Population genetic polymorphisms of pharmacogenes in Zimbabwe, a potential guide for the safe and efficacious use of medicines in people of African ancestry.

OBJECTIVE: Pharmacogenomics (PGx) is a clinically significant factor in the safe and efficacious use of medicines. While PGx knowledge is abundant for other populations, there are scarce PGx data on African populations and is little knowledge on drug-gene interactions for medicines used to treat diseases common in Africa. The aim of this study was to use a custom-designed open array to genotype clinically actionable variants in a Zimbabwean population. This study also identified some of the commonly used drugs in Zimbabwe and the associated genes involved in their metabolism. METHODS: A custom-designed open array that covers 120 genetic variants was used to genotype 522 black Zimbabwean healthy volunteers using TaqMan-based single nucleotide polymorphism genotyping. Data were also accessed from Essential Drugs' List in Zimbabwe (EDLIZ), and the medicines were grouped into the associated biomarker groups based on their metabolism. We also estimated the national drug procurement levels for medicines that could benefit from PGx-guided use based on the data obtained from the national authorities in Zimbabwe. RESULTS: The results demonstrate the applicability of an open-array chip in simultaneously determining multiple genetic variants in an individual, thus significantly reducing cost and time to generate PGx data. There were significantly high frequencies of African-specific variants, such as the CYP2D6*17 and *29 variants and the CYP2B6*18 variant. The data obtained showed that the Zimbabwean population exhibits PGx variations in genes important for the safe and efficacious use of drugs approved by the EDLIZ and are procured at significantly large amounts annually. The study has established a cohort of genotyped healthy volunteers that can be accessed and used in the conduct of clinical pharmacogenetic studies for drugs entering a market of people of predominantly African ancestry. CONCLUSION: Our study demonstrated the potential benefit of integrating PGx in Zimbabwe for the safe and efficacious use of drugs that are commonly used.

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress, and extracellular vesicle release in vascular smooth muscle cells.

AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms. METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine-induced human primary VSMC calcification, and increased osteogenic gene expression (Runx2, Osx, BSP, and OPN) and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide, we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels. CONCLUSION: In this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

Clinically relevant enantiomer specific R- and S-praziquantel pharmacokinetic drug-drug interactions with efavirenz and ritonavir.

We conducted a clinical study to determine the effect of efavirenz and ritonavir on the pharmacokinetics of R- and S-PZQ in healthy male participants. This was toward evaluating the risk of drug-drug interactions, which may occur after PZQ administration to HIV patients on efavirenz or ritonavir containing regimens. A non-randomized, open-label, single-dose, one sequence crossover study with 2 arms was conducted. We gave 26 healthy volunteers a single oral dose of 40 mg/kg PZQ followed by a daily oral dose of either 400 mg efavirenz or 100 mg ritonavir for 14 consecutive days. On day 14, they ingested a single 40 mg/kg dose of PZQ. We measured plasma levels up to 12 h on day 1 and day 14. Samples were analyzed by LC-MS. Pharmacokinetic analysis was conducted in WinNonlin to determine the primary endpoints (plasma T1/2 , Cmin , and AUC). Efavirenz had a significant effect on the pharmacokinetics of PZQ (p < .05), reducing the AUC by 4-fold (1213.15 vs. 281.35 h·ng/ml for R-PZQ and 5669 vs. 871.84 h·ng/ml for S-PZQ). Ritonavir had no significant effect on R-PZQ but increased the AUC 2-fold for S-PZQ (p < .05) (4154.79 vs. 7291.05 h·ng/ml). Using PZQ in HIV patients needs investigation, as there is a risk of both treatment failure and adverse effects because of induction and inhibition, respectively.

Healthcare Professionals' Knowledge of Pharmacogenetics and Attitudes Towards Antimicrobial Utilization in Zambia: Implications for a Precision Medicine Approach to Reducing Antimicrobial Resistance.

Introduction: Sub-Saharan Africa and other low- and middle-income countries (LMICs) have the highest rates of antimicrobial resistance (AMR) driven by high rates of antimicrobial utilization. This is a concern as AMR appreciably increases morbidity, mortality and costs. Pharmacogenetics (PGx) and precision medicine are emerging approaches to combat AMR. Consequently, as a first step there is a need to assess AMR knowledge and attitudes, and knowledge of PGx, among healthcare professionals and use the findings to guide future interventions. Methodology: We conducted a cross-sectional study involving 304 healthcare professionals at tertiary hospitals in Lusaka, Zambia. Structural Equation Modeling (SEM) was used to analyze relationships among latent variables. Results: Overall correctness of answers concerning AMR among healthcare professionals was 60.4% (7/11). Knowledge of pharmacogenetics was low (38%). SEM showed that high AMR knowledge score correlated with a positive attitude toward combating AMR (p < 0.001). Pharmacists had relatively higher AMR knowledge scores (mean = 7.67, SD = 1.1), whereas nurses had lower scores (mean = 5.57, SD = 1.9). A minority of respondents [31.5% (n = 95)] indicated that poor access to local antibiogram data promoted AMR, with the majority [56.5% (n = 190)] responding that poor adherence to prescribed antimicrobials can lead to AMR. Pharmacists had the highest scores for attitude (mean = 5.60, SD = 1.6) whereas nurses had the lowest scores (mean = 4.02, SD = 1.4). Conclusion: AMR knowledge and attitudes, as well as knowledge on PGx among healthcare professionals in Zambia, is sub-optimal and has the potential to affect the uptake of precision medicine approaches to reduce AMR rates. Educational and positive behavioral change interventions are required to address this and in future, we will be seeking to introduce these to improve the use of antimicrobials.

A critical role for thioredoxin-interacting protein in diabetes-related impairment of angiogenesis.

Impaired angiogenesis in ischemic tissue is a hallmark of diabetes. Thioredoxin-interacting protein (TXNIP) is an exquisitely glucose-sensitive gene that is overexpressed in diabetes. As TXNIP modulates the activity of the key angiogenic cytokine vascular endothelial growth factor (VEGF), we hypothesized that hyperglycemia-induced dysregulation of TXNIP may play a role in the pathogenesis of impaired angiogenesis in diabetes. In the current study, we report that high glucose-mediated overexpression of TXNIP induces a widespread impairment in endothelial cell (EC) function and survival by reducing VEGF production and sensitivity to VEGF action, findings that are rescued by silencing TXNIP with small interfering RNA. High glucose-induced EC dysfunction was recapitulated in normal glucose conditions by overexpressing either TXNIP or a TXNIP C247S mutant unable to bind thioredoxin, suggesting that TXNIP effects are largely independent of thioredoxin activity. In streptozotocin-induced diabetic mice, TXNIP knockdown to nondiabetic levels rescued diabetes-related impairment of angiogenesis, arteriogenesis, blood flow, and functional recovery in an ischemic hindlimb. These findings were associated with in vivo restoration of VEGF production to nondiabetic levels. These data implicate a critical role for TXNIP in diabetes-related impairment of ischemia-mediated angiogenesis and identify TXNIP as a potential therapeutic target for the vascular complications of diabetes.

Reduced metal ion concentrations in atherosclerotic plaques from subjects with type 2 diabetes mellitus.

AIMS: Transition metal ions have been implicated in atherosclerosis. The goal of this study was to investigate whether metal ion levels were higher in people with diabetes, in view of their increased risk of aggravated atherosclerosis. METHODS AND RESULTS: Absolute concentrations of iron, copper, zinc and calcium, and products of protein and lipid oxidation were quantified in atherosclerotic lesions from subjects with (T2DM, n=27), without Type 2 diabetes (nonDM, n=22), or hyperglycaemia (HG, n=17). Iron (P<0.05), zinc (P<0.01) and calcium (P=0.01) were lower in T2DM compared to nonDM subjects. Copper levels were comparable. A strong correlation (r=0.618; P<0.001) between EPR-detectable and total iron in nonDM patients was not seen in T2DM. X-ray fluorescence microscopy revealed "hot spots" of iron in both T2DM and nonDM. Calcium and zinc co-localised and levels correlated strongly. F(2)-isoprostanes (P<0.05) and di-Tyr/Tyr ratio (P<0.025), oxidative damage markers were decreased in T2DM compared to nonDM, or HG. CONCLUSION: Advanced atherosclerotic lesions from T2DM subjects unexpectedly contained lower levels of transition metal ions, and protein and lipid oxidation products, compared to nonDM and HG. These data do not support the hypothesis that elevated metal ion levels may be a major causative factor in the aggravated atherosclerosis observed in T2DM patients.

Dityrosine, 3,4-dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on milk proteins with globular and flexible structures as a result of riboflavin-mediated photo-oxidation.

Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals, with this species suggested to arise from oxidation of 3,4-dihydroxyphenylalanine (DOPA). The yield of this radical was lower in samples containing ß-casein than in samples containing only globular proteins. Consistent with this observation, the yield of DOPA detected on ß-casein was lower than that on two globular proteins, BSA and ß-lactoglobulin. In contrast, samples with ß-casein gave higher yields of dityrosine than samples containing BSA and ß-lactoglobulin. These results indicate that the flexible structure of ß-casein favors radical-radical termination of tyrosyl radicals to give dityrosine, whereas the less flexible structure of globular proteins decreases the propensity for tyrosyl radicals to dimerize, with this resulting in higher yields of DOPA and its secondary radical.

Accumulation of zinc in human atherosclerotic lesions correlates with calcium levels but does not protect against protein oxidation.

OBJECTIVE: Oxidized lipids and proteins, as well as decreased antioxidant levels, have been detected in human atherosclerotic lesions, with oxidation catalyzed by iron and copper postulated to contribute to lesion development. Zinc has been postulated to displace iron from critical sites and thereby protect against damage. In this study, metal ion and protein oxidation levels were quantified in human carotid and abdominal artery specimens containing early-to-advanced lesions, to determine whether zinc concentrations correlate inversely with iron levels and protein oxidation. METHODS AND RESULTS: Metal ions were quantified by EPR and inductively coupled plasma mass spectroscopy. Native and oxidized protein side-chains were quantified by high-performance liquid chromatography. Elevated levels of zinc ( approximately 6-fold) were detected in advanced lesions compared to healthy tissue or early lesions. Zinc did not correlate negatively with iron or copper levels suggesting that zinc does not displace these metal ions. Highly significant positive correlations (P<0.005) were detected between zinc and calcium levels. CONCLUSIONS: Zinc did not correlate with low iron levels and reduced protein oxidation. These data indicate that zinc does not prevent protein oxidation in advanced lesions. The reported protective effect of zinc accumulation is proposed to be associated with lesion calcification.

Smoking-induced monocyte dysfunction is reversed by vitamin C supplementation in vivo.

OBJECTIVE: The role of antioxidants in preventing vascular disease remains controversial. Vascular endothelial growth factor (VEGF-A) is important for endothelial and monocyte function. This study investigated the negative effects of smoking on monocyte migratory responsiveness to VEGF-A and the usefulness of vitamin C to prevent smoking-induced monocyte dysfunction. METHODS AND RESULTS: The chemotactic response of isolated monocytes from a cohort of 17 non-smokers and 10 smokers toward VEGF-A was assessed. VEGF-A significantly stimulated the migration of monocytes in non-smokers; the monocytes from smokers failed to respond to VEGF-A. Repeated analysis after 2 weeks of vitamin C intake (2 g/d) showed a fully restored VEGF-A-induced monocyte migration in smokers. VEGF-A serum levels were not altered by vitamin C. VEGF-A-inducible kinase activity was intact in monocytes from smokers as assessed by in vitro kinase assay. Monocyte dysfunction can be mimicked in vitro by challenging monocytes with a range of reactive oxygen species (ROS). CONCLUSIONS: Stimulation of monocyte migration by VEGF-A was severely attenuated in smokers, and the deficit observed was surmounted by vitamin C supplementation. The negative effects of smoking on monocyte function may translate into adverse impacts on VEGF-A-dependent repair processes such as arteriogenesis. These results propose a causative role of oxidative stress in smoking-induced monocyte dysfunction.

Concentrations of iron correlate with the extent of protein, but not lipid, oxidation in advanced human atherosclerotic lesions.

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.

Direct detection and quantification of transition metal ions in human atherosclerotic plaques: evidence for the presence of elevated levels of iron and copper.

OBJECTIVE: The involvement of transition metals in atherosclerosis is controversial. Some epidemiological studies have reported a relationship between iron (Fe) and cardiovascular disease, whereas others have not. Experimental studies have reported elevated levels of iron and copper (Cu) in diseased human arteries but have often used methods that release metal ions from proteins. METHODS AND RESULTS: In this study, we have used the minimally invasive technique of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICPMS) to quantify iron and copper in ex vivo healthy human arteries and carotid lesions. The EPR spectra detected are characteristic of nonheme Fe(III) complexes. Statistically elevated levels of iron were detected in the intima of lesions compared with healthy controls (0.370 versus 0.022 nmol/mg tissue for EPR, 0.525 versus 0.168 nmol/mg tissue by ICPMS, P<0.05 in each cases). Elevated levels of copper were also detected (7.51 versus 2.01 pmol/mg tissue, lesion versus healthy control, respectively, P<0.05). Iron levels did not correlate with the gender or age of the donor, or tissue protein or calcium levels, but cholesterol levels correlated positively with iron accumulation, as measured by EPR. CONCLUSIONS: These data support the hypothesis that iron accumulates in human lesions and may contribute to disease progression.