Preliminary Screening and Study of Key Gene Expression in Endometrial Window Period.

Background: Implantation is a highly coordinated event involving both embryonic and endometrial participation. The endometrium expresses a complex array of proteins during the menstrual cycle many of which help to define a period of receptivity collectively known as the "window of implantation." Objective: Using high-throughput RNA sequencing technology analysis to find differentially expressed genes before and after the endometrial window, and search for key marker genes of the membrane implantation window. Design: This was a retrospective study. Setting: This study was performed in the Department of Obstetrics and Gynecology, Taizhou People's Hospital. Participants: Fifty patients with repeated implantation failure in in vitro fertilization were selected and were divided into (1) the normal window group (36 cases); (2) the window forward group (8 cases); and (3) the window backward group (6 cases) based on endometrial biopsy findings. Interventions: Using RNA sequencing technology combined with biological information analysis tools to analyze the differentially-expressed genes in 9 samples. Gene Ontology databases were used for the functional annotation of these differentially-expressed genes. Kyoto Encyclopedia of Genes and Genomes analysis was used to draw a signal path diagram. Primary Outcome Measures: (1) Screening of differentially-expressed genes and (2) functional analysis of the differential genes. Results: A total of 22 differentially-expressed genes related to endometrial receptivity were obtained by transcriptome sequencing. Seven of the 22 differentially-expressed genes have been shown to have a close relationship with the endometrial receptive window period. Further, it was proved that the Wnt signaling pathway and mitogen-activated protein kinase signaling pathway were closely related to endometrial receptivity. Conclusions: The present study identified a series of key genes and pathways that may be involved in the endometrial window period, providing an experimental and theoretical basis for exploring the personalized embryo transfer program.

Azepino-indazoles as calcitonin gene-related peptide (CGRP) receptor antagonists.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown clinically to be effective treatments for migraine. Zavegepant (BHV-3500, BMS-742413) is a high affinity antagonist of the CGRP receptor (hCGRP Ki = 0.023 nM) that has demonstrated efficacy in the acute treatment of migraine with intranasal delivery in a Phase 2/3 trial, despite showing low oral bioavailability in rats (FPO = 1.7%). Using zavegepant as a template, we sought to improve oral bioavailability through a series of azepinones which were designed in an attempt to reduce the number of rotatable bonds. These efforts led to the discovery of compound 21 which was able to mostly maintain high affinity binding (hCGRP Ki = 0.100 nM) and in vivo efficacy in the marmoset facial blood flow assay, while greatly improving oral bioavailability (rat FPO = 17%).

Erenumab (AMG 334), a monoclonal antagonist antibody against the canonical CGRP receptor, does not impair vasodilatory or contractile responses to other vasoactive agents in human isolated cranial arteries.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a neuronal transmitter present in intracranial sensory nerves, where it is involved in migraine pathophysiology as well as other biological functions. Recently, the fully human monoclonal antibody erenumab (AMG 334), which targets the canonical calcitonin gene-related peptide receptor, showed significant prophylactic efficacy and favourable safety in phase II and III clinical trials for episodic and chronic migraine and is now approved for migraine prevention in several countries. OBJECTIVE: Given that calcitonin gene-related peptide can mediate vasodilation, we investigated the effect of erenumab on vasoactive responses in the presence or absence of various vasodilatory and vasocontractile mediators in a model using isolated human cerebral and meningeal arteries. METHODS: Ring segments of human isolated cerebral and meningeal arteries were mounted in a sensitive myograph. On arterial segments pre-contracted with 30 mM potassium chloride, vasoactive responses to calcitonin gene-related peptide were studied in the presence of different concentrations of erenumab. At the maximal tested inhibitory concentration of erenumab (100 nM), functional arterial relaxation in response to nicardipine or substance P, and the contractile responses to sumatriptan and dihydroergotamine were examined. RESULTS: 30 mM potassium chloride produced a stable contraction of the vessel segments and calcitonin gene-related peptide induced a concentration-dependent relaxation. We observed that (i) erenumab had no direct contractile or relaxant effects per se (by itself), (ii) pre-treatment with erenumab antagonized the calcitonin gene-related peptide-induced relaxation in a competitive manner, (iii) the relaxant responses to nicardipine or substance P were unaffected in the presence of erenumab and (iv) the contraction induced by sumatriptan or dihydroergotamine was not modified by erenumab. CONCLUSION: Our findings demonstrate that erenumab, while not associated with vasoactive properties per se, specifically inhibits calcitonin gene-related peptide-induced relaxation of cranial arteries without impacting vasodilatory responses or contractile responses of endogenous or pharmacological vasoactive compounds.

PAC1 receptor mRNA and protein distribution in rat and human trigeminal and sphenopalatine ganglia, spinal trigeminal nucleus and in dura mater.

BACKGROUND: To further understand the role of pituitary adenylate cyclase-activating polypeptide 1 (PAC1) receptors in headache disorders, we mapped their expression in tissues of the trigemino-autonomic system by immunohistochemistry and in situ hybridization. METHODS: To optimize screening for monoclonal antibodies suitable for immunohistochemistry on formalin-fixed, paraffin-embedded tissues, we developed a new enzyme-linked immunosorbent assay using formalin-fixed, paraffin-embedded cells overexpressing human PAC1 receptors. 169G4.1 was selected from these studies for analysis of rat and human tissues and chimerized onto a mouse backbone to avoid human-on-human cross-reactivity. Immunoreactivity was compared to PAC1 receptor mRNA by in situ hybridization in both species. RESULTS: 169G4.1 immunoreactivity delineated neuronal cell bodies in the sphenopalatine ganglion in both rat and human, whereas no staining was detected in the trigeminal ganglion. The spinal trigeminal nucleus in both species showed immunoreactivity as especially strong in the upper laminae with both cell bodies and neuropil being labelled. No immunoreactivity was seen in either rat or human dura mater vessels. In situ hybridization in both species revealed mRNA in sphenopalatine ganglion neurons and the spinal trigeminal nucleus, a weak signal in the trigeminal nucleus and no signal in dural vessels. CONCLUSION: Taken together, these data support a role for PAC1 receptors in the trigemino-autonomic system as it relates to headache pathophysiology.

Characterisation of vasodilatory responses in the presence of the CGRP receptor antibody erenumab in human isolated arteries.

BACKGROUND: Migraine is associated with activation of the trigeminovascular system, release of calcitonin gene-related peptide (CGRP) and dilation of dural arteries. Novel treatments target calcitonin gene-related peptide or its receptor, which are present in all vascular beds, raising cardiovascular concerns. Erenumab is a human CGRP-receptor antibody approved for the prophylactic treatment of migraine. METHODS: We characterised the relaxant responses to CGRP in the absence and presence of erenumab (1 µM) in isolated human middle meningeal, internal mammary and (proximal and distal) coronary arteries. Furthermore, in human internal mammary arteries from cardiovascularly-compromised patients, we assessed the pharmacological specificity of erenumab by investigating whether the vasodilatory responses to acetylcholine, sodium nitroprusside, pituitary adenylate cyclase activating polypeptide-38 (PACAP), vasoactive intestinal peptide and nicardipine, along with the vasoconstrictor responses to dihydroergotamine, were modified by erenumab. RESULTS: Calcitonin gene-related peptide induced concentration-dependent vasodilatory responses in all vessels studied that were significantly antagonised by erenumab. In human internal mammary arteries from cardiovascularly-compromised patients, the responses to acetylcholine, sodium nitroprusside, PACAP, vasoactive intestinal peptide, nicardipine and dihydroergotamine were unaffected by erenumab. CONCLUSION: Erenumab inhibits calcitonin gene-related peptide-induced vasodilatory responses in human middle meningeal arteries, human internal mammary arteries and human coronary arteries. Moreover, erenumab shows functional specificity as no interaction was observed with the relaxant responses to several vasodilators, nor the dihydroergotamine-dependent vasoconstrictor responses.

Nonclinical safety evaluation of erenumab, a CGRP receptor inhibitor for the prevention of migraine.

Calcitonin gene-related peptide (CGRP) and its receptor have been implicated as a key mediator in the pathophysiology of migraine. Thus, erenumab, a monoclonal antibody antagonist of the CGRP receptor, administered as a once monthly dose of 70 or 140 mg has been approved for the preventive treatment of migraine in adults. Due to the species specificity of erenumab, the cynomolgus monkey was used in the pharmacology, pharmacokinetics, and toxicology studies to support the clinical program. There were no effects of erenumab on platelets in vitro (by binding, activation or phagocytosis assays). Specific staining of human tissues with erenumab did not indicated any off-target binding. There were no erenumab-related findings in a cardiovascular safety pharmacology study in cynomolgus monkeys or in vitro in human isolated coronary arteries. Repeat-dose toxicology studies conducted in cynomolgus monkeys at dose levels up to 225 mg/kg (1 month) or up to 150 mg/kg (up to 6 months) with twice weekly subcutaneous (SC) doses showed no evidence of erenumab-mediated adverse toxicity. There were no effects on pregnancy, embryo-fetal or postnatal growth and development in an enhanced pre-postnatal development study in the cynomolgus monkey. There was evidence of placental transfer of erenumab based on measurable serum concentrations in the infants up to 3 months post birth. The maternal and developmental no-observed-effect level (NOEL) was the highest dose tested (50 mg/kg SC Q2W). These nonclinical data in total indicate no safety signal of concern to date and provide adequate margins of exposure between the observed safe doses in animals and clinical dose levels.

Effects of monoclonal antagonist antibodies on calcitonin gene-related peptide receptor function and trafficking.

BACKGROUND: Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated. METHODS: Here we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy. RESULTS: Using this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model. CONCLUSION: These data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.

Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor.

Therapeutic agents that block the calcitonin gene-related peptide (CGRP) signaling pathway are a highly anticipated and promising new drug class for migraine therapy, especially after reports that small-molecule CGRP-receptor antagonists are efficacious for both acute migraine treatment and migraine prevention. Using XenoMouse technology, we successfully generated AMG 334, a fully human monoclonal antibody against the CGRP receptor. Here we show that AMG 334 competes with [(125)I]-CGRP binding to the human CGRP receptor, with a Ki of 0.02 nM. AMG 334 fully inhibited CGRP-stimulated cAMP production with an IC50 of 2.3 nM in cell-based functional assays (human CGRP receptor) and was 5000-fold more selective for the CGRP receptor than other human calcitonin family receptors, including adrenomedullin, calcitonin, and amylin receptors. The potency of AMG 334 at the cynomolgus monkey (cyno) CGRP receptor was similar to that at the human receptor, with an IC50 of 5.7 nM, but its potency at dog, rabbit, and rat receptors was significantly reduced (>5000-fold). Therefore, in vivo target coverage of AMG 334 was assessed in cynos using the capsaicin-induced increase in dermal blood flow model. AMG 334 dose-dependently prevented capsaicin-induced increases in dermal blood flow on days 2 and 4 postdosing. These results indicate AMG 334 is a potent, selective, full antagonist of the CGRP receptor and show in vivo dose-dependent target coverage in cynos. AMG 334 is currently in clinical development for the prevention of migraine.

Synthesis and SAR of calcitonin gene-related peptide (CGRP) antagonists containing substituted aryl-piperazines and piperidines.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide implicated in the pathophysiology of migraine. In the course of seeking CGRP antagonists with improved oral bioavailability, metabolic stability, and pharmacokinetic properties, lower molecular weight, structurally simpler piperidine and piperazine analogs of BMS-694153 were prepared. Several were found to have nM binding affinity in vitro. The synthesis and SAR of these substituted piperidine and piperazine CGRP antagonists are discussed.

Effects of Shenlian extract on experimental atherosclerosis in ApoE-deficient mice based on ultrasound biomicroscopy.

BACKGROUND: This study directly and dynamically investigated the effects of SL extract (i.e., a combination of Radix Salviae miltiorrhizae and Andrographis paniculata extract) on plaque progression in vivo by high resolution ultrasound biomicroscopy (UBM). METHODS: An atherosclerosis model was established by placing a perivascular collar on the right common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice. Thickness, plaque area and local blood flow were observed by UBM, pathological changes were observed by histochemical staining, and lipid levels were measured by respective commercially available kits. RESULTS: Compared with the model group, the SL extract groups showed reduced wall thickness of the aortic arch (GC: P = 0.001, P = 0.002, and P < 0.001; LC: P < 0.001, P < 0.001, and P < 0.001; BC: P = 0.027, P = 0.017, and P = 0.003; respectively), which presented with retarded plaque progression of the cartoid artery with concordantly increased blood flow (P = 0.002 and P < 0.001) as visualized in vivo by UBM. Histological analysis confirmed the reduction of carotid atherosclerosis. CONCLUSIONS: The SL extract inhibited the formation of atherosclerotic plaques in an ApoE-/- mice model by UBM analysis, and did so by effects that ameliorated local blood flow and improved blood lipid levels.

Flavonoids casticin and chrysosplenol D from Artemisia annua L. inhibit inflammation in vitro and in vivo.

BACKGROUND: The aim of our experiments was to investigate the anti-inflammatory properties of casticin and chrysosplenol D, two flavonoids present in Artemisia annua L. METHODS: Topical inflammation was induced in ICR mice using croton oil. Mice were then treated with casticin or chrysosplenol D. Cutaneous histological changes and edema were assessed. ICR mice were intragastrically administrated with casticin or chrysosplenol D followed by intraperitoneal injection of lipopolysaccharide (LPS). Mouse Raw264.7 macrophage cells were incubated with casticin or chrysosplenol D. Intracellular phosphorylation was detected, and migration was assessed by trans-well assay. HT-29/NFκB-luc cells were incubated with casticin or chrysosplenol D in the presence or absence of LPS, and NF-κB activation was quantified. RESULTS: In mice, administration of casticin (0.5, 1 and 1.5µmol/cm(2)) and chrysosplenol D (1 and 1.5µmol/cm(2)) inhibited croton oil-induced ear edema (casticin: 29.39-64.95%; chrysosplenol D: 37.76-65.89%, all P<0.05) in a manner similar to indomethacin (0.5, 1 and 1.5µmol/cm(2); 55.63-84.58%). Casticin (0.07, 0.13 and 0.27mmol/kg) and chrysosplenol D (0.07, 0.14 and 0.28mmol/kg) protected against LPS-induced systemic inflammatory response syndrome (SIRS) in mice (all P<0.05), in a manner similar to dexamethasone (0.03mmol/kg). Casticin and chrysosplenol D suppressed LPS-induced release of IL-1 beta, IL-6 and MCP-1, inhibited cell migration, and reduced LPS-induced IκB and c-JUN phosphorylation in Raw264.7 cells. JNK inhibitor SP600125 blocked the inhibitory effect of chrysosplenol D on cytokine release. CONCLUSIONS: The flavonoids casticin and chrysosplenol D from A. annua L. inhibited inflammation in vitro and in vivo.

Therapeutic Monoclonal Antibodies: What Headache Specialists Need to Know.

BACKGROUND: Monoclonal antibodies (mAbs) are now an important part of the treatment armamentarium for a wide range of conditions including cancer, autoimmune diseases, inflammatory diseases of the joint and bowel, transplant rejection, and multiple sclerosis. Significant progress over the last 30 years in the development of therapeutic mAbs has resulted in improved efficacy and safety. Monoclonal antibodies approved for the treatment of neurological illnesses so far are limited to use in multiple sclerosis. Several therapeutic mAbs have completed phase 2 clinical trials for migraine prevention, and there are phase 3 trials underway for migraine prophylaxis and for cluster headache at the time of this writing. AIM: The purpose of this review is to discuss the characteristics of mAbs, including their mechanism of action and safety profile, and briefly describe the mAbs being evaluated for the prevention of migraine and cluster headaches. SUMMARY: Monoclonal antibodies have several features that distinguish them from small molecules, including very high selectivity, relatively long half-life that generally allows for once or twice monthly dosing, and significantly reduced potential for drug-drug interactions or other nontarget related toxicities. The clinical development of mAbs that target calcitonin gene-related peptide and its receptor is underway and will evaluate this promising new drug class for the prevention of migraine and cluster headache.

[Protection of Shenlian extracts to PM2.5 infected RAW 264.7 cell damage].

The aim of this research is to investigate the protection of PM2.5 infected RAW264.7 cell by traditional Chinese medicine (TCM)--Shenlian(SL) extracts and to establish the damage model. We use cell growth, cell damage and oxidative stress related markers, and inflammatory cytokines as observation index to evaluate the protection of PM2.5 infected RAW264.7 by SL extract. The results showed that 50 mg x L(-1) PM2.5 could cause cell particle deposition, inhibit the growth of cells, and significantly increase the cell supernatant of LDH, NO release quantity and intracellular reactive oxygen species (ROS) level during 4 h and 24 h. In the intervention of SL extract 50, 25, 10 mg x L(-1), the particle deposition of RAW264.7 cells, cell supernatant of LDH, NO, IL(-1) beta release, MCP-1 was significantly decreased, the SOD activity increased significantly. It shows that SL extracts of PM2.5 infected RAW264.7 cell damage has obvious protective effect, the effect may be related to the direct protection of cells, reduce oxidative stress and inflammatory injury.

Serendipitous oxidation product of BIBN4096BS: a potent CGRP receptor antagonist.

An oxidation product (5) formed during the synthesis of BIBN-4096BS (1) was found to be a potent CGRP antagonist (IC50=0.11nM). While 5 was found to be ten-fold less potent than 1, another analog 8 with lower molecular weight containing the oxidized fragment demonstrated twenty-fold higher activity than its parent 7. Alternative conditions which preclude the formation of the oxidation product are described. The activities of 1, 5, 7 and 8 in functional cAMP assay are also discussed.

[Pathogenesis of plaque destabilization induced by PM2.5 exposure and coping strategies].

With the increasingly more serious environmental pollution in China in recent years, effective intervention with PM25-induced health risks has become a major scientific issue to be addressed urgently in medical research field in China. NOD-like receptors (NLRs) are a family of cytoplasmic pattern-recognition receptors that have critical roles in innate immunity. On the basis of study progresses in international cardiovascular disease research "Fine particulate matter exposure is a modifiable risk factor for the morbidity and mortality of cardiovascular diseases", and with reference to the current understanding of pulmonary inflammation and oxidative stress in PM2.5-induced acute coronary syndrome, this study intended to investigate whether intracellular pattern recognition NL-RP3 plays a important role in the inital event of PM2.5 induced vessel inflammation as a foreign matter in the process of plaque destabilization and to thoroughly explore the underlying mechanisms responsible for PM2.5-induced acute cardiovascular events. On the other hand, it also studies the feasibility of using traditional Chinese medicine to treat plaque destabilization cause by PM2.5 exposure and discuss it's pathogenesis and intervention strategy based on TCM theory. This paper in order to provide scientific basis for social focal issues in public health proactively and offers the references for relevant research.

Preparation of imidazoles as potent calcitonin gene-related peptide (CGRP) antagonists.

Several new potent CGRP receptor antagonists have been prepared in which the amide bond of lead compound 1 has been replaced by bioisosteric imidazole moieties. Substitution at N-1 of the imidazole was optimized to afford compounds with comparable potency to that of lead 1. Conformational restraint of the imidazole to form tetrahydroimidazo[1,5-a]pyrazine 43 gave substantially improved permeability.

The synthesis and SAR of calcitonin gene-related peptide (CGRP) receptor antagonists derived from tyrosine surrogates. Part 2.

Various substituted indazole and benzoxazolone amino acids were investigated as d-tyrosine surrogates in highly potent CGRP receptor antagonists. Compound 3, derived from the 7-methylindazole core, afforded a 30-fold increase in CGRP binding potency compared with its unsubstituted indazole analog 1. When dosed at 0.03mg/kg SC, compound 2 (a racemic mixture of 3 and its (S)-enantiomer) demonstrated robust inhibition of CGRP-induced increases in mamoset facial blood flow up to 105min. The compound possesses a favorable predictive in vitro toxicology profile, and good aqueous solubility. When dosed as a nasal spray in rabbits, 3 was rapidly absorbed and showed good intranasal bioavailability (42%).

Discovery of (R)-N-(3-(7-methyl-1H-indazol-5-yl)-1-(4-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide (BMS-742413): a potent human CGRP antagonist with superior safety profile for the treatment of migraine through intranasal delivery.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.

Unexpected thrombocytopenia and anemia in cynomolgus monkeys induced by a therapeutic human monoclonal antibody.

Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥ 50 mg/kg had unexpected acute thrombocytopenia (nadir ~3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab')2 portion of mAbY.1 nor the F(ab')2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.