With the beginning of the Human Genome Project, an emerging field of science was brought to the forefront of the pharmaceutical community. Pharmacogenetics facilitates optimization of the current patient-centered care model and pharmacotherapy as a whole. Utilizing these ever-expanding branches of science to nonsteroidal anti-inflammatory drugs (NSAIDs) can provide novel opportunities to affect patient care. With a wide range of NSAID choices available as treatment options for relieving pain and/or reducing inflammation or fever, a more systematic way of selecting the ideal agent for the patients based upon their genetic information could spare them from a potentially permanent health-care condition. Furthermore, if a patient possesses or lacks certain alleles, serious adverse events can be anticipated and avoided. The tailoring of drug therapy can be achieved using the published data and cutting-edge genetic testing to attain a higher standard of care for patients.

Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition.

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Purpose: The scientific knowledge of how genes affect disease expression and evolution can facilitate more effective environmental and drug therapy interventions delivered by health care professionals. The purpose of this paper is to a) describe the role of genetic science in healthcare; b) explore genotype determinants for environmental and pharmacological interventions; c) and analyze ethical dilemmas, barriers to access, and allocation of resources based on genotype. Methods: A review of literature was conducted from the disciplines of nursing, medicine, psychology, and sociology using the CINAHL, Ebsco Host, Medline, and PsychINFO databases. The search was limited to peer reviewed, full text article in English that dated from 1987 to 2011. Inclusion criteria were articles describing environmental, pharmacologic, and nutritional influence on genetic expression. Forty-five articles on genetic intervention were chosen for further review, in addition to five book publications which met inclusion criteria. Many of the sources retrieved were obtained from the biomedical sciences and published in the last decade, owing to more recent innovations in genetic discovery. Results: Disease and treatment must be approached according to genetic profiles for effectiveness and to increase health outcomes. Several variations were found regarding response to pharmaceuticals, as well as environmental exposures, based on genotype. Conclusions: Health care has been practiced using a "universal protocol" approach; however, as the literature reveals, each individual genotype must be taken into account to provide optimal care.

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The Cytochrome P450 family of enzymes is responsible for the majority of Phase I metabolism, and has been identified as an important source of pharmacokinetic variation in therapeutic responses. CYP2C19 and CYP2D6, metabolising >35% of commonly prescribed medications, are two of the most important pharmacogenetic markers that have been studied with the aim of improving treatment response and reducing adverse drug reactions. The Food and Drug Administration (FDA) approved AmpliChip CYP450 Test (AmpliChip) was compared to a previously developed PCR-RFLP platform and a newly developed XLPCR+ Sequencing platform for the ability to identifying genotype and predicting phenotype for CYP2C19 and CYP2D6 respectively. The AmpliChip was found not to be genotypically comprehensive enough for evaluating CYP2C19 genotype, not robust enough for determining CYP2D6 genotype and inaccurate in predicting phenotype for both. The XLPCR+ Sequencing method identified three novel alleles and one sub-variant. Advances in online column-switching solid phase extraction generated a rapid and robust LCMS/ MS method for simultaneously quantifying the probe drugs omeprazole (CYP2C19 substrate), dextromethorphan (CYP2D6 substrate) and their metabolites. Antimodes were identified for phenotypic cut-offs which offered measured phenotype for comparison to predicted phenotype. Omeprazole metabolism by CYP2C19 correlated well with predicted phenotype in a demographically representative South African cohort. There are concerns regarding the use of omeprazole as a probe drug as participants predicted to be ultrarapid metabolisers for CYP2C19 had similar rates to extensive metabolisers. Regardless of this concern, decreased metabolism was assigned to the CYP2C19*15 for the first time. CYP2D6 predicted phenotype correlated very well with measured phenotype, validating the suitability of dextromethorphan use for measuring CYP2D6 metabolism. Substrate modified activity score using 0.5 to predict intermediate metabolisers fine-tuned the XLPCR+ Sequencing platform for phenotype prediction. This finding, along with observations in CYP2C19 metabolism of omeprazole, highlights the importance of substrate specific phenotype prediction strategies. Controversially, attempts to associate CYP2D6 phenotype prediction with risperidone-related adverse drug reactions has yielded conflicting results. The XL-PCR+Sequencing platform was able to discount this association by predicting a variety of metabolisers in a pilot cohort selected to be experiencing risperidone-related adverse drug reactions. The comprehensive capability of the XL-PCR+Sequencing allowed for the identification of an additional novel allele in this cohort. The data presented in thisthesis has provided insight into the relationship between predicted and measured phenotype for CYP2C19 and CYP2D6 in the South African population. The XL-PCR+Sequencing platform can be used for future research or can be applied to improve treatment outcome. The LC-MS/MS method developed could be used for future evaluations of predicted and measured phenotype with the ability to be adjusted for therapeutic drug monitoring. This thesis advances pharmacogenetics of CYP2C19 and CYP2D6 for use in the South African population. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: see item for full text / AFRIKAANSE OPSOMMING: sien item vir volteks

Thesis (MSC)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: South Africa is shouldering the highest burden of HIV-infection. Inter-individual differences in response to antiretroviral treatment (ART) and the limited availability of second and third-line ART regimens call for optimising first-line ART in South African populations. Measuring antiretroviral drug levels in patients may be of clinical value as an intermediate indicator of treatment response and may moreover serve to assess the genetic variation underlying differential drug exposure. This study aimed to determine the effect of SNPs in the CYP2B6 gene and efavirenz (EFV) levels measured in hair on ART outcomes in females of two South African populations. Female Xhosa (XH) (n = 81) and Mixed Ancestry (MA) (n = 53) patients receiving the first-line regimen component EFV for at least three months donated saliva for genomic DNA extraction and 20 strands of hair for determination of EFV concentrations by high performance liquid chromatography. Regulatory and exonic regions in the CYP2B6 gene, which codes for the major metabolising enzyme of EFV, were subjected to bi-directional sequence analysis in 15 XH and 15 MA individuals to assess common genetic variation in these populations. Out of 45 single nucleotide polymorphisms (SNPs) identified, 17 SNPs of known or predicted functional importance in EFV metabolism, including four novel SNPs, were genotyped in the entire patient cohort by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. All SNPs were tested for Hardy-Weinberg equilibrium (HWE) and maximum likelihood haplotypes and assessed for an association with EFV levels measured in hair, likelihood of developing adverse drug reactions (ADRs) and virological response to EFV-based treatment. After correcting for age and ethnicity, homozygous carriers of c.516G>T (CYP2B6*6) had significantly increased EFV levels (p = 0.0021; mean: 12.0 ng/mg; IQR: 3.95 – 6.99 ng/mg; n = 12), as did heterozygotes of c.983T>C (CYP2B6*18) (p = 0.0005; mean: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). No CYP2B6*18 homozygotes were detected. No association between EFV levels and virological response was evident (p = 0.8467), but CYP2B6*6 predicted increased odds of virological failure (VL > 80 copies/ml) after correcting for adherence, race, age, weight, time on treatment, baseline CD4, smoking, alcohol and WHO disease stage (p = 0.0328). Carriers of the CYP2B6*1 allele had increased odds (OR = 5) of favourable treatment outcome (VL < 80 copies/ml). In accordance with other studies, this study provides evidence that genetically predisposed poor metabolisers of EFV may be at increased risk of virological failure, possibly following non-adherence. Concurrently, these patients may be more vulnerable to adverse drug reactions and are more frequent in the XH (13%) than MA (4%). These results should be verified in larger patient cohorts, but contribute to a better understanding of the effect of genetic factors on EFV exposure and ART outcome in two South African populations. The outcomes of this study may thus provide recommendations for prospective studies and impact future clinical decisions. / AFRIKAANSE OPSOMMING: Suid-Afrika dra die grootste las van MIV-infeksies. Inter-individuele verskille in reaksie op anti-retrovirale terapie (ART) en die beperkte beskikbaarheid van tweede- en derde-linie ART-reekse regverdig die optimisering van eerste-linie ART in Suid-Afrikaanse bevolkings. Meting van antiretrovirale middel-vlakke in pasiënte, as ‘n intermediêre aanduiding van reaksie op behandeling, kan van kliniese belang wees en kan ook die waarde van die bepaling van genetiese variasie, onderliggend aan differensiële blootstelling aan middels, bepaal. Die doel van hierdie studie is om die effek van enkel-nukleotied polimorfismes (SNPs) in die CYP2B6-geen en efavirenz (EFV)-vlakke in hare op ART-uitkoms te bepaal in vroue van twee Suid-Afrikaanse bevolkingsgroepe. Vroulike Xhosa (XH) (n = 81) en Gemengde Herkoms (GH) (n = 53) pasiënte wat EFV as deel van eerste-linie ART vir ten minste drie maande ontvang het, het speekselmonsters vir genomiese DNA-ekstraksie en 20 hare vir die bepaling van EFV-konsentrasies deur hoë werkverrigting vloeistofchromatografie (“HPLC”) geskenk. Regulatoriese en eksoniese areas in die CYP2B6-geen, wat vir die vernaamste metaboliserende ensiem van EFV kodeer, is deur middel van tweerigting-volgordebepalings-analise in 15 XH en 15 GH individue ondersoek om gemeenskaplike genetiese variasie in hierdie bevolkings te bepaal. Uit ‘n totaal van 45 SNPs wat geïdentifiseer is, is 17 SNPs wat bekende of voorspelde belangrike rolle in EFV-metabolisme speel, insluitend vier nuwe SNPs, ondersoek. Hierdie SNPs is in die volledige pasiënt-kohort gegenotipeer deur polimerase-ketting reaksie gebaseerde restriksie fragment lengte-polimorfisme (PKR-RFLP) analise. Alle SNPs is getoets vir Hardy-Weinberg-ewewig (HWE) en maksimum waarskynlikheidshaplotipes en is geassesseer vir assosiasie met EFV-vlakke gemeet in hare, die waarskynlikheid om ongunstige reaksies tot die middel te ontwikkel en virologiese reaksie op EFV-gebaseerde behandeling. Nadat vir ouderdom en herkoms gekorrigeer is, het homosigotiese draers van c.516G>T (CYP2B6*6) beduidend verhoogde EFV-vlakke (p = 0.0021; gemiddeld: 12.0 ng/mg; IQR: 3.95 – 6.99; n=12) getoon, so ook heterosigote vir c.983T>C (CYP2B6*18) (p = 0.0005; gemiddeld: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). Geen CYP2B6*18 homosigote is gevind nie. Daarbenewens is geen duidelike assosiasie tussen EFV-vlakke en virologiese reaksie gevind nie (p = 0.8467), maar CYP2B6*6 het verhoogde waarskynlikheid op virologiese mislukking (VL > 80 kopieë/ml) getoon nadat daar vir mddel-getrouheid, ras, ouderdom, gewig, tydsduur van behandeling, basis-CD4, rook, alkohol en Wêreld Gesondheids Organisasie siekte-fase gekorrigeer is (p = 0.0328). Draers van die CYP2B6*1-alleel het verhoogde waarskynlikheid (OR = 5) op gunstige behandelingsuitkomste getoon (VL < 80 kopieë/ml). In ooreenstemming met ander studies verskaf hierdie studie bewyse dat pasiënte wat geneties geneig is tot stadige metabolisme van EFV ‘n hoër risiko kan hê vir virologiese mislukking, wat moontlik ‘n gevolg is van middel-ontrouheid. Hierdie pasiënte kan ook meer geneig wees tot vatbaarheid vir ongunstige middel-reaksie en kom meer voor in die XH (13%) as in die MA (4%). Hierdie resultate moet in groter pasiënt-kohorte gestaaf word, maar dra by tot ‘n beter begrip van die effek van genetiese faktore op blootstelling aan EFV en ART-uitkoms in twee Suid-Afrikaanse bevolkings. Die uitkomste van hierdie studie kan dus as aanbevelings gebruik word vir voornemende studies en ook toekomstige kliniese besluite beïnvloed. / The Medical Research Fund (MRC) for funding this project. The University Centre for Studies in Namibia (TUCSIN) and Deutscher Akademischer Austausch-Dienst (DAAD) for financial support