Publications by authors named "Frank Sinner"

70 Publications

Dietary spermidine improves cognitive function.

Cell Rep 2021 Apr;35(2):108985

Department of Internal Medicine I, Gastroenterology, Hepatology, Endocrinology and Metabolism, Medical University of Innsbruck, 6020 Innsbruck, Austria.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.
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http://dx.doi.org/10.1016/j.celrep.2021.108985DOI Listing
April 2021

OFM-recirculation and OFM-suction: advanced in-vivo open flow microperfusion (OFM) methods for direct and absolute quantification of albumin in interstitial fluid.

Biomed Phys Eng Express 2021 Apr 12;6(6):065031. Epub 2021 Apr 12.

HEALTH-Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H, Neue Stiftingtalstrasse 2, 8010 Graz, Austria.

Objective: To implement OFM-recirculation and OFM-suction capable of direct and absolute in-vivo quantification of albumin in the ISF of pigs.

Approach: OFM-recirculation and OFM-suction were used to collect ISF in-vivo in pigs and lymph was collected from the same pigs after OFM sampling. Blood was collected before and after OFM sampling, plasma was isolated and mean albumin plasma concentrations per pig were used to yield albumin ISF-to-plasma ratios. We characterized the quality of the collected undiluted ISF via (1) stable albumin ISF-to-plasma ratio in OFM-recirculation and in OFM-suction samples, (2) comparison of albumin ISF-to-plasma ratios from OFM-recirculation and OFM-suction and (3) comparison of normalized albumin concentrations in the ISF and lymph.

Main Results: Both advanced OFM methods were successfully implemented and albumin was quantified from the collected ISF samples. OFM-recirculation reached stable albumin ISF-to-plasma ratios after 20 recirculation cycles. Absolute ISF albumin concentrations were 11.2 mg ml (OFM-recirculation) and 14.2 mg ml (OFM-suction). Albumin ISF-to-plasma ratios were 0.39 ± 0.04 (OFM -recirculation) and 0.47 ± 0.1 (OFM-suction).

Significance: Knowledge of the ISF protein content is of major importance when assessing PK/PD effects, especially of highly protein bound drugs. Up to now, only blood albumin values have been available to determine the degree of protein binding in several tissues. OFM-recirculation and OFM-suction allow direct, absolute quantification of albumin in ISF for the first time and enable investigation of the degree of protein binding of a drug directly in its target tissue.
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http://dx.doi.org/10.1088/2057-1976/abc3a7DOI Listing
April 2021

Evaluating Dermal Pharmacokinetics and Pharmacodymanic Effect of Soft Topical PDE4 Inhibitors: Open Flow Microperfusion and Skin Biopsies.

Pharm Res 2020 Nov 13;37(12):243. Epub 2020 Nov 13.

LEO Pharma Global Research & Development, Industriparken 55, 2750, Ballerup, Denmark.

Purpose: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants.

Methods: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement.

Results: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations.

Conclusions: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.
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http://dx.doi.org/10.1007/s11095-020-02962-1DOI Listing
November 2020

Variability of Skin Pharmacokinetic Data: Insights from a Topical Bioequivalence Study Using Dermal Open Flow Microperfusion.

Pharm Res 2020 Sep 28;37(10):204. Epub 2020 Sep 28.

HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H, Neue Stiftingtalstrasse 2, 8010, Graz, Austria.

Purpose: Dermal open flow microperfusion (dOFM) has previously demonstrated its utility to assess the bioequivalence (BE) of topical drug products in a clinical study. We aimed to characterize the sources of variability in the dermal pharmacokinetic data from that study.

Methods: Exploratory statistical analyses were performed with multivariate data from a clinical dOFM-study in 20 healthy adults evaluating the BE, or lack thereof, of Austrian test (T) and U.S. reference (R) acyclovir cream, 5% products.

Results: The overall variability of logAUC values (CV: 39% for R and 45% for T) was dominated by inter-subject variability (R: 82%, T: 91%) which correlated best with the subject's skin conductance. Intra-subject variability was 18% (R) and 9% (T) of the overall variability; skin treatment sites or methodological factors did not significantly contribute to that variability.

Conclusions: Inter-subject variability was the major component of overall variability for acyclovir, and treatment site location did not significantly influence intra-subject variability. These results support a dOFM BE study design with T and R products assessed simultaneously on the same subject, where T and R treatment sites do not necessarily need to be next to each other. Localized variation in skin microstructure may be primarily responsible for intra-subject variability.
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http://dx.doi.org/10.1007/s11095-020-02920-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522073PMC
September 2020

Electronic Diabetes Management System Replaces Paper Insulin Chart: Improved Quality in Diabetes Inpatient Care Processes Due to Digitalization.

J Diabetes Sci Technol 2021 Mar 16;15(2):222-230. Epub 2020 Sep 16.

Department of Internal Medicine, Division of Endocrinology and Diabetology, Medical University of Graz, Austria.

Background: GlucoTab, an electronic diabetes management system (eDMS), supports healthcare professionals (HCPs) in inpatient blood glucose (BG) management at point-of-care and was implemented for the first time under routine conditions in a regional hospital to replace the paper insulin chart.

Method: To investigate quality of the eDMS for inpatients with type 2 diabetes mellitus a monocentric retrospective before-after evaluation was conducted. We compared documentation possibilities by assessing a blank paper chart vs the eDMS user interface. Further quality aspects were compared by assessing filled-in paper charts ( = 106) vs filled-in eDMS documentation ( = 241). HCPs ( = 59) were interviewed regarding eDMS satisfaction.

Results: The eDMS represented an improvement of documentation possibilities by offering a more structured and comprehensive user interface compared to the blank paper chart. The number of good diabetes days averaged to a median value of four days in both groups (paper chart: 4.38 [0-7] vs eDMS: 4.38 [0-7] days). Median daily BG was 170 (117-297) mg/dL vs 168 (86-286) mg/dL and median fasting BG was 152 (95-285) mg/dL vs 145 (69-333) mg/dL, and 0.1% vs 0.4% BG values <54 mg/dL were documented. Diabetes documentation quality improved when using eDMS, for example, documentation of ordered BG measurement frequency (1% vs 100%) and ordered BG targets (0% vs 100%). HCPs stated that by using eDMS errors could be prevented (74%), and digital support of work processes was completed (77%). Time saving was noted by 8 out of 11 HCPs and estimated at 10-15 minutes per patient day by two HCPs.

Conclusions: The eDMS completely replaced the paper chart, showed comparable glycemic control, was positively accepted by HCPs, and is suitable for inpatient diabetes management.
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http://dx.doi.org/10.1177/1932296820957043DOI Listing
March 2021

Study protocol for assessing the user acceptance, safety and efficacy of a tablet-based workflow and decision support system with incorporated basal insulin algorithm for glycaemic management in participants with type 2 diabetes receiving home health care: A single-centre, open-label, uncontrolled proof-of-concept study.

Contemp Clin Trials Commun 2020 Sep 17;19:100620. Epub 2020 Jul 17.

JOANNEUM RESEARCH Forschungsgesellschaft mbH, HEALTH, Institute for Biomedicine and Health Sciences, Graz, Austria.

Introduction: Diabetes management can be especially complex for older adults who receive health care at home. Thus, international guidelines recommend basal-insulin regimens due to simpler handling and low hypoglycaemia risk. A basal-insulin algorithm (including basal-plus) was developed to also include participant's health status and subsequently implemented into a tablet-based workflow and decision support system, GlucoTab@MobileCare. This study protocol describes a proof-of-concept study to investigate user acceptance, safety and efficacy of the GlucoTab@MobileCare system in participants receiving home health care.

Methods: The open-label, single-centre, uncontrolled study will recruit a maximum of ten participants with insulin treated type-2-diabetes (age ≥18 years) who receive home health care. During a three month study period participants will receive basal- or basal-plus-insulin therapy once daily as suggested by the GlucoTab@MobileCare system. Statistical analysis will be conducted on an intention-to-treat basis. The primary endpoint is the percentage of tasks (BG measurements, insulin dose calculations, insulin injections) that were performed according to GlucoTab@MobileCare suggestions relative to the total of suggested tasks. Secondary endpoints include user acceptance, safety and efficacy parameters. The study was approved by the ethics committee and regulatory authorities. Before obtaining written informed consent, all participants will receive oral and written information about all aspects of the study. Results will be published in a peer-reviewed journal and at diabetes and geriatric conferences.

Discussion: Potential implications may be improved quality and safety of basal-insulin therapy in older adults as well as support for health-care-providers in daily routine including evidence-based knowledge.

Trial Registration: German Clinical Trials Register (DRKS00015059).
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http://dx.doi.org/10.1016/j.conctc.2020.100620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399114PMC
September 2020

Alternate Day Fasting Improves Physiological and Molecular Markers of Aging in Healthy, Non-obese Humans.

Cell Metab 2019 09 27;30(3):462-476.e6. Epub 2019 Aug 27.

Division of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, Austria; Center for Biomarker Research in Medicine (CBmed), Graz, Austria; HEALTH Institute for Biomedicine and Health Sciences, JOANNEUM RESEARCH Forschungsgesellschaft mbH, Neue Stiftingtalstraße 2, Graz, Austria.

Caloric restriction and intermittent fasting are known to prolong life- and healthspan in model organisms, while their effects on humans are less well studied. In a randomized controlled trial study (ClinicalTrials.gov identifier: NCT02673515), we show that 4 weeks of strict alternate day fasting (ADF) improved markers of general health in healthy, middle-aged humans while causing a 37% calorie reduction on average. No adverse effects occurred even after >6 months. ADF improved cardiovascular markers, reduced fat mass (particularly the trunk fat), improving the fat-to-lean ratio, and increased β-hydroxybutyrate, even on non-fasting days. On fasting days, the pro-aging amino-acid methionine, among others, was periodically depleted, while polyunsaturated fatty acids were elevated. We found reduced levels sICAM-1 (an age-associated inflammatory marker), low-density lipoprotein, and the metabolic regulator triiodothyronine after long-term ADF. These results shed light on the physiological impact of ADF and supports its safety. ADF could eventually become a clinically relevant intervention.
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http://dx.doi.org/10.1016/j.cmet.2019.07.016DOI Listing
September 2019

Insulin Distribution in Human Adipose Tissue via a Novel Insulin Infusion Catheter.

Diabetes Technol Ther 2019 12 3;21(12):740-744. Epub 2019 Oct 3.

Joanneum Research Forschungsgesellschaft mbH, HEALTH-Institute for Biomedicine and Health Sciences, Graz, Austria.

Continuous subcutaneous insulin infusion (CSII) is a widely used treatment for diabetes patients. Insulin infusion sets (CSII-catheters) are continuously optimized regarding size, handling and safety, but recurring dysfunction (kinking or occlusion), due to different user situations, behavior or chain of events, demand new ways to improve the functionality and safety in patients experiencing these issues. A novel CSII-catheter design (Lantern) features additional lateral perforations, which guarantee functionality even in case of kinking or occlusion. This study aimed to compare functionality, insulin distribution, and failure rate of Lantern and standard catheters using excised human adipose tissue samples. Novel Lantern CSII-catheters (open and artificially occluded) and commercially available standard CSII-catheters were inserted into adipose tissue samples. A mixture of insulin and contrast agent was infused as single bolus (7 IU) with an insulin infusion pump at highest flow rate (1 IU/s). Microtomography images and surface-to-volume ratios were used to assess insulin distribution and depot volume indicating the functionality of CSII-catheters. Failure rate was measured by flow-stop alerts of the pump. We found no difference in the volume of insulin depots compared with the nominal volume of 70 μL. Surface-to-volume ratios showed no significant difference among CSII-catheters. None of the catheters triggered any flow-stop alarm. The novel Lantern CSII-catheter design achieved similar insulin distribution as commercially available CSII-catheters. Moreover, functionality of Lantern CSII-catheters was guaranteed during occlusion, which is an improvement compared with standard CSII-catheters. We conclude that the novel CSII-catheter design has the potential to provide a valuable contribution to patient well-being and safety.
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http://dx.doi.org/10.1089/dia.2019.0195DOI Listing
December 2019

The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species.

Nat Commun 2019 02 19;10(1):651. Epub 2019 Feb 19.

Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM U 1138, Paris, France.

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.
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http://dx.doi.org/10.1038/s41467-019-08555-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381180PMC
February 2019

LC/MS/MS analyses of open-flow microperfusion samples quantify eicosanoids in a rat model of skin inflammation.

J Lipid Res 2019 04 29;60(4):758-766. Epub 2019 Jan 29.

Joanneum Research Forschungsgesellschaft mbH, Institute for Biomedicine and Health Sciences, Graz, Austria. Electronic address:

Eicosanoids are lipid-mediator molecules with key roles in inflammatory skin diseases, such as psoriasis. Eicosanoids are released close to the source of inflammation, where they elicit local pleiotropic effects and dysregulations. Monitoring inflammatory mediators directly in skin lesions could provide new insights and therapeutic possibilities. Here, we analyzed dermal interstitial fluid samples obtained by dermal open-flow microperfusion in a rat model of skin inflammation. We developed a solid-phase extraction ultra-HPLC/MS/MS method to reliably and precisely analyze small-volume samples and quantified 11 eicosanoids [thromboxane B, prostaglandin (PG) E, PGD, PGF, leukotriene B, 15-HETE, 12-HETE, 5-HETE, 12-hydroxyeicosapentaenoic acid, 13-HODE, and 17-hydroxydocosahexaenoic acid]. Our method achieved a median intraday precision of approximately 5% and interday precision of approximately 8%. All calibration curves showed excellent linearity between 0.01 and 50 ng/ml ( > 0.980). In the rat model, eicosanoids were significantly increased in imiquimod-treated inflamed skin sites compared with untreated control sites. Oral treatment with an anti-inflammatory glucocorticoid decreased eicosanoid concentrations. These results show that a combination of tissue-specific sampling with LC/MS analytics is well suited for analyzing small sample volumes from minimally invasive sampling methods such as open-flow microperfusion or microdialysis to study local inflammation and the effect of treatments in skin diseases.
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http://dx.doi.org/10.1194/jlr.M087221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446707PMC
April 2019

Comparison of cerebral Open Flow Microperfusion and Microdialysis when sampling small lipophilic and small hydrophilic substances.

J Neurosci Methods 2019 01 25;311:394-401. Epub 2018 Sep 25.

JOANNEUM RESEARCH Forschungsgesellschaft mbH, HEALTH - Institute for Biomedicine and Health Sciences, Neue Stiftingtalstraße 2, 8010 Graz, Austria. Electronic address:

Background: Assessment of drug concentration in the brain interstitial fluid (ISF) is crucial for development of brain active drugs, which are mainly small, lipophilic substances able to cross the blood-brain barrier (BBB). We aimed to compare the applicability of cerebral Open Flow Microperfusion (cOFM) and Microdialysis (MD) to sample the lipophilic substance amitriptyline (AMI), its metabolites Hydroxyamitriptyline (HYA), Nortriptyline (NOR), Amitriptyline-N-Oxide (ANO), deuterated water (DO) and the hydrophilic substance sodium fluorescein (Naf) in brain ISF. NEW METHOD: cOFM has been refined to yield increased spatial resolution and performance.

Comparison Of Cofm And Md And Results: Performance of cOFM and MD was assessed by in vivo AUC ratios of probe samples (AUC/AUC) and the in vivo relative recovery of DO (RR). Adsorption of AMI and Naf to MD and cOFM was assessed by the in vitro relative recovery (RR) prior to the in vivo experiments. The in vivo AUC ratio of AMI and RR was about two times higher for cOFM than for MD (AUC/AUC = 2.0, RR(cOFM)/RR(MD) = 2.1). cOFM detected all investigated AMI metabolites except NOR. MD did not detect HYA, NOR, ANO and Naf. In vitro adsorption of AMI and Naf to the MD membrane was strong (RR = 4.4%, RR = 1.5%) but unspecific adsorption to cOFM was negligibly small (RR = 98% and RR = 98%).

Conclusions: cOFM showed better performance when sampling AMI and its metabolites, Naf and DO, and had an about two times higher RR than MD. MD did not detect HYA, NOR, ANO and Naf, most likely due to membrane adsorption.
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http://dx.doi.org/10.1016/j.jneumeth.2018.09.024DOI Listing
January 2019

Development of a Protocol for Automated Glucose Measurement Transmission Used in Clinical Decision Support Systems Based on the Continua Design Guidelines.

Stud Health Technol Inform 2018 ;248:132-139

HEALTH - JOANNEUM RESEARCH Forschungsgesellschaft mbH, Graz, Austria.

Background: A fast and accurate data transmission from glucose meter to clinical decision support systems (CDSSs) is crucial for the management of type 2 diabetes mellitus since almost all therapeutic interventions are derived from glucose measurements.

Objectives: Aim was to develop a prototype of an automated glucose measurement transmission protocol based on the Continua Design Guidelines and to embed the protocol into a CDSS used by healthcare professionals.

Methods: A literature and market research was performed to analyze the state-of-the-art and thereupon develop, integrate and validate an automated glucose measurement transmission protocol in an iterative process.

Results: Findings from literature and market research guided towards the development of a standardized glucose measurement transmission protocol using a middleware. The interface description to communicate with the glucose meter was illustrated and embedded into a CDSS.

Conclusion: A prototype of an interoperable transmission of glucose measurements was developed and implemented in a CDSS presenting a promising way to reduce medication errors and improve user satisfaction.
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June 2018

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra-high-performance liquid-high-resolution tandem mass spectrometry for topical bioequivalence evaluation.

Biomed Chromatogr 2018 Jun 27;32(6):e4194. Epub 2018 Feb 27.

Joanneum Research Forschungsgesellschaft mbH, HEALTH Institute for Biomedicine and Health Sciences, Graz, Austria.

Time-concentration curves for the topical anti-viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high-throughput ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1-25 ng/mL for a sample volume of just 20 μL and employed cation-exchange solid-phase extraction in a fully automated sample treatment procedure. Short- and long-term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.
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http://dx.doi.org/10.1002/bmc.4194DOI Listing
June 2018

Assessment of skin permeability to topically applied drugs by skin impedance and admittance.

Physiol Meas 2017 Nov 2;38(11):N138-N150. Epub 2017 Nov 2.

Joanneum Research, HEALTH-Institute for Biomedicine and Health Sciences, Graz, Austria.

Objective: Pharmacokinetic and pharmacodynamic studies of topically applied drugs are commonly performed by sampling of interstitial fluid with dermal open flow microperfusion and subsequent analysis of the samples. However, the reliability of results from the measured concentration-time profile of the penetrating drug suffers from highly variable skin permeability to topically applied drugs that is mainly caused by inter- and intra-subject variations of the stratum corneum. Thus, statistically significant results can only be achieved by performing high numbers of experiments. To reduce the expenditures needed for such high experiment numbers we aimed to assess the correlation between skin permeability and skin impedance/skin admittance.

Approach: We performed an ex vivo drug penetration study with human skin, based on the hypothesis that inter-subject variations of the respective concentration-time profiles can be correlated with variations of the passive electrical properties of the skin. Therefore, skin impedance and skin admittance were related to the skin permeability to the model drug Clobetasol-17-proprionate.

Main Results: The measured low frequency skin impedance and the skin admittance correlated linearly with the drug concentration-time profiles from dermal sampling.

Significance: Skin permeability can be assessed by measuring the passive electrical properties of the skin, which enables correction of skin permeability variations. This allows reduction of experiment numbers in future pharmacokinetic and pharmacodynamic studies with human skin ex vivo and in vivo and leads to diminished study costs.
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http://dx.doi.org/10.1088/1361-6579/aa904eDOI Listing
November 2017

A comprehensive approach to qualify and validate the essential parameters of an in vitro release test (IVRT) method for acyclovir cream, 5.

Int J Pharm 2018 Jan 19;535(1-2):217-227. Epub 2017 Sep 19.

Joanneum Research Forschungsgesellschaft mbH, Health - Institute for Biomedicine and Health Sciences, Neue Stiftingtalstr. 2, 8010 Graz, Austria.

The rate of release of an active pharmaceutical ingredient (API) from a topical semisolid dosage form can be influenced by its physical and structural properties. An In Vitro Release Test (IVRT) is an established method to characterize this rate of API release and compare the underlying sameness in product quality characteristics. The purpose of this work was to validate an IVRT method to compare acyclovir cream, 5% products. However, despite widespread use of the IVRT since 1997, there has been no established approach to validate an IVRT method. Our approach included: 1) qualification of the diffusion cell apparatus, 2) qualification of the laboratory, 3) validation of the HPLC analytical method, and 4) validation of numerous critical parameters of the IVRT method, itself, and resulted in a comprehensive and successful IVRT method validation. Subsequent to the IVRT validation work described here, the U.S. Food and Drug Administration (FDA) drafted a guidance on the development and validation of an IVRT method for acyclovir cream, 5%. Although there are notable differences between our approach and the approach in that guidance, this report illustrates how many of the same essential qualification parameters and validation concepts were considered and systematically addressed in our approach to IVRT validation.
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http://dx.doi.org/10.1016/j.ijpharm.2017.09.049DOI Listing
January 2018

Quantification of Basal Insulin Peglispro and Human Insulin in Adipose Tissue Interstitial Fluid by Open-Flow Microperfusion.

Diabetes Technol Ther 2017 05 22;19(5):305-314. Epub 2017 Mar 22.

1 HEALTH-Institute for Biomedicine and Health Sciences , Joanneum Research Forschungsgesellschaft mbH, Graz, Austria .

Background: Restoration of the physiologic hepatic-to-peripheral insulin gradient may be achieved by either portal vein administration or altering insulin structure to increase hepatic specificity or restrict peripheral access. Basal insulin peglispro (BIL) is a novel, PEGylated basal insulin with a flat pharmacokinetic and glucodynamic profile and altered hepatic-to-peripheral action gradient. We hypothesized reduced BIL exposure in peripheral tissues explains the latter, and in this study assessed the adipose tissue interstitial fluid (ISF) concentrations of BIL compared with human insulin (HI).

Methods: A euglycemic glucose clamp was performed in patients with type 1 diabetes during continuous intravenous (IV) infusion of BIL or HI, while the adipose ISF insulin concentrations were determined using open-flow microperfusion (OFM). The ratio of adipose ISF-to-serum concentrations and the absolute steady-state adipose ISF concentrations were assessed using a dynamic no-net-flux technique with subsequent regression analysis.

Results: Steady-state BIL concentrations in adipose tissue ISF were achieved by ∼16 h after IV infusion. Median time to reach steady-state glucose infusion rate across doses ranged between 8 and 22 h. The average serum concentrations (coefficient of variation %) of BIL and HI were 11,200 pmol/L (23%) and 425 pmol/L (15%), respectively. The ISF-to-serum concentration ratios were 10.2% for BIL and 22.9% for HI.

Conclusions: This study indicates feasibility of OFM to measure BIL in ISF. The observed low ISF-to-serum concentration ratio of BIL is consistent with its previously demonstrated reduced peripheral action.
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http://dx.doi.org/10.1089/dia.2016.0384DOI Listing
May 2017

Cerebral open flow microperfusion (cOFM) an innovative interface to brain tissue.

Drug Discov Today Technol 2016 Jun 2;20:19-25. Epub 2016 Dec 2.

Joanneum Research GmbH, HEALTH - Institute for Biomedicine and Health Sciences, Neue Stiftingtalstrasse 2, 8010 Graz, Austria; Medical University of Graz, Department of Internal Medicine, Division of Endocrinology and Diabetology, Auenbruggerplatz 15, 8036 Graz, Austria.

Cerebral open flow microperfusion (cOFM) is a new in-vivo technique for continuous sampling of the interstitial fluid in brain tissue. cOFM can be used to monitor substance transport across the blood-brain barrier (pharmacokinetics) and to investigate metabolic changes in brain tissue after drug application (pharmacodynamics). The possibility of long-term implantation into the brain makes cOFM an outstanding tool in the development of brain relevant pharmaceutics.
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http://dx.doi.org/10.1016/j.ddtec.2016.07.003DOI Listing
June 2016

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Nanomedicine 2017 04 21;13(3):1289-1300. Epub 2016 Nov 21.

Department of Neuroscience, School for Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands. Electronic address:

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.
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http://dx.doi.org/10.1016/j.nano.2016.11.009DOI Listing
April 2017

An Untargeted Metabolomics Approach to Characterize Short-Term and Long-Term Metabolic Changes after Bariatric Surgery.

PLoS One 2016 1;11(9):e0161425. Epub 2016 Sep 1.

JOANNEUM RESEARCH Forschungsgesellschaft mbH HEALTH Institute for Biomedicine and Health Sciences, Graz, Austria.

Bariatric surgery is currently one of the most effective treatments for obesity and leads to significant weight reduction, improved cardiovascular risk factors and overall survival in treated patients. To date, most studies focused on short-term effects of bariatric surgery on the metabolic profile and found high variation in the individual responses to surgery. The aim of this study was to identify relevant metabolic changes not only shortly after bariatric surgery (Roux-en-Y gastric bypass) but also up to one year after the intervention by using untargeted metabolomics. 132 serum samples taken from 44 patients before surgery, after hospital discharge (1-3 weeks after surgery) and at a 1-year follow-up during a prospective study (NCT01271062) performed at two study centers (Austria and Switzerland). The samples included 24 patients with type 2 diabetes at baseline, thereof 9 with diabetes remission after one year. The samples were analyzed by using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS, HILIC-QExactive). Raw data was processed with XCMS and drift-corrected through quantile regression based on quality controls. 177 relevant metabolic features were selected through Random Forests and univariate testing and 36 metabolites were identified. Identified metabolites included trimethylamine-N-oxide, alanine, phenylalanine and indoxyl-sulfate which are known markers for cardiovascular risk. In addition we found a significant decrease in alanine after one year in the group of patients with diabetes remission relative to non-remission. Our analysis highlights the importance of assessing multiple points in time in subjects undergoing bariatric surgery to enable the identification of biomarkers for treatment response, cardiovascular benefit and diabetes remission. Key-findings include different trend pattern over time for various metabolites and demonstrated that short term changes should not necessarily be used to identify important long term effects of bariatric surgery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008721PMC
August 2017

Open Flow Microperfusion as a Dermal Pharmacokinetic Approach to Evaluate Topical Bioequivalence.

Clin Pharmacokinet 2017 01;56(1):91-98

HEALTH-Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft mbH, Neue Stiftingtalstrasse 2, 8010, Graz, Austria.

Background: The availability of generic topical dermatological drug products is constrained by the limited methods established to assess topical bioequivalence (BE). A novel cutaneous pharmacokinetic approach, dermal open-flow microperfusion (dOFM), can continuously assess the rate and extent to which a topical drug becomes available in the dermis, to compare in vivo dermal bioavailability (BA) and support BE evaluations for topical products.

Objective: To evaluate whether dOFM is an accurate, sensitive, and reproducible in vivo method to characterize the intradermal BA of acyclovir from 5 % acyclovir creams, comparing a reference (R) product either to itself or to a different test (T) product.

Methods: In a single-center clinical study, R or T products were applied to six randomized treatment sites on the skin of 20 healthy human subjects. Two dOFM probes were inserted in each treatment site to monitor the intradermal acyclovir concentration for 36 h. Comparative BA (of R vs. R and T vs. R) was evaluated based on conventional BE criteria for pharmacokinetic endpoints (area under the curve and maximum dermal concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80-1.25.

Results: The positive control products (R vs. R) were accurately and reproducibly confirmed to be bioequivalent, while the negative control products (T vs. R) were sensitively discriminated not to be bioequivalent.

Conclusions: dOFM accurately, sensitively, and reproducibly characterized the dermal BA in a manner that can support BE evaluations for topical acyclovir 5 % creams in a study with n = 40 (20 subjects in this study).
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http://dx.doi.org/10.1007/s40262-016-0442-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222896PMC
January 2017

β-Defensin 2 is a responsive biomarker of IL-17A-driven skin pathology in patients with psoriasis.

J Allergy Clin Immunol 2017 Mar 5;139(3):923-932.e8. Epub 2016 Aug 5.

Novartis Institutes for BioMedical Research Shanghai, China. Electronic address:

Background: IL-17A is a key driver of human autoimmune diseases, particularly psoriasis.

Objective: We sought to determine the role of IL-17A in psoriasis pathogenesis and to identify a robust and measurable biomarker of IL-17A-driven pathology.

Methods: We studied 8 healthy subjects and 8 patients with psoriasis before and after administration of secukinumab, a fully human anti-IL-17A mAb, and used a combination of classical techniques and a novel skin microperfusion assay to evaluate the expression of 170 proteins in blood, nonlesional skin, and lesional skin. For validation, we also tested stored sera from 601 patients with a variety of autoimmune diseases.

Results: IL-17A was specifically expressed in lesional compared with nonlesional psoriatic skin (9.8 vs 0.8 pg/mL, P < .001). Proteomic and gene transcription analyses revealed dysregulated antimicrobial peptides, proinflammatory cytokines, and neutrophil chemoattractants, levels of which returned to normal after treatment with secukinumab. β-Defensin 2 (BD-2) was identified as a biomarker of IL-17A-driven pathology by comparing protein expression in patients with psoriasis versus that in healthy subjects (5746 vs 82 pg/mL in serum, P < .0001; 2747 vs <218 pg/mL in dermis, P < .001), responsiveness to secukinumab therapy, and synergistic induction by IL-17A and TNF-α in epidermal keratinocytes. In a validation set of sera from 601 patients with autoimmune diseases thought to be IL-17A driven, we found that BD-2 levels are most highly increased in patients with psoriatic skin lesions, and in patients with psoriasis, BD-2 levels correlated well with IL-17A levels (r = 0.70, n = 199, P < .001) and Psoriasis Area and Severity Index scores (r = 0.53, n = 281, P < .001).

Conclusion: IL-17A is a primary driver of skin pathology in patients with psoriasis, and serum BD-2 is an easily measurable biomarker of IL-17A-driven skin pathology.
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http://dx.doi.org/10.1016/j.jaci.2016.06.038DOI Listing
March 2017

Determination of (2)H-enrichment of rat brain interstitial fluid and rat plasma by headspace-gas-chromatography - quadrupole-mass-spectrometry.

Anal Biochem 2016 09 9;509:130-134. Epub 2016 Jul 9.

Health - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H, Neue Stiftingtalstrasse 2, 8010, Graz, Austria.

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.
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http://dx.doi.org/10.1016/j.ab.2016.07.010DOI Listing
September 2016

Kinetics of Clobetasol-17-Propionate in Psoriatic Lesional and Non-Lesional Skin Assessed by Dermal Open Flow Microperfusion with Time and Space Resolution.

Pharm Res 2016 09 6;33(9):2229-38. Epub 2016 Jun 6.

HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H, Neue Stiftingtalstrasse 2, 8010, Graz, Austria.

Purpose: To evaluate the kinetics of topically applied clobetasol-17-propionate (CP-17) in lesional and non-lesional psoriatic skin when released from a commercially available low-strength cream using in vivo dermal open-flow microperfusion (dOFM).

Methods: Twelve patients received Dermovate® cream (CP-17, 0.05%) on small lesional and non-lesional skin test sites for 14 days, once daily. On day 1 and 14, dOFM samples were continuously taken in the dermis for 24 h post-dose and analyzed by LC-MS/MS. Probe depths were assessed by 50 MHz ultrasound scanning.

Results: Mixed-effects modelling identified skin condition, treatment duration and probe-depth as kinetics determining variables. The time- and depth-resolved intradermal data revealed (i) slower penetration of CP-17 into lesional than into non-lesional skin, (ii) normalized (faster) skin penetration after repeated dosing, and (iii) no CP-17 accumulation within the dermis independently of the skin condition.

Conclusions: Intradermal investigation of a highly lipophilic drug released from low-strength cream was successfully performed by using dOFM and timely and spatially, i.e., probe-depth dependent, resolved kinetic data were delivered. These data support the assumption that the thickened psoriatic stratum corneum might act as trap compartment which lowers the skin penetration rate for lipophilic topical drugs.
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http://dx.doi.org/10.1007/s11095-016-1960-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967091PMC
September 2016

Detection of Peptide-based nanoparticles in blood plasma by ELISA.

PLoS One 2015 21;10(5):e0126136. Epub 2015 May 21.

Department of Neuroscience, School for Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands.

Aims: The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.

Materials & Methods: A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology.

Results: The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.

Conclusions: We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126136PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440766PMC
February 2016

IPO: a tool for automated optimization of XCMS parameters.

BMC Bioinformatics 2015 Apr 16;16:118. Epub 2015 Apr 16.

Joanneum Research Forschungsgesellschaft m.b.H., HEALTH, Institute for Biomedicine and Health Sciences, Graz, Austria.

Background: Untargeted metabolomics generates a huge amount of data. Software packages for automated data processing are crucial to successfully process these data. A variety of such software packages exist, but the outcome of data processing strongly depends on algorithm parameter settings. If they are not carefully chosen, suboptimal parameter settings can easily lead to biased results. Therefore, parameter settings also require optimization. Several parameter optimization approaches have already been proposed, but a software package for parameter optimization which is free of intricate experimental labeling steps, fast and widely applicable is still missing.

Results: We implemented the software package IPO ('Isotopologue Parameter Optimization') which is fast and free of labeling steps, and applicable to data from different kinds of samples and data from different methods of liquid chromatography - high resolution mass spectrometry and data from different instruments. IPO optimizes XCMS peak picking parameters by using natural, stable (13)C isotopic peaks to calculate a peak picking score. Retention time correction is optimized by minimizing relative retention time differences within peak groups. Grouping parameters are optimized by maximizing the number of peak groups that show one peak from each injection of a pooled sample. The different parameter settings are achieved by design of experiments, and the resulting scores are evaluated using response surface models. IPO was tested on three different data sets, each consisting of a training set and test set. IPO resulted in an increase of reliable groups (146% - 361%), a decrease of non-reliable groups (3% - 8%) and a decrease of the retention time deviation to one third.

Conclusions: IPO was successfully applied to data derived from liquid chromatography coupled to high resolution mass spectrometry from three studies with different sample types and different chromatographic methods and devices. We were also able to show the potential of IPO to increase the reliability of metabolomics data. The source code is implemented in R, tested on Linux and Windows and it is freely available for download at https://github.com/glibiseller/IPO . The training sets and test sets can be downloaded from https://health.joanneum.at/IPO .
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http://dx.doi.org/10.1186/s12859-015-0562-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404568PMC
April 2015

Modeling non-hereditary mechanisms of Alzheimer disease during apoptosis in yeast.

Microb Cell 2015 Mar 20;2(4):136-138. Epub 2015 Mar 20.

Institute of Molecular Biosciences, NAWI Graz, University of Graz, 8010 Graz, Austria. ; BioTechMed-Graz, 8010 Graz, Austria.

Impaired protein degradation and mitochondrial dysfunction are believed to contribute to neurodegenerative disorders, including Alzheimer disease (AD). In patients suffering from non-hereditary AD, UBB, the frameshift variant of ubiquitin B, accumulated in neurons affected by neurofibrillary tangles, which is a pathological hallmark. We established a yeast model expressing high levels of UBB, and could demonstrate that UBB interfered with both the ubiquitin-proteasome system (UPS) and mitochondrial function. More precisely, UBB promoted the mitochondrion-localized production of the basic amino acids arginine, ornithine, and lysine, which we identified as the decisive toxic event culminating in apoptosis. Inducing the UPS activity at mitochondria prevented the lethal basic amino acid accumulation and avoided UBB-triggered cell loss. The arginine/ornithine metabolism is altered in brains of AD patients, and VMS1, the mitochondrion-specific UPS component, co-existed with UBB in neurofibrillary tangles. Therefore, our data suggest that aberrant basic amino acid synthesis is a crucial link between UPS dysfunction and mitochondrial damage during AD progression.
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http://dx.doi.org/10.15698/mic2015.04.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348975PMC
March 2015

Accumulation of Basic Amino Acids at Mitochondria Dictates the Cytotoxicity of Aberrant Ubiquitin.

Cell Rep 2015 Mar 5;10(9):1557-1571. Epub 2015 Mar 5.

Institute of Molecular Biosciences, NAWI Graz, University of Graz, 8010 Graz, Austria; BioTechMed-Graz, 8010 Graz, Austria. Electronic address:

Neuronal accumulation of UBB, a frameshift variant of ubiquitin B, is a hallmark of Alzheimer's disease (AD). How UBB contributes to neuronal dysfunction remains elusive. Here, we show that in brain regions of AD patients with neurofibrillary tangles UBB co-exists with VMS1, the mitochondrion-specific component of the ubiquitin-proteasome system (UPS). Expression of UBB in yeast disturbs the UPS, leading to mitochondrial stress and apoptosis. Inhibiting UPS activity exacerbates while stimulating UPS by the transcription activator Rpn4 reduces UBB-triggered cytotoxicity. High levels of the Rpn4 target protein Cdc48 and its cofactor Vms1 are sufficient to relieve programmed cell death. We identified the UBB-induced enhancement of the basic amino acids arginine, ornithine, and lysine at mitochondria as a decisive toxic event, which can be reversed by Cdc48/Vms1-mediated proteolysis. The fact that AD-induced cellular dysfunctions can be avoided by UPS activity at mitochondria has potentially far-reaching pathophysiological implications.
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http://dx.doi.org/10.1016/j.celrep.2015.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407011PMC
March 2015