1,721,095 research outputs found
Population pharmacokinetic modelling for dose optimization of esomeprazole to treat early-onset preeclampsia
Get PDFRationale Esomeprazole is a proton pump inhibitor with preclinical efficacy data showing it lowers concentrations of soluble fms like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng), pathognomonic biomarkers identified in preeclampsia. A randomized controlled trial, Preeclampsia Intervention with Esomeprazole (PIE) trial, was conducted in South African women diagnosed with early-onset preeclampsia to investigate efficacy, but it found no change in clinical outcome or biomarker concentrations. It was hypothesized that the 40 mg daily oral dose was not enough to achieve therapeutic exposure. This study investigated the pharmacokinetics of esomeprazole in patients with early- onset preeclampsia with the aim to optimize the dose for future clinical trials. Methods Pharmacokinetic data from ten pregnant patients with early-onset preeclampsia from the PIE trial, median (range) age 30 (21-43) years, weight 98.8 (56-126) kg, and gestational age 29 (26- 31) weeks, were included for model development. In addition, pharmacokinetic data from non- pregnant healthy volunteers consisted of a pooled dataset of 26 male and female subjects, median (range) age of 21 (18-27) years and weight 69 (54-89) kg, who received 40 mg esomeprazole daily. Analysis of the pharmacokinetic data in pregnant patients was performed using nonlinear mixed-effects modelling with allometric scaling on clearance (CL) and volume of distribution (Vd). Metabolite to parent area under the time-concentration curve (AUCsulf/AUCeso and AUChyd/AUCeso) ratios were compared between pregnant and non-pregnant to assess metabolic changes in pregnancy. Simulations were performed with the model to determine the nonlinear increase in AUC with higher doses and with repeated dosing in the pregnant patients. Simulation results were compared with the preclinical target unbound concentration (0.917 mg/L) and preclinical target unbound AUC0-24 (9.29 mg·h/L). Results A one compartment pharmacokinetic model with first-order elimination and transit compartment absorption best described the data. Model estimated apparent CL and apparent Vd (95% CI) were 19.2 (14.2-26) L/h and 44.2 (29.9-56.6) L, respectively. Median AUCsulf/AUCeso (IQR) for pregnant patients, 2.00 (1.35-2.61) , was significantly higher than that for non-pregnant subjects on day1, 0.700 (0.636-1.00) , and day5, 1.18 (0.981- 1.58) . Median AUChyd/AUCeso (IQR) for pregnant patients, 0.0543 (0.0500-0.0914) , was not significantly different from that of non-pregnant subjects on day5, 0.0777 (0.0569-0.108) but lower than that of non-pregnant subjects on day1, 0.188 (0.156- 0.227). Simulation results showed that predicted steady state unbound Cmax is between 0.0949 and 0.398 mg/L while the predicted unbound AUC0-24 in pregnant patients with the highest dose of esomeprazole used clinically, i.e.120 mg BID, is between 0.696 and 2.92 mg·h/L. Discussion/Conclusion Model estimated CL/F and Vd/F are higher than values previously reported by other population pharmacokinetic models. AUCm/AUCp comparisons showed that esomeprazole metabolism in pregnancy appears to have shifted to the CYP3A4 pathway. This means that the nonlinear AUC increase expected with dose escalation and with repeated dosing are not as significant as in nonpregnant. Simulations indicate that pregnant patients are unlikely to achieve the target concentration and exposure with the highest dose of esomeprazole registered. Further research is necessary to determine the target site of action of esomeprazole in preeclampsia, and the pharmacokinetic metric that correlates with efficacy
Screening of South African marine filamentous actinobacteria for antitubercular compounds
Get PDFMultidrug-resistant tuberculosis (MDR-TB) is a rampant problem across the globe, perpetuated by the current antimicrobial resistance challenge. To lift the burden, novel antitubercular drugs are needed to treat TB more effectively. The filamentous actinobacteria are a viable source of potential novel antitubercular drugs especially with the advent of technological advancements providing the means to overcome the issues that hampered natural product drug discovery in the past. For example, molecular networking can rapidly dereplicate and verify novel compounds, streamlining the pipeline of drug discovery by allowing antibiotic-producing actinobacteria to be investigated more easily as possible drug sources. Microbial diversity has been linked to the chemical diversity of secondary metabolites and therefore unique actinobacteria have greater potential to produce novel antibiotics. South Africa is one of the most biodiverse countries in the world, however, the antibiotic potential of marine microorganisms has not been fully investigated. This project aimed to screen South African marine filamentous actinobacteria for antitubercular activity and purify the active compound(s) from the most active strains. Nineteen strains which had shown preliminary activity in a previous study were selected for screening, cultured in various liquid media according to their previously demonstrated activity, and extracted. Methanol and ethyl acetate were used to extract the produced secondary metabolites, which were submitted for screening against Mycobacterium tuberculosis (Mtb) in vitro. Streptomyces strain GGUI#16 grown in R2A was identified as a potential novel antibiotic producer as it showed activity against Mtb with an average MIC90 of 5.1 μg/mL, which was lower than the average MIC90 of 5.6 μg/mL seen for isoniazid, a first-line TB drug. GGUI#16 grown in R2A was therefore selected for large scale cultivation and purification by solid phase extraction and high-pressure liquid chromatography. Through a series of bioassay-guided fractionation techniques, the active fractions were successfully isolated and screened against Mtb in vitro. The average MIC90 activity of the crude extract decreased from 5.1 μg/mL to 1.56 μg/mL for the semi-purified compound. Following high resolution mass spectrometry and GNPS analyses, no known compounds were identified in the active fractions – suggesting the active compound(s) may be novel. Future research should aim at increasing the purity of the compound, performing structural elucidation, and conducting preclinical tests to further evaluate the active compound's antitubercular properties
Determination of biomarkers for toxicity and antiretroviral adherence in hair in South African patients
Get PDFBackground: Substance abuse is one of the many factors associated with poor levels of antiretroviral adherence and is also prevalent among HIV-infected individuals. Ethyl glucuronide, a minor metabolite of alcohol, is a stable biomarker in hair that can be used to detect and monitor alcohol consumption over long time periods. Drugs of abuse are also detected in hair. Hair provides a longer window of drug detection compared to blood and urine. Recently, hair has also been studied as an alternative matrix for adherence monitoring and concentrations of antiretrovirals in hair have been shown to be closely correlated with virologic outcomes. This study investigated the impact of substance abuse on adherence among HIV-infected patients attending an antiretroviral therapy clinic in Cape Town by measuring drug concentrations in hair. Efavirenz levels in hair were also measured to investigate the usefulness of using hair analysis as a method of adherence monitoring within the South African context. Method: This study describes the development and validation of three liquid chromatography tandem mass spectrometry methods of hair analysis. The first method developed was for the quantification of ethyl glucuronide in 20 mg samples of hair. This method was validated over the calibration range 7.5 - 480 pg/mg. Secondly, a qualitative method was developed to screen hair samples for amphetamine, methamphetamine, cocaine, benzoylecgonine, cocaethylene and methaqualone. The final method developed was for the quantification of efavirenz in 0.2 mg samples of hair. This method was validated over the calibration range 0.625 - 40 ng/mg. The validated methods were applied to 257 samples of hair collected from 135 HIV-infected patients during visits to the clinic at weeks 16, 32 and 48. The results generated from the analysis of the hair samples were analysed in the context of additional adherence measurements collected for a related randomized controlled study. Results: Analysis of the hair samples for ethyl glucuronide demonstrated that 27% of the samples analysed contained levels above 30 pg/mg which is the cutoff value suggested by the Society of Hair Testing to identify heavy drinkers. The results also show limitations to using the CAGE alcohol abuse screening tool which had a poor sensitivity of only 28.8%. Eight (5.9%) out of the 135 participants were identified to be chronic drug users, and of these five (62.5%) were identified to be heavy drinkers as well. The most commonly abused drug identified in the screen was methaqualone. The median efavirenz levels at weeks 16, 32 and 48 were 5.52 ng/mg (IQR: 3.60 - 9.77), 5.75 ng/mg (IQR: 3.21 - 8.18) and 4.89 ng/mg (IQR: 3.10 - 7.94) respectively. Participants with the poor CYP2B6 metaboliser genotype had significantly higher median efavirenz hair concentrations compared to participants with intermediate and extensive genotypes (P < 0.0001). Efavirenz levels in hair and plasma samples were strongly correlated throughout the study (Spearman correlation coefficients: 0.672 - 0.741, all P values < 0.0001). Substance abuse had no impact on adherence measured by an electronic adherence monitoring device. No significant correlation was observed between adherence and levels of efavirenz in hair. Conclusions: Methods of hair analysis were developed and successfully applied to hair samples in the context of better understanding the impact of substance abuse on adherence. The results from the analysis of the hair samples provided insight into the prevalence of substance abuse among HIV-infected patients. The strong correlation observed between levels of efavirenz in hair and plasma suggest that, in this subset of HIV-infected patients, a single plasma concentration was as good an adherence measure as a hair concentration. The hair analysis methods developed and validated in this study are novel in South Africa and demonstrate the potential of this matrix to be used in various contexts within the country
Determination of total, unbound, and intracellular concentrations of the antiretroviral drugs Efavirenz, Lopinavir, and Ritonavir
Get PDFEfavirenz, lopinavir, and ritonavir are antiretroviral drugs used for the treatment of HIV in South Africa. Plasma concentrations of these drugs are routinely monitored to ensure efficacy, minimise adverse effects, and adjust dosing. However, variability exists in patient treatment response and tolerability, which cannot always be explained by the therapeutic drug monitoring results. This may be due to variability in the amount of drug reaching the target site within the HIV-infected cells. Therefore, intracellular drug concentrations could provide a more accurate depiction of drug exposure. An alternative to intracellular drug concentrations could be the quantitation of drug not bound to plasma proteins as this is the portion able to diffuse into tissues and cells to exert a therapeutic effect. A method is described for the quantification of intracellular efavirenz, lopinavir, and ritonavir from one million human peripheral blood mononuclear cells. In addition, the quantification of unbound efavirenz, lopinavir, and ritonavir from human plasma using ultracentrifugation is demonstrated, including a novel surrogate matrix. The two methods were validated according to the United States Food and Drug Administration and European Medicines Agency guidelines and proven to be accurate, precise, and reproducible. Both methods were submitted to the United States National Institute of Allergy and Infectious Diseases' Clinical Pharmacology Quality Assurance group for review and have been approved for use on clinical samples. A proof-of-concept correlation study of intracellular, unbound, and total drug concentrations is described using blood samples from six HIV-positive patients. A further patient unresponsive to lopinavir treatment, despite total plasma concentrations within the normal therapeutic range, was also evaluated. Paired plasma and cell samples indicated that the drug reached the target site within the cells, eliminating a possible cause of treatment failure. These findings show the utility and validity of these methods in a clinical setting to provide an overall view of treatment response and support their novel application in individualised patient care in South Africa
Development and validation of selective and sensitive LC-MS/MS Methods for determination of para-aminosalicyclic acid and cycloserine/terizidone applicable to clinical studies for the treatment of tuberculosis
Get PDFA method was validated for the quantification of para-aminosalicylic acid (PAS) in human plasma. The technique consisted of a protein precipitation extraction, followed by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) detection. Rilmenidine was used as the internal standard (ISTD). Analyte mean extraction yields determined were ~100.3% (CV % = 3.3). The extraction procedure was followed by liquid chromatographic separation using a Phenomenex Synergi Hydro-RP (150 x 2.0 mm, 4µm) analytical column. An isocratic mobile phase containing methanol, water and formic acid (40:59.8:0.2, v/v/v) was used at a flow-rate of 300 µl per minute. The retention times for PAS and rilmenidine were, ~2.4 and ~1.6 minutes, respectively. An AB Sciex API 3000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 154.1 and m/z 181.2 to the product ions m/z 80.2 and m/z 95.2 for PAS and the ISTD, respectively. Electro Spray Ionisation (ESI) was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for PAS over the range 0.391 – 100 µg/ml, based on peak area ratios. A 1:1 and 1:4 dilution of the QC Dilution sample showed that concentrations of up to 160 µg/ml of PAS in plasma could be analysed reliably when diluted into the calibration range. Endogenous matrix components were found to have an insignificant effect on the reproducibility of the method, when human plasma originating from eight different sources were analysed. PAS was found to be stable in human plasma for 21 months kept at ~-80°C, for up to 21 hours at room temperature and when subjected to 3 freeze-thaw cycles. Stock solutions of PAS in methanol were stable for 2 days when stored at ~80°C and for 24 hours when stored at room temperature, ~4°C and ~-20°C. Plasma extracts of the analyte/ISTD ratio were shown to be stable on instrument over a period of ~55 hours. Reinjection reproducibility experiments indicated that an assay batch may be re-injected within 58 hours. Quantification of PAS in plasma was not significantly affected by the presence of haemolysed blood (2%) in plasma and when Lithium Heparin was used as anti-coagulant instead of K3EDTA. The best marker for terizidone pharmacokinetics is the analysis of cycloserine, a small polar drug with limited potential for absorbing UV that makes it difficult to analyse. A method was validated for the quantification of cycloserine in human plasma, and consisted of a protein precipitation extraction and derivatization, followed by high performance liquid chromatography with MS/MS detection. No ISTD was used as no suitable match could be found. The mean extraction yield determined was ~77% (CV% = 10.7). The extraction procedure was followed by liquid chromatographic separation using a Gemini NX C18 (50 x 2.0 mm, 5µ) analytical column. An isocratic mobile phase containing acetonitrile, water and formic acid (30:69.9:0.1, v/v/v) was used at a flow-rate of 300 µl per minute. The retention time for cycloserine was ~ 1.5 minutes. An AB Sciex API 3000 mass spectrometer at unit resolution in the MRM mode was used to monitor the transition of the protonated precursor ion m/z 335.9 to the product ion m/z 157.2 for cycloserine. ESI was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for cycloserine over the range 0.313 – 40.0 µg/ml, based on peak areas. A 1:4 dilution of the QC Dilution sample showed that concentrations of up to 64.0 µg/ml of cycloserine in plasma could be analysed reliably when diluted into the calibration range and no carry over peaks were observed. Endogenous matrix components were found to have no effect on the reproducibility of the method when human plasma originating from six different sources was analysed. Cycloserine was found to be stable in human plasma for up to 18 hours at room temperature, and when subjected to 3 freeze-thaw cycles. Stock solutions of cycloserine in water and methanol were stable for 10 days when stored at ~ -80°C and for 18 hours when stored at room temperature, ~ 4°C and ~ -20°C. Long term stability in plasma has been proven for 17 months at -80°C. Plasma extracts of the analyte were shown to be stable on instrument over a period of ~ 29 hours. Reinjection reproducibility experiments indicate that an assay batch may be re-injected within 29 hours. Cycloserine is stable in whole blood (on ice) for up to 30 minutes. Both validated methods presented performed well on clinical samples generated from a multi drug resistant TB (MDR-TB) research study in children dosed with PAS and terizidone
Screening of actinobacteria for novel antimalarial compounds
Get PDFThe success of our first-line antimalarial treatments is threatened by increased drug resistance in Plasmodium parasites. This makes the development of novel drugs critical to combat malaria. Historically, natural products have been an excellent source of novel antimalarial compounds and thus are an ideal place to search for potential drugs. Filamentous members of the bacterial phylum, Actinobacteria, are well-known antibiotic producers, but their antimalarial potential has not been well investigated. This makes these actinobacteria a potentially valuable source of novel antimalarial compounds. To evaluate the antimalarial potential of the filamentous actinobacteria, uncharacterized environmental actinobacterial strains from the Meyers laboratory culture collection, as well as the type strains of new actinobacterial species identified and characterized in the Meyers laboratory, were screened for antiplasmodial activity against drug-sensitive Plasmodium falciparum, NF54. Liquid cultures were extracted using the mid-polar solvent, ethyl acetate, with the aim of discovering drug-like molecules that can be administered orally. Thirty-one strains of actinobacteria belonging to eight genera (Actinomadura, Amycolatopsis, Gordonia, Kribbella, Micromonospora, Nocardia, Nonomuraea, and Streptomyces) were screened revealing fourteen active strains. Eight strains were identified for further study as the displayed antiplasmodial efficacy matching predefined criteria. Of these eight candidates, Streptomyces strain PR3 was selected, as it showed excellent antiplasmodial efficacy, no cytotoxicity against Chinese Hamster Ovary (CHO) or liver HepG2 cell lines, no haemotoxicity, and was easy to culture. Bioassay-guided fractionation of the crude extracts of strain PR3, supported by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis, was conducted to isolate and identify the compounds responsible for the antiplasmodial activity. During purification by solid phase extraction (SPE), a novel class of compounds was isolated. The structure of these compounds was elucidated by HRMS and NMR analysis and determined to be a series of crown ethers with a methylated backbone. These methylated crown ethers (MCE) were not produced by strain PR3, but by the cyclization of polypropylene glycol (PPG) oligomers from Amberlite® XAD-16N 20–60 mesh resin under aqueous conditions. The MCEs displayed weak antiplasmodial activity against P. falciparum NF54, without cytotoxicity against the Chinese Hamster Ovary, HepG2 cell lines, nor human erythrocytes. To the author's knowledge, the MCEs are novel compounds, and this is the first time the cyclization of PPG oligomers into crown ethers has been reported. As the MCEs were not responsible for strain PR3's potent antiplasmodial activity, further study was conducted. Using the Global Natural Product Social molecular networking (GNPS) workflow, genome mining, and NMR analysis, it was revealed that the cyclodepsipeptides, valinomycin, montanastatin, and nine other novel analogues were responsible for the high antiplasmodial activity detected. A review of the literature revealed that the structure of four of these analogues had been predicted, based on MS/MS and the biosynthesis of valinomycin. Using the same described biosynthetic logic and MS/MS analysis, two new cyclodepsipeptides, compounds 1054 and 1068, were elucidated. Unfortunately, chromatographic systems developed were unable to purify the cyclodepsipeptides, and individual evaluation of their antiplasmodial efficacy and host selectivity was not possible. The fraction containing the cyclodepsipeptides exhibited strong antiplasmodial activity against the drug-sensitive, NF54 and multidrug-resistant K1, strains of P. falciparum. No cytotoxicity was displayed against the CHO cell line and no haemotoxicity was seen against human erythrocytes. Moderate toxicity was exhibited against the liver HepG2 cell line; however, the selectivity index of the cyclodepsipeptides suggested that they are selectively targeting the Plasmodium parasites. Overall, these results are positive, and further study of the individual cyclodepsipeptides is warranted. During the investigation, discrepancies were noticed between different fractions in terms of antiplasmodial activity. These fractions contained both the MCEs and, cyclodepsipeptides along with a range of impurities, yet they displayed potent antiplasmodial activity. Further study suggested that combination of the MCEs and cyclodepsipeptides elicits a synergistic response and improves antiplasmodial efficacy. This was determined independently using two models, the fixed-ratio isobologram method and the CompuSyn programme based on the massaction law principle. The workflow developed during this investigation demonstrates how new technologies can be used to dereplicate and elucidate bioactive natural products. This workflow can be utilized to continue this research and identify new natural products that can combat malari
The Development and Validation of a Direct LC-MS/MS Assay for the Determination of Tenofovir-diphosphate in Dried Blood Spots for the Analysis of Clinical Samples
Get PDFTenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis
Preclinical pharmacokinetic evaluation of novel antimalarial and antituberculosis drug leads
Get PDFPreclinical pharmacokinetics relies on efficient and accurate screening to select clinical candidates from early leads. Poor pharmacokinetic interpretation can disadvantage drug discovery by promoting inadequate compounds and expelling potential drug candidates. Objectives of this project included pharmacokinetic evaluation of antimalarial and anti-tuberculosis lead compounds with techniques aimed at improving preclinical pharmacokinetic outcomes. This included mechanistic pharmacokinetic approaches such as non-linear mixed effects (NLME) modelling in comparison with traditional non-compartmental analysis. Where appropriate, pharmacokinetic methods were expanded to include organ distribution and capsule dosing in mice to bridge our techniques from discovery to early development. Three benzoxazole amodiaquine analogues possessing equipotent in vitro antiplasmodial activity and showed diverse in vivo efficacy in a malaria mouse model. Evaluation of their respective pharmacokinetics in mice showed their in vivo exposures could translate to in vivo efficacy. Retrospective PK/PD simulations point to a time above IC50 drive in efficacy. Pharmacokinetic evaluation of an aminopyridine antimalarial compound in its cyclodextrin inclusion complex revealed a pH dependent increase in solubility that reduced variance, likely due to favoured intestinal absorption. Investigation of two novel fusidic acid C-3 ester prodrugs aimed at repositioning fusidic acid for tuberculosis, showed high concentrations of the rodent specific 3-epifusidic acid metabolite that greatly reduced exposure of fusidic acid in mice. Further organ distribution studies showed a prodrug strategy is still viable for repositioning fusidic acid for tuberculosis, but that rodent models are inappropriate for further evaluation. NLME modelling successfully provided unique mechanistic and mathematical insight of pharmacokinetic profiles of new leads. The level of interpretation on pharmacology parameters improved and aided in understanding why drug leads are likely to fail or succeed, assisting future compound optimisation
Development and validation of liquid chromatography mass spectrometry (L/C/MS/MS) assay for the determination of plasma 4betahydroxycholestrol and cholesterol in HIV infected children in Africa
No full textIncludes bibliographical references4β-hydroxycholesterol (4β-OHC) is a metabolite of cholesterol formed by Cytochrome (CYP) 3A4/5/7 enzymes. It has recently been proposed as an endogenous biomarker forCYP3A4/5/7 activity. This may be useful in prediction of drug-drug interactions and other metabolic processes affected by regulators of CYP3A activity. The aim of this study was to develop and validate an LC/MS/MS assay for the determination of 4β-OHC in human plasma and use 4β-OHC as a biomarker of CYP3A4/5/7 metabolism in HIV-infected children with and without treatment in Africa. Determination of 4β-OHC from plasma was performed by saponification and derivatisation reaction processes followed by high performance liquid chromatography with MS/MS detection on an AB Sciex Qtrap 5500 mass spectrometer. Since 4β-OHC is an endogenous metabolite in human plasma, a stable isotope labelled (SIL) analogue, 4β-OHC-D7, was used as a surrogate analyte for the preparation of calibration standards and quality controls. A second SIL analogue, 4β-OHC-D4 was used as the internal standard. The transitions of the protonated derivatised products were monitored atm/z 613, 620 and 617 to the product ions m/z 490, 497 and 494 for 4β-OHC, 4β-OHC-D7and 4β-OHC-D4 respectively. The calibration curve fitted a quadratic (weighted by1/concentration2) regression over the range 2-500 ng/ml. Validation accuracy and precision statistics summary for three consecutive runs were between 98.9% and 103%, and 3.5%and 12% respectively of all quality controls. The assay's recovery, selectivity and analyte stability were established. The validated assay was successfully applied on clinical samples, where 4β-OHC was used as a biomarker to investigate the levels of CYP3A induction in HIV-infected children with and without treatment containing non-nucleoside reverse transcriptase inhibitors (NNRI).It was found that plasma 4β-OHC concentrations at baseline were significantly lower in children belonging to the naïve group compared to nevirapine (NVP) and efavirenz (EFV)groups. When NVP and EFV groups were compared at non-baseline treatment weeks, the median 4β-OHC concentrations were significantly higher in EFV group than the NVP group. Regarding the effect of time on treatment, a significant increase in 4β-OHC concentrations was observed from baseline to each of the non-baseline weeks in naïve group. Conversely, in the NVP group, there was a significant decrease in 4β-OHC concentrations from baseline to each of the non-baseline weeks. Time did not show any significant effect on 4β-OHC concentrations in EFV group. Furthermore, at baseline, age, sex and weight did not affect 4β-OHC concentrations in all the three groups. This study has provided a method that would be utilised to determine plasma 4β-OHC concentrations using relatively small volumes - typical of samples taken from children. The results of this study suggest that children on antiretroviral therapy (ART) are at risk of effects of CYP3A induction, as indicated by the increase of 4β-OHC concentrations in the NVP and EFV groups. Additionally, prolonged use of the ART may activate some nuclear receptors that regulate CYP3A enzyme activity thereby negatively affecting, for example, the regulation of lipid and glucose metabolism. The developed method may therefore be useful in predicting drug-drug interactions in the context of multiple therapy and may also be used in predicting other metabolic processes affected by regulators of CYP3A activity. Further prospective studies with larger sample sizes are required to confirm and build on the evidence shown in this study
A pharmacokinetic and antimalarial efficacy evaluation of pyridodibemequines and their metabolites
Get PDFThe recurring challenge of the emergence of drug resistance necessitates the continual development of improved antimalarial treatments, which can target parasite strains that display reduced susceptibility towards current therapy. Consequently, a novel series of dual functioning pyridodibemequine (PDBQ) compounds were designed with the intention of reversing resistance in chloroquine-resistant (CQR) strains of Plasmodium falciparum. These hybrid molecules integrate a 4-amino-7-chloroquinoline antiplasmodial core with a modified dibenzylmethylamine side chain, which interacts with the CQR mutant P. falciparum chloroquine resistance transporter (PfCRT) to hinder the efflux of chloroquine (CQ) from its site of action, thereby reversing CQ resistance. The parent compounds of the PDBQ series, which differ in the ortho-, meta-, and para-orientation of the dibenzylmethylamine side chain, displayed favourable in vitro potency against chloroquine-sensitive (CQS) and CQR strains of P. falciparum; however, they were shown to be metabolically labile. Structure elucidation demonstrated that all formed PDBQ metabolites retained the 4-amino-7-chloroquinoline pharmacophore of the parent. Thus, the major metabolites, M1 and M2, generally conserved the in vitro antimalarial activity and selectivity of the parent compounds. Mechanistic studies revealed that the antiplasmodial activity of the PDBQ parent compounds and major metabolites primarily results from the inhibition of haemozoin formation, culminating in a toxic accumulation of ferriprotoporphyrin IX. The parents and major metabolites exhibited minimal toxicity against a mammalian cell line, and the metabolites are proposed to display reduced systemic toxicity, and human ether-a-go-go-related gene liability compared to the parent compounds. Furthermore, the major metabolites generally exhibited similar or improved in vitro solubility, permeability, lipophilicity, and metabolic stability compared to the parent compounds. Therefore, given their favourable in vitro characteristics, the major active metabolites of each parent PDBQ compound were further evaluated in this project to determine their potential as early preclinical antimalarial lead candidates. The proof of concept study presented herein investigated the in vivo pharmacokinetics (PK) of the PDBQ series of parents and major metabolites in a healthy murine model to allow the rational selection of candidates to be evaluated for their in vivo antimalarial efficacy and PK in a P. falciparum-infected murine model. For the PK studies in healthy and malaria-infected mice, analyte detection from whole blood was achieved using high-performance liquid chromatography coupled to tandem mass spectrometry. The bioanalytical methods were developed and partially validated based on a fit-for-purpose approach, which ensured that the generated PK concentration data was reliable and accurate. Lastly, given the significance of combination therapy in delaying the onset of drug resistance, fixed-dose ratio isobologram analyses were performed to probe the potential of the lead candidates to be used synergistically with an antimalarial partner drug possessing a distinct mechanism of action to haemozoin inhibition. Additionally, the ability of the lead candidates to reverse CQ resistance was evaluated in a CQR strain of P. falciparum. A comparative PK study was performed in a healthy murine model to determine whether the parent PDBQ compound should be used as a strategy to deliver the active metabolites or whether the active major metabolite should be directly administered. This was achieved by oral administration of the parent PDBQ compound and subsequent quantification of the parent compound and formation of the major metabolites. In addition, each pre-synthesised major metabolite was orally administered, at the equivalent parent dose, to characterise the PK profile of the individual active metabolite. These PK studies revealed that the overall oral exposure of the antimalarial pharmacophore was markedly greater after direct administration of the preformed metabolite compared to the cumulative oral exposure of the parent and formed metabolites after administration of the parent PDBQ compound. Furthermore, the metabolites attained higher maximal concentrations and maintained circulating concentrations which favourably exceeded their respective in vitro half-maximal inhibitory concentration at 24 h post-oral administration compared to the parent compounds at the equivalent oral dose. These findings substantiated the direct administration of the preformed PDBQ major metabolite over the parent compound. From the series of PDBQ metabolite derivatives, compounds 43M1 and 47M1 displayed the highest maximal concentrations of 8 ± 1 and 9.4 ± 0.5 μM, respectively and the greatest oral exposures of 62 ± 3 and 93 ± 9 µM.h, respectively, after single 20 mg/kg oral administrations of either 43M1 or 47M1. Given their encouraging PK profiles, 43M1 and 47M1 were selected to progress to the subsequent phase of the study which evaluated their in vivo antimalarial efficacy and pharmacokinetic/pharmacodynamic (PK/PD) relationship in a P. falciparum-infected humanised murine model. 43M1 and 47M1 were efficacious against asexual intraerythrocytic P. falciparum infection in humanised mice, where both compounds displayed a 98% reduction in parasitaemia after 4 consecutive daily oral administrations of 20 mg/kg of either 43M1 or 47M1 compared to the untreated control. The PK/PD analysis revealed dose-dependent reductions in parasitaemia; and the doses required to produce 90% of the maximal parasiticidal response (ED90) were 12 and 7.7 mg/kg for 43M1 and 47M1, respectively. Additionally, the oral exposures required to achieve the effect at the ED90 were 6.2 and 18.6 µM.h for 43M1 and 47M1, respectively. 43M1 or 47M1 demonstrated overall in vitro antimalarial synergy with dihydroartemisinin or atovaquone and additivity with methylene blue in CQS and CQR strains of P. falciparum. 43M1 or 47M1 with mefloquine or lumefantrine displayed in vitro antimalarial synergy in a CQS strain of P. falciparum; however, in a CQR strain, antagonism and additivity were displayed with mefloquine and lumefantrine, respectively. Additionally, 43M1 and 47M1 were unable to potentiate the in vitro antiplasmodial activity of CQ in a CQR strain of P. falciparum which suggested that, unlike the parent PDBQ compound, the metabolite did not possess the ability to reverse CQ resistance. The promising in vivo antimalarial efficacy of 43M1 and 47M1 against P. falciparum and their prospective in vitro antimalarial synergy with dihydroartemisinin and atovaquone underlines the potential of 43M1 and 47M1 for further development as preclinical antimalarial candidates
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