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The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Introduction Malaria is a major health concern and affects over 300 million people a year. Accordingly, there is an urgent need for new efficacious anti-malarial drugs. A major challenge in developing new anti-malarial drugs is to design active molecules that have preferable drug-like characteristics. These “drug-like” characteristics include physiochemical properties that affect drug absorption, distribution, metabolism, and excretion (ADME). Compounds with poor ADME profiles will likely fail in vivo due to poor pharmacokinetics and/or other drug delivery related issues. There have been numerous assays developed in order to pre-screen compounds that would likely fail in further development due to poor absorption properties including PAMPA, Caco-2, and MDCK permeability assays. Methods The use of cell-based permeability assays such as Caco-2 and MDCK serve as surrogate indicators of drug absorption and transport, with the two approaches often used interchangeably. We sought to evaluate both approaches in support of anti-malarial drug development. Accordingly, a comparison of both assays was conducted utilizing apparent permeability coefficient (Papp) values determined from liquid chromatography/tandem mass spectrometry (LC-MS) analyses. Results Both Caco-2 and MDCK permeability assays produced similar Papp results for potential anti-malarial compounds with low and medium permeability. Differences were observed for compounds with high permeability and compounds that were P-gp substrates. Additionally, the utility of MDCK-MDR1 permeability measurements was demonstrated in probing the role of P-glycoprotein transport in Primaquine–Chloroquine drug–drug interactions in comparison with in vivo pharmacokinetic changes. Discussion This study provides an in-depth comparison of the Caco-2 and MDCK-MDR1 cell based permeability assays and illustrates the utility of cell-based permeability assays in anti-malarial drug screening/development in regard to understanding transporter mediated changes in drug absorption/distribution.

A high-performance liquid chromatographic method was developed to quantitate the plasma concentrations of the individual enantiomers of a candidate 8-aminoquinoline antimalarial agent WR 238,605 (I). The method employed one-step liquid extraction of a 0.5-ml plasma sample followed by direct injection of the extract through a chiral column and detection by fluorescence. Quantification was achieved using an internal standard. The limit of quantification was 10 ng/ml for each enantiomer. The method is sufficiently sensitive to quantitate the plasma concentrations of both enantiomers for 30 days following a single oral dose of 400 mg of the antimalarial agent administered as the racemic succinate salt to healthy human male volunteers. In nearly all samples taken 12 h to 30 days post-dose from three subjects, the difference in the plasma concentrations of the two enantiomers is less than 10%.

The 8-aminoquinoline drug primaquine (PQ) is currently the only drug in use against the persistent malaria caused by the hypnozoite-forming strains P. vivax and P. ovale. However, despite decades of research, its complete metabolic profile is still poorly understood. In the present study, the metabolism of PQ was evaluated by incubating the drug with pooled human hepatocytes cultured in vitro as well as with recombinant cytochrome P450 (CYP) iso- enzymes, monoamine oxidases (MAO), and flavin-containing monooxygenases (FMO). Targeted LC-MS/MS analysis of hepatocyte incubations using chemical inhibitors indicated that PQ was predominantly metabolized by CYPs 3A4, 1A2 and 2D6, MAO-A, -B and FMO-3. Confirmation of these results was sought by incubation of PQ with the corresponding recombinant enzymes. Small amounts of carboxyprimaquine (CPQ), the major observed PQ metabolite in vivo, were detected in recombinant MAO-A incubations along with another peak at m/z 261, and no significant formation of CPQ with any other recombinant enzymes was observed. Incubations with all recombinant enzymes identified as potentially active towards PQ from the hepatocyte-based assay resulted in significant parent loss over the course of 1 h. These results suggest that several enzymes, including CYPs in combination with FMOs and MAOs, play a role in the overall metabolism of PQ and indicate a major role for MAO-A. Future studies to elucidate the potential role in cytotoxicity and/or efficacy of the PQ metabolite observed at m/z 261, as observed in MAO-A isoenzyme studies, are needed.

In the presence of amine-containing sulfhydryl compounds, binding of heat-transformed cytosolic rat liver glucocorticoid receptor complex (GRC) to double-stranded calf thymus DNA-coated cellulose and to rat liver chromatin was enhanced up to 10-fold. These observations were made under conditions when a maximum of 8% of the total GRC bound to DNA in the absence of test compound. Compounds which did not contain both a sulfhydryl and amine group were inactive. Phosphorothioate derivatives of the active sulfhydryl compounds were also inactive. However, pretreatment of the phosphorothioate compounds with alkaline phosphatase restored activity. Upon centrifugation at 8800g, amine-containing disulfide compounds at millimolar concentrations caused considerable sedimentation of the GRC in the absence of DNA-coated cellulose or chromatin and no apparent increase in GRC binding to DNA or chromatin. Amine-containing disulfide compounds at micromolar concentrations did not cause heavy sedimentation of the GRC and enhanced binding of the GRC to DNA-coated cellulose up to 9.5-fold. Thus, diaminosulfhydryl compounds and the disulfide 1,18-diamino-6,13-diaza-9,10-dithiaoctadecane (WR 149,024) possess both the ability to restore and preserve the steroid binding capacity of the glucocorticoid receptor and to enhance binding of the GRC to DNA and chromatin.

A rapid, sensitive and simple method was developed for the quantitation of the plasma concentration of N 4 -[2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxy] -8-quinolinyl]-1,4-pentanediamine, a new antimalarial active against Plasmodium vivax . N 4 -(5-Hexoxy-6-methoxy-4-methyl-8-quinolinyl)-1,4-pentanediamine diphosphate, a similar 8-aminoquinoline, was used as an internal standard. The method involves sample clean-up by a prepacked cyano solid-phase column followed by reversed-phase liquid chromatography and oxidative electrochemical detection at +0.95 V. The assay has been validated to 5 ng/ml of plasma and is sensitive to 1 ng/ml of plasma. The results of a pilot study assessing the relative oral bioavailability of two different salt forms of the new antimalarial in dogs show the usefulness of the method for animal and human pharmacokinetic studies.

The purpose of this work was to examine whether the ability of dithiothreitol to preserve the steroid-binding capacity of glucocorticoid receptors in subcellular preparations is specific or a general property of sulfhydryl compounds and selected phosphorothioate and disulfide derivatives. A further goal was to see if this effect could be demonstrated in intact cells. The ability to preserve the steroid-binding capacity of the glucocorticoid receptor is not a universal property of all sulfhydryl compounds since many of the compounds tested were inactive. The steroid-binding capacity of the glucocorticoid receptor of the 100,000 g supernatant of rat liver homogenate is preserved/restored by sulfhydryl compounds containing a mercaptoethylamine or mercaptopropylamine subunit. However, small changes in the structure of the sulfhydryl compound such as the rearrangement of a methylene group significantly alter its effectiveness. All of the phosphorothioates examined are derivatives of active sulfhydryl compounds and are effective in preserving steroidbinding. The extent of metabolism of the phosphorothioates and their failure to restore steroidbinding capacity after short-time exposure to receptor preparations are consistent with the sulfhydryl form being the active form of the phosphorothioates. S -2-(3-Amino-propylamino)ethylphosphorothioic acid (WR 2721) preserved steroid-binding capacity in isolated intact rat hepatocytes down to 25 μM demonstrating that concentrations obtainable in whole animals are effective with intact cells. Disulfide derivatives of active sulfhydryl compounds are either immediately toxic or ineffective except for 1,18-diamino-6,13-diaza-9,10-dithiaoctadecane (WR 149,024) which is more effective than its corresponding sulfhydryl. The demonstration that some sulfhydryl-forming compounds preserve the steroid-binding capacity of glucocorticoid receptors in intact cells at potentially physiologically obtainable concentrations suggests a potential role for these or similar compounds to bolster the efficacy of conventional glucocorticoid therapy.