Intramuscular vaccination against SARS-CoV-2 transiently induces neutralizing IgG rather than IgA in the saliva.

The mucosal immunity is crucial for restricting SARS-CoV-2 at its entry site. Intramuscularly applied vaccines against SARS-CoV-2 stimulate high levels of neutralizing Abs in serum, but the impact of these intramuscular vaccinations on features of mucosal immunity is less clear. Here, we analyzed kinetic and functional properties of anti-SARS-CoV-2 Abs in the saliva after vaccination with BNT162b2. We analyzed a total of 24 healthy donors longitudinally for up to 16 months. We found that specific IgG appeared in the saliva after the second vaccination, declined thereafter and reappeared after the third vaccination. Adjusting serum and saliva for the same IgG concentration revealed a strong correlation between the reactivity in these two compartments. Reactivity to VoCs correlated strongly as seen by ELISAs against RBD variants and by live-virus neutralizing assays against replication-competent viruses. For further functional analysis, we purified IgG and IgA from serum and saliva. In vaccinated donors we found neutralizing activity towards authentic virus in the IgG, but not in the IgA fraction of the saliva. In contrast, IgA with neutralizing activity appeared in the saliva only after breakthrough infection. In serum, we found neutralizing activity in both the IgA and IgG fractions. Together, we show that intramuscular mRNA vaccination transiently induces a mucosal immunity that is mediated by IgG and thus differs from the mucosal immunity after infection. Waning of specific mucosal IgG might be linked to susceptibility for breakthrough infection.

The relationship between blood-brain barrier dysfunction and neurocognitive impairments in first-episode psychosis: findings from a retrospective chart analysis.

BACKGROUND: Even before the onset of psychotic symptoms, individuals with schizophrenia display cognitive impairments. Simultaneously, increasing amounts of individuals exhibit dysfunction of the blood-brain barrier (BBB). However, the impact of BBB dysfunction on neurocognitive impairment in people with first-episode psychosis has not yet been investigated. AIMS: To advance understanding of said relationship, we considered one of the largest first-episode psychosis cohorts with cerebrospinal fluid parameters available, and investigated whether BBB dysfunction is related to working memory, working speed and attention. METHOD: We conducted a retrospective chart review of 121 in-patients diagnosed with a first episode of a schizophrenia spectrum disorder. Patients underwent neurocognitive testing and a lumbar puncture within routine clinical care. To define BBB dysfunction, albumin cerebrospinal fluid/serum quotients, immunoglobulin G ratios and oligoclonal band types were evaluated, and gender-specific differences investigated. Neurocognitive functioning was assessed by the Wechsler Adult Intelligence Scale, Test of Attentional Performance and Repeatable Battery for the Assessment of Neuropsychological Status. We performed simple and multiple linear regression analyses to interpret associations of interest. RESULTS: Of those tested, 16% showed an alteration in albumin quotients and 12% had an oligoclonal band type indicating BBB dysfunction. Notably, male patients were more likely to have an increased albumin quotient and a higher immunoglobulin G ratio than female patients. We found no significant association between BBB dysfunction and neurocognitive assessments. CONCLUSIONS: The hypothesised relationship between BBB and neurocognitive impairments was not detectable in our retrospective cohort. Further cerebrospinal fluid-based studies with a longitudinal assessment of cognitive functioning and disease trajectory are urgently needed.

Blood-brain barrier dysfunction and folate and vitamin B12 levels in first-episode schizophrenia-spectrum psychosis: a retrospective chart review.

Vitamin deficiency syndromes and blood-brain barrier (BBB) dysfunction are frequent phenomena in psychiatric conditions. We analysed the largest available first-episode schizophrenia-spectrum psychosis (FEP) cohort to date regarding routine cerebrospinal fluid (CSF) and blood parameters to investigate the association between vitamin deficiencies (vitamin B12 and folate) and BBB impairments in FEP. We report a retrospective analysis of clinical data from all inpatients that were admitted to our tertiary care hospital with an ICD-10 diagnosis of a first-episode F2x (schizophrenia-spectrum) between January 1, 2008 and August 1, 2018 and underwent a lumbar puncture, blood-based vitamin status diagnostics and neuroimaging within the clinical routine. 222 FEP patients were included in our analyses. We report an increased CSF/serum albumin quotient (Qalb) as a sign of BBB dysfunction in 17.1% (38/222) of patients. White matter lesions (WML) were present in 29.3% of patients (62/212). 17.6% of patients (39/222) showed either decreased vitamin B12 levels or decreased folate levels. No statistically significant association was found between vitamin deficiencies and altered Qalb. This retrospective analysis contributes to the discussion on the impact of vitamin deficiency syndromes in FEP. Although decreased vitamin B12 or folate levels were found in approximately 17% of our cohort, we found no evidence for significant associations between BBB dysfunction and vitamin deficiencies. To strengthen the evidence regarding the clinical implications of vitamin deficiencies in FEP, prospective studies with standardized measurements of vitamin levels together with follow-up measurements and assessment of symptom severity in addition to CSF diagnostics are needed.

Antibodies Against Glutamic Acid Decarboxylase 65 Are Locally Produced in the CSF and Arise During Affinity Maturation.

BACKGROUND AND OBJECTIVES: Antibodies (Abs) against the cytoplasmic protein glutamic acid decarboxylase 65 (GAD65) are detected in patients with neurologic syndromes together referred to as GAD65-Ab spectrum disorders. The response of some of these patients to plasma exchange or immunoglobulins indicates that GAD65-Abs could contribute to disease pathogenesis at least at some stages of disease. However, the involvement of GAD65-reactive B cells in the CNS is incompletely understood. METHODS: We studied 7 patients with high levels of GAD65-Abs and generated monoclonal Abs (mAbs) derived from single cells in the CSF. Sequence characteristics, reactivity to GAD65, and the role of somatic hypermutations of the mAbs were analyzed. RESULTS: Twelve CSF-derived mAbs were generated originating from 3 patients with short disease duration, and 7/12 of these mAbs (58%) were GAD65 reactive in at least 1 detection assay. Four of 12 (33%) were definitely positive in all 3 detection assays. The intrathecal anti-GAD65 response was polyclonal. GAD65-Abs were mostly of the IgG1 subtype and had undergone affinity maturation. Reversion of 2 GAD65-reactive mAbs to their corresponding germline-encoded unmutated common ancestors abolished GAD65 reactivity. DISCUSSION: GAD65-specific B cells are present in the CNS and represent a sizable fraction of CSF B cells early in the disease course. The anti-GAD65 response in the CSF is polyclonal and shows evidence of antigen-driven affinity maturation required for GAD65 recognition. Our data support the hypothesis that the accumulation of GAD65-specific B cells and plasma cells in the CSF is an important feature of early disease stages.

Persistence of functional memory B cells recognizing SARS-CoV-2 variants despite loss of specific IgG.

Although some COVID-19 patients maintain SARS-CoV-2-specific serum immunoglobulin G (IgG) for more than 6 months postinfection, others eventually lose IgG levels. We assessed the persistence of SARS-CoV-2-specific B cells in 17 patients, 5 of whom had lost specific IgGs after 5-8 months. Differentiation of blood-derived B cells in vitro revealed persistent SARS-CoV-2-specific IgG B cells in all patients, whereas IgA B cells were maintained in 11. Antibodies derived from cultured B cells blocked binding of viral receptor-binding domain (RBD) to the cellular receptor ACE-2, had neutralizing activity to authentic virus, and recognized the RBD of the variant of concern Alpha similarly to the wild type, whereas reactivity to Beta and Gamma were decreased. Thus, differentiation of memory B cells could be more sensitive for detecting previous infection than measuring serum antibodies. Understanding the persistence of SARS-CoV-2-specific B cells even in the absence of specific serum IgG will help to promote long-term immunity.

Cerebrospinal Fluid Pathologies in Schizophrenia-Spectrum Disorder-A Retrospective Chart Review.

BACKGROUND: The role of inflammatory processes in the etiology of schizophrenia is increasingly being investigated. A link between psychosis and inflammation measured with different biomarkers has been reported in the literature and needs to be further explored. To investigate the presence of inflammatory biomarkers in first-episode psychosis (FEP) we analyzed the largest available FEP cohort to date regarding routine CSF and blood diagnostics. METHODS: We report a retrospective analysis of clinical data from all inpatients that were admitted to our tertiary care hospital with a ICD-10 diagnosis of F2x (schizophrenia-spectrum) between January 1, 2008 and August 1, 2018 and underwent a lumbar puncture. RESULTS: A total of n = 314 FEP patients were included in our sample. 42.7% patients (134/314) showed cerebrospinal fluid (CSF) alterations. Oligoclonal bands in the CSF were present in 21.8% of patients (67/307) with 12.4% (27/217) of patients presenting OCBs type 2 or 3. 15.8% (49/310) of our cohort revealed signs of blood-brain-barrier (BBB) dysfunction with increased albumin ratios. Mean serum CRP levels were 2.4 mg/l (SD = 9.5). CRP elevation was present in 116/280 cases (41.4%). CONCLUSIONS: This large retrospective analysis on FEP cohort greatly enriches the clinical data available on this population and contributes to the discussion around inflammation in psychosis. Of note, even though several inflammatory alterations were found both in CSF and in blood tests, we found no evidence for a significant relationship between peripheral inflammation and inflammatory CSF. Furthermore, no significant relationship between CSF alterations and peripheral inflammation measured with CRP could be established.

Cerebrospinal fluid abnormalities in first- and multi-episode schizophrenia-spectrum disorders: impact of clinical and demographical variables.

Multiple lines of evidence indicate that immunological and inflammatory alterations contribute at least in a subgroup to the pathophysiology of schizophrenia. In this retrospective chart review, we investigated whether clinical factors contribute to altered cerebrospinal fluid (CSF) findings in schizophrenia-spectrum disorders. Clinical data from electronic medical records of patients with psychotic disorders (ICD-10: F20-F29) who received routine CSF diagnostics at the Department of Psychiatry and Psychotherapy, LMU Munich, Germany, were included. Chi² tests for dichotomous outcomes and independent t tests for continuous outcomes were used to compare differences between groups. A total of 331 patients were included in the analyses (43.2% female and 56.8% male). The mean age was 37.67 years (±15.58). The mean duration of illness was 71.96 months (±102.59). In all, 40% (128/320) were first-episode psychosis (FEP) patients and 60% (192/320) were multi-episode psychosis (MEP) patients. Elevated CSF protein levels were found in 19.8% and elevated CSF/serum albumin ratios (QAlb) in 29.4% of the cases. Pleocytosis was found in 6.1% of patients. MEP patients showed significantly higher mean QAlb compared with FEP patients (t(304.57) = -2.75, p = 0.006), which did not remain significant after correcting for age. QAlb elevation occurred more frequently in men (X2(1) = 14.76, p = <0.001). For treatment resistance, family history, and cMRI alterations, no significant differences in CSF-related outcomes were detected. Our work extends other retrospective cohorts confirming a relevant degree of CSF alterations in schizophrenia-spectrum disorders and shows the difficulty to relate these alterations to clinical and disease course trajectories. More research is needed to develop treatment response predictors from CSF analyses.

High-resolution serum proteome trajectories in COVID-19 reveal patient-specific seroconversion.

A deeper understanding of COVID-19 on human molecular pathophysiology is urgently needed as a foundation for the discovery of new biomarkers and therapeutic targets. Here we applied mass spectrometry (MS)-based proteomics to measure serum proteomes of COVID-19 patients and symptomatic, but PCR-negative controls, in a time-resolved manner. In 262 controls and 458 longitudinal samples of 31 patients, hospitalized for COVID-19, a remarkable 26% of proteins changed significantly. Bioinformatics analyses revealed co-regulated groups and shared biological functions. Proteins of the innate immune system such as CRP, SAA1, CD14, LBP, and LGALS3BP decreased early in the time course. Regulators of coagulation (APOH, FN1, HRG, KNG1, PLG) and lipid homeostasis (APOA1, APOC1, APOC2, APOC3, PON1) increased over the course of the disease. A global correlation map provides a system-wide functional association between proteins, biological processes, and clinical chemistry parameters. Importantly, five SARS-CoV-2 immunoassays against antibodies revealed excellent correlations with an extensive range of immunoglobulin regions, which were quantified by MS-based proteomics. The high-resolution profile of all immunoglobulin regions showed individual-specific differences and commonalities of potential pathophysiological relevance.

Effects of Natalizumab Therapy on Intrathecal Immunoglobulin G Production Indicate Targeting of Plasmablasts.

OBJECTIVES: To evaluate the long-term effects of natalizumab (NTZ) on different features of intrathecal immunoglobulin (Ig) synthesis in patients with multiple sclerosis (MS) and to quantify the expression of α4-integrin in stages of B-cell maturation. METHODS: We combined a cross-sectional (49 NTZ-treated MS patients, mean treatment duration 5.1 years, and 47 untreated MS patients) and a longitudinal study (33 patients with MS before and during NTZ, mean treatment duration: 4.8 years), analyzing paired serum and CSF samples for IgG, IgA, and IgM levels, reactivity against selected viruses (measles virus, rubella virus, and varicella zoster virus [MRZ] reaction), and oligoclonal bands (OCBs). Banding patterns before and after therapy were directly compared by isoelectric focusing in 1 patient. In addition, we determined the expression of α4-integrin by FACS analysis on blood-derived B-cell subsets (plasmablasts, memory B cells, and naive B cells) of healthy controls. RESULTS: In serum, NTZ decreased IgM and IgG, but not IgA, levels. IgM hypogammaglobulinemia occurred in 28% of NTZ-treated patients. In CSF, NTZ treatment resulted in a strong reduction of intrathecally produced IgG and, to a lesser extent, IgA, whereas IgM indices [(Ig CSF/Serum)/(Albumin CSF/Serum)] remained largely unchanged. Reduction of the IgG index correlated with NTZ treatment duration, as did serum IgM and IgA levels. MRZ reaction was unchanged and OCB persisted. Direct comparison of OCB pattern before and after NTZ revealed the persistence of individual bands. α4-Integrin expression was highest on plasmablasts (CD19+CD38+CD27+). CONCLUSION: Our data indicate that NTZ reduces short-lived plasmablasts in the CNS compartment but has little effect on locally persisting long-lived plasma cells.

SARS-CoV-2 antibody immunoassays in serial samples reveal earlier seroconversion in acutely ill COVID-19 patients developing ARDS.

OBJECTIVES: During the COVID-19 pandemic, SARS-CoV-2 antibody testing has been suggested for (1) screening populations for disease prevalence, (2) diagnostics, and (3) guiding therapeutic applications. Here, we conducted a detailed clinical evaluation of four Anti-SARS-CoV-2 immunoassays in samples from acutely ill COVID-19 patients and in two negative cohorts. METHODS: 443 serum specimens from serial sampling of 29 COVID-19 patients were used to determine clinical sensitivities. Patients were stratified for the presence of acute respiratory distress syndrome (ARDS). Individual serum specimens from a pre-COVID-19 cohort of 238 healthy subjects and from a PCR-negative clinical cohort of 257 patients were used to determine clinical specificities. All samples were measured side-by-side with the Anti-SARS-CoV-2-ELISA (IgG), Anti-SARS-CoV-2-ELISA (IgA) and Anti-SARS-CoV-2-NCP-ELISA (IgG) (Euroimmun AG, Lübeck, Germany) and the Elecsys Anti-SARS-CoV-2 ECLIA (Roche Diagnostics International, Rotkreuz, Switzerland). RESULTS: Median seroconversion occurred earlier in ARDS patients (8-9 days) than in non-ARDS patients (11-17 days), except for EUR N-IgG. Rates of positivity and mean signal ratios in the ARDS group were significantly higher than in the non-ARDS group. Sensitivities between the four tested immunoassays were equivalent. In the set of negative samples, the specificity of the Anti-SARS-CoV-2-ELISA (IgA) was lower (93.9%) compared to all other assays (≥98.8%) and the specificity of Anti-SARS-CoV-2-NCP-ELISA (IgG) was lower (98.8%) than that of Elecsys Anti-SARS-CoV-2 (100%). CONCLUSIONS: Serial sampling in COVID-19 patients revealed earlier seroconversion and higher signal ratios of SARS-CoV-2 antibodies as a potential risk marker for the development of ARDS, suggesting a utility for antibody testing in acutely diseased patients.

Retrospective mass spectrometric analysis of wastewater-fed mesocosms to assess the degradation of drugs and their human metabolites.

Temporary rivers become dependent on wastewater effluent for base flows, which severely impacts river ecosystems through exposure to elevated levels of nutrients, dissolved organic matter, and organic micropollutants. However, biodegradation processes occurring in these rivers can be enhanced by wastewater bacteria/biofilms. Here, we evaluated the attenuation of pharmaceuticals and their human metabolites performing retrospective analysis of 120 compounds (drugs, their metabolites and transformation products) in mesocosm channels loaded with wastewater effluents twice a week for a period of 31 days. Eighteen human metabolites and seven biotransformation products were identified with high level of confidence. Compounds were classified into five categories. Type-A: recalcitrant drugs and metabolites (diclofenac, carbamazepine and venlafaxine); Type-B: degradable drugs forming transformation products (TPs) (atenolol, sitagliptin, and valsartan); Type-C: drugs for which no known human metabolites or TPs were detected (atorvastatin, azithromycin, citalopram, clarithromycin, diltiazem, eprosartan, fluconazole, ketoprofen, lamotrigine, lormetazepam, metformin, telmisartan, and trimethoprim); Type-D: recalcitrant drug metabolites (4-hydroxy omeprazole sulfide, erythro/threo-hydrobupropion, and zolpidem carboxylic acid); Type-E: unstable metabolites whose parent drug was not detectable (norcocaine, benzolylecgonine, and erythromycin A enol ether). Noteworthy was the valsartan acid formation from valsartan with transient formation of TP-336.

Travel-associated neurological disease terminated in a postmortem diagnosed atypical HSV-1 encephalitis after high-dose steroid therapy - a case report.

BACKGROUND: Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment. CASE PRESENTATION: A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient's condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem. CONCLUSIONS: This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur.

Evaluating the diagnostic utility of new line immunoassays for myositis antibodies in clinical practice: a retrospective study.

BACKGROUND: Myositis-associated antibodies (MAA) and myositis-specific antibodies (MSA) are detected in patients with idiopathic inflammatory myopathies (IIM); their role as diagnostic biomarkers is however still debated. The aim of our study was to assess the utility of MAA/MSA assessed by new line immunoassays in detecting myositis among neuromuscular patients. METHODS: We retrospectively analysed sera samples obtained from patients tested for myositis antibodies with the "Euroline: Autoimmune Inflammatory Myopathies 16Ag" and "myositis profile 3" kits (Mi-2, TIF1γ, MDA5, NXP2, SAE1, Jo-1, SRP, PL-7/12, EJ, OJ, Ro-52, Ku, PM-Scl75/100). First symptom, CK, EMG, muscle biopsy and diagnosis were also analysed. Using logistic regression analysis, two diagnostic models were built to evaluate the diagnostic power of MAA/MSA in distinguishing myositis patients from controls and other myopathies. RESULTS: 1229 patients were identified. 141 patients had a bioptic confirmed IIM; other diagnoses included: myopathy (n = 357), other neuromuscular diseases (n = 144) and no neuromuscular diseases (n = 587). The specificity was 95% for MSA and 89% for MAA, the sensitivity 20% and 22%, respectively. MAA showed no use in differentiating myositis patients from controls, whereas MSA had limited effect (OR = 5.165), compared to other variables as EMG (OR = 47.755) or CK > 2000 U/L (OR = 45.307). MSA were, however, the most useful parameter differentiating IIM from non-IIM patients (OR = 7.259), better than CK > 2000 U/L (OR = 4.033) and MAA (OR = 2.737). CONCLUSIONS: Line immunoassays for myositis antibodies show high specificity but low sensitivity. Their usefulness as diagnostic biomarkers widely depends on the clinical settings. Our study suggests that MSA/MAA should be used for confirmatory and differential diagnosis rather than for screening purposes in inflammatory myopathies.

Case report: de novo ANCA-associated vasculitis after kidney transplantation treated with rituximab and plasma exchange.

BACKGROUND: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis causes end-stage renal failure in up to a third of cases even with treatment. The disease recurs occasionally after kidney transplantation, but new onset of ANCA-associated vasculitis after transplantation is highly unusual. The use of rituximab or plasmapheresis for de novo disease after transplantation has not previously been reported. CASE PRESENTATION: Routine post-transplant follow-up for a 66-year old asymptomatic woman revealed a rise in creatinine from 1.8 to 2.6 mg/dl and increased proteinuria. She had received a cadaveric kidney transplant 20 months previously for end-stage autosomal dominant polycystic kidney disease. Renal allograft biopsy unexpectedly demonstrated pauci-immune glomerulonephritis with extracapillary proliferation and interstitial inflammation. Concurrent serum tested strongly positive for ANCA specific to proteinase 3 (PR3), but stored pre- and post-transplantation serum samples tested negative. These findings established a diagnosis of de novo ANCA-associated vasculitis in the renal allograft. We started treatment with high-dose corticosteroid and rituximab. Despite this, serum creatinine continued to rise and glomerulonephritis remained active in a repeat biopsy. Escalation of the treatment with seven sessions of plasmapheresis led to a temporary improvement in creatinine. No further features of vasculitis emerged and PR3-ANCA titres declined. However, multiple infections complicated the recovery period and were associated with progressive loss of renal transplant function. Four months after the index presentation, transplant function became insufficient and dialysis was restarted. CONCLUSIONS: De novo ANCA-associated vasculitis after renal transplantation is exceptionally rare. It poses a significant risk to graft survival even in the context of intensified immunosuppression. Management relies on clinical evidence from populations with native renal function, yet post-transplant patients may be at increased risk of treatment-related adverse events. Precautions against these risks are crucial in the delivery of care.

Absence of cerebrospinal fluid antineuronal antibodies in schizophrenia spectrum disorders - CORRIGENDUM.

This notice describes a correction to the above mentioned paper.

Absence of cerebrospinal fluid antineuronal antibodies in schizophrenia spectrum disorders.

Antibody-mediated encephalitis has been discussed as one possible cause for isolated psychotic syndromes. Mostly based on serum samples, findings have been controversial. We present the results of a retrospective study of 124 clinically diagnosed psychotic patients without documented relevant neurological symptoms. All were tested for different antineuronal antibodies in cerebrospinal fluid (CSF) while 81 received serum testing. Antineuronal antibodies in CSF were negative across the sample. 3.7% showed low positive serum antibodies. Our findings highlight the importance of a deeper discussion about the relevance of low positive serum antibodies without concurrent findings in CSF or clinical signs for autoimmune encephalitis.Declaration of interestNone.

4-Amino-7,8-dihydro-1,6-naphthyridin-5(6 H)-ones as Inhaled Phosphodiesterase Type 4 (PDE4) Inhibitors: Structural Biology and Structure-Activity Relationships.

Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with subnanomolar enzymatic potencies. X-ray crystallography confirmed the binding mode of this novel template. We achieved compounds with double-digit picomolar enzymatic potencies through further structure-based design by targeting both the PDE4 enzyme metal-binding pocket and occupying the solvent-filled pocket. A strategy for lung retention and long duration of action based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung neutrophilia model by suspension microspray and dry powder administration. Suspension microspray of potent compounds showed in vivo efficacy with a clear dose-response. Despite sustained lung levels, dry powder administration performed much less well and without proper dose-response, highlighting clear differences between the two formulations. This indicates a deficiency in the low aqueous solubility strategy for long duration lung efficacy.

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry.

The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment. Graphical abstract Studies of metabolism of carbamazepine and ibuprofen in fish.