Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

What works in appraisal meetings for newly graduated doctors? - and what doesn't?

BACKGROUND: In Denmark a national formal advisory program (NFAP) is mandatory in Postgraduate Medical Education (PGME). According to this, an educational advisor is assigned to each doctor in every clinical rotation to guide and oversee the work and learning progress of the trainee. This study explores why newly graduated trainees evaluated the appraisal meetings in the advisory program as either beneficial (successes) or not beneficial (non-successes). METHODS: Inspired by the Success Case Method, a survey was conducted among all 129 doctors employed in their first six-month clinical rotation of postgraduate medical education (PGY1) in the Central Denmark Region. A cluster analysis resulted in a group with eight successes respectively seven non-successes. Semi-structured interviews were conducted with six successes and five non-successes. RESULTS: In contrast to non-successes, the successes had longer appraisal meetings and their advisor introduced them to purpose and process of meetings including use of the personal learning plan. Successes received feedback on clinical skills, overall global performance and career plans. The successes perceived their advisors as prepared, skilled and motivated and the advisor acted as a contact person. To the successes, the appraisal meetings fostered clarification of and reflections on educational goals, progress and career as well as self-confidence and a sense of security. CONCLUSION: Success with appraisal meetings seemed to depend on advisor's skills and motivation including willingness to prioritize time for this task. The results from this study indicate the importance of faculty development. It also raises the question if all doctors should serve as advisors or if this task should be assigned to the most motivated candidates.

Radiofrequency catheter ablation in 246 children with supraventricular tachyarrhythmia.

INTRODUCTION: Radiofrequency catheter ablation (RFA) is the treatment of choice for a variety of cardiac arrhythmias in adults. RFA is considered effective and is associated with few complications. We aimed to review the characteristics and outcomes of invasive electrophysiological study (EPS) and RFA in children with supraventricular tachyarrhythmia. METHODS: Consecutive patients younger than 16 years of age undergoing EPS and possible RFA from January 2009 to September 2018 at Aarhus University Hospital (uptake three million people) were reviewed retrospectively. Procedural and outcome data were collected from patient charts and from the Danish Ablation Database. Numbers (%) or median (range) are reported. RESULTS: A total of 304 patients (148 girls) underwent EPS (352 procedures). RFA was performed in 246 patients (279 procedures), aged 13 (1-15) years and weighing 46 (6-99) kg. Treatment success was achieved in 195 (79%) of the initial procedures. Using more than one procedure, 227 (92%) patients were free from arrhythmia after 89 (26-143) months of follow-up. The procedure time was 60 (22-222) min. and ablation time 2 (1-23) min. Major complications occurred in two cases. One patient developed transient superior vena cava syndrome and one patient developed an atrioventricular block requiring pacemaker implantation. CONCLUSIONS: RFA may be performed in children with a high success rate and a low but not negligible risk of complications. FUNDING: none. TRIAL REGISTRATION: Approval was obtained from the Danish Data Protection Agency (1-16-02-430-13).

Chronic medicine users' self-managing medication with information - A typology of patients with self-determined, security-seeking and dependent behaviors.

BACKGROUND: Information on medicines is key for safety and quality of care in long-term treatment courses with medicines. Little is known on how patients self-manage medication with information, and how interactions with health professionals influence such self-managing. OBJECTIVE: The objective of this study was to investigate how patients manage long-term medication with information, and how interactions with health professionals influence this managing, with the aim of developing a typology of patients' practices for managing with information. A secondary objective was to generate theoretical reflections on patients' roles in establishing resilience in health care systems. METHODS: Qualitative interviews with 15 chronic medicine users. A Safety-II-approach was used to obtain knowledge of what worked for medicine users, at the same time as acknowledging hindrances. Data were analyzed using thematic analysis and Halkiers' method for ideal-typologizing. RESULTS: Four types of practices for managing medication with information were identified, distinguished by patients' ways of self-managing on their own and through relations with health professionals: Ideal-type I: Self-determined and highly self-managing; Ideal-type II: Security-seeking and self-managing; Ideal-type III: Dependent with limited self-managing; Ideal-type IV: Co-managing with close family. The findings suggest that patients with a high degree of self-managing medication with information have good chances for facilitating quality of medical treatment. For patients who are more dependent on oral information from health professionals, the character of dialogue facilitated or hindered their self-managing. All patients had the best options for managing medication when being recognized by health professionals through dialogues. CONCLUSION: A typology of 4 types of managing practices was developed, characterized by patients' different abilities to self-manage medication with information and their relations to health professionals. Recognizing patients' different behaviors for managing medication with information is important for maximizing treatment quality of long-term medical treatment in a modern and resilient healthcare system.

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

A SWOT analysis of how the youngest doctors perceive the formal Danish educational advisory programme.

INTRODUCTION: A national formal advisory programme (NFAP) was introduced in Denmark in 1998. This study investigates the implementation of the NFAP and identifies areas to improve. METHODS: In March 2017, a survey was conducted among all 129 doctors employed in the first rotation of postgraduate medical education in the Central Denmark Region. A priority chart was created to appoint strengths, weaknesses, op-portunities and threats (SWOT). RESULTS: The response rate was 67%. The questionnaire showed good reliability and discriminant validity. Almost all respondents had completed the recommended appraisal meetings and a personal learning plan, both of which - in contrast to the NFAP's coherence to everyday clinical practice - showed to have much influence on the overall value of the NFAP. Strengths found were that appraisal meetings and learning plans support the development of clinical competencies, the latter identifying learning objectives and how to achieve them. Threats identified included learning plans that were not prepared sufficiently early, were not regularly adjusted and that did not describe when each learning objective is to be achieved, or when, by whom or how assessment will take place. CONCLUSIONS: Appraisal meetings and learning plans seem to be well implemented and to support the development of clinical competencies. Even so, improvements are needed, particularly to ensure an earlier preparation, inclusion of plans for assessment and regular adjustment of the learning plans. FUNDING: The study was funded by the Central Denmark Region. TRIAL REGISTRATION: Data collection was approved by the Danish Health Data Authority.

Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice.

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

Prevalence and geographical distribution of insulin pump therapy in the Central Denmark Region and its association with metabolic parameters.

AIMS: Insulin treatment in type 1 diabetes encompasses multiple daily insulin injections (MDI) or continuous subcutaneous insulin infusion (CSII). Both population-based studies and comparative studies regarding CSII use are sparse. The aim of the current study was to describe the prevalence and distribution of CSII use among adults with type 1 diabetes in the Central Denmark Region and to compare metabolic control in CSII-treated patients to those treated with MDI. METHODS: A database was constructed using the Danish Adult Diabetes Registry in 2014/2015 in combination with an audit of the patients' medical records. RESULTS: 3909 adults with type 1 diabetes patients were included. The proportion of patients treated with CSII differed significantly between the 8 regional hospitals from 12.0% to 31.1%. CSII users had a significantly lower HbA1c compared to MDI treated patients (7.6% (60 mmol/mol) versus 8.0% (64 mmol/mol)) in unadjusted analyses. After adjustment for clinically relevant characteristics the difference between CSII and MDI-treated patients was attenuated, but remained statistically significant. CONCLUSION: The distribution of CSII differed markedly between hospitals and CSII users had better glycemic control, even after adjustment for sex, age, BMI, diabetes duration, smoking, use of lipid-lowering and blood pressure-lowering medication.

Locked nucleic acid: modality, diversity, and drug discovery.

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.

Nineteen Years of Adult Congenital Heart Surgery in a Single Center.

BACKGROUND: Adults with congenital heart disease are a growing population. We describe surgical interventions, short- and long-term mortality and morbidity, and risk factors for adverse events in a population-based cohort. METHODS: Patients over or equal to 18 years with congenital heart disease who underwent cardiac surgery at Aarhus University Hospital, Denmark, from 1994 to 2012 were included in the study. Diagnoses, surgical procedures, postoperative complications, and survival were identified in hospital databases, medical records, and the Danish Civil Registration System. RESULTS: Four hundred seventy-four surgeries were performed in 445 adults (50% men). The median age was 39 years (range 18-83). Thirty-nine percent had previous surgical or catheter-based interventions. Thirty-day and in-hospital mortality were 1.1%. Postoperative complications occurred in 50% of cases, most were minor such as temporary arrhythmias and pneumonia. Major complications included postoperative bleeding necessitating intervention (6%), stroke (2%), and acute temporary renal failure (1%). Multivariate analysis identified RACHS-1 categories over or equal to 3 compared to category 1 (odds ratio (OR) = 2.3; 95% confidence interval (CI): 1.5-3.7), New York Heart Association functional class III and IV compared to class I (OR = 2.2; 95% CI: 1.3-3.7) and age at surgery (OR = 1.03, 95% CI: 1.01-1.04), as risk factors for adverse events. Survival during a median follow-up of 7.8 years (range 0 days-21.4 years) was 85% (95% CI: 80%-89%). CONCLUSION: Adults with congenital heart disease constitute a growing population with the need for cardiac surgery. Postoperative complications are frequent but early and late mortality are low.

[The effect of Christmas joy on the mood among medical doctors - a randomized, blinded intervention study].

INTRODUCTION: Each December Santa's elves spread Christmas joy (JN). Laughter and humour may influence health and stress level. No other study has investigated the effect of JN on the good spirit (DGH) among healthcare professionals. MATERIAL AND METHODS: We performed a single-centre blinded intervention study with crossover at three hospital departments. JN intervention of three days was randomized. Median ± standard deviation was given. The level of significance was p < 0.05. RESULTS: During a four-week period, we made 24 observations (response rate 67). The laugh index increased from 0.02 in November to 0.03 in December (without JN) and further to 0.05 with JN. At one department, the rise was significant. At a department without morning coffee, the DGH level raised after JN intervention corresponding to the level at the departments with morning coffee before JN intervention. CONCLUSION: Christmas atmosphere tended to increase DGH at the morning conferences. JN tended to have an additive effect. JN exposure may be beneficial. FUNDING: The study did not receive any funding. TRIAL REGISTRATION: The trial was not registered and was kept secret for the participants in accordance with the tradition of Santa's elves.

Photohemolysis Sensitized by the Furocoumarin Derivative Alloimperatorin and its Hydroperoxide Photooxidation Product.

The dark and photosensitized effects of alloimperatorin methyl ether 1 (hereafter simply alloimperatorin) and its photooxygenation product alloimperatorin hydroperoxide 2 were investigated on human erythrocytes. The results reveal that the furocoumarin 1 photosensitizes efficiently the hemolysis of erythrocytes. The rate of photohemolysis increases on raising the temperature of the postirradiated incubation from 4°C to 37°C. Thermal activation of the photohemolysis and inhibition by 2,6-di-tert-butyl-p-cresol (BHT) suggest that the furocoumarin 1 photosensitizes lipid peroxidation, increasing permeability in the erythrocyte membrane. The hydroperoxide 2 induces dark and photosensitized hemolysis more efficiently than the furocoumarin 1. The rate of hemolysis induced by 2 increases with the incubation temperature and decreases in the presence of tert-butanol and BHT. The hydroperoxide 2 photosensitizes the formation of lipid peroxidation products as shown by the reaction with thiobarbituric acid. This process is diminished by BHT. Our data imply that the photohemolysis sensitized by the furocoumarin 1 is caused by the in situ-formed photooxygenation product 2. Such hydroperoxides are potent hemolytic agents in the dark and especially on photosensitization.

Hepatotoxic potential of therapeutic oligonucleotides can be predicted from their sequence and modification pattern.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

[The communication with patients about analogue substitution is complex]. / Kommunikation med patienter om analog medicinsubstitution er kompleks.

Information about "effect" was considered more important than "price" on analogue substitution by a group of patients using the original medicine, esomeprazol, and having mixed experiences with generics. The word "cheap" implied negative associations to the expected effect and was associated with perception of more side effects. Patients adapted individually to substitution, e.g. by self-payment and -dosing.Furthermore they expressed themselves in methaphors, which was a figure lacking in written information to them. This qualitative study supports the need for, as well as more knowledge on, patient-oriented information and -dialogue on analogue substitution of medicines.

PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

Clinical evaluation of the accuracy and precision of the CDI 500 in-line blood gas monitor with and without gas calibration.

During cardiopulmonary bypass blood gases can be analyzed with laboratory equipment or with an in-line monitor giving instant results. The manufacturer of the CDI 500 in-line blood gas monitor recommends gas calibration before use. In acute cases there may not be time to perform a gas calibration. We hypothesized that after calibration against laboratory results, the CDI values of pH, pO2, and pCO2 will keep the same level of accuracy, whether the CDI has been gas calibrated or not. We performed a prospective randomized observational study using a study group without gas calibration (29 patients) and a control group with gas calibration (29 patients). Blood sampling was done at the beginning of bypass, and 30 minutes later. After each blood sample the CDI was in-vivo calibrated to the values simultaneously obtained from the ABL. Before in-vivo calibration values from the CDI without gas calibration were significantly different from the ABL-values in accuracy as well as precision, whereas the results from the gas calibrated CDI were largely consistent with the ABL. Before in-vivo calibration, the CDI without gas calibration was completely unreliable. After in-vivo calibration there was no statistical difference between the values of the CDI with and without calibration. We recommend gas calibration of the CDI before use in the period before in-vivo calibration.

3-Month Results from Denmark within the Globally Prospective and Observational Study to Evaluate Insulin Detemir Treatment in Type 1 and Type 2 Diabetes: The PREDICTIVE Study.

PREDICTIVEtrade mark is a large, multi-national, open-label, prospective, observational study to assess the efficacy and safety of insulin detemir in clinical practice. We report 3-month follow-up data from 389 patients with type 1 (n = 312) and type 2 (n = 77) diabetes from Denmark. Insulin detemir improved glycemic control in type 1 patients, with decreases in mean HbA1c (-0.2%, p = 0.0026), fasting glucose (-1.7 mmol/l, p = 0.0033) and within-patient fasting glucose variability (-0.6 mmol/l, p = 0.0472). Non-significant reductions in glycemic parameters were observed in type 2 patients (-0.3% for HbA1c and -2.7 mmol/l for fasting glucose). There was a decrease in mean body weight in both type 1 and type 2 patients (-0.6 kg, p = 0.025 and -1.0 kg, p = 0.0361, respectively). Three patients (0.8%) reported 4 serious adverse drug reactions, including major hypoglycemia. The incidence of major hypoglycemic episodes was reduced from 3.9/patient-years at baseline to 0.4/patient-years at follow-up in type 1 patients (p < 0.0001), and from 1.0 to 0.0/patient-years in type 2 patients (p = 0.1250). In addition, the mean incidence of total and nocturnal hypoglycemic episodes was reduced in both type 1 (-37.4 and -17.7/patient-years, p < 0.0001 for both) and type 2 patients (-17.7 and -7.8/patient-years, p = 0.0012 and p = 0.0020, respectively). The observations from the Danish cohort of the PREDICTIVE study support the overall findings of PREDICTIVE, i.e. insulin detemir improves glycemic control, with a reduced risk of hypoglycemia and no weight gain.

Overnight versus 24 hours of continuous subcutaneous insulin infusion as supplement to oral antidiabetic drugs in type 2 diabetes.

BACKGROUND: Basal continuous subcutaneous insulin infusion (CSII) therapy at a fixed rate may effectively improve glycemic control in patients with type 2 diabetes when oral antidiabetic treatment fails. Regimens of simple constant subcutaneous delivery of insulin may provide theoretical advantages in type 2 diabetes. METHODS: Ten subjects with type 2 diabetes who obtained insufficient glycemic control on oral antidiabetic drugs were included. Following an initial control day, two periods of 3 days with CSII of a rapid-acting insulin analogue, 1.5 IU/h (dose obtained from a preceding study), for 8 hours overnight and for 24 hours, respectively, were carried out in random order. Profiles of serum insulin aspart, serum endogenous insulin, and plasma glucose were recorded. RESULTS: Compared to the control day, an 8-hour overnight insulin infusion during a 3-day period improved fasting plasma glucose (FPG) (mean differences +/- SEM; Delta59.0 +/- 10.1 mg/dl; p < 0.01) and 2-hour postprandial plasma glucose (PPPG) (Delta57.8 +/- 10.6 mg/dl; p < 0.01) after breakfast. Compared to an 8-hour overnight infusion, a 24-hour infusion further improved all three PPPG values after breakfast, lunch, and dinner (Delta28.8 +/- 8.1 mg/dl, Delta30.6 +/- 8.1 mg/dl, and Delta35.1 +/- 7.9 mg/dl; p < 0.01). During insulin infusion, only one hypoglycemic episode with PG <55.8 mg/dl and mild symptoms was recorded. CONCLUSION: Continuous subcutaneous insulin infusion with a rapid-acting insulin analogue at a fixed rate of 1.5 IU/h, either overnight or for 24 hours, improved glycemic control without safety concerns in patients with type 2 diabetes who had secondary failure to oral antidiabetic drugs. The effect on FPG was similar for both treatments, whereas the effect on PPPG was superior when insulin was infused during the entire 24 hours.

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation.

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T(1) plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.