Pharmacology of N-desmethylclozapine Lameh, Jelveh; Burstein, Ethan S; Taylor, Eve ...
Pharmacology & therapeutics (Oxford)
115, Številka:
2
Journal Article
Recenzirano
Currently available treatments for schizophrenia have limited efficacy and are generally poorly tolerated. However, among these antipsychotic agents, clozapine stands apart in having generally ...superior motoric tolerability and efficacy. One intriguing possibility, based on clinical correlations, receptor activity profiles and studies with animal models predictive of antipsychotic or cognitive action is that the activity of N-desmethylclozapine (NDMC), a major metabolite of clozapine, may, at least in part, underlie the unique efficacy of clozapine. In this review we compare the pharmacological properties of NDMC to those of clozapine and consider how they may contribute to the overall clinical properties of clozapine. We also consider whether NDMC, in its own right, might be a superior antipsychotic drug.
Abstract
While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for ...single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents.
FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21).
Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores.
In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance.
Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 853.
The role of neuropeptide FF (NPFF) and its analogs in pain modulation is ambiguous. Although NPFF was first characterized as an antiopioid peptide, both antinociceptive and pronociceptive effects ...have been reported, depending on the route of administration. Currently, two NPFF receptors, termed FF1 and FF2, have been identified and cloned, but their roles in pain modulation remain elusive because of the lack of availability of selective compounds suitable for systemic administration in in vivo models. Ligand-binding studies confirm ubiquitous expression of both subtypes in brain, whereas only FF2 receptors are expressed spinally. This disparity in localization has served as the foundation of the hypothesis that FF1 receptors mediate the pronociceptive actions of NPFF. We have identified novel small molecule NPFF receptor agonists and antagonists with varying degrees of FF2/FF1 functional selectivity. Using these pharmacological tools in vivo has allowed us to define the roles of NPFF receptor subtypes as pertains to the modulation of nociception. We demonstrate that selective FF2 agonism does not modulate acute pain but instead ameliorates inflammatory and neuropathic pains. Treatment with a nonselective FF1/FF2 agonist potentiates allodynia in neuropathic rats and increases sensitivity to noxious thermal and to non-noxious mechanical stimuli in normal rats in an FF1 antagonist-reversible manner. Treatment with FF1 antagonists reversed established mechanical allodynia, indicating the possibility of increased NPFF tone through FF1 receptors. In conclusion, we provide evidence for the opposing roles of NPFF receptors and highlight selective FF2 agonism and/or selective FF1 antagonism as potential targets warranting further investigation.
The recent discovery of allosteric potentiators and agonists of the muscarinic M
1 receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe ...the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC
50 7.6–7.7) and efficacious (90–98% of carbachol) muscarinic M
1 receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M
1 receptor over the M
2, M
3, M
4 and M
5 muscarinic receptor subtypes. Using GTPγS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M
1 receptor activation since it was not observed in M
1 knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M
1 receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M
1 selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.
The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) ...(ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of (3)Hketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzoa,dcyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.
Although there have been compelling advances in the cancer immunotherapy space recently in the form of chimeric antigen receptor (CAR) modified T-cells and checkpoint inhibitors, advanced tools to ...explore the therapeutic mechanisms of their combination are not adequately developed or widely available. To address this growing need, we developed a robust quantitative fluorescent immunohistochemistry platform using multiplex AQUA (Automated Quantitative Analysis) technology to evaluate checkpoint inhibitor expression, enumerate CAR T cells and determine the interaction between tumor cells and immune cells via novel co-localization algorithms. We explored utility of this method both in preclinical- and clinical model systems. In an immunodeficient mouse model of B-cell lymphoma, we evaluated homing of CAR T cells to malignant B-cells in primary lymphoid organs. We determined the phenotype and functional status of the CAR T cells via multiplex analyses of CD4, CD8, PD1 and FOXP3 expression. Additionally, to enable combination immunotherapies in Diffuse Large B-Cell Lymphoma (DLBCL) setting, we explored prevalence of adaptive immune resistance mechanisms in the form of PD1 and PD-L1 expression in immune- and tumor cell compartments via landmarks created by cytoplasmic and nuclear stains in both primary and secondary biopsies from DLBCL patients (n = 63). To support patient selection for CAR T trials, we quantified expression and prevalence of relevant tumor antigens that could not be scored reproducibly by traditional methods to yield objective cut points. We anticipate utilization of these quantitative multiplexed IHC methods for optimal selection of patients into upcoming novel combination immunotherapy trials
Tran:Genoptix: Employment. Scott:Genoptix: Employment. Lee:Genoptix: Employment. Singh:Novartis: Employment. Cogan:Novartis: Employment. Bordeaux:Genoptix: Employment. Jennifer:Genoptix: Employment. Lameh:Genoptix: Employment. Tribouley:Novartis: Employment. Kassim:Novartis: Employment. Tangri:Genoptix Inc., a Novartis company: Employment. Dakappagari:Genoptix Inc., a Novartis company: Employment.
Abstract
Introduction: There is an urgent need for deep targeted sequencing for detection of varieties of somatic aberrations, including single nucleotide variants (SNV), insertions/deletions ...(indels), loss of heterozygosity (LOH), and copy number variants (CNV) in heterogeneous FFPE cancer patient specimens. We have developed a Next Generation Sequencing (NGS)-based assay for deep sequencing of a number of selected oncogenes and tumor suppressor genes for testing of clinical breast cancer samples. The panel includes those genes, which are commonly mutated in cancers (e.g. breast and ovarian cancer), based on The Cancer Genome Atlas studies and are potentially indicative of responses to experimental compounds undergoing clinical trials. Importantly, the assay can be used in FFPE samples which pose challenges because of high variability in quality and often suboptimal quantities.
Methods: To minimize FFPE sample input requirement, we optimized multiple assay steps. DNA was extracted from macrodissected FFPE tissue, and randomly fragmented. The DNA library was constructed and subject to target capture and enrichment before being sequenced on the Illumina MiSeq® systems. To analytically qualify the assays, we tested well-characterized cell lines and reference HapMap cell lines. Furthermore, to confirm the performance of the NGS results in clinical FFPE samples, we compared the results generated using orthogonal methodologies, e.g. SNVs using a custom Ion AmpliSeq™ panel (Life Technologies) on Ion Personal Genome Machine® (PGM), or CNVs using real-time PCR assays or custom Nanostring nCounter® CNV assays (NanoString Technologies). To establish the lower limit of detection, we used mixed DNA samples from FFPE cell lines and from HapMap DNA and further tested Horizon FFPE reference DNA containing mutations at defined allele frequencies.
Results: For FFPE DNA extraction, we selected Cobas® FFPE kit (Roche), and Maxwell® CSC DNA FFPE kit (Promega) for higher yield and better purity. For library construction, we chose a KAPA streamlined library preparation kit. For target capture, we used custom xGEN® DNA probes (Integrated DNA Technologies) for improved flexibility in assay optimization, reduced reagent cost, and minimizing batch-to-batch variability. We were able to detect SNVs for >99% of exons selected down to a 5% sensitivity level using about 100 ng FFPE DNA input. Indels, CNV and LOH could also be detected reliably.
Conclusions: We developed a targeted NGS assay that could be used in clinical trials and meets the dual challenge of limited DNA amounts and/or variable quality often associated with FFPE clinical specimens and the detection of low level mutations. Considering the recent recognition of Illumina NGS platform by FDA, this approach can represent a path forward to assist in cancer patient assessment, clinical trial recruitment and care.
Citation Format: Wenge Shi, Christine Chin, Tingdong Tang, Loretta Hipolito, Preethi Srinivasan, Derek Chiang, David Peng, Emmanuelle Di Tomaso, Shabnam Tangri, Jelveh Lameh, Reinhold Pollner. Development of a clinical targeted next generation sequencing test for challenging formalin-fixed paraffin-embedded (FFPE) cancer samples. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2014-1892
Abstract
HER2 is a prognostic and predictive marker for breast cancer patients and its expression is routinely evaluated by immunochemistry (IHC). Scoring of IHC slides is prone to operator ...subjectivity and equivocal results make selection of appropriate therapy difficult, ultimately affecting patient outcomes. We describe development and validation of a reproducible quantitative immunofluorescence assay to accurately assess HER-2 levels in Formalin-Fixed Paraffin-Embedded (FFPE) breast cancer specimens by AQUA Technology that avoids the ambiguity of equivocal results and enables critical treatment decisions.
Methods: A tissue microarray (n=80) composed of breast cancer cases with known IHC and fluorescence in situ hybridization (FISH) status was used to characterize and optimize assay performance for three HER-2 antibody clones; A0485, CB11, and SP3. Eight dilutions of each antibody were tested with four different antigen retrieval conditions. A total of 108 assay conditions were evaluated by receiver operator characteristic (ROC) analysis for sensitivity and specificity. Equal weight was given to sensitivity and specificity to select the most robust assay. The top six assay conditions were then assessed using 45 whole tissue breast cancer specimens to identify one condition with highest sensitivity and specificity. The selected assay condition was evaluated on an additional 90 breast cancer specimens (training set) annotated for IHC and FISH scores to determine the binary cut point for the HER-2 AQUA assay®, the cut point was then confirmed on a validation set composed of 90 independent breast cancer specimens. The final assay was analytically validated in accordance with College of American Pathologists (CAP) guidelines utilizing over 120 independent breast cancer specimens.
Results: Based on an evaluation of over 400 breast cancer specimens with nearly equal distribution of 0, 1+, 2+ and 3+ cases, HER-2 antibody clone, SP3, which recognizes the extracellular domain of the receptor, clearly segregated HER2 positive specimens from HER2 negative breast cancer cases.
Conclusion: The availability of a highly reproducible quantitative binary test facilitates rapid treatment decisions with appropriate HER-2 targeting biologics on the market.
Citation Format: Lakshmi Krupa Chandrasekaran, Jennifer Bordeaux, Sue Beruti, Naveen Dakappagari, Mike Nerenberg, Jelveh Lameh, Armin Graber, David Rimm, Bruce Robbins, Nagesh Rao. Development of a binary diagnostic immunofluorescence assay by AQUA® technology for accurate detection of HER-2 levels in breast cancer specimens. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2014-2843
We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of ...aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.
