Integrating Pharmacokinetic-Pharmacodynamic Modeling and Physiologically Based Pharmacokinetic Modeling to Optimize Human Dose Predictions for Plasmodium falciparum Malaria: a Chloroquine Case Study.

The translation of a preclinical antimalarial drug development candidate to the clinical phases should be supported by rational human dose selection. A model-informed strategy based on preclinical data, which incorporates pharmacokinetic-pharmacodynamic (PK-PD) properties with physiologically based pharmacokinetic (PBPK) modeling, is proposed to optimally predict an efficacious human dose and dosage regimen for the treatment of Plasmodium falciparum malaria. The viability of this approach was explored using chloroquine, which has an extensive clinical history for malaria treatment. First, the PK-PD parameters and the PK-PD driver of efficacy for chloroquine were determined through a dose fractionation study in the P. falciparum-infected humanized mouse model. A PBPK model for chloroquine was then developed for predicting the drug's PK profiles in a human population, from which the human PK parameters were determined. Lastly, the PK-PD parameters estimated in the P. falciparum-infected mouse model and the human PK parameters derived from the PBPK model were integrated to simulate the human dose-response relationships against P. falciparum, which subsequently allowed the determination of an optimized treatment. The predicted efficacious human dose and dosage regimen for chloroquine were comparable to those recommended clinically for the treatment of uncomplicated, drug-sensitive malaria, which provided supportive evidence for the proposed model-based approach to antimalarial human dose predictions.

Spiropyrimidinetriones: a Class of DNA Gyrase Inhibitors with Activity against Mycobacterium tuberculosis and without Cross-Resistance to Fluoroquinolones.

Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg2+-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed in vitro cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a recA reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.

Toward New Transmission-Blocking Combination Therapies: Pharmacokinetics of 10-Amino-Artemisinins and 11-Aza-Artemisinin and Comparison with Dihydroartemisinin and Artemether.

As artemisinin combination therapies (ACTs) are compromised by resistance, we are evaluating triple combination therapies (TACTs) comprising an amino-artemisinin, a redox drug, and a third drug with a different mode of action. Thus, here we briefly review efficacy data on artemisone, artemiside, other amino-artemisinins, and 11-aza-artemisinin and conduct absorption, distribution, and metabolism and excretion (ADME) profiling in vitro and pharmacokinetic (PK) profiling in vivo via intravenous (i.v.) and oral (p.o.) administration to mice. The sulfamide derivative has a notably long murine microsomal half-life (t1/2 > 150 min), low intrinsic liver clearance and total plasma clearance rates (CLint 189.4, CLtot 32.2 ml/min/kg), and high relative bioavailability (F = 59%). Kinetics are somewhat similar for 11-aza-artemisinin (t1/2 > 150 min, CLint = 576.9, CLtot = 75.0 ml/min/kg), although bioavailability is lower (F = 14%). In contrast, artemether is rapidly metabolized to dihydroartemisinin (DHA) (t1/2 = 17.4 min) and eliminated (CLint = 855.0, CLtot = 119.7 ml/min/kg) and has low oral bioavailability (F) of 2%. While artemisone displays low t1/2 of <10 min and high CLint of 302.1, it displays a low CLtot of 42.3 ml/min/kg and moderate bioavailability (F) of 32%. Its active metabolite M1 displays a much-improved t1/2 of >150 min and a reduced CLint of 37.4 ml/min/kg. Artemiside has t1/2 of 12.4 min, CLint of 673.9, and CLtot of 129.7 ml/kg/min, likely a reflection of its surprisingly rapid metabolism to artemisone, reported here for the first time. DHA is not formed from any amino-artemisinin. Overall, the efficacy and PK data strongly support the development of selected amino-artemisinins as components of new TACTs.

Investigating Sulfoxide-to-Sulfone Conversion as a Prodrug Strategy for a Phosphatidylinositol 4-Kinase Inhibitor in a Humanized Mouse Model of Malaria.

The in vivo antimalarial efficacies of two phosphatidylinositol 4-kinase (PI4K) inhibitors, a 3,5-diaryl-2-aminopyrazine sulfoxide and its corresponding sulfone metabolite, were evaluated in the NOD-scid IL2Rγnull (NSG) murine malaria disease model of Plasmodium falciparum infection. We hypothesized that the sulfoxide would serve as a more soluble prodrug for the sulfone, which would lead to improved drug exposure with oral dosing. Both compounds had similar efficacy (90% effective dose [ED90], 0.1 mg kg-1 of body weight) across a quadruple-dose regimen. Pharmacokinetic profiling revealed rapid sulfoxide clearance via conversion to sulfone, with sulfone identified as the major active metabolite. When the sulfoxide was dosed, the exposure of the sulfone achieved was as much as 2.9-fold higher than when the sulfone was directly dosed, thereby demonstrating that the sulfoxide served as an effective prodrug for the treatment of malaria.

Artemisone and Artemiside Are Potent Panreactive Antimalarial Agents That Also Synergize Redox Imbalance in Plasmodium falciparum Transmissible Gametocyte Stages.

The emergence of resistance toward artemisinin combination therapies (ACTs) by the malaria parasite Plasmodium falciparum has the potential to severely compromise malaria control. Therefore, the development of new artemisinins in combination with new drugs that impart activities toward both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular, those of resistant parasites, is urgently required. We define artemisinins as oxidant drugs through their ability to oxidize reduced flavin cofactors of flavin disulfide reductases critical for maintaining redox homeostasis in the malaria parasite. Here we compare the activities of 10-amino artemisinin derivatives toward the asexual and gametocyte stages of P. falciparum parasites. Of these, artemisone and artemiside inhibited asexual and gametocyte stages, particularly stage V gametocytes, in the low-nanomolar range. Further, treatment of both early and late gametocyte stages with artemisone or artemiside combined with the pro-oxidant redox partner methylene blue displayed notable synergism. These data suggest that modulation of redox homeostasis is likely an important druggable process, particularly in gametocytes, and this finding thereby enhances the prospect of using combinations of oxidant and redox drugs for malaria control.

In Vitro and In Vivo Pharmacokinetics of Aminoalkylated Diarylpropanes NP085 and NP102.

Malaria remains a great burden on humanity. Although significant advances have been made in the prevention and treatment of malaria, malaria control is now hindered by an increasing tolerance of the parasite to one or more drugs within artemisinin combination therapies; therefore, an urgent need exists for development of novel and improved therapies. The University of the Free State Chemistry Department previously synthesized an antimalarial compound, NP046. In vitro studies illustrated an enhanced efficacy against Plasmodium falciparum However, NP046 showed low bioavailability. Efforts to enhance the bioavailability of NP046 have resulted in the synthesis of a number of aminoalkylated diarylpropanes, including NP085 and NP102. Pharmacokinetic studies were conducted in C57BL/6 mice, with 15 mg/kg NP085 or NP102 administered orally and the 5 mg/kg NP085 or NP102 administered intravenously. Blood samples were collected by means of tail bleeding at predetermined time intervals. Drug concentrations were determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and subsequently pharmacokinetic modeling was done for both compounds. NP085 and NP102 were incubated in vitro with human and mouse liver microsomes. Both compounds were also subjected to a parallel artificial membrane permeation assay. In vitro studies of NP085 and NP102 illustrated that both of the compounds are rapidly absorbed and undergo rapid hepatic metabolism. The maximum concentration of drug (Cmax) obtained following oral administration of NP085 and NP102 was 0.2 ± 0.4 and 0.7 ± 0.3 µM, respectively; the elimination half-life of both compounds was 6.1 h. NP085 and NP102 showed bioavailability levels of 8% and 22%, respectively.

Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials.

BACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5 % v/v, pH 7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.

Efficacy and pharmacokinetic evaluation of a novel anti-malarial compound (NP046) in a mouse model.

BACKGROUND: Even though malaria is a completely preventable and treatable disease, it remains a threat to human life and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. According to the World Malaria Report 2013, in 2012 there were an estimated 207 million malaria cases and 627,000 deaths. Thus, the discovery and development of new, effective anti-malarial drugs are required. To achieve this goal, the Department of Chemistry at the University of the Free State has synthesized a number of novel amino-alkylated chalcones and analogues, which showed in vitro anti-malarial activity against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The lead compound (NP046) was selected for a comprehensive pharmacokinetic (PK) and in vivo efficacy evaluation in a mouse model. METHODS: In vivo efficacy: Water solutions of NP046 were administered orally at 50 and 10 mg/kg using oral gavage and IV at 5 and 1 mg/kg via the dorsal penile vein to Plasmodium berghei (ANKA strain) infected male C57BL/6 mice (n = 5), once a day for four days. Blood samples were collected via tail bleeding in tubes containing phosphate buffer saline (PBS) on day five to determine the % parasitaemia by flow cytometry.In vivo PK: NP046 solutions in water were administered orally (50 and 10 mg/kg) and IV (5 mg/kg) to male C57BL/6 mice (n = 5). Blood samples were collected via tail bleeding into heparinized tubes and analysed using a validated LC-MS/MS assay. Data obtained from the concentration-time profile was evaluated using Summit PK software to determine the PK parameters of NP046. RESULTS: NP046 inhibited parasite growth for the oral and IV groups. Better parasite growth inhibition was observed for the IV group. The PK evaluation of NP046 showed low oral bioavailability (3.2% and 6% at 50 mg/kg and 10 mg/kg dose, respectively and a moderate mean half-life ranging from 3.1 to 4.4 hours. CONCLUSION: Even though the oral bioavailability of NP046 is low, its percentage parasite growth inhibition is promising, but in order to improve the oral bioavailability, structure-activity-relationship (SAR) optimization studies are currently being conducted.

The development and validation of an LC-MS/MS method for the determination of a new anti-malarial compound (TK900D) in human whole blood and its application to pharmacokinetic studies in mice.

BACKGROUND: Malaria is one of the most lethal and life-threatening killer infectious diseases in the world, and account for the deaths of more than half a million people annually. Despite the remarkable achievement made in preventing and eradicating malaria, it still remains a threat to the public health and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. Therefore, the need to develop new anti-malarial drugs is crucial. The chemistry department at the University of Cape Town synthesized a number of new CQ-like derivatives (TK-series), and evaluated them for in vitro activity against both CQ-sensitive and -resistant Plasmodium falciparum strains, and for general cytotoxicity against a Chinese Hamster Ovarian (CHO) mammalian cell line. The lead compounds from the TK-series were selected for a comprehensive pharmacokinetic (PK) evaluation in a mouse model. METHODS: A sensitive LC-MS/MS assay was developed for the quantitative determination of TK900D. Multiple reaction monitoring (MRM) in the positive ionization mode was used for detection. The analyte and the internal standard (TK900E) were isolated from blood samples by liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved with a Phenomenex® Kinetex C18 (100 × 2.0 mm id, 2.6 µm) analytical column, using a mixture of 0.1% formic acid and acetonitrile (50:50; v/v) as the mobile phase. The method was fully validated over concentrations that ranged from 3.910 to 1000 ng/ml, and used to evaluate the PK properties of the lead compounds in a mouse model. RESULTS: The assay was robust, with deviation not exceeding 11% for the intra- and inter-run precision and accuracy. Extraction recovery was consistent and more than 60%. PK evaluation showed that TK900D and TK900E have moderate oral bioavailability of 30.8% and 25.9%, respectively. The apparent half-life ranged between 4 to 6 h for TK900D and 3.6 to 4 h for TK900E. CONCLUSION: The assay was sensitive and able to measure accurately low drug levels from a small sample volume (20 µl). PK evaluation showed that the oral bioavailability was moderate. Therefore, from a PK perspective, the compounds look promising and can be taken further in the drug development process.

hERG, Plasmodium Life Cycle, and Cross Resistance Profiling of New Azabenzimidazole Analogues of Astemizole.

Toward addressing the cardiotoxicity liability associated with the antimalarial drug astemizole (AST, hERG IC50 = 0.0042 µM) and its derivatives, we designed and synthesized analogues based on compound 1 (Pf NF54 IC50 = 0.012 µM; hERG IC50 = 0.63 µM), our previously identified 3-trifluoromethyl-1,2,4-oxadiazole AST analogue. Compound 11 retained in vitro multistage antiplasmodium activity (ABS PfNF54 IC50 = 0.017 µM; gametocytes PfiGc/PfLGc IC50 = 1.24/1.39 µM, and liver-stage PbHepG2 IC50 = 2.30 µM), good microsomal metabolic stability (MLM CLint < 11 µL·min-1·mg-1, EH < 0.33), and solubility (150 µM). It shows a ∼6-fold and >6000-fold higher selectivity against human ether-á-go-go-related gene higher selectively potential over hERG relative to 1 and AST, respectively. Despite the excellent in vitro antiplasmodium activity profile, in vivo efficacy in the Plasmodium berghei mouse infection model was diminished, attributable to suboptimal oral bioavailability (F = 14.9%) at 10 mg·kg-1 resulting from poor permeability (log D7.4 = -0.82). No cross-resistance was observed against 44 common Pf mutant lines, suggesting activity via a novel mechanism of action.

2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase ß (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

A Water-Soluble Polymer-Lumefantrine Conjugate for the Intravenous Treatment of Severe Malaria.

Uncomplicated malaria is effectively treated with oral artemisinin-based combination therapy (ACT). Yet, there is an unmet clinical need for the intravenous treatment of the more fatal severe malaria. There is no combination intravenous therapy for uncomplicated due to the nonavailability of a water-soluble partner drug for the artemisinin, artesunate. The currently available treatment is a two-part regimen split into an intravenous artesunate followed by the conventional oral ACT . In a novel application of polymer therapeutics, the aqueous insoluble antimalarial lumefantrine is conjugated to a carrier polymer to create a new water-soluble chemical entity suitable for intravenous administration in a clinically relevant formulation . The conjugate is characterized by spectroscopic and analytical techniques, and the aqueous solubility of lumefantrine is determined to have increased by three orders of magnitude. Pharmacokinetic studies in mice indicate that there is a significant plasma release of lumefantrine and production its metabolite desbutyl-lumefantrine (area under the curve of metabolite is ≈10% that of the parent). In a Plasmodium falciparum malaria mouse model, parasitemia clearance is 50% higher than that of reference unconjugated lumefantrine. The polymer-lumefantrine shows potential for entering the clinic to meet the need for a one-course combination treatment for severe malaria.

The evaluation of ADME and pharmacokinetic properties of decoquinate derivatives for the treatment of malaria.

The emergence of Plasmodium falciparum (Pf) parasite strains tolerant of the artemisinin component and resistant to the other drug component in artemisinin combination therapies (ACTs) used for treatment now markedly complicates malaria control. Thus, development of new combination therapies are urgently required. For the non-artemisinin component, the quinolone ester decoquinate (DQ) that possesses potent activities against blood stage Pf and acts on a distinct target, namely the Pf cytochrome bc 1 complex, was first considered. However, DQ has poor drug properties including high lipophilicity and exceedingly poor aqueous solubility (0.06 µg/ml), rendering it difficult to administer. Thus, DQ was chemically modified to provide the secondary amide derivative RMB005 and the quinoline O-carbamate derivatives RMB059 and RMB060. The last possesses sub-nanomolar activities against multidrug resistant blood stages of Pf, and P. berghei sporozoite liver stages. Here we present the results of ADME analyses in vitro and pharmacokinetic analyses using C57BL/6 mice. The amide RMB005 had a maximum mean whole blood concentration of 0.49 ± 0.02 µM following oral administration; however, the area under the curve (AUC), elimination half-life (t1/2) and bioavailability (BA) were not significantly better than those of DQ. Surprisingly, the quinoline O-carbamates which can be recrystallized without decomposition were rapidly converted into DQ in human plasma and blood samples. The maximum concentrations of DQ reached after oral administration of RMB059 and RMB060 were 0.23 ± 0.05 and 0.11 ± 0.01 µM, the DQ elimination half-lives were 4.79 ± 1.66 and 4.66 ± 1.16 h, and the DQ clearance were 19.40 ± 3.14 and 21.50 ± 3.38 respectively. Under these assay conditions, the BA of DQ could not be calculated Overall although RMB059 and -060 are labile in physiological medium with respect to the DQ parent, the potential to apply these as prodrugs is apparent from the current data coupled with their ease of preparation.

Novel 3-Trifluoromethyl-1,2,4-oxadiazole Analogues of Astemizole with Multi-stage Antiplasmodium Activity and In Vivo Efficacy in a Plasmodium berghei Mouse Malaria Infection Model.

Iterative medicinal chemistry optimization of an ester-containing astemizole (AST) analogue 1 with an associated metabolic instability liability led to the identification of a highly potent 3-trifluoromethyl-1,2,4-oxadiazole analogue 23 (PfNF54 IC50 = 0.012 µM; PfK1 IC50 = 0.040 µM) displaying high microsomal metabolic stability (HLM CLint < 11.6 µL·min-1·mg-1) and > 1000-fold higher selectivity over hERG compared to AST. In addition to asexual blood stage activity, the compound also shows activity against liver and gametocyte life cycle stages and demonstrates in vivo efficacy in Plasmodium berghei-infected mice at 4 × 50 mg·kg-1 oral dose. Preliminary interrogation of the mode of action using live-cell microscopy and cellular heme speciation revealed that 23 could be affecting multiple processes in the parasitic digestive vacuole, with the possibility of a novel target at play in the organelles associated with it.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

Antimalarial Benzimidazole Derivatives Incorporating Phenolic Mannich Base Side Chains Inhibit Microtubule and Hemozoin Formation: Structure-Activity Relationship and In Vivo Oral Efficacy Studies.

A novel series of antimalarial benzimidazole derivatives incorporating phenolic Mannich base side chains at the C2 position, which possess dual asexual blood and sexual stage activities, is presented. Structure-activity relationship studies revealed that the 1-benzylbenzimidazole analogues possessed submicromolar asexual blood and sexual stage activities in contrast to the 1H-benzimidazole analogues, which were only active against asexual blood stage (ABS) parasites. Further, the former demonstrated microtubule inhibitory activity in ABS parasites but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/kg. Generally, the compounds were noncytotoxic to mammalian cells.

The Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside.

Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5-2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and In Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model.

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure-activity and structure-property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.

The palladacycle complex AJ-5 induces apoptotic cell death while reducing autophagic flux in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anti-cancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.