1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis.

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.

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.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

The In Vitro and In Vivo Effects of Hypoxis hemerocallidea on Indinavir Pharmacokinetics: Modulation of Efflux.

Hypoxis hemerocallidea (African potato) is a popular medicinal plant that has been used traditionally for the treatment of various disorders. Some HIV/AIDS patients use this traditional medicine together with their antiretroviral therapy. This study aimed to determine the impact of selected H. hemerocallidea materials (i.e., a commercial product, an aqueous extract, and biomass reference plant material) on the bidirectional permeability of indinavir across Caco-2 cell monolayers as well as the bioavailability of indinavir during an acute, single administration study in Sprague-Dawley rats. All of the selected H. hemerocallidea test materials demonstrated inhibition effects on indinavir efflux across Caco-2 cell monolayers, albeit to different extents. An increase in the bioavailability of indinavir was obtained in vivo when administered concomitantly with the H. hemerocallidea materials, albeit not statistically significantly. The change in bioavailability directly correlated with the in vitro permeability results. It can therefore be concluded that the change in permeability and bioavailability of indinavir was caused by efflux inhibition and this effect was dependent on the type of H. hemerocallidea material investigated, which was found to be in the following order: commercial product > aqueous extract > reference plant material. The clinical significance of the combined effect of efflux and metabolism inhibition by H. hemerocallidea should be determined in another in vivo model that expresses the cytochrome P450 3A4 enzyme.

Identification of Fast-Acting 2,6-Disubstituted Imidazopyridines That Are Efficacious in the in Vivo Humanized Plasmodium falciparum NODscidIL2Rγ null Mouse Model of Malaria.

Optimization of a chemical series originating from whole-cell phenotypic screening against the human malaria parasite, Plasmodium falciparum, led to the identification of two promising 2,6-disubstituted imidazopyridine compounds, 43 and 74. These compounds exhibited potent activity against asexual blood stage parasites that, together with their in vitro absorption, distribution, metabolism, and excretion (ADME) properties, translated to in vivo efficacy with clearance of parasites in the PfSCID mouse model for malaria within 48 h of treatment.

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies.

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

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.

Evaluation of Isolated Fractions of Aloe vera Gel Materials on Indinavir Pharmacokinetics: In vitro and in vivo Studies.

Aloe vera is a plant with a long history of traditional medicinal use and is consumed in different products, sometimes in conjunction with prescribed medicines. A. vera gel has shown the ability to modulate drug absorption in vitro. The aim of this study was to fractionate the precipitated polysaccharide component of A. vera gel based on molecular weight and to compare their interactions with indinavir pharmacokinetics. Crude polysaccharides were precipitated from a solution of A. vera gel and was fractionated by means of centrifugal filtration through membranes with different molecular weight cut-off values (i.e. 300 KDa, 100 KDa and 30 KDa). Marker molecules were quantified in the aloe leaf materials by means of nuclear magnetic resonance spectroscopy and the average molecular weight was determined by means of gel filtration chromatography linked to multi-angle-laser-light scattering and refractive index detection. The effect of the aloe leaf materials on the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers as well as indinavir metabolism in LS180 cells was measured. The bioavailability of indinavir in the presence and absence of the aloe leaf materials was determined in Sprague-Dawley rats. All the aloe leaf materials investigated in this study reduced the TEER of Caco-2 cell monolayers, inhibited indinavir metabolism in LS 180 cells to different extents and changed the bioavailability parameters of indinavir in rats compared to that of indinavir alone. These indinavir pharmacokinetic modulation effects were not dependent on the presence of aloverose and also not on the average molecular weight of the isolated fractions.

Herb-Drug Pharmacokinetic Interactions: Transport and Metabolism of Indinavir in the Presence of Selected Herbal Products.

Patients receiving anti-retroviral drug treatment are sometimes simultaneously taking herbal remedies, which may result in pharmacokinetic herb-drug interactions. This study aimed to determine if pharmacokinetic interactions exist between selected commercially available herbal products (i.e., Linctagon Forte(®), Viral Choice(®) and Canova(®)) and indinavir in terms of in vitro transport and metabolism. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in the presence and absence of the selected herbal products and verapamil (positive control). Metabolism of indinavir was determined in LS180 cells in the presence and absence of the selected herbal products as well as ketoconazole (positive control). The secretory transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte(®) and Viral Choice(®) when compared to that of indinavir alone. Canova(®) only slightly affected the efflux of indinavir compared to that of the control group. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir's bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made.

Syntheses and in Vitro Antiplasmodial Activity of Aminoalkylated Chalcones and Analogues.

A series of readily synthesized and inexpensive aminoalkylated chalcones and diarylpropane analogues (1-55) were synthesized and tested against chloroquinone-sensitive (D10 and NF54) and -resistant (Dd2 and K1) strains of Plasmodium falciparum. Hydrogenation of the enone to a diarylpropane moiety increased antiplasmodial bioactivity significantly. The influence of the structure of the amine moiety, A-ring substituents, propyl vs ethyl linker, and chloride salt formation on further enhancing antiplasmodial activity was investigated. Several compounds have IC50 values similar to or better than chloroquine (CQ). The most active compound (26) had an IC50 value of 0.01 µM. No signs of resistance were detected, as can be expected from compounds with structures unrelated to CQ and other currently used antimalarial drugs. Toxicity tests (in vitro CHO cell assay) gave high SI indices.

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.

Synthesis and in vitro and in vivo pharmacological evaluation of new 4-aminoquinoline-based compounds.

A new class of 4-aminoquinolines was synthesized and evaluated in vitro for antiplasmodial activity against both the chloroquine-sensitive (3D7) and -resistant (K1 and W2) strains. The most active compounds 3c-3e had acceptable cytotoxicity but showed strong inhibition toward a panel of cytochrome P450 enzymes in vitro. Pharmacokinetic studies on 3d and 3e in mice showed that they had moderate half-life (4-6 h) and low oral bioavailability. The front runner compound 3d exhibited moderate inhibition of the malaria parasite on P. berghei infected mice following oral administration (5 mg/kg), achieving reduction of parasitemia population by 47% on day 7.