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.

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.

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.

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.

Characterization of the chloroquine-induced mouse model of pruritus using an automated behavioural system.

Pruritus is a major symptom of several dermatological diseases but has limited therapeutic options available. Animal models replicating the pathophysiology of pruritus are needed to support the development of new drugs. Induction of pruritus by chloroquine (CQ) in mice is widely used, although, as with similar models, it has low throughput and does not distinguish between antipruritic effects and confounding factors such as sedation. To overcome these issues, we incorporated into the model an automated system that measures both scratching and locomotor behaviour simultaneously. We combined this system with the determination of CQ levels in different tissues to understand the impact of the route of CQ administration on the pruritogenic response. We concluded that whereas oral CQ does not induce pruritus due to insufficient skin levels, the bell-shaped curve of pruritus observed following subcutaneous administration is due to toxicity at high doses. We validated the model with several drugs currently used in humans: nalfurafine, aprepitant, cyproheptadine and amitriptyline. By comparing the effects of the drugs on both scratching and locomotor activity, we concluded that nalfurafine and aprepitant can exhibit efficacy at doses devoid of central effects, whereas central effects drove the efficacy of the other two drugs. This was further confirmed using non-brain-penetrant drugs. Moreover, as anticipated, anti-inflammatory drugs showed no efficacy. In conclusion, the use of an automated integrated behavioural assessment in CQ-induced pruritus makes the assay suitable for screening purposes and allows for a correct interpretation of the antipruritic effect of the compounds evaluated.

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.

Using MALDI-TOF MS imaging and LC-HRMS for the investigation of the degradation of polycaprolactone diol exposed to different wastewater treatments.

Polymers are used in high amounts in a wide range of applications from biomedicine to industry. Because of the growing awareness of the increasing amounts of plastic wastes in the aquatic environment during recent years, the evaluation of their biodegradability deserves special attention. In the past, most efforts were dedicated to studying the biodegradation of polyesters in soil and compost, while very little research has been conducted on their fate in wastewater. Here, we assessed the ability of bacterial communities residing in the aerobic and denitrification tank from a wastewater treatment plant (WWTP) to degrade the polymeric ester polycaprolactone diol (PCLD; average molecular weight of 1250 Da). Following the incubation of the solid polymer in WWTP tanks, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to provide evidence for hydrolytic reactions and to study differences in the spatial degradation on the PCLD surface. It was demonstrated that regardless of the wastewater type, the chemical structure on the PCLD surface underwent modifications after 7 days of exposure. Apart from the parent PCLD peak series in MALDI-MSI mass spectra, the presence of a second oligomer series with mass peaks spaced by m/z 114 (as in PCLD) was observed. It was proposed to correspond to polycaprolactone (PCL) originating from the hydrolytic cleavage of the diethylene glycol from PCLD. Their ion masses were detected at m/z 104 below the PCLD peaks and their structures were proposed as PCL cyclized oligomers. Differences in the spatial distribution of low MW ions (<800) between the aerobic and denitrifying exposed samples in MALDI MSI were also noticeable. While the ions at m/z 221.1, 247.1 and 449.2 predominated in the aerobic exposed sample, those at m/z 475.5 and 677.4 were characteristic of the denitrifying one. The MALDI-MSI measurements in the low mass range were complemented with LC-HRMS analysis to determine plausible structures of the major degradation products. Ten transformation products (TPs) were detected in the denitrifying wastewater experiment, five of them were the result of ester hydrolysis forming caprolactone oligomers (TPs 220, 334, 448, 562, and 676) while the other series corresponded to formation of PCL chain with a terminal diethylene glycol, likewise formed by ester hydrolysis (TPs 246, 360, 474, 588, and 702). Graphical abstract Investigation of the polymer degradation in WWTPs by MALDI-MSI and LC-HRMS.

Pharmacological characterization of CRTh2 antagonist LAS191859: Long receptor residence time translates into long-lasting in vivo efficacy.

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTh2) is a G protein-coupled receptor expressed on the leukocytes most closely associated with asthma and allergy like eosinophils, mast cells, Th2-lymphocytes and basophils. At present it is clear that CRTh2 mediates most prostaglandin D2 (PGD2) pro-inflammatory effects and as a result antagonists for this receptor have reached asthma clinical studies showing a trend of lung function improvement. The challenge remains to identify compounds with improved clinical efficacy when administered once a day. Herein we described the pharmacological profile of LAS191859, a novel, potent and selective CRTh2 antagonist. In vitro evidence in GTPγS binding studies indicate that LAS191859 is a CRTh2 antagonist with activity in the low nanomolar range. This potency is also maintained in cellular assays performed with human eosinophils and whole blood. The main differentiation of LAS191859 vs other CRTh2 antagonists is in its receptor binding kinetics. LAS191859 has a residence time half-life of 21h at CRTh2 that translates into a long-lasting in vivo efficacy that is independent of plasma levels. We believe that the strategy behind this compound will allow optimal efficacy and posology for chronic asthma treatment.

A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response.

Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The aim of this study was to characterize the in vitro and in vivo profile of LAS189386. The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model. LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation (IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without affecting systemic mast cells. The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.

Structure-activity relationships (SAR) and structure-kinetic relationships (SKR) of pyrrolopiperidinone acetic acids as CRTh2 antagonists.

Pyrrolopiperidinone acetic acids (PPAs) were identified as highly potent CRTh2 receptor antagonists. In addition, many of these compounds displayed slow-dissociation kinetics from the receptor. Structure-kinetic relationship (SKR) studies allowed optimisation of the kinetics to give potent analogues with long receptor residence half-lives of up to 23 h. Low permeability was a general feature of this series, however oral bioavailability could be achieved through the use of ester prodrugs.

Structure-activity relationships (SAR) and structure-kinetic relationships (SKR) of bicyclic heteroaromatic acetic acids as potent CRTh2 antagonists I.

A knowledge-based design strategy led to the discovery of several new series of potent and orally bioavailable CRTh2 antagonists where a bicyclic heteroaromatic ring serves as the central core. Structure-kinetic relationships (SKR) opened up the possibility of long receptor residence times.

Structure-activity relationships (SAR) and structure-kinetic relationships (SKR) of bicyclic heteroaromatic acetic acids as potent CRTh2 antagonists II: lead optimization.

Extensive structure-activity relationship (SAR) and structure-kinetic relationship (SKR) studies in the bicyclic heteroaromatic series of CRTh2 antagonists led to the identification of several molecules that possessed both excellent binding and cellular potencies along with long receptor residence times. A small substituent in the bicyclic core provided an order of magnitude jump in dissociation half-lives. Selected optimized compounds demonstrated suitable pharmacokinetic profiles.

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.

2-(1H-Pyrazol-4-yl)acetic acids as CRTh2 antagonists.

High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antagonists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar inhibitors.

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.

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.

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.

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.

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.