Patent Application
20220142957
The present disclosure describes a eutectic composition for use in the treatment of cancer, comprising a nonsteroidal anti-inflammatory drug and a terpene.
Deep eutectic solvents (DES) have emerged in the last decade as a new class of ionic liquid (IL) analogues. Although DES share many characteristics with ILs, the terms are not interchangeable and DES offers several other advantages that turns them into a viable alternative to ILs. Contrary to ILs, DES fully obey the green chemistry metrics being less toxic, often biodegradable and no waste is generated upon their production. Furthermore, DES are cheaper to produce since the raw materials have lower cost and the synthesis is very simple and with high purity as compared with other designer solvents. DES are obtained by mixing two or more components which at certain molar ratio supress the melting point of their chief compounds. This depression in temperature is the result of charge delocalization occurring via hydrogen bonding between the components of the mixture. The properties of DES can be tuned by changing the molar ratio and/or nature of hydrogen bond donor (HBD) and the hydrogen bond acceptor (HBA), which in turn influence the position and the number of the hydrogen bonds. All these attractive features have positioned DES as attractive and advanced designer solvents with a wide range of applications including extraction, carbon dioxide capture, electrochemistry, biocatalysis and biomedical applications. In the biomedical field it has been reported that DES improve the solubility, permeation and absorption of model active pharmaceutical ingredients (API's). When one of the components of the DES is an API the system is called therapeutic deep eutectic solvents (THEDES).
Fatty acids, terpenes and nonsteroidal anti-inflammatory drugs (NSAIDs) have already been reported to have an effect on the inflammation process. Capric acid (“CA”), is known to act against oxidative stress and pro-inflammatory cytokines. Menthol is capable of reducing IL-1β at chronic colonic inflammation. Ibuprofen (“IBU”) is a commercialized anti-inflammatory compound also associated with tumour reduction.
The scientific publication by N. González and H. Sumano on “Design of Two Liquid Ibuprofen-Poloxamer-Limonene or Menthol Preparations for Dermal Administration” discloses a method of preparing an ibuprofen-limonene mixture with enhanced transdermal permeation preparation. The enhanced transdermal permeation effect is due to the addition of Poloxamer 407 to the mixture. The scientific publication does not disclose an ibuprofen-limonene mixture whereby the enhanced cell permeation property observed is attributed to the mixture being a deep eutectic system.
Document CN 108186620 A discloses the use of ibuprofen in the preparation of antitumor drugs. Specifically, the document discloses the use of a specific concentration (10% to 19%) of ibuprofen as an injection for the treatment of pancreatic cancer. The document does not disclose the use of ibuprofen together with limonene as an anti-tumour drug mixture.
These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.
This disclosure relates to the thermophysical properties of different therapeutic deep eutectic systems (“THEDES”) for use in the treatment or therapy of cancer.
Carcinogenesis is a phenomenon not only restricted to the abnormal growth of cells but also includes angiogenesis and inflammation processes which play an important role in tumour progression. The bioactivity of these emerging THEDES solvents is not yet well explored, nonetheless, literature has already reported cytotoxicity of ammonium- and choline chloride-based DES for several cancer cell lines. This disclosure describes the potential of naturally occurring molecules such as terpenes and fatty acids in anticancer therapies.
Limonene (“LIM”) is a cyclic monoterpene studied at preclinical and clinical levels due to its chemopreventive and chemotherapeutic activities against several types of cancer such as lung, breast, gastric and prostate etc. It is generally recognized as safe (GRAS) for use in the food industry as a flavouring agent. Its high lipophilicity contributes to a favourable cellular absorption, specifically at the intestinal level, allowing good bioavailability in the systemic circulation. In colorectal cancer, limonene has been reported to induce apoptosis via mitochondrial pathway and affects the PI3K/Akt signalling pathway (survival and apoptosis).
One of the aspects of the present disclosure is the development of novel THEDES based on LIM due to its well-known anticancer properties. LIM was combined with different compounds, including menthol, IBU and CA due to their well-known anti-inflammatory properties. CA also increases the bioavailability of the API.
An aspect of the present disclosure described a eutectic composition for use in the treatment or therapy of cancer, comprising a nonsteroidal anti-inflammatory drug and a terpene.
In an embodiment for better results, the nonsteroidal anti-inflammatory drug may be selected from a list consisting of ibuprofen, flurbiprofen, ketoprofen, acetylsalicylic acid or mixtures thereof, preferably ibuprofen.
In an embodiment for better results, the molar ratio of anti-inflammatory drug:terpene may vary from 1:1-1:8. Preferably, the molar ratio of anti-inflammatory drug:terpene varies from 1:4-1:6; more preferably 1:3-1:6.
In an embodiment for better results, the molar ratio of ibuprofen:terpene varies from 1:4-1:6.
In an embodiment for better results, the molar ratio of anti-inflammatory drug:limonene varies, from 1:4-1:6.
In an embodiment for better results, the terpene may be selected from a list consisting of: limonene, carvacrol, thymol, safranal, linalool, myrcene, perilyl alcohol, borneol, geraniol, pinene, citronellol, or mixtures thereof, preferably limonene.
In an embodiment for better results, the composition of the present disclosure may comprise the combination of:
ibuprofen and limonene; ibuprofen and menthol; ibuprofen and carvacrol; ibuprofen and thymol; ibuprofen and safranal; ibuprofen and linalool; ibuprofen and myrcene; ibuprofen and perilyl alcohol; ibuprofen and borneol; ibuprofen and geraniol; ibuprofen and pinene; ibuprofen and citronellol; flurbiprofen and limonene; flurbiprofen and carvacrol; flurbiprofen and thymol; flurbiprofen and safranal; flurbiprofen and linalool; flurbiprofen and perilyl alcohol; flurbiprofen and geraniol; flurbiprofen and citronellol; ketoprofen and carvacrol; ketoprofen and thymol; ketoprofen and safranal; ketoprofen and linalool; ketoprofen and perilyl alcohol; ketoprofen and geraniol; ketoprofen and citronellol; acetylsalicylic acid and safranal; acetylsalicylic acid and linalool; acetylsalicylic acid and geraniol; acetylsalicylic acid and citronellol; or mixtures thereof.
In an embodiment for better results, the composition of the present disclosure preferably comprising ibuprofen and limonene, or ibuprofen and perylil alcohol.
In an embodiment for better results, the molar ratio of ibuprofen:limonene varies from 1:3-1:8, preferably from 1:4-1:6.
In an embodiment for better results, the ibuprofen concentration is preferably from 0.15-0.75 mM, preferably from 0.25-0.5 mM.
In an embodiment for better results, the limonene concentration is preferably from 0.5-3 mM, preferably from 1-2 mM.
In an embodiment, the composition is an injectable composition or an oral composition.
Another aspect of the present disclosure is the use of the eutectic composition of the present subject-matter for use in medicine or as a medicament, or veterinary. Preferably, for use in cancer therapy or treatment.
Another aspect of the present disclosure is the use of the composition as an anti-proliferator for cancer cells.
Another aspect of the present disclosure is a method of inhibiting the proliferation of cancer cells in a subject, the method comprising administering the eutectic composition of the present subject-matter.
In one embodiment, different formulations based on LIM were prepared. The formulations that led to a pasty-like solid at room temperature (“RT”, 20° C.) were readily discarded. Combinations based on CA:LIM (1:1), menthol:LIM (1:1), IBU:LIM (1:4) and IBU:LIM (1:8) were the ones that succeed in the formation of a transparent liquid solution which indicates the loss of lattice arrangement.
In another embodiment, thermal behaviour was evaluated. The evaluation of the thermal behaviour confirmed the formation of a eutectic mixture (
The hypothesis of having similar or different effects between THEDES and a simple mixture of the compounds has also been raised.
In one embodiment, a direct mixture of IBU with LIM in culture medium was performed and antiproliferative activity assessed, where it showed to be totally different from IBU:LIM (1:4) (
In another embodiment, the anticancer properties of IBU:LIM (1:4) were evaluated analysing the effects on cell cycle, apoptosis, intracellular reactive oxidative species (“ROS”) and nitric oxide (“NO”) production.
In one embodiment, IBU:LIM (1:4) was not capable of inhibiting cell cycle or apoptosis. This suggests that the antiproliferative effect of IBU:LIM (1:4) may not be due to the cell death mechanism involving caspase-3 dependent apoptosis or cell cycle arrest. Assessing intracellular ROS and NO production, the results indicate that the lowest concentration of IBU:LIM (1:4) (0.25+1 mM) protected HT29 cells from oxidative stress, demonstrating anti-inflammatory effects by inhibiting ROS and NO production (
In one embodiment, the apoptosis was not induced by caspase-3 cascade as shown in
Ibuprofen has been reported as a compound easily absorbed by passive transport associated to pH gradient at the intestinal gut. Terpenes, such as LIM, are known to stimulate paracellular transport of bigger molecules by interacting with tight junctions, however, its own transport is mainly passive.
In one embodiment, the eutectic composition of IBU:LIM surprisingly inhibit cancer proliferation without compromising the viability of other cells.
In one embodiment, transportation studies were performed using Caco-2 cell model differentiated in a transwell plate. The maintenance of Transepithelial Electrical Resistance (TEER) values reinforces the non-cytotoxicity of the concentration applied. The results obtained indicate a transcellular transport of IBU:LIM (1:4). While 40% of the DES was observed in either the basal or apical side, the remaining 60% was accumulated inside the cells or metabolized, which provides an indication that cells are able to metabolize or retain the DES in their interior. Moreover, this can confirm the effectiveness IBU:LIM (1:4) action.
In one embodiment, the results suggest that IBU:LIM (1:4) has different effects depending on the dose, being the mechanism of action completely different from isolated IBU and LIM. Therefore, these evidences demonstrate the effectiveness of the IBU:LIM (1:4) as compared to the individual compounds. Indeed, this THEDES comprises the protective and anti-inflammatory properties of IBU and the anticancer properties of LIM. Thus, it was surprisingly observed that using IBU:LIM (1:4) it is possible to decrease the cell cytotoxicity associated with LIM and increase the solubility of IBU. This shows the potential of this system as a drug delivery system.
The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.
The present disclosure is further described, in particular, using embodiments of the disclosure. Therefore, the disclosure is not limited to the descriptions and illustrations provided. These are used so that the disclosure is sufficiently detailed and comprehensive. Moreover, the intention of the drawings is for illustrative purposes and not for the purpose of limitation.
In an embodiment, different THEDES based on limonene (LIM) were developed to unravel the anticancer potential of such systems.
In one embodiment, THEDES mixtures were prepared. The reagents used in the preparation of THEDES were S-Limonene (LIM, ref.218367 Sigma Aldrich), Ibuprofen (IBU, ref. I4883, Sigma Aldrich), Capric acid (CA, ref. A14788.30, Sigma Aldrich), and menthol (ref.M2772, Sigma Aldrich). The LIM:IBU, CA:LIM, menthol:LIM systems were prepared at different molar ratios. The systems were prepared by gently mixing the two components at the given molar ratio. The mixture was heated to 40° C., with constant stirring, until a clear liquid solution was formed.
In one embodiment, thermal properties were evaluated using Differential Scanning calorimetry (DSC). The DSC experiments were performed in a thermal analyser (“TA”) DSC Q100 model (Thermal analysis & analysers, USA), using the different formulation in a TA aluminium pan. The temperature program for CA and CA:LIM comprises a heating step from −20° C. to 100° C. at 5° C. min−1, an isothermal step of 2 min at 100° C. and a cooling step to 20° C. at 5° C. min−1. For menthol thermograms the samples were equilibrated at 40° C. for 5 min followed by cooling to −40° C., an isothermal period for 5 min and heating to 120° C. at 5° C. min−1. The temperature programme for IBU based THEDES comprise a heating step from −20° C. to 120° C. at 5° C. min−1, an isothermal step of 2 min at 120° C. and a cooling step from 120° C. to 20° C. at 5° C. min−1. All measurements were performed under a nitrogen atmosphere (purge gas flux of ca. 50 mL min−1).
In one embodiment, solubility measurements were performed using IBU in powder or THEDES form (IBU:LIM at a molar ratio of 1:4 and 1:8). Briefly, an excess of API in powder and in THEDES form was added to a Hank's Balanced Salt Solution (HBSS, ref 14025-092, Alfagene) solution (≈37° C.) and stirred for 24 h. The determination of the API solubility was quantified by UV-vis spectroscopy at 265 nm in a microplate reader (BIO-TEK, SYNERGY HT).
In one embodiment, human Caco-2 and HT29 cell lines were obtained from Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ, Germany) and American Type Culture Collection (ATCC, USA), respectively. These cell lines were cultured in RPMI 1640 medium supplemented with 10% of heat-inactivated Fetal Bovine Serum (FBS). In the case of Caco-2, 1% Penicillin-Streptomycin (PS) was also added to the medium. Cells were maintained at 37° C. with 5% CO2 in a humidified incubator and routinely grown as a monolayer in 75 cm2 culture flasks. The cell culture medium and supplements were purchased from Invitrogen (Gibco, Invitrogen Corporation, UK).
In one embodiment, cytotoxicity assay was performed using confluent and non-differentiated Caco-2 cells. The assay was performed as previously described by Rodrigues et al. Briefly, Caco-2 cells were seeded into 96-well plates at a density of 2×104 cells/well and allowed to grow for 7 days, with medium renewal every 48 hours. On day 7, cells were incubated with the samples diluted in culture medium. Cells incubated only with culture medium were considered as control. After 24 hours, cells were washed once with PBS (Sigma-Aldrich, USA) and cell viability was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) containing MTS reagent, according to manufacturer's instructions. The absorbance was measured at 490 nm using a Spark® 10M Multimode Microplate Reader (Tecan Trading AG, Switzerland) and cell viability was expressed in terms of percentage of living cells relative to the control. At least three independent experiments were performed in triplicate.
In one embodiment, the antiproliferative effect of THEDES and standard compounds was evaluated in HT29 cells. Briefly, cells were seeded at a density of 1×104 cells/well in 96-well culture plates. After 24 hours cells were incubated with different concentrations of the samples diluted in culture medium. Cells incubated only with culture medium were considered as control. Cell proliferation was measured after 24 hours using MTS reagent, as mentioned above. Results were expressed in terms of percentage of living cells relative to the control. At least three independent experiments were performed in triplicate.
In one embodiment, cell transport studies were conducted. Caco-2 cells were seeded in 12 mm i.d. Transwell® inserts (polycarbonate membrane, 0.4 μm pore size, Corning Costar Corporation) in 12-well plates at a density of 2.24×105 cell/mL. Cells were allowed to grow and differentiate to confluent monolayers for 21-24 days post seeding by changing the medium three times a week. Transepithelial Electrical Resistance (TEER) of grown cells in Transwell was measured using EVOM™ voltmeter (WPI, Germany). Only monolayers with a TEER value higher than 700 Ωcm2 were used for experiments. For the assays, cells were washed two times with HBSS and the compounds diluted in HBSS were added to the apical side. Transepithelial transport was followed at several time points (0, 15, 30, 60, 120, 180, 240 minutes and 24 hours) where 200 μL was collected from the basolateral side. Samples were analysed by HPLC method as described above and concentration of compounds calculated. Results were expressed in terms of percentage of compounds at basolateral and apical sides and uptake by the cells. Three independent experiments were performed in duplicate.
In one embodiment, High Performance Liquid Chromatography (“HPLC”) analysis of samples resulting from transepithelial transport studies were performed using a Waters Alliance HPLC system (2695, Waters, Milford, Mass., USA) coupled to a photodiode array detector (2996, Waters, Ireland). Separation was carried out in a reversed-phase column LiChrospher 100 RP-18 (5 μm) LiChroCART 250-4 (Merck Millipore, Kenilworth, N.J., USA) in isocratic mode, with a mobile phase formed by 60:40 (% v/v) acetonitrile:water acidified with phosphoric acid (pH 2.5). The injection volume was set at 40 μL, the flow rate at 1.0 mL/min and the column temperature at a constant temperature of 40° C. Transepithelial transport was followed as a function of time by detection of IBU at 221 nm. Empower Pro (2002) software was used for data acquisition.
In one embodiment, cell cycle assessment was conducted. HT29 cell line was seeded at a density of 1×106 cells in a 25 cm2 culture flask for 24 hours. Then, cells were incubated another 24 hours with different concentrations of the samples diluted in culture medium and culture medium alone (as control). For flow cytometry analysis of DNA content, bare nuclei were prepared. Briefly, cells were detached from the culture flasks with trypsin-EDTA 0.5% and washed one time with cold PBS. Finally, 1×106 cells were re-suspended in 1 mL of staining solution containing 50 mg/mL of Propidium Iodide (Sigma-Aldrich, USA), 1.5% (v/v) of Triton X-100 (Sigma-Aldrich, USA), 0.700 U/mL of DNase/protease-free Ribonuclease A (Thermo Scientific, USA) and 0.01 M of NaCl, followed by incubation at room temperature in the dark for 2 hours. Cell cycle was assessed by flow cytometry, using a CyFlow space (Partec GmbH) instrument, registering 30.000 events/sample.
In one embodiment, apoptosis was evaluated by caspase-3 activity using NucView488™ and MitoView633™ Apoptosis Assay Kit (Biotium, USA) that allows staining of living cells by a far-red fluorescent dye MitoView633™ and the intracellular caspase-3/7 activity and cells nuclei morphological changes by bright green fluorescent dye NucView488™. HT29 cells were seeded at a density of 40.000 cell/cm2 at 24-well plate for 24 hours. Then, cells were incubated another 24 hours with different concentrations of the samples diluted in culture medium and in culture medium alone (as control). Staining was performed with 200 μL/well of culture medium containing 1 μL of NucView488™ and 1 μL of MitoView633™ for 2 hours. Cells were observed under fluorescence microscope (Leica DM6000, Germany) and image analysis performed using ImageJ software.
In one embodiment, intracellular ROS level were assessed according to the method described by Wolf and Liu (2007) with slight alterations. HT29 cells were seeded at a density of 40.000 cell/cm2 in a 24-well plate. After 24 hours, cells were incubated for 1 hour with different concentrations of the samples diluted in culture medium and in culture medium alone (as control). Cells were then washed two times with PBS, thereafter 600 μL of dichlorofluorescin-diacetate (DCFH-DA 25 uM) was added and the cells were allowed to incubate for 1 hour. Fluorescence was measured using a Microplate Fluorimeter FLx800 (Bioteck Instruments) (excitation and emission wavelengths of 485 nm and 528 nm, respectively). Results were expressed in terms of percentage of fluorescence intensity relative to the control. Three independent experiments were performed in triplicate.
In one embodiment, Griess assay was conducted. In order to evaluate the inhibition of nitric oxide (NO) species produced by HT29 treated with THEDES or isolated compounds, culture media was assessed using Griess reaction. A mixture of 50:50 (v/v) of Griess reagent (1% sulphanilamide, 0.1% N-(L-naphthyl)-ethylene diamine dihydrochloride and 2% H3PO4) and cell supernatant was prepared and the absorbance of the coloured solution was measured at 540 nm using a Spark® 10M Multimode Microplate Reader (Tecan Trading AG, Switzerland). Percentage of NO produced was expressed relative to the control. Three independent experiments were performed in triplicate.
In one embodiment, all data were expressed as mean±Standard Deviation (SD). GraphPad Prism 6 software was used to calculate EC50 values (the concentration of sample necessary to decrease 50% of cell population) and to analyse significant differences between data set through One-Way Analysis of Variance (ANOVA) following Dunnett's multiple comparison tests. A p-value<0.05 was considered statistically significant.
In one embodiment, THEDES were prepared by gently mixing capric acid (CA), menthol or ibuprofen (IBU) with LIM. Successful eutectic mixtures were obtained for menthol:LIM (1:1), CA:LIM (1:1), IBU:LIM (1:4) and IBU:LIM(1:8). LIM-based THEDES were evaluated in terms of their cytotoxicity and antiproliferative effects. The results indicate that all the THEDES present antiproliferative properties, but IBU:LIM; preferably IBU:LIM (1:4), surprisingly inhibit HT29 proliferation without compromising cell viability. Therefore, IBU:LIM (1:4) was the formulation selected for further assessment of anticancer properties. The results suggest that the mechanism of action of LIM:IBU is different from isolated IBU and LIM. Thus, the eutectic composition, IBU:LIM comprises the protective and anti-inflammatory properties of IBU allied to the anticancer properties of LIM.
In one embodiment, a novel THEDES based on LIM was developed due to LIM's well-known anticancer properties. LIM was combined with different compounds, including menthol, IBU and CA, due to their well-known anti-inflammatory properties. CA also increases the bioavailability of the API. Different formulations based on LIM were prepared. Formulations that led to a pasty-like solid at RT were readily discarded. Combinations based on CA:LIM (1:1), menthol:LIM (1:1), IBU:LIM (1:4) and IBU:LIM (1:8) were the ones that succeed in the formation of a transparent liquid solution. This indicates the loss of lattice arrangement. The evaluation of the thermal behaviour confirmed the formation of a eutectic mixture (
In one embodiment, LIM was mixed with different compounds (i.e. myristic acid (MA), menthol, CA, IBU) to obtain novel eutectic formulations with pharmaceutical applications that may boost the use of THEDES in cancer treatments. The different molar ratios are listed in Table 1. Using CA:LIM at a molar ratio of 1:1; 1:2 and 2:1 a clear and transparent liquid without any precipitate was obtained at RT after 1-2 hours. Similarly, a clear liquid was obtained using menthol:LIM at a molar ratio of 1:1, 1:2 and 2:1 where it was observed that there were no insoluble particles visible to the naked eye. Combining IBU with LIM in different molar ratios, only 1:8 gives rise to a transparent liquid at RT, whereas using a molar ratio of 1:4, a liquid mixture with almost imperceptible crystals was obtained at RT.
In one embodiment, the eutectic formulations were further studied by Differential Scanning Colorimetry (“DSC”). The DSC analysis (
In one embodiment, the cytotoxicity and antiproliferative effects of LIM-based THEDES were assessed (
In one embodiment, CA:LIM (1:1) and menthol:LIM (1:1) systems showed similar antiproliferative activity as compared to isolated LIM (Table 2). Using IBU:LIM (1:4) and IBU:LIM (1:8) eutectic systems, a behaviour distinct from the single components was observed. Although all eutectic mixtures showed antiproliferative effect, only IBU:LIM (1:4) was selected for further bioactivity evaluation. IBU:LIM (1:4) antiproliferative effect was compared with the simple mixture of IBU and LIM without THEDES preparation.
As
In one embodiment, the potential of LIM based THEDES to increase the solubility of IBU was assessed. As a non-steroidal anti-inflammatory compound, IBU has a very low solubility in water (≈2 mg mL−1). Thus, the solubility of IBU in physiological-like condition (i.e., HBSS at 37° C.) in powder and THEDES form was quantitatively determined (
In one embodiment, the bioactivity of THEDES was evaluated. Two non-cytotoxic concentrations of IBU:LIM (1:4) were used for analysis of cell cycle, apoptosis, intracellular ROS and NO production. As
In one embodiment, the effect over oxide species production was assessed. IBU:LIM (1:4) was capable of protecting HT29 cells from oxidative stress by decreasing the production of basal intracellular ROS relatively to the control (
In one embodiment, the selected combinations of THEDES were used to assess cell cytotoxicity and antiproliferative effects. The similar behaviour between LIM and CA:LIM (1:1) and menthol:LIM (1:1) (
In one embodiment, the effect of IBU:LIM (1:4), ibuprofen and limonene on ROS accumulation (
In one embodiment, IBU:LIM (1:4) cell transport in differentiated Caco-2 transwell cell model was analysed (
In an embodiment, other TEDES prepared combining an NSAIDs and a terpene can be obtained, as listed on Table 3. The systems were prepared by gently mixing the two components at the given molar ratio. The mixture was heated to 40° C., with constant stirring, until a clear liquid solution was formed. From the table it can be observed that different combinations were successfully obtained using ibuprofen, flurbiprofen and ketoprofen. In which concerns acetylsalicylic acid, fewer combinations were successfully achieved. In a further embodiment, the cytotoxicity (Caco-2 cell line) and antiproliferative effects (HT29 cells) of said THEDES, prepared from NSAIDs and terpenes, were assessed (Table 4).
The above described embodiments are combinable.
The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 115383 | Mar 2019 | PT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/052708 | 3/23/2020 | WO | 00 |
