Non-adherence to cardiometabolic medication as assessed by LC-MS/MS in urine and its association with kidney and cardiovascular outcomes in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Non-adherence to medication is a frequent barrier in the treatment of patients with type 2 diabetes mellitus, potentially limiting the effectiveness of evidence-based treatments. Previous studies have mostly relied on indirect adherence measures to analyse outcomes based on adherence. The aim of this study was to use LC-MS/MS in urine-a non-invasive, direct and objective measure-to assess non-adherence to cardiometabolic drugs and analyse its association with kidney and cardiovascular outcomes. METHODS: This cohort study includes 1125 participants from the PROVALID study, which follows patients with type 2 diabetes mellitus at the primary care level. Baseline urine samples were tested for 79 cardiometabolic drugs and metabolites thereof via LC-MS/MS. An individual was classified as totally adherent if markers for all drugs were detected, partially non-adherent when at least one marker for one drug was detected, and totally non-adherent if no markers for any drugs were detected. Non-adherence was then analysed in the context of cardiovascular (composite of myocardial infarction, stroke and cardiovascular death) and kidney (composite of sustained 40% decline in eGFR, sustained progression of albuminuria, kidney replacement therapy and death from kidney failure) outcomes. RESULTS: Of the participants, 56.3% were totally adherent, 42.0% were partially non-adherent, and 1.7% were totally non-adherent to screened cardiometabolic drugs. Adherence was highest to antiplatelet and glucose-lowering agents and lowest to lipid-lowering agents. Over a median (IQR) follow-up time of 5.10 (4.12-6.12) years, worse cardiovascular outcomes were observed with non-adherence to antiplatelet drugs (HR 10.13 [95% CI 3.06, 33.56]) and worse kidney outcomes were observed with non-adherence to antihypertensive drugs (HR 1.98 [95% CI 1.37, 2.86]). CONCLUSIONS/INTERPRETATION: This analysis shows that non-adherence to cardiometabolic drug regimens is common in type 2 diabetes mellitus and negatively affects kidney and cardiovascular outcomes.

Timing of Interleukin-4 Stimulation of Macrophages Determines Their Anti-Microbial Activity during Infection with Salmonella enterica Serovar Typhimurium.

Priming of macrophages with interferon-gamma (IFNγ) or interleukin-4 (IL-4) leads to polarisation into pro-inflammatory or anti-inflammatory subtypes, which produce key enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), respectively, and in this way determine host responses to infection. Importantly, L-arginine is the substrate for both enzymes. ARG1 upregulation is associated with increased pathogen load in different infection models. However, while differentiation of macrophages with IL-4 impairs host resistance to the intracellular bacterium Salmonella enterica serovar Typhimurium (S.tm), little is known on the effects of IL-4 on unpolarised macrophages during infection. Therefore, bone-marrow-derived macrophages (BMDM) from C57BL/6N, Tie2Cre+/-ARG1fl/fl (KO), Tie2Cre-/-ARG1fl/fl (WT) mice were infected with S.tm in the undifferentiated state and then stimulated with IL-4 or IFNγ. In addition, BMDM of C57BL/6N mice were first polarised upon stimulation with IL-4 or IFNγ and then infected with S.tm. Interestingly, in contrast to polarisation of BMDM with IL-4 prior to infection, treatment of non-polarised S.tm-infected BMDM with IL-4 resulted in improved infection control whereas stimulation with IFNγ led to an increase in intracellular bacterial numbers compared to unstimulated controls. This effect of IL-4 was paralleled by decreased ARG1 levels and increased iNOS expression. Furthermore, the L-arginine pathway metabolites ornithine and polyamines were enriched in unpolarised cells infected with S.tm and stimulated with IL-4. Depletion of L-arginine reversed the protective effect of IL-4 toward infection control. Our data show that stimulation of S.tm-infected macrophages with IL-4 reduced bacterial multiplication via metabolic re-programming of L-arginine-dependent pathways.

A rapid and convenient sample preparation method for the analysis of cannabinoids in oral fluid samples.

Cannabis is the world's most used illegal drug. The main psychoactive component of cannabis is Δ9-tetrahydrocannabinol (THC). To aid the identification of cannabis-impaired individuals, a simple but effective workflow for reliable quantification of THC and its metabolites in oral fluid samples collected with the Greiner Bio-One Saliva Collection System is presented. Sampling involves rinsing the oral cavity with an extraction solution containing a citrate buffer stimulating salivary flow. Sample processing targeted the cannabinoid fraction interacting with proteins and other insoluble constituents that can be separated by centrifugation. Approximately 50% of the total amount of cannabinoids included in the oral fluid was recovered from the obtained pellet by extraction with acetonitrile. Liquid chromatography-tandem mass spectrometry was used for cannabinoid quantification. Fitness of the developed workflow for application in forensic and clinical cannabis testing was demonstrated by evaluating multiple performance parameters, including selectivity, linearity, limits of quantification (LOQs), accuracy, precision, matrix effects, extraction recoveries, process efficiencies and stability. Furthermore, sensitivity and specificity of the developed oral fluid-based cannabis test was demonstrated by analysing 195 samples collected either from opioid addicts or persons suspected of driving under the influence of drugs. The accuracy of identifying a person with the presence of THC in blood was found to be 97.9%.

Human tissue distribution of caspofungin.

Tissue concentrations of caspofungin were determined in nine clinically relevant tissues taken during routine autopsy of 20 patients who had died during caspofungin treatment or within 23 days of cessation. The highest levels were achieved in liver, with concentrations ranging from ≤0.50 to 91.5 µg/g (0.60 µg/g 21 days after the last administration), followed by spleen (<0.25-46.3 µg/g), kidney (<0.25-33.6 µg/g) and lung (<0.25-31.0 µg/g). Intermediate concentrations were found in pancreas, skeletal muscle, thyroid and myocardium. The lowest concentrations were found in brain; caspofungin was only detectable in six of 17 samples. Caspofungin concentrations exceeded the minimum inhibitory concentration values of pathogenic Candida spp. in most of the tissue samples taken from patients who had died during treatment, except in brain samples. These findings warrant clinical outcome studies to establish the optimal treatment for deep-seated candidiasis, and support the current recommendations against echinocandins for treatment of fungal meningoencephalitis.

In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples.

Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro-generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.

Tofacitinib-Induced Modulation of Intestinal Adaptive and Innate Immunity and Factors Driving Cellular and Systemic Pharmacokinetics.

OBJECTIVE: By interfering with multiple cytokines, human Janus kinase inhibitors (JAKis) are of growing importance in the treatment of malignant and inflammatory conditions. Although tofacitinib has demonstrated efficacy as the first-in-class JAKi in ulcerative colitis many aspects concerning its mode of action and pharmacokinetics remain unresolved. DESIGN: We studied tofacitinib's impact on various primary human innate and adaptive immune cells. In-depth in vivo studies were performed in dextran sodium sulfate-induced colitis in mice. Immune populations were characterized by flow cytometry and critical transcription factors and effector cytokines were analyzed. Pharmacokinetics of tofacitinib was studied by liquid chromatography-tandem mass spectrometry. RESULTS: Tofacitinib inhibited proliferation in CD4+ and CD8+ T cells along with Th1 and Th17 differentiation, while Th2 and regulatory T cell lineages were largely unaffected. Monocytes and macrophages were directed toward an anti-inflammatory phenotype and cytokine production was suppressed in intestinal epithelial cells. These findings were largely reproducible in murine cells of the inflamed mucosa in dextran sulfate sodium colitis. Short-term treatment with tofacitinib had little impact on the mouse microbiota. Strikingly, the degree of inflammation and circulating tofacitinib levels showed a strong positive correlation. Finally, we identified inflammation-induced equilibrative nucleoside transporters as regulators of tofacitinib uptake into leukocytes. CONCLUSIONS: We provide a detailed analysis of the cell-specific immune-suppressive effects of the JAKis tofacitinib on innate and adaptive immunity and reveal that intestinal inflammation critically impacts tofacitinib's pharmacokinetics in mice. Furthermore, we describe an unappreciated mechanism-namely induction of equilibrative nucleoside transporters-enhancing baseline cellular uptake that can be inhibited pharmaceutically.

Analysis of cannabis seizures by non-targeted liquid chromatography-tandem mass spectrometry.

Due to the popularity of recreational cannabis use, contamination of this drug with diverse classes of chemicals, including pesticides, mycotoxins, and synthetic cannabinoids, has been identified as major threat for public health. For the detection of these compounds in seized cannabis, a screening workflow involving non-targeted liquid chromatography-tandem mass spectrometry (LCMS/MS) was developed. A Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method was used for the extraction of small bioorganic molecules from ground dried material. Instrumental analysis involved chromatographic separation of compounds and subsequent mass spectrometric detection. Collection of MS and MS/MS information was accomplished by data-dependent acquisition. Compound identification was primarily based on matching acquired MS/MS-spectra to several thousands of reference spectra stored in multiple libraries. Additionally, for selected cannabinoid and pesticide standards, a retention time library was developed. Performance of the workflow was evaluated for 182 pesticides. All tested pesticides were detectable at 5000 µg/kg, 94 % at 500 µg/kg, and 50 % at 50 µg/kg. The workflow was applied to the screening of seized cannabis samples. 41 out of 93 analysed samples (44 %) were tested positive for one or more contaminants impairing quality and/or safety of the material. The detected contaminants included a synthetic cannabinoid (5F-MDMB-PINACA), fifteen pesticide residues (boscalid, carbendazim, chlorantraniliprole, chlorpyrifos, chlorotoluron, cyprodinil, diflubenzuron, ethiofencarb sulfoxide, hexythiazox, iprodione, metalaxyl, pyrimethanil, terbutryn, thiophanate methyl, and trifloxystrobin), and a mycotoxin (sterigmatocystin).

Monitoring drug consumption in Innsbruck during coronavirus disease 2019 (COVID-19) lockdown by wastewater analysis.

The coronavirus disease 2019 (COVID-19) has developed into a serious pandemic with millions of cases diagnosed worldwide. To fight COVID-19 pandemic, over 100 countries instituted either a full or partial lockdown, affecting billions of people. In Tyrol, first lockdown measures were taken on 10 March 2020. On 16 March 2020, a curfew went into force which ended on 1 May 2020. On 19 March 2020, Tyrol as a whole was placed in quarantine which ended on 7 April 2020. The governmental actions helped reducing the spread of COVID-19 at the cost of significant effects on social life and behaviour. Accordingly, to provide a comprehensive picture of the population health status not only input from medical and biological sciences is required, but also from other sciences able to provide lifestyle information such as drug use. Herein, wastewater-based epidemiology was used for studying temporal trends of licit and illicit drug consumption during lockdown and quarantine in the area of the Tyrolean capital Innsbruck (174,000 inhabitants). On 35 days between 12 March 2020 and 15 April 2020, loads of 23 markers were monitored in wastewater. Loads determined on 292 days between March 2016 and January 2020 served as reference. During lockdown, changes in the consumption patterns of recreational drugs (i.e. cocaine, amphetamine, 3,4-methylenedioxymethamphetamine, methamphetamine, and alcohol) and pharmaceuticals for short-term application (i.e. acetaminophen, codeine, and trimethoprim) were detected. For illicit drugs and alcohol, it is very likely that observed changes were linked to the shutdown of the hospitality industry and event cancelation which led to a reduced demand of these compounds particularly on weekends. For the pharmaceuticals, further work will be necessary to clarify if the observed declines are indicators of improved population health or of some kind of restraining effect that reduced the number of consultations of medical doctors and pharmacies.

Cerebrospinal Fluid Drug Concentrations and Clinical Outcome of Patients with Neoplastic Meningitis Treated with Liposomal Cytarabine.

BACKGROUND AND OBJECTIVE: Liposomal cytarabine is a slow-release formulation for intrathecal application in patients with neoplastic meningitis. Although standard dosing intervals range from 2 to 4 weeks, it is unclear whether sustained cytotoxic cerebrospinal fluid (CSF) concentrations can be achieved beyond 14 days from drug injection. The objective of this study was to assess CSF and plasma concentrations of liposomal cytarabine more than 2 weeks after lumbar drug administration and to correlate those findings with clinical outcome. METHODS: 66 matched CSF and plasma drug concentrations were analyzed by a validated liquid chromatography-tandem mass spectrometry method starting at day 13 from lumbar drug injection in 19 patients with neoplastic meningitis treated with liposomal cytarabine. CSF drug concentrations were correlated with clinical outcome. RESULTS: Overall response rate was 63.2% (12/19). 100% (9/9) of patients with positive CSF cytology at diagnosis achieved cytological complete remission, and none of the patients (0/19) experienced on-drug disease progression. In responding patients with controlled systemic disease, CNS-specific progression-free survival was 14 months (n = 4; range 5-25 months). The median CSF concentration of free cytarabine was 156 ng/ml (range 5-4581 ng/ml) and 146 ng/ml (range 5-353 ng/ml) in samples withdrawn at days 13-16 and at days 25-29 after intrathecal drug injection, respectively. Free cytarabine concentrations > 100 ng/ml were detected in 58.8% (20/34) and 53.3% (7/13) of the CSF samples obtained at days 13-16 and days 25-29, respectively. CSF drug concentrations did not differ significantly between responding and nonresponding patients. CONCLUSION: Liposomal cytarabine permits prolonged CSF drug exposure, with cytotoxic cytarabine concentrations that are detectable for 4 weeks in the majority of patients. The preserved clinical activity seen in patients with inferior CSF drug concentrations (< 100 ng/ml) suggests that maintaining lower cytarabine concentrations for a longer period of time may be similarly effective as using short peak concentrations.

A validated workflow for drug detection in oral fluid by non-targeted liquid chromatography-tandem mass spectrometry.

Oral fluid is recognized as an important specimen for drug testing. Common applications are monitoring in substance abuse treatment programs, therapeutic drug monitoring, pain management, workplace drug testing, clinical toxicology, and driving under the influence of drugs (DRUID). In this study, we demonstrate that non-targeted LC-MS/MS with subsequent compound identification by tandem mass spectral library search is a valuable tool for comprehensive detection and confirmation of drugs in oral fluid samples. The workflow developed involves solid-phase extraction and chromatographic separation on reversed phase materials. Mass spectrometric detection is accomplished on a quadrupole-quadrupole-time-of-flight instrument operated with data-dependent acquisition control. The workflow was optimized for 500 µl of neat oral fluid collected with the Greiner Bio-One saliva collection system. The fitness of the developed method was tested and proven by analyzing blank and spiked samples as well as 59 authentic patient samples. We could demonstrate that compounds with logP values in the range 0.5-5.5 are efficiently detected at low nanograms per milliliter concentrations. The true positive and true negative rates of automated library search were equal or close to 100%. The beauty of the non-targeted LC-MS/MS approach is the ability to detect compounds hardly included in routinely applied targeted assays, and this was demonstrated by detecting the synthetic opioid U-47700 in two patient samples. Graphical abstract ᅟ.

Annotating Nontargeted LC-HRMS/MS Data with Two Complementary Tandem Mass Spectral Libraries.

Tandem mass spectral databases are indispensable for fast and reliable compound identification in nontargeted analysis with liquid chromatography⁻high resolution tandem mass spectrometry (LC-HRMS/MS), which is applied to a wide range of scientific fields. While many articles now review and compare spectral libraries, in this manuscript we investigate two high-quality and specialized collections from our respective institutes, recorded on different instruments (quadrupole time-of-flight or QqTOF vs. Orbitrap). The optimal range of collision energies for spectral comparison was evaluated using 233 overlapping compounds between the two libraries, revealing that spectra in the range of CE 20⁻50 eV on the QqTOF and 30⁻60 nominal collision energy units on the Orbitrap provided optimal matching results for these libraries. Applications to complex samples from the respective institutes revealed that the libraries, combined with a simple data mining approach to retrieve all spectra with precursor and fragment information, could confirm many validated target identifications and yield several new Level 2a (spectral match) identifications. While the results presented are not surprising in many ways, this article adds new results to the debate on the comparability of Orbitrap and QqTOF data and the application of spectral libraries to yield rapid and high-confidence tentative identifications in complex human and environmental samples.

Impact of different hydrophobic ion pairs of octreotide on its oral bioavailability in pigs.

The objective of this study was to investigate the impact of different hydrophobic ion pairs (HIP) on the oral bioavailability of the model drug octreotide in pigs. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate and docusate differing in lipophilicity. These hydrophobic ion pairs were incorporated in self-emulsifying drug delivery systems (SEDDS) based on BrijO10, octyldodecanol, propylene glycol and ethanol in a concentration of 5mg/ml. SEDDS were characterized regarding size distribution, zeta potential, stability towards lipase, log DSEDDS/release medium and mucus diffusion behavior. The oral bioavailability of octreotide was evaluated in pigs via LC-MS/MS analyses. Most efficient ion pairing was achieved at a molar ratio of 1:3 (peptide: surfactant). SEDDS containing the octreotide-deoxycholate, -decanoate and -docusate ion pair exhibited a mean droplet size of 152nm, 112nm and 191nm and a zeta potential of -3.7, -4.6 and -5.7mV, respectively. They were completely stable towards degradation by lipase and showed a log DSEDDS/release medium of 1.7, 1.8 and 2.7, respectively. The diffusion coefficient of these SEDDS was in the range of 0.03, 0.11 and 0.17×10-9cm2/s, respectively. In vivo studies with these HIPs showed no improvement in the oral bioavailability in case of octreotide-decanoate. In contrast, octreotide-deoxycholate and octreotide-docusate SEDDS resulted in a 17.9-fold and 4.2-fold higher bioavailability vs. CONTROL: According to these results, hydrophobic ion pairing could be identified as a key parameter for SEDDS to achieve high oral bioavailability.