Patent Application
20200148718
The present invention relates to extracts of purified 20-hydroxyecdysone (20E), and their therapeutic use, in particular for the improvement of muscular function, by increasing mass and muscular force as well as mobility in mammals, 20E having anabolic effects in mammals.
More particularly the invention allows to improve and/or prevent the degradation of muscular function in sarcopenic mammals or in mammals having been immobilised namely after a surgical procedure or suffering from muscular dystrophies namely of genetic origin.
Moreover, the invention allows to combat cardio-metabolic diseases including cardiovascular diseases and diabetes, via the metabolic effects of 20E.
Phytoecdysones represent an important family of polyhydroxylated sterols. These molecules are produced by various species of plants (ferns, gymnosperms, angiosperms) and participate in the defence of these plants against pests.
Given their abundance in nature, and the difficulty of their chemical synthesis, the phytoecdysones proposed in various food supplements are extracted from plants cultivated for this purpose. These plants produce a complex cocktail of phytoecdysones (e.g. Bathori et al., 1999; Kokoska & Jankovska, 2009) and their extracts contain for the most part 20E, but also a set of phytoecdysones called minor, as well as components belonging to other chemical families, the sum of the phytoecdysones representing according to the preparations from 5 to 90% of the extracts.
The nature of the minor phytoecdysones depends on the plants used as the raw material (Hunyadi et al., 2016), on the season in which the harvest is carried out, or even on the region in which they are cultivated (soil, climate) and is therefore capable of varying substantially from one batch to another. These variations are progressively attenuated during the purification.
The data of the literature describes 20E as slightly cytotoxic but, if this applies to the pure molecule, the same is not true for incompletely purified preparations.
With the goal of taking advantage of the beneficial effects of 20E on muscular quality in the context of a drug, it is necessary to have available a pharmaceutical-grade extract of 20E.
A pharmaceutical-grade extract of 20E (≥97%) from an extract purified to 90% is currently only obtained by the implementation of chromatographic methods (BPLC, HPLC). These methods, like that disclosed by Toth and Bathori (2008), are adapted for the production of quantities of extracts of approximately several grams.
The present invention is aimed at an extract of a plant or of a plant part, said plant being chosen from the plants containing at least 0.5% 20-hydroxyecdysone by dry weight of said plant, characterised in that said extract comprises at least 95%, and preferably at least 97%, 20-hydroxyecdysone.
The extract according to the invention, also called BIO101 below, is in a remarkable manner free of impurities, such as minor compounds, capable of affecting the safety, the availability or the effectiveness of a pharmaceutical use of said extract.
According to one feature of the invention, the impurities absent from BIO101 are compounds with 19 or 21 atoms of carbon, such as Rubrosterone, Dihydrorubrosterone or Poststerone.
The plant from which BIO101 is produced is advantageously chosen from Stemmacantha carthamoides (also called Leuzea carthamoides), Cyanotis arachnoidea and Cyanotis vaga. The invention is aimed more particularly at obtaining BIO101 from the roots of these plants.
The invention is especially interested in an extract of roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, 20-hydroxyecdysone.
Via the extract according to the invention, the 20E is purified to pharmaceutical grade and BIO101 can be used for various pharmaceutical uses.
Advantageously, BIO101 is intended to be used in order to improve and/or prevent the degradation of the muscular function of sarcopenic mammals or mammals that have been immobilised, namely after a surgical procedure, or suffering from muscular dystrophies, namely of genetic origin. Moreover, the use of BIO101 allows to combat cardio-metabolic diseases including cardiovascular diseases and diabetes, via the metabolic effects of 20E.
BIO101 is intended to be used in the treatment of muscular or cardio-metabolic illnesses.
Very particularly, the invention relates to an extract of roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, 20-hydroxyecdysone for its use in the treatment and/or the prevention of sarcopenia.
Likewise, the invention relates very particularly to an extract of roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, 20-hydroxyecdysone for its use in the treatment and/or the prevention of muscular dystrophies in particular of genetic origin.
The invention also relates very particularly to an extract of roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, 20-hydroxyecdysone for its use in the treatment and/or the prevention of the loss of mass and muscular function after an immobilisation, namely in a post-surgical context.
More particularly, BIO101 is used at a rate of an administration of 200 to 1000 mg/day, in one or more doses, to a patient.
The invention further relates to a composition comprising as an active agent the extract according to the invention (BIO101).
The composition preferably contains between 200 and 1000 mg of active agent (BIO101).
The invention also concerns a method for preparing an extract according to the invention, from a preparation of approximately 90% pure 20-hydroxyecdysone, characterised in that it comprises the following steps:
This purification involves a process of recrystallisation appropriate for this molecule and capable of being carried out on the industrial scale.
Advantageously, the filtration of step a) is carried out via a 0.2 μm particle filter.
The partial concentration of step a) is advantageously carried out by vacuum distillation, at a temperature of approximately 50° C., in the presence of MeOH.
The step f) of drying is carried out under vacuum at a temperature of approximately 50° C.
Advantageously, the method for preparing an extract that is the object of the invention allows to obtain a pharmaceutical-grade extract of 20E. The method for preparing an extract that is the object of the invention allows to work on the industrial scale.
Other advantages, goals and particular features of the present invention will be clear from the following non-limiting description of at least one specific embodiment of the devices that are objects of the present invention, with respect to the appended drawings, in which:
The present description is non-limiting.
1. Method for Purification by Recrystallisation
The method was developed while starting from a preparation of 20E extracted from Stemmacantha carthamoides, but it can apply to preparations of 20E coming from other plants.
A data sheet for 90%-pure 20E usable as an initial preparation is described in the appendix.
The process of purification, illustrated in
The solution obtained is then heated to 45-50° C., until a light brown colouration appears. The solution is then filtered through a 0.2 μm particle filter in a second reactor. The particle filter is rinsed with 5 L of MeOH, in said second reactor, then the solution is concentrated to a volume of 25 L by vacuum distillation at a temperature of 50° C.
80 L of acetone are added as an anti-solvent at 45-50° C. The crystallisation of the 20E is caused by cooling to 15-20° C. and by stirring for 16 to 18 hours at 15-25° C., then by cooling to 0-5° C. and by stirring for at least 4 hours. The precipitate is filtered and then it is washed with 20 L of acetone previously cooled to 0-5° C., and it is dried in a current of gaseous nitrogen for at least 1.5 hours.
The second reactor is loaded with the product thus obtained, which is again placed in suspension in 20 L of water in order to eliminate the residual acetone. The suspension is stirred at 15-25° C. for 2 hours, then it is filtered through the particle filter and the material is washed with 20 L of purified water before drying it under vacuum at a temperature less than or equal to 50° C.
2. Analysis of the Finished Product
The finished product (BIO101) was analysed by HPLC-MS/MS. The minor components were isolated by HPLC from the mother liquors and then analysed via mass spectrometry and NMR spectrometry. It turns out that the significant components present in a quantity greater than or equal to 0.05% in dry weight of BIO101 are exclusively phytoecdysones. The values reported in table 1 below correspond to averages over four distinct batches of raw extract of roots (Stemmacantha carthamoides) and four distinct batches of finished product. With regard to the data on the raw extract of roots, the values are compared to the sum of the phytoecdysones observed via HPLC, but in fact the 20E only represents 15-21% by weight in the dried residue of the alcoholic extract of the dried roots (n=2).
It is noted that at the end of all of the steps of the purification method, certain phytoecdysones were eliminated. Moreover, a new compound appeared: stachysterone B. The latter corresponds to a dehydration product resulting from the loss of the tertiary hydroxyl in position 14 of the 20E. This compound is partly eliminated during the purification, and no other new product appears during the method.
With regard to the four eliminated molecules, these are molecules resulting from a cutting of the side chain of the 20E and which are thus 21C (poststerone) or 19C (rubrosterone, dihydrorubrosterone) molecules. Contrary to 20E, these molecules are capable of interacting with the nuclear receptors of the androgens or of the oestrogens (Lapenna et al., 2015) and it is therefore crucial to eliminate them in order to prevent any risk of interference or even of adverse effects of the finished product. This point is even more important since these molecules, having a reduced polarity with respect to the 20E, have a bioavailability much higher than the latter and are therefore proportionally much better (approximately twenty times better) bioavailable.
3. Characterisation of the BIO101
Table 2 below presents the characterisation of the extract obtained above.
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4. Stability of the BIO101
The monitoring of the stability of the BIO101 stored at 25° C. and 60% relative humidity reveals the absence of an appearance of impurities after one year.
The same result is obtained for BIO101 stored at 30° C. and 65% relative humidity.
The product obtained thus turns out to be very stable, it does not degrade and does not generate undesirable impurities.
Forced-degradation tests, on samples of BIO101 at 40° C. and 75% relative humidity for 6 months, confirm the excellent behaviour of the BIO101, precisely due to its very high concentration of active principle with respect to the quantity of minor compounds.
5. Elements of Regulatory Toxicology
Works carried out with preparations of partially purified 20E coming from Pfaffia glomerate turned out to be genotoxic and cytotoxic (Neves et al., 2016a,b). This is due to the presence of other compounds present in alcoholic extracts of the plant (De Souza et al., 2005) and incompletely eliminated in the preparation of 20E used.
In terms of genotoxicity, the results obtained by following the ICH and OCDE rules in force in vitro and in vivo, on BIO101, were negative. In animals, no toxicity was observed at the highest dose tested, or 1000 and 1500 mg/kg respectively in rats and dogs chronically exposed for 28 days. Thus, the purified 20E obtained according to the method described above (BIO101) is not toxic, even at a high dose. The battery of “safety pharmaco” tests (behaviour, SNC, respiratory function, hERG test and cardiac telemetry) confirms this point.
The chronic administration of BIO101 orally in two GLP trials in rats and in dogs for 26 weeks confirmed the excellent tolerance of the product as well as its safety profile.
6. Pharmacokinetic Trials in Humans
In humans, Simon & Koolman (1989), then Brandt (2003) and finally Bolduc et al. (2008) measured the kinetics of urinary elimination of the partially purified 20E, after oral administration, respectively at the doses of 0.2 mg/kg*day, 10 mg/kg*day and 434 mg/day, either in a single dose, or over 5 days. These works bring to light a low oral bioavailability of this molecule.
The trials carried out in animals and humans with BIO101 confirm the low bioavailability of this molecule after oral administration. They show that the ingested molecule is totally eliminated after 48 hours, mainly through the faeces.
In the context of a phase I clinical trial, the plasma concentrations of BIO101 were measured after single administrations (SAD) of 100, 350, 700 and 1400 mg/day in individuals on an empty stomach. Repeated administrations (MAD) over 14 days were then carried out with a single daily dose of 350 mg or with two daily doses (morning and evening) of 350 mg and 450 mg. These administrations of BIO101 to various cohorts of volunteers (n=6) allowed to determine the main pharmacokinetic parameters of BIO101: Cmax (maximum plasma concentration of BIO101 after single or repeated), Cmin (minimum plasma concentration of BIO101 after single or repeated administration), Tmax (time necessary to reach the maximum plasma concentration of BIO101 after single or repeated administration), and AUC (Area under curve) synonymous with level of plasma exposure. These results are presented in table 3 below, and the values correspond to the arithmetic means (CV %) except for Tmax where these are the extreme values.
The average profile of the cohorts of the SAD is presented in
Table 4, below, illustrates the pharmacokinetic parameters after repeated administrations of BIO101. This table shows that the repeated administrations have very similar profiles between the first and the fourteenth day, in accordance with an absence of plasma accumulation of the ingested compound as well as with the absence of induction of mechanisms of detoxication with the doses used.
The values correspond to the arithmetic means (CV %) except for Tmax where these are the extreme values. N=8 unless otherwise indicated; n=number of subjects for this observation; NA=does not apply. * A subject left the trial after 10 days.
These results are compatible with a chronic use of BIO101, which has also been validated by the absence of toxicity after a daily administration for 6 months at doses of 1000 mg/kg*d in rats and 1500 mg/kg*d in dogs.
7. Biological Activity of BIO101
While the anabolising effects of the 20E present in commercial preparations have already been demonstrated in young animals, it is not known whether the same is true in an old context, which is accompanied by a reduction in the mass of the muscles (sarcopenia) and especially in their performance (dynapenia), which cause mobility problems. BIO101 was administered chronically to old mice and the maximum running speed of the animals was measured and compared to that of old animals and non-treated adults. This trial demonstrated that the anabolic/hypertrophic effects of BIO101 translate into a functional improvement characterised by an increase in the maximum running speed.
Adult (12 months) and old (22 months) C57Bl6/J female mice were used. A group of old mice received BIO101 orally for 13 weeks at the dose of 50 mg/kg*day and another group the vehicle. The adult mice were exposed to the vehicle. All the animals were exposed to a hypercaloric diet rich in lipids over the entire duration of the experimentation (4442 kcal/kg; proteins 19.8%, lipids 23% and carbohydrates 39.5%). After 13 weeks of treatment, the animals were tested for their functional capacity (activity in toto). They were subjected to a running exercise and the maximum running speed was recorded.
The results are illustrated in
Importantly, it is demonstrated here that the treatment of the old animals with BIO101 compensates for a large part of the significant loss of running speed due to the ageing of the animals. Indeed, there is a significant overlap of the 95% confidence intervals of the groups old+BIO101 (95% CI=0.471 at 0.558 m/s) and Untreated adult (95% CI=0.503 at 0.602 m/s). The treatment with BIO101 thus allows to limit the functional loss related to ageing to 7%, instead of the 20% observed in the untreated old animals.
These works demonstrate that the beneficial properties of the active ingredient of BIO101 on muscular function (Chermnykh et al., 1988; Gorelick-Feldman et al., 2008; Toth et al., 2008) apply in the context of old mammals, and that the chronic treatment of old animals with BIO101 can significantly compensate for the functional losses due to ageing.
More widely, and given the properties of BIO101 on muscular function, the use of BIO101 can thus be proposed in order to treat illnesses that have the consequence of deterioration of said muscular function. These illnesses include, but are not limited to sarcopenia, muscular dystrophies, namely of genetic origin and the loss of muscular mass and function after an immobilisation, namely in a post-surgical context.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1753775 | Apr 2017 | FR | national |
| 1758071 | Aug 2017 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/061060 | 4/30/2018 | WO | 00 |
