Development and validation of an LC-MS/MS method for quantifying diamorphine and its major metabolites 6-monoacetylmorphine, morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma.

Diamorphine, commonly known as heroin, is a semi-synthetic opioid analgesic. In the context of heroin-assisted treatment for opioid-dependent patients, diamorphine is mostly administered intravenously. However, recent attention has shifted towards intranasal administration as a better-tolerated alternative to the intravenous route. Here, we developed and validated a rapid bioanalytical method for the simultaneous quantification of diamorphine and its major metabolites 6-monoacetylmorphine, morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A straightforward protein precipitation extraction step was used for sample preparation. Chromatographic analyte separation was achieved using a Kinetex EVO C18 analytical column and a mobile phase gradient comprising an aqueous solution of ammonium hydrogen carbonate and methanol supplied with formic acid. Employing positive electrospray ionization and scheduled multiple reaction monitoring, we established a quantification range of 1-1,000 ng/mL for all analytes. Our validation results demonstrate a mean intra-assay accuracy of 91-106% and an intra-assay precision (CV) between 2 and 9% for all analytes and over three validation runs. The method exhibits a high extraction recovery (> 87%) and a negligible matrix effect (99-125%). Furthermore, no interferences with endogenous plasma compounds were detected. Lastly, we applied the method to assess the plasma concentrations of an opioid-dependent patient after the intranasal administration of diamorphine in a clinical study. In summary, we have successfully developed a rapid, highly reliable, and straightforward bioanalytical method for quantifying diamorphine and its metabolites in low amounts of clinical plasma samples.

Acute effects of intravenous DMT in a randomized placebo-controlled study in healthy participants.

N,N-dimethyltryptamine (DMT) is distinct among classic serotonergic psychedelics because of its short-lasting effects when administered intravenously. Despite growing interest in the experimental and therapeutic use of intravenous DMT, data are lacking on its clinical pharmacology. We conducted a double-blind, randomized, placebo-controlled crossover trial in 27 healthy participants to test different intravenous DMT administration regimens: placebo, low infusion (0.6 mg/min), high infusion (1 mg/min), low bolus + low infusion (15 mg + 0.6 mg/min), and high bolus + high infusion (25 mg + 1 mg/min). Study sessions lasted for 5 h and were separated by at least 1 week. Participant's lifetime use of psychedelics was ≤20 times. Outcome measures included subjective, autonomic, and adverse effects, pharmacokinetics of DMT, and plasma levels of brain-derived neurotropic factor (BDNF) and oxytocin. Low (15 mg) and high (25 mg) DMT bolus doses rapidly induced very intense psychedelic effects that peaked within 2 min. DMT infusions (0.6 or 1 mg/min) without a bolus induced slowly increasing and dose-dependent psychedelic effects that reached plateaus after 30 min. Both bolus doses produced more negative subjective effects and anxiety than infusions. After stopping the infusion, all drug effects rapidly decreased and completely subsided within 15 min, consistent with a short early plasma elimination half-life (t1/2α) of 5.0-5.8 min, followed by longer late elimination (t1/2ß = 14-16 min) after 15-20 min. Subjective effects of DMT were stable from 30 to 90 min, despite further increasing plasma concentrations, thus indicating acute tolerance to continuous DMT administration. Intravenous DMT, particularly when administered as an infusion, is a promising tool for the controlled induction of a psychedelic state that can be tailored to the specific needs of patients and therapeutic sessions.Trial registration: ClinicalTrials.gov identifier: NCT04353024.

Liquid chromatography-tandem mass spectrometry method for the bioanalysis of N,N-dimethyltryptamine (DMT) and its metabolites DMT-N-oxide and indole-3-acetic acid in human plasma.

The indole alkaloid N,N-dimethyltryptamine (DMT) induces psychedelic effects in humans. In addition to ceremonial and recreational use, DMT is subject to clinical investigations. Sensitive bioanalytical methods are required to assess the pharmacokinetics of DMT and its metabolites in human plasma. Here, a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMT and its major metabolites indole-3-acetic acid (IAA) and DMT-N-oxide (DMT-NO) was developed and validated. As IAA is an endogenous component of human plasma, 13C6-IAA was used to determine IAA concentrations. After simple protein precipitation with methanol, analytes were separated on a pentafluorophenyl column. A gradient consisting of 0.1% (v/v) formic acid in a methanol-water mixture was applied for analyte separation. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range of the assay was 0.25-250 ng/mL for DMT, 0.1-100 ng/mL for DMT-NO, and 25-25,000 ng/mL for 13C6-IAA. The intra- and inter-assay accuracy was 93-113% for all analytes at all quality control levels, with coefficient of variation ≤ 11%. All analytes were stable under storage conditions relevant for the analysis of large batches of study samples. The validated method was capable of assessing pharmacokinetic (PK) parameters of DMT and its metabolites in study participants intravenously perfused with 1 mg/min DMT for 90 min. Overall, the developed method is easy-to-use, has a short run time, and qualifies for PK and metabolism studies of DMT in clinical settings.

Differences in clinical features associated with cannabis intoxication in presentations to European emergency departments according to patient age and sex.

OBJECTIVE: To investigate if clinical features associated with acute cannabis intoxication in patients presenting to Emergency Departments for medical assistance differ according to patient age and sex. METHODS: We analysed presentations in the Euro-DEN Plus dataset from 2014 to 2019 in which cannabis was the only drug involved (except for alcohol), and age, sex and alcohol co-ingestion had been recorded. Age was considered as categorical (five groups; <20, 20-29, 30-39, 40-49 and ≥50 years), and sex as binary variable (male/female). We evaluated 12 key clinical features recorded during emergency department (ED) care. Risks of presenting with each of these clinical features according to patient age and sex were calculated by logistic regression models, and adjusted for sex, age and alcohol co-ingestion. RESULTS: 4,268 of 43,633 Euro-DEN presentations (9.8%) fulfilled the inclusion criteria (median age: 26 years (IQR = 20-34), 70% male, 52% co-ingested alcohol). The frequency of clinical features was: anxiety 28%, vomiting 24%, agitation 23%, palpitations 14%, reduced consciousness 13%, acute psychosis 9%, hallucinations 9%, chest pain 7%, headache 6%, hypotension 4%, hypertension 3% and seizures 2%. Patients younger than 20 years more frequently had vomiting (34.7% of cases), reduced consciousness (21.5%), and headache (10.8%); and less frequently acute psychosis (5.5%). Patients older than 49 years more often had hypotension (6.5%) and less frequently vomiting (20%), anxiety (14%), agitation (14%) and reduced consciousness (10%). Males more frequently presented with hypertension (3.7 vs. 1.5%; OR = 2.311, 95%CI = 1.299-3.816), psychosis (10.4 vs 6.3%; 1.948, 1.432-2.430), chest pain (8.1 vs 4.5%; 1.838, 1.390-2.430) and seizures (2.5 vs 1.4%; 1.805, 1.065-3.060), and less frequently with vomiting (21.8 vs 28.2%; 0.793, 0.677-0.930), anxiety (25.4 vs 32.3%; 0.655, 0.561-0.766) and hypotension (2.9 vs 5.8%; 0.485, 0.350-0.671). CONCLUSIONS: The prevalence of some clinical features typically associated with acute cannabis intoxication differed according to age and sex. The causes for these differences should be further investigated in order to better understand the pathophysiology of cannabis-related acute toxicity, and they may be relevant particularly for developing prevention campaigns and for treatment in specific sex and/or age groups.

Association of mannose-binding lectin, ficolin-2 and immunoglobulin concentrations with future exacerbations in patients with chronic obstructive pulmonary disease: secondary analysis of the randomized controlled REDUCE trial.

BACKGROUND: The innate and adaptive immune system is involved in the airway inflammation associated with acute exacerbations in patients with chronic obstructive pulmonary disease (COPD). We evaluated the association of mannose-binding lectin (MBL), immunoglobulin (Ig) and ficolin-2 concentrations with COPD exacerbations and according to the glucocorticoid treatment duration for an index exacerbation. METHODS: Post-hoc analysis of the randomized, double-blind, placebo-controlled REDUCE trial of 5 vs. 14 days of glucocorticoid treatment for an index exacerbation. MBL, ficolin-2 and total IgG/IgA and subclass concentrations were determined in stored samples drawn (n = 178) 30 days after the index exacerbation and associated with the risk of re-exacerbation during a 180-day follow-up period. RESULTS: IgG and subclass concentrations were significantly lower after 14 days vs. 5 days of glucocorticoid treatment. Patients with higher MBL concentrations were more likely to suffer from a future exacerbation (multivariable hazard ratio 1.03 per 200 ng/ml increase (95% confidence interval (CI) 1.00-1.06), p = 0.048), whereas ficolin-2 and IgG deficiency were not associated. The risk was most pronounced in patients with high MBL concentrations, IgG deficiency and 14 days of glucocorticoid treatment pointing towards an interactive effect of MBL and IgG deficiency in the presence of prolonged glucocorticoid treatment duration [Relative excess risk due to interaction 2.13 (95% CI - 0.41-4.66, p = 0.10)]. IgG concentrations were significantly lower in patients with frequent re-exacerbations (IgG, 7.81 g/L vs. 9.53 g/L, p = 0.03). CONCLUSIONS: MBL modified the short-term exacerbation risk after a recent acute exacerbation of COPD, particularly in the setting of concurrent IgG deficiency and recent prolonged systemic glucocorticoid treatment. Ficolin-2 did not emerge as a predictor of a future exacerbation risk.

Epitope-Specific Anti-C1q Autoantibodies in Systemic Lupus Erythematosus.

Objective: In patients with systemic lupus erythematosus (SLE) complement C1q is frequently targeted by autoantibodies (anti-C1q), that correlate best with active renal disease. Anti-C1q bind to largely unknown epitopes on the collagen-like region (CLR) of this highly functional molecule. Here we aimed at exploring the role of epitope-specific anti-C1q in SLE patients. Methods: First, 22 sera of SLE patients, healthy controls and anti-C1q positive patients without SLE were screened for anti-C1q epitopes by a PEPperMAP® microarray, expressing CLR of C1q derived peptides with one amino acid (AA) shift in different lengths and conformations. Afterwards, samples of 378 SLE patients and 100 healthy blood donors were analyzed for antibodies against the identified epitopes by peptide-based ELISA. Relationships between peptide-specific autoantibodies and SLE disease manifestations were explored by logistic regression models. Results: The epitope mapping showed increased IgG binding to three peptides of the C1q A- and three of the C1q B-chain. In subsequent peptide-based ELISAs, SLE sera showed significantly higher binding to two N-terminally located C1q A-chain peptides than controls (p < 0.0001), but not to the other peptides. While anti-C1q were associated with a broad spectrum of disease manifestations, some of the peptide-antibodies were associated with selected disease manifestations, and antibodies against the N-terminal C1q A-chain showed a stronger discrimination between SLE and controls than conventional anti-C1q. Conclusion: In this large explorative study anti-C1q correlate with SLE overall disease activity. In contrast, peptide-antibodies are associated with specific aspects of the disease suggesting epitope-specific effects of anti-C1q in patients with SLE.

Local and Systemic Concentrations of Pattern Recognition Receptors of the Lectin Pathway of Complement in a Cohort of Patients With Interstitial Lung Diseases.

Background: The role of the lectin pathway of complement in the pathogenesis of interstitial lung diseases (ILDs) is largely unknown. Pattern recognition receptors (PRR) of the lectin pathway are involved in the clearance of apoptotic cells either via activation of the complement system or as direct opsonins. As recent findings suggest a role of apoptosis in the development of pulmonary fibrosis, the influence of plasma lectins has lately been considered in various ILDs, but data on local concentrations in the lungs are lacking. This study investigated the role of mannose-binding lectin (MBL), ficolin-2 and ficolin-3 in ILD patients with a focus on idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Methods: A case control study was conducted involving 80 patients with different forms of ILD as well as 40 control patients undergoing routine flexible bronchoscopy with bronchoalveolar lavage (BAL). Plasma and BAL fluid (BALF) levels of MBL, ficolin-2 and ficolin-3 as well as complement split products C4d and C5a (only in BALF) were measured by enzyme-linked immunosorbent assays. Eight single-nucleotide polymorphisms (SNPs) of MBL and ficolin-2 were determined by genotyping and tested for their association with ILDs. Results: We included 35, 35, 10, and 40 patients with sarcoidosis, idiopathic pulmonary fibrosis (IPF), other ILD, and a control group, respectively. BALF but not plasma levels of the three PRR were significantly elevated in sarcoidosis patients compared to a control group without ILD (MBL: median 66.8 vs. 24.6 ng/ml, p = 0.02, ficolin-2: 140 vs. 58.8 ng/ml, p = 0.01, ficolin-3: 2523 vs. 1180 ng/ml, p = 0.02), whereas the frequency of the investigated SNPs was similar. In line, complement split products were markedly elevated in BALF of sarcoidosis patients (C4d, median 97.4 vs. 0 ng/ml, p = 0.10; C5a, 23.9 vs. 9.1 ng/ml, p = 0.01). There was a weak positive correlation of BALF ficolin-3 with serum neopterin, a marker of sarcoidosis activity. In IPF patients, we observed numerically higher MBL plasma and BALF levels (plasma, median 1511 vs. 879 ng/ml, p = 0.44; BALF, 37.5 vs. 24.6 ng/ml, p = 0.7) as well as lower ficolin-2 plasma levels (plasma 1111 vs. 1647 ng/ml, p = 0.11). Ficolin-2 plasma levels were inversely correlated with the forced vital capacity (r = 0.55, p = 0.1). Conclusion: This is the first study to simultaneously assess systemic and local lectin pathway protein levels in ILD patients. Our data suggest an involvement of PRR of the lectin pathway in the pathogenesis of sarcoidosis given the significantly higher BALF levels compared to a control group. Additional analyses in a larger patient cohort are required to confirm or refute a potential effect of local and/or systemic ficolin-2 levels in IPF patients.

Impact of inflammation on adverse cardiovascular events in patients with acute coronary syndromes.

AIMS: Inflammation is a key factor in the long-term outcome of acute coronary syndromes (ACS). The aim of the present study was to evaluate inflammatory markers in patients with ACS as predictors for major adverse cardiovascular events (MACE) and hard events. METHODS: This study included 1548 patients with ACS. C-reactive protein (CRP), white blood count (WBC), and their subtypes were analyzed during hospitalization. Receiver operator characteristic (ROC) and Kaplan-Meier survival curves were used to assess the predictive value and hard events (nonfatal myocardial infarction and cardiac death) and MACE (hard events, hospitalization for cardiac causes, late revascularization and stroke) were obtained during 30 days. RESULTS: ROC analysis of CRP and WBC to predict adverse events revealed cut-offs of 47.5 ng/l and 16.6 × 10/µl for MACE and 93.5 ng/l and 16.6 × 10/µl for hard events. The cumulative adverse event rates were significantly higher in patients with increased CRP (≥47.5 ng/l; 17 versus 4%, P < 0.001) and WBC (≥16.6 × 10/µl; 21 versus 5%, P < 0.001) for MACE and with elevated CRP (≥93.5 ng/l; 16 versus 2%, P < 0.001) and WBC (≥16.6 × 10/µl; 18 versus 2%, P < 0.001) for hard events, demonstrating highest event rates with elevation of both inflammatory markers: (28 versus 5%, P < 0.001) for MACE and (26 versus 2%, P < 0.001) for hard events. Analysis of CRP and WBC further revealed a substantial negative correlation with left ventricular function (P < 0.001). Moreover, markers of myocardial damage were significantly elevated in patients with abnormal CRP or WBC (P < 0.001). CONCLUSION: Inflammatory markers such as CRP and WBC alone and, particularly, in combination are strong and independent predictors of outcome in patients with ACS.