Efficacy of oral 20-hydroxyecdysone (BIO101), a MAS receptor activator, in adults with severe COVID-19 (COVA): a randomized, placebo-controlled, phase 2/3 trial.

Background: SARS-CoV-2 binding to ACE2 is potentially associated with severe pneumonia due to COVID-19. The aim of the study was to test whether Mas-receptor activation by 20-hydroxyecdysone (BIO101) could restore the Renin-Angiotensin System equilibrium and limit the frequency of respiratory failure and mortality in adults hospitalized with severe COVID-19. Methods: Double-blind, randomized, placebo-controlled phase 2/3 trial. Randomization: 1:1 oral BIO101 (350 mg BID) or placebo, up to 28 days or until an endpoint was reached. Primary endpoint: mortality or respiratory failure requiring high-flow oxygen, mechanical ventilation, or extra-corporeal membrane oxygenation. Key secondary endpoint: hospital discharge following recovery (ClinicalTrials.gov Number, NCT04472728). Findings: Due to low recruitment the planned sample size of 310 was not reached and 238 patients were randomized between August 26, 2020 and March 8, 2022. In the modified ITT population (233 patients; 126 BIO101 and 107 placebo), respiratory failure or early death by day 28 was 11.4% lower in the BIO101 (13.5%) than in the placebo (24.3%) group, (p = 0.0426). At day 28, proportions of patients discharged following recovery were 80.1%, and 70.9% in the BIO101 and placebo group respectively, (adjusted difference 11.0%, 95% CI [-0.4%, 22.4%], p = 0.0586). Hazard Ratio for time to death over 90 days: 0.554 (95% CI [0.285, 1.077]), a 44.6% mortality reduction in the BIO101 group (not statistically significant). Treatment emergent adverse events of respiratory failure were more frequent in the placebo group. Interpretation: BIO101 significantly reduced the risk of death or respiratory failure supporting its use in adults hospitalized with severe respiratory symptoms due to COVID-19. Funding: Biophytis.

RAR Inhibitors Display Photo-Protective and Anti-Inflammatory Effects in A2E Stimulated RPE Cells In Vitro through Non-Specific Modulation of PPAR or RXR Transactivation.

N-retinylidene-N-retinylethanolamine (A2E) has been associated with age-related macular degeneration (AMD) physiopathology by inducing cell death, angiogenesis and inflammation in retinal pigmented epithelial (RPE) cells. It was previously thought that the A2E effects were solely mediated via the retinoic acid receptor (RAR)-α activation. However, this conclusion was based on experiments using the RAR "specific" antagonist RO-41-5253, which was found to also be a ligand and partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ. Moreover, we previously reported that inhibiting PPAR and retinoid X receptor (RXR) transactivation with norbixin also modulated inflammation and angiogenesis in RPE cells challenged in the presence of A2E. Here, using several RAR inhibitors, we deciphered the respective roles of RAR, PPAR and RXR transactivations in an in vitro model of AMD. We showed that BMS 195614 (a selective RAR-α antagonist) displayed photoprotective properties against toxic blue light exposure in the presence of A2E. BMS 195614 also significantly reduced the AP-1 transactivation and mRNA expression of the inflammatory interleukin (IL)-6 and vascular endothelial growth factor (VEGF) induced by A2E in RPE cells in vitro, suggesting a major role of RAR in these processes. Surprisingly, however, we showed that (1) Norbixin increased the RAR transactivation and (2) AGN 193109 (a high affinity pan-RAR antagonist) and BMS 493 (a pan-RAR inverse agonist), which are photoprotective against toxic blue light exposure in the presence of A2E, also inhibited PPARs transactivation and RXR transactivation, respectively. Therefore, in our in vitro model of AMD, several commercialized RAR inhibitors appear to be non-specific, and we propose that the phototoxicity and expression of IL-6 and VEGF induced by A2E in RPE cells operates through the activation of PPAR or RXR rather than by RAR transactivation.

BIO101 stimulates myoblast differentiation and improves muscle function in adult and old mice.

BACKGROUND: Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo. METHODS: The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50 mg/kg/day) adult mice (3 months) for 28 days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14 weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests. RESULTS: Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P < 0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity). CONCLUSIONS: Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.

A Phase 1 study for safety and pharmacokinetics of BIO101 (20-hydroxyecdysone) in healthy young and older adults.

BACKGROUND: Sarcopenia is an age-related skeletal muscle disorder characterized by loss of muscle mass and strength leading to mobility disability. 20-Hydroxyecdysone (20E) is a polyhydroxylated plant steroid that demonstrates pharmacological effects in many disease animal models including ageing/sarcopenia. BIO101 is a 20E purified investigational drug (≥97%) that previously demonstrated good toxicology profiles in rat and dog. BIO101 is evaluated in healthy young and older adults in a Phase 1 study. METHODS: This study is a Single Ascending Dose (SAD) followed by a 14-day Multiple Ascending Dose (MAD). In SAD, BIO101 was administered orally to 16 young adults at doses from 100 to 1400 mg and to 8 older adults (age ≥65 years) at 1400 mg. In MAD, doses of 350 mg once daily (qd), 350 mg twice daily (bid) and 450 mg bid were administered to 10 older adults. The primary objective was to evaluate safety and pharmacokinetics (PK), including dosing of circulating metabolites. Pharmacodynamic effects were investigated with regard to myostatin, procollagen-III-amino-terminal propeptide (PIIINP), myoglobin, creatine-kinase Muscle Brain (CKMB), renin and aldosterone plasma/serum levels. RESULTS: BIO101 showed a good safety profile with only mild to moderate adverse events and a satisfactory pharmacokinetic profile. In SAD, at 100 mg to 1400 mg, mean Cmax and areas under the curve increased less than dose-proportionally. Mean half-life was short (2.4-4.9 h), and mean renal clearance was comparable in all doses (4.05-5.05 L/h). Mean plasma exposure was slightly lower in older adults (22% lower for Cmax and 13%-15% lower for AUCs) compared with young subjects. In MAD, 350 and 450 mg bid led to a slight accumulation over 14 days (mean ratio of accumulation [Rac] of 1.31 in both cohorts). Reduction of biomarkers (myoglobin, CK-MB) mean serum levels (vs. baseline) was observed at 450 mg bid. Two major metabolites of 20E (14-deoxy-20-hydroxyecdysone and 14-deoxypoststerone) were identified and quantified. CONCLUSIONS: BIO101 shows a good safety and pharmacokinetic profile that led to the selection of doses for the subsequent interventional clinical trials of Phase 2 in age-related sarcopenia (SARA-INT) and Phase 3 in Covid-19 (COVA).

Anti-Inflammatory Effects and Photo- and Neuro-Protective Properties of BIO203, a New Amide Conjugate of Norbixin, in Development for the Treatment of Age-Related Macular Degeneration (AMD).

9'-cis-norbixin (norbixin/BIO201) protects RPE cells against phototoxicity induced by blue light and N-retinylidene-N-retinylethanolamine (A2E) in vitro and preserves visual functions in animal models of age-related macular degeneration (AMD) in vivo. The purpose of this study was to examine the mode of action and the in vitro and in vivo effects of BIO203, a novel norbixin amide conjugate. Compared to norbixin, BIO203 displays improved stability at all temperatures tested for up to 18 months. In vitro, BIO203 and norbixin share a similar mode of action involving the inhibition of PPARs, NF-κB, and AP-1 transactivations. The two compounds also reduce IL-6, IL-8, and VEGF expression induced by A2E. In vivo, ocular maximal concentration and BIO203 plasma exposure are increased compared to those of norbixin. Moreover, BIO203 administered systemically protects visual functions and retinal structure in albino rats subjected to blue-light illumination and in the retinal degeneration model of Abca4-/- Rdh8-/- double knock-out mice following 6 months of oral complementation. In conclusion, we report here that BIO203 and norbixin share similar modes of action and protective effects in vitro and in vivo. BIO203, with its improved pharmacokinetic and stability properties, could be developed for the treatment of retinal degenerative diseases such as AMD.

The Distribution of Phytoecdysteroids among Terrestrial Vascular Plants: A Comparison of Two Databases and Discussion of the Implications for Plant/Insect Interactions and Plant Protection.

Phytoecdysteroids are a class of plant secondary compounds which are present in a wide diversity of vascular plant species, where they contribute to a reduction in invertebrate predation. Over the past 55 years, a significant body of heterogeneous literature on the presence, identities and/or quantities of ecdysteroids in plant species has accumulated, resulting in the compilation of a first database, the Ecdybase Literature Survey (ELS; 4908 entries, covering 2842 species). A second extensive database on the distribution of ecdysteroids in vascular plants is available as the Exeter Survey (ES; 4540 entries, covering 4155 species), which used standardised extraction and analysis methods to survey seeds/spores. We compare the usefulness of these two databases to provide information on the occurrence of phytoecdysteroids at the order/family levels in relation to the recent molecular classifications of gymnosperms, pteridophytes/lycophytes and angiosperms. The study, in conjunction with the other published literature, provides insights into the distribution of phytoecdysteroids in the plant world, their role in plant protection in nature and their potential future contribution to crop protection. Furthermore, it will assist future investigations in the chemotaxonomy of phytoecdysteroids and other classes of plant secondary compounds.

High performance Reversed-Phase Thin-Layer Chromatography-Desorption electrospray ionisation - time of flight high resolution mass spectrometric detection and imaging (HPTLC/DESI/ToFMS) of phytoecdysteroids.

Reversed-phase high performance thin-layer chromatography (RP-HPTLC) on C18 bonded silica gel was combined with desorption electrospray ionization (DESI) and high resolution time of flight mass spectrometry (HRToFMS) to detect, characterize and image (MSI) phytoecdysteroids (plant-derived insect moulting hormones) in ethanolic extracts of members of the Silene plant family. As seen previously for silica gel, DESI provided a simple and convenient method for recovering polar polyhydoxysteroids from RP-HPTLC plates for the purposes of both the MS and MSI of extracts obtained from three species of the Silene family (Silene otites, S. nutans and S. viridiflora). Using RP-HPTLC/DESI/MSI/HRToFMS a number of ecdysteroids, including 20-hydroxyecdysone, polypodine-B, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone were identified in these extracts. Differences were noted in the mass spectra obtained depending upon both the stationary phase on which they were separated, and the temperatures used in the heated transfer line used for introduction into the ion source. Ecdysteroids detected after chromatography on C18 bonded silica showed increased fragmentation due to water loss compared to those imaged from silica. In addition, the benefits of the additional resolution provided by 2-dimensional TLC for increasing spectral quality compared to a 1-dimensional separation are demonstrated.

Structural analysis of unstable norbixin isomers guided by pure shift nuclear magnetic resonance.

We report the analysis of complex samples obtained during the microwave irradiation/heating of norbixin, which has been identified as a potential therapeutic target for age-related macular degeneration (AMD). In this context, identifying the different isomers that are obtained during its degradation is of primary importance. However, this characterization is challenging because, on the one hand, some of these isomers are unstable, and on the other hand, the 1 H spectra of these isomeric mixtures are poorly resolved. We could successfully apply 1D pure shift experiments to obtain ultrahigh-resolution 1 H nuclear magnetic resonance (NMR) spectra of the norbixin isomer samples and exploit their information content to analyze complementary 2D NMR data and describe accurately their isomeric composition.

20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERß receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17ß-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

A comparison between virus- versus patients-centred therapeutic attempts to reduce COVID-19 mortality.

Since December 2019, coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has changed our lives. Elderly and those with comorbidities represent the vast majority of patients hospitalized with severe COVID-19 symptoms, including acute respiratory disease syndrome and cardiac dysfunction. Despite a huge effort of the scientific community, improved treatment modalities limiting the severity and mortality of hospitalized COVID-19 patients are still required. Here, we compare the effectiveness of virus- and patients-centred strategies to reduce COVID-19 mortality. We also discuss the therapeutic options that might further reduce death rates associated with the disease in the future. Unexpectedly, extensive review of the literature suggests that SARS-CoV-2 viral load seems to be associated neither with the severity of symptoms nor with mortality of hospitalized patients with COVID-19. This may explain why, so far, virus-centred strategies using antivirals aiming to inhibit the viral replicative machinery have failed to reduce COVID-19 mortality in patients with respiratory failure. By contrast, anti-inflammatory treatments without antiviral capacities but centred on patients, such as dexamethasone or Tocilizumab®, reduce COVID-19 mortality. Finally, since the spike protein of SARS-CoV-2 binds to angiotensin converting enzyme 2 and inhibits its function, we explore the different treatment options focussing on rebalancing the renin-angiotensin system. This new therapeutic strategy could hopefully further reduce the severity of respiratory failure and limit COVID-19 mortality in elderly patients.

A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -ß/δ, -γ, and RXR antagonists and by norbixin.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -ß/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

20-Hydroxyecdysone, from Plant Extracts to Clinical Use: Therapeutic Potential for the Treatment of Neuromuscular, Cardio-Metabolic and Respiratory Diseases.

There is growing interest in the pharmaceutical and medical applications of 20-hydroxyecdysone (20E), a polyhydroxylated steroid which naturally occurs in low but very significant amounts in invertebrates, where it has hormonal roles, and in certain plant species, where it is believed to contribute to the deterrence of invertebrate predators. Studies in vivo and in vitro have revealed beneficial effects in mammals: anabolic, hypolipidemic, anti-diabetic, anti-inflammatory, hepatoprotective, etc. The possible mode of action in mammals has been determined recently, with the main mechanism involving the activation of the Mas1 receptor, a key component of the renin-angiotensin system, which would explain many of the pleiotropic effects observed in the different animal models. Processes have been developed to produce large amounts of pharmaceutical grade 20E, and regulatory preclinical studies have assessed its lack of toxicity. The effects of 20E have been evaluated in early stage clinical trials in healthy volunteers and in patients for the treatment of neuromuscular, cardio-metabolic or respiratory diseases. The prospects and limitations of developing 20E as a drug are discussed, including the requirement for a better evaluation of its safety and pharmacological profile and for developing a production process compliant with pharmaceutical standards.

The complex metabolism of poststerone in male rats.

Ecdysteroids are not endogenous to mammals, but are normal components of the food intake of many mammalian species consuming phytoecdysteroid-containing plants. The most frequently encountered phytoecdysteroid is 20-hydroxyecdysone (20E). Several pharmaceutical effects have been observed after ecdysteroid injection or ingestion, but it is not clear to what extent metabolites generated in the mammalian body contribute to these effects. The C21-ecdysteroid poststerone (Post) is a metabolite of 20E in rodents. Post analogues are key intermediates in the metabolism of exogenous ecdysteroids possessing a C20/22-diol. The pharmacokinetics, bioavailability and metabolism of Post have been assessed in male rats after ingestion and injection. The bioavailability of Post is significantly greater than that of 20E and the presence of an efficient entero-hepatic cycle allows Post to be effectively metabolised to a wide range of metabolites which are excreted mainly in the faeces, but also to some extent in the urine. Several of the major metabolites in the bile have been identified unambiguously as 3-epi-poststerone, 16α-hydroxypoststerone, 21-hydroxypoststerone and 3-epi-21-hydroxypoststerone. Conjugates are also present. Parallels are drawn to the metabolism of endogenous vertebrate steroid hormones, to which Post bears more similarity than 20E.

Developing new drugs that activate the protective arm of the renin-angiotensin system as a potential treatment for respiratory failure in COVID-19 patients.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached pandemic proportions with negative impacts on global health, the world economy and human society. The clinical picture of COVID-19, and the fact that Angiotensin converting enzyme 2 (ACE2) is a receptor of SARS-CoV-2, suggests that SARS-CoV-2 infection induces an imbalance in the renin-angiotensin system (RAS). We review clinical strategies that are attempting to rebalance the RAS in COVID-19 patients by using ACE inhibitors, angiotensin receptor blockers, or agonists of angiotensin-II receptor type 2 or Mas receptor (MasR). We also propose that the new MasR activator BIO101, a pharmaceutical grade formulation of 20-hydroxyecdysone that has anti-inflammatory, anti-fibrotic and cardioprotective properties, could restore RAS balance and improve the health of COVID-19 patients who have severe pneumonia.

Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease.

Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4-/- Rdh8-/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'-cis-norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4-/- Rdh8-/- mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4-/- Rdh8-/- mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.

Small-scale analysis of phytoecdysteroids in seeds by HPLC-DAD-MS for the identification and quantification of specific analogues, dereplication and chemotaxonomy.

INTRODUCTION: Phytoecdysteroids are analogues of arthropod steroids occurring in plants. They contribute to invertebrate deterrence. A wide diversity of ecdysteroids occurs in phytoecdysteroid-containing plant species, sometimes in high amounts. Ecdysteroids demonstrate potentially useful pharmaceutical actions in mammals. OBJECTIVES: Establish reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC-MS/MS) and RP-HPLC-DAD-MS (diode array detector mass spectrometry) methods for the separation, identification and quantification of ecdysteroids to screen for species containing significant amounts of 20-hydroxyecdysone (20E) and other useful ecdysteroids. MATERIALS AND METHODS: Micro-extracts of seed samples (ca. 30 mg) in 50% ethanol were subjected to RP-SPE (solid-phase extraction) purification prior to analysis by RP-HPLC-MS/MS and RP-HPLC-DAD-MS. The method was initially applied to genera (Amaranthus, Centaurea, Lychnis, Ourisia, Serratula, Silene and Trollius) where high-accumulating species had been previously encountered. Seeds of 160 randomly selected species, many of which have not previously been assessed, were then analysed. HPLC-MS/MS with a short analysis time initially identifies ecdysteroid-positive extracts and quantifies 20E. The positive extracts (20 ng 20E) are then analysed by HPLC-MS/MS with a longer analysis time to identify and quantify 17 common phytoecdysteroids and, finally, HPLC-DAD-MS (0.1-0.25 µg 20E) is used to obtain UV- and MS-spectra to confirm identifications or as a basis for characterisation of partially identified or novel analogues. RESULTS: Lychnis coronaria, Silene fimbriata and Silene hookeri ecdysteroids are characterised for the first time and those of Cucubalus baccifer and Ipheion uniflorum are more extensively characterised. CONCLUSIONS: The procedure provides a rapid/sensitive method for screening small plant samples for the presence, quantification and identification of ecdysteroids. It permits ready dereplication of samples, identifying extracts containing large amounts or novel analogues.

Beyond AREDS Formulations, What Is Next for Intermediate Age-Related Macular Degeneration (iAMD) Treatment? Potential Benefits of Antioxidant and Anti-inflammatory Apocarotenoids as Neuroprotectors.

Age-related macular degeneration (AMD) is the commonest cause of severe visual loss and blindness in developed countries among individuals aged 60 and older. AMD slowly progresses from early AMD to intermediate AMD (iAMD) and ultimately late-stage AMD. Late AMD encompasses either neovascular AMD (nAMD) or geographic atrophy (GA). nAMD is defined by choroidal neovascularization (CNV) and hemorrhage in the subretinal space at the level of the macula. This induces a rapid visual impairment caused by the death of photoreceptor cells. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibodies is the standard treatment of nAMD but adds to the burden of patient care. GA is characterized by slowly expanding photoreceptor, and retinal pigment epithelium (RPE) degeneration patches progressively leading to blindness. There is currently no therapy to cure GA. Late AMD continues to be an unmet medical need representing a major health problem with millions of patients worldwide. Oxidative stress and inflammation are recognized as some of the main risk factors to developing late AMD. The antioxidant formulation AREDS (Age-Related Eye Disease Studies), contains ß-carotene, which has been replaced by lutein and zeaxanthin in AREDS2, are given to patients with iAMD but have a limited effect on the incidence of nAMD and GA. Thus, to avoid or slowdown the development of late stages of AMD (nAMD or GA), new therapies targeting iAMD are needed such as crocetin obtained through hydrolysis of crocin, an important component of saffron (Crocus sativus L.), and norbixin derived from bixin extracted from Bixa orellana seeds. We have shown that these apocarotenoids preserved more effectively RPE cells against apoptosis following blue light exposure in the presence of A2E than lutein and zeaxanthin. In this review, we will discuss the potential use of apocarotenoids to slowdown the progression of iAMD, to reduce the incidence of both forms of late AMD.

The effects of curcumin, mangiferin, resveratrol and other natural plant products on aminopeptidase B activity.

Aminopeptidase B (Ap-B) is a Zn2+-aminopeptidase of the M1 family which is implicated, in conjunction with the nardilysin endoprotease, in the generation of miniglucagon, a peptide involved in the maintenance of glucose homeostasis. Other in vivo physiological roles have been established for this vertebrate enzyme, such as the processing of Arg-extended forms of human insulin and cholecystokinin 9 and the degradation of viral epitopes in the cytoplasm. Among M1 family members, Ap-B is phylogenetically close to leukotriene A4 hydrolase (LTA4H), a bi-functional aminopeptidase also able to transform LTA4 in LTB4 (a lipid mediator of inflammation). As the activities of LTA4H are reported to be inhibited by resveratrol, a polyphenolic molecule from red wine, the effect of this molecule was investigated on the Ap-B activity. Several other active phenolic compounds produced in plants were also tested. Among them, curcumin and mangiferin are the most effective inhibitors. Dixon analysis indicates that curcumin is a non-competitive inhibitor with a Ki value of 46 µmol.L-1. Dixon and Lineweaver-Burk representations with mangiferin show a mixed non-competitive inhibition with Ki' and Ki values of 194 µmol.L-1 and 105 µmol.L-1, respectively. At 200 µmol.L-1, no significant effect was observed with caffeic, chlorogenic, ferulic, salicylic and sinapic acids as well as with resveratrol. Analyses on the 3D-structure of LTA4H with resveratrol (pdb: 3FTS) and the Ap-B 3D-model allow hypothesis to explain theses results.

Size matters! Aurora A controls Drosophila larval development.

In metazoans, organisms arising from a fertilized egg, the embryo will develop through multiple series of cell divisions, both symmetric and asymmetric, leading to differentiation. Aurora A is a serine threonine kinase highly involved in such divisions. While intensively studied at the cell biology level, its function in the development of a whole organism has been neglected. Here we investigated the pleiotropic effect of Aurora A loss-of-function in Drosophila larval early development. We report that Aurora A is required for proper larval development timing control through direct and indirect means. In larval tissues, Aurora A is required for proper symmetric division rate and eventually development speed as we observed in central brain, wing disc and ring gland. Moreover, Aurora A inactivation induces a reduction of ecdysteroids levels and a pupariation delay as an indirect consequence of ring gland development deceleration. Finally, although central brain development is initially restricted, we confirmed that brain lobe size eventually increases due to additive phenotypes: delayed pupariation and over-proliferation of cells with an intermediate cell-identity between neuroblast and ganglion mother cell resulting from defective asymmetric neuroblast cell division.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.