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).

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.

Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats.

Phytoecdysteroids are molecules derived from sterol metabolism and found in many plants. They display a wide array of pharmacological effects on mammals (e.g. anabolic, anti-diabetic). Although these effects have been long established, the molecular targets involved remain to be identified. Like endogenous steroid hormones and bile acids, which are biochemically related, ingested or injected phytoecdysteroids undergo a set of reactions in mammals leading to the formation of numerous metabolites, only some of which have been so far identified, and it is presently unknown whether they represent active metabolites or inactivation products. In the large intestine, ecdysteroids undergo efficient 14-dehydroxylation. Other changes (reductions, epimerization, side-chain cleavage) are also observed, but whether these occur in the liver and/or large intestine is not known. The purpose of this study was to investigate the pharmacokinetics of 20-hydroxyecdysone (20E), the most common phytoecdysteroid, when administered to mice and rats, using, when required, tritium-labelled molecules to permit metabolic tracking. Bioavailability, the distribution of radioactivity and the kinetics of formation of metabolites were followed for 24-48 hours after ingestion and qualitative and quantitative analyses of circulating and excreted compounds were performed. In mice, the digestive tract always contains the majority of the ingested 20E. Within 30 min after ingestion, 20E reaches the large intestine, where microorganisms firstly remove the 14-hydroxyl group and reduce the 6-one. Then a very complex set of metabolites (not all of which have yet been identified) appears, which correspond to poststerone derivatives formed in the liver. We have observed that these compounds (like bile acids) undergo an entero-hepatic cycle, involving glucuronide conjugation in the liver and subsequent deconjugation in the intestine. Despite the very short half-life of ecdysteroids in mammals, this entero-hepatic cycle helps to maintain their plasma levels at values which, albeit low (≤0.2 µM), would be sufficient to evoke several pharmacological effects. Similar 20E metabolites were observed in mice and rats; they include in particular 14-deoxy-20E, poststerone and 14-deoxypoststerone and their diverse reduction products; the major products of this metabolism have been unambiguously identified. The major sites of metabolism of exogenous ecdysteroids in mammals are the large intestine and the liver. The entero-hepatic cycle contributes to the metabolism and to maintaining a low, but pharmacologically significant, concentration of ecdysteroids in the blood for ca. 24 h after ingestion. These data, together with parallel in vitro experiments provide a basis for the identification of 20E metabolite(s) possibly involved in the physiological effects associated with ecdysteroids in mammals.

Bioavailability and structural study of 20-hydroxyecdysone complexes with cyclodextrins.

20-Hydroxyecdysterone - (2ß,3ß,5ß,22R)-2,3,14,20,22,25-hexahydroxycholest-7-en-6-one was isolated in satisfactory yield using ethanol extraction from the aerial part of Silene wolgensis (Hornem.) Otth; sometimes Silene wolgensis (Willd.) Bess. ex Spreng. The complexation of the phytoecdysteroid with ß-cyclodextrin was studied by NMR spectroscopy. By studying the changes in chemical shifts of protons of substrates and receptors it was found that ecdysterone interacts with cyclodextrins to form supramolecular inclusion complexes of stoichiometric composition of 1:1 or 1:2. Ecdysterone-ß-cyclodextrin complexes exhibit 100 times higher solubility in water than the parent compound.

Evaluating the effect of 20-hydroxyecdysone (20HE) on mechanistic target of rapamycin complex 1 (mTORC1) signaling in the skeletal muscle and liver of rats.

Phytoecdysteroids such as 20-hydroxyecdysone (20HE) are nutritional supplements marketed as enhancers of lean body mass. In this study the impact of 20HE ingestion on protein kinase B/Akt-mechanistic target of rapamycin complex 1 signaling in the skeletal muscle and liver of male rats was found to be limited. Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested. Additional work is necessary to clarify 20HE mechanism of action in vivo.

[Research on relationship between tissue quantitative distribution of 3H-Achyranthes bidentata ecdysterone and channel-tropism of herbal drugs in mice].

OBJECTIVE: To study the relationship between tissue quantitative distribution and pharmacokinetics of 3H-achyranthes bidentata ecdysterone and the channel-tropism of herbal drugs in mice. METHOD: 3H-achyranthes bidentata ecdysterone was used as a tracer agent and injected into mice by the caudal vein. In 36 hours, the contents of the tracer agent of samples involving 9 different tracing phases and organ or tissue were determined in order to observe the dynamic quantitative distribution and excretion and pharmacokinetics of 3H-achyranthes bidentata ecdysterone and to understand the channel-tropism of herbal drugs achyranthes bidentata. RESULT: 3H-achyranthes bidentata ecdysterone of same organs in different tracing phases and the contents of 3H-achyranthes bidentata ecdysterone in same tracing phases of different organs were significantly different (P<0.01). 3H-achyranthes bidentata ecdysterone was mainly distributed, in the liver, kidney, adrenal gland, small intestine and lung. The concentration-time profiles of achyranthes bidentata ecdysterone in rats injected into mice by the caudal vein were shown to fit a two-compartment open model with half-lives of (778.65 +/- 12.36) min, the elimination of achyranthes bidentata ecdysterone from plasma was found to be in accord with linear kinetics. CONCLUSION: The above mentioned selective distribution of 3H-achyranthes bidentata ecdysterone basically coincides with the meridian affinity and zang fu selection of the traditional Chinese medicine drug Achyranthes bidentata. This study will provide a scientific basis for the channel-tropism of A. bidentata.

Detection and identification of 20-hydroxyecdysone metabolites in calf urine by liquid chromatography-high resolution or tandem mass spectrometry measurements and establishment of their kinetics of elimination after 20-hydroxyecdysone administration.

Ecdysteroids, which are steroid hormones in invertebrates, but are also present in plants, could be potentially used as anabolic agents in food-producing animals because of their growth-promoting properties. In this context, the metabolism of 20-hydroxyecdysone (20E) has been investigated in cattle in order to efficiently control its potential misuse. The analytical procedure involves purification on two solid-phase extraction cartridges (SPE octadecylsilyl and SPE silica) prior to detection based on liquid chromatography coupled to high resolution mass spectrometry in negative electrospray ionization mode (LC-(ESI-)-HRMS). Each new signal appearing on full-scan HRMS (30,000) during the analysis was investigated by tandem mass spectrometry (MS/MS). Comparison of the mass spectra pattern between 20E and potential metabolites has given informations on the chemical structures of the metabolites. This targeted approach, combining HRMS and MS(n) measurements on a linear trap in tandem with an orbital trap, allowed us to elucidate the structure of several 20E metabolites in calf urine: 14-deoxy-20-hydroxyecdysone, 20,26-dihydroxyecdysone and 14-deoxy-20,26-dihydroxyecdysone, the last of which had never previously been reported in bovine. The kinetics of elimination of these metabolites were investigated, and two of them were monitored by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) measurements over 6 days after 20E administration to calves, thus increasing the time-window for detection after 20E administration and thereby allowing for more efficient control of its misuse.

Detection of 20-hydroxyecdysone in calf urine by comparative liquid chromatography/high-resolution mass spectrometry and liquid chromatography/tandem mass spectrometry measurements: application to the control of the potential misuse of ecdysteroids in cattle.

Ecdysteroids, which are steroid hormones in invertebrates, but which are also present in plants, could potentially be used as anabolic agents in food-producing animals. The control of ecdysteroid misuse in cattle relies on the development of an efficient method for their detection in biological matrices at trace levels (microg L(-1)). In order to propose an analytical procedure dedicated to the identification of excreted 20-hydroxyecdysone (20E) in urine and faecal samples of breeding animals, a comparative study of the spectrometric behaviour of these compounds was carried out both by LC/(ESI-)/HRMS(n) (hybrid linear ion trap - orbital trap) and by LC/(ESI+)/MS/MS (triple quadrupole). This study revealed the formation of a large number of product ions both in positive and negative ion mode, corresponding to losses of water molecules and specific cleavages on the side chain. The sample preparation consisted of sequential purification on two solid-phase extraction cartridges (SPE octadecylsilyl and SPE silica). The detection limits were around 0.5 microg L(-1) in the selected reaction monitoring (SRM) mode and recoveries above 60% were obtained. The method was successfully applied to the analysis of real samples collected from calves treated with 60 mg 20E over 4 days. Analysis of the samples allowed the investigation of the kinetics of elimination of 20E in calf urine and determination of the time-frame for the control of potential abuse.

Diversity of detoxification pathways of ingested ecdysteroids among phytophagous insects.

The metabolic pathways of ingested ecdysteroids have been investigated in three insect species, the aphid Myzus persicae and two Lepidoptera, Plodia interpunctella and Ostrinia nubilalis. M. persicae produces mainly a 22-glucoside conjugate, whereas P. interpunctella eliminates a mixture of 20E and its 3-oxo and 3-epi derivatives, both in free form and as conjugates with various fatty acids. O. nubilalis only produces fatty acyl ester conjugates. These data point out the great diversity of detoxification mechanisms used by phytophagous insects in order to overcome the potential harmful effects of ecdysteroids present in their food.

Biotransformations of 20-hydroxyecdysone and analogues by Curvularia lunata NRRL 2178.

20-Hydroxyecdysone, the arthropod moulting hormone, was biotransformed by the fungus Curvularia lunata NRRL 2178 to the rare ecdysteroid, 2-dehydro-3-epi-20-hydroxyecdysone, and the novel 3alpha,9alpha-cyclo ecdysteroid analogue, (20R,22R)-3beta,14alpha,20,22,25-pentahydroxy-3alpha,9alpha-cyclo-5beta-cholest-7-en-2,6-dione in 14 and 44% yields, respectively. Ponasterone A and pterosterone were similarly biotransformed to the corresponding 2-dehydro-3-epi- and 3alpha,9alpha-cyclo-analogues.

Study of excretion of ecdysterone in human urine.

A study of excretion in human urine of ecdysterone, which is the active component of several over-the-counter supplements such as "Ecdysten", reportedly used by athletes, is presented. The study was performed after oral administration of 20 mg of ecdysterone. The collected urine samples were prepared using the standard screening extraction procedure for the free and conjugated fraction of anabolic steroids, and analyzed by gas chromatography (GC) coupled with quadrupole mass spectrometry (MS) and also with high-resolution mass spectrometry (HRMS). Two ecdysterone metabolites were identified and detected along with unchanged ecdysterone. Accurate mass measurements were made for diagnostic ions, including the molecular ion of the main metabolite of ecdysterone, deoxyecdysone, which, to our knowledge, has not previously been reported in the literature. These accurate mass measurements support the proposed fragmentation scheme.

Identification and ecdysteroid antagonist activity of three oligostilbenes from the seeds of Carex pendula (Cyperaceae).

Methanolic extracts of seeds of several (Carex species were found to antagonise the action of 20-hydroxyecdysone in the Drosophila melanogaster microplate-based B(II) cell bioassay. Bioassay-guided HPLC analysis of seeds of Carex pendula (drooping sedge) provided one previously unknown tetrastilbene (cis-miyabenol A) and two known oligostilbenes (kobophenol B and cis-miyabenol C) as the biologically active compounds (EC50 values were 31, 37 and 19 microM, respectively, vs. 5 x 10(-8) M 20-hydroxyecdysone). The structures and relative stereochemistries of these compounds were deduced by comprehensive ID- and 2D-NMR experiments. These compounds are isolated from Carex pendula for the first time. In vitro experiments with dipteran and lepidopteran ecdysteroid receptor proteins demonstrate that the oligostilbenes are able to compete with radiolabelled ecdysteroid ([3H]ponasterone A) for occupancy of the ligand binding site. IC50/Ki values are similar to the EC50 values obtained in the B(II) bioassay.

20-Hydroxyecdysone release from biodegradable devices: the effect of size and shape.

20-Hydroxyecdysone (20-OH) is a natural compound with many demonstrated effects on the physiological functions of vertebrates, particularly increased protein synthesis. Our study sought a suitable dosage form with continuous release of the drug lasting several weeks for implantation into agricultural animals. Biodegradable microparticles and implants of poly(L-lactic) and poly(DL-lactic) acids were prepared. Oligomers of these materials were synthesized, and a method of melting the binary mixture of the oligomer and 20-OH was employed. The particles were prepared simply by grinding the solidified block of the melt and sieving. Implants were prepared by extruding the melt into silicone tubes, removing the solidified content, and cutting into cylinders of 2 mm diameter and various lengths. A new method of preparation of hollow cylinders by aspirating air into silicone tubes filled with the melt was developed. The experiments demonstrated stability of 20-OH during heat treatment. Release of the active ingredient was tested in static in vitro conditions, analogous to those at the site of implantation, and prolonged drug release was obtained with both types of implant. The hollow implants gave release rates nearest to ideal zero-order kinetics and would appear most appropriate for testing in vivo.

Ecdysone-inducible gene expression in mammalian cells and transgenic mice.

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.