Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Analysis of Immune Checkpoint Drug Targets and Tumor Proteotypes in Non-Small Cell Lung Cancer.

New therapeutics targeting immune checkpoint proteins have significantly advanced treatment of non-small cell lung cancer (NSCLC), but protein level quantitation of drug targets presents a critical problem. We used multiplexed, targeted mass spectrometry (MS) to quantify immunotherapy target proteins PD-1, PD-L1, PD-L2, IDO1, LAG3, TIM3, ICOSLG, VISTA, GITR, and CD40 in formalin-fixed, paraffin-embedded (FFPE) NSCLC specimens. Immunohistochemistry (IHC) and MS measurements for PD-L1 were weakly correlated, but IHC did not distinguish protein abundance differences detected by MS. PD-L2 abundance exceeded PD-L1 in over half the specimens and the drug target proteins all displayed different abundance patterns. mRNA correlated with protein abundance only for PD-1, PD-L1, and IDO1 and tumor mutation burden did not predict abundance of any protein targets. Global proteome analyses identified distinct proteotypes associated with high PD-L1-expressing and high IDO1-expressing NSCLC. MS quantification of multiple drug targets and tissue proteotypes can improve clinical evaluation of immunotherapies for NSCLC.

Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry.

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Supratherapeutic Psychotropic Drug Levels in the Emergency Department and Their Association with Delirium Duration: A Preliminary Study.

OBJECTIVES: Polypharmacy is associated with delirium, but the mechanisms for this connection are unclear. Our goal was to determine the frequency of supratherapeutic psychotropic drug levels (SPDLs) in older hospitalized patients and if it is associated with the duration of emergency department (ED) delirium. DESIGN: Secondary analysis of a prospective cohort study. SETTING: Tertiary care academic medical center. PARTICIPANTS: ED patients 65 years or older who were admitted to the hospital. MEASUREMENTS: Delirium was assessed in the ED and during the first 7 days of hospitalization using the modified Brief Confusion Assessment Method. Drug concentrations were determined in serum samples collected at enrollment via a novel platform based on liquid chromatography-tandem mass spectrometry capable of identifying and quantitating 78 clinically approved medications including opioids, benzodiazepines, antidepressants, antipsychotics, and amphetamines. Patients with serum psychotropic drug concentrations above established reference ranges were considered supratherapeutic and have a SPDL. We performed proportional odds logistic regression to determine if SPDLs were associated with ED delirium duration adjusted for confounders. Medical record review was performed to determine if the doses of medications associated with SPDLs were adjusted at hospital discharge. RESULTS: A total of 158 patients were enrolled; of these, 66 were delirious in the ED. SPDLs were present in 11 (17%) of the delirious and 4 (4%) of the non-delirious ED patients. SPDLs were significantly associated with longer ED delirium duration (adjusted proportional odds ratio = 6.0; 95% confidence interval = 2.1-17.3) after adjusting for confounders. Of the 15 medications associated with SPDLs, 9 (60%) were prescribed at the same or higher doses at the time of hospital discharge. CONCLUSION: SPDLs significantly increased the odds of prolonged ED delirium episodes. Approximately half of the medications associated with SPDLs were continued after hospital discharge at the same or higher doses. J Am Geriatr Soc 67:2387-2392, 2019.

Medication Exposure in Highly Adherent Psychiatry Patients.

Medication exposure is dependent upon many factors, the single most important being if the patient took the prescribed medication as indicated. To assess medication exposure for psychotropic and other medication classes, we enrolled 115 highly adherent psychiatry patients prescribed five or more medications. In these patients, we measured 21 psychotropic and 38 nonpsychotropic medications comprising a 59 medication multiplex assay panel. Strict enrollment criteria and reconciliation of the electronic health record medication list prior to study initiation produced a patient cohort that was adherent with 91% of their prescribed medications as determined by comparing medications detected empirically in blood to the electronic health record medication list. In addition, 13% of detected medications were not in the electronic health record medication list. We found that only 53% of detected medications were within the literature-derived reference range with 41% below and 6% above the reference range specific to each medication. When psychotropic medications were analyzed near trough-level, only sertraline was found to be within the literature-derived reference range for all patients tested. Concentrations of the remaining medications indicated extensive exposure below the reference range. This is the first study to empirically and comprehensively assess medication exposure obtained in comorbid polypharmacy patients, minimizing the important behavioral factor of adherence in the study of medication exposure. These data indicate that low medication exposure is extensive and must be considered when therapeutic issues arise, including the lack of response to medication therapy.

Assessment of Patient Medication Adherence, Medical Record Accuracy, and Medication Blood Concentrations for Prescription and Over-the-Counter Medications.

Importance: Inaccurate medication records and poor medication adherence result in incomplete knowledge of therapy for patients. Objective: To study accuracy of medical records and patient adherence by measuring blood concentrations of medications. Design, Setting, and Participants: This cross-sectional study validated a serum-based liquid chromatography-tandem mass spectrometry assay to simultaneously quantify 263 medications used for acute and chronic conditions. The assay panel was applied to 3 clinical patient cohorts: residual serum from 1000 randomly selected samples sent for routine clinical chemistry testing between April 8 and October 6, 2015 (residuals cohort), 50 prospectively enrolled patients in a gastroenterology clinic between March 1 and March 15, 2016, who were prescribed more than 5 medications (gastroenterology care cohort), and a convenience cohort of 296 patients with hypertension who sought care in an emergency department (ED care cohort) between July 1, 2012, and April 25, 2013. Integrated data analysis of the cohorts was performed from August 22 to November 29, 2017. Main Outcomes and Measures: Medication serum concentrations, electronic health record medication lists, and predicted drug interactions. Results: Of the 1346 total samples, 1000 came from the residuals cohort (640 women and 360 men; median age, 60 years [interquartile range (IQR), 44-71 years]), 50 from the gastroenterology care cohort (30 women and 20 men; median age, 66 years [IQR, 62-70 years]), and 296 from the ED care cohort (160 women and 136 men; median age, 59 years [IQR, 52-66 years]). Median medication adherence, defined as the subset of detected medications from the prescription record, was 83% (IQR, 50%-100%) in the residuals cohort, 100% (IQR, 84%-100%) in the gastroenterology care cohort, and 78% (IQR, 57%-100%) in the ED care cohort. Patients adherent to 1 medication were more often adherent to other medications. Among patients prescribed 3 medications or more, there were no significant associations between medication adherence and sex or number of prescribed medications, and there was a modest association between adherence and age. By comparing detected vs prescribed medications, we detected a median of 0 (IQR, 0-2) medications per patient that were not listed in the electronic health record in the residuals cohort, 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the gastroenterology care cohort, and 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the ED care cohort. A total of 435 patients (43.5%) in the residuals cohort had no discrepancy between the electronic health record and detected medication lists, 22 patients (44.0%) in the gastroenterology care cohort had no discrepancy between the electronic health record and detected medication lists, and 41 patients (13.9%) in the ED care cohort had no discrepancy between the electronic health record and detected medication lists. Half of adverse drug reaction alerts occurred among medications detected without prescription. Conclusions and Relevance: Comprehensive medication monitoring offers promise to improve adherence, the accuracy of medical records, and the safety for patients with polypharmacy.

Medication adherence, medical record accuracy, and medication exposure in real-world patients using comprehensive medication monitoring.

BACKGROUND: Poor adherence to medication regimens and medical record inconsistencies result in incomplete knowledge of medication therapy in polypharmacy patients. By quantitatively identifying medications in the blood of patients and reconciling detected medications with the medical record, we have defined the severity of this knowledge gap and created a path toward optimizing medication therapy. METHODS AND FINDINGS: We validated a liquid chromatography-tandem mass spectrometry assay to detect and/or quantify 38 medications across a broad range of chronic diseases to obtain a comprehensive survey of patient adherence, medical record accuracy, and exposure variability in two patient populations. In a retrospectively tested 821-patient cohort representing U.S. adults, we found that 46% of medications assessed were detected in patients as prescribed in the medical record. Of the remaining medications, 23% were detected, but not listed in the medical record while 30% were prescribed to patients, but not detected in blood. To determine how often each detected medication fell within literature-derived reference ranges when taken as prescribed, we prospectively enrolled a cohort of 151 treatment-regimen adherent patients. In this cohort, we found that 53% of medications that were taken as prescribed, as determined using patient self-reporting, were not within the blood reference range. Of the medications not in range, 83% were below and 17% above the lower and upper range limits, respectively. Only 32% of out-of-range medications could be attributed to short oral half-lives, leaving extensive exposure variability to result from patient behavior, undefined drug interactions, genetics, and other characteristics that can affect medication exposure. CONCLUSIONS: This is the first study to assess compliance, medical record accuracy, and exposure as determinants of real-world treatment and response. Variation in medication detection and exposure is greater than previously demonstrated, illustrating the scope of current therapy issues and opening avenues that warrant further investigation to optimize medication therapy.

Managing Psychotropic Medications in Complex, Real-World Patients Using Comprehensive Therapeutic Drug Monitoring.

There are multiple treatment options for depression, anxiety, psychosis, and other psychiatric disorders, and psychiatry patients are often comorbid with complex, polypharmacy treatment regimens. Unlike cardiovascular disease and diabetes, there are no readily available biomarkers to gauge treatment success with psychotropic medications, often resulting in subjective determination of medication therapy effectiveness. The physiochemical properties of psychiatric medications in general lend themselves to quantitative measurement in blood, offering an avenue to optimize treatment for each patient. Herein, we describe a novel application that employs comprehensive therapeutic drug monitoring of both psychiatric and nonpsychiatric medications to holistically personalize therapy for complex psychiatry patients.

Metabolism and Distribution of Clozapine-N-oxide: Implications for Nonhuman Primate Chemogenetics.

The use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in neuroscience has rapidly expanded in rodent studies but has lagged behind in nonhuman primate (NHP) experiments, slowing the development of this method for therapeutic use in humans. One reason for the slow adoption of DREADD technology in primates is that the pharmacokinetic properties and bioavailability of clozapine-n-oxide (CNO), the most commonly used ligand for human muscarinic (hM) DREADDs, are not fully described in primates. We report an extensive pharmacokinetic study using subcutaneous (SC) administration of CNO in five adult rhesus monkeys. CNO reached maximal plasma and cerebrospinal fluid (CSF) concentrations within 2 h after injection, with an observed dose-dependent increase in levels following a 3 and 10 mg/kg SC dose. Since CSF concentrations were below values predicted from unbound plasma concentrations, we investigated whether CNO was restricted from the CNS through active transport at the blood-brain barrier. In vitro assessment demonstrated that CNO is a substrate for P-glycoprotein (Pgp; efflux ratio, 20), thus providing a likely mechanism limiting CNO levels in the CNS. Furthermore, CNO is metabolized to the psychoactive compounds clozapine and n-desmethylclozapine in monkeys. The concentrations of clozapine detected in the CSF are sufficient to activate several types of receptor (including the hM-DREADDs). Our results suggest that CNO metabolism and distribution may interfere with reproducibility and interpretation of DREADD-related experiments in NHPs and calls for a re-evaluation of the use of CNO in DREADD-related experiments in NHPs along with the need to test alternative compounds.

Discovery of (S)-2-cyclopentyl-N-((1-isopropylpyrrolidin2-yl)-9-methyl-1-oxo-2,9-dihydro-1H-pyrrido[3,4-b]indole-4-carboxamide (VU0453379): a novel, CNS penetrant glucagon-like peptide 1 receptor (GLP-1R) positive allosteric modulator (PAM).

A duplexed, functional multiaddition high throughput screen and subsequent iterative parallel synthesis effort identified the first highly selective and CNS penetrant glucagon-like peptide-1R (GLP-1R) positive allosteric modulator (PAM). PAM (S)-9b potentiated low-dose exenatide to augment insulin secretion in primary mouse pancreatic islets, and (S)-9b alone was effective in potentiating endogenous GLP-1R to reverse haloperidol-induced catalepsy.

Discovery of a highly selective PLD2 inhibitor (ML395): a new probe with improved physiochemical properties and broad-spectrum antiviral activity against influenza strains.

Further chemical optimization of the halopemide-derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80-fold PLD2 selective allosteric inhibitor (cellular PLD1, IC50 >30,000 nM; cellular PLD2, IC50 =360 nM). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).

Enzymatic conversion of 6-nitroquinoline to the fluorophore 6-aminoquinoline selectively under hypoxic conditions.

There is substantial interest in small molecules that can be used to detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl moieties are useful components in the design of hypoxia-selective imaging agents and prodrugs because one-electron reductases can convert the nitroaryl group to nitroso, hydroxylamino, and amino metabolites selectively under low oxygen conditions. Here, we describe the in vitro, cell free metabolism of a pro-fluorescent substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analyses provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed intermediate in this reduction process, 6-hydroxylaminoquinoline (6), is fluorescent and can be efficiently converted by xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy samples. In addition, the compound may find use as a fluorogenic probe for the detection of hypoxia in tumor models. The occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.

The role of aldehyde oxidase and xanthine oxidase in the biotransformation of a novel negative allosteric modulator of metabotropic glutamate receptor subtype 5.

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of ¹8O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because ¹8O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu4), including N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu4 PAMsexhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu4 in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu4 PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with L-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of L-DOPA, suggesting that mGlu4 PAMs may provide L-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu4 PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either L-DOPA or A2A antagonists.

Design, synthesis, and biological evaluation of halogenated N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)benzamides: discovery of an isoform-selective small molecule phospholipase D2 inhibitor.

Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to the lipid second messenger phosphatidic acid. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms. Inhibition of PLD enzymatic activity leads to increased cancer cell apoptosis, decreased cancer cell invasion, and decreased metastasis of cancer cells; therefore, the development of isoform-specific, PLD inhibitors is a novel approach for the treatment of cancer. Previously, we developed potent dual PLD1/PLD2, PLD1-specific (>1700-fold selective), and moderately PLD2-preferring (>10-fold preferring) inhibitors. Here, we describe a matrix library strategy that afforded the most potent (PLD2 IC(50) = 20 nM) and selective (75-fold selective versus PLD1) PLD2 inhibitor to date, N-(2-(1-(3-fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)-2-naphthamide (22a), with an acceptable DMPK profile. Thus, these new isoform-selective PLD inhibitors will enable researchers to dissect the signaling roles and therapeutic potential of individual PLD isoforms to an unprecedented degree.