Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1ß, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1ß, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1ß and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

Activation of HCA2 regulates microglial responses to alleviate neurodegeneration in LPS-induced in vivo and in vitro models.

BACKGROUND: Previous studies have shown a close association between an altered immune system and Parkinson's disease (PD). Neuroinflammation inhibition may be an effective measure to prevent PD. Recently, numerous reports have highlighted the potential of hydroxy-carboxylic acid receptor 2 (HCA2) in inflammation-related diseases. Notably, the role of HCA2 in neurodegenerative diseases is also becoming more widely known. However, its role and exact mechanism in PD remain to be investigated. Nicotinic acid (NA) is one of the crucial ligands of HCA2, activating it. Based on such findings, this study aimed to examine the effect of HCA2 on neuroinflammation and the role of NA-activated HCA2 in PD and its underlying mechanisms. METHODS: For in vivo studies, 10-week-old male C57BL/6 and HCA2-/- mice were injected with LPS in the substantia nigra (SN) to construct a PD model. The motor behavior of mice was detected using open field, pole-climbing and rotor experiment. The damage to the mice's dopaminergic neurons was detected using immunohistochemical staining and western blotting methods. In vitro, inflammatory mediators (IL-6, TNF-α, iNOS and COX-2) and anti-inflammatory factors (Arg-1, Ym-1, CD206 and IL-10) were detected using RT-PCR, ELISA and immunofluorescence. Inflammatory pathways (AKT, PPARγ and NF-κB) were delineated by RT-PCR and western blotting. Neuronal damage was detected using CCK8, LDH, and flow cytometry assays. RESULTS: HCA2-/- increases mice susceptibility to dopaminergic neuronal injury, motor deficits, and inflammatory responses. Mechanistically, HCA2 activation in microglia promotes anti-inflammatory microglia and inhibits pro-inflammatory microglia by activating AKT/PPARγ and inhibiting NF-κB signaling pathways. Further, HCA2 activation in microglia attenuates microglial activation-mediated neuronal injury. Moreover, nicotinic acid (NA), a specific agonist of HCA2, alleviated dopaminergic neuronal injury and motor deficits in PD mice by activating HCA2 in microglia in vivo. CONCLUSIONS: Niacin receptor HCA2 modulates microglial phenotype to inhibit neurodegeneration in LPS-induced in vivo and in vitro models.

Do dimethyl fumarate and nicotinic acid elicit common, potentially HCA2 -mediated adverse reactions? A combined epidemiological-experimental approach.

AIM: Dimethyl fumarate and nicotinic acid activate the hydroxy-carboxylic acid receptor 2 (HCA2 ) and induce flushing. It is not known whether HCA2 mediates other adverse drug reactions (ADRs) to these two substances. This study aims to compare ADRs associated with dimethyl fumarate and nicotinic acid, and to discuss whether they are HCA2 -mediated. METHODS: We identified spontaneous reports of suspected ADRs to dimethyl fumarate and nicotinic acid in the European Adverse Drug Reaction Database (EudraVigilance). These reports were analysed at different hierarchical levels of the Medical Dictionary for Regulatory Activities (MedDRA). In addition, we screened murine organs for HCA2 expression. RESULTS: Similarities in the ADR profile of dimethyl fumarate and nicotinic acid included "gastrointestinal signs and symptoms" (odds ratio [OR] 0.8 [0.6-1.1]), "hepatobiliary investigations" (OR 1.3 [0.7-2.5]) and "anxiety disorders and symptoms" (OR 0.9 [0.3-2.2]) in High Level Group Terms; "diarrhoea (excluding infective)" (OR 1.2 [0.7-1.8]) and "liver function analyses" (OR 1.3 [0.7-2.6]) in High Level Terms; and "diarrhoea" (OR 1.2 [0.7-2.0]) and "vomiting" (OR 0.9 [0.4-1.7]) in Preferred Terms. In analogy, HCA2 was expressed in the gastrointestinal tract, liver and central nervous system (CNS) of murine organs. A discrepant ADR profile was seen for "lymphopenia" (n = 777) at the preferred term level (only reported for dimethyl fumarate) and "blood glucose increased" (more often reported for nicotinic acid; OR 0.1 [0.0-0.5]). CONCLUSION: The gastrointestinal ADRs common to both substances may be mediated by HCA2 . Other ADRs not common to both substances are compound or indication-specific reactions and likely do not involve HCA2 .

Dual action by fumaric acid esters synergistically reduces adhesion to human endothelium.

OBJECTIVE: Dimethyl fumarate (DMF) is prescribed against relapsing-remitting multiple sclerosis (MS). Here, we investigated the effects of DMF and monomethyl fumarate (MMF), its metabolite in vivo, at the (inflamed) blood-brain barrier (BBB). METHODS: Effects of fumaric acid esters were analyzed using primary human brain-derived microvascular endothelial cells (HBMECs) in combination with peripheral blood mononuclear cells (PBMCs) derived from DMF-treated MS patients. RESULTS: MMF-binding to brain endothelium cells leads to activation of nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2)-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1). This might be mediated via the G-protein-coupled receptor (GPCR) hydroxycarboxylic acid receptor 2 (HCA2), a known molecular target of MMF, as we could demonstrate its expression and regulation on HBMECs. DMF treatment in vivo led to a strongly reduced expression of VCAM-1's ligand very late antigen 4 (VLA-4) by selectively reducing integrin high-expressing memory T cells of MS patients, potentially due to inhibition of their maturation by reduced trans-localization of NFκB. CONCLUSION: DMF-mediated VCAM-1 downregulation on the endothelial side and reduction in T cells with a migratory phenotype on the lymphocyte side result in a synergistic reduction in T-cell adhesion to activated endothelium and, therefore, to reduced BBB transmigration in the setting of MS.

Synthesis and evaluation of (E)-2-(5-phenylpent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate derivatives as HCA2 receptor agonists.

Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are connected by enyne (compounds 2a-f), but-1-yne (compounds 4a-j), and phenylethylene (compounds 5a-f) linkers as HCA2 full agonists were designed and their functional activity using cAMP assay and binding affinity using radioligand (3H-niacin) binding assay were evaluated. In general, compounds of all three series exhibit similar HCA2 binding and activation profile. However, the activity is strongly dependent on the substituent at the aromatic part of the structure. Among the structures evaluated, the highest affinity and potency in all series were exhibited by compounds containing 4-hydroxy and/or 2-chloro or 2-fluoro substituents. The most active compounds in the enyne and but-1-yne series in the cAMP assay are 2-fluoro,4-hydroxy and 2-chloro,4-hydroxy phenyl derivatives 2f, 4f, and 4g showing potency similar to the previously described 4-hydroxy-biphenyl analogue 5c.

Affinity and kinetics study of anthranilic acids as HCA2 receptor agonists.

Structure-affinity relationship (SAR) and structure-kinetics relationship (SKR) studies were combined to investigate a series of biphenyl anthranilic acid agonists for the HCA2 receptor. In total, 27 compounds were synthesized and twelve of them showed higher affinity than nicotinic acid. Two compounds, 6g (IC50=75nM) and 6z (IC50=108nM) showed a longer residence time profile compared to nicotinic acid, exemplified by their kinetic rate index (KRI) values of 1.31 and 1.23, respectively. The SAR study resulted in the novel 2-F, 4-OH derivative (6x) with an IC50 value of 23nM as the highest affinity HCA2 agonist of the biphenyl series, although it showed a similar residence time as nicotinic acid. The SAR and SKR data suggest that an early compound selection based on binding kinetics is a promising addition to the lead optimization process.

Synthesis and evaluation of (E)-2-(acrylamido)cyclohex-1-enecarboxylic acid derivatives as HCA1, HCA2, and HCA3 receptor agonists.

2-(3-(Naphthalen-2-yl)propanamido)cyclohex-1-enecarboxylic acid and its 6-hydroxynaphthalen-2-yl analogue are well-known hydroxyl-carboxylic acid (HCA) receptor HCA2 agonists. A series of novel aryl derivatives of 2-amidocyclohex-1-ene carboxylic acid that contained rigidity elements, such as an E-double bond, triple bond, and trans or cis-substituted cyclopropane rings, instead of the saturated ethane linker in the amide part of the molecules were designed and synthesized, and the derivatives' potency for the activation of HCA1, HCA2, and HCA3 receptors by 3'-5'-cyclic adenosine monophosphate (cAMP) assay were evaluated. The SAR studies revealed that the rigidifying of appropriate molecules enabled modulation of the potency and selectivity of the HCA2 receptor activation.

Stimulation of the Hydroxycarboxylic Acid Receptor 2 With the Ketone Body 3-Hydroxybutyrate and Niacin in Patients With Chronic Heart Failure: Hemodynamic and Metabolic Effects.

Background The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output (CO) in patients with heart failure through unknown mechanisms. 3-OHB activates the hydroxycarboxylic acid receptor 2 (HCA2), which increases prostaglandins and suppresses circulating free fatty acids. We investigated whether the cardiovascular effects of 3-OHB involved HCA2 activation and if the potent HCA2-stimulator niacin may increase CO. Methods and Results Twelve patients with heart failure with reduced ejection fraction were included in a randomized crossover study and examined by right heart catheterization, echocardiography, and blood sampling on 2 separate days. On study day 1, patients received aspirin to block the HCA2 downstream cyclooxygenase enzyme, followed by 3-OHB and placebo infusions in random order. We compared the results with those of a previous study in which patients received no aspirin. On study day 2, patients received niacin and placebo. The primary end point was CO. 3-OHB increased CO (2.3 L/min, P<0.01), stroke volume (19 mL, P<0.01), heart rate (10 bpm, P<0.01), and mixed venous saturation (5%, P<0.01) with preceding aspirin. 3-OHB did not change prostaglandin levels, neither in the ketone/placebo group receiving aspirin nor the previous study cohort. Aspirin did not block 3-OHB-induced changes in CO (P=0.43). 3-OHB decreased free fatty acids by 58% (P=0.01). Niacin increased prostaglandin D2 levels by 330% (P<0.02) and reduced free fatty acids by 75% (P<0.01) but did not affect CO. Conclusions The acute increase in CO during 3-OHB infusion was not modified by aspirin, and niacin had no hemodynamic effects. These findings show that HCA2 receptor-mediated effects were not involved in the hemodynamic response to 3-OHB. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04703361.

Hydroxycarboxylic acid receptor 2 (HCA2) agonists induce NET formation and MMP-9 release from bovine polymorphonuclear leukocytes.

Periparturient cows are commonly fed diets supplemented with Niacin (nicotinic acid, NA) because of its anti-lipolytic properties. NA confers its anti-lipolytic effects by activating the hydroxycarboxylic acid 2 receptor (HCA2). HCA2 is also activated by the ketone body beta-hydroxybutyrate (BHB) and circulating BHB levels are elevated in postpartum dairy cows. The HCA2 receptor is highly expressed in bovine polymorphonuclear leukocytes (PMN) and could link metabolic and innate immune responses in cattle. We investigated how HCA2 agonists affected bovine PMN function in vitro. We studied different PMN responses, such as granule release, surface expression of CD11b and CD47, generation of neutrophil extracellular traps (NETs), and apoptosis. NA, BHB, and 4,4aR,5,5aR-tetrahydro-1H-cyclopropa [4,5] cyclopenta [1,2-c] pyrazole-3-carboxylic acid (MK-1903) treatment triggered the release of matrix metalloproteinase 9 (MMP-9), a component of the tertiary granule, from neutrophils. Additionally, all HCA2 agonists induced NETs formation but did not affect surface expression of CD11b and CD47. Finally, none of the HCA2 agonists triggered apoptosis in bovine PMN. This information will give new insights into the potential role of the HCA2 receptor in the bovine innate immune response.

Combining metabolic phenotype determination with metabolomics and transcriptional analyses to reveal pathways regulated by hydroxycarboxylic acid receptor 2.

BACKGROUND: The adaptation of cellular metabolism is considered a hallmark of cancer. Oncogenic signaling pathways support tumorigenesis and cancer progression through the induction of certain metabolic phenotypes associated with altered regulation of key metabolic enzymes. Hydroxycarboxylic acid receptor 2 (HCA2) is a G protein-coupled receptor previously shown to act as a tumor suppressor. Here, we aimed to unveil the connection between cellular metabolism and HCA2 in BT-474 cells. Moreover, we intend to clarify how well this metabolic phenotype is reflected in transcriptional changes and metabolite levels as determined by global metabolomics analyses. METHODS: We performed both, siRNA mediated knockdown of HCA2 and stimulation with the HCA2-specific agonist monomethyl fumarate. Seahorse technology was used to determine the role of HCA2 in BT-474 breast cancer cell metabolism and its potential to induce a switch in the metabolic phenotype in the presence of different energy substrates. Changes in the mRNA expression of metabolic enzymes were detected with real-time quantitative PCR (RT-qPCR). Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolic profiling was used to determine changes in metabolite levels. RESULTS: Knockdown or stimulation of HCA2 induced changes in the metabolic phenotype of BT474 cells dependent on the availability of energy substrates. The presence of HCA2 was associated with increased glycolytic flux with no fatty acids available. This was reflected in the increased mRNA expression of the glycolytic enzymes PFKFB4 and PKM2, which are known to promote the Warburg effect and have been described as prognostic markers in different types of cancer. With exogenous palmitate present, HCA2 caused elevated fatty acid oxidation and likely lipolysis. The increase in lipolysis was also detectable at the transcriptional level of ATGL and the metabolite levels of palmitic and stearic acid. CONCLUSIONS: We combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA2, for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.

Role of HCA2 in Regulating Intestinal Homeostasis and Suppressing Colon Carcinogenesis.

Hydroxycarboxylic acid receptor 2 (HCA2) is vital for sensing intermediates of metabolism, including ß-hydroxybutyrate and butyrate. It also regulates profound anti-inflammatory effects in various tissues, indicating that HCA2 may serve as an essential therapeutic target for mediating inflammation-associated diseases. Butyrate and niacin, endogenous and exogenous ligands of HCA2, have been reported to play an essential role in maintaining intestinal homeostasis. HCA2, predominantly expressed in diverse immune cells, is also present in intestinal epithelial cells (IECs), where it regulates the intricate communication network between diet, microbiota, and immune cells. This review summarizes the physiological role of HCA2 in intestinal homeostasis and its pathological role in intestinal inflammation and cancer.

The Specificity and Broad Multitarget Properties of Ligands for the Free Fatty Acid Receptors FFA3/GPR41 and FFA2/GPR43 and the Related Hydroxycarboxylic Acid Receptor HCA2/GPR109A.

The paradigm of ligand-receptor interactions postulated as "one compound-one target" has been evolving; a multi-target, pleiotropic approach is now considered to be realistic. Novel series of 1,4,5,6,7,8-hexahydro-5-oxoquinolines, pyranopyrimidines and S-alkyl derivatives of pyranopyrimidines have been synthesized in order to characterise their pleiotropic, multitarget activity on the FFA3/GPR41, FFA2/GPR43, and HCA2/GPR109A receptors. Hexahydroquinoline derivatives have been known to exhibit characteristic activity as FFA3/GPR41 ligands, but during this study we observed their impact on FFA2/GPR43 and HCA2/GPR109A receptors as well as their electron-donating activity. Oxopyranopyrimidine and thioxopyranopyrimidine type compounds have been studied as ligands of the HCA2/GPR109A receptor; nevertheless, they exhibited equal or higher activity towards FFA3/GPR41 and FFA2/GPR43 receptors. S-Alkyl derivatives of pyranopyrimidines that have not yet been studied as ligands of GPCRs were more active towards HCA2/GPR109A and FFA3/GPR41 receptors than towards FFA2/GPR43. Representative compounds from each synthesized series were able to decrease the lipopolysaccharide-induced gene expression and secretion of proinflammatory cytokines (IL-6, TNF-α) and of a chemokine (MCP-1) in THP-1 macrophages, resembling the effect of HCA2/GPR109A ligand niacin and the endogenous ligand propionate. This study revealed groups of compounds possessing multitarget activity towards several receptors. The obtained data could be useful for further development of multitarget ligands.

Subcutaneous administration of ß-hydroxybutyrate improves learning and memory of sepsis surviving mice.

Post-sepsis cognitive impairment is one of the major sequelae in sepsis survivors. Its prevention remains clinically challenging. Here we tested the effects and underlying mechanisms of exogenous ß-hydroxybutyrate (BHB) on post-sepsis cognitive impairment. We found that subcutaneous administration of BHB increased survival and body weight recovery of sepsis mice and improved learning and memory of sepsis surviving mice in a cecal ligation and perforation-induced sepsis model. Additionally, the improvement of learning and memory of sepsis surviving mice was still detected even if BHB was administrated at the late stage of sepsis. In contrast, glucose solution did not show similar effects. Mechanistically, subcutaneous administration of BHB increased the BHB level of hippocampus, and limited neuroinflammation and neuroplasticity damage in sepsis mice. Intracerebroventricular administration of BHB also alleviated neuroinflammation and cognitive impairment of sepsis surviving mice. In the coculture of neurons, astrocytes, and BV2 cells (a microglial cell line), knocking down the expression of microglial HCA2 (BHB receptor) via a specific shRNA reduced the protection of BHB to lipopolysaccharide-induced inflammatory response and neuron damage more significantly than knocking down neuronal MCT2 (BHB transporter). These data showed that (1) BHB was a potential pharmacological adjunct treatment for prevention of post-sepsis cognitive impairment and (2) inhibiting neuroinflammation via HCA2 was an important mechanism.

Anti-inflammatory effects of the hydroxycarboxylic acid receptor 2.

The hydroxycarboxylic acid receptors (HCA1-3) are a family of G-protein-coupled receptors that are critical for sensing endogenous intermediates of metabolism. All three receptors are predominantly expressed on adipocytes and mediate anti-lipolytic effects. In addition to adipocytes, HCA2 is highly expressed on immune cells, including macrophages, monocytes, neutrophils and dermal dendritic cells, among other cell types. The endogenous ligand for HCA2 is beta-hydroxybutyrate (ß-OHB), a ketone body produced by the liver through ß-oxidation when an individual is in a negative energy balance. Recent studies demonstrate that HCA2 mediates profound anti-inflammatory effects in a variety of tissues, indicating that HCA2 may be an important therapeutic target for treating inflammatory disease processes. This review summarizes the roles of HCA2 on inflammation in a number of tissues and clinical states.

Activation of the niacin receptor HCA2 reduces demyelination and neurofilament loss, and promotes functional recovery after spinal cord injury in mice.

After spinal cord injury (SCI), there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound, which is believed to derail healing and compromize organ functions. Thus, agents which are able to modulate macrophage phenotypes may provide significant benefits to SCI patients. In the present study, we demonstrate that the niacin receptor HCA2 is specifically expressed on the cell surface of M1 but not M2 macrophages. Treatment of M1 macrophages with niacin (300µM) resulted in down-regulation of the p65 NF-κB phosphorylation, associated with a marked decrease in the levels of M1 markers, including CD86, IL-12, and IL-6, and a significant increase in the expressions of M2 markers, such as CD206, IL-10, and IL-13, suggesting that niacin causes a shift of M1 to M2. Moreover, treatment of the M1-oligodendrocyte precursor cell (OPC) co-cultures with niacin markedly promoted the expression of myelin binding protein (MBP). After SCI in C57/BL6 mice for a week, a marked accumulation of M1 macrophages, which expressed HCA2 receptor, was evident in the wound. Treatment of the SCI mice with niacin (100mg/kg) resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function. Thus, HCA2 receptor may serve as a therapeutic target to promote post-SCI recovery.

The role of the C-terminus of the human hydroxycarboxylic acid receptors 2 and 3 in G protein activation using Gα-engineered yeast cells.

In the present study we focused our attention on the family of hydroxycarboxylic acid (HCA) receptors, a GPCR family of three members, of which the HCA2 and HCA3 receptors share 95% high sequence identity but differ considerably in C-terminus length with HCA3 having the longest tail. The two receptors were expressed and analysed for their activation profile in Saccharomyces cerevisiae MMY yeast strains that have different G protein Gα subunits. The hHCA2 receptor was promiscuous in its G protein coupling preference. In the presence of nicotinic acid the hHCA2 receptor activated almost all G protein pathways except Gαq (MMY14). However, the Gα protein coupling profile of the hHCA3 receptor was less promiscuous, as the receptor only activated Gαi1 (MMY23) and Gαi3 (MMY24) pathways. We then constructed two mutant receptors by 'swapping' the short (HCA2) and long (HCA3) C-terminus. The differences in HCA2 and HCA3 receptor activation and G protein selectivity were not controlled, however, by their C-terminal tails, as we observed only minor differences between mutant and corresponding wild-type receptor. This study provides new insights into the G protein coupling profiles of the HCA receptors and the function of the receptor's C terminus, which may be extended to other GPCRs.

GSK256073 acutely regulates NEFA levels via HCA2 agonism but does not achieve durable glycaemic control in type 2 diabetes. A randomised trial.

This study investigated safety and efficacy of GSK256073, an in vitro potent, selective GPR109A agonist, for treatment of subjects with type 2 diabetes mellitus (Type 2 DM) poorly controlled with metformin alone. Patients with Type 2 DM (n=94) were enroled into this randomised, double-blind (sponsor unblinded), placebo-controlled, parallel group trial. Participants received placebo for two weeks before being randomised (2:2:2:2:1:1) to receive doses of GSK256073 5mg twice-daily (BID), 10mg once-daily (QD), 25mg BID, 50mg QD or placebo BID or QD in addition to their current metformin treatment. The primary efficacy endpoint was change from baseline in glycosylated haemoglobin (HbA1c) at week 12. The safety profile of GSK256073 did not significantly differ from that of placebo. Decreases from baseline in HbA1c were observed in all treatment groups but were not statistically significant compared to placebo; at week 12 a maximum decrease of 0.30% from placebo was reached in the GSK256073 50mg QD group. On day 2, GSK256073 significantly decreased non-esterified fatty acid (NEFA) (0-12h) concentrations but pharmacological activity was lost (5mg BID, 10mg QD, 25mg BID) or reduced (50mg QD) at week 6. Drug exposure demonstrated 2-fold accumulation over 6 weeks. The primary efficacy objective of the study was not met. GSK256073 did not improve HbA1c concentrations at week 12. Despite sustained drug exposure, the ability of the HCA2 agonist to suppress plasma NEFA concentrations waned over time and targeted effects on glucose oxidation and insulin sensitivity subsided.

Discovery and characterization of GSK256073, a non-flushing hydroxy-carboxylic acid receptor 2 (HCA2) agonist.

Niacin has been used for many years in the treatment of dyslipidemia due to its ability to decrease serum levels of triglycerides and low-density lipoprotein cholesterol and to increase levels of high density lipoprotein cholesterol. However, niacin causes severe flushing resulting in poor patient compliance. The discovery of hydroxy-carboxylic acid receptor 2 (HCA2) as a high affinity receptor for niacin has opened avenues to investigate the mechanism of action of niacin, and to potentially discover agonists which maintain the antilipolytic effects of niacin accessed by a decrease in circulating non-esterified fatty acids (NEFA) and thereby perhaps the lipid/lipoprotein effects, but avoid the flushing effects. Here we describe the strategy we implemented to identify such compounds. This approach resulted in the discovery of GSK256073, a highly potent HCA2 agonist, which produced similar NEFA lowering effects to niacin in preclinical models (rat and guinea pig). A guinea pig model was used to predict flushing, via an increase in ear temperature, and GSK256073 was found to have a minimal effect in this model. These preclinical models appeared to be predictive of human response, since in a first-time-in-human study, GSK256073 displayed long lasting NEFA and triglyceride lowering effects in healthy male subjects, which were not associated with flushing. GSK256073 can be used as a pharmacological tool to better understand the role of HCA2 in lipid metabolism.

Niacin attenuates the production of pro-inflammatory cytokines in LPS-induced mouse alveolar macrophages by HCA2 dependent mechanisms.

Niacin has been reported to have potent anti-inflammatory effects in LPS-induced acute lung injury. However, the molecular mechanism of niacin has not been fully understood. The aim of the present study was to investigate the effects of niacin on the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß in LPS-induced mouse alveolar macrophages and explore its underlying mechanism. Mouse alveolar macrophages were incubated in the presence or absence of various concentrations of niacin (1, 10, 100 µmol/l) 1h before LPS (1 µg/ml) challenge. The results showed that niacin reduced the levels of TNF-α, IL-6 and IL-1ß in LPS-challenged alveolar macrophages. Furthermore, NF-κB activation was inhibited by niacin through blocking the phosphorylation of NF-κB p65 and IκBα. In addition, silencing HCA2 abrogated the effect of niacin on the production of pro-inflammatory cytokines. These findings suggested that niacin attenuated the LPS-induced pro-inflammatory cytokines possibly mediated by HCA2 in LPS-challenged alveolar macrophages.

Screening of Different Organs of Rats for HCA2 Receptor mRNA.

Interest in hydroxy - carboxylic acid 2 (HCA2) receptor has been raised since it is the target of antidyslipidemic drug nicotinic acid. The present study aimed to evaluate the presence of mRNA of this receptor in different organs of laboratory rat. Twenty two different organs of rats including mesenteric fat, epididymis (head, body and tail), testis, ovary, xiphoid process, liver, adrenal gland, femoral head, proximal epiphyseal and metaphyseal bone marrow of femur, esophagus, glandular stomach, forestomach, intestines, colons, heart, spleen, kidney, trachea, lung, skeletal muscle (quadriceps), cerebrum and cerebellum were removed and examined for HCA2 mRNA by RT- PCR method. The mRNA for HCA2 receptor was detected in all analyzed tissues. In conclusion, the different organs of rat express HCA2 receptor mRNA which makes a proper animal model for future studies on the physiological and pharmacological roles of this receptor in vivo.