Regulation of gene expression of the 25-Hydroxyvitamin D 1α-Hydroxylase (CYP27B1) promoter : study of a transgenic mouse model.

The enzyme 25-hydroxyvitamin D la-hydroxylase or CYP27Bl is the key enzyme in the two-step activation process by which vitamin D is converted to its biologically active form 1,25-dihydroxyvitamin D (1,25D). The actions of a number of regulators on the renal CYP27B1 enzyme activity have been recognized for some years, although the underlying molecular mechanisms remain largely unknown and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated as yet. In order to identify the regulatory regions through which these factors control CYP27B1 expression in the kidney in vivo and to study the spatial and temporal expression of the CYP27B1 gene during development, a transgenic mouse model was established. This model was developed using pro-nuclear injection of a DNA construct containing the firefly luciferase reporter gene under the control of the 1541 bp region of the human CYP27B1 promoter. Following pro-nuclear injection, three transgenic founders were obtained and bred to establish three independent transgenic lines. In all three lines, a very similar expression pattern of the luciferase reporter gene was detected. High levels of luciferase activity were detected in the kidney, brain, testis, skin and bone. Lower levels of luciferase activity were detected in heart, lung, liver, distal small intestine, skeletal muscle and spleen extracts. No reporter gene expression could be detected in the proximal small intestine. This animal model was used to identify the ability of the 1541 bp promoter region of the CYP27B1 gene to respond in the kidney to a number of physiological challenges including dietary calcium, vitamin D and the immunomodulator LPS. In addition, the temporal expression of the reporter gene was studied by sacrificing animals at 6 different time points (2, 4, 6, 8, 12 and 64 weeks of age). The functionality of the CYP27B1 promoter was verified by comparing the regulation of the expression of the reporter gene with that of the endogenous CYP27B1 gene. The expression of endogenous CYP27B1 mRNA levels was therefore determined using Real-Time RT-PCR. The expression of the reporter gene and the endogenous CYP27B1 mRNA levels in the kidney were increased during early development (2 week old animals) and fell with increasing age. Reporter gene expression and CYP27BI mRNA levels were down-regulated in response to increasing amounts of dietary calcium in a dosedependent manner. Vitamin D-deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. However, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting that other regulatory elements are required to maximize the effect of vitamin D-deficiency. LPS administration did not affect the expression of either luciferase or the endogenous CYP27B1 gene in the kidney. Immunohistochemistry was used to identify the cell-specific location of the luciferase and the endogenous CYP27B1 protein in the kidney in kidney sections of vitamin D-deficient animals. Both luciferase protein and the endogenous CYP27B1 protein were identified in the proximal tubular cells of the kidney. The regulation of the expression of the reporter gene was also studied in the transgenic mouse model in a number of extra-renal tissues that have been shown to express CYP27Bl and to be responsive to 1,25D. These tissues include heart, liver, lung, femora, bone marrow, skeletal muscle, testis, skin, brain, spleen and proximal and distal small intestine. Although in most tissues, the expression of luciferase was highest in the 2 week old animals and fell with increasing age, in the testis, the expression levels were low in the developing animals and increased with increasing age. No physiological significant effects were detected in any of the extra-renal tissues examined in response to dietary calcium and vitamin D, suggesting that these factors control CYP27Bl expression in a kidney-specific manner. In addition, no physiologically significant effect of the LPS administration could be detected in these tissues. Future studies employing transgenic animals which express transgenic constructs containing both the CYP27Bl promoter and upstream and/or intronic sequences are required to identify the factors that regulate CYP27Bl expression in the different tissues and to delineate the DNA regulatory regions through which these factors exert their effects in vivo.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Patterns of High-Dose and Long-Term Proton Pump Inhibitor Use: A Cross-Sectional Study in Six South Australian Residential Aged Care Services

Aim: While proton pump inhibitors (PPIs) are generally considered safe and well tolerated, frail older people who take PPIs long term may be susceptible to adverse events. This study characterized PPI use and determined factors associated with high-dose use among older adults in residential aged care services (RACSs).Methods: A cross-sectional study of 383 residents of six South Australian RACSs within the same organization was conducted. Clinical, diagnostic, and medication data were collected by study nurses. The proportions of residents who took a PPI for > 8 weeks and without documented indications were calculated. Factors associated with high-dose PPI use compared to standard/low doses were identified using age- and sex-adjusted logistic regression models.Results: 196 (51%) residents received a PPI, with 45 (23%) prescribed a high dose. Overall, 173 (88%) PPI users had documented clinical indications or received medications that can increase bleeding risk. Three-quarters of PPI users with gastroesophageal reflux disease or dyspepsia had received a PPI for > 8 weeks. High-dose PPI use was associated with increasing medication regimen complexity [odds ratio (OR) 1.02; 95% confidence interval (CI) 1.01–1.04 per one-point increase in Medication Regimen Complexity Index score] and a greater number of medications prescribed for regular use (OR 1.11; 95% CI 1.01–1.21 per additional medication).Conclusions: Half of all residents received a PPI, of whom the majority had documented clinical indications or received medications that may increase bleeding risk. There remains an opportunity to review the continuing need for treatment and consider “step-down” approaches for high-dose PPI users.</p

The benefits and harms of deprescribing

• Currently, the strongest evidence for benefit of deprescribing is from cohort and observational studies of withdrawal of specific medication classes that have shown better patient outcomes, mainly through resolution of adverse drug reactions.• Deprescribing is the process of trial withdrawal of inappropriate medications.• More evidence is needed regarding negative, nonreversible effects of ceasing use of certain classes of medication, such as acetylcholinesterase inhibitors.• Cessation of use has not been studied for many medication classes, and large-scale randomised controlled trials of systematic deprescribing are required before the true benefits and harms can be known.• Additional potential benefits of deprescribing include reduced financial costs and improved adherence with other medications.• The harms of ceasing medication use include adverse drug withdrawal reactions, pharmacokinetic and pharmacodynamic changes and return of the medical condition. These can be minimised with proper planning (ie, tapering), monitoring after withdrawal, and reinitiation of the medication if the condition returns

Development and validation of the patients' attitudes towards deprescribing (PATD) questionnaire

Background There is a large amount of research into and promotion of rational prescribing, but there is a comparative lack of investigation into deprescribing. The success of deprescribing is likely to be dependent on both medical and patient factors. Objective The aim of this study was to develop and validate a tool to capture the views and beliefs of patients regarding cessation of medications. Setting Participants were recruited from a multidisciplinary clinic at the Royal Adelaide Hospital and Hampstead Rehabilitation Centre. Methods The patients' attitudes towards deprescribing (PATD) questionnaire was developed through expert opinion and piloting. Psychometric testing included face, content and criterion validity, sensitivity and test-retest reliability. Results A final 15 item questionnaire was produced. Through piloting, expert review and gamma rank correlation with the previously validated beliefs about medicines questionnaire, the PATD was determined to be valid. Test-retesting resulted in a total concordance of 71.3 % (95 % confidence interval, 64.1-78.5 %). Conclusion The PATD has acceptable psychometric properties and has potential for future use in research and practice to not only determine patients' willingness towards deprescribing, but also uncover what beliefs may influence this

People's attitudes, beliefs, and experiences regarding polypharmacy and willingness to deprescribe

The objective of this research was to capture people’s attitudes, beliefs, and experiences regarding the number of medications they are taking and their feelings about stopping medications. Participants were administered the 15-item Patients’ Attitudes Towards Deprescribing (PATD) questionnaire. Participants were individuals aged 18 and older (median 71.5) taking at least one regular prescription medication; 100 participants completed all items of the questionnaire, 65 of whom were aged 65 and older.