Browsing by Author "Norman, Jennifer"

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    Open Access
    Detectable HIV-1 in semen in individuals with very low blood viral loads
    (2020-03-05) Kariuki, Samuel M; Selhorst, Philippe; Norman, Jennifer; Cohen, Karen; Rebe, Kevin; Williamson, Carolyn; Dorfman, Jeffrey R
    Abstract Background Several reports indicate that a portion (5–10%) of men living with HIV-1 intermittently shed HIV-1 RNA into seminal plasma while on long term effective antiretroviral therapy (ART). This is highly suggestive of an HIV-1 reservoir in the male genital tract. However, the status of this reservoir in men living with HIV-1 who are not under treatment is underexplored and has implications for understanding the origins and evolution of the reservoir. Finding Forty-three HIV-1 positive, antiretroviral therapy naïve study participants attending a men’s health clinic were studied. Semen viral loads and blood viral loads were generally correlated, with semen viral loads generally detected in individuals with blood viral loads > 10,000 cp/ml. However, we found 1 individual with undetectable viral loads (<20cp/ml) and 2 individuals with very low blood viral load (97 and 333cp/ml), but with detectable HIV-1 in semen (485–1157 copies/semen sample). Blood viral loads in the first individual were undetectable when tested three times over the prior 5 years. Conclusions Semen HIV-1 viral loads are usually related to blood viral loads, as we confirm. Nonetheless, this was not true in a substantial minority of individuals suggesting unexpectedly high levels of replication in the male genital tract in a few individuals, despite otherwise effective immune control. This may reflect establishment of a local reservoir of HIV-1 populations.
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    Open Access
    Determination of Rifapentine and 25-O-desacetyl Rifapentine from 100 µl human breastmilk by LC-MS/MS using protein precipitation and solid phase extraction
    (2019) Mkhize, Buyisile; Wiesner, Lubbe; Kellermann, Tracy; Norman, Jennifer
    There is currently no information available on the transfer of the second-line anti-TB drug, rifapentine and its metabolite, into breastmilk. The subsequent implications to the breastfed infant, as well as consequences of long-term exposure to potentially sub-therapeutic drug levels with regards to the development of drug resistant bacteria is therefore not known. A liquid chromatography method with detection by mass spectrometry (LC-MS/MS) is described for the quantification of rifapentine and its metabolite, 25-O-desacetyl rifapentine in human breastmilk, using rifampicin-d3 as an internal standard. An AB Sciex 4000 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 877.5, m/z 835.4 and m/z 827.4 to the product ions m/z 151.1, m/z 453.2 and m/z 151.200 for rifapentine, 25-Odesacetyl rifapentine and rifampicin-d3, respectively. Ions were produced using Electro spray ionisation (ESI) in the positive ionisation mode. An Agilent Poroshell 120 EC-C18 (4.6 x 50 mm, 2.7 μm) column was used for chromatographic separation using an isocratic method of acetonitrile containing 0.1% formic acid and water containing 10% methanol and 0.1% formic acid (55:45, v/v), at a flow rate of 450 µl per minute. The retention times for rifapentine, 25- O-desacetyl rifapentine and rifampicin-d3 were ≈2.67, ≈1.88 and ≈1.96 minutes, respectively. The method was developed and validated according to FDA guidelines. The extraction method consisted of a combination of protein precipitation and C18 solid phase extraction. Rifapentine and 25-O-desacetyl rifapentine showed no significant carry over on the Agilent autosampler. The method was reproducible when analysed with human breastmilk from six different sources from Western Cape Maternity Breastmilk Bank. Rifapentine mean extraction yield was 84.2% (%CV = 1.7) and that of 25-O-desacetyl rifapentine was 71.1% (%CV = 10.8). Rifapentine had a mean process efficiency of 80.4% (%CV = 4.7) and that of 25-O-desacetyl rifapentine was 95.7% (%CV = 5.7). Intra- and inter day validations over 3 days were performed. The calibration curves fit a quadratic regression with 1/x weighting over a concentration range of 2 - 2000 ng/ml for both rifapentine and 25-Odesacetyl rifapentine based on the analyte/internal standard peak area ratios, the accuracy ranged from 92.9% to 105.5% for both rifapentine and 25-O-desacetyl rifapentine standards. The Quality Controls accuracy ranged from 97.4% to 106.0% for both rifapentine and 25-Odesacetyl rifapentine. Stock solutions were shown to be stable for 69 days at -80°C. v Rifapentine and 25-O-desacetyl rifapentine were stable in human breastmilk for up to 72 hours at approximately -80°C and -20°C, on benchtop for ≈4.5 hours on ice and after three freeze-thaw cycles. Rifapentine and 25-O-desacetyl rifapentine were shown to be stable on the autosampler over a period of approximately 48 hours after which the entire batch could be reinjected. Autosampler stability revealed a decrease in peak area ratios, indicating that a partial batch cannot be reinjected after 48 hours in case of instrument failure. This method will be utilized in the analysis of patient samples from a clinical study in South Africa in breastfeeding women with tuberculosis.
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    Potent in vivo anti-malarial activity and representative snapshot pharmacokinetic evaluation of artemisinin-quinoline hybrids
    (BioMed Central Ltd, 2013) Lombard, Marli; N'Da, David; Tran Van Ba, Christophe; Wein, Sharon; Norman, Jennifer; Wiesner, Lubbe; Vial, Henri
    BACKGROUND:Because Plasmodium falciparum displays increase tolerance against the recommended artemisinin combination therapies (ACT), new classes of anti-malarial drugs are urgently required. Previously synthesized artemisinin-aminoquinoline hybrids were evaluated to ascertain whether the potent low nanomolar in vitro anti-plasmodial activity would carry over in vivo against Plasmodium vinckei. A snapshot pharmacokinetic analysis was carried out on one of the hybrids to obtain an indication of the pharmacokinetic properties of this class of anti-malarial drugs. METHODS: In vitro activity of hybrids 2 and 3 were determined against the 3D7 strain of P. falciparum. Plasmodium vinckei-infected mice were treated with hybrids 1 - 3 for four days at a dosage of 0.8mg/kg, 2.5mg/kg, 7.5mg/kg or 15mg/kg intraperitoneally (ip), or orally (per os) with 2.7mg/kg, 8.3mg/kg, 25mg/kg or 50mg/kg. Artesunate was used as reference drug. A snapshot oral and IV pharmacokinetic study was performed on hybrid 2. RESULTS: Hybrids 1 - 3 displayed potent in vivo anti-malarial activity with ED50 of 1.1, 1.4 and <0.8mg/kg by the ip route and 12, 16 and 13mg/kg per os, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15mg/kg via ip route and at 50mg/kg by oral route for hybrid 1 and 2, whereas artesunate was only able to provide a complete cure at 30mg/kg ip and 80mg/kg per os. CONCLUSIONS: These compounds provide a new class of desperately needed anti-malarial drug. Despite a short half-life and moderate oral bioavailability, this class of compounds was able to cure malaria in mice at very low dosages. The optimum linker length for anti-malarial activity was found to be a diaminoalkyl chain consisting of two carbon atoms either methylated or unmethylated.
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    Simultaneous quantification of first-line anti-tuberculosis drugs and metabolites in human plasma
    (2019) Mazanhanga, Marian Tafadzwa; Wiesner, Lubbe; Joubert, Anton; Norman, Jennifer
    Tuberculosis (TB) currently kills more people than any other infectious disease worldwide, the highest burden being in Africa and Asia (1). Therapy recommended for drug sensitive TB consists of a cocktail of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB), all given in a 2-month intensive phase, followed by only INH and RIF in a 4-month continuation phase. Clinical studies seeking to optimize dosing, gain more knowledge on the pharmacokinetics and pharmacodynamics of the drugs and compare current therapy to alternative regimens are required (2, 3). Therapeutic drug monitoring (TDM) is frequently carried out in cases responding poorly to therapy (3, 4). Both clinical studies and TDM require bioanalytical methods for quantifying drug concentrations in biological fluids. Several methods have been developed, mostly analysing individual drugs but a few analyse combinations. Ideally, quantification of all four drugs in one method is desirable as it is economical and allows high throughput. A method was developed and validated for the quantification of first line anti-tuberculosis drugs EMB, INH, PZA and RIF and the metabolites N-acetyl isoniazid (AcINH) and 25- desacetyl rifampicin (desRIF). Sample preparation consisted of protein precipitation, followed by high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) detection. Deuterated internal standards for each analyte (AcINH-d4, desRIF-d3, EMB-d4, INH-d4, PZA-15N,d3 and RIF-d3) were used. Mean recoveries of the analytes from plasma were as follows: AcINH 106.5%, DesRIF 123.2%, EMB 105.3%, INH 110.1%, PZA 132.0% and RIF 127.7%. Sample preparation was followed by reverse phase liquid chromatography on an Agilent 1200 series HPLC system using an Agilent Poroshell 120 EC- C18 2.7µm, 4.6 X 50mm analytical column. Separation of all analytes was achieved using a mobile phase gradient consisting of an aqueous mobile phase A (0.05% formic acid in water) and an organic mobile phase B (0.05% formic acid in a mixture of methanol and acetonitrile, 1:1). A T-junction splitter was used to reduce the mobile phase flow to the ion source by about 30%. Retention times for AcINH, desRIF, EMB, INH, PZA and RIF were 2.45, 5.40, 1.75, 2.22, 4.30 and 5.68 minutes respectively. An AB Sciex API 4000 triple quadrupole mass spectrometer at unit mass resolution in the multiple reaction monitoring (MRM) mode was used for detection, monitoring the following transitions for the six analytes: AcINH 180 → 121, desRIF 784 → 752, EMB 205 → 116, INH 138 → 79, PZA 124 → 81 and RIF 823 → 792. An electrospray ionisation (ESI) source in the positive ion mode was used to couple the mass spectrometer to the LC system. Accuracy and precision were assessed over three consecutive and independent runs. The calibration curves fit quadratic regressions for all analytes, with weighting of 1/x (where x=concentration) for all except PZA which had a weighting of 1/x2 over the calibration range. Calibration ranges in µg/ml were as follows: AcINH 0.050 – 12.5, desRIF 0.040 – 10.0, EMB 0.020 – 5.00, INH 0.100 – 25.0, PZA 0.32. – 80.0 and RIF 0.120 – 30.0, based on peak area ratios. A 1:4 dilution of the QC Dilute sample showed that concentrations of up to 20.0 µg/ml for AcINH, 16.0 µg/ml for desRIF, 8.00 µg/ml for EMB, 40.0 µg/ml for INH, 128 µg/ml for PZA and 48.0 µg/ml for RIF in plasma could be analysed reliably when diluted into the calibration range. No significant carry-over was observed for all analytes. The method was shown to be reproducible when human plasma samples from six different sources were analysed and endogenous matrix components had no significant effect on the assay. All analytes were stable in plasma for at least four hours on ice, and when subjected to three freeze-thaw cycles. Reinjection reproducibility experiments showed that all analytes except PZA could be reliably analysed by re-injecting an entire batch after about 48 hours. Quantification of AcINH, INH and RIF was not significantly affected by 2% hemolysis of sample while desRIF, EMB and PZA were significantly affected. Data was analysed using Analyst ® version 1.6.2 software. With wide calibration ranges, the assay is suitable for both routine TDM and PK studies. Concurrent analysis of metabolites allows inferences to be made on the PK of the two main TB drugs. The total run time of 6.5 minutes per sample combined with the simple sample preparation procedure, the method is more economical on both time and resources than single analyte assays.