Validation of an Isavuconazole High-Performance Liquid Chromatography Assay in Plasma for Routine Therapeutic Drug Monitoring Applications.

BACKGROUND: Isavuconazole is a triazole antifungal agent for treatment of invasive aspergillosis and mucormycosis. At present, it is unclear whether a therapeutic drug monitoring (TDM) of isavuconazole would be necessary. The aim of the investigation was to validate a high-performance liquid chromatography (HPLC) assay for routine applications. METHODS: An HPLC assay for routine determination of isavuconazole in plasma has been adapted and validated. The assay used the reagents and HPLC column provided by the ChromSystems HPLC Kit for TDM of itraconazole, posaconazole, and voriconazole. Isocratic flow rate was set at 1.0 mL/min. Detection was performed using a fluorescence detector with excitation wavelength set at 261 nm and emission wavelength set at 366 nm. RESULTS: The assay was linear between 0.15 and 10 mg/L with intraday and interday imprecision and accuracy <10% (<20% at lower limit of quantification). The method was applied to routine TDM of 7 patients after hematopoietic stem cell transplantation (n = 31 samples). In these patients, trough levels ranged from 0.45 to 3.06 mg/L (median 1.44 mg/L). CONCLUSIONS: A robust and simple HPLC assay of isavuconazole in plasma for routine TDM applications is presented.

Risk approximation in decision making: approximative numeric abilities predict advantageous decisions under objective risk.

Many decision situations in everyday life involve mathematical considerations. In decisions under objective risk, i.e., when explicit numeric information is available, executive functions and abilities to handle exact numbers and ratios are predictors of objectively advantageous choices. Although still debated, exact numeric abilities, e.g., normative calculation skills, are assumed to be related to approximate number processing skills. The current study investigates the effects of approximative numeric abilities on decision making under objective risk. Participants (N = 153) performed a paradigm measuring number-comparison, quantity-estimation, risk-estimation, and decision-making skills on the basis of rapid dot comparisons. Additionally, a risky decision-making task with exact numeric information was administered, as well as tasks measuring executive functions and exact numeric abilities, e.g., mental calculation and ratio processing skills, were conducted. Approximative numeric abilities significantly predicted advantageous decision making, even beyond the effects of executive functions and exact numeric skills. Especially being able to make accurate risk estimations seemed to contribute to superior choices. We recommend approximation skills and approximate number processing to be subject of future investigations on decision making under risk.

Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli.

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain--using chromosomal gene knock-in techniques--that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.

Fluorescence-based monitoring of ribosome assembly landscapes.

BACKGROUND: Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. RESULTS: We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution. CONCLUSION: This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.

Evaluation of limited sampling models for prediction of oral midazolam AUC for CYP3A phenotyping and drug interaction studies.

BACKGROUND: The area under the concentration-time curve (AUC) after oral midazolam administration is commonly used for cytochrome P450 (CYP) 3A phenotyping studies. The aim of this investigation was to evaluate a limited sampling strategy for the prediction of AUC with oral midazolam. METHODS: A total of 288 concentration-time profiles from 123 healthy volunteers who participated in four previously performed drug interaction studies with intense sampling after a single oral dose of 7.5 mg midazolam were available for evaluation. Of these, 45 profiles served for model building, which was performed by stepwise multiple linear regression, and the remaining 243 datasets served for validation. Mean prediction error (MPE), mean absolute error (MAE) and root mean squared error (RMSE) were calculated to determine bias and precision RESULTS: The one- to four-sampling point models with the best coefficient of correlation were the one-sampling point model (8 h; r (2) = 0.84), the two-sampling point model (0.5 and 8 h; r (2) = 0.93), the three-sampling point model (0.5, 2, and 8 h; r (2) = 0.96), and the four-sampling point model (0.5,1, 2, and 8 h; r (2) = 0.97). However, the one- and two-sampling point models were unable to predict the midazolam AUC due to unacceptable bias and precision. Only the four-sampling point model predicted the very low and very high midazolam AUC of the validation dataset with acceptable precision and bias. The four-sampling point model was also able to predict the geometric mean ratio of the treatment phase over the baseline (with 90 % confidence interval) results of three drug interaction studies in the categories of strong, moderate, and mild induction, as well as no interaction. CONCLUSION: A four-sampling point limited sampling strategy to predict the oral midazolam AUC for CYP3A phenotyping is proposed. The one-, two- and three-sampling point models were not able to predict midazolam AUC accurately.

Intraindividual long term stability and response corridors of cytokines in healthy volunteers detected by a standardized whole-blood culture system for bed-side application.

BACKGROUND: The variation of immune cell activities over time is an immanent property of the human immune system, as can be measured by the stimulated secretion of cytokines in cell cultures. However, inter-individual variability is considerably higher. Especially the latter is the major reason why it has not been possible to establish international standard values for cytokines as was possible for other parameters, such as leukocyte sub-population numbers. In this trial, a highly standardized whole-blood culture model (TrueCulture®), developed to characterise drug effects on cells of the human immune system in clinical trials, was used to analyse cytokine patterns in the blood samples of 12 healthy subjects over a period of one month. METHODS: After an overnight fast, 12 healthy subjects donated blood three times a week on three consecutive days over a period of 4 weeks. TruCulture® blood collection and whole-blood culture systems were used to measure whole-blood leukocyte stimulation. The levels of IL-2, IL-5, IL-13, IL-6, IL-8, IL-10, IFNγ, and MCP-1 in the culture supernatants were quantified by sandwich ELISA. RESULTS: The pattern of cytokine concentrations in the supernatants of the stimulated whole-blood cultures was highly individual, but considerably stable over the whole observation period of 4 weeks. CONCLUSIONS: By using TruCulture® it seems feasible to determine subject-specific cytokine reference patterns, for example under healthy conditions, or before starting an experimental treatment, e.g. during a clinical trial, against which changes in the behaviour of the immune system can be detected more accurately in future.

Adverse drug reactions in Germany: direct costs of internal medicine hospitalizations.

PURPOSE: German hospital reimbursement modalities changed as a result of the introduction of Diagnosis Related Groups (DRG) in 2004. Therefore, no data on the direct costs of adverse drug reactions (ADRs) resulting in admissions to departments of internal medicine are available. The objective was to quantify the ADR-related economic burden (direct costs) of hospitalizations in internal medicine wards in Germany. METHODS: Record-based study analyzing the patient records of about 57,000 hospitalizations between 2006 and 2007 of the Net of Regional Pharmacovigilance Centers (Germany). All ADRs were evaluated by a team of experts in pharmacovigilance for severity, causality, and preventability. The calculation of accurate person-related costs for ADRs relied on the German DRG system (G-DRG 2009). Descriptive and bootstrap statistical methods were applied for data analysis. RESULTS: The incidence of hospitalization due to at least 'possible' serious outpatient ADRs was estimated to be approximately 3.25%. Mean age of the 1834 patients was 71.0 years (SD 14.7). Most frequent ADRs were gastrointestinal hemorrhage (n = 336) and drug-induced hypoglycemia (n = 270). Average inpatient length-of-stay was 9.3 days (SD 7.1). Average treatment costs of a single ADR were estimated to be approximately €2250. The total costs sum to €434 million per year for Germany. Considering the proportion of preventable cases (20.1%), this equals a saving potential of €87 million per year. CONCLUSIONS: Preventing ADRs is advisable in order to realize significant nationwide savings potential. Our cost estimates provide a reliable benchmark as they were calculated based on an intensified ADR surveillance and an accurate person-related cost application.

Sleeve Gastrectomy: A Procedure in a State of Flux.

Originally, sleeve gastrectomy (SG) was part of biliopancreatic diversion (BPD), which is the most effective bariatric procedure for super obese patients. As BPD is a complex procedure with substantial morbidity and mortality, attempts were undertaken to split the procedure into two steps. SG became the first step in a staged BPD procedure assuming that this less-invasive operation would reduce the surgical risk in super obese patients. The second step - the duodenal switch - was thus postponed until after a major weight loss. Several studies have since been published showing data that, besides the successful weight loss, the procedure itself had a positive effect on obesity-associated comorbidities. Sleeve gastrectomy has gained widespread acceptance as a primary and definite bariatric procedure. SG has become an innovative tool in the battle against obesity. Although several variations of SG have been described, standardization is paramount for optimal results. Of particular interest are: the minimally invasive access, the dissection, preservation of the antrum, position of staple-line and buttress material, as well as the size of the bougie and the extent of fundal resection. This article describes the different procedural and technical aspects of the operation. In addition, it will line out how we transferred our skills and experience in single incision laparoscopic surgery (SILS) from cholecystectomy and sigmoid resection to sleeve gastrectomy.

Periprosthetic Fungal Infections in Severe Endoprosthetic Infections of the Hip and Knee Joint-A Retrospective Analysis of a Certified Arthroplasty Centre of Excellence.

The treatment of periprosthetic joint infections (PJI), and especially of re-infections, poses a highly complex problem in orthopaedic surgery. While fungal infections are rare, they present a special challenge. The therapy is often protracted and based on limited evidence. A total of 510 hip and knee revision surgeries were analysed for the occurrence of bacterial and fungal PJI. In patients with PJI, the duration of the hospital stay and the incidence of disarticulation of the infected joint were recorded. Out of the analysed revision arthroplasties, 43.5% were due to PJI. Monomicrobial infection occurred in 55.2%, dual microbial infection in 21.4%, and polymicrobial (≥3 different bacterial or fungal species) infection in 17.2% of the cases. Overall, Candida species were detected in 12.4% cases. Candida albicans was the main fungal pathogen. In 6.9% of cases, disarticulation of the joint was the only option to control PJI. The detection of polymicrobial infection more than doubled in follow-up revisions and there was a strong association between detection of Candida infection and disarticulation (OR 9.39). The majority of fungal infections were mixed infections of bacteria and Candida albicans. The choice of a biofilm penetrating antimycotic, e.g., caspofungin, together with a sufficient standard procedure for detection and surgical treatment can help to control the infection situation. Fungal infection often proves to be more difficult to treat than anticipated and is more frequent than expected.

No clinically relevant CYP3A induction after St. John's wort with low hyperforin content in healthy volunteers.

OBJECTIVE: Induction of CYP3A by St. John's wort (SJW) products with high hyperforin content is well described. Since CYP3A induction is mediated by hyperforin in a concentration-dependent manner, and SJW preparations differ significantly in hyperforin content, the aim of the study was to evaluate the effect of an SJW powder with low hyperforin content on CYP3A function. METHODS: Twenty healthy male volunteers received an SJW powder with low hyperforin content for 2 weeks. Midazolam plasma concentration time profiles were characterized after a single oral dose of 7.5 mg midazolam on the day before and on the 14th day of SJW medication. RESULTS: Midazolam AUC(0-infinity) slightly decreased from 124.0 +/- 62.5 ng/ml.h at baseline to 105.6 +/- 53.2 ng/ml.h after SJW (P < 0.05), representing a mean 11.3% decrease (95% CI: -22.8 to 0.21). No significant change in midazolam C(max), t(1/2) and t(max) was observed. For all pharmacokinetic parameters, the 90% CI for the geometric mean ratio of treatment over baseline were within the no-effect boundaries of 0.70-1.43. CONCLUSION: Administration of an SJW product with low hyperforin content resulted in a mild induction of CYP3A not considered clinically relevant.

Facets of impulsivity and related aspects differentiate among recreational and unregulated use of Internet pornography.

BACKGROUND AND AIMS: Unregulated Internet pornography (IP) use is discussed as a clinically significant disorder. Because of its primarily rewarding nature, IP is a predestinated target for addictive behaviors. However, not every user develops an unregulated usage pattern. In fact, most users tend to use IP recreationally. Impulsivity-related constructs have been identified as promoters of addictive behaviors. It is unclear whether these impulsivity-related constructs are specific for unregulated IP use or also play a role in recreational but frequent behaviors. In this study, we investigated impulsive tendencies (trait impulsivity, delay discounting, and cognitive style), craving toward IP, attitude regarding IP, and coping styles in individuals with recreational-occasional, recreational-frequent, and unregulated IP use. METHODS: A total of 1,498 heterosexual males participated in an online survey. Groups of individuals with recreational-occasional use (n = 333), recreational-frequent use (n = 394), and unregulated use (n = 225) of IP were identified by screening instruments. RESULTS: Craving and attitude regarding IP as well as delay discounting and cognitive and coping styles differed between groups. Individuals with unregulated use showed the highest scores for craving, attentional impulsivity, delay discounting, and dysfunctional coping, and lowest scores for functional coping and need for cognition. Recreational-frequent users had the most positive attitude toward IP. Motor and non-planning impulsivity did not differ between groups. DISCUSSION AND CONCLUSIONS: The results indicate that some facets of impulsivity and related factors such as craving and a more negative attitude are specific for unregulated IP users. The results are also consistent with models on specific Internet use disorders and addictive behaviors.

Decision support in patients with mild Alzheimer's disease.

INTRODUCTION: Making advantageous decisions is a key competence of individuals of all ages. However, previous studies reported a reduction of this competence in patients with neurodegenerative diseases such as Alzheimer's disease, which is explained by impairments of executive functions such as cognitive flexibility or working memory. While previous findings from healthy participants with reduced executive functions showed that support can improve decision making under risk, the study at hand aimed to investigate this effect in patients with mild Alzheimer's disease (mAD). METHOD: A group of elderly individuals diagnosed with mAD (n = 14; mean Mini-Mental State Examination, MMSE = 24.14, SD = 3.18) and a group of healthy age-matched controls (n = 14; mean MMSE = 29.29, SD = 1.98) performed the Game of Dice Task (GDT) three times (t0, t1, t2) with intervals of five to nine days between each: The standard GDT plus other neurocognitive tasks (t0), the GDT with decision support (t1), and again the standard GDT (t2). RESULTS: At any time, mAD patients made more disadvantageous decisions than controls. However, the decision-making performance of mAD patients improved significantly with decision support. Interestingly, when the standard GDT was played again (t2), mAD patients' performance remained similar to the performance in the GDT with decision support (t1). GDT performance correlated consistently with executive function measures in the control group, but only at t0 in the mAD group. CONCLUSIONS: The findings indicate that supportive information about the riskiness of options can compensate for mAD-related deficits in decision making under risk. Thus, decision support can improve the quality of mAD patients' decisions. Further, it may prevent mAD patients from making highly risky decisions in similar situations in the future. The persistence of decision support should be further investigated as it has relevant implications for everyday decisions that include risks.

Approximate Number Processing Skills Contribute to Decision Making Under Objective Risk: Interactions With Executive Functions and Objective Numeracy.

Research on the cognitive abilities involved in decision making has shown that, under objective risk conditions (i.e., when explicit information about possible outcomes and risks is available), superior decisions are especially predicted by executive functions and exact number processing skills, also referred to as objective numeracy. So far, decision-making research has mainly focused on exact number processing skills, such as performing calculations or transformations of symbolic numbers. There is evidence that such exact numeric skills are based on approximate number processing (ANP) skills, which enable quick and accurate processing of non-symbolic numbers (e.g., Chen and Li, 2014). Very few studies, however, have investigated ANP skills in the context of risky decision making and have analyzed direct associations among the aforementioned sub functions. Possible interactions between the closely related skills have not been considered. The current study (N = 128) examines interactions of ANP skills with executive functions and objective numeracy, in predicting risky choice behavior. ANP skills are represented by the accuracy in a dot-comparison task. Decision making is measured by two versions of the Game of Dice Task (GDT), which place different emphases on the reflection of potential risks. The results show two-way as well as three-way interactions between the measures of ANP skills, executive functions, and objective numeracy in predicting risky decisions in both GDT versions. The riskiest decisions were most frequently made in case of low scores in all of the three competencies, while good performance in any one of them resulted in significant reductions of disadvantageous decisions. The findings indicate that high ANP skills can positively affect choice behavior in individuals who have weaknesses in reflectively attributed skills, namely executive functions and objective numeracy. Potential compensatory effects and mechanisms of ANP in decision making are discussed.

Effect of St John's wort dose and preparations on the pharmacokinetics of digoxin.

BACKGROUND AND OBJECTIVE: St John's wort preparations vary in composition, main constituents, formulation, and daily dose administered. The aim of the study was to evaluate the possible pharmacokinetic interaction of marketed St John's wort formulations and doses with digoxin. METHODS: A randomized, placebo-controlled, parallel-group study was performed in 96 healthy volunteers in 3 study parts. A 7-day loading phase with digoxin was followed by 14 days of comedication with placebo or one of 10 St John's wort products varying in dose and formulation. The pharmacokinetics of digoxin was determined before comedication and on day 14 of comedication. RESULTS: Comedication comprised traditionally used Hypericum products; 2 g powder without hyperforin, tea, juice, oil extract, and placebo had no significant interaction with digoxin nor did hyperforin-free extract (Ze 117) or low daily doses of hyperforin-containing Hypericum powder (1 g, 0.5 g). However, comedication with the high-dose hyperforin-rich extract LI 160 resulted in a reduction of digoxin area under the curve from time 0 to 24 hours (AUC(0-24)) of -24.8% (95% confidence interval [CI], -28.3 to -21.3), a reduction in digoxin maximal plasma concentration (C(max)) of -37% (95% CI, -42 to -32), and a reduction in digoxin plasma concentration at 24 hours after previous dosing (C(trough)) of -19% (95% CI, -27 to -11). Comedication with 4 g Hypericum powder with comparable hyperforin content resulted in a reduction in digoxin AUC(0-24) of -26.6% (95% CI, -37.3 to -15.9), a reduction in digoxin C(max) of -38% (95% CI, -48 to -18), and a reduction in digoxin C(trough) of -19% (95% CI, -27 to -10). Two grams of Hypericum powder with half the hyperforin content resulted in a less prominent reduction in AUC(0-24) of -17.7% (95% CI, -21.6 to -13.7), C(max) (-21%; 95% CI, -40 to -2), and C(trough) (-13%; 95% CI, -21 to -5). CONCLUSIONS: The interaction of St John's wort and digoxin varies within St John's wort preparations and doses and seems to be correlated with the dose, particularly of hyperforin.

The extent of induction of CYP3A by St. John's wort varies among products and is linked to hyperforin dose.

OBJECTIVE: Induction of CYP3A by St. John's wort (SJW) extracts with high hyperforin (HYF) content is well described. Since SJW products vary in the amount of HYF and other main constituents, the aim of the study was to evaluate the effect on CYP3A function of SJW preparations with a range from very low to high HYF content. METHODS: Forty-two male, healthy volunteers were randomized into six parallel SJW medication groups with varying composition especially with regard to HYF content. Midazolam plasma concentration profiles were characterized after a single oral dose of 7.5 mg midazolam on the day before and on the 14th day of SJW medication. RESULTS: All SJW preparations tested resulted in a decrease in midazolam AUC, although the extent of the effect differed. The extract LI 160 (HYF 41 mg/day) decreased midazolam AUC0-12h by 79.4% (95% CI -88.6; -70.1), which was significantly greater than the effect by any other medication (p<0.05). SJW powder tablets 2.7 g/day (HYF 12 mg/day) resulted in a midazolam AUC0-12h decrease of 47.9% (95% CI -59.7;-36.2), while 2.7 g/day SJW powder tablets that were almost devoid of HYF (0.13 mg/day) reduced midazolam AUC0-12h by only 21.1% (95% CI -33.9; -8.3). Considering all six SJW medications tested, the extent of midazolam AUC decrease correlated significantly with increasing HYF dose (r=-0.765, p<0.001), but not with hypericin dose (r=-0.067; p=0.673). CONCLUSION: The extent of induction of CYP3A varies among St. John's wort products and depends on hyperforin dose.

Pharmacokinetics of piperacillin-tazobactam in anuric intensive care patients during continuous venovenous hemodialysis.

The pharmacokinetics of piperacillin-tazobactam were investigated in eight anuric intensive care patients treated by continuous venovenous hemodialysis (CVVHD). The elimination half-life of piperacillin was 4.3 +/- 1.2 h, and that of tazobactam was 5.6 +/- 1.3 h. The contribution of CVVHD to the overall elimination was relevant (>25%) for both drugs.