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GDC‐0334 is a novel small molecule inhibitor of transient receptor potential cation channel member A1 (TRPA1), a promising therapeutic target for many nervous system and respiratory diseases. The pharmacokinetic (PK) profile and pharmacodynamic (PD) effects of GDC‐0334 were evaluated in this first‐in‐human (FIH) study. A starting single dose of 25 mg was selected based on integrated preclinical PK, PD, and toxicology data following oral administration of GDC‐0334 in guinea pigs, rats, dogs, and monkeys. Human PK and PK‐PD of GDC‐0334 were characterized after single and multiple oral dosing using a population modeling approach. The ability of GDC‐0334 to inhibit dermal blood flow (DBF) induced by topical administration of allyl isothiocyanate (AITC) was evaluated as a target‐engagement biomarker. Quantitative models were developed iteratively to refine the parameter estimates of the dose‐concentration‐effect relationships through stepwise estimation and extrapolation. Human PK analyses revealed that bioavailability, absorption rate constant, and lag time increase when GDC‐0334 was administered with food. The inhibitory effect of GDC‐0334 on the AITC‐induced DBF biomarker exhibited a clear sigmoid‐Emax relationship with GDC‐0334 plasma concentrations in humans. This study leveraged emerging preclinical and clinical data to enable iterative refinement of GDC‐0334 mathematical models throughout the FIH study for dose selection in subsequent cohorts throughout the study. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? GDC‐0334 is a novel, small molecule TRPA1 inhibitor and a pharmacokinetic‐pharmacodynamic (PK‐PD) modeling strategy could be implemented in a systematic and step‐wise manner to build and learn from emerging data for early clinical development. WHAT QUESTION DID THIS STUDY ADDRESS? Can noncompartmental and population‐based analyses be used to describe the PK and PD characteristics of GDC‐0334 in preclinical and clinical studies? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? GDC‐0334 exposure generally increased with dose in rats, dogs, and monkeys. The starting dose (25 mg) in the clinical study was determined based on the preclinical data. GDC‐0334 exhibited linear PK in humans and the bioavailability was increased with food. The inhibitory effect of GDC‐0334 on dermal blood flow induced by the TRPA1 agonist allyl isothiocyanate in humans indicates a clear PK‐PD relationship. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The models developed based on TRPA1 agonist‐induced dermal blood flow inhibition data can be used to predict PK‐PD relationships in future preclinical and clinical studies evaluating new drug entities that target TRPA1.

Background and purpose Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker. Experimental approach Allyl isothiocyanate (AITC), capsaicin or citric acid were applied to ears of wildtype and Trpa1-knock out (Trpa1 KO) rats, and changes in dermal blood flow (DBF) were measured by laser speckle contrast imaging. In humans, the DBF, pain and itch responses to 5-20% AITC applied to the forearm were measured and safety was evaluated. Reproducibility of the DBF, pain and itch responses to topically applied 10% and 15% AITC were assessed at two visits separated by 13-15 days. DBF changes were summarized at 5-minute intervals as areas under the curve (AUC) and maxima. Intraclass correlation coefficient (ICC) was calculated to assess arm-arm and period-period reproducibility. Key results AITC- and citric acid-induced DBF were significantly reduced in Trpa1 KO rats compared to wildtype (90 ± 2% and 65 ± 11% reduction, respectively), whereas capsaicin response did not differ. In humans, each AITC concentration significantly increased DBF compared to vehicle with the maximal increase occurring 5 minutes post application. Ten percent and 15% AITC were selected as safe and effective stimuli. AUC from 0 to 5 minutes was the most reproducible metric of AITC-induced DBF across arms (ICC = 0.92) and periods (ICC = 0.85). Subject-reported pain was more reproducible than itch across visits (ICC = 0.76 vs 0.17, respectively). Conclusion and implications AITC-induced DBF is a suitable target engagement biomarker of TRPA1 activity for preclinical and clinical studies of TRPA1 antagonists.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

BACKGROUND Checkpoint inhibitor therapy has demonstrated overall limited efficacy in the treatment of GBM. Mechanisms of resistance to checkpoint blockade need to be better elucidated. Analysis of the tumor microenvironment is critical to identify correlates of response to immune checkpoint blockade. 60 newly diagnosed GBM patients unselected for MGMT status underwent treatment with concurrent atezolizumab with radiation therapy and TMZ followed by adjuvant atezolizumab and TMZ (NCT03174197). Clinical data has been reported previously. METHODS Tissue image immunoprofiling was conducted using 2 multiplex immunofluorescence (mIF) panels against; CD3, CD8, PD-1, PD-L1, Granzyme B, FOXP3, CD45RO, CD68, and GFAP antibodies. PDL-1 (Clone SP263) malignant cells expression was assessed by immunohistochemistry. Correlations between mIF biomarkers co-expressions, IHC PD-L1, and clinical outcome including OS, radiographic response, and PFS were evaluated. RESULTS Of 60 patients enrolled, image immunoprofiling was performed successfully on pre-treatment tissue in 48 patients. 20 of 60 patients underwent re-resection for suspected recurrent disease of which 10 patients had immunoprofiling performed successfully on pre and post treatment samples. An analysis of CD3CD8+ cytotoxic T lymphocytes was consistent with prior work, showing no or relatively low levels at baseline, and no association with clinical outcome. PDL-1 expression by IHC, at thresholds of >1% or >5%, was not associated with clinical outcome. Tumors with a higher number of GFAP-expressing cancer cells had a significantly lower tumor response (p< 0.05) and median OS (430 vs. 799 days, p< 0.01). CONCLUSIONS For newly diagnosed GBM patients treated with standard of care radiation and temozolomide in combination with atezolizumab, T-cell levels and PDL-1 expression were not predictive of outcome. GFAP may represent a novel predictive biomarker of overall survival. Ongoing studies to evaluate the gut microbiome and tumor genomic (WES, CNA) and transcriptomic (RNAseq) features of these and matched tumors are underway.

Synthesis of several 7-hydroxy oxysterols and their potential roles as signaling molecules in the innate and adaptive immune responses is discussed. Discovery of a new, fluorinated, synthetic analog of the 7α,25-dihydroxycholesterol-the endogenous ligand of GPR 183 (EBI2), a G-protein coupled receptor highly expressed upon Epstein-Barr virus infection is described. Fluoro oxysterol 12 showed good metabolic stability while maintaining excellent EBI2 agonist activity.

Previous research suggests animals may integrate temporal information into mental representations, or temporal maps. We examined the parameters under which animals integrate temporal information in three appetitive conditioning experiments. In Experiment 1 the temporal relationship between 2 auditory cues was established during sensory preconditioning (SPC). Subsequently, rats were given first order conditioning (FOC) with one of the cues. Results showed integration of the order of cues between the SPC and FOC training phases. In subsequent experiments we tested the hypothesis that quantitative temporal information can be integrated across phases. In Experiment 2, SPC of two short auditory cues superimposed on a longer auditory cue was followed by FOC of either one of the short cues, or of the long cue at different times in the cue. Contrary to our predictions we did not find evidence of integration of temporal information across the phases of the experiment and instead responding to the SPC cues in Experiment 2 appeared to be dominated by generalization from the FOC cues. In Experiment 3 shorter auditory cues were superimposed on a longer duration light cue but with asynchronous onset and offset of the superimposed cues. There is some evidence consistent with the hypothesis that quantitative discrimination of whether reward should be expected during the early or later parts of a cue could be integrated across experiences. However, the pattern of responding within cues was not indicative of integration of quantitative temporal information. Generalization of expected times of reward during FOC seems to be the dominant determinant of within-cue response patterns in these experiments. Consequently, while we clearly demonstrated the integration of temporal order in the modulation of this dominant pattern we did not find strong evidence of integration of precise quantitative temporal information. This article is part of a Special Issue entitled: Associative and Temporal Learning.

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

Neuronal coding of stimulus-to-action sequences are believed to involve the release of dopamine (DA) and norepinephrine (NE). The electrochemical similarity of these monoamines, however, confounds real-time measurements of their release. Here we report the creation of cell-based neurotransmitter fluorescent-engineered reporters (CNiFERs) that utilize the specificity of G-protein coupled receptors (GPCRs) to discriminate nanomolar concentrations of DA and NE. CNiFERs were implanted into frontal cortex of mice to measure the timing of neurotransmitter release during classical conditioning using two-photon microscopy. The onset of DA release correlated with that of licking and monotonically shifted from the time of the reward toward that of the cue. In contrast, concurrent release of NE did not correlate with licking or the cue. This new generation of CNiFERs provides unique tools to assess the release of monoamines. The molecular design of these CNiFERs may be generalized to realize CNiFERs for any molecule that activates a GPCR.

The unfolded protein response (UPR) is a conserved, intracellular signaling pathway activated by endoplasmic reticulum (ER) stress. In mammalian cells, the UPR is controlled by three ER-resident transmembrane proteins: inositol-requiring enyzme-1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6), by which cytoprotective mechanisms are initiated to restore ER functions. However, if cellular homeostasis is not restored by the UPR's initial events, UPR signaling triggers apoptotic cell death, which correlates with the pathogenesis of a wide range of human diseases. The intrinsic function of the UPR in regulating cell survival and death suggests its importance as a mechanistic link between ER stress and disease pathogenesis. Understanding UPR regulatory molecules or signaling pathways involved in disease pathogenesis is critical to establishing therapeutic strategies. For this purpose, several experimental tools have been developed to evaluate individual UPR components. In this chapter, we present methods to monitor and quantify activation of individual UPR signaling pathways in mammalian cells and tissues, and we review strategies to artificially and selectively activate individual UPR signaling pathways using chemical-genetic approaches.

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.

In three between-groups blocking experiments with rats, two concurrent and one forward, several common control procedures were employed: Reinforced trials with the putative blocking stimulus were either omitted entirely (Kamin control), replaced by unsignaled reinforcements (Wagner control), or replaced by reinforced trials with a different stimulus (C+ control). In each experiment, parallel treatments with the target stimulus absent during training served to examine the possibility that differential responding in tests with the target stimulus might be traced solely to differential exposure to the nontarget stimuli. In Experiment 1, responding by a concurrent blocking group during the test was no different than responding by a Kamin control group, and responding by a Wagner control group was greater than that of either of the other groups--a pattern of results, mirrored in the performance of the target-absent groups, that could be attributed to the elevation of contextual excitation by unsignaled reinforcement. In Experiment 2, responding in the test by a concurrent blocking group was no different than that by a C+ control group. In Experiment 3, a finding of less responding by a forward blocking group than by a C+ control group when the target stimulus was present during training, but not when it was absent, provided plausible evidence of blocking.