Inhibition of the in vitro growth of Plasmodium falciparum by acyclic nucleoside phosphonates.

Forty-eight acyclic nucleoside phosphonates (putative prodrugs of acyclic nucleoside triphosphate inhibitors of DNA replication) have been evaluated for in vitro antiplasmodial activity. Only certain purine derivatives with a hydroxyl group attached to the acyclic sugar moiety displayed antiplasmodial activity. The two most active analogs were (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine ((S)-HPMPA, IC50=0.18+/-0.07 microM) and (S)-3-deaza-HPMPA (IC50=0.29+/-0.08 microM). Their cyclic derivatives, containing an ester bond between the phosphonate and the hydroxyl group, were slightly less active. All tested compounds that lacked the hydroxyl group, including potent antiretrovirus analogs such as 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and the (S)-HPMPA derivatives (R)-PMPA and (S)-FPMPA, did not show any activity, even at very high concentrations ( >250 microM). Similarly, pyrimidine analogs of (S)-HPMPA, such as (S)-HPMPT, (S)-HPMPU and the anti-herpesvirus analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine ((S)-HPMPC), were devoid of any antiplasmodial activity. In addition, 11 acyclic nucleoside (non-phosphorylated) analogs--which in contrast to the acyclic nucleoside phosphonates require the presence of a monophosphorylating enzyme for the first activation step--were tested. None of them inhibited the growth of the parasite. In short three chemical entities seem to be imperative for antiplasmodial activity: a purine base, a hydroxyl group in the acyclic side chain and a phosphonate group terminating this chain.

Disposition of the acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine.

The acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] has been shown to be active against pathogens, like hepatitis B viruses and Plasmodium parasites, that infect parenchymal liver cells. (S)-HPMPA is therefore an interesting candidate drug for the treatment of these infections. To establish effective therapeutic protocols for (S)-HPMPA, it is essential that the kinetics of its hepatic uptake be evaluated and that the role of the various liver cell types be examined. In the present study, we investigated the disposition of (S)-HPMPA and assessed its hepatic uptake. Rats were intravenously injected with [3H](S)-HPMPA, and after an initial rapid distribution phase (360 +/- 53 ml/kg of body weight), the radioactivity was cleared from the circulation with a half-life of 11.7 +/- 1.4 min. The tissue distribution of [3H](S)-HPMPA was determined at 90 min after injection (when >99% of the dose cleared). Most (57.0% +/- 1.1%) of the injected [3H](S)-HPMPA was excreted unchanged in the urine. The radioactivity that was retained in the body was almost completely recovered in the kidneys and the liver (68.4% +/- 2.5% and 16.1% +/- 0.4% of the radioactivity in the body, respectively). The uptake of [3H](S)-HPMPA by the liver occurred mainly by parenchymal cells (92.1% +/- 3.4% of total uptake by the liver). Kupffer cells and endothelial cells accounted for only 6.1% +/- 3.5% and 1.8% +/- 0.8% of the total uptake by the liver, respectively. Preinjection with probenecid reduced the hepatic and renal uptake of [3H](S)-HPMPA by approximately 75%, which points to a major role of a probenecid-sensitive transporter in the uptake of (S)-HPMPA by both tissues. In conclusion, we show that inside the liver, (S)-HPMPA is mainly taken up by parenchymal liver cells. However, the level of uptake by the kidneys is much higher, which leads to nephrotoxicity. An approach in which (S)-HPMPA is coupled to carriers that are specifically taken up by parenchymal cells may increase the effectiveness of the drug in the liver and reduce its renal toxicity.

In vivo and in vitro antiplasmodial activities of some plants traditionally used in Guatemala against malaria.

We present an evaluation of the antiplasmodial and cytotoxic effects of four plants commonly used in Guatemalan folk medicine against malaria. Methanol extracts of Simarouba glauca D. C., Sansevieria guineensis Willd, Croton guatemalensis Lotsy, and Neurolaena lobata (L.)R.Br. significantly reduced parasitemias in Plasmodium berghei-infected mice. Dichloromethane fractions were screened for their cytotoxicities on Artemia salina (brine shrimp) larvae, and 50% inhibitory concentrations were determined for Plasmodium falciparum in in vitro cultures. Both chloroquine-susceptible and -resistant strains of P. falciparum were significantly inhibited by these extracts. Of all dichloromethane extracts, only the S. glauca cortex extract was considered to be toxic to nauplii of A. salina in the brine shrimp test.

Antimalarial and toxic effects of the acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine in Plasmodium berghei-infected mice.

Plasmodium berghei-infected mice died with low levels of parasitemia after repeated intraperitoneal administration (five times at 15 mg kg of body weight-1 every other day) of the in vitro active antimalarial acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]. Toxicological studies showed that the main cause of death resulted from (S)-HPMPA-induced nephrotoxicity. Although concomitant intraperitoneal administration of the tubular epithelium transport blocker probenecid prevented (S)-HPMPA-induced toxicity, mice eventually died with a high level of parasitemia, despite repeated administration of high doses of (S)-HPMPA. The short half-life of (S)-HPMPA in plasma combined with the insusceptibility of the nonreplicative stages of the parasite to (S)-HPMPA could explain this failure to eradicate all parasites. Indeed, a low but sustained (calculated) level of 200 nM (S)-HPMPA in plasma completely cured P. berghei-infected mice. However, these mice, which received a total dose of only 28 mg kg-1 administered via osmotic pumps for 7 days, died because of the toxicity of the drug. These findings indicate that nephrotoxicity hinders the use of (S)-HPMPA as a drug against blood stage parasites. An alternative application of (S)-HPMPA as a potent prophylactic drug is discussed.

Simple, fast, and accurate fluorometric method to determine drug susceptibility of Plasmodium falciparum in 24-well suspension cultures.

An in vitro test which quantifies drug inhibition of Plasmodium falciparum replication by measuring the fluorescence intensity of Hoechst 33258 dye bound to DNA is described. The procedure does not require expensive reagents or equipment and can be completed in less than 10 min. The assay was highly accurate and sensitive: cultures with as few as 0.4% schizont-infected erythrocytes could reliably be analyzed. The method was not biased by the actual parasite stage used; i.e., the amount of fluorescence detected in a sample of a culture of mature schizonts equaled the amount detected with the ring form culture derived from these schizonts. Even the presence of large proportions of free merozoites, which are easily neglected in microscopic estimates, did not bias the results. Furthermore, measurement of the chloroquine susceptibility of the multidrug-resistant K1 strain and the chloroquine-susceptible NF54 strain showed that the method is most suitable for quantifying the drug resistance of P. falciparum.

The effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine on nuclear and organellar DNA synthesis in erythrocytic schizogony in malaria.

The very effective (ID50 = 47 nM) and selective antimalarial compound (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) abruptly arrests Plasmodium falciparum-cultured schizonts at concentrations between 1 and 10 x ID50 as soon as their DNA content reaches 8 times that of the haploid ringform stage. Even very high HPMPA concentrations do not inhibit the first 2-3 rounds of schizogonic DNA replication. Also, in the presence of HPMPA, replication of the 6-kb mitochondrial and 35-kb chloroplast-like DNA proceeds normally and in close concert with each other, both to a 16-fold amount within 5 h during the trophozoite stage. Hence the in in vitro assays HPMPApp-sensitive plasmodial DNA polymerase gamma-like enzyme (IC50 = 1 microM)--assumed to be involved in mitochondrial DNA replication--is not the target of HPMPA in vivo (living parasites), nor seems to be the DNA polymerization activities of the--in vitro also HPMPA-sensitive (IC50 = 38 microM)--DNA polymerase alpha or of any other nuclear DNA polymerase of Plasmodium. In vitro assays demonstrated that HPMPApp does not act as an alternative substrate for plasmodial polymerases, contradicting the suggestion that the observed delayed inhibition of plasmodial schizogony might be the result of DNA strand breakage caused by HPMPApp incorporation. Neither do results support the idea that the HPMPA-induced arrest of DNA replication might be due to chain termination as a result of such incorporation. We investigated whether arrest of DNA replication by HPMPA in schizonts could be explained by inhibition of the DNA synthesis rate limiting ribonucleotide reductase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)