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1.
Cephalalgia ; 44(1): 3331024231226186, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38215228

ABSTRACT

BACKGROUND: The trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) is identified as an essential element in migraine pathogenesis. METHODS: In vitro and in vivo studies evaluated pharmacologic properties of the CGRP receptor antagonist atogepant. Radioligand binding using 125I-CGRP and cyclic adenosine monophosphate (cAMP) accumulation assays were conducted in human embryonic kidney 293 cells to assess affinity, functional potency and selectivity. Atogepant in vivo potency was assessed in the rat nitroglycerine model of facial allodynia and primate capsaicin-induced dermal vasodilation (CIDV) pharmacodynamic model. Cerebrospinal fluid/brain penetration and behavioral effects of chronic dosing and upon withdrawal were evaluated in rats. RESULTS: Atogepant exhibited high human CGRP receptor-binding affinity and potently inhibited human α-CGRP-stimulated cAMP responses. Atogepant exhibited significant affinity for the amylin1 receptor but lacked appreciable affinities for adrenomedullin, calcitonin and other known neurotransmitter receptor targets. Atogepant dose-dependently inhibited facial allodynia in the rat nitroglycerine model and produced significant CIDV inhibition in primates. Brain penetration and behavioral/physical signs during chronic dosing and abrupt withdrawal were minimal in rats. CONCLUSIONS: Atogepant is a competitive antagonist with high affinity, potency and selectivity for the human CGRP receptor. Atogepant demonstrated a potent, concentration-dependent exposure/efficacy relationship between atogepant plasma concentrations and inhibition of CGRP-dependent effects.


Subject(s)
Calcitonin Gene-Related Peptide , Piperidines , Pyridines , Pyrroles , Receptors, Calcitonin Gene-Related Peptide , Spiro Compounds , Humans , Rats , Animals , Calcitonin Gene-Related Peptide/metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide Receptor Antagonists/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists/therapeutic use , Hyperalgesia/drug therapy
2.
J Dermatol ; 50(10): 1321-1329, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37455419

ABSTRACT

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

3.
Bioorg Med Chem ; 63: 116743, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35436748

ABSTRACT

The voltage-gated sodium channel Nav1.7 is an attractive target for the treatment of pain based on the high level of target validation with genetic evidence linking Nav1.7 to pain in humans. Our effort to identify selective, CNS-penetrant Nav1.7 blockers with oral activity, improved selectivity, good drug-like properties, and safety led to the discovery of 2-substituted quinolines and quinolones as potent small molecule Nav1.7 blockers. The design of these molecules focused on maintaining potency at Nav1.7, improving selectivity over the hERG channel, and overcoming phospholipidosis observed with the initial leads. The structure-activity relationship (SAR) studies leading to the discovery of (R)-(3-fluoropyrrolidin-1-yl)(6-((5-(trifluoromethyl)pyridin-2-yl)oxy)quinolin-2-yl)methanone (ABBV-318) are described herein. ABBV-318 displayed robust in vivo efficacy in both inflammatory and neuropathic rodent models of pain. ABBV-318 also inhibited Nav1.8, another sodium channel isoform that is an active target for the development of new pain treatments.


Subject(s)
Pain , Sodium Channels , Humans , Pain/drug therapy , Pain Management , Protein Isoforms , Sodium Channels/metabolism , Structure-Activity Relationship
4.
Sci Rep ; 9(1): 17675, 2019 11 27.
Article in English | MEDLINE | ID: mdl-31776355

ABSTRACT

Foxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed RORγt and produced IL-17A. Genesis of this population was attenuated by a RORγt inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+RORγt+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+RORγt+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.


Subject(s)
Cell Plasticity/drug effects , Dermatitis/metabolism , Interleukin-23/pharmacology , Psoriasis/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Cells, Cultured , Dermatitis/pathology , Disease Models, Animal , Forkhead Transcription Factors/metabolism , Interleukin-17/metabolism , Interleukin-23/administration & dosage , Mice , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Psoriasis/chemically induced , Psoriasis/pathology , T-Lymphocytes, Regulatory/drug effects
5.
Sci Rep ; 9(1): 9089, 2019 06 24.
Article in English | MEDLINE | ID: mdl-31235749

ABSTRACT

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.


Subject(s)
Interleukin-1/antagonists & inhibitors , Psoriasis/drug therapy , Small Molecule Libraries/pharmacology , Animals , Gene Expression Regulation/drug effects , Humans , Mice , Psoriasis/metabolism , Psoriasis/pathology , Skin/drug effects , Skin/pathology , Small Molecule Libraries/therapeutic use
6.
J Dermatol ; 46(6): 482-497, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31062408

ABSTRACT

The interleukin (IL)-23/IL-17 axis plays a central role in the pathogenesis of psoriasis and is elevated in lesional psoriatic skin. Different murine models have been developed to mimic this pathophysiology each carrying specific merits and limitations. In an attempt to address some of these limitations, B10.RIII mice received a single hydrodynamic injection of IL-23 minicircles (MC) to induce hepatic transcription and the endogenous production of IL-23. Plasma and ear IL-23 levels were dose-dependently (0.3-3 µg) increased in MC injected mice and were sustained over the 14-day study duration. Beginning on day 7 post-injection, mice developed dose-related ear inflammation, histologically confirmed increases in epidermal and dermal area, as well as enhanced neutrophil and macrophage content. Flow cytometry demonstrated increased levels of granulocytes, T cells and monocytes/macrophages in the ear skin, with T cells identified as the main cellular source of IL-17A. Evaluation of mRNA and protein showed time-dependent, increased levels of the IL-23/IL-17 pathway and inflammatory/microbial cytokines/chemokines in the ear which differed kinetically from circulating levels. An anti-IL-23p40 antibody was assessed following both prophylactic administration and administration once the disease was established. Prophylactic dosing completely prevented the development of the ear phenotype across endpoints. Treatment administration showed a dose-related response, with a maximum inhibition of 64-94%, depending on endpoint. These data demonstrate that the IL-23 MC model is a useful approach to study IL-23/IL-17-driven skin inflammation and may facilitate preclinical assessment of novel therapies.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Interleukin-17/immunology , Interleukin-23/immunology , Psoriasis/immunology , Animals , DNA, Circular/administration & dosage , DNA, Circular/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Gene Transfer Techniques , Humans , Interleukin-17/metabolism , Interleukin-23/antagonists & inhibitors , Interleukin-23/genetics , Male , Mice , Psoriasis/blood , Psoriasis/drug therapy , Psoriasis/pathology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Skin/immunology , Skin/pathology , Treatment Outcome
7.
Sci Rep ; 9(1): 5310, 2019 03 29.
Article in English | MEDLINE | ID: mdl-30926837

ABSTRACT

Psoriasis is an immune-mediated inflammatory skin disease that affects millions worldwide. Studying immune cells involved in psoriasis pathogenesis is essential to identify effective and safe therapeutics for the disease. Using human psoriasis skin, activated macrophages were observed in both lesional and non-lesional skin, but were elevated in lesional skin. Activation of the IL-23/IL-17 pathway is integral to the development of psoriasis. To further characterize the monocyte/macrophage (Mon/Mac) population when the IL-23 pathway is activated, a murine model of intradermal injection of IL-23 was used. Flow cytometry revealed that Mon/Mac cells were the dominant immune population, particularly late in the model, highlighted by strong presence of Ly6ChiMHC IIhi cells. The Mon/Mac cells were also shown to have high expression for TNFα but not IL-17A. Prophylactic dosing of a CSF-1R inhibitor to deplete Mon/Mac cells significantly reduced several inflammatory mediators from the skin tissue suggesting a pathogenic role for Mon/Mac. Treatment dosing of the inhibitor produced a less robust effect. Mon/Mac cells were also differentiated by levels of Ki67 and TNFα expression. These data point to an important contribution of Mon/Mac cells in IL-23 related skin inflammation and suggest that these cells are a significant player in the underlying pathophysiology of psoriasis.


Subject(s)
Macrophages/immunology , Macrophages/metabolism , Monocytes/immunology , Monocytes/metabolism , Psoriasis/etiology , Psoriasis/metabolism , Biomarkers , Cytokines/metabolism , Dermatitis/etiology , Dermatitis/metabolism , Dermatitis/pathology , Disease Susceptibility , Humans , Immunohistochemistry , Interleukin-23/metabolism , Macrophage Activation/immunology , Psoriasis/pathology
8.
Exp Dermatol ; 28(2): 113-120, 2019 02.
Article in English | MEDLINE | ID: mdl-30417427

ABSTRACT

Psoriasis vulgaris (PV) results from activation of IL-23/Th17 immune pathway and is further amplified by cytokines/chemokines from skin cells. Among skin-derived pro-inflammatory cytokines, IL-36 family members are highly upregulated in PV patients and play a critical role in general pustular psoriasis. However, there is limited data showing crosstalk between the IL-23 and IL-36 pathways in PV. Herein, potential attenuation of skin inflammation in the IL-23-induced mouse model of psoriasiform dermatitis by functional inhibition of IL-36 receptor (IL-36R) was interrogated. Anti-mouse IL-36R monoclonal antibodies (mAbs) were generated and validated in vitro by inhibiting IL-36α-induced secretion of CXCL1 from NIH 3T3 cells. Antibody target engagement was demonstrated by inhibition of CXCL1 production in a novel acute model of IL-36α systemic injection in mice. In addition, anti-IL-36R mAbs inhibited tissue inflammation and inflammatory gene expression in an IL-36α ear injection model of psoriasiform dermatitis demonstrating engagement of the target in the ear skin. To elucidate the possible role of IL-36 signalling in IL-23/Th17 pathway, the ability of anti-IL-36R mAbs to inhibit skin inflammation in an IL-23 ear injection model was assessed. Inhibiting the IL-36 pathway resulted in significant attenuation of skin thickening and psoriasis-relevant gene expression. Taken together, these data suggest a role for IL-36 signalling in the IL-23/Th17 signalling axis in PV.


Subject(s)
Antibodies, Monoclonal/immunology , Dermatitis/immunology , Inflammation/immunology , Interleukins/immunology , Psoriasis/immunology , Receptors, Interleukin/antagonists & inhibitors , Animals , Antibodies, Monoclonal/therapeutic use , Chemokine CXCL1/metabolism , Cytokines/metabolism , Dermatitis/therapy , Disease Models, Animal , Female , Gene Expression Profiling , Inflammation/metabolism , Interleukin-1/immunology , Interleukin-23/pharmacology , Ligands , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Psoriasis/therapy , Rats , Rats, Sprague-Dawley , Receptors, Interleukin/immunology , Receptors, Interleukin-1/immunology , Signal Transduction , Skin/metabolism , Skin/pathology , Th17 Cells/cytology
9.
J Neurophysiol ; 120(4): 1893-1905, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30067136

ABSTRACT

This overview compares the activity of wide dynamic range (WDR) and nociceptive specific (NS) neurons located in the deep dorsal horn across different rat models of pathological pain and following modulation by diverse pharmacology. The data were collected by our group under the same experimental conditions over numerous studies to facilitate comparison. Spontaneous firing of WDR neurons was significantly elevated (>3.7 Hz) in models of neuropathic, inflammation, and osteoarthritic pain compared with naive animals (1.9 Hz) but was very low (<0.5 Hz) and remained unchanged in NS neurons. WDR responses to low-intensity mechanical stimulation were elevated in neuropathic and inflammation models. WDR responses to high-intensity stimuli were enhanced in inflammatory (heat) and osteoarthritis (mechanical) models. NS responses to high-intensity stimulation did not change relative to control in any model examined. Several therapeutic agents reduced both evoked and spontaneous firing of WDR neurons (e.g., TRPV1, TRPV3, Nav1.7, Nav1.8, P2X7, P2X3, H3), other targets affected neither evoked nor spontaneous firing of WDR neurons (e.g., H4, TRPM8, KCNQ2/3), and some only modulated evoked (e.g, ASIC1a, Cav3.2) whereas others decreased evoked but affected spontaneous activity only in specific models (e.g., TRPA1, CB2). Spontaneous firing of WDR neurons was not altered by any peripherally restricted compound or by direct administration of compounds to peripheral sites, although the same compounds decreased evoked activity. Compounds acting centrally were effective against this endpoint. The diversity of incoming/modulating inputs to the deep dorsal horn positions this group of neurons as an important intersection within the pain system to validate novel therapeutics. NEW & NOTEWORTHY Data from multiple individual experiments were combined to show firing properties of wide dynamic range and nociceptive specific spinal dorsal horn neurons across varied pathological pain models. This high-powered analysis describes the sensitization following different forms of injury. Effects of diverse pharmacology on these neurons is also summarized from published and unpublished data all recorded under the same conditions to facilitate comparison. This comprehensive overview describes the function and utility of these neurons.


Subject(s)
Evoked Potentials , Neuralgia/physiopathology , Posterior Horn Cells/physiology , Analgesics/pharmacology , Animals , Hot Temperature/adverse effects , Male , Membrane Transport Modulators/pharmacology , Neuralgia/etiology , Neuralgia/pathology , Nociception , Osteoarthritis/complications , Osteoarthritis/physiopathology , Posterior Horn Cells/classification , Posterior Horn Cells/drug effects , Rats , Rats, Sprague-Dawley
10.
J Pharmacol Toxicol Methods ; 94(Pt 1): 34-43, 2018.
Article in English | MEDLINE | ID: mdl-29684554

ABSTRACT

INTRODUCTION: The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS: Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS: Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION: Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.


Subject(s)
Adenine/administration & dosage , Fibrosis/chemically induced , Kidney Diseases/chemically induced , Kidney/drug effects , Administration, Oral , Animals , Biomarkers/urine , Fibrosis/metabolism , Fibrosis/urine , Glomerular Filtration Rate/drug effects , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/urine , Kidney/metabolism , Kidney Diseases/metabolism , Kidney Diseases/urine , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
11.
J Orthop Res ; 2018 Feb 11.
Article in English | MEDLINE | ID: mdl-29430715

ABSTRACT

Osteoarthritis (OA) is a degenerative form of arthritis that can result in loss of joint function and chronic pain. The pathological pain state that develops with OA disease involves plastic changes in the peripheral and central nervous systems, however, the cellular mechanisms underlying OA are not fully understood. We characterized the medial meniscal tear (MMT) surgical model and the intra-articular injection of monosodium iodoacetate (MIA) chemical model of OA in rats. Both models produced histological changes in the knee joint and associated bones consistent with OA pathology. Both models also increased p38 activation in the L3, but not L4 dorsal root ganglia (DRG), increased tyrosine hydroxylase immunostaining in the L3 DRG indicating sympathetic sprouting, and increased phosphorylated (p)CREB in thalamic neurons. In MIA-OA, but not MMT-OA rats, p38 and pERK were increased in the spinal cord, and pCREB was enhanced in the prefrontal cortex. Using in vivo electrophysiology, elevated spontaneous activity and increased responsiveness of wide dynamic range neurons to stimulation of the knee was found in both models. However, a more widespread sensitization was observed in the MIA-OA rats as neurons with paw receptive fields spontaneously fired at a greater rate in MIA-OA than MMT-OA rats. Taken together, the MIA and MMT models of OA share several common features associated with histopathology and sensitization of primary somatosensory pathways, but, observed differences between the models highlights unique consequences of the related specific injuries, and these differences should be considered when choosing an OA model and when interpreting data outcomes. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

12.
J Basic Clin Physiol Pharmacol ; 29(3): 281-290, 2018 Jun 27.
Article in English | MEDLINE | ID: mdl-29397387

ABSTRACT

BACKGROUND: Losartan, a blocker of the angiotensin II type I receptor, is an important part of the standard of care for diabetic nephropathy (DN). The obese ZSF-1 rats display many aspects of the clinical features of human Type II DN. The current study was designed to examine the treatment effects of losartan on obese ZSF-1 rats and to evaluate the impact of the onset of dosing on efficacy. METHODS: The rats (7-10 weeks) underwent a right uninephrectomy (Unx) or sham surgery. Losartan (3, 10, 30 mg/kg) was dosed 3 or 9 weeks post-Unx and continued for 12 weeks. RESULTS: Treatment with losartan reduced urinary protein excretion and blood lipids (triglyceride and cholesterol) dose-dependently in both studies. The glomerular filtration rate (GFR) was significantly lower in obese ZSF-1 rats compared with those in lean rats, and losartan was efficacious against this endpoint, in particular with the earlier onset of treatment. Losartan also decreased tubulointerstitial fibrosis, and similar to GFR, earlier treatment conferred beneficial actions even at the lowest dose of 3 mg/kg. Several urinary biomarkers were elevated in the obese ZSF-1 rats, but the levels of sTNFR1, TIMP-1, L-FABP and KIM-1 were the only markers decreased by losartan. CONCLUSIONS: Losartan was renoprotective in the ZSF-1 rats with DN, improving both the pathological and functional parameters of the disease. Importantly, the data also highlight the importance of treatment at earlier stages of the disease for protecting against decline in the GFR and the development of fibrosis.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Diabetic Nephropathies/prevention & control , Losartan/pharmacology , Obesity/drug therapy , Angiotensin II Type 1 Receptor Blockers/administration & dosage , Animals , Biomarkers/metabolism , Cholesterol/blood , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/drug therapy , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Glomerular Filtration Rate/drug effects , Losartan/administration & dosage , Male , Rats , Triglycerides/blood
13.
J Am Soc Nephrol ; 28(12): 3616-3626, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28827403

ABSTRACT

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Kidney Diseases/drug therapy , Kidney/drug effects , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Disease Models, Animal , Extracellular Matrix/metabolism , Fibronectins/chemistry , Fibrosis/drug therapy , Humans , Hybridomas/metabolism , Kidney/diagnostic imaging , Kidney/pathology , Male , Mice , Tomography, Emission-Computed, Single-Photon , Ureter/pathology
14.
J Neurophysiol ; 118(2): 904-916, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28468993

ABSTRACT

TRPV3 is a nonselective cation channel activated by temperatures above 33°C and is reported to be localized in keratinocytes and nervous tissue. To investigate a role for TRPV3 in pain modulation, we conducted a series of in vivo electrophysiological studies on spinal and brain nociceptive neurons. Structurally diverse TRPV3 receptor antagonists reduced responses of spinal wide dynamic range (WDR) neurons to low-intensity mechanical stimulation in neuropathic rats, but only CNS-penetrant antagonists decreased elevated spontaneous firing. Injections of an antagonist into the neuronal receptive field, into the L5 dorsal root ganglion, or intracerebroventricularly (ICV) attenuated the evoked firing, but only ICV injections reduced spontaneous activity. Intraspinal injections did not affect either. Spinal transection blocked the effect on spontaneous but not evoked firing after systemic delivery of a TRPV3 antagonist. Systemic administration of an antagonist to neuropathic rats also impacted the firing of On- and Off-cells in the rostral ventromedial medulla in a manner consistent with dampening nociceptive signaling. An assessment of nonevoked "pain," an EEG-measured pain-induced sleep disturbance induced by hind paw injections of CFA, was also improved with CNS-penetrant TRPV3 antagonists but not by an antagonist with poor CNS penetration. Antagonism of TRPV3 receptors modulates activity of key classes of neurons in the pain pathway in a manner consistent with limiting pathological nociceptive signaling and was mediated by receptors in the periphery and brain. Blockade of TRPV3 receptors is likely an effective means to alleviate mechanical allodynia and nonevoked pain. However, the latter will only be obtained by blocking supraspinal TRPV3 receptors.NEW & NOTEWORTHY Recent studies have linked TRPV3 to pain modulation, and much of this work has focused on its role in the skin-primary afferent interface. In this electrophysiological study, we demonstrate that receptor antagonists modulate evoked signals through peripheral mechanisms but blockade of supraspinal TRPV3 receptors contributes to dampening both evoked and nonevoked "pain" through descending modulation. Thus, the full therapeutic potential of TRPV3 antagonists may only be realized with the ability to access receptors in the brain.


Subject(s)
Brain/metabolism , Ganglia, Spinal/metabolism , Nociceptive Pain/metabolism , Nociceptors/metabolism , Spinal Cord/metabolism , TRPV Cation Channels/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Analgesics, Non-Narcotic/chemistry , Analgesics, Non-Narcotic/pharmacology , Animals , Brain/drug effects , Disease Models, Animal , Ganglia, Spinal/drug effects , Male , Membrane Transport Modulators/chemistry , Membrane Transport Modulators/pharmacology , Nociception/drug effects , Nociception/physiology , Nociceptive Pain/drug therapy , Nociceptors/drug effects , Rats, Sprague-Dawley , Sleep/drug effects , Sleep/physiology , Spinal Cord/drug effects , TRPV Cation Channels/antagonists & inhibitors , Tissue Culture Techniques
15.
Am J Nephrol ; 44(5): 339-353, 2016.
Article in English | MEDLINE | ID: mdl-27736813

ABSTRACT

BACKGROUND: Obese ZSF-1 rats display many features of human type II diabetes including nephropathy (DN). The study aimed to further understand the relevance of this model to DN, for which glomerular filtration rate (GFR), renal fibrosis and several urinary/tissue biomarkers was followed over 24 weeks in ZSF-1 rats. METHODS: Intact/sham or uninephrectomized male and female ZSF-1 rats were studied. GFR was measured by transdermal clearance of fluorescein isothiocyanate-sinistrin. Urine was collected every 2-4 weeks for biomarker analysis. Renal tissue was examined histologically for fibrosis and for levels of inflammatory and fibrotic genes. RESULTS: Male obese ZSF-1 rats demonstrated metabolic syndrome and proteinuria. Female counterparts were hyperlipidemic with delayed proteinuria, but were not hyperglycemic. Kidney hyperfiltration was observed in male obese rats in weeks 2-4 after surgery, and subsequently declined to levels significantly lower than controls. Tubulointerstitial/glomerular fibrosis in male obese rats was significantly elevated by week 12 post surgery and continued to expand in the ensuing weeks, particularly in uninephrectomized rats. Female rats had less severe fibrosis. Except for epidermal growth factor which decreased, the levels of several key inflammatory, injury and fibrotic factors were elevated in both tissue (mRNA) and urine (protein) of male obese rats. CONCLUSION: Male obese ZSF-1 rats represent an important DN model, manifesting key pathophysiological features including metabolic syndrome, proteinuria, progressive tubular and glomerular fibrosis, and transient hyperfiltration followed by progressive decline in renal function. Uninephrectomy significantly accelerated disease progression. Females were less severe in disease manifestation. Several urinary and tissue biomarkers were identified in the male obese rats that tracked with disease progression.


Subject(s)
Diabetic Nephropathies/physiopathology , Disease Models, Animal , Glomerular Filtration Rate , Kidney/pathology , Animals , Biomarkers/urine , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/complications , Diabetic Nephropathies/etiology , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Female , Fibrosis , Gene Expression Profiling , Male , Nephrosclerosis/etiology , Nephrosclerosis/pathology , Rats
16.
Eur J Pharmacol ; 788: 1-11, 2016 Oct 05.
Article in English | MEDLINE | ID: mdl-27288879

ABSTRACT

Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on "quality of life" measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Basement Membrane/diagnostic imaging , Cytoprotection/drug effects , Kidney/cytology , Kidney/injuries , Podocytes/drug effects , Protein Kinase Inhibitors/pharmacology , Animals , Fibrosis , Kidney/drug effects , Kidney/pathology , Male , Mice , Podocytes/cytology , Podocytes/metabolism , Puromycin Aminonucleoside/toxicity , Rats
17.
J Med Chem ; 59(10): 4926-47, 2016 05 26.
Article in English | MEDLINE | ID: mdl-27077528

ABSTRACT

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.


Subject(s)
Cyclobutanes/chemical synthesis , Cyclobutanes/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , TRPV Cation Channels/antagonists & inhibitors , Calcium/metabolism , Cyclobutanes/chemistry , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Molecular Conformation , Pyridines/chemistry , Structure-Activity Relationship , TRPV Cation Channels/metabolism
18.
Eur J Pharmacol ; 745: 234-42, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25446431

ABSTRACT

There is strong pharmacological, biological, and genetic evidence supporting the role of N-type calcium channels (CaV2.2) in nociception. There is also human validation data from ziconotide, the CaV2.2-selective peptidyl inhibitor used clinically to treat refractory pain. Unfortunately, ziconotide utility is limited by its narrow therapeutic window and required intrathecal route of administration. A major focus has been placed on identifying state-dependent CaV2.2 inhibitors to improve safety margins. Much less attention, however, has been given to characterizing the kinetics of CaV2.2 inhibitors as a means to further differentiate compounds and maximize therapeutic potential. Here we provide a detailed characterization of the CaV2.2 inhibitor T4 in terms of its state-dependence, use-dependence, kinetics, and mechanism of inhibition. Compound T4 displayed a >20-fold difference in potency when measured under inactivating conditions (IC50=1.1 µM) as compared to closed-state conditions (IC50=25 µM). At 3 µM, T4 produced a 15-fold hyperpolarizing shift in the inactivation curve for CaV2.2 while having no effect on channel activation. To assess the kinetic properties of T4 in a more physiological manner, its inhibition kinetics were assessed at 32°C using 2 mM Ca(2+) as the charge carrier. Surprisingly, the repriming rate for CaV2.2 channels at hyperpolarized potentials was similar in both the presence and absence of T4. This was in contrast to other compounds which markedly delayed repriming. Furthermore, T4 inhibited CaV2.2 channels more potently when channel inactivation was driven through a tonic sub-threshold depolarization rather than through a use-dependent protocol, despite similar levels of inactivation.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type/drug effects , Calcium Channels, N-Type/metabolism , Triazines/pharmacology , Animals , Cell Line , Humans , Kinetics , Patch-Clamp Techniques , Rats , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism
19.
Biochem Pharmacol ; 89(4): 536-44, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24726441

ABSTRACT

Activation of T-type Ca²âº channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²âº channels in chronic pain is supported by gene knockdown studies showing that decreased Ca(v)3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²âº channel blocker. ABT-639 blocks recombinant human T-type (Ca(v)3.2) Ca²âº channels in a voltage-dependent fashion (IC50 = 2 µM) and attenuates LVA currents in rat DRG neurons (IC50 = 8 µM). ABT-639 was significantly less active at other Ca²âº channels (e.g. Ca(v)1.2 and Ca(v)2.2) (IC50 > 30 µM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED50 = 2 mg/kg, p.o.). ABT-639 (10-100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced). [corrected]. ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100-300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca(v)3.2) channels in chronic pain states.


Subject(s)
Calcium Channel Blockers/therapeutic use , Calcium Channels, T-Type/metabolism , Disease Models, Animal , Heterocyclic Compounds, 2-Ring/therapeutic use , Nerve Tissue Proteins/antagonists & inhibitors , Neuralgia/drug therapy , Nociceptive Pain/drug therapy , Peripheral Nerves/drug effects , Sulfonamides/therapeutic use , Animals , Behavior, Animal/drug effects , Biological Availability , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Calcium Channel Blockers/adverse effects , Calcium Channel Blockers/pharmacokinetics , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/chemistry , Calcium Channels, T-Type/genetics , Cells, Cultured , Chronic Pain/drug therapy , Chronic Pain/metabolism , Dose-Response Relationship, Drug , Heterocyclic Compounds, 2-Ring/adverse effects , Heterocyclic Compounds, 2-Ring/pharmacokinetics , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuralgia/metabolism , Nociceptive Pain/metabolism , Peripheral Nerves/cytology , Peripheral Nerves/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sulfonamides/adverse effects , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology
20.
J Pain ; 15(4): 387.e1-14, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24374196

ABSTRACT

UNLABELLED: Voltage-gated Ca(2+) channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca(2+) channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca(2+) channels with similar potency (IC50 = 1-2 µM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene-related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant-induced inflammatory, and chronic constrictive injury of sciatic nerve-induced, neuropathic pain models with ED50 values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval = 2.2-3.5, 3.7-10, and 5.5-12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal-regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant-inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations. PERSPECTIVE: The present results demonstrate that the neuronal Ca(2+) channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.


Subject(s)
Analgesics/pharmacology , Azabicyclo Compounds/pharmacology , Calcium Channel Blockers/pharmacology , Leucine/analogs & derivatives , Neurons/metabolism , Nociception/drug effects , Spinal Cord/drug effects , Animals , Disease Models, Animal , Immunohistochemistry , Leucine/pharmacology , Male , Neurons/drug effects , Pain/metabolism , Patch-Clamp Techniques , Rats, Sprague-Dawley , Spinal Cord/metabolism
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