Oluseye O. Bolaji

Establishment of a biobank and pharmacogenetics database of African populations

Pharmacogenetics and genomics research has experienced great advances over the past decade as witnessed by the completion of the human genome in 2003 (www.genome.gov/HGP). The field has been driven by the belief that understanding the human genome, that of pathogens, and interindividual genetic variability would result in radical advances in medicine. Anticipated measurable end points include increased targets for which drug discovery campaigns could be initiated, increased understanding of human susceptibility to disease and variability in drug response hence development of diagnostic tools to realize individualized treatment where drugs would be given to patients in whom they are predicted to work and at doses predicted to be safe.1 Toward the realization of this biomedical paradigm, Biobanking and Pharmacogenetics Databasing have become well established in developed countries (www.biobanks.se, www.icelandbio.com, www.ukbiobank.ac.uk). Little has, however, been done in developing countries.2 Starting with a workshop organized by the African Institute of Biomedical Science and Technology (www.aibst.com) in 2003 on Pharmacogenetics of Drug Metabolism in Nairobi, Kenya, a number of African scientists initiated a consortium for the biobanking and pharmacogenetics databasing of African populations. We here report the results of the first phase of this initiative that has seen research groups from five different African countries with collaborative support from leading experts in Europe and America establish a biobank of blood and DNA from nine ethnic groups from across the African continent. The biobank of anonymous samples has been used to establish baseline frequency distribution of SNPs of genes important in drug metabolism, hence the initiation of a pharmacogenetics database (http://www.aibst.com/biobank.html).

High-performance liquid chromatographic method for the determination of drotaverine in human plasma and urine

A simple and sensitive HPLC method for the determination of drotaverine in human plasma and urine has been developed. Alkalinized plasma or urine was extracted with organic solvent and the basic components in the organic phase were back-extracted into 0.1 M HCl. An aliquot of the aqueous layer was injected onto the column and the eluent was monitored at 254 nm. Separation was performed on a C18-column with 0.02 M sodium dihydrogen phosphate-methanol (30:70, v/v) containing perchlorate ion at pH 3.2 as mobile phase. Drotaverine was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios and plasma concentrations and the intra- and inter-assay coefficients of variation were always < 10%. The lowest limit of detection (signal-to-noise ratio 3:1) was 6 ng/ml. The method is suitable for therapeutic monitoring and pharmacokinetic studies of drotaverine in humans as well as in animal models.

Pharmacokinetics of quinine in African patients with acute falciparum malaria.

The pharmacokinetics of quinine were studied in six Nigerian patients during acute uncomplicated falciparum malaria and convalescent periods. An oral dose of 10 mg/kg quinine dihydrochloride administered 8‐hourly for 7 days gave parasite and fever clearance times of 36.0 ± 16.6 h and 18.0 ± 6.4 h, respectively. From the individual quinine plasma profiles the mean plasma concentration of quinine at the time of parasite clearance was estimated as 4.5 ± 1.1 μg/ml. Plasma quinine levels during malaria rose rapidly reaching a peak around the second and third days and declining thereafter as patients improved clinically. In acute malaria plasma quinine levels were more than two-fold higher than in convalescence; the mean AUC(0-12) in malaria was 37.9 ± 14.7 μg.h/ml compared to 17.9 ± 8.5μg.h/ml in convalescence. The apparent oral clearance (CL/F) and volume of distribution (Vd/F) duri ng the acute phase of the malaria (1.9 ± 0.7 ml/min/kg and 1.8 ± 0.9 l/kg, respectively) were significantly lower than in convalescence (4.5 ± 2.1 ml/min/kg and 4.2 ± 3.2 l/kg). The present data suggest that malaria parasites in African patients are still very sensitive to quinine and that the current dosage of quinine is effective for the treatment of acute falciparum malaria in African patients without augmenting therapy with any other drug such as tetracycline or sulphadoxine-pyrimethamine. It also confirms that malaria significantly alters the pharmacokinetics of quinine in humans.

Column liquid chromatographic analysis of quinine in human plasma, saliva and urine.

A new simple, selective and reproducible high-performance liquid chromatographic method for the determination of quinine in plasma, saliva and urine is described. The ion-pair method was carried out on a reversed-phase C18 column, using perchlorate ion as the counter ion and ultraviolet detection at 254 nm. Quinine was well resolved from its major metabolite, 3-hydroxyquinine, and the internal standard, primaquine. The limit of detection was 10 ng/ml and the recovery was greater than 90% from the three biological fluids.

Pharmacokinetics and bioavailability of drotaverine in humans

SummaryThe pharmacokinetics and bioavailability of drotaverine was studied in 10 healthy volunteers after administration of single 80 mg oral and intravenous doses of the HCl salt of the drug, in a crossover fashion. Plasma and urine samples were analyzed for the unchanged drug by HPLC. The pharmacokinetic parameters, such as elimination half-life, plasma clearance, renal clearance and apparent volume of distribution, were not influenced by the route of drug administration. The drug was mainly eliminated by non-renal routes since renal clearance accounted for only 0.31±0.13% of the total plasma clearance. The absolute bioavailability was variable and ranged from 24.5–91% with a mean of 58.2±18.2% (mean ±SD). It is suggested that the high variation in the bioavailability of drotaverine HCl after oral administration may result in significant interindividual differences in therapeutic response.

Development, validation and clinical application of a novel method for the quantification of efavirenz in dried breast milk spots using LC-MS/MS

OBJECTIVES This manuscript describes the development, validation and clinical application of a novel method for the quantification of the antiretroviral drug efavirenz in dried breast milk spots using LC-MS. METHODS Dried breast milk spots were prepared by spotting 30 μL of human breast milk on each circle of Whatman 903 Protein Saver cards. Chromatographic separation was achieved on a reverse-phase C18 column with 1 mM ammonium acetate in water/acetonitrile using a solvent gradient at a flow rate of 400 μL/min and detection was by TSQ Quantum Access triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. The method was applied to characterize the breast milk pharmacokinetic profile of efavirenz in HIV-positive nursing mothers receiving regimens containing 600 mg of efavirenz once daily. RESULTS The assay was validated over the concentration range 50-7500 ng/mL. Accuracy ranged between 95.2% and 102.5% and precision ranged between 1.05% and 9.53%. The average recovery of efavirenz from dried breast milk spots was 106.4% and the matrix effect was 8.14%. Stability of efavirenz in dried breast milk spots and processed samples at room temperature, -40°C and -80°C was demonstrated. In the pharmacokinetic study, the mean (SD) AUC0-24, Cmax and Cmin of efavirenz in breast milk were 59,620 ng·h/mL (17,440), 4527 ng/mL (1767) and 1261 ng/mL (755.9), respectively. The mean (range) milk-to-plasma concentration ratio over the dosing interval was 0.78 (0.57-1.26). CONCLUSIONS The dried breast milk spot method is simple, robust, accurate and precise, and can be used in settings with limited resources.

Evidence for increased metabolism of chloroquine during the early third trimester of human pregnancy

Objective  To examine the possibility of a different extent of chloroquine (CQ) metabolism in human pregnancy by determining blood level profiles of the drug and its major metabolite, desethylchloroquine (CQM).

Validation and clinical application of a method to quantify nevirapine in dried blood spots and dried breast-milk spots

OBJECTIVES The validation and clinical application of an LC-MS/MS method for the quantification of nevirapine in dried blood spots (DBS) and dried breast-milk spots (DBMS) are presented. METHODS DBS and DBMS were prepared from 50 and 30 μL of nevirapine-spiked whole blood and human breast milk, respectively. Chromatographic separation was achieved on a reverse-phase C18 column with 0.1% formic acid in water/acetonitrile using a solvent gradient programme at a flow rate of 400 μL/min, and detection was by a TSQ Quantum Access triple quadrupole mass spectrometer. The clinical application was evaluated in HIV-positive nursing mothers and their breastfed infants. RESULTS The assay was validated over the concentration range 50-10,000 ng/mL. Accuracy ranged from 93.3% to 113.4% and precision ranged from 1.9% to 12.0%. The mean (percentage coefficient of variation) recovery of nevirapine from DBS and DBMS was ≥ 70.7% (≤ 8.2) and the matrix effect was ≤ 1.04 (≤ 6.1). Nevirapine was stable in DBS and DBMS for ≥ 15 months at room temperature and -80°C. Mean (SD) AUC0-12, Cmax and Cmin in maternal plasma versus breast milk were 57,808 ng · h/mL (24,315) versus 55,817 ng · h/mL (22,368), 6140 ng/mL (2605) versus 5231 ng/mL (2215) and 4334 ng/mL (1880) versus 4342 ng/mL (2245), respectively. The milk-to-plasma concentration ratio over the dosing interval was 0.94 (0.15). Infant plasma concentrations 2 and 8 h after maternal dosing were 580.6 ng/mL (464.7-1607) and 584.1 ng/mL (381.5-1570), respectively. CONCLUSIONS These methods further extend opportunities for conducting clinical pharmacokinetic studies in nursing mother-infant pairs, especially in resource-limited settings.

Sensitive high performance liquid chromatographic method for the determination of proguanil and its metabolites, cycloguanil and 4-chlorophenylbiguanide in biological fluids

A new simple, sensitive, cost-effective and reproducible high performance liquid chromatographic (HPLC) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 4chlorophenylbiguanide (4-CPB) in urine and plasma is described. The extraction procedure is a simple three-step process that has eliminated the need for costly extraction and evaporation equipment. The mobile phase consisted largely of buffer, making the method cheap to run. The calibration plots were linear over the concentration range up to 3.0 µg/ml PG, CG and 4-CPB in urine and concentration range up to 1000 ng/ml in plasma. The correlation coefficients (r) were of the order of 0.99 and above for PG and 4-CPB and 0.98 for CG. The ion pair method was carried out on a 5 µ reversed-phase C-18 column, using perchlorate ion as the counter ion and ultra violet detection at 254 nm. The method was reproducible with coefficient of variation for PG, CG and 4-CPB, being less than 10% in urine and plasma. PG was well resolved from its metabolites, CG and 4-CPB, and the internal standard, pyrimethamine. The limit of detection of PG was 10 ng/ml and the recovery was greater than 90% in urine and plasma. The analytical method therefore, exhibits good precision and sensitivity and is one of the few methods that can detect PG and the two metabolites CG and 4-CPB. The analytical method developed in this study was used to determine PG bioavailability after rectal administration in humans.

Characterization of the principal metabolite of quinine in human urine by 1H-n.m.r. spectroscopy

1. The major metabolite of quinine in human urine, which is also the sole metabolite in human plasma and saliva, has been identified and characterized by chemical ionization mass spectrometry and 1H-n.m.r. spectrometry. 2. The mass spectrum showed that an oxygen atom is incorporated in the quinuclidine nucleus, and the exact position of the oxidation was established from the n.m.r. spectrum to be at the C-3 position.