Benjamin U. Ebeshi

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).

Traditional Herbal Management of Sickle Cell Anemia: Lessons from Nigeria

Background. Patients in West Africa where sickle cell anemia (SCA) is endemic have for ages been treated with natural products, especially herbs, as, is still the case in rural communities. Objective. In this paper we look closely at some of these herbs to see if there are any lessons to be learnt or clues to be found for optimizing the treatments based on them, as had been done in the case of NIPRISAN, which was developed from herbs in Nigeria based on Yoruba Medicine. Methods. Select publications on SCA, its molecular biology and pathology, and actual and experimental cases of herbal treatment were perused in search of molecular clues that can be linked to chemical constituents of the herbs involved. Results. The study revealed that during the last 2-3 decades, much progress was made in several aspects of SCA pharmacology, especially the approval of hydroxyurea. As for SCA herbalism, this paper revealed that antisickling herbs abound in West Africa and that the most promising may yet be found. Three new antisickling herbs (Entandrophragma utile, Chenopodium ambrosioides, and Petiveria alliacea) were reported in May 2011. At NIPRD, where NIPRISAN was developed, three other recipes are currently awaiting development. Conclusion. The study raised the hope that the search in the Tropics for more effective herbal recipes for managing sickle cell anaemia will be more fruitful with time and effort.

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.

Essential Oils in Ginger, Hops, Cloves, and Pepper Flavored Beverages-A Review.

ABSTRACT In the West, sugar-based, ginger flavored beverages may contain hops, other flavorings, fruit juices, and varying levels of ethanol. Ginger ales contain 0.5%v/v; ginger beers >0.5%; and alcoholic ginger beers 0.5 ≤ 11%. Ales are carbonated by pressurized CO2, while beers and alcoholic beers are carbonated by yeast or ginger beer plant (GBP). In Africa, grain-based beverages include “fura da nono,” “kunu,” and “akamu,” which are spiced with one or more flavorings including ginger, black pepper, clove, chili pepper, or Aframomum alligator peppers. Spices have flavor because they contain essential oils (EOs), which are composed of aroma-active compounds (AACs). The benefits and toxicities of spices are ascribed to their EOs/AACs contents. Aim: Given the toxic potentials of EOs/AACs vis-à-vis their benefits, this review aimed to investigate the means by which the levels of EOs/AACs in spiced beverages are regulated. Methodology: The benefits and liabilities of key EOs/AACs of spices were identified and described. The methods for assaying them in raw materials and beverages were also identified. Results: There was a dearth of data on the levels of EOs/AACs in both raw and finished goods. Moreover, their assay methods were found to be tedious and costly. The implications of these findings on regulation are discussed. Conclusions: Owing to the practical difficulties in assaying flavors in beverages, both manufacturers and regulators should focus on: (i) the wholesomeness of raw materials; and (ii) good manufacturing practice (GMP). However, studies aimed at developing more robust methods for flavor should continue.

Diversity, utility, analytical methods and use implications of aroma-active compounds from select angiosperm families.

Introduction: An “aroma-active compound” (AAC) has a “flavor”ie: a “distinct taste and odor”. An example is menthol. All aromatic plants (APs), including some medicinal plants, such as Mentha×piperita (Family Lamiaceae), produce a group of fat-soluble secondary metabolites called “essential oils” (EOs) for various ecophysiological reasons. An EO has Review Article European Journal of Medicinal Plants, 4(9): 1046-1086, 2014 1047 a “flavor” because it contains one or more AACs. A typical EO is a complex mixture of several AACs, with wide ranging, dose-dependent pharmacological/ toxic effects. Owing to their complexity and variability, many EOs need to be standardized to ISO’s criteria. Professional use of EOs/ AAPs in food and drugs is controlled by good manufacturing practice (GMP). Aim: Given the immense diversities in sources, chemical structures, and bioactivities of EOs/ AACs, which are greatly patronized in foods and drugs, this review focused on their: i) sources in plants, beneficial attributes and liabilities; and ii) chemistry and analytical methods, in order to gain a better insight into their regulation in foods and drugs. Methodology: Using the 2009 Angiosperm Phylogenic Grouping (APG) of plants as a guide, pertinent literature was perused to ascertain: i) the taxa of APs; ii) their EOs/ AAPs; and iii) the methods for analyzing EOs/ AACs in raw materials (RMs) and finished products (FPs). Results: The literature revealed scores of AACs with varying health implications. But their levels in samples are usually unknown, or extremely hard to ascertain, owing to costs and complexities of the methods used. Conclusions: Given the wide ranging effects of EOs/ AAPs vis-a-vis the dearth of data on their levels in samples, it is recommended that their regulation in FPs should focus on: i) controlling the wholesomeness of RMs; and ii) on enforcing strict GMP in using such RMs. Meanwhile relevant agencies should sponsor research into more cost-effective methods.

Herbal Drug Development from Traditional Formulations: Refocusing Pharmaceutics and Posology for Accelerated Validation

Background: The World Health Organization (WHO) recommended that the toxicity data of a traditional medicine (TM) product that has been in use for 20 years or more without untoward effects should be determined, as the first step in its research and development Review Article British Journal of Pharmaceutical Research, 4(12): 1451-1476, 2014 1452 (R&D). Such data in conjunction with efficacy data would be used to develop an appropriate dosage form of the product. A key objective in researching such a product is to validate the basis of the therapy, including the formula. Such validation, and any attempt to modernize the product, should be guided by an understanding of the traditional know-how. The Nigerian National Institute for Pharmaceutical Research and Development (NIPRD) utilized this approach in developing Niprisan, an antisickling drug, based on a TM product used since antiquity in Yoruba Medicine. Aim: This article aimed to advocate the continuance and improvement of the WHO model of herbal drug research and regulation (HDRR) as the most logical approach for adoption by researchers and regulators. Methodology: NIPRD’s adoption of the WHO model since 1989 was reviewed in parallel with trends in herbal drug research worldwide; and within the contexts of regulatory practices by Nigeria’s National Agency for Food and Drug Administration and Control (NAFDAC) and the European Medicines Evaluation Agency (EMEA), with a view to identifying more effective strategies within the WHO paradigm for HDRR. Conclusion: Drug regulatory agencies (DRAs) like NAFDAC require effective laws, policies and quality management systems (QMS) to execute their mandates effectively. On the other hand, NIPRD’s output depends upon proper actions by a seasoned and responsive DRA. Therefore, noting that NIPRD and NAFDAC were both created by military decrees in 1989 and 1992 respectively, rather than by parliament acts, it is recommended that in addition to instituting more effective laws and policies to regulate NAFDAC, both NIPRD and NAFDAC need to adopt and implement suitable QMS for self-regulation, eg: ISO 9001 for whole organizations; and ISO/IEC 17250 for the laboratories.

Simple Reversed-Phase High Performance Liquid Chromatographic Estimation of the Antiretroviral Agent Efavirenz from Human Plasma

Aims: Sequel to the resurgence of TB co-infection in HIV/AIDS patients in sub-Saharan Africa, efavirenz has become an important component of the highly active antiretroviral treatment (HAART). The objective of this study therefore is to provide a simple reversedphase high performance liquid chromatographic (HPLC) method for the determination of efavirenz in human plasma. Study Design: Method development and experimental study. Place and Duration of Study: School of Pharmacy, University of Nairobi, Nairobi, Kenya, between October 2009 and September 2010. Methodology: A 500μl drug-free plasma sample was each placed in six different centrifuge tubes (2ml) and varying aliquots of the stock solution (100μg/ml) of efavirenz were spiked and vortexed for 60sec to give concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16μg/ml for calibration standards and 2.0, 4.0, 8.0 and 16.0μg/ml for quality control Research Article British Journal of Pharmaceutical Research, 4(1): 145-157, 2014 146 samples. The off-column sample pretreatment was carried out by protein precipitation using ice-cold acetonitrile. The samples were chromatographed in a phenomenex (C18) 5μm particle size column with 250x4.6mm I.D and UV detection at 254nm using a mobile phase, which was made up of a mixture of solutions A and B. Both consisted of acetonitrile, 25mM ammonium acetate buffer and glacial acetic acid in proportions of 90:10:0.1 and 10:90:0.1(v/v), respectively. The analytical technique was validated for precision, accuracy and analyte recovery. Results: The calibration plot for efavirenz was found to be linear over the concentration range of 0.5 to 16.0μg/ml with the regression line equation obtained as y=26842x–409.4 and the regression coefficient (R=0.999), which allows for accurate reading of the concentrations of the test samples. The RSD (%) in intraday and interday assays ranged from 0.44 to 0.78%. Accuracy ranged from 92 to 110% and the recovery was >97%. Conclusion: This new HPLC method is simple, reproducible and cost-effective and can be used for therapeutic drug monitoring of efavirenz in HIV/AIDS patients on HAART as demonstrated in this study.