs in combination therapy. Amodiaquine showed marked synergism when combined with each of the three partner compounds at concentration ratios between 90 and 9x10(-7), including the therapeutically relevant range. The interaction profiles of desethylamodiaquine with quinine and artemisinin also showed predominantly synergism over a wide range of concentration ratios between 70 and 9x10(-7). The responses to all combinations exhibited signs of strain-specificity, but such phenomena were usually observed outside the therapeutic range of the concentration ratios. Synergism was generally more marked with increasing EC values, i.e. at concentrations expected to be therapeutically effective and thus clinically relevant. Even trace quantities of amodiaquine were able to potentiate the activity of structurally unrelated antimalarial drugs.

Synergism between amodiaquine and its major metabolite, desethylamodiaquine, against Plasmodium falciparum in vitro.

The in vitro activity of the prodrug amodiaquine and its metabolite monodesethyl-amodiaquine has been studied for three strains of Plasmodium falciparum: LS-2, LS-3, and LS-1. Both compounds showed significant activity against all three strains; the activity of amodiaquine was slightly higher than that of the metabolite. By use of a checkerboard design, interaction studies with both compounds yielded evidence of significant synergism; means of the sums of the fractional inhibitory concentrations were 0.0392 to 0.0746 for strain LS-2, 0.1567 to 0.3102 for strain LS-3, and 0.025 to 0.3369 for strain LS-1. In further investigations, the interaction of amodiaquine with monodesethyl-amodiaquine was tested at clinically relevant concentrations of both compounds. In these studies, involving amodiaquine at picomolar and femtomolar concentrations, the compound was found to exert high potentiating activity on monodesethyl-amodiaquine. This interaction produced mean ratios of observed to expected activity of 0.0505 to 0.0642 for strain LS-2, 0.0882 to 0.3820 for strain LS-3, and 0.0752 to 0.2924 for strain LS-1. The synergistic activity was most marked at monodesethyl-amodiaquine/amodiaquine ratios up to 100,000:1 but was still evident at higher ratios.

In vitro activity of quinolines against Plasmodium falciparum in Gabon.

The assessment of drug sensitivity of Plasmodium falciparum to antimalarial drugs is of vital interest for malaria endemic regions. We conducted a follow-up study to monitor the in vitro activity of the most commonly used quinolines against fresh P. falciparum isolates in Lambaréné, Gabon by measuring schizont maturation inhibition in 2002. Mean 50% effective concentration levels for chloroquine, quinine, and mefloquine were 5.5micromol/l blood, 286nmol/l blood medium mixture (BMM), and 1.1micromol/l blood, respectively. All isolates (n=40) were found to be highly resistant to chloroquine. One isolate was resistant to mefloquine and five isolates were presenting borderline-resistance. All isolates were inhibited by quinine concentrations below the threshold of resistance (n=43). Besides the observation of an increasing number of borderline resistant isolates to mefloquine, an extremly high parasite resistance to chloroquine-still officially the first line antimalarial in Gabon-seems to be of particular concern.

In vitro activity and interaction of clindamycin combined with dihydroartemisinin against Plasmodium falciparum.

Combination regimens are considered a valuable tool for the fight against drug-resistant falciparum malaria. This study was conducted to evaluate the antimalarial potential of clindamycin in combination with dihydroartemisinin in continuously cultured and in freshly isolated Plasmodium falciparum parasites, measuring the inhibition of Plasmodium falciparum histidine-rich protein II synthesis. Interaction analysis revealed a synergistic or additive mode of interaction at various concentration ratios in all continuously cultured parasites at the 50% effective concentration (EC(50)) level. Antagonism was not found for any of the culture-adapted parasites. In fresh P. falciparum isolates, a fixed clindamycin-dihydroartemisinin combination exhibited additive activity at the EC(50) and EC(90) levels. The drug mixture showed no significant activity correlation to other commonly used antimalarials. The clindamycin-dihydroartemisinin combination appears to be a promising candidate for clinical investigation.

Sensitivity of Plasmodium vivax to chloroquine in Sa Kaeo Province, Thailand.

Following a recent, abrupt local increase in the incidence of vivax malaria, a study was conducted in order to evaluate the efficacy of chloroquine for the treatment of 26 adult patients with acute vivax malaria in Sa Kaeo Province, Thailand. The chloroquine sensitivity of Plasmodium vivax has been assessed in parallel, using a growth inhibition method. Blood samples for the in vitro tests were taken prior to the administration of the standard treatment with chloroquine--in total 25 mg base/kg over 3 days--and primaquine 0.25 mg base/kg once daily for 14 days. The efficacy has been assessed according to the WHO standard in vivo test. The cure rate was 100%. No recrudescence was observed during the follow-up period of 28 days. The mean fever clearance time (FCT) was 40 h, the mean parasite clearance time (PCT) was 49 h. Mean IC(50) and IC(90) of the parasites were 28 and 171 nM, respectively. These results show that local P. vivax is still sensitive to chloroquine. The epidemic outbreak was therefore obviously not due to the presence of chloroquine-resistant P. vivax.

Plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine.

In the scenario of drug-resistant Plasmodium falciparum malaria combination therapy represents an effective approach. Artemisinin and its derivatives are of special interest because they represent the most effective group of compounds against multidrug-resistant malaria with a rapid onset of action and a short half-life. Interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine were therefore investigated against three strains of P. falciparum using a 48-h in vitro culture assay. Two of the strains were chloroquine sensitive and one was partially chloroquine resistant. Observed effective concentrations (O) of the combined compounds at different concentration ratios were calculated for different degrees of inhibition (EC50, EC90, EC99) and compared to expected calculated effective concentrations (E) using a probit method. Synergism with mean O/E EC90 values of 0.25 and 0.8 were found with the combination of artemisinin and the two Mannich bases, amodiaquine and pyronaridine, respectively, whereas chloroquine showed addition with a mean value of 1.2. Although both amodiaquine and chloroquine are 4-aminoquinolines, their interaction with artemisinin appears to be different. The combination of artemisinin with amodiaquine represents an important option for the treatment of falciparum malaria.

In vitro interactions of artemisinin with atovaquone, quinine, and mefloquine against Plasmodium falciparum.

The interactions of artemisinin with atovaquone, quinine, and mefloquine were investigated in three Plasmodium falciparum strains (strains F-32, FCR-3, and K-1) by an in vitro culture assay. The parasites were cultured for 48 h in the presence of different concentrations and proportions of two drugs at a time in a checkerboard design. The response parameters were determined, and the sums of the fractional inhibitory concentrations (sigmaFICs) of the drug combinations were calculated for different degrees of inhibition (50% effective concentration [EC50], EC90, and EC99). Within therapeutically relevant molar ratios (19 to 200), the combination of quinine and artemisinin showed mean sigmaFICs of 1.71 at the EC50, 0.36 at the EC90, and 0.13 at the EC99, indicating increasing synergism. Within the range of molar ratios of 4.3 to 50, the combination of mefloquine and artemisinin yielded mean sigmaFCIs of 0.93, 0.44, and 0.31 at the EC50, EC90, and EC99, respectively, indicating synergism. The atovaquone combination showed additive activity to synergism at atovaquone/artemisinin proportions considered relevant to the in vivo situation, i.e., between 4.3 and 200, with the mean sigmaFICs decreasing from 1.34 at the EC50 to 0.85 and 0.23 at the EC90 and EC99, respectively. Interstrain differences in the degree of drug interaction were seen with the three strains for all combinations. Synergism was most consistent with quinine.

Reassessment of the resistance of Plasmodium falciparum to chloroquine in Gabon: implications for the validity of tests in vitro vs. in vivo.

Increasing resistance of Plasmodium falciparum to antimalarial drugs presents a major risk factor for people living in endemic areas of tropical Africa. In Lambaréné, Gabon, regular surveillance of chloroquine sensitivity of P. falciparum in vitro has been carried out since 1992 using the WHO standard microtest. Results indicated that from 1994 onwards chloroquine resistance in vitro decreased significantly and that by 2000, about 70% of parasite isolates seemed to be sensitive to chloroquine in vitro. In 2001, we conducted a clinical study to reassess the efficacy of chloroquine in vivo for the treatment of uncomplicated P. falciparum malaria. Twenty-six patients aged 4-15 years were included in this study. Most unexpectedly, the study demonstrated high-grade resistance to chloroquine in vivo (failure rate on day 28 of 100%). As a consequence, tests of parasite susceptibility to chloroquine in vitro were repeated using the same protocol except for the replacement of previously used commercially available predosed WHO culture plates by independently dosed plates. All tested P. falciparum isolates were highly resistant to chloroquine, correlating well with our clinical findings. We concluded that high level resistance of P. falciparum to chloroquine persists in the study area. Neglect or absence of quality controls of essential test material can lead to invalid study results and wrong conclusions and should always be suspected in the case of major fluctuations in the sensitivity patterns of an antimalarial drug in vitro. In addition, our results highlight the supreme value of tests in vivo in providing reliable estimates of the efficacy of an antimalarial in a specific area.

Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand.

Antibiotics with antimalarial activity may offer an interesting alternative for the treatment of multidrug-resistant falciparum malaria. Azithromycin, a relatively recent semisynthetic derivative of erythromycin, was tested for its in vitro activity against fresh isolates of Plasmodium falciparum. As the reportedly slow onset of action of azithromycin suggests its combination with fast-acting substances, such as artemisinin-derivatives, dihydroartemisinin (DHA) was tested parallel as a possible combination partner. The effective concentrations found for azithromycin in this study (EC(50) = 29.3 micromol/l, EC(90) = 77.1 micromol/l blood medium mixture (BMM)) are comparable to those of other antimalarials in the antibiotics class and are considerably higher than those found for mefloquine or quinine. The absence of an activity correlation between azithromycin and chloroquine, quinine and artemisinin emphasises the independence of azithromycin drug response from the sensitivity to these drugs. A weak activity correlation (rho(EC90) = 0.352; p = 0.028), which could point to a potential cross-sensitivity but is probably of little clinical importance, was found with mefloquine above the EC(50) level. Provided that further clinical trials support the combination of these drugs, DHA may offer an interesting combination partner for azithromycin owing to its rapid onset of action and the comparatively low effective concentrations (EC(50) = 1.65 nmol/l, EC(90) = 7.10 nmol/l BMM). This combination may serve as an interesting alternative for tetracycline and doxycycline, which cannot be used in pregnant women and children, and exhibit phototoxicity. Nevertheless, the relatively high cost of this combination, as well as the controversial reports of the clinical efficacy, may limit the usefulness of azithromycin in malaria therapy and require an adjustment of previously used treatment regimens.

Desbutyl-benflumetol, a novel antimalarial compound: in vitro activity in fresh isolates of Plasmodium falciparum from Thailand.

Desbutyl-benflumetol (DBB) is a novel antimalarial compound closely related to benflumetol (lumefantrine), of which it is a putative metabolite. The in vitro response of Plasmodium falciparum to DBB was studied in Mae Hong Son and Mae Sot, in northwest Thailand, in 1997 and 1998. In total, 155 fresh isolates were successfully tested using the World Health Organization standard in vitro microtest system (Mark II). The mean 50% effective concentration (EC(50)) and 90% effective concentration of DBB were 6.36 and 31.09 nmol/liter, respectively. The comparison of the activity of DBB and benflumetol yielded a highly significant potency ratio of 4.52, corresponding to a more than four times higher efficacy of DBB. A considerable potency difference was found between isolates from Mae Hong Son and those from Mae Sot, reflecting lesser sensitivity in the area with marked resistance to mefloquine and quinine. This observation is also supported by a highly significant activity correlation with benflumetol (P < 0.001) and to a similar degree with mefloquine (P < 0.001), reflecting a close relationship of DBB with the class II aryl amino alcohol blood schizontocides. A less distinct association was also found with artemisinin, which was significant only at the EC(50) level, and there was no correlation at all with chloroquine. DBB is a promising antimalarial compound that merits further investigation in order to define its practical therapeutic potential.

Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. It is therefore responsible for significant morbidity and mortality in a number of countries. Infections with E. histolytica are treated with nitroimidazoles, primarily with metronidazole. At this time, there is a lack of useful alternative classes of substances for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) such as hexadecyl-PC (miltefosine) were originally developed as antitumor agents, but recently they have been successfully used for the treatment of visceral leishmaniasis in humans. We examined hexadecyl-PC and several other alkyl-PCs with longer alkyl chains, with and without double bond(s), for their activity against two strains of E. histolytica. The compounds with the highest activity were oleyl-PC, octadecyl-PC, and nonadecenyl-PC, with 50% effective concentrations for 48 h of treatment between 15 and 21 microM for strain SFL-3 and between 73 and 98 microM for strain HM-1:IMSS. We also tested liposomal formulations of these alkyl-PCs and miltefosine. The alkyl-PC liposomes showed slightly lower activity, but are expected to be well tolerated. Liposomal formulations of oleyl-PC or closely related alkyl-PCs could be promising candidates for testing as broad-spectrum antiprotozoal and antitumor agents in humans.

Declining chloroquine resistance of Plasmodium falciparum in Lambaréné, Gabon from 1992 to 1998.

Plasmodium falciparum malaria continues to threaten human populations in the tropics and travellers in endemic areas. Drug resistance of the parasite is a major problem in treating this devastating disease. In a prospective trial we investigated the in vitro sensitivity of Plasmodium falciparum to chloroquine, quinine and mefloquine in the Albert Schweitzer Hospital in Lambaréné, Gabon every second year from 1992 to 1998. We used the standard WHO in vitro sensitivity assay. Parasite sensitivity to quinine and mefloquine remained stable over the years. However, parasite resistance to chloroquine decreased highly significantly with the change in local malaria treatment policy. In 1992, 100% of parasite isolates showed resistance to chloroquine, whereas in 1998 only 45% were found resistant.

In vivo-in vitro model for the assessment of clinically relevant antimalarial cross-resistance.

Cross-resistance may be considered one of the most important factors leading to decreased drug susceptibility of Plasmodium falciparum. The study aimed to determine whether clinically relevant cross-sensitivity of P. falciparum existed between artemisinin and mefloquine. Seventy-six patients with falciparum malaria were admitted and treated with artemisinin derivatives. Treatment response parameters were assessed and in vitro drug sensitivity tests were performed with artemisinin, mefloquine, quinine, and chloroquine. Distinct in vitro cross-sensitivity between artemisinin and mefloquine was observed (p = 0.604; P < 0.001). To assess the relevance of this finding for clinical cross-resistance, we used an analytical model based on the relation of in vivo treatment response parameters (fever, parasite and symptom clearance) to a single reference drug with in vitro drug sensitivity data of several other drugs. Artemisinin (R = 0.554; P = 0.009) and mefloquine (R = 0.615; P = 0.002) in vitro drug sensitivities were equally well reflected in the in vivo treatment response to artemisinin, thereby suggesting the clinical relevance of in vitro cross-sensitivity.

Antiparasitics - a review of the recent patent literature.

A total of 45 patents relating to chemical substances with antiparasitic activity appeared during the last quarter of 1999 and the first quarter of 2000. While ten were concerned with specific formulations of registered drugs, nine alluded to the antiparasitc activity in a general way but without substantiating such effect. The majority of the 26 remaining patents reflected the further exploration of chemical groups with known antiparasitic activity. All patents covering antimalarials relate to artemisinin analogs, some of which may offer pharmacodynamic advantages over currently available commercial products. Only two patents concern candidate compounds against human and animal infections by members of the Trypanosomatidae family. In contrast, ectoparasitoses of livestock animals received greater attention with new developments in the area of avermectins, nodulosporins, marcfortines and paraherquamides, with pyrazole analogs obviously reflecting a major veterinary medical interest. As a result of molecular biological research, two new approaches to antiparasitic therapy were identified and two new screening methods were developed for application in antiparasitic and antifungal screening.

Mefloquine concentration profiles during prophylactic dose regimens.

A pharmacokinetic study with (malaria) prophylactic doses of mefloquine hydrochloride was conducted in 12 healthy adult subjects (Caucasians), 6 females and 6 males, mean age 29.2 +/- 6.4 years, mean weight 70.6 +/- 13.4 kg. Doses of 250 mg mefloquine were administered on days 0, 1, 7, 14, 21 and 28. Six subjects received a further 5 weekly doses of 250 mg mefloquine, the others 5 further weekly doses of 125 mg. After the third dose the protective threshold mefloquine concentration in blood plasma was achieved in all subjects. In female subjects, mean Cmin ss, Cmax ss and AUCd 0-35 were significantly higher than in males. After the fifth dose, mean Cmax in females reached 1692 ng/ml (4.48 mumol/l), equivalent to a high therapeutic concentration. This is apparently due to a generally lower body weight and a narrower volume of distribution in women. Adverse reactions were significantly more frequent in women than in men. Headache, anorexia, insomnia and vertigo were the most common side effects. The lesser tolerability of mefloquine in females may be due to the higher drug concentrations in this group. This may indicate the need for appropriate adjustment of the prophylactic dose regimen of mefloquine in females.

In vitro sensitivity of Plasmodium falciparum and clinical response to lumefantrine (benflumetol) and artemether.

AIMS: To assess the sensitivity of 103 Plasmodium falciparum isolates to a combination of lumefantrine (benflumetol) and artemether (CGP 56697), with the objective of determining a correlation between in vitro drug sensitivity and therapeutic outcome. METHODS: Patients suffered from uncomplicated falciparum malaria and came from areas of Thailand affected by multidrug resistance. CGP 56697 was given in the form of tablets containing 20 mg artemether and 120 mg lumefantrine. The standard dose regimen, 4 doses of 4 tablets over 48 h, was compared with two lower dose regimens (4 x 2 tablets and 3 x 4 tablets). RESULTS: The parasites showed high resistance to chloroquine, fairly advanced resistance to mefloquine and compromised sensitivity to quinine. Sensitivity to artemisinin and lumefantrine prior to treatment was similar in all treatment groups. The 4 x 4 tablet regimen was more effective than the other regimens in coping with infections with relatively low sensitivity to artemisinin and/or lumefantrine. The EC90 for artemisinin is an important determinant of treatment success. Parasite density at the start of treatment was identified as another critical predictor of treatment outcome. CONCLUSIONS: The results indicate that parasite exposure to the drugs may have been inadequate and/or too short in the cases of treatment failure, particularly marked in the lower dose regimens. This could probably be remedied by expanding the dose regimen in areas affected by multidrug resistance and in the case of relatively high parasitaemia.