A prospective study to analyse the concentration of octenidine in hand wounds after disinfection by LC-MS/MS.

Toxic reactions can appear after pressurised flushing of soft tissue with octenidine (OCT) containing disinfectants. Their use for surgical disinfection could complicate the diagnosis of possible contamination. In patients with open lacerations of their hand's subcutaneous tissue samples were taken before and after surgical disinfection with Octenisept® and analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In 16 out of 20 tissue samples, OCT was detected after disinfection (lower limit of quantification (LLOQ)=10 pg/mL/mg). The concentration of OCT was below the LLOQ, estimation of mean of 0.6 pg/mL/mg (0.22-0.98 pg/mL/mg, 95%-CI) before disinfection and mean of 179.4 pg/mL/mg (13.35-432.0 pg/mL/mg, 95%-CI) after disinfection. This study shows that the disinfection of open wounds with Octenisept® leads to a quantifiable concentration of OCT in open wounds. In cases of suspected OCT-mediated toxic reaction, the use of antiseptics containing OCT should be avoided.

Population pharmacokinetic analyses of regorafenib and capecitabine in patients with locally advanced rectal cancer (SAKK 41/16 RECAP).

AIMS: Locally advanced rectal cancer (LARC) is an area of unmet medical need with one third of patients dying from their disease. With response to neoadjuvant chemo-radiotherapy being a major prognostic factor, trial SAKK 41/16 assessed potential benefits of adding regorafenib to capecitabine-amplified neoadjuvant radiotherapy in LARC patients. METHODS: Patients received regorafenib at three dose levels (40/80/120 mg once daily) combined with capecitabine 825 mg/m2 bidaily and local radiotherapy. We developed population pharmacokinetic models from plasma concentrations of capecitabine and its metabolites 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine as well as regorafenib and its metabolites M-2 and M-5 as implemented into SAKK 41/16 to assess potential drug-drug interactions (DDI). After establishing parent-metabolite base models, drug exposure parameters were tested as covariates within the respective models to investigate for potential DDI. Simulation analyses were conducted to quantify their impact. RESULTS: Plasma concentrations of capecitabine, regorafenib and metabolites were characterized by one and two compartment models and absorption was described by parallel first- and zero-order processes and transit compartments, respectively. Apparent capecitabine clearance was 286 L/h (relative standard error [RSE] 14.9%, interindividual variability [IIV] 40.1%) and was reduced by regorafenib cumulative area under the plasma concentration curve (median reduction of 45.6%) as exponential covariate (estimate -4.10 × 10-4 , RSE 17.8%). Apparent regorafenib clearance was 1.94 L/h (RSE 12.1%, IIV 38.1%). Simulation analyses revealed significantly negative associations between capecitabine clearance and regorafenib exposure. CONCLUSIONS: This work informs the clinical development of regorafenib and capecitabine combination treatment and underlines the importance of studying potential DDI with new anticancer drug combinations.

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry.

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7-97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. Graphical Abstract.

Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection.

OBJECTIVE: The dyslipidemia of type 2 diabetes mellitus has multiple etiologies and impairs lipoprotein functionality, thereby increasing risk for cardiovascular disease. High-density lipoproteins (HDLs) have several beneficial effects, notably protecting the heart from myocardial ischemia. We hypothesized that glycation of HDL could compromise this cardioprotective effect. APPROACH AND RESULTS: We used in vitro (cardiomyocytes) and ex vivo (whole heart) models subjected to oxidative stress together with HDL isolated from diabetic patients and nondiabetic HDL glycated in vitro (methylglyoxal). Diabetic and in vitro glycated HDL were less effective (P<0.05) than control HDL in protecting from oxidative stress. Protection was significantly, inversely correlated with the degree of in vitro glycation (P<0.001) and the levels of hemoglobin A1c in diabetic patients (P<0.007). The ability to activate protective, intracellular survival pathways involving Akt, Stat3, and Erk1/2 was significantly reduced (P<0.05) using glycated HDL. Glycation reduced the sphingosine-1-phosphate (S1P) content of HDL, whereas the S1P concentrations of diabetic HDL were inversely correlated with hemoglobin A1c (P<0.005). The S1P contents of in vitro glycated and diabetic HDL were significantly, positively correlated (both <0.01) with cardiomyocyte survival during oxidative stress. Adding S1P to diabetic HDL increased its S1P content and restored its cardioprotective function. CONCLUSIONS: Our data demonstrate that glycation can reduce the S1P content of HDL, leading to increased cardiomyocyte cell death because of less effective activation of intracellular survival pathways. It has important implications for the functionality of HDL in diabetes mellitus because HDL-S1P has several beneficial effects on the vasculature.

Specific GAG ratios in the diagnosis of mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) screening is tedious and still performed by analysis of total glycosaminoglycans (GAG) using 1,9-dimethylmethylene blue (DMB) photometric assay, although false positive and negative tests have been reported. Analysis of differentiated GAGs have been pursued classically by gel electrophoresis or more recently by quantitative LC-MS assays. Secondary elevations of GAGs have been reported in urinary tract infections (UTI). In this manuscript, we describe the diagnostic accuracy of urinary GAG measurements by LC-MS for MPS typing in 68 untreated MPS and mucolipidosis (ML) patients, 183 controls and 153 UTI samples. We report age-dependent reference values and cut-offs for chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS) and specific GAG ratios. The use of HS/DS ratio in combination to GAG concentrations normalized to creatinine improves the diagnostic accuracy in MPS type I, II, VI and VII. In total 15 samples classified to the wrong MPS type could be correctly assigned using HS/DS ratio. Increased KS/HS ratio in addition to increased KS improves discrimination of MPS type IV by excluding false positives. Some samples of UTI patients showed elevation of specific GAGs, mainly CS, KS and KS/HS ratio and could be misclassified as MPS type IV. Finally, DMB photometric assay performed in MPS and ML samples reveal four false negative tests (sensitivity of 94%). In conclusion, specific GAG ratios in complement to quantitative GAG values obtained by LC-MS enhance discrimination of MPS types. Exclusion of patients with UTI improve diagnostic accuracy in MPS IV but not in other types.

A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

Introduction: The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. Methods: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. Results: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. Conclusion: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.

Methylation metabolism in sepsis and systemic inflammatory response syndrome.

We have recently shown that sepsis leads to alterations of methylation metabolism in a rodent model. In this study we analyzed methylation metabolism and DNA methylation in human sepsis. Patients treated in one of the Intensive Care Units (ICU) at the University Hospital Bonn diagnosed with sepsis or systemic inflammatory response syndrome (n = 12) and patients who were treated due to traumatic brain injury, or stroke without clinical or laboratory signs of sepsis or major inflammation (n = 22) were included. Blood samples were taken two times a week, until ICU treatment was discontinued. Deproteinized plasma was used for simultaneous determination of the ubiquitous methyl-group donor S-adenosylmethionine (SAM) and its demethylated residue, S-adenosylhomocysteine (SAH), by using stable isotope dilution tandem mass spectrometry. Homocysteine (Hcys), hydrolyzation product of SAH, was determined by fully automated particle-enhanced immunonephelometry, and global DNA-methylation was measured by liquid chromatography tandem mass spectrometry. SAM (p < 0.001) and SAH (p < 0.001) plasma levels were higher in septic patients suggesting an increased cellular release of SAM and SAH in septic patients. The SAM/SAH ratio was decreased in septic patients (p = 0.002). There were no differences in homocysteine plasma levels (p = 0.32) or global leukocyte DNA methylation between septic and non-septic patients (p = 0.21) suggesting that sepsis-induced changes in methylation metabolism do not affect homocysteine plasma levels or the availability of SAM-derived methyl groups for DNA methylation. Sepsis and systemic inflammatory response syndrome induce considerable changes of methylation metabolism without apparent functional consequences on homocysteine plasma levels or DNA methylation. Further studies may explore the clinical relevance of the observed changes.

Pharmacokinetics of Faster and Standard Insulin Aspart During Fully Closed-Loop Insulin Delivery in Type 2 Diabetes.

Background: Faster insulin aspart is a novel formulation of insulin aspart aiming to accelerate its subcutaneous absorption. The aim of this study was to compare pharmacokinetics of faster insulin aspart versus standard insulin aspart in adults with type 2 diabetes during closed-loop insulin delivery. Methods: We assessed the pharmacokinetics of faster and standard insulin aspart from data obtained in a randomized double-blind crossover study evaluating fully closed-loop insulin delivery in adults with type 2 diabetes (n = 13, age 59 ± 10 years, BMI 34.5 ± 9.1 kg/m2, HbA1c 7.7% ± 1.2% [60 ± 13 mmol/mol]). Blood samples were collected every 15-30 min for 10 h to determine plasma insulin aspart concentration using liquid chromatography mass spectrometry. Time to peak plasma concentration (Tmax) was calculated using a two-compartment model. Results:Tmax was 68.7 ± 21.6 min for faster aspart and 89.7 ± 31.8 min for aspart (mean paired difference faster aspart minus aspart -15.5 min, 95% CI [-31.6 to 0.6 min], P = 0.06). Metabolic clearance rate did not differ between the two insulins (P = 0.61). Insulin amount delivered during closed-loop with faster aspart positively correlated with Tmax (rS = 0.73, P = 0.01), whereas no statistically significant correlation was found with body mass index (BMI), weight or HbA1C (all P > 0.18). Conclusion: In conclusion, Tmax tended to be shorter for faster aspart versus aspart during fully automated closed-loop insulin delivery and positively correlated with the amount of insulin delivered.

Delivery of Rapamycin Using In Situ Forming Implants Promotes Immunoregulation and Vascularized Composite Allograft Survival.

Vascularized composite allotransplantation (VCA), such as hand and face transplantation, is emerging as a potential solution in patients that suffered severe injuries. However, adverse effects of chronic high-dose immunosuppression regimens strongly limit the access to these procedures. In this study, we developed an in situ forming implant (ISFI) loaded with rapamycin to promote VCA acceptance. We hypothesized that the sustained delivery of low-dose rapamycin in proximity to the graft may promote graft survival and induce an immunoregulatory microenvironment, boosting the expansion of T regulatory cells (Treg). In vitro and in vivo analysis of rapamycin-loaded ISFI (Rapa-ISFI) showed sustained drug release with subtherapeutic systemic levels and persistent tissue levels. A single injection of Rapa-ISFI in the groin on the same side as a transplanted limb significantly prolonged VCA survival. Moreover, treatment with Rapa-ISFI increased the levels of multilineage mixed chimerism and the frequency of Treg both in the circulation and VCA-skin. Our study shows that Rapa-ISFI therapy represents a promising approach for minimizing immunosuppression, decreasing toxicity and increasing patient compliance. Importantly, the use of such a delivery system may favor the reprogramming of allogeneic responses towards a regulatory function in VCA and, potentially, in other transplants and inflammatory conditions.

Local release of tacrolimus from hydrogel-based drug delivery system is controlled by inflammatory enzymes in vivo and can be monitored non-invasively using in vivo imaging.

BACKGROUND: Local drug delivery systems that adjust the release of immunosuppressive drug in response to the nature and intensity of inflammation represent a promising approach to reduce systemic immunosuppression and its side effects in allotransplantation. Here we aimed to demonstrate that release of tacrolimus from triglycerol monostearate hydrogel is inflammation-dependent in vivo. We further report that by loading the hydrogel with a near-infrared dye, it is possible to monitor drug release non-invasively in an in vivo model of vascularized composite allotransplantation. MATERIALS AND METHODS: Inflammation was induced by local challenge with lipopolysaccharides in naïve rats 7 days after injection of tacrolimus-loaded hydrogel in the hind limb. Tacrolimus levels in blood and tissues were measured at selected time points. A near-infrared dye was encapsulated in the hydrogel together with tacrolimus in order to monitor hydrogel deposits and drug release in vitro and in vivo in a model of vascularized composite allotransplantation. RESULTS: Injection of lipopolysaccharides led to increased blood and skin tacrolimus levels (p = 0.0076, day 7 vs. day 12 in blood, and p = 0.0007 in treated limbs, 48 h after injection compared to controls). Moreover, lipopolysaccharides-injected animals had higher tacrolimus levels in treated limbs compared to contralateral limbs (p = 0.0003 for skin and p = 0.0053 for muscle). Imaging of hydrogel deposits and tacrolimus release was achieved by encapsulating near-infrared dye in the hydrogel for 160 days. The correlation of tacrolimus and near-infrared dye release from hydrogel was R2 = 0.6297 and R2 = 0.5619 in blood and grafts of transplanted animals respectively and R2 = 0.6066 in vitro. CONCLUSIONS: Here we demonstrate the inflammation-responsiveness of a tacrolimus-loaded hydrogel in vivo. Moreover, we show that encapsulating a near-infrared dye in the hydrogel provides a reliable correlation of tacrolimus and dye release from the hydrogel, and an accessible non-invasive method for monitoring drug release from hydrogel deposits.

Local Injections of Tacrolimus-loaded Hydrogel Reduce Systemic Immunosuppression-related Toxicity in Vascularized Composite Allotransplantation.

BACKGROUND: Routine application of vascularized composite allotransplantation is hampered by immunosuppression-related health comorbidities. To mitigate these, we developed an inflammation-responsive hydrogel for local immunosuppression. Here, we report on its long-term effect on graft survival, immunological, and toxicological impact. METHODS: Brown Norway-to-Lewis rat hindlimb transplantations were treated either systemically with daily injections of 1 mg/kg tacrolimus (TAC) or with subcutaneous intragraft injections of hydrogel containing 7 mg TAC, every 70 days. Animals were monitored for rejection or other pathology for 280 days. Systemic and graft TAC levels, regulatory T cells, and donor cell chimerism were measured periodically. At endpoint, markers for kidney, liver, and metabolic state were compared to naive age-matched rats. RESULTS: Both daily systemic TAC and subcutaneous intragraft TAC hydrogel at 70-day intervals were able to sustain graft survival longer than 280 days in 5 of 6 recipients. In the hydrogel group, 1 graft progressed to grade 3 rejection at postoperative day 149. In systemic TAC group, 1 animal was euthanized due to lymphoma on postoperative day 275. Hydrogel treatment provided stable graft and reduced systemic TAC levels, and a 4 times smaller total TAC dose compared with systemic immunosuppression. Hydrogel-treated animals showed preserved kidney function, absence of malignancies or opportunistic infections and increased hematopoietic chimerism compared with systemic immunosuppression. CONCLUSIONS: Our findings demonstrate that localized immunosuppression with TAC hydrogel is a long-term safe and reliable treatment. It may reduce the burden of systemic immunosuppression in vascularized composite allotransplantation, potentially boosting the clinical application of this surgical intervention.

Intrauterine valproate exposure is associated with alterations in hippocampal cell numbers and folate metabolism in a rat model of valproate teratogenicity.

PURPOSE: Valproate is one of the most commonly used anticonvulsive drugs. Despite its significant benefits, the teratogenicity of valproate is a relevant problem in the treatment of women of childbearing age. In addition to major congenital malformations, such as neural tube defects, reduced intelligence and attention after intrauterine valproate exposure are reported. Until now the mechanisms of teratogenicity of VPA are poorly understood and concepts how to reduce valproate teratogenicity are lacking. METHODS: In a rat model of valproate teratogenicity we examined hippocampal cell structure in 4 week old animals with a stereological approach. As potential mechanisms of VPA teratogenicity we examined histone acetylation by western blotting and metabolites of the folate metabolism as well as global DNA methylation by tandem mass spectrometry in the brain and liver tissue of newborn pups (p0). RESULTS: We found an increase in the number of neurons in the hippocampal areas CA1/2 (p=0.018) and CA3 (p=0.022), as well as a decreased number of astrocytes in CA1/2 (p=0.004) and CA3 (p=0.003) after intrauterine VPA exposure, as a possible indication of altered cell differentiation during intrauterine VPA exposure. Valproate exposure was also associated with an increase in 5-methyl-tetrahydrofolate (THF) (p=0.002) and a decrease in 5-10-methenyl-THF in the brain of newborn pups, as well as a reduced homocysteine plasma level (p<0.001). The described changes in hippocampal cell numbers and folate metabolism were only significant after high-dose intrauterine VPA exposure indicating a dose-dependent effect. VPA exposure was not associated with changes in histone acetylation or global DNA methylation in brain tissue in newborn pups. CONCLUSION: This study shows that intrauterine VPA exposure is associated with changes in hippocampal cell numbers in the CA1/2 and CA3 region and in folate metabolism.

Alcohol abuse and cigarette smoking are associated with global DNA hypermethylation: results from the German Investigation on Neurobiology in Alcoholism (GINA).

Recent studies have shown that smoking and alcoholism may be associated with altered DNA methylation and that alcohol consumption might induce changes in DNA methylation by altering homocysteine metabolism. In this monocenter study, we included 363 consecutive patients referred for hospitalization for alcohol detoxification treatment. Blood samples were obtained on treatment days 1, 3, and 7 for measurement of global DNA methylation in leukocytes by liquid chromatography tandem mass spectrometry. Genomic DNA was used for genotyping the following seven genetic variants of homocysteine metabolism: cystathionine beta-synthase (CBS) c.844_855ins68, dihydrofolate-reductase (DHFR) c.594 + 59del19bp, methylenetetrahydrofolate-reductase (MTHFR) c.677C > T and c.1298A > C, methyltetrahydrofolate-transferase (MTR) c.2756A > G, reduced folate carrier 1 (RFC1) c.80G > A, and transcobalamin 2 c.776C > G. Multivariate linear regression showed a positive correlation of global DNA methylation with alcohol consumption and smoking on day 1 of hospitalization. DNA methylation was not correlated with homocysteine or vitamin plasma levels, nor with the tested genetic variants of homocysteine metabolism. This suggests a direct effect of alcohol consumption and smoking on DNA methylation, which is not mediated by effects of alcohol on homocysteine metabolism.

Improving reconstituted HDL composition for efficient post-ischemic reduction of ischemia reperfusion injury.

BACKGROUND: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. OBJECTIVE: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. METHODS AND RESULTS: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. CONCLUSION: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.