Raloxifene encapsulated spanlastic nanogel for the prevention of bone fracture risk via transdermal administration: Pharmacokinetic and efficacy study in animal model.

This research work is to evaluate spanlastic-loaded raloxifene (RLX) nanogel administration via the transdermal route to avoid its hepatic metabolism and to enhance the bioavailability for better management of osteoporosis. RLX-loaded spanlastic nanogel was prepared and characterized for its viscosity, pH, spreadability, and texture profile. The formulation was applied on the skin surface of the animal for pharmacokinetic evaluation, and later, the efficacy of the formulation was assessed in ovariectomized female Wistar rats. The nanogel was obtained with a viscosity (2552.66 ± 30.61 cP), pH (7.1 ± 0.1), and spreadability (7.1 ± 0.2 cm). The texture properties, cohesiveness, and adhesiveness of the nanogel showed its suitability for transdermal application. Nanogel showed no sign of edema and erythema in the skin irritation test which revealed its safety for transdermal application. The t1/2 obtained for RLX-spanlastic nanogel (37.02 ± 0.59 h) was much higher than that obtained for RLX-oral suspension (14.43 h). The relative bioavailability was found to be 215.96% for RLX-spanlastic nanogel, and the drug and formulation did not show any toxicity in any of the vital organs, as well as no hematological changes occurring in blood samples. In microarchitectural measurement, RLX-spanlastic nanogel exhibited no unambiguous deviations along with improved bone mineral density compared to the RLX suspension treated group. Transdermal administration of RLX-spanlastic nanogel showed significant improvement of drug bioavailability (approx. twice to oral administration) without any toxic effect in the treated rats. Hence, spanlastic nanogel could be a better approach to deliver RLX via transdermal route for the management of osteoporosis.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes.

Currently, the development of selective fluorescent probes toward targeted enzymes is still a great challenge, due to the existence of numerous isoenzymes that share similar catalytic capacity. Herein, a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s) for cytochrome P450 (CYP) which are key enzymes involving in metabolism of endogenous substances and drugs. In the first-stage of our filtering approach, near-infrared (NIR) fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system. In the second stage of our filtering approach, these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s). Using our double-filtering approach, probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2, respectively, to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis. These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.

Human Recombinant Relaxin (Serelaxin) as Anti-fibrotic Agent: Pharmacology, Limitations and Actual Perspectives.

Relaxin (recombinant human relaxin-2 hormone; RLX-2; serelaxin) had raised expectations as a new medication for fibrotic diseases. A plethora of in vitro and in vivo studies have offered convincing demonstrations that relaxin promotes remodeling of connective tissue extracellular matrix mediated by inhibition of multiple fibrogenic pathways, especially the downstream signaling of transforming growth factor (TGF)-ß1, a major pro-fibrotic cytokine, and the recruitment and activation of myofibroblasts, the main fibrosis-generating cells. However, all clinical trials with relaxin in patients with fibrotic diseases gave inconclusive results. In this review, we have summarized the molecular mechanisms of fibrosis, highlighting those which can be effectively targeted by relaxin. Then, we have performed a critical reappraisal of the clinical trials performed to date with relaxin as an anti-fibrotic drug, in order to highlight their key points of strength and weakness and to identify some future opportunities for the therapeutic use of relaxin, or its analogues, in fibrotic diseases and pathologic scarring which, in our opinion, deserve to be investigated.

Relaxin abrogates genomic remodeling of the aged heart.

"Healthy" aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in clinical trials, Relaxin-therapy for 2-days reduced mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short lifespan (2-3h) but long-lasting protective actions suggested that relaxin acts at a genomic level to reverse maladaptive remodeling in AF, HF and aging. Our recent studies showed that a 2-week treatment with Relaxin (0.4mg/kg/day) of aged (24months old F-344 rats) increases the expression of voltage-gated Na+ channels (mRNA, Nav1.5 and INa), connexin-43, abrogates inflammatory and immune responses and reverses myocardial fibrosis and cellular hypertrophy of the aged hearts. Relaxin acts directly at a wide range of cell types in the cardiovascular system that express its cognate GPCR receptor, RXFP1. RNA-seq analysis of young and aged hearts with and without Relaxin treatment revealed that "normal" aging altered the expression of ~10% of genes expressed in the ventricles, including: ion channels, components of fibrosis, hemodynamic biomarkers, immune and inflammatory responses which were reversed by Relaxin. The extensive cardiovascular remodeling caused by Relaxin was mediated through the activation of the Wnt/ß-catenin signaling pathway which was otherwise suppressed by in adult cardiomyocytes intracellular by cytosolic Dickkopf1 (Dkk1). Wnt/ß-catenin signaling is a mechanism that can explain the pleiotropic actions of Relaxin and the marked reversal of genomic changes that occur in aged hearts.

The application of nanoparticles in cancer immunotherapy: Targeting tumor microenvironment.

The tumor development and metastasis are closely related to the structure and function of the tumor microenvironment (TME). Recently, TME modulation strategies have attracted much attention in cancer immunotherapy. Despite the preliminary success of immunotherapeutic agents, their therapeutic effects have been restricted by the limited retention time of drugs in TME. Compared with traditional delivery systems, nanoparticles with unique physical properties and elaborate design can efficiently penetrate TME and specifically deliver to the major components in TME. In this review, we briefly introduce the substitutes of TME including dendritic cells, macrophages, fibroblasts, tumor vasculature, tumor-draining lymph nodes and hypoxic state, then review various nanoparticles targeting these components and their applications in tumor therapy. In addition, nanoparticles could be combined with other therapies, including chemotherapy, radiotherapy, and photodynamic therapy, however, the nanoplatform delivery system may not be effective in all types of tumors due to the heterogeneity of different tumors and individuals. The changes of TME at various stages during tumor development are required to be further elucidated so that more individualized nanoplatforms could be designed.

Transcriptional evaluation of relaxin and endothelin-1 axis in heart failure patients: First evidence of its involvement during left ventricular assist device support.

BACKGROUND: Left ventricular assist devices (LVAD) are implanted in patients with end-stage heart failure (ESHF) as a mechanical support for the failing myocardium, which is characterized by an activation of the neuro-hormonal system, with release of vasoactive mediators, such as endothelin (ET)-1 and relaxin (RLX)-2. The aim of this study was to evaluate whether LVAD is able to modulate the RLX-2 and ET-1 system expression in ESHF patients. METHODS: Cardiac tissue was collected from ESHF patients before LVAD implantation (pre-LVAD group, n = 22), at the time of cardiac transplantation with concomitant LVAD removal (post-LVAD group, n = 6), and from stable HF patients on medical therapy at the time of cardiac transplantation (HTx group, n = 7). The expression of RLX-2, ET-1 system and inflammatory markers (IL-8, IL-6, TNF-α) were evaluated by Real-Time PCR. RESULTS: RLX-2 mRNA resulted similar in pre-LVAD and HTx, but it was significantly increased in post-LVAD (p = 0.02/p = 0.01 respectively). A similar trend was observed for ET-1 and ET-converting enzyme (ECE)-1 while no significant difference was observed for ET-receptors. A positive correlation was found between ET-1 and ET-A (p = 0.031) and ECE-1 (p < 0.0001). The inflammatory markers resulted activated in all the three groups. A significant correlation between RLX-2 and ET-1 in pre-LVAD, as well as between RLX-2 and IL-8/IL-6, was found. CONCLUSIONS: Our research investigates for the first time the involvement of RLX-2 and ET-1 system in ESHF patients supported by LVAD, demonstrating their potential ability to partially recover the failing myocardium, indicating their possible clinical role as biomarkers or pharmacological agents in LVAD patients. TRANSLATIONAL ASPECT: The study of novel biomarkers in patients supported by continuous axial flow devices may be a starting point analysis applicable to patients with centrifugal flow devices.

Combined administration on You-Gui Yin and low-dose Raloxifene partially attenuates the bone loss in ovariectomized mice through the proliferation and osteogenic differentiation of bone marrow stromal cells.

BACKGROUND: Osteoporosis is a systemic skeletal disease of fragility fractures due to the loss of mass and deterioration of the microarchitecture of bone. PURPOSE: The aim of the study was to assess the osteogenic effects and the underlying mechanisms of the combined administration of You-Gui Yin (YGY) and Raloxifene hydrochloride (RLX) in ovariectomized (OVX) mice. METHODS: First, a classic animal model was used to mimic postmenopausal osteoporosis through the removal of the ovary of mice. Second, the OVX mice were administered YGY, RLX, and YGY + RLX for 12 weeks. Next, the bone microtomographic histomorphometry and bone mineral density (BMD) were assessed by micro-CT, and the biochemical markers of procollagen type I N-terminal propeptide (P1NP) and beta-isomerized C-telopeptide (ß-CTX) in serum were assessed. Finally, primary bone marrow stromal cells (BMSCs) were isolated from the tibia and cultured to evaluate cell proliferation and osteogenic differentiation. RESULTS: The results showed that BMD on the YGY + RLX group was higher than that on the RLX group (p < 0.05) and did not have a significant difference when compared with the sham group. Notably, the YGY + RLX group had a dramatically increased trabecular number (Tb.N) compared with that of the YGY group (p < 0.05). Moreover, the BV/TV (bone volume/total volume) and Tb.N in the YGY + RLX group were higher than that in the RLX group (p < 0.05), and the Tb.Sp (trabecular separation) was lower than that in the RLX group (p < 0.05). Moreover, the serum level of P1NP from the YGY + RLX group dramatically increased when compared with that from the YGY and RLX groups (YGY group: p < 0.05; RLX groups: p < 0.01). Notably, there was no significant difference between the YGY and YGY + RLX groups. In addition, cell proliferation from the co-administration of YGY and RLX was clearly higher than a single use of YGY and RLX (p < 0.01, respectively). The ALP/BCA (alkaline phosphatase/bicinchoninic acid) in the YGY + RLX group was higher than that in the RLX group (p < 0.01). CONCLUSION: Overall, co-administered YGY and RLX could partially attenuate bone loss and were more effective than individually using either one; this outcome might be associated with the proliferation and osteogenic differentiation of BMSCs.

Cardioprotective actions of relaxin.

Relaxin is a hormone of pregnancy first discovered for its ability to induce ligament relaxation in nonpregnant guinea pig and is important for softening of the birth canal during parturition, decidualization, implantation, nipple development and increased maternal renal perfusion, glomerular filtration, and cardiac output. Subsequently, relaxin has been shown to exert multiple beneficial cardiovascular effects during pathological events such as hypertension, atrial fibrillation, heart failure and myocardial infarction, including suppression of arrhythmia and inflammation, and reversal of fibrosis. Despite extensive studies, the mechanisms underlying relaxin's effects are not well understood. Relaxin signals primarily through its G protein coupled receptor, the relaxin family peptide receptor-1, to activate multiple signaling pathways and this review summarizes our understanding of these pathways as they relate to the cardioprotective actions of relaxin, focusing on relaxin's anti-fibrotic, anti-arrhythmic and anti-inflammatory properties. Further, this review includes a brief overview of relaxin in clinical trials for heart failure and progress in the development of relaxin mimetics.

Similar pro-NT and pro-RLX2 levels after preeclampsia and after uncomplicated pregnancy.

OBJECTIVE: Women are at increased risk of developing cardiovascular disease (CVD) after preeclampsia. Proneurotensin 1-117 (pro-NT) and prorelaxin 2 connecting peptide (pro-RLX2) have recently emerged as potential biomarkers for CVD risk in women. We assessed pro-NT and pro-RLX2 levels in women with and without a history of preeclampsia. STUDY DESIGN: 339 women with a history of early-onset preeclampsia and 327 women with an uncomplicated pregnancy underwent cardiovascular screening 10 years after delivery (the Preeclampsia Risk EValuation in FEMales (PREVFEM) cohort). MAIN OUTCOME MEASURES: Pro-NT, a stable fragment of the neurotensin precursor, was assessed in the whole cohort. Pro-RLX2, the stable connecting peptide of the relaxin 2 prohormone, was assessed in a subset of this cohort, consisting of 27 women with a history of preeclampsia and 23 healthy controls. Associations between biomarker levels and traditional CVD risk factors in the preeclampsia and control group were assessed by Pearson's correlation coefficient. RESULTS: We found no differences in pro-NT and pro-RLX2 levels between the preeclampsia and control group. Pro-NT levels were associated with higher HbA1c levels (r=0.113, p-value 0.045) and with BMI (r=0.124, p-value 0.027), but only in the control group. Pro-RLX2 was related to current smoking and triglyceride levels in women with a history of preeclampsia and related to LDL-cholesterol in women with an uncomplicated pregnancy. CONCLUSIONS: Pro-NT and pro-RLX2 levels were comparable in women 10 years after preeclampsia and women with an uncomplicated pregnancy. The role of pro-NT and pro-RLX2 in CVD development after preeclampsia should be further investigated.

Upcoming drugs for the treatment of preeclampsia in pregnant women.

Preeclampsia is a pregnancy-specific multisystem disorder, complicating 2 - 8% of pregnancies, and represents a leading cause of maternal and perinatal morbidity and mortality. Recent investigations have elucidated the understanding of its underlying pathogenic mechanisms. However, despite these advances, therapeutic approaches are still severely limited. Ongoing lines of research indicate some potential novel therapeutic options, targeting the etiopathogenic pathways and, thus, offering hope for effective pharmacologic interventions to be available in the near future. In this editorial, we will give an updated overview of Preeclampsia pathogenesis and promising emerging therapeutic options.

Comparison of chemical compositions and osteoprotective effects of different sections of velvet antler.

ETHNOPHARMACOLOGICAL RELEVANCE: Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA. MATERIALS AND METHODS: Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats. RESULTS: Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1 and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells. CONCLUSION: Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler.

Tissue-selective estrogen complexes for postmenopausal women.

Although hormone therapy using estrogens plus progestogens (EPT) is effective for the management of menopausal symptoms (e.g., vasomotor symptoms and vulvar/vaginal atrophy) and prevention/treatment of postmenopausal osteoporosis, EPT is associated with safety and tolerability concerns. A new alternative to EPT is the tissue selective estrogen complex (TSEC), which partners a selective estrogen receptor modulator (SERM) with one or more estrogens and is designed to treat menopausal symptoms and prevent postmenopausal osteoporosis without the tolerability concerns associated with EPT. The first TSEC to reach advanced clinical development is a combination of the SERM bazedoxifene (BZA) with conjugated estrogens (CE). BZA has been shown to inhibit the stimulatory activity of CE on uterine tissue and breast in vitro and in vivo. In clinical studies, BZA/CE treatment has been associated with significant improvements in menopausal symptoms including hot flushes and vulvar/vaginal atrophy and significant increases in bone mineral density, coupled with reductions in bone turnover marker levels and improvements in sleep and health-related quality of life. Additionally, BZA/CE has been shown to have a neutral effect on endometrial and breast tissue because BZA inhibits the stimulatory effects of estrogens in tissue-selective fashion in these 2 organs. Taken together, results of these preclinical and clinical studies indicate that the benefits of estrogens for treating menopausal symptoms are maintained with BZA/CE without endometrial or breast stimulation, resulting in a safe and effective treatment for symptomatic postmenopausal women.

A water-soluble selenoxide reagent as a useful probe for the reactivity and folding of polythiol peptides.

A water-soluble selenoxide (DHS(ox)) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS(ox) would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS(ox) was extensively studied at pH 4-10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS(ox) (375 mV at 25 °C) compared to l-cystine supported the high ability of DHS(ox) for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pK a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT(red)) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.