Synthesis and antimalarial activity in vitro and in vivo of a new ferrocene-chloroquine analogue

The antimalarial activities of ferrocenic compounds mimicking chloroquine and active upon chloroquine-resistant strains of Plasmodium falciparum were evaluated. Four 7-chloro-4-[[[2-[(N,N-substituted amino)methyl]ferrocenyl]methyl]amino]quinoline derivatives have been synthesized; one of them, 1a, showed high potent antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and was 22 times more potent against schizontocides than chloroquine in vitro against a drug-resistant strain of P. falciparum.     

Antiplasmodial triterpene from Vernonia brasiliana

The hexane extract from leaves of Vernonia brasiliana (L.) Druce (Compositae) was active in vitro against Plasmodium falciparum and in vivo in mice infected with Plasmodium berghei. This extract was subjected to a bioassay-guided fractionation protocol based on the in vitro model. Lupeol was identified as a compound responsible for the activity, inhibiting the P.falciparum growth by 45% when tested at 25 micrograms/ml. However, this triterpene was inactive in vivo when 15 mg/kg were administered per os during four consecutive days to mice infected with P.berghei. beta-Amyrin and germanicol, isolated from the same fraction that yielded lupeol, were inactive in the in vitro assay.     

The immunomodulating effect of a new polyamine (the MAP-1987) administered with chloroquine in plasmodia infected mice

The biological activity of a new synthetic polypeptide, the MAP-1987 was proved in the rodent malaria system. The administration of 4 micrograms/kg of MAP-1987 prevents the haemolysis of the Plasmodium berghei infected erythrocytes but not the Plasmodium vinckei infected ones. The MAP-1987 given alone changes neither the survival time of the infected mice nor the rate of parasitaemia. The chloroquine given alone increases the survival time of the mice infected with P. berghei under the standardized experimental condition but later the animals die with a low rate of parasitaemia. Chloroquine administered together with MAP-1987 definitely cures the P. berghei infected animals. This activity is unique and specific and it does not apply to P. vinckei infection.     

Reversal of chloroquine resistance in murine malaria parasites by prostaglandin derivatives

An oligomeric ester of prostaglandin B2 (OC-5186) was found to reverse chloroquine resistance in the murine malarial parasite Plasmodium berghei. When mice were infected with either chloroquine-sensitive or -resistant P. berghei on day 0 (by intraperitoneal injection of 1 x 10(6) parasitized erythrocytes), they died before day 23. When treated with 15 mg/kg/day of chloroquine for the first four days of infection, all mice infected with the sensitive-strain survived, while all those infected with the resistant strain died before day 23. When OC-5186 (3-12 mg/kg/day) was administered in combination with chloroquine for the first four days, 60% of the animals infected with the resistant strain survived. The differences in the survival rate between the group treated with chloroquine only and the group treated with a combination of drugs (chloroquine plus 3-12 mg/kg/day of OC-5186) were significant. There was also a significant inhibition of parasitemia in the group treated with the combination of drugs. The combinations of chloroquine and a monomer ester of prostaglandin B2 (OC-5181) had some antimalarial activity, but the differences between the chloroquine-treated group and the combination treatment group were not significant in terms of both the parasitemia and the survival rate. Another oligomeric ester of prostaglandin E1 (MR-356) as well as unesterified monomer prostaglandins (PGA2 and PGB2) were ineffective by themselves and in combination with chloroquine.     

Gamma delta T-cell function in pathogenesis of cerebral malaria in mice infected with Plasmodium berghei ANKA

Mice depleted of gammadelta T cells by monoclonal antibody treatment and infected with Plasmodium berghei ANKA did not develop cerebral malaria (CM). In striking contrast, delta0/0 mice infected with P. berghei developed CM despite their gammadelta T-cell deficiency. gammadelta T cells appear to be essential for the pathogenesis of CM in mice having experienced normal ontogeny but not in mice genetically deprived of gammadelta T cells from the beginning of life.     

Bisquinolines. 2. Antimalarial N,N-bis(7-chloroquinolin-4-yl)heteroalkanediamines

N,N-Bis(7-chloroquinolin-4-yl)heteroalkanediamines 1-11 were synthesized and screened against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. These bisquinolines had IC50 values from 1 to 100 nM against P. falciparum in vitro. Six of the 11 bisquinolines were significantly more potent against the chloroquine-resistant W2 clone compared to the chloroquine-sensitive D6 clone. For bisquinolines 1-11 there was no relationship between the length of the bisquinoline heteroalkane bridge and antimalarial activity and no correlation between in vitro and in vivo antimalarial activities. Bisquinolines with alkyl ether and piperazine bridges were substantially more effective than bisquinolines with alkylamine bridges against P. berghei in vivo. Bisquinolines 1-10 were potent inhibitors of hematin polymerization with IC50 values falling in the narrow range of 5-20 microM, and there was a correlation between potency of inhibition of hematin polymerization and inhibition of parasite growth. Compared to alkane-bridged bisquinolines (Vennerstrom et al., 1992), none of these heteroalkane-bridged bisquinolines had sufficient antimalarial activity to warrant further investigation of the series.     

Antimalarial activity of novel arylene bis(methylketone) compounds

Because of the spread of drug-resistant Plasmodium species, there is an urgent need for novel effective antimalarial agents. A series of arylene bis(methylketone) compounds were screened in vitro against a number of Plasmodium falciparum clones and in vivo against Plasmodium berghei. 2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (Cytokine Network Inc. [CNI]-H0294) was the most effective of the compounds in vitro, with an IC50 of 1.5-4.0 microM against parasite clones with a wide range of sensitivities to chloroquine and pyrimethamine. Other compounds in the series had in vitro IC50 values of 20-25 microM. In a 4-day test for suppression of P. berghei parasitemia in vivo, 50 mg/kg/day CNI-H0294 significantly decreased parasitemia by >90%. The compound was found to have low toxicity in mice, with an LD50 of 590 +/- 66 mg/kg intraperitoneally, and rapid plasma kinetics. These results show that CNI-H0294 has considerable antimalarial activity and merits further study.     

Synthesis and antiprotozoal activities of some new triazine derivatives including a new antitrypanosomal agent: SIPI-1029

Two series of compounds, 1,2-dihydro-2,2-dimethyl-4, 6-diamino-1-(omega-haloalkyloxy)-s-triazines and O, O'-bis (4, 6-diamino-1, 2-dihydro-2, 2-disubstituted-s-triazin-l-yl) alkanediols were synthesized and tested against Plasmodium berghei and Trypanosoma evansi in mice. Most title compounds showed good antimalarial activity and compounds IIc-e showed good antitrypanosomal effect. After further studies on pharmacology, toxicology, pharmacokinetics and efficacy on infected cattles compound IIe (SIPI-1029, T-46) was shown to be a new highly active antitrypanosomal agent with low toxicity and long half life in plasma.     

A pathogenic role of IL-12 in blood-stage murine malaria lethal strain Plasmodium berghei NK65 infection

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-gamma, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-alpha, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-gamma. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-gamma in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-gamma production rather than the protection.     

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.     

Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum

A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions. 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices. A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods. Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position. In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM). Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme. The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM. It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above. In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM. Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay. The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P. falciparum in vitro (IC50 20 nM) and P. falciparum-derived topoisomerase II. However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.     

Effects of Plasmodium berghei infection on arteether metabolism and disposition

Arteether (AE) is primarily deethylated to dihydroqinghaosu (DQHS) in rats and humans. Conversion of AE to DQHS was impaired in microsomes from rats infected with Plasmodium berghei. The Km for AE was 175.1 +/- 49.1 and 124.4 +/- 115.1 mumol/l, and Vmax was 2.24 +/- 0.45 and 1.22 +/- 0.67 nmol AE formed/mg protein/min in control and infected microsomes (p < 0.05), respectively. Calculated intrinsic clearance (CLint = initial Vmax/Km) for AE was only 4% lower in infected microsomes. Apparent pharmacokinetic parameter estimates for AE using the isolated perfused rat liver demonstrated no differences (p > 0.05) in volume of distribution, clearance, and half-life between normal and infected animals. Malaria infection resulted in decreased biliary excretion of free AE and DQHS. The majority of AE is eliminated via biliary excretion of conjugated DQHS, which is approximately 500-fold higher than free DQHS and 75-fold higher than free AE on a molar basis.     

Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the full-length 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.     

Chloroquine-resistant Plasmodium vivax in Papua New Guinea

An Australian expatriate on regular weekly antimalarial prophylaxis with chloroquine base and Maloprim developed symptomatic Plasmodium vivax infection which failed to respond adequately to 600 mg of chloroquine base. More ominously, a resident of the Highlands region of Papua New Guinea contracted vivax malaria which failed to be cleared by 2400 mg chloroquine base administered over 4 d. Both patients had achieved appropriate blood and plasma concentrations of chloroquine after treatment. Chloroquine-resistant P. vivax is now a clinical fact in Papua New Guinea.     

Orally active, hydrolytically stable, semisynthetic, antimalarial trioxanes in the artemisinin family

In only three chemical operations, natural trioxane lactone artemisinin (1) was converted into a series of C-10 carbon-substituted 10-deoxoartemisinin compounds 4-9. The three steps involved lactone reduction, replacement of the anomeric lactol OH by F using diethylaminosulfur trifluoride, and finally boron trifluoride-promoted substitution of F by aryl, heteroaryl, and acetylide nucleophiles. All of these C-10 nonacetal, chemically robust, enantiomerically pure compounds 4-9 have high antimalarial potencies in vitro against Plasmodium falciparum malaria parasites, and furans 5a and 5b and pyrrole 7a are antimalarially potent also in vivo even when administered to rodents orally.     

Acute Plasmodium chabaudi chabaudi malaria infection induces antibodies which bind to the surfaces of parasitized erythrocytes and promote their phagocytosis by macrophages in vitro

CBA/Ca mice infected with 5 x 10(4) Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks ( approximately 40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P. c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P. c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P. c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P. c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P. c. chabaudi CB) did not mediate the same effect against P. c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.     

Effect of tryptophan-N-formylated gramicidin on growth of Plasmodium berghei in mice

The effect of tryptophan-N-formylated gramicidin (NFG) on the growth of Plasmodium berghei in mice was tested in three different experiments. NFG was shown to be capable of inhibiting the growth of the parasite in a dose-dependent way, although its action did not result in elimination of the parasite and was only temporary, preventing mice from early death, presumably due to cerebral malaria, but not from fatal generalized malaria. Intriguingly, a similar observation was made with two other drugs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, an inhibitor of viral and eukaryotic DNA polymerases, and the presumed topoisomerase II inhibitor, a bisquaternary quinolinium salt. A rise in the level of parasitemia after 8 days, despite continued treatment, was not due to parasite-induced reticulocytosis, as demonstrated in experiments in which this condition was induced artificially. NFG was added in the form of lipid vesicles in which the peptide had been incorporated. The inhibitory action of NFG was not modulated by the lipid composition of the vesicles. Control experiments did not demonstrate any toxicity of NFG when it was administered in lipid vesicles. The main observation is that NFG is able to inhibit the growth of a malaria parasite in vivo at concentrations that are well tolerated by the host.     

Inhibition of nitric oxide interrupts the accumulation of CD8+ T cells surrounding Plasmodium berghei-infected hepatocytes

The elimination of liver-stage malaria parasites by nitric oxide (NO)-producing hepatocytes is regulated by T cells. Both CD8+ and CD4+ T cells, which surround infected hepatocytes, are evident by 24 h after sporozoite challenge in Brown Norway rats previously immunized with irradiated Plasmodium berghei sporozoites. While the number of CD4+ T cells remained the same beyond 24 h postchallenge, the number of CD8+ T cells increased three- and sixfold by 31 and 44 h, respectively. This increase in the number of CD8+ T cells correlated with a decrease in the number of intrahepatic parasites. In immunized rats, intrahepatic parasites were reduced in number by 31 h after sporozoite challenge and cleared from the liver by 44 h, as visualized by P. berghei-specific DNA in situ hybridization. If immunized rats were treated with aminoguanidine, a substrate inhibitor of NO synthase, at the time of challenge, liver-stage protection was blocked, as shown by the increase in parasite liver burden. Further histological examination of infected livers from immunized animals treated with aminoguanidine revealed fewer and smaller cellular infiltrates surrounding the infected hepatocytes, and the number of CD8+ T cells that normally accumulate within the infiltrates was drastically reduced. Consequently, the infected hepatocytes were not cleared from the liver. We hypothesize that the early production of NO may promote the influx and/or enhance local proliferation of malaria parasite-specific CD8+ T cells or a CD8+ T-cell subset which is required for parasite clearance.     

Plasmodium falciparum and Plasmodium yoelii: effect of the iron chelation prodrug dexrazoxane on in vitro cultures

To determine if an iron-chelating prodrug that must undergo intracellular hydrolysis to bind iron has antimalarial activity, we examined the action of dexrazoxane on Plasmodium falciparum cultured in human erythrocytes and P. yoelii cultured in mouse hepatocytes. Dexrazoxane was recently approved to protect humans from doxorubucin-induced cardiotoxicity. Using the fluorescent marker calcein, we confirmed that the iron-chelating properties of dexrazoxane are directly related to its ability to undergo hydrolysis. As a single agent, dexrazoxane inhibited synchronized cultures of P. falciparum in human erythrocytes only at suprapharmacologic concentrations (> 200 microM). In combination with desferrioxamine B, dexrazoxane in pharmacologic concentrations (100-200 microM) moderately potentiated inhibition by approximately 20%. In contrast, pharmacologic concentrations of dexrazoxane (50-200 microM) as a single agent inhibited the progression of P. yoelli from sporozoites to schizonts in cultured mouse hepatocytes by 45 to 69% (P < 0.001). These results are consistent with the presence of a dexrazoxane-hydrolyzing enzyme in hepatocytes but not in erythrocytes or malaria parasites. Furthermore, these findings suggest that dexrazoxane must be hydrolyzed to an iron-chelating intermediate before it can inhibit the malaria parasite, and they raise the possibility that the iron chelator prodrug concept might be exploited to synthesize new antimalarial agents.