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Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 (

Recent mouse knockout studies identified adapter protein-2 associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. Potent small-molecule inhibitors of AAK1 have been identified and show efficacy in various rodent pain models. (

BackgroundThe costimulatory receptor CD137 (also known as 4-1BB and TNFRSF9) plays an important role in sustaining effective cytotoxic T cell immune responses and its agonism has been investigated as a cancer immunotherapy. In clinical trials, the systemic administration of the 1st generation CD137 agonist monotherapies, utomilumab and urelumab, were suspended due to either low anti-tumor efficacy or hepatotoxicity mediated by recognized epitope on CD137 and FcγR ligand-dependent clustering.MethodsM9657, a bispecific antibody was engineered a tetravalent bispecific antibody (mAb2) format with the Fab portion binding to the tumor antigen Mesothelin (MSLN) and a modified CH2-CH3 domain as Fc antigen binding (Fcab) portion binding to CD137. M9657 has a human IgG1 backbone with LALA mutations to abrogate the binding to Fcγ receptor. The biological characteristics and activities of M9657 were investigated in a series of in vitro assays and the in vivo efficacy was investigated in syngeneic tumor models with FS122m, a murine-reactive surrogate with the same protein structure of M9657.ResultsM9657 binds efficiently to both human and Cynomolgus CD137 as well as MSLN. In the cellular functional assay, M9657 displayed MSLN- and TCR/CD3 interaction (signal 1)-dependent cytokine release and tumor cell cytotoxicity associated with Bcl-XL activation and immune memory formation. FS122m demonstrated potent MSLN- and dose- dependent in vivo anti-tumor efficacy (figure 1). Comparing with 3H3, a Urelumab surrogate Ab, FS122m displayed an improved therapeutic window with significantly lower for on-target /off-tumor toxicity.ConclusionsTaken together, M9657 exhibits a promising developability profile as a tumor-targeted immune agonist with potent anti-cancer activity, but without systemic immune activation.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.Abstract 757 Figure 1FS122m displayed dose-dependent anti-tumor efficacy

Background: T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) is an inhibitory receptor expressed on lymphocytes and has recently emerged as a target in cancer immunotherapy. M6223 is a fully human antagonistic anti-TIGIT antibody in immunoglobulin (Ig) G1 format with Fc-mediated effector function. Preclinical studies demonstrated that M6223 could induce an anti-tumor immune response by complementary mechanisms including but not limited to direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. M6223 showed dose-dependent anti-tumor efficacy in multiple preclinical tumor models. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β "trap") fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti-PD-L1), was designed to target two key immunosuppressive pathways in the tumor microenvironment. Methods: To attenuate the tumor immunosuppressive microenvironment, the anti-tumor immunity and efficacy of combining M6223 with bintrafusp alfa was investigated in syngeneic models in humanized mice with huTIGIT knock-in sequences. Results: M6223 combined with bintrafusp alfa significantly enhanced anti-tumor efficacy and extended survival compared with either monotherapies or the combination of M6223 with anti-PD-L1. M6223 combined with bintrafusp alfa stimulated higher CD8+ T cells and natural killer cells infiltration, proliferation and cytotoxicity in tumor microenvironment in comparison to the combination of M6223 with anti-PD-L1. The ratio of CD8+ T cells to regulatory T cells and the ratio of CD226 to TIGIT were significantly increased, indicating the conversion of the tumor microenvironment from an immuno-suppressive phenotype to a more immune-permissive phenotype. With the combination therapy, bintrafusp alfa was the main driver promoting CD8+ T cell proliferation, infiltration, and cytotoxicity. Alternatively, adding M6223 to bintrafusp alfa may decrease the risk of immune resistance caused by elevated TIGIT expression triggered by bintrafusp alfa treatment. Conclusion: These complementary mechanisms of M6223 and bintrafusp alfa orchestrate antitumor activity in preclinical tumor models. Currently, M6223 and bintrafusp alfa combination is being investigated in a Phase I clinical trial (NCT04457778) in patients with metastatic or locally advanced solid unresectable tumors. Citation Format: Chunxiao Xu, Sireesha Yalavarthi, Clotilde Bourin, Christie Kelton, Joern-Peter Halle, Jacques Moisan. Anti-tumor immunity and efficacy of combination treatment of M6223 and bintrafusp alfa versus the combination of M6223 and anti-PD-L1 in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 593.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Nav1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and NaV1.7 potency associated with 29.

BackgroundM6223 is a fully human antagonistic anti-T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) antibody in IgG1 format with Fc-mediated effector function.MethodsThe ability of M6223 to block the interaction of TIGIT with its ligands, CD155 and CD112, and the interaction of TIGIT with CD226 was determined by a flow cytometry-based binding assay. The anti-tumor efficacy, immune profile, and effector function of M6223 were investigated in syngeneic tumor models in huTIGIT knock-in mice. M6223 was either formatted with an effector competent mouse IgG2c constant region (M6223-muIgG2c) or formatted with effector null mouse IgG1-D256A constant region (M6223-muIgG1) as two versions of chimeric antibodies for the in vivo studies.ResultsM6223 dose-dependently blocked the binding of TIGIT to its ligands, including CD155 and CD112, thereby inhibiting a TIGIT-mediated immunosuppressive pathway. In addition, M6223 interrupted the interaction of TIGIT with the costimulatory receptor CD226. By blocking the interactions, the chimeric protein M6223-muIgG2c showed anti-tumor efficacy in multiple tumor models, including an MC38 tumor model (figure 1), and generated tumor antigen-specific long-term protective immunity in immunocompetent huTIGIT knock-in mice. M6223 monotherapy dose-dependently elevated the ratio of CD8+ cytotoxic T cells to regulatory T cells and the ratio of CD226 to TIGIT expression in immune cells in the tumor microenvironment. We also found that M6223 selectively depleted suppressive and exhausted TIGIT+ immune cell subsets and the anti-tumor activity of effector null M6223-muIgG1 was significantly lost (pAbstract 326 Figure 1M6223-muIgG2c displayed dose-dependent anti-tumor efficacy. M6223-muIgG2c displayed dose-dependent anti-tumor efficacy in an MC38 tumor model in hTIGIT knock-in mice.ConclusionsGiven that TIGIT blockade can inhibit an immunosuppressive pathway as well as remove the suppression on a costimulatory pathway, M6223 has the potential to induce an anti-tumor immune response by three complementary mechanisms: direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. Our data demonstrate that these complementary mechanisms orchestrate the anti-tumor activity of M6223. A Phase I, first-in-human clinical trial (NCT04457778) is underway to determine the safety, tolerability, maximum tolerated dose and recommended dose for expansion of M6223 as a single agent (Part 1A) and in combination with bintrafusp alfa (Part 1B) in patients with metastatic or locally advanced solid unresectable tumors.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.

CD4 T cells include multiple sublineages orchestrating a broad range of effector activities during the initiation, promotion, and progression of carcinogenesis. Although regulatory T cells (Tregs) are well-characterized to promote tumor progression, the impact of effector CD4+ T cell subsets (Teff) in anti-tumor immunity is less well known. Further, the relative contribution of Teff and Treg cell subsets in different syngeneic tumor models has not been characterized. We observed that CD4 depletion had significantly different impact of tumor growth across different syngeneic tumor models: no effect on MC38 tumor growth; enhanced tumor growth in CT26.KSA tumor model; while almost complete tumor regression in the EMT-6 tumor model. We hypothesized that each tumor model has a vastly different composition in the CD4 T cell compartment. To test this, we characterized expression of activation and CD4 T cell differentiation markers as well as cytokine production from CD4 TILs in each of the syngeneic tumor models. We found that while effector CD4 TILs (FoxP3−CD4+) from MC38 and CT26.KSA tumors expressed high levels activation and differentiation markers, Teff from EMT-6 tumors expressed very low levels of T cell activation and differentiation markers, suggesting the Teff in EMT-6 tumors are largely dysfunctional. Ex vivo restimulation of tumor samples with anti-CD3/anti-CD28 promoted IFNγ production in CD4 TILs from MC38 and CT26.KSA tumor models, but not from EMT-6 tumor models, further supporting that Teff from MC38 and CT26.KSA tumors remain highly active, while CD4 TILs from EMT-6 tumors are dysfunctional. When looking at the Treg compartment, the frequency of Tregs in CT26.KSA tumors was reduced compared to MC38 and EMT-6 tumors. Further, Tregs in MC38 and EMT-6 tumors expressed very high levels of CD69, a marker of highly immunosuppressive Tregs, while Tregs in CT26.KSA tumors expressed lower levels of CD69, suggesting that these Tregs may be less immunosuppressive. In summary, the CD4 T cell compartment is vastly different across CT26.KSA, MC38 and EMT-6 syngeneic tumor models, resulting in very different impact of tumor growth control. Citation Format: Chunxiao Xu, Lindsay Webb, Sireesha Yalavarthi, Clotilde Bourin, Jacques Moisan. Composition of CD4 T cell subsets and impact on tumor growth control across mouse syngeneic tumor models [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P077.

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Triazolopyridine ethers with mGlu2 positive allosteric modulator (PAM) activity are disclosed. The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided. The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule BMT-133218 ((+)-7m). After oral administration at 10mg/kg, BMT-133218 demonstrated full reversal of PCP-stimulated locomotor activity and prevented MK-801-induced working memory deficits in separate mouse models. Also, reversal of impairments in executive function were observed in rat set-shifting studies at 3 and 10mg/kg (p.o.). Extensive plasma protein binding as the result of high lipophilicity likely limited activity at lower doses. Optimized triazolopyridine ethers offer utility as mGlu2 PAMs for the treatment of schizophrenia and merit further preclinical investigation.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Since zwitterionic benzenesulfonamide Na

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Na

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.

Attentional set shifting, a measure of executive function, is impaired in schizophrenia patients. Current standard of care has little therapeutic benefit for treating cognitive dysfunction in schizophrenia; therefore, novel drugs and animal models for testing novel therapies are needed. The NMDA receptor antagonist, MK-801, produces deficits in a rat maze-based set-shifting paradigm, an effect which parallels deficits observed on tests of executive function in schizophrenia patients. Alpha7 nicotinic acetylcholine receptor (nAChR) agonists, currently under clinical development by several companies, show promise in treating cognitive symptoms in schizophrenia patients and can improve cognition in various animal models. The objectives of the present study were to determine whether the MK-801 deficit in set shifting could be reproduced in a drug discovery setting and to determine whether cognitive improvement could be detected for the first time in this task with alpha7 nAChR agonists. The data presented here replicate findings that a systemic injection of the NMDA receptor antagonist MK-801 can induce a deficit in set shifting in rats. Furthermore, the deficit could be reversed by the atypical antipsychotic clozapine as well as by several alpha7 nAch receptor agonists (SSR-180711, PNU-282987, GTS-21) with varying in vitro properties. Results indicate that the MK-801 set-shift assay is a useful preclinical tool for measuring prefrontal cortical function in rodents and can be used to identify novel mechanisms for the potential treatment of cognitive deficits in schizophrenia.

Acetylcholinesterase inhibitors (AChEIs) are approved to treat the symptoms of mild to moderate Alzheimer’s disease by restoring acetylcholine levels at synapses where the neurotransmitter has been depleted due to neurodegeneration. This assumption is challenged by more recent clinical studies suggesting the potential for disease-modifying effects of AChEIs as well as in vitro studies showing neuroprotective effects. However, few preclinical studies have assessed whether the improvement of cognitive symptoms may be mediated by reductions in Abeta or Tau pathology. The objective of the present study was to determine whether short-duration treatment with donepezil could improve spatial learning and memory in transgenic mice overexpressing mutant human amyloid precursor protein (hAPP) and presenilin 1 (PS1) (Dewachter et al., J Neurosci 20(17):6452–6458, 2000) after amyloid pathology has fully developed, consistent with early stages of Alzheimer’sdisease in humans. In parallel, the effect of donepezil treatment on brain amyloid, Tau, and glial endpoints was measured. This study showed a significant improvement in reference memory in hAPP/PS1 mice along with dose-dependent reductions in brain amyloid-β (Aβ). These results suggest that the observed cognitive improvement produced by donepezil in Alzheimer’s disease may be due, at least in part, to reduction of brain Aβ.

Triazolopyridine ethers with mGlu

A series of novel oxime carbamates have been identified as potent inhibitors of the key regulatory enzyme of the endocannabinoid signaling system, fatty acid amide hydrolase (FAAH). In this Letter, the rationale behind the discovery and the biological evaluations of this novel class of FAAH inhibitors are presented. Both in vitro and in vivo results of selected targets are discussed, along with inhibition kinetics and molecular modeling studies.(1).

Amitriptyline and gabapentin are the primary treatments for painful diabetic neuropathy (PDN), and it is clear that they produce beneficial effects, but there are questions about these treatments that have not been adequately addressed. For example, although there is a growing consensus that the therapeutic effects of amitriptyline in pain patients are independent of its effects on mood, it is not clear that amitriptyline has specific and direct effects on pain. There is also a fairly broad consensus that gabapentin is safe and well tolerated, but the side-effect profile of gabapentin has not been adequately assessed in pain populations. The rat streptozotocin (STZ) model of PDN was used (a) to assess the effects of amitriptyline on objective, quantitative measures of tactile allodynia, a common type of pain in PDN patients, and (b) to assess the side effects of gabapentin using measures of motor/ambulatory and cognitive function. Amitriptyline did not attenuate STZ-induced mechanical allodynia, even after chronic administration of high doses. Gabapentin produced robust anti-allodynic effects but also produced deficits in tests of motor/ambulatory and cognitive functions. The present experiments suggest that the beneficial effects of amitriptyline in PDN may not be a result of anti-allodynic efficacy and that gabapentin produces robust anti-allodynic effects but may also produce significant motor and cognitive deficits even at or near the lowest effective doses. These findings challenge the consensus opinions about these primary treatments for PDN and suggest that their therapeutic and adverse effects should be explored further in pain patients.

A class of bisarylimidazole derivatives are identified as potent inhibitors of the enzyme fatty acid amide hydrolase (FAAH). Compound 17 (IC(50)=2 nM) dose-dependently (0.1-10mg/kg, iv) potentiates the effects of exogenous anandamide (1 mg/kg, iv) in a rat thermal escape test (Hargreaves test), and shows robust antinociceptive activity in animal models of persistent (formalin test) and neuropathic (Chung model) pain. Compound 17 (20 mg/kg, iv) demonstrates activity in the formalin test that is comparable to morphine (3mg/kg, iv), and is dose-dependently inhibited by the CB1 antagonist SR141716A. In the Chung model, compound 17 shows antineuropathic effects similar to high-dose (100 mg/kg) gabapentin. FAAH inhibition shows potential utility for the clinical treatment of persistent and neuropathic pain.

Recent findings suggest that Alzheimer's dementia may be mediated by soluble beta amyloid (Abeta) more than the deposits of aggregated, insoluble Abeta, and vulnerability to cognitive deficits after scopolamine challenge may help identify AD even in patients that are still pre-symptomatic. The objectives of the present experiments were to determine if vulnerability to cognitive deficits after scopolamine challenge is related to levels of soluble Abeta, and if levels of soluble Abeta are more closely related to cognitive deficits than levels of insoluble Abeta, even in aged, transgenic mice, after they have developed very high levels of insoluble Abeta. Aged F-344 rats and young mice over-expressing the Swedish mutation in the human amyloid precursor protein (APPsw; Tg2576+) had elevated levels of soluble Abeta, and were more vulnerable to scopolamine challenge in the Morris water maze (MWM), relative to young rats and Tg2576- mice; but, among individual animals, higher levels of soluble Abeta were not correlated with vulnerability to scopolamine. On the other hand, in aged Tg2576+ mice, cognitive deficits were related to levels of soluble Abeta, not insoluble Abeta, despite the fact that the levels of insoluble Abeta were thousands of times higher than the levels of soluble Abeta. The results of the present experiments suggest that vulnerability to cognitive deficits after scopolamine challenge is not related to elevated levels of soluble Abeta, but that high levels of soluble Abeta are more closely correlated with cognitive deficits than the amount insoluble Abeta, even after large amounts of aggregated, insoluble Abeta have been deposited.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.