Acute, subchronic oral toxicity, and genotoxicity evaluations of LPM570065, a new potent triple reuptake inhibitor.

In the current study, to support the safety of LPM570065 as a new potent triple reuptake inhibitors (TRIs), LPM570065 was investigated through a single- and 13-week repeated-dose oral toxicity evaluation and mutagenicity assays. In an acute toxicity evaluation, Sprague-Dawley (SD) rats were single administration at dose of 500, 1000 and 2000 mg/kg. The results suggested that two (2/20) and seven (7/20) animals were died in the 1000 and 2000 mg/kg group, respectively. In contrast, there were no treatment-related effects at a dose of 500 mg/kg. In a 13-week toxicity evaluation, SD rats were given 30, 100, or 300 mg/kg LPM570065 for 13 successive weeks and then allowed a 4-week recovery period. Impermanent salivation was found at each of the doses, and an impermanent minor body weight decrease was noted in the 300 mg/kg males (P < 0.05). Notably, serum prolactin levels were lowered by 43.25% and 78.65% in the male rats in 100 and 300 mg/kg groups, respectively (P < 0.05). Further, the serum testosterone was elevated by 37% in the 30 and 100 mg/kg males. In conclusion, the maximum tolerated dose (MTD) was 500 mg/kg and the lethal dose was 1000 mg/kg in SD rats after a single administration of LPM570065. In 13-week repeated-dose oral toxicity, the no-observed-adverse-effect level (NOAEL) of LPM570065 was greater than 300 mg/kg for rats. Moreover, LPM570065 was not mutagenic or clastogenic. According to this result it can be concluded that the MTD of LMP570065 is approximately up to 3000 mg/person/day in clinic, and the effects of LMP570065 on sexual function also should be considered.

Toxicity effects of a novel potent triple reuptake inhibitor, LPM570065, on the fertility and early embryonic development in Sprague-Dawley rats.

Selective serotonin reuptake inhibitors (SSRIs) have developed as novel antidepressants and have been determined to possess higher efficacy and less adverse effects compared to other antidepressants. Our previous studies have showed that LPM570065, a new potent TRI, is relatively nontoxic in acute, subchronic toxicity and genotoxicity evaluations. In the current study, toxicity of LPM570065 was further evaluated on the fertility and early embryonic development in Sprague-Dawley rats. A total of 264 rats were treated with various concentrations of LPM570065 (30 mg/kg, 100 mg/kg, and 300 mg/kg) or used as control. Females rats were treated for two consecutive weeks, followed by mating via cohabitation up to the 7th gestation day (GD). The male rats were treated for four consecutive weeks, which were followed by first mating with treated female rats. Then, all males were treated up to the 9th week and followed by second mating with non-treated female rats, and were sacrificed. All surviving pregnant females were euthanized on GD 15. We evaluated the following parameters, namely, mortality, toxicity symptoms, body weight, amount of food consumed, sexual cycle, mating behavior, pregnancy, sperm production, gross necropsy, and weight of organs. Excessive salivation was observed post treatment in nearly all females and males in the 100 and 300 mg/kg LPM570065 treatment groups. Body weight gain was decreased in gravid rats treated with 300 mg/kg LPM570065 during GD 0-6 (P < 0.05). The application of 300 mg/kg of LPM57006 to male rats induced a decrease in implantation sites and lower fertility rates (P < 0.05). However, sperm concentration and count were higher in the LPM570065-treated groups (30 mg/kg, 100 mg/kg, and 300 mg/kg) compared to the controls. Moreover, duration of mating significantly decreased to 37.5% after nine weeks of LPM570065 treatment at a concentration of 300 mg/kg (P < 0.05). In conclusion, the no observable adverse effect level (NOAEL) was established at 100 mg/kg and 300 mg/kg for female and male rats, respectively. The NOAEL for fertility and early embryonic development was established at 300 mg/kg and 100 mg/kg for female and male rats, respectively.

Protocatechuic aldehyde ameliorates experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway.

An abnormal high mobility group box 1 (HMGB1) activation and a decrease in receptor for advanced glycation end-product (RAGE) play a key role in the pathogenesis of pulmonary fibrosis. Protocatechuic aldehyde (PA) is a naturally occurring compound, which is extracted from the degradation of phenolic acids. However, whether PA has anti-fibrotic functions is unknown. In this study, the effects of PA on the transforming growth factor-ß1 (TGF-ß1)-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the apoptosis of human type I alveolar epithelial cells (AT I), on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis in vivo were investigated. PA treatment resulted in a reduction of EMT in A549 cells with a decrease in vimentin and HMGB, an increase of E-cadherin and RAGE, a reduction of HLF-1 proliferation with a decrease of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor (PDGF). Apoptosis of AT I was attenuated with an increase of RAGE. PA ameliorated BLM-induced pulmonary fibrosis in rats with a reduction of histopathological scores and collagen deposition, and a lower FGF-2, PDGF, α-smooth muscle actin (α-SMA) and HMGB1 expression, whereas higher RAGE was found in BLM-instilled lungs. Through the decrease of HGMB1 and the regulation of RAGE, PA reversed the EMT, inhibited HLF-1 proliferation as well as reduced apoptosis in AT I, and prevented pulmonary fibrosis in vivo. Collectively, our results demonstrate that PA prevents experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway.

Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury.

Curculigoside A has shown protective effects against rat cortical neuron damage in vivo. However, the molecular mechanisms through which Curculigoside A affords this protection are unclear. In the present study, we sought to elucidate the mechanisms of angiogenesis in rat aortic endothelial cells (RAEC), rat aortic smooth muscle cells (RASMC) as well as a rat model of cerebral ischemia and reperfusion injury following treatment with Curculigoside A. We examined the role of Curculigoside A on RAEC and RASMC proliferation, migration, and tube formation in vitro and in a cerebral ischemia and reperfusion injury rat model. We used the recombinant Dickkopf (DKK)-1 protein, a Wnt/ß-catenin inhibitor, and the recombinant WIF-1 protein, a Wnt5a antagonist to determine mechanisms. In addition, we measured leakage of the blood-brain barrier (BBB) and tested for angiogenesis associated proteins. Our data suggest that Curculigoside A induces angiogenesis in vitro by increasing proliferation, migration and tube formation in RAEC and RASMC. The increase in Curculigoside A-induced proliferation and tube formation was counteracted by DKK-1 and WIF-1. Curculigoside A increased expression of VEGF, p-VEGFR, p-CREB, Egr-3, VCAM-1, Ang1 and Tie2 while prohibiting BBB leakage in cerebral ischemia and reperfusion injured rats. However, Cyclosporine A, a CREB inhibitor, reduced the expression of p-CREB, Egr-3, VCAM-1, Ang1 and Tie2. These data suggest that Curculigoside A induces cell proliferation and angiogenesis through the Wnt5a/ß-catenin and VEGF/CREB/Egr-3/VCAM-1 signaling axis and promotes maturation and stability of new blood vessels via increasing Ang1 and Tie-2 expression.

Ponatinib ameliorates pulmonary fibrosis by suppressing TGF-ß1/Smad3 pathway.

TGF-ß1/Smad3 pathway plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, including lung fibroblasts proliferation and epithelial cell aberrant activation. Ponatinib is a multi-targeted tyrosine-kinase inhibitor. However, whether Ponatinib has anti-fibrotic functions is unknown. In this study, the effects of Ponatinib on TGF-ß1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1), on the apoptosis of human type I alveolar epithelial cells (AT I) in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis was investigated in vivo. Treatment with Ponatinib resulted in a reduction of EMT in A549 cells with a decrease in vimentin and p-Smad3, whereas an increase in E-cadherin. Apoptosis of AT I was attenuated with an increase in the Bcl-2/Bax ratio. HLF-1 proliferation was reduced with a decrease in PDGF-BB and FGF-2 expressions. Treatment with Ponatinib resulted in an amelioration of the BLM-induced pulmonary fibrosis in rats with reductions of the pathological score, collagen deposition, p-Smad3, α-SMA, PDGF-BB and FGF-2 expression. In summary, Ponatinib reversed the EMT, inhibited the apoptosis of AT I, as well as HLF-1 proliferation and prevented pulmonary fibrosis by suppressing the TGF-ß1/Smad3 pathway.

Synthesis and evaluation of neuroactive steroids with novel pharmacophore at C-21 let identify a compound with advantageous PK profile and higher EC50 and Emax as PAM on GABAA receptor.

Zuranolone (SAGE-217) is a neuroactive steroid (γ-aminobutyric acid)A (GABAA) receptor positive allosteric modulator (PAM) as the first oral drug approved by the FDA in 2023, which is used to treat patients with postpartum depression (PPD). SAGE-217 has a "black box" warning with impairing ability to drive or engage in other potentially hazardous activities. In addition, SAGE-217 can cause CNS depressant effects such as somnolence and confusion, suicidal thoughts and behavior and embryo-fetal toxicity. Based on the structure-activity relationship (SAR) of SAGE-217, a total of 28 neuroactive steroids with novel pharmacophore at C-21 modulated SAGE-217 derivatives were designed and synthesized. The biological activities were evaluated by both synaptic α1ß2γ2 GABAA receptor and extrasynaptic α4ß3δ GABAA receptor cell assays. The optimal compound S28 exhibited much more potent potency and similar efficacy at extrasynaptic GABAA receptor than SAGE-217. Different from above, compound S28 exhibited similar potency and lower efficacy at synaptic GABAA receptor than SAGE-217, which were consistent with the analysis of molecular docking and dynamics simulation results. The appropriate lower efficacy at synaptic GABAA receptor of compound S28 might contribute to reduce the side effects of excessive sedation. Furthermore, compound S28 was demonstrated to have excellent in vivo pharmacokinetic (PK) parameters, robust in vivo pharmacodynamic (PD) effects and good safety profiles. Therefore, compound S28 represents a potentially promising treatment of PPD candidate that warrants further investigation.

Rosmarinic acid protects against experimental diabetes with cerebral ischemia: relation to inflammation response.

BACKGROUND: Inflammatory activation plays a vital role in the pathophysiological mechanisms of stroke, exerting deleterious effects on the progression of tissue damage and may lead to the vascular damage in diabetes. The objectives of this study were to determine the effects of rosmarinic acid (RA) on a cultured neuronal cell line, SH-SY5Y in vitro and experimental ischemic diabetic stroke in vivo. METHODS: For oxygen-glucose deprivation (OGD) and tumor necrosis factor-α (TNF-α) stimulated SH-SY5Y cell line in vitro, SH-SY5Y cells were incubated with RA. For an in vivo experiment, diabetic rats were subjected to middle cerebral artery occlusion (MACO) for 40 minutes followed by reperfusion for 23 h. RESULTS: Treatment of SH-SY5Y cells with RA reduced the OGD-induced apoptosis and cytotoxicity, blocked TNF-α-induced nuclear transcription factor κB (NF-κB) activation, and decreased high-mobility group box1 (HMGB1) expression. At doses higher than 50 mg/kg, RA produced a significant neuroprotective potential in rats with ischemia and reperfusion (I/R). RA (50 mg/kg) demonstrated significant neuroprotective activity even after delayed administration at 1 h, 3 h and 5 h after I/R. RA 50 mg/kg attenuated histopathological damage, decreased brain edema, inhibited NF-κB activation and reduced HMGB1 expression. CONCLUSION: These data show that RA protects the brain against I/R injury with a favorable therapeutic time-window by alleviating diabetic cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and its protective effects may involve HMGB1 and the NF-κB signaling pathway.

Sapanisertib attenuates pulmonary fibrosis by modulating Wnt5a/mTOR signalling.

Sapanisertib is an orally bioavailable ATP-dependent high-potential raptor-mTOR (TORC1) inhibitor with antineoplastic activity. Here, the impact of sapanisertib was assessed on transforming growth factor-ß1 (TGF-ß1)-treated L929 and A549 cells and on a rat model of bleomycin pulmonary fibrosis. First, in A549 cells treated with TGF-ß1, sapanisertib significantly suppressed the TGF-ß1-induced epithelial-mesenchymal transition, with elevated and reduced E-cadherin and vimentin expression, respectively. In L929 cells treated with TGF-ß1, sapanisertib significantly blocked the TGF-ß1-induced cell proliferation, with decreases in the extracellular matrix-related proteins collagens I and III and smooth muscle actin and in the mechanism-related proteins hypoxia-inducing factor, mTOR, p70S6K, and Wnt5a. Compared with bleomycin alone, continuous gavage administration of sapanisertib for 14 days reduced pathological scores in bleomycin-induced pulmonary fibrosis rats, with decreases in collagen deposition and in the same proteins as in L929 and A549 cells. Accordingly, our findings show that sapanisertib can ameliorate experimental pulmonary fibrosis by inhibiting Wnt5a/mTOR/HIF-1α/p70S6K.

Paritaprevir ameliorates experimental acute lung injury in vitro and in vivo.

Paritaprevir is a potent inhibitor of the NS3/4A protease used to treat chronic hepatitis C virus infection. However, its therapeutic effect on acute lung injury (ALI) remains to be elucidated. In this study, we investigated the effect of paritaprevir on a lipopolysaccharide (LPS)-induced two-hit rat ALI model. The anti-ALI mechanism of paritaprevir was also studied in human pulmonary microvascular endothelial (HM) cells following LPS-induced injury in vitro. Administration of 30 mg/kg paritaprevir for 3 days protected rats from LPS-induced ALI, as reflected by the changes in the lung coefficient (from 0.75 to 0.64) and lung pathology scores (from 5.17 to 5.20). Furthermore, the levels of the protective adhesion protein VE-cadherin and tight junction protein claudin-5 increased, and the cytoplasmic p-FOX-O1 and nuclear ß-catenin and FOX-O1 levels decreased. Similar effects were observed in vitro with LPS-treated HM cells, including decreased nuclear ß-catenin and FOX-O1 levels and higher VE-cadherin and claudin-5 levels. Moreover, ß-catenin inhibition resulted in higher p-FOX-O1 levels in the cytoplasm. These results suggested that paritaprevir could alleviate experimental ALI via the ß-catenin/p-Akt/ FOX-O1 signaling pathway.

Subacute toxicity evaluations of LPM3480392 in rats, a full µ-opioid receptor biased agonist.

Opiates produce analgesia via G-protein signaling, and adverse effects, such as respiratory depression and decreased bowel motility, by ß-arrestin pathway. Oliceridine, a G protein-biased MOR agonist, only presents modest safety advantages as compared to other opiates in clinical trials, possibly due to its limited bias. Our previous study shown that LPM3480392, a full MOR biased agonist, is selective for the Gi pathway over the ß-arrestin-2. In the present article, we evaluated the subacute toxicity of LPM3480392 in rats. The rats were administered with control article or LPM3480392 0.6, 1.2 or 2.4 mg/kg/day for 4 consecutive weeks followed by a 4-week recovery phase. Intravenous infusion was conducted at tail vein at 0.2, 0.4 or 0.8 mg/kg/day with a dosing volume of 10 mL/kg and 5 min/rat/dose, three times a day with an interval of approximately 4 h. The concomitant toxicokinetics study was conducted. Two unscheduled rats at 2.4 mg/kg/day died with no clear cause. For the scheduled necropsy, the major effects were associated with the MOR agonist-related pharmacodynamic properties of LPM3480392 (e.g., increased activity, increased muscle tone; decreased food consumption and body weight gain; and clinical chemistry changes related with decreased food consumption) in three LPM3480392 groups. In addition, LPM3480392 at 2.4 mg/kg/day also induced deep respiration and histopathology changes in testis and epididymis in sporadic individual rats. However, different from other opiates, LPM3480392 presents weak/no immunosuppression and the decreased adrenal gland weight, which may be due to LPM3480392' full MOR bias. At the end of recovery phase, all findings were recovered to some extent or completely. In the toxicokinetics study, the dose-dependent elevation of drug exposure was observed, which partly explained the toxicity of high dose. In summary, LPM3480392 has exhibited good safety characteristics in this subacute toxicity study in rats.

Reproductive and developmental toxicity assessments of LPM3480392, a novel mu opioid receptor biased agonist in rats.

Opioids remain the most powerful analgesics for moderate to severe pain but their clinical use, misuse and abuse has been an alarming medical problem, especially for those users at child-bearing age. Mu-opioid receptor (MOR) biased agonists have been suggested as superior alternatives with better therapeutic ratios. We recently discovered and characterized a novel MOR biased agonist, LPM3480392, which demonstrates robust analgesic effect, favorable pharmacokinetic performance, and mild respiratory suppression in vivo. To understand the safety profile of LPM3480392 on the reproductive system and embryonic development, this study evaluated the effects of LPM3480392 on the fertility and early embryonic development, embryo-fetal development, and pre- and postnatal development in rats. Results showed that LPM3480392 had mild effects on parental male and female animals, accompanied by subtle early embryonic loss and delayed ossification of fetal development during organogenesis period. In addition, although minor effects were found on normal developmental milestones and behaviors in the pups, there was no evidence of malformed effect. In conclusion, these results suggest that LPM3480392 has a favorable safety profile with only minor effects on the reproductive and developmental outcomes in animals, which support the development of LPM3480392 as a novel analgesic.

Forsythoside B protects against experimental sepsis by modulating inflammatory factors.

The present study investigated the effects of Forsythoside B on an experimental model of sepsis induced by caecal ligation and puncture (CLP) in rats and elucidated the potential mechanism in cultured RAW 264.7 cells. Results showed that Forsythoside B concentration-dependently down-regulated the levels of TNF-α, IL-6 and high-mobility group-box 1 protein (HMGB1) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, inhibited the IκB kinase (IKK) pathway and modulated nuclear factor (NF)- κB. Intravenous injection (i.v.) of Forsythoside B alone or plus Imipenem reduced serum levels of TNF-α, IL-6, HMGB1, triggering receptor expressed on myeloid cells (TREM-1) and endotoxin, while the serum level of IL-10 was up-regulated and myeloperoxidase (MPO) in lung, liver and small intestine was reduced. Meanwhile, i.v. of Forsythoside B alone or plus Imipenem reduced CLP-induced lethality in rats. These data indicated that the antisepsis effect of Forsythoside B is mediated by decreasing local and systemic levels of a wide spectrum of inflammatory mediators. Its antisepsis mechanism may be that Forsythoside B binds to LPS and reduces the biological activity of serum LPS, and inhibits NF-κB activition. Our studies enhance the case for the use of Forsythoside B in sepsis. Forsythoside B itself has promise as a therapy for the treatment of sepsis in humans.

Antifibrotic effects of protocatechuic aldehyde on experimental liver fibrosis.

BACKGROUND: Recent studies have demonstrated that transforming growth factor-ß1 (TGF-ß1) and connective transforming growth factor (CTGF) are associated with the pathophysiology of liver fibrosis. We isolated protocatechuic aldehyde, the major degradation of phenolic acids. OBJECTIVE: This study was carried out to investigate the potential antifibrotic effect of Protocatechuic aldehyde (PA) on experimental liver fibrosis in vitro and in vivo, and to explore its possible mechanism. MATERIALS AND METHODS: Cell proliferation was determined. Type I collagen, type III collagen, transforming growth factor-ß1 (TGF-ß1) and connective transforming growth factor (CTGF) were measured by ELISA kits in TNF-α stimulated HSCs. In the carbon tetrachloride (CCL(4))-induced rat liver fibrosis model, liver fibrosis grade and histopathological changes were evaluated, and biochemical indicators were determined. Furthermore, immunostaining and Western blot analysis were used to detect hepatic TGF-ß1 and CTGF expression in liver tissue. RESULTS: Overall, our results indicated that PA inhibits HSCs proliferation, inhibits the levels of TGF-ß1, CTGF, type I collagen and type III collagen in TNF-α stimulated HSCs. Treatment of PA causes a significant reduction in fibrosis grade, ameliorates biochemical indicators and histopathological morphology, and reduces liver TGF-ß1 and CTGF expression in rat model of CCL(4)-induced liver fibrosis. CONCLUSION: These findings suggest that PA has potentially conferring antifibrogenic effects.

The protective role of raltegravir in experimental acute lung injury in vitro and in vivo.

Disruption of pulmonary endothelial permeability and associated barrier integrity increase the severity of acute respiratory distress syndrome (ARDS). This study investigated the potential ability of the human immunodeficiency virus-1 (HIV-1) integrase inhibitor raltegravir to protect against acute lung injury (ALI) and the underlying mechanisms. Accordingly, the impact of raltegravir treatment on an in vitro lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMEC) model of ALI and an in vivo LPS-induced two-hit ALI rat model was examined. In the rat model system, raltegravir treatment alleviated ALI-associated histopathological changes, reduced microvascular permeability, decreased Evans blue dye extravasation, suppressed the expression of inflammatory proteins including HMGB1, TLR4, p-NF-κB, NLRP3, and MPO, and promoted the upregulation of protective proteins including claudin 18.1, VE-cadherin, and aquaporin 5 as measured via western blotting. Immunohistochemical staining further confirmed the ability of raltegravir treatment to reverse LPS-induced pulmonary changes in NLRP3, claudin 18.1, and aquaporin 5 expression. Furthermore, in vitro analyses of HPMECs reaffirmed the ability of raltegravir to attenuate LPS-induced declines in VE-cadherin and claudin 18.1 expression while simultaneously inhibiting NLRP3 activation and reducing the expression of HMGB1, TLR4, and NF-kB, thus decreasing overall vascular permeability. Overall, our findings suggested that raltegravir may represent a viable approach to treating experimental ALI that functions by maintaining pulmonary microvascular integrity.

Verdiperstat attenuates acute lung injury by modulating MPO/µ-calpain/ß-catenin signaling.

Verdiperstat, a myeloperoxidase (MPO) inhibitor, is a well-known drug used for the treatment of multisystem atrophy. However, its therapeutic effect on acute lung injury (ALI) remains to be elucidated. In this study, the effect of verdiperstat on lipopolysaccharide (LPS)-induced two-hit rat ALI model was studied in vivo. Subsequently, to explore the anti-ALI mechanism of verdiperstat, an LPS-induced injury in human pulmonary microvascular endothelial cells (HMs) was studied in vitro. The continuous administration of verdiperstat at 120 mg/kg for 3 days exerted a protective effect on the LPS-induced two-hit rat ALI model, as reflected by the change in the lung coefficient and lung pathology scores from 0.72 to 0.61 and 6.08 to 4.37, respectively. Furthermore, the values of protective adhesion protein VE-cadherin and tight junction protein claudin 5 changed from 0.42 to 0.97 and 0.25 to 0.72, but MPO, the ratio of N-µ-calpain to µ-calpain, and the distribution of ß-catenin in the nucleus changed from 3.04 to 2.17, 0.62 to 0.38 and 2.25 to 0.76, respectively. LPS-induced HMs in vitro also showed similar results, including lower MPO and the distribution of ß-catenin in the nucleus, but higher VE-cadherin claudin 5 and N-µ-calpain. Moreover, MPO inhibition resulted in lower µ-calpain activation and lower ß-catenin in the nucleus. Our cumulative results suggest that verdiperstat alleviates ALI by strengthening VE-cadherin and claudin 5 through the inhibition of MPO/µ-calpain/ß-catenin activation.

Synthesis, biological, and structural explorations of a series of µ-opioid receptor (MOR) agonists with high G protein signaling bias.

Biased agonism refers to the ability of compounds to drive preferred signaling pathways and avoid adverse signaling pathways in a ligand-dependent manner for some G-protein-coupled receptors. It is thought that the separation of therapeutic efficacy (e.g., analgesia) from adverse effects (e.g., respiration depression) can be achieved through the design of biased MOR agonists and one example is the recently approved MOR biased agonist oliceridine (TRV130). However, oliceridine only demonstrates modest beneficial effects as compared to other opioids in terms of therapeutic/adverse effect balance. One possibility attributable to the modest success of oliceridine is its limited bias, and as such developing MOR ligands with a more biased agonism profile could in theory further improve the beneficial effects of the ligands. Here, we rationally designed and synthesized a series of derivatives as potent highly biased MOR agonists (19a-v) through the modification and structure-activity relationship study of TRV130. This novel synthetic molecule, LPM3480392 (19m), demonstrated improved in vitro biased agonism (EC50 = 0.35 nM, Emax = 91.4%) with no measured ß-arrestin recruitment (EC50 > 30000 nM, Emax = 1.6%), good brain penetration (B/P ratio = 4.61, 0.25 h post-IV dosing 2.0 mg/kg), a favorable pharmacokinetic profile (distribution volume = 10766 mL/kg, t1/2 = 1.9 h) and produced potent antinociceptive effect with reduced respiratory suppression (sO2(%) = 92.17, 0.32 mg/kg, SC) as compared to TRV130. LPM3480392 has completed preclinical studies and is currently under clinical development (CTR20210370) as an analgesic for the treatment of moderate to severe pain.

Discovery of the thieno[2,3-d]pyrimidine-2,4-dione derivative 21a: A potent and orally bioavailable gonadotropin-releasing hormone receptor antagonist.

The gonadotropin releasing hormone receptor (GnRH-R) is a G protein-coupled receptor (GPCR) belonging to the rhodopsin family. GnRH-R antagonists suppress testosterone to castrate level more rapidly than gonadotropin releasing hormone agonists but lack the flare phenomenon often seen during the early period of GnRH-R agonist treatment. Recently orgovyx (relugolix) was approved as the first oral GnRH-R antagonist for the treatment of advanced prostate cancer. However, orgovyx has demonstrated poor pharmacokinetic profile with low oral bioavailability and high efflux. Here, we rationally designed and synthesized a series of derivatives (13a-m, 21a-i) through the modification and structure-activity relationship study of relugolix, which led to the discovery of 21a as a highly potent GnRH-R antagonist (IC50 = 2.18 nM) with improved membrane permeability (Papp, A-B = 0.98 × 10-6 cm/s) and oral bioavailability (F % = 44.7). Compound 21a showed high binding affinity (IC50 = 0.57 nM) and potent in vitro antagonistic activity (IC50 = 2.18 nM) at GnRH-R. 21a was well tolerated and efficacious in preclinical studies to suppress blood testosterone levels, which merits further investigation as a candidate novel GnRH-R antagonist for clinical studies.

Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling.

Tirbanibulin (KX-01) is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward the Src kinase. This study assessed the impact of KX-01 on cobalt chloride (CoCl2)-treated L929 cells and bleomycin (BLM)-induced pulmonary fibrosis in rats to evaluate the efficacy of this compound in vitro and in vivo, respectively. In CoCl2-treated L929 cells, KX-01 significantly reduced the expression of smooth muscle actin (α-SMA), collagen I, collagen III, hypoxia inducing factor (HIF-1α), signal transducers and transcriptional activators (p-STAT3), and p-Src. In BLM-induced pulmonary fibrosis rats, KX-01 reduced pathological scores, collagen deposition, α-SMA, collagen I, collagen III, p-Src, HIF-1α, and p-STAT3. Overall, these findings revealed that KX-01 can alleviate experimental pulmonary fibrosis via suppressing the p-SRC/p-STAT3 signaling pathways.

Pharmacological Characterization of Toludesvenlafaxine as a Triple Reuptake Inhibitor.

Toludesvenlafaxine hydrochloride dihydrate is a novel chemical entity and a potential triple monoamine reuptake inhibitor. This study characterized the in vitro triple reuptake inhibition activity, antidepressant-like activity in animals, and pharmacokinetic profiles in rats of toludesvenlafaxine. Binding affinity was determined using human serotonin transporter (SERT) protein, norepinephrine transporter (NET) protein and dopamine transporter (DAT) protein, and the reuptake inhibition was determined using Chinese hamster ovary cells expressing human SERT, NET and DAT. The antidepressant-like activity was examined in rat chronic unpredictable mild stress model and olfactory bulbectomized model. In rats, the tissue distribution and pharmacokinetic parameters were determined. Toludesvenlafaxine had high binding affinity on SERT, NET and DAT, and significantly inhibited the reuptake of serotonin (IC50 = 31.4 ± 0.4 nM), norepinephrine (IC50 = 586.7 ± 83.6 nM) and dopamine (IC50 = 733.2 ± 10.3 nM) in vitro. Toludesvenlafaxine demonstrated significant antidepressant-like effects in rat models at 8-16 mg/kg. In addition, toludesvenlafaxine significantly reduced serum corticosterone and significantly increased testosterone levels in rats. Toludesvenlafaxine was quickly absorbed and converted to O-desvenlafaxine (ODV) after oral administration, both of which were selectively distributed into the hypothalamus with high concentration. Plasma ODV exposure was proportionally related to the doses after oral dosing. These results suggest that toludesvenlafaxine is a triple reuptake inhibitor with relatively fast-acting antidepressant-like activity and good therapeutic profile including improvement of anhedonia and sexual function.

Inhibition of nuclear factor-κB by 6-O-acetyl shanzhiside methyl ester protects brain against injury in a rat model of ischemia and reperfusion.

BACKGROUND: Recent studies have demonstrated an inflammatory response associated with the pathophysiology of cerebral ischemia. The beneficial effects of anti-inflammatory drugs in cerebral ischemia have been documented. When screening natural compounds for drug candidates in this category, we isolated 6-O-acetyl shanzhiside methyl ester (ND02), an iridoid glucoside compound, from the leaves of Lamiophlomis rotata (Benth.) Kudo. The objectives of this study were to determine the effects of ND02 on a cultured neuronal cell line, SH-SY5Y, in vitro, and on experimental ischemic stroke in vivo. METHODS: For TNF-α-stimulated SH-SY5Y cell line experiments in vitro, SH-SY5Y cells were pre-incubated with ND02 (20 µM or 40 µM) for 30 min and then incubated with TNF-α (20 ng/ml) for 15 min. For in vivo experiments, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion for 23 h. RESULTS: ND02 treatment of SH-SY5Y cell lines blocked TNF-α-induced nuclear factor-κB (NF-κB) and IκB-α phosphorylation and increased Akt phosphorylation. LY294002 blocked TNF-α-induced phosphorylation of Akt and reduced the phosphorylation of both IκB-α and NF-κB. At doses higher than 10 mg/kg, ND02 had a significant neuroprotective effect in rats with cerebral ischemia and reperfusion (I/R). ND02 (25 mg/kg) demonstrated significant neuroprotective activity even after delayed administration 1 h, 3 h and 5 h after I/R. ND02, 25 mg/kg, attenuated histopathological damage, decreased cerebral Evans blue extravasation, inhibited NF-κB activation, and enhanced Akt phosphorylation. CONCLUSION: These data show that ND02 protects brain against I/R injury with a favorable therapeutic time-window by alleviating cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and that these protective effects may be due to blocking of neuronal inflammatory cascades through an Akt-dependent NF-κB signaling pathway.