Patent Grant
11207411
The present invention relates to a stabilized pharmaceutical preparation of R27T, the preparation comprising a human interferon beta variant (R27T), an acetate buffer, arginine, trehalose, Poloxamer 188, and methionine.
Interferons (IFNs) exhibit anti-viral activity as a type of cytokine and have a function of suppressing cell proliferation and regulating innate immune responses. IFNs are classified into IFN-α, IFN-β and IFN-γ according to cellular origin (leukocytes, fibroblasts, T cells). Among them, interferon-beta (IFN-β) is a spherical protein having 5 alpha-helices (α-helices), which is 22 kD in size, and the size becomes 18 kD when the sugar chain is removed. Studies on the clinical application of IFN-β have been actively conducted, IFN-β has been particularly highlighted as an agent for mitigating, reducing, or treating symptoms of multiple sclerosis, and further, through various studies, study results that IFN-β is effective for treating cancer, autoimmune diseases and viral infectious diseases, diseases associated with HIV, hepatitis C, rheumatoid arthritis, and the like with various immunological activities such as anti-viral activity, cell growth suppression, lymphocyte cytotoxicity increasing activity, immunoregulatory activity, activity of inducing or suppressing differentiation of target cells, activation of macrophages, activity for increasing production of cytokines, activity for increasing effects of cytotoxic T cells, and activity for increasing natural killing cells have been reported.
Human IFN-β is also a type of glycoprotein, and since the sugar chain moiety linked to the protein plays an important role in the activity of the protein, in the case of a glycoprotein, the activity of the protein may increase when a sugar chain is added. That is, it is known that protein glycosylation may affect a lot of biochemical characteristics such as stability, solubility, intracellular trafficking activity, pharmacokinetics and antigenicity.
Thus, an example has been reported in which a sugar chain is introduced into natural human IFN-β as a glycoprotein to prepare a human IFN-β variant whose activity or function is increased or improved (Korean Patent No. 10-0781666). The human IFN-β variant R27T used in the present invention is a recombinant human IFN-β variant (hereinafter, referred to as rhINF-β) designed by substituting arginine (Arg) at position 27 with threonine (Thr) for additional glycosylation of position 25 of IFN-β 1a, and exhibits effects of increasing stability, decreasing a protein aggregation tendency, and increasing half-life when compared with the wild-type INF-β 1a (Rebif). That is, R27T is a biobetter of rhINF-β produced for additional glycosylation through site-directed mutagenesis.
Meanwhile, one of the major issues in the development of protein drugs is to produce a product having a long shelf life for 24 months or more by imparting sufficient chemical, physical and biological stability. However, due to various intrinsic susceptibilities in a protein degradation pathway, the complexity of protein structures having various levels, such as macromolecules and secondary, tertiary, and quaternary structures of proteins, it still remains a difficult problem to achieve high stability.
30 years or more have passed since the first approval and successful production of insulin as the first recombinant peptide hormone in 1982, and subsequently, numerous successful cases of recombinant protein/peptide drugs have been reported until now. However, in the process of developing biopharmaceuticals, particularly in the formulation of biopharmaceuticals, difficulties are still faced due to various factors such as protein aggregation, physicochemical instability, low half-life, low solubility, and pharmacokinetic properties.
In particular, protein aggregation is one of the major issues easily occurring in almost all biopharmaceutical processes, but the reason is that therapeutic proteins are structurally/thermodynamically unstable in solution during storage. Since therapeutic proteins are susceptible to structural changes caused by various factors during purification, processing, and storage, the aforementioned problems may be exacerbated when proteins are exposed to high temperature, maximum/minimum pH, shear strain, and surface adsorption. In addition, since protein-based biopharmaceuticals have the possibility of physical degradation such as formation of insoluble particles due to unfolding, aggregation, and non-native aggregation, optimization of a preparation system is required such as a stable pH range, an appropriate buffer system, and the development of excipients in order to avoid protein aggregation or physical degradation and maximize stability.
Therefore, the present inventors tried to develop a stabilized pharmaceutical preparation with enhanced storage and thermodynamic/structural stability of the human IFN-β variant.
In order to solve the problems in the related art, as a result of intensive studies to develop a pharmaceutical preparation capable of enhancing the stability of R27T which is a human interferon beta variant, the present inventors developed a stabilized R27T preparation having a novel composition in which storage and thermodynamic/structural stability are improved and the formation of an R27T protein aggregate is decreased as compared to existing preparations, thereby completing the present invention based on this finding.
Therefore, an object of the present invention is to provide a stabilized pharmaceutical preparation of a human interferon beta variant (R27T) having a novel composition.
However, a technical problem to be achieved by the present invention is not limited to the aforementioned problem, and other problems that are not mentioned may be clearly understood by those skilled in the art from the following description.
To achieve the object of the present invention, the present invention provides a stabilized pharmaceutical preparation of a human interferon beta variant (R27T), comprising the following:
(a) a human interferon beta variant (R27T),
(b) an acetate buffer at a concentration of 5 to 100 mM,
(c) arginine at a concentration of 10 to 150 mM,
(d) trehalose at a concentration of 50 to 300 mM,
(e) Poloxamer 188 at a concentration of 0.1 to 10 mg/mL, and
(f) methionine at a concentration of 0.5 to 5 mM.
As an embodiment of the present invention, the human interferon beta variant (R27T) may include an N-linked sugar chain at an asparagine residue which is amino acid 25 by substituting arginine which is amino acid 27 of the human interferon beta with threonine.
As another embodiment of the present invention, the acetate buffer may be included at a concentration of 10 to 30 mM.
As still another embodiment of the present invention, the acetate buffer may have a pH within a range of 3.6 to 4.4.
As yet another embodiment of the present invention, the arginine may be included at a concentration of 50 to 100 mM.
As yet another embodiment of the present invention, the trehalose may be included at a concentration of 150 to 250 mM.
As yet another embodiment of the present invention, the Poloxamer 188 may be included at a concentration of 0.1 to 1 mg/mL.
As yet another embodiment of the present invention, the methionine may be included at a concentration of 0.5 to 2 mM.
As yet another embodiment of the present invention, the stabilized pharmaceutical preparation may have a hydrogen ion concentration index (pH) within a range of 3.6 to 4.4.
As yet another embodiment of the present invention, the stabilized pharmaceutical preparation may be for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis.
As yet another embodiment of the present invention, the stabilized pharmaceutical preparation may be for oral or parenteral administration.
As yet another embodiment of the present invention, the stabilized pharmaceutical preparation may be a liquid or lyophilized formulation.
Further, the present invention provides a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis, the composition comprising a pharmaceutically effective amount of the stabilized preparation.
In addition, the present invention provides a method for preventing or treating one or more diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis, the method comprising a step of administering a pharmaceutically effective amount of the stabilized preparation to an individual.
Furthermore, the present invention provides a use of the stabilized preparation for preventing or treating one or more diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis.
The stabilized R27T pharmaceutical preparation according to the present invention is obtained by the development of a preparation having a novel composition through the substitution of mannitol with trehalose in the composition of a preparation containing mannitol, which was previously studied by the present inventors. It was confirmed that the preparation can remedy a disadvantage of the protein aggregate increase due to the mixing and addition of mannitol and arginine HCl and can improve thermodynamic/structural stability and the resulting stability during long-term storage, and thus it is expected that the preparation can be advantageously used in the prevention, alleviation, and treatment of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, rheumatoid arthritis, and the like.
The present invention relates to a stabilized pharmaceutical preparation of R27T, the preparation comprising a human interferon beta variant (R27T), an acetate buffer, arginine, trehalose, Poloxamer 188, and methionine.
Hereinafter, the present invention will be described in detail.
The present inventors selected a 20 mM acetate buffer with a pH of 3.8±0.2 using a fundamental buffer system which is optimized for R27T through prior studies to develop a stabilized pharmaceutical preparation of a human interferon beta variant R27T, and confirmed through experiments in which various excipients for enhancing the stability of R27T were added that a preparation prepared by adding mannitol, arginine HCl, Poloxamer 188, and methionine could enhance the stability of R27T as compared to existing preparations. However, the mixed composition of mannitol and arginine HCl has a disadvantage in that when the mixed composition is stored at high concentration and high temperature, stability is decreased as with existing preparations, and in order to overcome the disadvantage, the present inventors developed a stabilized pharmaceutical preparation of R27T having a new composition capable of enhancing the stability of R27T by screening an excipient capable of replacing mannitol.
Therefore, the present invention provides a stabilized pharmaceutical preparation of a human interferon beta variant (R27T), comprising a human interferon beta variant (R27T), an acetate buffer at a concentration of 5 to 100 mM, arginine at a concentration of 10 to 150 mM, trehalose at a concentration of 50 to 300 mM, Poloxamer 188 at a concentration of 0.1 to 10 mg/mL, and methionine at a concentration of 0.5 to 5 mM.
In an embodiment of the present invention, after a R27T preparation having the same composition was prepared by using trehalose, sucrose, and xylitol in order to find a mannitol substitute capable of enhancing the storage stability of R27T as compared to the aforementioned R27T preparation prepared by adding mannitol, the stability of each preparation was determined after each preparation was stored at low concentration. As a result, it was confirmed that when trehalose was used, storage stability and reversibility were significantly increased, and that even when sucrose and xylitol were used, the reversibility of the preparation was relatively increased (see Example 2).
In another embodiment of the present invention, as a result of determining the stability and anti-viral activity of the R27T preparation to which trehalose or xylitol was added as an excipient by replacing mannitol based on the result of Example 2, it was confirmed that a preparation prepared by adding trehalose during storage at high temperature had the best stability against heat and the best stability according to an increase in concentration, and exhibited thermodynamic/structural stability and anti-viral activity similar to those of a preparation using mannitol (see Example 3).
In still another embodiment of the present invention, as a result of analyzing the stability of the preparation through the change in protein aggregate in a composition in which mannitol or trehalose was mixed with arginine, it was confirmed that mannitol increased the protein aggregation when mixed with arginine as compared to the case where arginine was not present, whereas trehalose rather decreased protein aggregation in a mixed composition with arginine (see Example 4).
Through the results of the embodiments of the present invention, it was confirmed that the novel R27T stabilized pharmaceutical preparation of the present invention using trehalose as an excipient is excellent in stability as compared to existing R27T preparations using mannitol, and thus the novel R27T stabilized pharmaceutical preparation may be usefully used for the treatment of various diseases by using interferon beta.
As used herein, the term “interferon-beta (IFN-β)” refers to not only a human-originated fibroblast interferon obtained by isolation from a biological fluid or obtained from eukaryotic or prokaryotic host cells by a DNA recombinant technique, but also salts, functional derivatives, variants, analogs, and active fractions thereof. Preferably, IFN-β is intended to mean Interferon beta-1a.
As used herein, trehalose used as a substitute for mannitol refers to a non-reducing disaccharide where two molecules of D-glucose are linked via a 1→1 alpha bond, and chemically, there are three types of an α,α type, an α,β type, and a β,β type, and trehalose that may be naturally obtained is an α,α type. Trehalose was first discovered in ergot of rye, is present in large amounts in bacteria or yeast and was observed even in body fluids and eggs of insects, and is known to play an important role as an energy source or a storage carbohydrate of insects.
As used herein, the term “stabilized preparation” is a preparation such that the degree of degradation, denaturation, aggregation, and loss of biological activity of the proteins contained therein is controlled to an acceptable degree and is not increased to an unacceptable level over time. Preferably, the preparation maintains R27T activity to at least about 60%, preferably at least about 70%, and more preferably at least about 80% for up to 24 months.
As used herein, the term “buffer” refers to a solution of a compound having an effect of adjusting or maintaining the pH of a preparation such that the pH of the preparation falls within a preferred pH range. In the present invention, a buffer suitable for adjusting pH is not limited, but buffer may include phosphoric acid, acetic acid, citric acid, and histidine, is preferably an acetate buffer at a concentration of 5 to 100 mM, and more preferably, may be included at a concentration of 10 to 30 mM, even more preferably 20 mM.
In the present invention, the stabilized pharmaceutical preparation of R27T may be prepared by including: steps of dialyzing a solution including a human interferon variant R27T by using a solution including an acetate buffer at a concentration of 5 to 100 mM and an excipient; and filtering the dialysate.
In the present invention, as an excipient, arginine at a concentration of 10 to 150 mM, trehalose at a concentration of 50 to 300 mM, Poloxamer 188 at a concentration of 0.1 to 10 mg/mL, and methionine at a concentration of 0.5 to 5 mM may be added, and more preferably, arginine at a concentration of 50 to 100 mM, trehalose at a concentration of 150 to 250 mM, Poloxamer 188 at a concentration of 0.1 to 1 mg/mL, and methionine at a concentration of 0.5 to 2 mM may be added.
When the stabilized pharmaceutical preparation of R27T of the present invention is prepared as a “liquid formulation”, a preferred solvent is water or sterile water for injection, and may be a monodose or multidose. It is preferred that the liquid R27T preparation for multidose of the present invention includes a bacteriostatic agent such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal. The bacteriostatic agent is used in a content that may yield a concentration that is effective for maintaining an essentially bacteria free preparation (suitable for injection) over the multidose injection period, which is about 12 or 24 hours to about 12 days, preferably about 6 to 12 days. It is preferred that the bacteriostatic agent is present at a concentration of about 0.1% to about 2.0% (mass of the bacteriostatic agent/mass of the solvent).
The preparation of the present invention may optionally further include not only a diluent, an excipient, and a carrier, but also a physiologically/pharmaceutically acceptable additive, for example, a free-fluidizing agent, an emulsifier, a stabilizer, a preservative, a colorant, an antifoaming agent, and an anticaking agent.
The pharmaceutically acceptable carrier is not particularly limited, but may include physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, and the like.
Further, the present invention provides a method for treating a disease such as multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis by administering a pharmaceutically effective amount of a sterilized preparation to an individual.
As used herein, the “individual” refers to a subject in need of treatment for a disease, and more specifically, refers to a mammal such as a human or a non-human primate, a mouse, a rat, a dog, a cat, a horse, and a cow.
Regarding the “pharmaceutically effective amount”, it is obvious to those skilled in the art that the range thereof may be adjusted variously depending on a patient's body weight, age, gender, health status, diet, administration time, administration method, and excretion rate, severity of disease, and the like.
A preferred dosage of the preparation of the present invention varies depending on the condition and body weight of a patient, the degree of a disease, the form of drug, the administration route, and the duration, but may be appropriately selected by those skilled in the art. However, the preparation is administered preferably at 0.001 to 100 mg/kg of body weight daily, and more preferably at 0.01 to 30 mg/kg of body weight daily. The dose may be administered once a day or may be divided into several doses.
The preparation of the present invention may be administered to a mammal such as a rat, a mouse, livestock, and a human via various routes. The administration method is not limited, and the preparation of the present invention may be administered by oral, rectal, or intravenous, intramuscular, hypodermic, intrauterine, or intra-cerebroventricular injections.
Hereinafter, preferred Examples for helping the understanding of the present invention will be suggested. However, the following Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the following Examples.
1-1. Size Exclusion Chromatography (SEC)
An R27T sample was analyzed by using an Agilent high performance liquid chromatography system (Agilent HPLC 1260, Santa Clara, Calif., USA) equipped with a TSK-GEL G3000SWXL SEC column (TOSOH Bioscience, PA, USA) and a diode detector (DAD). In order to isolate water-soluble R27T particles, mobile-phase A (a 0.1% aqueous trifluoroacetic acid (TFA) solution, 100 mM NaCl) and mobile phase B (0.1% TFA acetonitrile, 100 mM NaCl) were flowed at a flow rate of 0.5 mL/min at a ratio of 4:6. A region analyzed as a peak of the multimer in the sample was calculated along with a region of the aqueous aggregate. The difference in total area of the R27T sample between the first measurement and the subsequent measurements (sum of the areas of all peak regions in the chromatogram result) was defined to be due to the formation of an insoluble aggregate during the measurements. The remaining proportions of the respective types (insoluble aggregates, monomers, and protein fragments) were calculated as a peak area compared to the initial time, and the storage period was graphed as the X-axis. In this case, the equation for calculating the remaining proportion is as follows.
Remaining amount (%)=(at−A0)×100
In the equation, at refers to the area of the protein peak at each time, and A0 refers to the initial area of each protein peak. The error bar refers to a standard deviation (SD) for three measurements.
1-2. Differential Scanning Calorimetry (DSC)
In order to perform the thermodynamic stability evaluation of the R27T sample, a VP-DSC Microcalorimeter (Microcal, Northampton, Mass., USA) was used. The experiment was performed at a heating rate of 1° C. per minute from 15° C. to 120° C. and repeated three times in total. The DSC experimental results were standardized by subtracting a baseline measured using a buffer finally measured and calculating the concentration of protein present in the sample.
The R27T measurement results set a baseline for zero adjustment of the result through a linear baseline adjustment. In this case, the procedure of setting the baseline for zero adjustment of the sample is complicated because the procedure is disturbed by the aggregation and precipitation phenomenon caused by heating, and the present invention allowed the most stable result to be obtained regardless of the type of sample or the determination of the user in the repeated experiments by selecting a prior baseline option.
The final calorimetric record was plotted with the excess specific heat (cal/° C.mol) on the Y axis and the temperature on the X axis (° C.). From these results, the protein transition temperature (Tm) was calculated.
1-3. Cytopathic Effect (CPE) Assay
In order to evaluate the anti-viral effect of each R27T stabilized preparation, A549 cells as a lung cancer cell line were aliquoted at 3×105 cells/mL into a 96-well plate, 100 μl of a medium including the cells was treated with 100 μl of each preparation, and the cells were cultured at 37° C. for 22 hours. After the supernatant was removed on day 2 and the cells were treated with 100 uL of encephalomyocarditis virus (EMCV) at a concentration of 1000 TCID50/mL and cultured at 37° C. for 22 hours, the titer as compared to the standard was calculated by removing the supernatant on day 3 and staining the viable cells to measure absorbance at 570 nm.
2-1. Preparation of R27T Preparation
The present inventors prepared a preparation for enhancing the stability of an interferon beta variant R27T by adding excipients of 20 mM acetate buffer with a pH of 3.8, mannitol, arginine HCl, Poloxamer 188, and methionine to the interferon variant through prior studies. Furthermore, the present invention intends to develop a preparation which is more stable than a mixed preparation to which mannitol and arginine HCl are added by discovering an excipient capable of substituting for mannitol used as an existing interferon stabilizer.
For this purpose, as shown in the following Table 1, a total of 10 types of R27T preparations were prepared by using the same composition and concentration as those of the other excipients along with the composition (1(F5)) of the preparation including mannitol prepared in prior studies, adding various concentrations (150, 200, or 250 mM) of trehalose known to be present in large amounts in bacteria or yeast and observed even in body fluids and eggs of insects as a non-reducing disaccharide where two molecules of D-glucose are linked via a 1→1 alpha bond instead of mannitol, or adding various concentrations (150, 200, or 250 mM) of sucrose or xylitol determined to substitute for mannitol in prior studies, and experiments for comparison and analysis of the stability of the preparation were performed.
2-2. SEC: Analysis of Amount of Monomer
In order to evaluate the storage stability of each R27T preparation prepared in Example 2-1, after the 10 preparations were refrigerated at a concentration of 0.1 mg/mL and 4° C. for 0, 1 week, 2 weeks, 3 weeks, and 3 months, respectively, the amount of a residual monomer was measured by using the size exclusion chromatography method in Example 1-1.
As a result, as illustrated in
3-1. SEC: Analysis of Amount of Monomer
Based on the results in Example 2, an additional stability analysis was performed mainly on the preparation using trehalose confirmed to enhance the cold-storage stability of the R27T preparation as compared to the case where mannitol was used. More specifically, the R27T preparation was prepared by adding mannitol at a concentration of 250 mM, or adding trehalose or xylitol at a concentration of 200 or 250 mM, and the detailed compositions of the preparations are shown in the following Table 2.
First, in order to evaluate the storage stability of the 5 R27T preparations, after each preparation was stored at a high concentration of 0.64 mg/mL and at a high temperature of 37° C. for 0, 14, 21, and 28 days, the amount of a monomer was measured by the method in Example 1-1.
As a result, as illustrated in
3-2. DSC: Analysis of Thermodynamic Stability
In addition to the analysis of the storage stability in Example 3-1, the thermodynamic/structural stability of R27T was evaluated by performing a DSC analysis on the 5 R27T preparations by the method in Example 1-2.
As a result, as illustrated in the following Table 3, it can be seen that in Preparations Nos. 1 to 3 to which mannitol or trehalose was added, Tm values higher than those of Preparations Nos. 4 and 5 to which xylitol was added were exhibited, Preparations Nos. 1 to 3 exhibited similar Tm values, and thus, the thermodynamic or structural stability of R27T was high in the 3 R27T preparations.
3-3. CPE Assay: Analysis of Anti-Viral Activity
In addition to the analyses of the stability in Examples 3-1 and 3-2, the CPE assay was performed by the method in Example 1-3 in order to compare the anti-viral activities of the respective R27T preparations.
As a result, as illustrated in the following Table 4, it was confirmed that in the case of Preparation No. 2 to which 200 mM trehalose was added, an anti-viral activity similar to that of the preparation to which mannitol was added was exhibited.
Through the Example results, it was confirmed that when the R27T preparation prepared by adding 200 mM trehalose was compared with the preparation prepared by adding 250 mM mannitol, storage stability, structural stability, and anti-viral activity were similar or enhanced, and it can be seen that trehalose at the above concentration had an advantage sufficient for replacing mannitol.
In the R27T preparation using 200 mM trehalose finally selected as an excipient capable of substituting for mannitol through Examples 2 and 3, it was analyzed whether there is a difference in storage stability of the preparation according to the presence or absence of arginine HCl. For this purpose, as illustrated in the following Table 5, a preparation (control-1) using only an acetate buffer and an R27T preparation (control-2) to which 250 mM mannitol was added without adding arginine HCl were used as controls, and a preparation (3) to which 50 mM arginine HCl and 250 mM mannitol were added together and preparations (4 and 5) to which 200 mM trehalose was added with or without the addition of 50 mM arginine HCl were used as experimental groups. After each preparation was stored at a low concentration (0.1 mg/mL) or a high concentration (0.64 mg/mL) at a high temperature of 37° C. for 1, 7, 14, and 28 days, the degree of protein aggregation was measured through the SEC analysis of Example 1-1.
As a result, as illustrated in
Through the result, it can be seen that the preparation to which trehalose and arginine HCl was added together had better stability than the mannitol-based preparation.
The above-described description of the present invention is provided for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described Examples are illustrative only in all aspects and are not restrictive.
The stabilized R27T pharmaceutical preparation provided by the present invention is a preparation having a novel composition through the substitution of mannitol with trehalose in the composition of a preparation containing mannitol, which was previously studied by the present inventors. It was confirmed that the preparation can remedy a disadvantage of the protein aggregate increase due to the mixing and addition of mannitol and arginine HCl and can improve thermodynamic/structural stability and the resulting stability during long-term storage, and thus the preparation can be advantageously used in the prevention, alleviation, and treatment of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, rheumatoid arthritis, and the like.
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| PCT/KR2017/010826 | 9/28/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2018/066891 | 4/12/2018 | WO | A |
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