Activity of piperaquine and other 4-aminoquinoline antiplasmodial drugs against chloroquine-sensitive and resistant blood-stages of Plasmodium falciparum. Role of beta-haematin inhibition and drug concentration in vacuolar water- and lipid-phases.

Chloroquine (CQ), a 4-aminoquinoline, accumulates in acidic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to beta-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and 4 and 5, representing halves of PQ, beta-haematin inhibitory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured pK(a)s and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH 7.4) into vacuolar water (pH 4.8) and lipid accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while 4 and 5 were 100 times less potent. CQ with two basic centres has a VAR of 143,482, while 4 and 5, with two basic centres of lower pK(a)s have VARs of 1287 and 1966. In contrast PQ and OH-PQ have four basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R. 5, PQ and OH-PQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to beta-haematin inhibition in vacuolar lipid.

Effects of piperaquine, chloroquine, and amodiaquine on drug uptake and of these in combination with dihydroartemisinin against drug-sensitive and -resistant Plasmodium falciparum strains.

Piperaquine is being developed as a long-acting component in artemisinin combination therapies. It was highly active in vitro and drug interaction studies showed that dihydroartemisinin combinations with piperaquine, chloroquine, and amodiaquine were indifferent tending toward antagonism. Competitive uptake of radiolabeled chloroquine and dihydroartemisinin in combination with other antimalarials was observed.

Identification of isoflavone derivatives as effective anticryptosporidial agents in vitro and in vivo.

We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein analogues (2-H isoflavones), 2-carboethoxy isoflavones, and the precursor deoxybenzoins were all evaluated. Excellent in vitro activity against Cryptosporidium parvum was observed for both classes of isoflavones in cell cultures, and the lead compound 19, RM6427, shows high in vivo efficacy against an experimental infection.

Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum.

A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed.

The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids.

BACKGROUND: The 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90 degrees difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown. METHODS: Ionization constants, octanol/water distribution and haematin interaction are examined for eight alkaloids to explain the influence of small structural differences on activity. RESULTS: Erythro isomers have a high distribution ratio of 55:1 from plasma to the erythrocyte membrane, while for the more basic threo epimers this is only 4.5:1. This gives an increased transfer rate of the erythro drugs into the erythrocyte and thence into the parasite vacuole where their favourable conformation allows interaction with haematin, inhibiting its dimerization strongly (90 +/- 7%) and thereby killing the parasite. The threo compounds not only enter more slowly but are then severely restricted from binding to haematin by the gauche alignment of their N1-H1 and C9-O12 bonds. Confirmatory molecular models allowed measurement of angles and bond lengths and computation of the electronic spectrum of a quinine-haematin complex. CONCLUSION: Differences in the antiplasmodial activity of the erythro and threo cinchona alkaloids may therefore be attributed to the cumulative effects of lipid/aqueous distribution ratio and drug-haematin interaction. Possible insights into the mechanism of chloroquine-resistance are discussed.

Hydroxychloroquine is much less active than chloroquine against chloroquine-resistant Plasmodium falciparum, in agreement with its physicochemical properties.

The 4-aminoquinoline drug hydroxychloroquine (HCQ) is reported to be as active as chloroquine (CQ) against falciparum malaria, and less toxic. Existing prophylactic regimens for areas where there is CQ-resistant malaria recommend CQ with proguanil as an alternative where none of the three preferred regimens (atovaquone-proguanil, doxycycline or mefloquine) is thought suitable. In such cases, toxicity is likely when CQ-proguanil is administered to persons being treated for autoimmune disease with daily HCQ. The question therefore arises whether in such circumstances HCQ could effectively replace the CQ component of the prophylactic combination. We confirmed similar activity of CQ and HCQ against CQ-sensitive Plasmodium falciparum, but found that whereas HCQ in vitro was 1.6 times less active than CQ in a CQ-sensitive isolate, it was 8.8 times less active in a CQ-resistant isolate. The result can also be predicted from an analysis of the physicochemical properties of CQ and HCQ. To give limited protective effect similar to 300 mg CQ base weekly against CQ-resistant P. falciparum would demand daily doses of HCQ above the recommended safe level. These observations contraindicate the use of HCQ in prophylaxis or treatment of CQ-resistant falciparum malaria. Where CQ-proguanil prophylaxis is the only option available in a patient on high-dose HCQ treatment, visiting a CQ-resistant area, replacement of the anti-inflammatory regimen by a daily CQ course at a suitable dose should be considered.

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria.

We report the first in vitro and genetic confirmation of Malarone (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone. Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.