Inactivation of Anopheles gambiae

Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters kinact and KI were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs

Oral lipid-based nanoformulation of tafenoquine enhanced bioavailability and blood stage antimalarial efficacy and led to a reduction in human red blood cell loss in mice.

Tafenoquine (TQ), a new synthetic analog of primaquine, has relatively poor bioavailability and associated toxicity in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. A microemulsion formulation of TQ (MTQ) with sizes <20 nm improved the solubility of TQ and enhanced the oral bioavailability from 55% to 99% in healthy mice (area under the curve 0 to infinity: 11,368±1,232 and 23,842±872 min·μmol/L) for reference TQ and MTQ, respectively. Average parasitemia in Plasmodium berghei-infected mice was four- to tenfold lower in the MTQ-treated group. In vitro antiplasmodial activities against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum indicated no change in half maximal inhibitory concentration, suggesting that the microemulsion did not affect the inherent activity of TQ. In a humanized mouse model of G6PD deficiency, we observed reduction in toxicity of TQ as delivered by MTQ at low but efficacious concentrations of TQ. We hereby report an enhancement in the solubility, bioavailibility, and efficacy of TQ against blood stages of Plasmodium parasites without a corresponding increase in toxicity

In Vitro interactionsof plant phenolic compounds and antiparasitic drugs with drug metabolising enzymes and transporters

Drug metabolising enzymes and transporters play a critical role in the pharmacokinetics and hence, absorption, distribution, metabolism, and excretion (ADME) of compounds and medicines. The objective of this thesis was to apply in vitro systems used in ADME studies to investigate interactions of pure compounds isolated from plants and of antiparasitic drugs with drug metabolising enzymes and transporters. In addition, drug metabolising enzymes and transporters are often studied in isolation and though this is simpler, the two biological systems are often coupled, thus, the Caco-2 cell line was also used to study enzyme-transporter coupling. Simple in vitro experimental systems were also compared to complex in vitro and in vivo systems. In this study, the inhibitory effects of 19 purified compounds isolated from plants, including diospyrin, geshoidin, and some flavonoids, on cytochrome P450s (CYPs) 1A2, 3A4, 2C9, 2C19 and 2D6 and glutathione transferases (GSTs) A1-1, M1-1 and P1-1, as well as p-glycoprotein (Pgp), was investigated in vitro. The effect of 21 antiparasitic drugs, such as chloroquine, cycloguanil and amodiaquine, on Pgp was also investigated. Inhibition of heterologously expressed human CYPs and GSTs was investigated using spectrophotometric, fluorescence and liquid chromatography-based assays. Eleven out of the 19 plant compounds inhibited at least one of the GSTs. Diospyrin was the most potent inhibitor with IC50 values in the range 0.1 - 0.5 μM. Diospyrin and geshoidin were further investigated and it was found that the predominant mode of GST inhibition was noncompetitive with respect to both glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Diospyrin, however, competitively inhibited A1-1 and M1-1 with respect to GSH and geshoidin displayed mixed inhibition toward A1-1 with respect to GSH. The Ki values for diospyrin with respect to both GSH and CDNB were in the range 0.08–0.6 μM and those for geshoidin were in the range 16– 173 μM. Diospyrin and geshoidin were also found to inactivate GSTP1-1 with diospyrin being a potent inactivator with KI of 0.7 μM, whereas geshoidin had a KI of 47 μM. Eight of the 19 plant compounds inhibited at least one of the CYPs. Diospyrin was found once again to be the most potent inhibitor with IC50 values in the range 0.4 - 2 μM. Diospyrin showed mixed inhibition toward CYPs 3A4, 2C9 and 2D6, with Ki values in the range 0.25 – 2 μM. For CYP1A2, the inhibition was non competitive with Ki 0.8 μM, compared to mixed inhibition of rat recombinant CYP1A with Ki 0.34 μM. Diospyrin was further investigated for its in vivo CYP1A inhibition properties in rats, and no evidence for in vivo CYP1A inhibition was found. The plant compounds and antiparasitic drugs were screened for interaction with Pgp based on inhibition of Pgp mediated [3H]-taxol transport in Caco-2 cells. Bidirectional transport of selected inhibitors was further evaluated to identify potential Pgp substrates using the Caco-2 cells. Of 21 antiparasitics tested, 14 were found to inhibit Pgp mediated [3H]-taxol with Kiapp values in the range 4–2000 μM. The antimalarial quinine was the most potent inhibitor with a Kiapp of 4 μM. Of the 12 natural compounds tested, 3 inhibited [3H]-taxol transport with Kiapp values in the range 50–400 μM. Quinine, amodiaquine, chloroquine, flavone, genistein, praziquantel, quercetin and thiabendazole were further investigated in bidirectional transport assays to determine whether they were substrates for Pgp. Transport of quinine in the secretory direction exceeded that in the absorptive direction and was saturable, suggesting quinine being a Pgp substrate. The rest of the compounds inhibiting Pgp showed no evidence of being Pgp substrates. Amodiaquine was further investigate because the transport experiments in Caco-2 cells showed low recovery of 30 % and rapid disappearance of the compound from the apical chamber. Compounds structurally similar to amodiaquine, and those affecting non-specific binding of amodiaquine or pH of the system, were tested to unravel the mechanism behind these observations. Chloroquine and ammonium chloride increased the transmonolayer permeability of amodiaquine and decreased its accumulation in Caco-2 cells, whereas bovine serum albumin (BSA) had no effect. Chloroquine and BSA decreased plastic binding whereas ammonium chloride had no effect. This suggests that amodiaquine is trapped in acidic cell compartments such as lysosomes. Amodiaquine was also trapped in rat intestinal tissue with permeability from the apical to basolateral direction significantly higher than in the opposite direction, suggesting an active uptake over the apical membrane of the rat tissue. Transport in rat jejunum was asymmetric with apparent active apical uptake. Rapid apical uptake in Caco-2 cells appeared to support this theory. However, screening of a range of potential inhibitors as a first step to identifying the potential transporter was not successful and further studies will be required. To study enzyme-transporter coupling in Caco-2 cells, the GST substrate monochlorobimane (MCB) was used. The Caco-2 cells displayed GST activity by metabolising MCB to the fluorescent conjugate glutathione-bimane (GSB). The cells were loaded with MCB which they conjugated to GSB, and the latter was effluxed into the apical and basolateral compartments with a greater amount being effluxed into the apical compartment. The multidrug resistance protein (MRP) inhibitor benzbromarone, inhibited GSB efflux at both the apical and basolateral membranes of the Caco-2 cells indicating the presence of GSB-transporting MRPs at both membranes. Ethacrynic acid, which inhibits both GSTs and MRPs reduced both apical and basolateral efflux as well as GSB formation. Diospyrin and ellagic acid which had been seen to be potent inhibitors of recombinant GSTs had no effect on MCB conjugation to GSB or on GSB efflux. In another experiment, Caco-2 cells were loaded with bromosulfophthalein (BSP) a known MRP substrate, and BSP was effluxed preferentially into the apical compartment. Addition of the MRP inhibitor benzbromarone resulted in decrease in apical efflux of BSP, and a corresponding increase in the basolateral efflux. This indicates a transporter-transporter interaction whereby the basolateral MRP has a lower affinity for BSP than the apical MRP, and upon inhibition of the apical MRP, the basolateral MRP takes over the efflux of BSP. In conclusion, a number of purified compounds isolated from plants and a number of antiparasitic drugs, in a range of concentrations, were found to be capable of inhibiting the major drug metabolising CYPs and the major drug transporter Pgp as well as GSTs, in vitro. Drug metabolising enzymes and transporters play an important role in pharmacokinetics. Interaction of the purified plant isolates and antiparasitic drugs with CYPs, GSTs and Pgp has implications for potentially harmful drug-drug interactions where the plant isolates contained in, for instance, a herbal medicine, may be taken concomitantly with prescribed medicines and where antiparasitic drugs may be taken in combination due to emerging resistance. The GST and Pgp inhibitors may also be potential chemomodulators in the case of multidrug resistance in cancer therapy. In the case of amodiaquine, the compound was trapped in acidic cell compartments due to its basicity, and the use of ammonium chloride rather than BSA in transport experiments with similar compounds is recommended for better prediction of permeability. Comparison of the different experimental methods used, i.e. in vitro recombinant human CYPs vs in vitro recombinant rat CYP; in vitro recombinant rat CYP vs in vivo rat CYP; in vitro recombinant GST vs cellular GST, and membrane transport in cells vs membrane transport in excised tissue, indicated that scaling of in vitro data to more complex in vivo effects should be done with caution. The research work also demonstrated the applicability of in vitro ADME assays routinely used in pharmaceutical companies to the evaluation of natural products and antiparasitic drugs

A review of the pharmacodynamic effect of chemo-herbal drug combinations therapy for cancer treatment

There is mounting evidence that cancer patients co-administer herbal drugs with chemotherapy, however, information on the pharmacodynamic (PD) effects of such combination therapy is scarce. Natural products including crude extracts, herbal formulas, and bioactive compounds from plants hold great potential to prevent and treat cancers. More importantly, some herbal drugs can reduce the incidence of chemotherapy-induced toxicity including oral mucositis, gastrointestinal toxicity, hepatotoxicity etc. This review focuses on the effectiveness of some herbal products as adjuvant therapy and describes the possible mechanisms of chemo-herbal drug PD interactions in enhancing the efficacy/ or reducing the side effects of chemotherapy. We also highlighted recent advances in preclinical in vitro and in vivo studies to establish the effectiveness of herbal medicine to enhance efficacy or counteract chemotherapy-induced side effects. In addition, we draw particular attention to the synergistic effects of chemo-herbal drug combination therapy to prevent and treat cancers using evidence from clinical trials. We concluded that herbal drugs hold great potential as adjuvant therapy for the prevention and treatment of chemotherapy-induced side effects. It is important to also highlight that the clinical evidence on chemo-herbal drug combination therapy is limited. There is an urgent need for an in-depth PD evaluation including the safety pharmacology of chemo-herbal drug combination therapy as well as reliable evidence from multicentre clinical trials to establish the beneficial or negative effects of chemo-herbal drug combination therapy in the ongoing fight against cancer

Development and evaluation of a reconstitutable dry suspension containing isoniazid for flexible pediatric dosing

Tuberculosis (TB) is a major cause of childhood death. Despite the startling statistics, it is neglected globally as evidenced by treatment and clinical care schemes, mostly extrapolated from studies in adults. The objective of this study was to formulate and evaluate a reconstitutable dry suspension (RDS) containing isoniazid, a first-line anti-tubercular agent used in the treatment and prevention of TB infection in both children and adults. The RDS formulation was prepared by direct dispersion emulsification of an aqueous-lipid particulate interphase coupled with lyophilization and dry milling. The RDS appeared as a cream-white free-flowing powder with a semi-crystalline and microparticulate nature. Isoniazid release was characterized with an initial burst up to 5 minutes followed by a cumulative release of 67.88% ± 1.88% (pH 1.2), 60.18% ± 3.33% (pH 6.8), and 49.36% ± 2.83% (pH 7.4) over 2 h. An extended release at pH 7.4 and 100% drug liberation was achieved within 300 min. The generated release profile best fitted the zero order kinetics (R2 = 0.976). RDS was re-dispersible and remained stable in the dried and reconstituted states over 4 months and 11 days respectively, under common storage condition

Preclinical assessment addressing intravenous administration of a [68Ga]Ga-PSMA-617 microemulsion : acute in vivo toxicity, tolerability, PET imaging, and biodistribution

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [68Ga]Ga-PSMA- 617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [68Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results.The Nuclear Technologies in Medicine and the Biosciences Initiative (NTeMBI) is funded by the Department of Science and Technology, the Department of Nuclear Medicine, University of Pretoria and the Council for Scientific and Industrial Research and the Carl and Emily Fuchs Foundation.http://www.mdpi.com/journal/moleculesam2021Nuclear Medicin

Preclinical Assessment Addressing Intravenous Administration of a [68Ga]Ga-PSMA-617 Microemulsion: Acute In Vivo Toxicity, Tolerability, PET Imaging, and Biodistribution

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [68Ga]Ga-PSMA-617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [68Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results