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An in vitro toolbox to accelerate anti-malarial drug discovery and development.

ABSTRACT: BACKGROUND:Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS:Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS:Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS:This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

SUBMITTER: Charman SA

PROVIDER: S-EPMC6941357 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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An in vitro toolbox to accelerate anti-malarial drug discovery and development.

Charman Susan A SA Andreu Alice A Barker Helena H Blundell Scott S Campbell Anna A Campbell Michael M Chen Gong G Chiu Francis C K FCK Crighton Elly E Katneni Kasiram K Morizzi Julia J Patil Rahul R Pham Thao T Ryan Eileen E Saunders Jessica J Shackleford David M DM White Karen L KL Almond Lisa L Dickins Maurice M Smith Dennis A DA Moehrle Joerg J JJ Burrows Jeremy N JN Abla Nada N

Malaria journal 20200102 1

<h4>Background</h4>Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing ...[more]

PMID: 31898492

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Project description:BackgroundThe erythrocytic stage of Plasmodium falciparum parasites in humans is clinically important, as the parasites at this growth stage causes malarial symptoms. Most of the currently available anti-malarial drugs target this stage, but the emergence and spread of parasites resistant to anti-malarial drugs are a major challenge to global eradication efforts; therefore, the development of novel medicines is urgently required. In this study, the in vitro anti-malarial activity of five current anti-malarial drugs (artemisinin, atovaquone, chloroquine, mefloquine, and pyrimethamine) and 400 compounds from the Pathogen Box provided by the Medicines for Malaria Venture on P. falciparum parasites was characterized using the XN-30 analyzer. Furthermore, the outcomes obtained using the analyser were classified according to the parasitaemias of total and each developmental stages.ResultsThe growth inhibition rate and the half-maximal (50%) inhibitory concentration (IC50) of the five current anti-malarial drugs were calculated from the parasitaemia detected using the XN-30 analyzer. Respective strains and drugs presented strongly fitted sigmoidal curves, and the median SD at all tested concentrations was 1.6, suggesting that the variation in values measured with the analyser was acceptably low for the comparison of drug efficacy. Furthermore, the anti-malarial activity of the 400 compounds from the Pathogen Box was tested, and 141 drugs were found to be effective. In addition, the efficacy was classified into 4 types (Type I, parasites were arrested or killed without DNA replication; Type II, parasites were arrested or killed similar to Type I, and the parasitaemia was apparently decreased; Type III, parasites progressed to trophozoite without sufficient DNA replication; and Type IV, parasites were arrested at late trophozoite or schizont after DNA replication).ConclusionThe current study demonstrates that the XN-30 analyzer objectively, reproducibly, and easily evaluated and characterized the anti-malarial efficacy of various compounds. The results indicate the potential of the XN-30 analyzer as a powerful tool for drug discovery and development in addition to its use as an important diagnostic tool.

Project description:Mechanistic within-host models relating blood anti-malarial drug concentrations with the parasite-time profile help in assessing dosing schedules and partner drugs for new anti-malarial treatments. A comprehensive simulation study to assess the utility of a stage-specific pharmacokinetic-pharmacodynamic (PK-PD) model for predicting within-host parasite response was performed.Three anti-malarial combination therapies were selected: artesunate-mefloquine, dihydroartemisinin-piperaquine, and artemether-lumefantrine. The PK-PD model included parameters to represent the concentration-time profiles of both drugs, the initial parasite burden and distribution across the parasite life cycle, and the parasite multiplication factor due to asexual reproduction. The model also included the maximal killing rate of each drug, and the blood drug concentration associated with half of that killing effect (in vivo EC50), derived from the in vitro IC50, the extent of binding to 0.5% Albumax present in the in vitro testing media, and the drugs plasma protein binding and whole blood to plasma partitioning ratio. All stochastic simulations were performed using a Latin-Hypercube-Sampling approach.The simulations demonstrated that the proportion of patients cured was highly sensitive to the in vivo EC50 and the maximal killing rate of the partner drug co-administered with the artemisinin derivative. The in vivo EC50 values that corresponded to on average 95% of patients cured were much higher than the adjusted values derived from the in vitro IC50. The proportion clinically cured was not strongly influenced by changes in the parameters defining the age distribution of the initial parasite burden (mean age of 4 to 16 hours) and the parasite multiplication factor every life cycle (ranging from 8 to 12 fold/cycle). The median parasite clearance times, however, lengthened as the standard deviation of the initial parasite burden increased (i.e. the infection became more asynchronous).This simulation study demonstrates that the PD effect predicted from in vitro growth inhibition assays does not accord well with the PD effect of the anti-malarials observed within the patient. This simulation-based PK-PD modelling approach should not be considered as a replacement to conducting clinical trials but instead as a decision tool to improve the design of a clinical trial during drug development.

Project description:BackgroundMultidrug-resistant Plasmodium is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.Presentation of the hypothesisAdvances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.Testing the hypothesisAvailability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the Plasmodium genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between Plasmodium and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to Plasmodium-infected RBCs (iRBCs) and different Plasmodium organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more Plasmodium cell membrane translocation peptide seems achievable.Implications of the hypothesisTargeting of the Plasmodium genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.

Project description:BackgroundQuinine (QN) remains the first line anti-malarial drug for the treatment of complicated malaria in Europe and Africa. The emergence of QN resistance has been documented. QN resistance is not yet a significant problem, but there is an urgent need to discover partners for use in combination with QN. The aim of the study was to assess the in vitro potentiating effects of atorvastatin (AVA), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with QN against Plasmodium falciparum and to evaluate whether the effects of AVA could be associated with gene copy number or mutations in genes involved in QN resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe.MethodsThe susceptibilities to combination of AVA with QN were assessed against 21 parasite strains using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfcrt, pfmdr1, pfmdr2, pfmrp genes. In addition, the number of DNNND, DDNHNDNHNN repeats in pfnhe-1 ms4760 and the ms4760 profile were determined for each strains of P. falciparum.ResultsAVA demonstrated synergistic effects in combination with QN against 21 P. falciparum strains. The QN IC50 was reduced by 5% (0% to 15%; 95%CI: 1%-8%), 10% (3% to 23%; 95%CI: 7%-14%) and 22% (14% to 40%; 95%CI: 19%-25%) in presence of AVA at concentrations of 0.1, 0.5 and 1.0 microM, respectively. These reductions were all significant (p < 0.009). The reduction in the QN IC50 in presence of AVA was not significantly correlated with the QN IC50 (r = 0.22, P = 0.3288) or the AVA IC50 (r = 0.03, P = 0.8946). The synergistic effect of AVA in combination with QN was not significantly associated with polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes that could be involved in QN resistance. The synergistic effect of AVA on QN responses was not significantly associated with pfmdr1 copy number (P = 0.0428).ConclusionThe synergistic effect of AVA in combination with QN was found to be unrelated to mutations occurring in transport protein genes involved in QN drug resistance. The different mechanisms of drug uptake and/or mode of action for AVA compared to the other anti-malarial drugs, as well as the AVA-mediated synergy of the anti-malarial effect of QN, suggests that AVA will be a good candidate for combinatorial malaria treatment. All of these observations support calls for both an in vivo evaluation with pharmacokinetic component and clinical trials of AVA as an anti-malarial therapy.

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An in vitro toolbox to accelerate anti-malarial drug discovery and development.

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An in vitro toolbox to accelerate anti-malarial drug discovery and development.

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