The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

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, -β/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.

Atrophic 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.

No abstract

Ajuga turkestanica ecdysteroids are characterised by the abundance of 11α-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11α-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11α-hydroxy and 11-deoxy forms.

Ecdysteroid: member of a class of polyhydroxylated steroids found in invertebrate animals (zooecdysteroids; moulting hormones), plants (phytoecdysteroids) and fungi (mycoecdysteroids). Over 500 structural analogues are currently known. Biosynthetically, they derive from C27-, C28- or C29-sterols. The most frequently encountered analogue (in arthropods and plants) is 20-hydroxyecdysone (2β,3β,14α, 20R,22R,25-hexahydroxycholest-7-en-6-one). In arthropods, ecdysteroids occur universally and regulate development by inducing moulting and reproduction, where their action is mediated by high-affinity binding to an intracellular member of the class of nuclear receptor (NR) proteins (ecdysteroid receptor; EcR) dimerised with a second NR (USP/RxR). This receptor complex binds to specific DNA promoter sites and regulates gene expression. In plants, ecdysteroids are a class of secondary compounds, occurring in varying amounts in certain species, but not all in others. Phytoecdysteroids are believed to contribute to the reduction of invertebrate predation by acting as feeding deterrents or endocrine disruptors. Ecdysteroids also possess a wide range of positive pharmacological effects in mammals, where the mode of action involves moderate-affinity binding to plasma-membrane-bound receptors and not interaction with the classical NRs for vertebrate steroid hormones.

IntroductionPhytoecdysteroids 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.ObjectivesEstablish 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 methodsMicro‐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.ResultsLychnis 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.ConclusionsThe 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.

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 doseproportionally. 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 14deoxypoststerone) 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).