Preclinical assessment and randomized Phase I study of CT-P63, a broadly neutralizing antibody targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant morbidity and mortality worldwide. Despite a successful vaccination programme, the emergence of mutated variants that can escape current levels of immunity mean infections continue. Herein, we report the development of CT-P63, a broad-spectrum neutralizing monoclonal antibody. In vitro studies demonstrated potent neutralizing activity against the most prevalent variants, including Delta and the BA.1 and BA.2 sub-lineages of Omicron. In a transgenic mouse model, prophylactic CT-P63 significantly reduced wild-type viral titres in the respiratory tract and CT-P63 treatment proved efficacious against infection with Beta, Delta, and Omicron variants of SARS-CoV-2 with no detectable infectious virus in the lungs of treated animals. A randomized, double-blind, parallel-group, placebo-controlled, Phase I, single ascending dose study in healthy volunteers (NCT05017168) confirmed the safety, tolerability, and pharmacokinetics of CT-P63. Twenty-four participants were randomized and received the planned dose of CT-P63 or placebo. The safety and tolerability of CT-P63 were evaluated as primary objectives. Eight participants (33.3%) experienced a treatment-emergent adverse event (TEAE), including one grade ≥3 (blood creatine phosphokinase increased). There were no deaths, treatment-emergent serious adverse events, TEAEs of special interest, or TEAEs leading to study drug discontinuation in the CT-P63 groups. Serum CT-P63 concentrations rapidly peaked before declining in a biphasic manner and systemic exposure was dose proportional. Overall, CT-P63 was clinically safe and showed broad-spectrum neutralizing activity against SARS-CoV-2 variants in vitro and in vivo.

The in vitro and in vivo efficacy of CT-P59 against Gamma, Delta and its associated variants of SARS-CoV-2.

The SARS-CoV-2 variant is rapidly spreading across the world and causes to resurge infections. We previously reported that CT-P59 presented its in vivo potency against Beta variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on Gamma, Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal studies. CT-P59 showed neutralization against Gamma, Delta, Epsilon, and Kappa variants in cells, with reduced susceptibility. The mouse challenge experiments with Gamma and Delta variants substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against Gamma and Delta variants infection, hinting that CT-P59 has therapeutic potential for patients infected with Gamma, Delta and its associated variants.

Brassinin, a brassica-derived phytochemical, regulates monocyte-to-macrophage differentiation and inflammatory responses in human monocytes and murine macrophages.

OBJECTIVES: The effects and molecular mechanisms of brassinin (BR), an indole phytoalexin from cruciferous vegetables, on monocyte-to-macrophage differentiation and inflammatory responses were investigated in this study. METHODS: Inflammatory responses from RAW264.7 cells and THP-1 were stimulated by lipopolysaccharide (1 µg/ml), and monocyte-to-macrophage differentiation of THP-1 was induced by phorbol myristate acetate (50 ng/ml). The production of inflammatory mediators was determined by ELISA, Western blot or real-time PCR. Reactive oxygen species were examined by DCFH-DA assay. KEY FINDINGS: Brassinin at 50 µm suppressed lipopolysaccharide-induced production of nitric oxide synthase, cyclooxygenase-2, prostaglandin E2 and reactive oxygen species by 90%, 69%, 52% and 41%, respectively, in RAW264.7 cells. In THP-1 cells, BR inhibited phorbol myristate acetate-induced monocyte-to-macrophage differentiation by suppressing cluster of differentiation molecule ß and CD36. In addition, BR suppressed translocation of nuclear factor 'kappa-light-chain-enhancer' of activated B cells (NF-κB) into the nucleus. However, BR activated the nuclear factor erythroid-derived 2-like 2 (Nrf2) and its target molecules hemoxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), with an increase in nuclear translocation of Nrf2. CONCLUSIONS: Brassinin suppressed monocyte-to-macrophage differentiation and inflammatory responses by differentially regulating Nrf2 and NF-κB signallings.

Parthenolide inhibits lipid accumulation via activation of Nrf2/Keap1 signaling during adipocyte differentiation.

The effects of parthenolide (PL), a sesquiterpene lactone obtained from feverfew plant, on lipid accumulation and signaling pathway in adipocytes were investigated. PL significantly inhibited lipid accumulation and adipogenic factors during adipogenesis. In particular, PL exerted its inhibitory effects in early adipogenic stage by regulating the early adipogenic factors. In addition, PL regulated the expression of adipokines; leptin, retinol binding protein, and resistin mRNAs were downregulated, whereas adiponectin gene expression was increased. Furthermore, PL significantly reduced intracellular reactive oxygen species (ROS) production during adipogenesis. This PL-mediated regulation of ROS production was associated with the regulation of nuclear factor erythroid 2-related factor (Nrf2)-kelch-like ECH-associated protein 1 (Keap1) pathway. PL effectively increased the abundance of Nrf2 and its target proteins, heme oxygenase-1 (HO-1) and NADPH dehydrogenase 1 (NQO1), by promoting the nuclear translocation of Nrf2, indicating that PL-mediated anti-adipogenic effects are associated with the Nrf2/Keap1 pathway.

Red ginseng extract regulates differentiation of monocytes to macrophage and inflammatory signalings in human monocytes.

This study was aimed to investigate the effect of red ginseng extract (RGE) on monocyte to macrophage differentiation and inflammatory signalings in THP-1 human monocytes. In HPLC analysis, RGE contained saponin level of 516 µg/mg (extract) with 14 ginsenosides. RGE effectively suppressed the monocyte-to-macrophage differentiation induced by phorbol 12-myristate 13-acetated (PMA) by inhibiting the THP-1 cell adhesion. This result is evidenced by the down-regulation of cluster of differentiation molecule ß (CD11ß) and CD36. RGE significantly reduced translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (78%), while cytosolic NF-κB was increased (53%), compared with LPS group. In addition, RGE significantly increased the protein abundance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its target protein, hemoxygenase-1 (HO-1), but, Kelch-like ECH-associated protein 1 (KEAP1), a negative regulator of Nrf2, was greatly decreased by RGE. Furthermore, RGE effectively mediated the regulation of Nrf2 level in nucleus and cytoplasm of THP-1.

Punicalagin, a Pomegranate-Derived Ellagitannin, Suppresses Obesity and Obesity-Induced Inflammatory Responses Via the Nrf2/Keap1 Signaling Pathway.

SCOPE: Punicalagin (PCG) is one of the most abundant phytochemicals found in pomegranates. The effects and mechanistic action of PCG on obesity and obesity-induced inflammatory and oxidant responses are investigated in vitro and in vivo. METHODS AND RESULTS: The effect of PCG on adipogenesis is examined using Oil red O staining. The effects and mechanism of action of PCG on inflammatory responses are determined in adipocyte-conditioned medium (ACM)-cultured macrophages, a cell-to-cell contact system, and a transwell system. The effects of PCG on obesity and obesity-induced inflammatory/oxidant responses are examined in high-fat diet (HFD)-fed mice. PCG effectively suppresses lipid accumulation in adipocytes and adipocyte-induced inflammatory responses in adipocyte-macrophage co-culture systems. Small interfering RNA (siRNA) transfection indicates that the PCG-mediated anti-inflammatory effect is exerted via the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1(Nrf2/Keap1) pathway. PCG administration results in a significant reduction in body and white adipose tissue (WAT) weights. PCG favorably regulates pro- and anti-inflammatory cytokines, downregulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Immunohistochemical (IHC) analysis demonstrates that PCG differentially modulates the distribution of complement component 3 receptor 4 subunit (CD11c) and cluster of differentiation 206 (CD206). PCG regulates the level of antioxidant and oxidant molecules by activating Nrf2/Keap1 signaling. CONCLUSIONS: PCG ameliorates obesity and obesity-induced inflammatory responses via activation of Nrf2/Keap1 signaling, suggesting that PCG has potential as an oral agent to control obesity-mediated diseases.

Parthenolide, a feverfew-derived phytochemical, ameliorates obesity and obesity-induced inflammatory responses via the Nrf2/Keap1 pathway.

Parthenolide (PL) is one of the most abundant sesquiterpene lactones found in the plant feverfew (Tanacetum parthenium (L.) Sch.Bip.). PL was investigated for its effect on obesity and obesity-induced inflammatory/oxidant responses in vitro and in vivo. An obesity-induced inflammatory response was induced in various co-culture systems using adipocytes (3T3-L1) and macrophages (RAW264.7) in vitro and the effect of PL and its mechanism of action were determined. PL effectively suppressed the adiposity-induced inflammatory responses by downregulating IL-6 (40-42%) and MCP-1 (26-37%) in 3T3-CM-cultured macrophages and contact co-culture system. PL also favorably regulated the dysregulations of adiponectin and resistin in macrophage-conditioned medium (RAW-CM)-cultured adipocytes. In transwell system of adipocyte and macrophage, PL was shown to upregulated Nrf2 and its target molecule, HO-1 by promoting nuclear translocation of Nrf2. In particular, in siRNA knockdown study, the PL-mediated anti-inflammatory response was exerted via the Nrf2/Keap1 pathway. In animal study using high-fat diet (HFD)-fed mice, PL-administered mice showed a significant reduction in body weight and white adipose tissues (WATs). This PL-mediated anti-obese effect was connected to anti-inflammatory responses with the regulation of inflammatory cytokines, and the downregulation of NF-κB and MAPKs. Furthermore, PL differentially modulated CD11c and CD206, which are pro-/anti-inflammatory phenotypes of ATMs, in stroma vascular fraction (SVF) and immunohistochemistry (IHC) staining analyses. PL also regulated the level of (anti)oxidant molecules with the activation of Nrf2/Keap1signaling. Taken together, PL inhibited obesity and obesity-induced inflammatory responses via the activation of Nrf2/Keap1 signaling, indicating a potential of PL as a functional agent to control obesity-related diseases.

Brassinin, a phytoalexin in cruciferous vegetables, suppresses obesity-induced inflammatory responses through the Nrf2-HO-1 signaling pathway in an adipocyte-macrophage co-culture system.

The aim of this study was to investigate the effect of brassinin (BR), a phytoalexin found in plants belonging to the Brassicaceae family, on the obesity-induced inflammatory response and its molecular mechanism in co-culture of 3T3-L1 adipocytes and RAW264.7 macrophages. BR effectively suppressed lipid accumulation by down-regulating the expression of adipogenic factors, which in turn, were regulated by early adipogenic factors such as CCAAT-enhancer-binding protein-ß and Kruppel-like factor 2. Production of inflammatory cytokines and reactive oxygen species, induced by adipocyte-conditioned medium, was significantly decreased in BR-treated cells. This effect of BR was more prominent in contact co-culture of adipocytes and macrophages with a 90% and 34% reduction in IL-6 and MCP-1 levels, respectively. BR also restored adiponectin expression, which was significantly reduced by culturing adipocytes in macrophage-conditioned medium. In the transwell system, BR increased the protein levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its target molecule, hemoxygenase-1 (HO-1), by 55%-93% and 45%-48%, respectively, and also increased Nrf2 translocation into the nucleus. However, knockdown of Nrf2 or HO-1 in RAW264.7 cells restored this BR-mediated inhibition of IL-6 and MCP-1 production. These results indicated that BR inhibited obesity-induced inflammation via the Nrf2-HO-1 pathway.

Red ginseng-derived saponin fraction suppresses the obesity-induced inflammatory responses via Nrf2-HO-1 pathway in adipocyte-macrophage co-culture system.

The aim of this study was to investigate the effect of saponin fraction (SF) from red ginseng on obesity-induced inflammatory response in a co-culture system of 3T3-L1 and RAW264.7 cells. HPLC analysis showed that SF contains more than 50% ginsenosides, and Rb1 was the most abundant ginsenoside [135.31 µg/mg (extract)]. The production of nitric oxide and cytokines, induced by adipocyte-conditioned medium (3T3-CM), was significantly decreased by SF. SF (100 µg/mL) suppressed the abundance of tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) by 78%, 40%, and 22%, respectively. This SF-mediated reduction in inflammatory cytokines was due to the suppression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation, and translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) into the nucleus. SF also regulated adipokine expression in adipocytes, which were stimulated by macrophage-conditioned medium (RAW-CM); adiponectin expression was upregulated (> 2-fold), while resistin was downregulated (40%). In the contact system of adipocytes and macrophages, SF significantly decreases MCP-1 (37%) and IL-6 (25%) production. In the transwell system, SF (100 µg/mL) significantly increased the abundance of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its target protein, hemoxygenase-1 (HO-1) by 1.5∼3.5-fold and 2.8∼3.6-fold, respectively, thus increasing Nrf2 translocation into nucleus. However, SF-mediated inhibitory effect on the release of IL-6 and MCP-1 cytokines was reversed in the Nrf2 or HO-1 knockdown condition. This result indicated that SF-mediated inhibition of obesity-induced inflammation was dependent on Nrf2 activation.

Dibenzoylmethane, a Component of Licorice, Suppresses Monocyte-to-Macrophage Differentiation and Inflammatory Responses in Human Monocytes and Mouse Macrophages.

The objective of this study was to investigate the effect of dibenzoylmethane (DBM) on monocyte-to-macrophage differentiation, the inflammatory response, and the resulting signaling in human monocytes and murine macrophage. DBM effectively inhibited the monocyte-to-macrophage differentiation induced by phorbol 12-myristate 13-acetate (PMA) through a reduction in adhesion of THP-1 cells. Cluster of differentiation molecule ß (CD11ß) and CD36, which are surface markers of macrophage differentiation, were downregulated by 80 and 74%, respectively. DBM also significantly inhibited lipopolysaccharide (LPS)-induced nitrite (NO) production through the downregulation of inducible oxide synthase (iNOS) in RAW264.7 cells. The abundance of cyclooxygenase-2 (COX-2), a pro-inflammatory protein, was also effectively decreased by DBM in a dose-dependent manner. DBM (50 µM) reduced the levels of COX-2 and iNOS by 81 and 78%, respectively. DBM significantly inhibited the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), an inflammatory transcription factor, into the nucleus. DBM-mediated increase of NF-κB translocation resulted from the DBM-induced suppression of the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). In contrast, DBM effectively increased the expression of nuclear factor E2-related factor 2 (Nrf2) and its target protein, hemeoxygenase-1 (HO-1). Nrf2 translocation into the nucleus was also significantly enhanced by DBM. Furthermore, DBM effectively inhibited the expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, and monocyte chemoattractant protein-1 (MCP-1). These results indicated that the DBM-mediated differential regulation of NF-κB and Nrf2, which are major transcription factors involved in inflammation, inhibited the expression of inflammatory cytokines.

Dibenzoylmethane Suppresses Lipid Accumulation and Reactive Oxygen Species Production through Regulation of Nuclear Factor (Erythroid-Derived 2)-Like 2 and Insulin Signaling in Adipocytes.

The aim of this study was to investigate the effects of dibenzoylmethane (1,3-diphenyl-1,3-propanedione, DBM) from licorice roots on lipid accumulation and reactive oxygen species (ROS) production in 3T3-L1 cells. DBM effectively inhibited lipid accumulation during adipogenesis, and its inhibitory effect was shown to be due to the down-regulation of adipogenic factors such as CCAAT-enhancer-binding protein-α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), and fatty acid-binding protein 4 (FABP4). DBM was observed to exert its inhibitory effect on lipid accumulation in the early adipogenic stage (days 0-2) by regulating early adipogenic factors including CCAAT-enhancer-binding protein-ß (C/EBPß) and Krueppel-like factor (KLF) 2. DBM significantly increased the translocation of nuclear factor (erythroid-derived 2)-like 2(Nrf2) into the nucleus, promoting the protein expression of its target gene, heme oxygenase-1 (HO-1). DBM significantly suppressed the insulin-mediated activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), which are components of insulin signaling. In addition, intracellular ROS production was effectively reduced by DBM treatment, which upregulated antioxidant genes such as glutathione peroxidase (Gpx), catalase (CAT), and superoxide dismutase 1 (SOD1). Furthermore, DBM significantly regulated the expression of the adipokines, resistin and adiponectin. This DBM-mediated regulation of lipid accumulation, ROS production, and adipokine production was shown to be involved in the regulation of the Nrf2 and insulin signaling.