Lysophosphatidic Acid Receptor 1 Antagonist Research Articles

Lysophosphatidic Acid Receptor 1 Plays a Pathogenic Role in Permanent Brain Ischemic Stroke by Modulating Neuroinflammatory Responses.

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in brain injury following a transient brain ischemic stroke. However, its role in permanent brain ischemic stroke remains unknown. To address this, we investigated whether LPA1 could contribute to brain injury of mice challenged by permanent middle cerebral artery occlusion (pMCAO). A selective LPA1 antagonist (AM152) was used as a pharmacological tool for this investigation. When AM152 was given to pMCAO-challenged mice one hour after occlusion, pMCAO-induced brain damage such as brain infarction, functional neurological deficits, apoptosis, and blood-brain barrier disruption was significantly attenuated. Histological analyses demonstrated that AM152 administration attenuated microglial activation and proliferation in injured brain after pMCAO challenge. AM152 administration also attenuated abnormal neuroinflammatory responses by decreasing expression levels of pro-inflammatory cytokines while increasing expression levels of anti-inflammatory cytokines in the injured brain. As underlying effector pathways, NF-κB, MAPKs (ERK1/2, p38, and JNKs), and PI3K/Akt were found to be involved in LPA1-dependent pathogenesis. Collectively, these results demonstrate that LPA1 can contribute to brain injury by permanent ischemic stroke, along with relevant pathogenic events in an injured brain.

Discovery of a Novel Orally Active, Selective LPA Receptor Type 1 Antagonist, 4-(4-(2-Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin-1-yl)-4-oxobutanoic Acid, with a Distinct Molecular Scaffold.

Lysophosphatidic acid receptor 1 (LPAR1) antagonists show promise as potentially novel antifibrotic treatments. In a human LPAR1 β-arrestin recruitment-based high-throughput screening campaign, we identified urea 19 as a hit with a LPAR1 IC50 value of 5.0 μM. Hit-to-lead activities revealed that one of the urea nitrogen atoms can be replaced by carbon and establish the corresponding phenylacetic amide as a lead structure for further optimization. Medicinal chemistry efforts led to the discovery of piperidine 18 as a potent and selective LPAR1 antagonist with oral activity in a mouse model of LPA-induced skin vascular leakage. The molecular scaffold of 18 shares no obvious structural similarity with any other LPAR1 antagonist disclosed so far.

Fibromyalgia Animal Models Using Intermittent Cold and Psychological Stress.

Fibromyalgia (FM) is a chronic pain condition characterized by widespread musculoskeletal pain and other frequent symptoms such as fatigue, sleep disturbance, cognitive impairment, and mood disorder. Based on the view that intermittent stress would be the most probable etiology for FM, intermittent cold- and intermittent psychological stress-induced generalized pain (ICGP and IPGP) models in mice have been developed and validated as FM-like pain models in terms of the patho-physiological and pharmacotherapeutic features that are shared with clinical versions. Both models show long-lasting and generalized pain and female-predominant sex differences after gonadectomy. Like many other neuropathic pain models, ICGP and IPGP were abolished in lysophosphatidic acid receptor 1 (LPAR1) knock-out mice or by LPAR1 antagonist treatments, although deciding the clinical importance of this mechanism depends on waiting for the development of a clinically available LPAR1 antagonist. On the other hand, the nonsteroidal anti-inflammatory drug diclofenac with morphine did not suppress hyperalgesia in these models, and this is consistent with the clinical findings. Pharmacological studies suggest that the lack of morphine analgesia is associated with opioid tolerance upon the stress-induced release of endorphins and subsequent counterbalance through anti-opioid NMDA receptor mechanisms. Regarding pharmacotherapy, hyperalgesia in both models was suppressed by pregabalin and duloxetine, which have been approved for FM treatment in clinic. Notably, repeated treatments with mirtazapine, an α2 adrenergic receptor antagonist-type antidepressant, and donepezil, a drug for treating Alzheimer's disease, showed potent therapeutic actions in these models. However, the pharmacotherapeutic treatment should be carried out 3 months after stress, which is stated in the FM guideline, and many preclinical studies, such as those analyzing molecular and cellular mechanisms, as well as additional evidence using different animal models, are required. Thus, the ICGP and IPGP models have the potential to help discover and characterize new therapeutic medicines that might be used for the radical treatment of FM, although there are several limitations to be overcome.

Integration of an LPAR1 Antagonist into Liposomes Enhances Their Internalization and Tumor Accumulation in an Animal Model of Human Metastatic Breast Cancer.

Lysophosphatidic acid receptor 1 (LPAR1) is elevated in breast cancer. The deregulation of LPAR1, including the function and level of expression, is linked to cancer initiation, progression, and metastasis. LPAR1 antagonists, AM095 or Ki16425, may be effective therapeutic molecules, yet their limited water solubility hinders in vivo delivery. In this study, we report on the synthesis of two liposomal formulations incorporating AM095 or Ki16425, embedded within the lipid bilayer, as targeted nanocarriers for metastatic breast cancer (MBC). The data show that the Ki16425 liposomal formulation exhibited a 50% increase in internalization by MBC mouse epithelial cells (4T1) and a 100% increase in tumor accumulation in a mouse model of MBC compared with that of a blank liposomal formulation (control). At the same time, normal mouse epithelial cells (EpH-4Ev) internalized the Ki16425 liposomal formulation 25% lesser than the control formulation. Molecular dynamics simulations show that the integration of AM095 or Ki16425 modified the physical and mechanical properties of the lipid bilayer, making it more flexible in these liposomal formulations compared with liposomes without drug. The incorporation of an LPAR1 antagonist within a liposomal drug delivery system represents a viable therapeutic approach for targeting the LPA-LPAR1 axis, which may hinder the progression of MBC.

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice

BackgroudThe frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks.MethodsICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses.ResultsAfter ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT).ConclusionsThis study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS’s neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

LPA1 antagonist BMS-986020 changes collagen dynamics and exerts antifibrotic effects in vitro and in patients with idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease with limited treatment options. A phase 2 trial (NCT01766817) showed that twice-daily treatment with BMS-986020, a lysophosphatidic acid receptor 1 (LPA1) antagonist, significantly decreased the slope of forced vital capacity (FVC) decline over 26 weeks compared with placebo in patients with IPF. This analysis aimed to better understand the impact of LPA1 antagonism on extracellular matrix (ECM)-neoepitope biomarkers and lung function through a post hoc analysis of the phase 2 study, along with an in vitro fibrogenesis model.MethodsSerum levels of nine ECM-neoepitope biomarkers were measured in patients with IPF. The association of biomarkers with baseline and change from baseline FVC and quantitative lung fibrosis as measured with high-resolution computed tomography, and differences between treatment arms using linear mixed models, were assessed. The Scar-in-a-Jar in vitro fibrogenesis model was used to further elucidate the antifibrotic mechanism of BMS-986020.ResultsIn 140 patients with IPF, baseline ECM-neoepitope biomarker levels did not predict FVC progression but was significantly correlated with baseline FVC and lung fibrosis measurements. Most serum ECM-neoepitope biomarker levels were significantly reduced following BMS-986020 treatment compared with placebo, and several of the reductions correlated with FVC and/or lung fibrosis improvement. In the Scar-in-a-Jar in vitro model, BMS-986020 potently inhibited LPA1-induced fibrogenesis.ConclusionsBMS-986020 reduced serum ECM-neoepitope biomarkers, which were previously associated with IPF prognosis. In vitro, LPA promoted fibrogenesis, which was LPA1 dependent and inhibited by BMS-986020. Together these data elucidate a novel antifibrotic mechanism of action for pharmacological LPA1 blockade.Trial registration ClinicalTrials.gov identifier: NCT01766817; First posted: January 11, 2013; https://clinicaltrials.gov/ct2/show/NCT01766817.

Mechanism of hepatobiliary toxicity of the LPA1 antagonist BMS-986020 developed to treat idiopathic pulmonary fibrosis: Contrasts with BMS-986234 and BMS-986278

In a Phase 2 clinical trial, BMS-986020, a lysophosphatidic acid receptor-1 (LPA1) antagonist, produced hepatobiliary toxicity (increased ALT, AST, and ALP; cholecystitis) and increases in plasma bile acids (BA). Nonclinical investigations conducted to identify a potential mechanism(s) for this toxicity examined BMS-986020 and two LPA1 antagonists structurally distinct from BMS-986020 (BMS-986234 and BMS-986278). BMS-986020 inhibited hepatic BA efflux transporters BSEP (IC50 1.8 μM), MRP3 (IC50 22 μM), and MRP4 (IC50 6.2 μM) and inhibited BA canalicular efflux in human hepatocytes (68% at 10 μM). BMS-986020 inhibited mitochondrial function (basal and maximal respiration, ATP production, and spare capacity) in human hepatocytes and cholangiocytes at ≥10 μM and inhibited phospholipid efflux in human hepatocytes (MDR3 IC50 7.5 μM). A quantitative systems toxicology analysis (DILIsym®), considering pharmacokinetics, BA homeostasis, mitochondrial function, oxidative phosphorylation, and reactive intermediates performed for BMS-986020 recapitulated clinical findings ascribing the effects to BA transporter and mitochondrial electron transport chain inhibition. BMS-986234 and BMS-986278 minimally inhibited hepatic BA transporters (IC50 ≥20 μM) and did not inhibit MDR3 activity (IC50 >100 μM), nor did BMS-986234 inhibit BA efflux (≤50 μM) or mitochondrial function (≤30 μM) (BMS-986278 not evaluated). Multiple mechanisms may be involved in the clinical toxicity observed with BMS-986020. The data indicate that this toxicity was unrelated to LPA1 antagonism since the mechanisms that likely influenced the adverse clinical outcome of BMS-986020 were not observed with equipotent LPA1 antagonists BMS-986234 and BMS-986278. This conclusion is consistent with the lack of hepatobiliary toxicity in nonclinical and clinical safety studies with BMS-986278.

Phase 2 trial design of BMS-986278, a lysophosphatidic acid receptor 1 (LPA1) antagonist, in patients with idiopathic pulmonary fibrosis (IPF) or progressive fibrotic interstitial lung disease (PF-ILD)

IntroductionIdiopathic pulmonary fibrosis (IPF) and non-IPF, progressive fibrotic interstitial lung diseases (PF-ILD), are associated with a progressive loss of lung function and a poor prognosis. Treatment with antifibrotic agents can...

Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).

Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis

Osteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA–LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

BMS-986020, a Specific LPA1 Antagonist, Provides Neuroprotection against Ischemic Stroke in Mice.

Stroke is a leading cause of death. Stroke survivors often suffer from long-term functional disability. This study demonstrated neuroprotective effects of BMS-986020 (BMS), a selective lysophosphatidic acid receptor 1 (LPA1) antagonist under clinical trials for lung fibrosis and psoriasis, against both acute and sub-acute injuries after ischemic stroke by employing a mouse model with transient middle cerebral artery occlusion (tMCAO). BMS administration immediately after reperfusion significantly attenuated acute brain injuries including brain infarction, neurological deficits, and cell apoptosis at day 1 after tMCAO. Neuroprotective effects of BMS were preserved even when administered at 3 h after reperfusion. Neuroprotection by BMS against acute injuries was associated with attenuation of microglial activation and lipid peroxidation in post-ischemic brains. Notably, repeated BMS administration daily for 14 days after tMCAO exerted long-term neuroprotection in tMCAO-challenged mice, as evidenced by significantly attenuated neurological deficits and improved survival rate. It also attenuated brain tissue loss and cell apoptosis in post-ischemic brains. Mechanistically, it significantly enhanced neurogenesis and angiogenesis in injured brains. A single administration of BMS provided similar long-term neuroprotection except survival rate. Collectively, BMS provided neuroprotection against both acute and sub-acute injuries of ischemic stroke, indicating that BMS might be an appealing therapeutic agent to treat ischemic stroke.

Inhibition of lysophosphatidic acid receptor 1 attenuates neuroinflammation via PGE2/EP2/NOX2 signalling and improves the outcome of intracerebral haemorrhage in mice

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in proinflammatory processes in the central nervous system by modulating microglia activation. The aim of this study was to explore the anti-inflammatory effects and neurological function improvement of LPA1 inhibition after intracerebral haemorrhage (ICH) in mice and to determine whether prostaglandin E2 (PGE2), E-type prostaglandin receptor 2 (EP2), and NADPH oxidase 2 (NOX2) signalling are involved in LPA1-mediated neuroinflammation. ICH was induced in CD1 mice by autologous whole blood injection. AM966, a selective LPA1 antagonist, was administered by oral gavage 1 h and 12 h after ICH. The LPA1 endogenous ligand, LPA was administered to verify the effect of LPA1 activation. To elucidate potential inflammatory mechanisms of LPA1, the selective EP2 activator butaprost was administered by intracerebroventricular injection with either AM966 or LPA1 CRISPR knockout (KO). Water content of the brain, neurobehavior, immunofluorescence staining, and western blot were performed. After ICH, EP2 was expressed in microglia whereas LPA1 was expressed in microglia, neurons, and astrocytes, which peaked after 24 h. AM966 inhibition of LPA1 improved neurologic function, reduced brain oedema, and suppressed perihematomal inflammatory cells after ICH. LPA administration aggravated neurological deficits after ICH. AM966 treatment and LPA1 CRISPR KO both decreased the expressions of PGE2, EP2, NOX2, NF-κB, TNF-α, IL-6, and IL-1β expressions after ICH, which was reversed by butaprost. This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signalling pathway in mice, which consequently improved neurobehavioral functions and alleviated brain oedema. LPA1 may be a promising therapeutic target to attenuate ICH-induced secondary brain injury.

Lysophosphatidic acid receptor 1 (LPA1) plays critical roles in microglial activation and brain damage after transient focal cerebral ischemia

BackgroundLysophosphatidic acid receptor 1 (LPA1) is in the spotlight because its synthetic antagonist has been under clinical trials for lung fibrosis and psoriasis. Targeting LPA1 might also be a therapeutic strategy for cerebral ischemia because LPA1 triggers microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed this possibility using a mouse model of transient middle cerebral artery occlusion (tMCAO).MethodsTo address the role of LPA1 in the ischemic brain damage, we used AM095, a selective LPA1 antagonist, as a pharmacological tool and lentivirus bearing a specific LPA1 shRNA as a genetic tool. Brain injury after tMCAO challenge was accessed by determining brain infarction and neurological deficit score. Role of LPA1 in tMCAO-induced microglial activation was ascertained by immunohistochemical analysis. Proinflammatory responses in the ischemic brain were determined by qRT-PCR and immunohistochemical analyses, which were validated in vitro using mouse primary microglia. Activation of MAPKs and PI3K/Akt was determined by Western blot analysis.ResultsAM095 administration immediately after reperfusion attenuated brain damage such as brain infarction and neurological deficit at 1 day after tMCAO, which was reaffirmed by LPA1 shRNA lentivirus. AM095 administration also attenuated brain infarction and neurological deficit at 3 days after tMCAO. LPA1 antagonism attenuated microglial activation; it reduced numbers and soma size of activated microglia, reversed their morphology into less toxic one, and reduced microglial proliferation. Additionally, LPA1 antagonism reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-κB activation, demonstrating its regulatory role of proinflammatory responses in the ischemic brain. Particularly, these LPA1-driven proinflammatory responses appeared to occur in activated microglia because NF-κB activation occurred mainly in activated microglia in the ischemic brain. Regulatory role of LPA1 in proinflammatory responses of microglia was further supported by in vitro findings using lipopolysaccharide-stimulated cultured microglia, showing that suppressing LPA1 activity reduced mRNA expression levels of proinflammatory cytokines. In the ischemic brain, LPA1 influenced PI3K/Akt and MAPKs; suppressing LPA1 activity decreased MAPK activation and increased Akt phosphorylation.ConclusionThis study demonstrates that LPA1 is a new etiological factor for cerebral ischemia, strongly indicating that its modulation can be a potential strategy to reduce ischemic brain damage.

Lysophosphatidic acid and its receptor LPA1 mediate carrageenan induced inflammatory pain in mice

Lysophosphatidic acid receptor 1 (LPA1) is one of six G protein-coupled receptors (GPCRs) activated by the bioactive lipid, lysophosphatidic acid (LPA). Previous studies have shown that LPA1 signaling plays a major role in the pathophysiology of neuropathic pain. It has also been shown that the inhibition of phospholipase A2, an enzyme upstream of LPA synthesis, reduces mechanical allodynia in experimental inflammatory orofacial pain. This suggests that the LPA-LPA1 axis may mediate inflammatory pain in addition to its known role in neuropathic pain, but this activity has not been reported. LPA1 signaling was disrupted in mice with both genetic and pharmacological approaches. Mice were then evaluated for behavioral and molecular characteristics of allodynia in a model for inflammatory orofacial pain. Pain behavior was significantly attenuated in LPA1 knockout mice relative to wild-type littermate controls. A similar significant attenuation in allodynia was observed when mice were treated with an LPA1 antagonist, AM095, following validation of its potency and selectivity. This was accompanied by a marked reduction in phosphorylated cAMP response element-binding protein (pCREB) labelling in the cerebral cortex. Interestingly, the reduction in allodynia was observed with central, but not systemic drug administration. Taken together, our findings indicate that LPA1 signaling in the central nervous system (CNS) plays a key role in mediating orofacial inflammatory pain, identifying LPA1 as a potential therapeutic target for treating inflammatory pain with a brain-penetrant drug.

Randomized, Double-Blind, Placebo-Controlled, Phase 2 Trial of BMS-986020, a Lysophosphatidic Acid Receptor Antagonist for the Treatment of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) causes irreversible loss of lung function. The lysophosphatidic acid receptor 1 (LPA1) pathway is implicated in IPF etiology. Safety and efficacy of BMS-986020, a high-affinity LPA1 antagonist, was assessed vsplacebo in a phase 2 study in patients with IPF. IM136003 was a phase 2, parallel-arm, multicenter, randomized, double-blind, placebo-controlled trial. Adults with IPF (FVC, 45%-90%; diffusing capacity for carbon monoxide, 30%-80%) were randomized to receive placebo or 600mg BMS-986020 (once daily [qd] or bid) for 26weeks. The primary end point was rate of change in FVC from baseline to week26. Of 143 randomized patients, 108 completed the 26-week dosing phase. Thirty-five patients discontinued prematurely. Patient baseline characteristics were similar between treatment groups (placebo: n= 47; 600mg qd: n= 48; 600mg bid: n= 48). Patients treated with BMS-986020 bid experienced a significantly slower rate of decline in FVC vsplacebo (-0.042 L; 95%CI,-0.106 to-0.022 vs-0.134 L; 95%CI,-0.201 to-0.068, respectively; P= .049). Dose-related elevations in hepatic enzymes were observed in both BMS-986020 treatment groups. The study was terminated early because of three cases of cholecystitis that were determined to be related to BMS-986020 after unblinding. BMS-986020 600mg bid treatment for 26weeks vsplacebo significantly slowed the rate of FVC decline. Both regimens of BMS-986020 were associated with elevations in hepatic enzymes. ClinicalTrials.gov; No.: NCT01766817; URL: www.clinicaltrials.gov.

Lysophosphatidic acid LPA1 and LPA3 receptors play roles in the maintenance of late tissue plasminogen activator-induced central poststroke pain in mice

We developed a mouse model for central post-stroke pain (CPSP), a centrally-originated neuropathic pain (NeuP). In this mode, mice were first injected with Rose Bengal, followed by photo-irradiation of left middle cerebral artery (MCA) to generate thrombosis. Although the MCA thrombosis was soon dissolved, the reduced blood flow remained for more than 24 h due to subsequent occlusion of microvessels. This photochemically induced thrombosis (PIT) model showed a hypersensitivity to the electrical stimulation of both sides of paw, but did not show any abnormal pain in popular thermal or mechanical nociception tests. When tissue-type plasminogen activator (tPA) was injected 6 h after the PIT stress, tPA-dependent hypersensitivity to the electrical paw stimulation and stable thermal and mechanical hyperalgesia on both sides for more than 17 or 18 days after the PIT treatment. These hyperalgesic effects were abolished in lysophosphatidic acid receptor 1 (LPA1)- and lysophosphatidic acid receptor 3 (LPA3)-deficient mice. When Ki-16425, an LPA1 and LPA3 antagonist was treated twice daily for 6 days consecutively, the thermal and mechanical hyperalgesia at day 17 and 18 were significantly reversed. The liquid chromatography–mass spectrometry (LC–MS/MS) analysis revealed that there is a significant increase in several species of LPA molecules in somatosensory S-I and medial dorsal thalamus (MD), but not in striatum or ventroposterior thalamus. All these results suggest that LPA1 and LPA3 signaling play key roles in the development and maintenance of CPSP.

Discovery of a Slow Tight Binding LPA1 Antagonist (ONO-0300302) for the Treatment of Benign Prostatic Hyperplasia.

Scaffold hopping from the amide group of lead compound ONO-7300243 (1) to a secondary alcohol successfully gave a novel chemotype lysophosphatidic acid receptor 1 (LPA1) antagonist 4. Wash-out experiments using rat isolated urethra showed that compound 4 possesses a tight binding feature to the LPA1 receptor. Further modification of two phenyl groups of 1 to pyrrole and an indane moiety afforded an optimized compound ONO-0300302 (19). Despite its high i.v. clearance, 19 inhibited significantly an LPA-induced increase of intraurethral pressure (IUP) in rat (3 mg/kg, p.o.) and dog (1 mg/kg, p.o.) over 12 h. Binding experiments with [3H]-ONO-0300302 suggest that the observed long duration action is because of the slow tight binding character of 19.

LPA1 receptor involvement in fibromyalgia-like pain induced by intermittent psychological stress, empathy

Treatment for fibromyalgia is an unmet medical need and its pathogenesis is still poorly understood. The present study demonstrated that intermittent psychological stress (IPS), or empathy causes generalized chronic abnormal pain with female predominance. The persistence of the pain phenotype was dependent on the unpredictability of the stressor. The pain was reversed by pregabalin (PGB), duloxetine (DLX) or mirtazapine (Mir), but not by diclofenac or morphine. Differential administration of these existing medicines revealed that the sites of PGB and Mir actions exist in the brain, but not in the spinal cord, while that of DLX is preferentially in the spinal cord. It is interesting to note that the intracerebroventricular injection of PGB or Mir showed potent analgesia for 24h or longer, though systemic injection of these medicines shows anti-hyperalgesia just for several hours. These results indicate that initial intense actions in the target brain may prevent the forthcoming development of pain memory. IPS-induced abnormal pain was prevented in mice deficient of lysophosphatidic acid receptor 1 (LPA1) gene, and completely cured by the repeated intrathecal treatments with LPA1 antagonist, AM966, which did not show acute action. All these results suggest that IPS model is an experimental animal model, which mimics the pathophysiology and pharmacotherapy in fibromyalgia in clinic, and LPA1 signaling plays crucial roles in the IPS-induced fibromyalgia-like abnormal pain.

AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin.

Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin.

An Lysophosphatidic Acid Receptors 1 and 3 Axis Governs Cellular Senescence of Mesenchymal Stromal Cells and Promotes Growth and Vascularization of Multiple Myeloma.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells and there is much interest in how MSCs contribute to the regulation of the tumor microenvironment. Whether MSCs exert a supportive or suppressive effect on tumor progression is still controversial, but is likely dependent on a variety of factors that are tumor-type dependent. Multiple myeloma (MM) is characterized by growth of malignant plasma cells in the bone marrow. It has been shown that the progression of MM is governed by MSCs, which act as a stroma of the myeloma cells. Although stroma is created via mutual communication between myeloma cells and MSCs, the mechanism is poorly understood. Here we explored the role of lysophosphatidic acid (LPA) signaling in cellular events where MSCs were converted into either MM-supportive or MM-suppressive stroma. We found that myeloma cells stimulate MSCs to produce autotaxin, an indispensable enzyme for the biosynthesis of LPA, and LPA receptor 1 (LPA1) and 3 (LPA3) transduce opposite signals to MSCs to determine the fate of MSCs. LPA3-silenced MSCs (siLPA3-MSCs) exhibited cellular senescence-related phenotypes in vitro, and significantly promoted progression of MM and tumor-related angiogenesis in vivo. In contrast, siLPA1-MSCs showed resistance to cellular senescence in vitro, and efficiently delayed progression of MM and tumor-related angiogenesis in vivo. Consistently, anti-MM effects obtained by LPA1-silencing in MSCs were completely reproduced by systemic administration of Ki6425, an LPA1 antagonist. Collectively, our results indicate that LPA signaling determines the fate of MSCs and has potential as a therapeutic target in MM. Stem Cells 2017;35:739-753.