Comparison of bioassay and high performance liquid chromatographic assay of artesunate and dihydroartemisinin in plasma.

The study was a comparison of bioassay and HPLC analysis of artesunate (ARTS) and dihydroartemisinin (DHA) in plasma. ARTS and DHA in plasma samples from patients treated with ARTS were quantified by HPLC and expressed as DHA. DHA-equivalents in the same plasma samples were measured using a standardised parasite culture technique. DHA concentrations estimated by both methods were highly correlated (bioassay=0.96 x HPLC+11.0; r2=0.92). At high concentrations (>12000 nmol/l) bioassay sometimes overestimated DHA. Bioassay of active drug in plasma correlates well with specific chemical analysis by HPLC. ARTS and DHA appear to account for the total antimalarial activity in plasma after ARTS administration.

Phenoxypropoxybiguanides, prodrugs of DHFR-inhibiting diaminotriazine antimalarials.

A total of 34 analogues of the biguanide PS-15 (5s), a prodrug of the diaminotriazine WR-99210 (8s), have been prepared. Several of them, such as 5b (PS-33) and 5m (PS-26), maintain or exceed the in vivo activity of PS-15 while not requiring the use of highly regulated starting materials. The putative diaminotriazine metabolites of these new analogues (compounds 8) have also been prepared and shown to maintain the activity against resistant P. falciparum strains. The structure-activity relationships of biguanides 5 and putative metabolites 8 are discussed.

Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site.

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc(1) (CYT bc(1)) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.

Structure-activity relationship analysis of substituted 4-quinolinamines, antagonists of immunostimulatory CpG-oligodeoxynucleotides.

On the basis of a systematic SAR analysis of substituted quinolines, a derivative 32 was synthesized that shows half-maximal inhibition of the immunostimulatory effect of CpG-oligodeoxynucleotides in vitro at the concentration of 0.24 nM.

In vitro activities of the biguanide PS-15 and its metabolite, WR99210, against cycloguanil-resistant Plasmodium falciparum isolates from Thailand.

The in vitro activities of the new biguanide PS-15 and its putative active metabolite, WR99210, were determined against seven different isolates or clones of Plasmodium falciparum. The mean 50% inhibitory concentrations of PS-15 and WR99210 were 1,015 and 0.06 ng/ml, respectively. WR99210 was up to 363 times more potent than cycloguanil, the active metabolite of proguanil, against cycloguanil-resistant parasites. The pronounced activity of WR99210 against multidrug-resistant P. falciparum indicates that further studies are required to determine the value of the prodrug, PS-15, as an antimalarial agent.

Plasma chloroquine concentrations in young and older malaria patients treated with chloroquine.

Plasma chloroquine (CQ) concentrations were measured by bioassay in young (0-4 years, n = 9) and older (5-60 years, n = 21) patients from Vanuatu infected with malaria following treatment with 25 mg/kg CQ over 3 days. CQ concentrations in young children tended to be lower than in older patients at days 2, 3, 4 and 7 after onset of treatment, with no drug present in two young children on day 3 and in one child on day 7. The greater difficulty experienced by young children to ingest all of their prescribed medication could have contributed to the lower CQ concentrations observed in the younger age group. The possibility that sub-therapeutic CQ concentrations are responsible for treatment failures in young children should be considered in areas where a high degree of CQ resistance has not yet been established. In such areas, the presence or prevalence of CQ-resistant infections should not be based on treatment failures observed in young children unless it can be confirmed that adequate blood CQ concentrations were achieved after treatment.

New quinoline di-Mannich base compounds with greater antimalarial activity than chloroquine, amodiaquine, or pyronaridine.

We have compared the ex vivo antimalarial activity of 12 new quinoline di-Mannich base compounds containing the 7-dichloroquinoline or 7-trifluoromethylquinoline nucleus with amodiaquine, chloroquine, and pyronaridine using the Saimiri-bioassay model. Each compound was administered orally (30 mg/kg of body weight) to three or more noninfected Saimiri sciureus monkeys, and serum samples were collected at various times after drug administration and serially diluted with drug-free (control) serum. In vitro activity against the multidrug-resistant K1 isolate of Plasmodium falciparum was determined in serum samples by measuring the maximum inhibitory dilution at which the treated monkey serum inhibited schizont maturation in vitro. Of the 12 Mannich bases tested, 8 were associated with levels of ex vivo antimalarial activity in serum greater than those of amodiaquine, chloroquine, or pyronaridine 1 to 7 days after drug administration. Further studies were carried out with four of these compounds, and the results showed that the areas under the serum drug concentration-time curves for the four compounds were between 7- and 26-fold greater than that obtained for pyronaridine. Activity against four multidrug-resistant strains of P. falciparum was also much greater in serum samples collected from monkeys after administration of these four compounds than in serum samples collected after administration of pyronaridine or chloroquine. These findings suggest that these four quinoline Mannich base compounds possess a very marked and prolonged antimalarial activity and that further studies should be performed to determine their value as antimalarial drugs.

Chloroquine bioassay of plasma specimens obtained from soldiers on chloroquine plus doxycycline for malaria prophylaxis.

In this study we describe the application of a bioassay for measuring chloroquine equivalent concentrations in plasma samples obtained from soldiers on chloroquine (300 mg weekly) and doxycycline (50 or 100 mg daily) for malaria prophylaxis. Chloroquine, its principal metabolite monodesethylchloroquine and doxycycline were also measured by high performance liquid chromatography (HPLC). Physiological concentrations of doxycycline did not interfere with the measurement of chloroquine equivalent concentrations. The correlation between bioassay and HPLC was rs = 0.88, with a median bioassay/HPLC chloroquine concentration ration of 1.1 (range 0.6-2.4, n = 26). The bioassay is a valuable method, particularly under field conditions, for measuring chloroquine concentrations and can be very helpful in distinguishing drug failures from either poor compliance or inadequate drug absorption.

In vivo-in vitro model to assess chloroquine activity in monkeys.

The bioassay technique and high-performance liquid chromatography (HPLC) method were used to establish chloroquine (CQ) concentration equivalents in serum samples collected from Aotus and Saimiri monkeys after administration of CQ. The results indicate some differences in CQ metabolism between the two simian species. They also indicate a strong positive relationship (rs = 0.96) between data obtained by the bioassay and HPLC technique. The findings suggest that the use of the bioassay in these small primates, using only a fraction of the serum required for HPLC analysis, should provide a useful mean for obtaining preliminary information about the degree and duration of serum antimalarial activity of promising experimental drugs. Apart from reducing the number of monkeys required for drug evaluation, this in vivo-in vitro model should also decrease the overall cost and duration of developing new antimalarial agents.

Chloroquine bioassay using malaria microcultures.

The in vitro microculture technique was used to develop a relatively simple bioassay for estimating chloroquine (CQ) in plasma or serum. Chloroquine concentrations were determined by multiplying the maximum inhibitory dilution of plasma/serum required to inhibit growth of the CQ-sensitive FC27 isolate of Plasmodium falciparum by the minimum inhibitory concentration of CQ against the same isolate. Human serum samples spiked with CQ gave similar measurements using both bioassay and high-performance liquid chromatography. The antimalarial activity of plasma or serum samples collected from 13 patients treated with CQ was equivalent to the sum of the combined activity of CQ and its metabolite, mono-desethylchloroquine. The concentration of these components using the bioassay could be expressed conveniently in terms of CQ concentration equivalents. This bioassay can be used to estimate drug concentrations without the use of sophisticated methods or equipment. Since it is based on the microculture technique, it can be easily carried out in conjunction with the drug susceptibility test to assess CQ treatment failures in malaria patients.

The in vitro and in vivo antimalarial activity of some Mannich bases derived from 4-(7'-trifluoromethyl-1',5'-naphthyridin-4'-ylamino)phenol, 2-(7'-trifluoromethyl-quinolin-4'-ylamino)phenol, and 4'-chloro-5-(7''-trifluoromethylquinolin-4''-ylamino)biphenyl -2-ols.

A series of di-Mannich base derivatives (4 and 5) from 4-(7'-trifluoromethyl-1',5'-naphthyridin-4'-ylamino)phenol and 2-(7'-trifluoromethylquinolin-4'-ylamino)phenol, respectively, and mono-Mannich base derivatives (6) from 4'-chloro-5-(7''-trifluoromethylquinolin-4''- ylamino)biphenyl-2-ol were assayed for activity against the chloroquine-sensitive (FCQ-27) isolate of cultured Plasmodium falciparum using the inhibition of uptake of radiolabelled hypoxanthine. All seven di-Mannich base derivatives (5) revealed a higher activity than chloroquine, whereas the di-Mannich base derivatives (4) were slightly less active (with some derivatives more active and some less active than chloroquine). The mono-Mannich base derivatives (6) were less active than chloroquine. Comparative tests of selected compounds of (4 and 5) using a morphological assay revealed no significant differences in activity between the chloroquine-sensitive (FCQ-27) and chloroquine-resistant (K-1) isolates. Selected di-Mannich bases (4 and 5) and the mono-Mannich bases 5-7''-bromo (and 7-trifluoromethyl)-1'',5''-naphthyridin-4''-ylamino)-3-(t- butylaminomethyl)-4'-chlorobiphenyl-2-ols (7, X = Br, CF3) markedly suppressed parasitaemia in Plasmodium vinckei vinckei infected mice when administered (i.p.) in a single dose of 200 mg kg-1.

An inexpensive and simple method for screening potential antimalarial drugs.

The visual in vitro test proved to be a very useful tool for screening a series of 54 chemical analogues for their antimalarial activity. The test relies on the formation of dark pigment precipitates after an alkaline solution is added to synchronous cultures of Plasmodium falciparum. Pigment is produced when a chemical compound is unable to prevent the maturation of rings to schizonts during incubation. However, in the presence of effective concentrations of a compound, maturation is inhibited and, consequently, pigment and precipitation are not observed. By using this procedure, 36 of the inactive or less active compounds could be excluded from assessment by the radioisotopic hypoxanthine incorporation test. This resulted in a substantial reduction in the overall cost of screening these compounds.

Inhibition of taurocholate and ouabain transport in isolated rat hepatocytes by cyclosporin A.

The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and alpha-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of alpha-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.