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Aims and methodsAccurate 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.ResultsTargeted 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 β-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.ConclusionsPathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

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

Background Although angiotensin-converting enzyme II inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) improve chronic heart failure (HF) outcomes, their potential harms and benefits in acute HF (AHF) is less clear. Study question We explored the relationship between ACEI or ARB plasma concentrations among patients with AHF with in-hospital change in estimated glomerular filtration rate (eGFR). Data sources and study design From August 2016-June 2017, patients with AHF prescribed an outpatient ACEI or ARB were enrolled before AHF treatment. All patients were given twice their home dose of diuretic intravenously and received clinical care at the discretion of the medical team. Of 61 patients in the parent study, saved plasma from 34 who were prescribed an outpatient ACEI or ARB was included in this substudy. Measures and outcomes Liquid chromatography-tandem mass spectrometry was performed to assess ACEI or ARB plasma concentrations before AHF treatment. Change in eGFR was computed using the Chronic Kidney Disease Epidemiology Collaboration equation, which adjusts for age, sex, and race; diuretic dose and enrollment eGFR were used to adjust for HF severity. Multiple linear regression adjusting for enrollment eGFR and diuretic dose was performed to examine the relationship between drug concentration (undetectable/low vs. in/above-range) and in-hospital change in eGFR. Results Of 34 patients with AHF, median age was 63 years (interquartile range, 58-78 years), 19 (55.9%) were women, median eGFR at enrollment was 55.6 mL/min (interquartile range, 35.2-75.3 mL/min), and for 11 (32.4%), no ACEI or ARB was detectable in plasma. Medication concentrations in- or above-reference range were associated with in-hospital decrease in eGFR of 8.3 mL/min (95% confidence interval, 15.3-1.3 mL/min decrease), after adjusting for enrollment eGFR and diuretic treatment. Conclusions Bioanalytical assessment of medication levels may be useful to guide in-hospital ACEI and ARB therapy for patients with AHF.

This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

This letter describes the further chemical optimization of VU0424238 (auglurant), an mGlu5 NAM clinical candidate that failed in non-human primate (NHP) 28 day toxicology due to accumulation of a species-specific aldehyde oxidase (AO) metabolite of the pyrimidine head group. Here, we excised the pyrimidine moiety, identified the minimum pharmacophore, and then developed a new series of saturated ether head groups that ablated any AO contribution to metabolism. Putative back-up compounds in this novel series provided increased sp3 character, uniform CYP450-mediated metabolism across species, good functional potency and high CNS penetration. Key to the optimization was a combination of matrix and iterative libraries that allowed rapid surveillance of multiple domains of the allosteric ligand.

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.

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu(5) negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu(5) NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu(5) assay, and an exemplar analog 27 was more than 60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.

Reliable quantitative assays for protein targets of immune checkpoint (IC) therapeutics are lacking and this complicates clinical development of IC drug combinations. Biomarker-blind clinical development of drug combinations may help explain high profile failures of late-stage trials for combination immunotherapies. Targeted mass spectrometry (MS) of IC drug targets in tumors offers a solution to this problem through precise quantitation of multiple IC targets, immune co-regulators and immune cell markers in the same tissue sample. Combination immunotherapies targeting PD-1/PD-L1 and TIGIT are in clinical development in solid tumors, but without the use of TIGIT biomarkers. We used MS to precisely quantify PD-1, PD-L1, TIGIT and their co-regulators in 100 primary non-small cell lung cancers (NSCLC) and matched tumor draining lymph node metastases (TDLN). PD-1, PD-L1 and PD-L2 were more abundant in TDLN than in tumors for approximately two-thirds of the cases. PD-L1 and PD-L2 were co-expressed in all tumors and TDLN and were quantified across a 46-fold and 15-fold abundance range, respectively. PD-L1 was more abundant than PD-L2 in approximately 80% of tumors and TDLN, potentially explaining differential responsiveness to PD-1 compared to PD-L1-targeted therapies. In contrast to PD-1/PD-L1, TIGIT was quantifiable in only 4% of tumors and 60% of TDLN, whereas the activating CD226 receptor was quantified in 47% of tumors and 93% of TDLN. All TDLN that expressed TIGIT also co-expressed CD226 at approximately 2- to 3-fold greater abundance than TIGIT. TIGIT ligands CD112 and CD155 were co-expressed in nearly all tumors and TDLN, with combined CD112/CD155 in greater abundance than TIGIT in TDLN. Quantitation of CD4, CD8, FOXP3 and CD56 in the same analyses enabled comparison of the IC targets and their co-regulators with abundance of cytotoxic T-cells, Tregs and NK cells. PD-1, PD-L1, and PD-L2 were measured in approximately a 15-fold, 8-fold, and 5-fold abundance range, respectively, in TIGIT-expressing TDLN. The data indicate that clinical biomarker assays for TIGIT should ideally include both TDLN and primary tumors. Verified co-expression of TIGIT and PD-1 or PD-L1 in TDLN provide a rationale for combination therapy, whereas lack of target expression should have negative predictive value. Targeted MS assay panels offer a powerful platform for quantifying IC drug targets and their functional partners, defining their co-expression in tumor specimens. This deployment of a new generation of biomarkers will enable a systematic prioritization and development of combination immunotherapies to transform therapeutic development from serendipity to predictability. Citation Format: Daniel C. Liebler, Matt Westfall, Salisha Hill, Ryan D. Morrison, Kevin J. Horgan, Alexander Haragan. Quantitative mass spectrometry analysis of the PD-L1 and TIGIT signaling axes in primary non-small cell lung cancers and lymph node metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB042.

The G protein-gated inwardly-rectifying potassium channels (GIRK, Kir3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5- yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent Kp > 0.6).

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.

Background/aims The prostaglandin E2 (PGE2) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype. Methods DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041. Results Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides. Conclusion Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.

Background and purpose G protein-gated inwardly rectifying K+ (Kir 3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal Kir 3 channels consist of Kir 3.1 and Kir 3.2 subtypes, while cardiac Kir 3 channels consist of Kir 3.1 and Kir 3.4 subtypes. Previously, we identified a family of urea-containing Kir 3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for Kir 3.1/3.2 relative to Kir 3.1/3.4 channels. Here, we characterize a non-urea activator, VU0810464, which displays nanomolar potency as a Kir 3.1/3.2 activator, improved selectivity for neuronal Kir 3 channels, and improved brain penetration. Experimental approach We used whole-cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac Kir 3 channel subtypes. We tested VU0810464 in vivo in stress-induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of Kir 3.1-containing Kir 3 channels, were performed to permit direct comparisons. Key results VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal Kir 3 channel activators, but VU0810464 was more selective for neuronal Kir 3 channels. VU0810464, like ML297, reduced stress-induced hyperthermia in a Kir 3-dependent manner in mice. ML297, but not VU0810464, decreased anxiety-related behaviour as assessed with the elevated plus maze test. Conclusion and implications VU0810464 represents a new class of Kir 3 channel activator with enhanced selectivity for Kir 3.1/3.2 channels. VU0810464 may be useful for examining Kir 3.1/3.2 channel contributions to complex behaviours and for probing the potential of Kir 3 channel-dependent manipulations to treat neurological disorders.

This letter describes progress towards an M(4) PAM preclinical candidate driven by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M(4) PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

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.

Background 2-Hydroxybenzylamine (2-HOBA) is a selective scavenger of dicarbonyl electrophiles that protects proteins and lipids from being modified by these electrophiles. It is currently being developed for use as a nutritional supplement to help maintain good health and protect against the development of conditions associated with dicarbonyl electrophile formation, such as the cognitive decline associated with Mild Cognitive Impairment and Alzheimer’s disease. Methods In this first-in-human study, the safety, tolerability, and pharmacokinetics of six ascending single oral doses of 2-HOBA acetate were tested in eighteen healthy human volunteers. Results Reported adverse events were mild and considered unlikely to be related to 2-HOBA. There were no clinically significant changes in vital signs, ECG recordings, or clinical laboratory parameters. 2-HOBA was fairly rapidly absorbed, with a tmax of 1–2 h, and eliminated, with a t1/2 of approximately 2 h. Both tmax and t1/2 were independent of dose level, while Cmax and AUC increased proportionally with dose level. Conclusions 2-HOBA acetate was safe and well-tolerated at doses up to 825 mg in healthy human volunteers, positioning it as a good candidate for continued development as a nutritional supplement. Trial registration This study is registered at ClinicalTrials.gov (NCT03176940). Electronic supplementary material The online version of this article (10.1186/s40360-018-0281-7) contains supplementary material, which is available to authorized users.

Dopaminergic medications ameliorate many of the motor impairments of Parkinson’s disease (PD). However, parkinsonism is often only partially reversed by these drugs, and they can have significant side effects. Therefore, a need remains for novel treatments of parkinsonism. Studies in rodents and preliminary clinical evidence have shown that T-type calcium channel (TTCC) antagonists have antiparkinsonian effects. However, most of the available studies utilized non-selective agents. We now evaluated whether systemic injections of the specific TTCC blocker ML218 have antiparkinsonian effects in MPTP-treated parkinsonian Rhesus monkeys. The animals were treated chronically with MPTP until they reached stable parkinsonism. In pharmacokinetic studies we found that ML218 reaches a peak CSF concentration 1–2 hrs after s.c. administration. In electrocardiographic studies, we found no effects of ML218 on cardiac rhythmicity. As expected, systemic injections of the dopamine precursor L-DOPA dose-dependently increased the movements in our parkinsonian animals. We then tested the behavioral effects of systemic injections of ML218 (1, 10 or 30 mg/kg) or its vehicle, but did not detect specific antiparkinsonian effects. ML218 (3 or 10 mg/kg) was also not synergistic with L-DOPA. Using recordings of electrocorticogram signals (in one animal), we found that ML218 increased sleep. We conclude that ML218 does not have antiparkinsonian effects in MPTP-treated parkinsonian monkeys, due at least in part, to the agent’s sedative effects.

This letter describes the further chemical optimization of the picolinamide-derived family of mGlu4 PAMs wherein we identified a 3-amino substituent to the picolinamide warhead that engendered potency, CNS penetration and in vivo efficacy. From this optimization campaign, VU0477886 emerged as a potent (EC50 = 95 nM, 89% Glu Max) mGlu4 PAM with an attractive DMPK profile (brain:plasma Kp = 1.3), rat CLp = 4.0 mL/min/kg, t1/2 = 3.7 h) and robust efficacy in our standard preclinical Parkinson's disease model, haloperidol-induced catalepsy (HIC).

Selective negative allosteric modulators (NAMs) of each of the group I metabotropic glutamate receptors (mGlu1 and mGlu5) have been well characterized in the literature and offer potential as therapeutics in several disorders of the central nervous system (CNS). Still, compounds that are potent mGlu1/5 NAMs with selectivity versus the other six members of the mGlu family as well as the balance of properties required for use in vivo are lacking. A medicinal chemistry effort centered on the identification of a lead series with the potential of delivering such compounds is described in this Letter. Specifically, a new class of pyrido[1′,2′:1,5]pyrazolo[4,3-d]pyrimidin-4-amines was designed as a novel isosteric replacement for 4-aminoquinazolines, and compounds from within this chemotype exhibited dual NAM activity at both group I mGlus. One compound, VU0467558 (29), demonstrated near equipotent activity at both receptors, selectivity versus other mGlus, a favorable ancillary pharmacology profile, and CNS exposure in rodents.

G protein-gated inwardly-rectifying potassium (GIRK) channels are potassium-selective ion channels. As their name suggests, GIRK channels are effectors of G(i/o) G protein-couple receptors whereby activation of these GPCRs leads to increased GIRK channel activity resulting in decreased cellular excitability. In this way, GIRK channels play diverse roles in physiology as effectors of G(i/o)-coupled GPCRs: peacemaking in the heart rate, modulation of hormone secretion in endocrine tissues, as well as numerous CNS functions including learning, memory and addiction/reward. Notably, GIRK channels are widely expressed along the spinothalamic tract and are positioned to play roles in both ascending and descending pain pathways. More notably, GIRK channel knockout and knock-down studies have found that GIRK channels play a major role in the action of opioid analgesics which act predominantly through G(i/o)-coupled, opioid-activated GPCRs (e.g. μ-opioid receptors). Recent advances in GIRK channel pharmacology have led to the development of small molecules that directly and selectively activate GIRK channels. Based on research implicating the involvement of GIRK channels in pain pathways and as effectors of opioid analgesics, we conducted a study to determine whether direct pharmacological activation of GIRK channels could produce analgesic efficacy and/or augment the analgesic efficacy morphine, an opioid receptor agonist capable of activating μ-opioid receptors as well as other opioid receptor subtypes. In the present study, we demonstrate that the small-molecule GIRK activator, VU0466551, has analgesic effects when dosed alone or in combination with sub-maximally effective doses of morphine.

2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.

BACKGROUND AND PURPOSE: G protein‐gated inwardly rectifying K(+) (K(ir)3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal K(ir)3 channels consist of K(ir)3.1 and K(ir)3.2 subtypes, while cardiac K(ir)3 channels consist of K(ir)3.1 and K(ir)3.4 subtypes. Previously, we identified a family of urea‐containing K(ir)3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for K(ir)3.1/3.2 relative to K(ir)3.1/3.4 channels. Here, we characterize a non‐urea activator, VU0810464, which displays nanomolar potency as a K(ir)3.1/3.2 activator, improved selectivity for neuronal K(ir)3 channels, and improved brain penetration. EXPERIMENTAL APPROACH: We used whole‐cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac K(ir)3 channel subtypes. We tested VU0810464 in vivo in stress‐induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of K(ir)3.1‐containing K(ir)3 channels, were performed to permit direct comparisons. KEY RESULTS: VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal K(ir)3 channel activators, but VU0810464 was more selective for neuronal K(ir)3 channels. VU0810464, like ML297, reduced stress‐induced hyperthermia in a K(ir)3‐dependent manner in mice. ML297, but not VU0810464, decreased anxiety‐related behaviour as assessed with the elevated plus maze test. CONCLUSION AND IMPLICATIONS: VU0810464 represents a new class of K(ir)3 channel activator with enhanced selectivity for K(ir)3.1/3.2 channels. VU0810464 may be useful for examining K(ir)3.1/3.2 channel contributions to complex behaviours and for probing the potential of K(ir)3 channel‐dependent manipulations to treat neurological disorders.

Employing a novel and proprietary comprehensive precision medicine clinical tool that is a LC/MS/MS-based platform (PrecisCMQ™), we analyzed the serum of HTN patients seeking emergency care (n=293) for the presence and quantity of 42 antihypertensive and cardiovascular medications. Patient self-reported adherence correlated with clinical tool-based adherence. Among patients prescribed > 3 medications, clinical tool-based adherence in patients (n=74) who indicate they never miss a dose was 74%, versus 60% in patients (n=69) who reported missed doses (p = 0.009, two-sided t-test; median prescribed medications = 3.5). Patients were considered adherent when 80% of prescribed medications were detected and categorized as nonadherent otherwise. Among patients with > 3 prescribed medications, we found adherent patients had lower SBP (9.7 mm Hg; 95% CI 1.8 - 17.6; p = 0.02) and lower DBP (6.9 mm Hg; 95% CI 1.9 - 12.1; p = 0.007) after adjusting for age, sex, BMI, and patient-reported adherence. No significant difference in BP was observed in patients prescribed < 3 medications. By comparing medication clinical tool-based detections to prescribed medications listed in the EHR, 12% of the detected medications (68/549) were not recorded in the patients’ EHR. After accounting for the above covariates and the number of medications in the EHR, we observed that the total number of detected medications, including prescriptions not in the EHR, significantly correlated with both SBP (attributed to 4.8% of variation; p = 0.01) and DBP (patients prescribed > 3medications; explaining 2.5% of BP variation; p = 0.05). Additionally, to assess whether patients with higher systemic medication concentrations had lower BP, we compared SBP and DBP to medication concentrations normalized to published reference ranges. After accounting for the above covariates and the number of detected medications, we observed a relationship between medication(s) concentration(s) and BP across all patients. Together, these results support the utility of clinical tool-based medication monitoring for assessing adherence and improving BP control in HTN 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.

Herein, we detail the optimization of the mGlu3 NAM, VU0650786, via a reductionist approach to afford a novel, simplified mGlu3 NAM scaffold that engenders potent and selective mGlu3 inhibition (mGlu3 IC50 = 245 nM, mGlu2 IC50 > 30 μM) with excellent central nervous system penetration (rat brain/plasma Kp = 1.2, Kp,uu = 0.40). Moreover, this new chemotype, exemplified by VU6010572, requires only four synthetic steps and displays improved physiochemical properties and in vivo efficacy in a mouse tail suspension test (MED = 3 mg/kg i.p.).

Preclinical evidence in support of the potential utility of mGlu5 NAMs for the treatment of a variety of psychiatric and neurodegenerative disorders is extensive, and multiple such molecules have entered clinical trials. Despite some promising results from clinical studies, no small molecule mGlu5 NAM has yet to reach market. Here we present the discovery and evaluation of N-(5-fluoropyridin-2-yl)-6-methyl-4-(pyrimidin-5-yloxy)picolinamide (27, VU0424238), a compound selected for clinical evaluation. Compound 27 is more than 900-fold selective for mGlu5 versus the other mGlu receptors, and binding studies established a Ki value of 4.4 nM at a known allosteric binding site. Compound 27 had a clearance of 19.3 and 15.5 mL/min/kg in rats and cynomolgus monkeys, respectively. Imaging studies using a known mGlu5 PET ligand demonstrated 50% receptor occupancy at an oral dose of 0.8 mg/kg in rats and an intravenous dose of 0.06 mg/kg in baboons.

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.

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.

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).

Herein we report the design and synthesis of a series of substituted pyrazolo[1,5-a]quinazolin-5(4H)-ones as negative allosteric modulators of metabotropic glutamate receptors 2 and 3 (mGlu2 and mGlu3, respectively). Development of this series was initiated by reports that pyrazolo[1,5-a]quinazoline-derived scaffolds can yield compounds with activity at group II mGlu receptors which are prone to molecular switching following small structural changes. Several potent analogues, including 4-methyl-2-phenyl-8-(pyrimidin-5-yl)pyrazolo[1,5-a]quinazolin-5(4H)-one (10b), were discovered with potent in vitro activity as dual mGlu2/mGlu3 NAMs, with excellent selectivity versus the other mGluRs.

Development of SAR in an aryl ether series of mGlu5 NAMs leading to the identification of tool compound VU0409106 is described in this Letter. VU0409106 is a potent and selective negative allosteric modulator of mGlu5 that binds at the known allosteric binding site and demonstrates good CNS exposure following intraperitoneal dosing in mice. VU0409106 also proved efficacious in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu5 antagonists as well as clinically efficacious anxiolytics.

Development of SAR in an octahydropyrrolo[3,4-c]pyrrole series of negative allosteric modulators of mGlu1 using a functional cell-based assay is described in this Letter. The octahydropyrrolo[3,4-c]pyrrole scaffold was chosen as an isosteric replacement for the piperazine ring found in the initial hit compound. Characterization of selected compounds in protein binding assays was used to identify the most promising analogs, which were then profiled in P450 inhibition assays in order to further assess the potential for drug-likeness within this series of compounds.

Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system disorders, including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 were discovered to exhibit in vivo efficacy in rodent models of psychosis, including PAMs possessing varying degrees of agonist activity (ago-PAMs), as well as PAMs devoid of agonist activity. However, previous studies revealed that ago-PAMs can induce seizure activity and behavioral convulsions, whereas pure mGlu5 PAMs do not induce these adverse effects. We recently identified a potent and selective mGlu5 PAM, VU0403602, that was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by coadministration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine. Consistent with potential adverse effects induced by ago-PAMs, we found that VU0403602 had significant allosteric agonist activity. Interestingly, inhibition of VU0403602 metabolism in vivo by a pan cytochrome P450 (P450) inactivator completely protected rats from induction of seizures. P450-mediated biotransformation of VU0403602 was discovered to produce another potent ago-PAM metabolite-ligand (M1) of mGlu5. Electrophysiological studies in rat hippocampal slices confirmed agonist activity of both M1 and VU0403602 and revealed that M1 can induce epileptiform activity in a manner consistent with its proconvulsant behavioral effects. Furthermore, unbound brain exposure of M1 was similar to that of the parent compound, VU0403602. These findings indicate that biotransformation of mGlu5 PAMs to active metabolite-ligands may contribute to the epileptogenesis observed after in vivo administration of this class of allosteric receptor modulators.

The G-protein activated, inward-rectifying potassium (K(+)) channels, "GIRKs", are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo- and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.

A multi-dimensional, iterative parallel synthesis effort identified a series of highly selective mGlu3 NAMs with sub-micromolar potency and good CNS penetration. Of these, ML337 resulted (mGlu3 IC50 = 593 nM, mGlu2 IC50 >30 μM) with B:P ratios of 0.92 (mouse) to 0.3 (rat). DMPK profiling and shallow SAR led to the incorporation of deuterium atoms to address a metabolic soft spot, which subsequently lowered both in vitro and in vivo clearance by >50%.

Cocaine is a powerful and highly addictive stimulant that disrupts the normal reward circuitry in the central nervous system (CNS), producing euphoric effects. Cocaine use can lead to acute and life threatening emergencies, and abuse is associated with increased risk for contracting infectious diseases. Though certain types of behavioral therapy have proven effective for treatment of cocaine addiction, relapse remains high, and there are currently no approved medications for the treatment of cocaine abuse. Evidence has continued to accumulate that indicates a critical role for the metabotropic glutamate receptor subtype 5 (mGlu5) in the modulation of neural circuitry associated with the addictive properties of cocaine. While the small molecule mGlu5 negative allosteric modulator (NAM) field is relatively advanced, investigation into the potential of small molecule mGlu5 NAMs for the treatment of cocaine addiction remains an area of high interest. Herein we describe the discovery and characterization of a potent and selective compound 29 (VU0463841) with good CNS exposure in rats. The utility of 29 (VU0463841) was demonstrated by its ability to attenuate drug seeking behaviors in relevant rat models of cocaine addiction.

An iterative parallel synthesis effort identified a PLD2 selective inhibitor, ML298 (PLD1 IC5020000 nM, PLD2 IC50 = 355 nM) and a dual PLD1/2 inhibitor, ML299 (PLD1 IC50 = 6 nM, PLD2 IC50 = 20 nM). SAR studies revealed that a small structural change (incorporation of a methyl group) increased PLD1 activity within this classically PLD2-preferring core and that the effect was enantiospecific. Both probes decreased invasive migration in U87-MG glioblastoma cells.

This letter describes the continued chemical optimization of the VU0453595 series of M1 positive allosteric modulators (PAMs). By surveying alternative 5,6- and 6,6-heterobicylic cores for the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-5-one core of VU453595, we found new cores that engendered not only comparable or improved M1 PAM potency, but significantly improved CNS distribution (Kps 0.3 to 3.1). Moreover, this campaign provided fundamentally distinct M1 PAM chemotypes, greatly expanding the available structural diversity for this valuable CNS target, devoid of hydrogen-bond donors.

The efficacy of positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 4 (mGlu4) in preclinical rodent models of Parkinson’s disease has been established by a number of groups. Here, we report an advanced preclinically characterized mGlu4 PAM, N-(3-chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (VU0418506). We detail the discovery of VU0418506 starting from a common picolinamide core scaffold and evaluation of a number of amide bioisosteres leading to the novel pyrazolo[4,3-b]pyridine head group. VU0418506 has been characterized as a potent and selective mGlu4 PAM with suitable in vivo pharmacokinetic properties in three preclinical safety species.

Utilizing a combination of high-throughput and multi-step synthesis, SAR in a novel series of M1 acetylcholine receptor antagonists was rapidly established. The efforts led to the discovery the highly potent M1 antagonists 6 (VU0431263), and 8f (VU0433670). Functional Schild analysis and radioligand displacement experiments demonstrated the competitive, orthosteric binding of these compounds; human selectivity data are presented.

Further chemical optimization of the MLSCN/MLPCN probe ML077 (KCC2 IC(50)=537 nM) proved to be challenging as the effort was characterized by steep SAR. However, a multi-dimensional iterative parallel synthesis approach proved productive. Herein we report the discovery and SAR of an improved novel antagonist (VU0463271) of the neuronal-specific potassium-chloride cotransporter 2 (KCC2), with an IC(50) of 61 nM and >100-fold selectivity versus the closely related Na-K-2Cl cotransporter 1 (NKCC1) and no activity in a larger panel of GPCRs, ion channels and transporters.

M(1) muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer's disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M(1) receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M(1) allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M(1) coupling to different signaling pathways including Ca(2+) and β-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M(1) to specific signaling pathways leads to selective actions on some but not all M(1)-mediated responses in brain circuits. These novel M(1) allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M(1)-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M(1) agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M(1) allosteric agonists can differentially regulate coupling of M(1) to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.

Herein we report the discovery and SAR of a novel metabotropic glutamate receptor 3 (mGlu(3)) NAM probe (ML289) with 15-fold selectivity versus mGlu(2). The mGlu(3) NAM was discovered via a 'molecular switch' from a closely related, potent mGlu(5) positive allosteric modulator (PAM), VU0092273. This NAM (VU0463597, ML289) displays an IC(50) value of 0.66 μM and is inactive against mGlu(5).

This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we arrived at a more highly M(1)-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selectivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound (ML012), is presented.

There is an increasing amount of literature data showing the positive effects on preclinical antiparkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu4). However, most of the data generated utilize compounds that have not been optimized for druglike properties, and as a consequence, they exhibit poor pharmacokinetic properties and thus do not cross the blood–brain barrier. Herein, we report on a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides with improved PK properties with excellent potency and selectivity as well as improved brain exposure in rodents. Finally, ML182 was shown to be orally active in the haloperidol induced catalepsy model, a well-established antiparkinsonian model.

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.

Introduction We report on a novel tandem liquid chromatography mass spectrometry (LC/MS/MS) method for the simultaneous bioanalytical assessment of blood levels for multiple commonly prescribed medications, including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB). Despite morbidity and mortality benefits of ACEI/ARBs, these medications may cause transient rise in creatinine and occasionally contribute to hypotension, which in turn may result in acute kidney injury (AKI). Previous studies of AKI in the setting of ACEI/ARBs have not assessed medication levels. Hypothesis We hypothesized ACEI/ARB plasma levels in/above reference range for patients with AHF treated by intravenous (IV) diuretics would be associated with increased risk of in-hospital AKI compared to undetectable/low ACEI/ARB plasma levels. Methods From June 2016 to June 2017, adults with AHF were approached for consent. Patients were excluded for: 1) no outpatient ACEI/ARB, 2) systolic blood pressure Results Mean age for the 37 patients was 65.9 years (sd 14.6), 20 (54.0%) were female, 11 (29.7%) were African American, 10 (32.3%) had an ejection fraction Figure ), and ACEI/ARB levels in/above reference range at the time of ED presentation were associated with an in-hospital rise in creatinine (beta 0.22, 95% confidence interval 0.02-0.42, P=0.03, adjusted for age, sex, and race). Conclusions Patients with AHF may be more likely to develop AKI in the hospital if they have ACEI/ARB blood levels in/above reference range at ED presentation. Bioanalytical assessment of ACEI/ARB levels may prove useful for guiding in-hospital medical therapy for AHF.

Marketed drugs cleared by aldehyde oxidase (AO) are few, with no known clinically relevant pharmacokinetic drug interactions associated with AO inhibition, whereas cytochrome P450 (P450) inhibition or induction mediates a number of clinical drug interactions. Little attention has been given to the consequences of coadministering a P450 inhibitor with a compound metabolized by both AO and P450. Upon discovering that VU0409106 (1) was metabolized by AO (to M1) and P450 enzymes (to M4-M6), we sought to evaluate the in vivo disposition of 1 and its metabolites in rats with attenuated P450 activity. Male rats were orally pretreated with the pan-P450 inactivator, 1-aminobenzotriazole (ABT), before an i.p. dose of 1. Interestingly, the plasma area under the curve (AUC) of M1 was increased 15-fold in ABT-treated rats, indicating a metabolic shunt toward AO resulted from the drug interaction condition. The AUC of 1 also increased 7.8-fold. Accordingly, plasma clearance of 1 decreased from 53.5 to 15.3 ml/min per kilogram in ABT-pretreated rats receiving an i.v. dose of 1. Consistent with these data, M1 formation in hepatic S9 increased with NADPH-exclusion to eliminate P450 activity (50% over reactions containing NADPH). These studies reflect possible consequences of a drug interaction between P450 inhibitors and compounds cleared by both AO and P450 enzymes. Notably, increased exposure to an AO metabolite may hold clinical relevance for active metabolites or those mediating toxicity at elevated concentrations. The recent rise in clinical drug candidates metabolized by AO underscores the importance of these findings and the need for clinical studies to fully understand these risks.