Patent Application
20180221403
The present embodiments generally relate to fibrosis treatment, and in particular to the use of dextran sulfate for treating, inhibiting or preventing cardiac fibrosis in a subject.
Fibrosis is a process involving formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Fibrosis can be a reactive, benign or pathological state. In response to injury the fibrosis process is sometimes referred to scarring.
Physiologically fibrosis involves depositing connective tissue, which can obliterate the architecture and function of the underlying organ or tissue. Fibrosis is similar to the process of scarring in that both involve stimulated cells laying down connective tissue, including collagen and glycosaminoglycans. Macrophages and damaged tissue release transforming growth factor beta (TGFβ) in response to, for instance, inflammation or tissue damage. This in turn stimulates the proliferation and activation of fibroblasts, which deposit connective tissue.
U.S. Pat. No. 5,605,938 discloses that biocompatible anionic polymers, including dextran sulfate with an average molecular weight of about 40,000 to 2,000,000 Da, can inhibit fibrosis, scar formation and surgical adhesions typically in connection with surgery. The anionic polymers are administered locally at the fibrotic lesions or can be soaked onto an organ or implant in the form of a viscous liquid or gel that preferably also comprises an adhesive protein containing dihydroxyphenylalanine (DOPA) and hydroxyl-containing amino acid residues.
CN 102973593 discloses the use of dextran sulfate in preparing a medicament for treating hepatic fibrosis. The document mentions that dextran sulfate inhibits the activation of astrocytes and promotes macrophages to secrete metalloproteinase.
It is a general objective to treat, inhibit or reduce cardiac fibrosis in a subject.
This and other objectives are met by embodiments as disclosed herein.
An aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, formulated for systemic administration to a subject for use in treating, inhibiting or preventing cardiac fibrosis in the subject.
Another aspect of the embodiments relates to use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament formulated for systemic administration for treatment, inhibition or prevention of cardiac fibrosis in a subject.
A further aspect of the embodiments relates to a method of treating, inhibiting or preventing cardiac fibrosis in a subject. The method comprises systemically administering dextran sulfate, or a pharmaceutically acceptable salt thereof, to the subject.
The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:
The present embodiments generally relate to fibrosis treatment, and in particular to the use of dextran sulfate for treating, inhibiting or preventing cardiac fibrosis in a subject.
The embodiments are based on the discovery that systemically administered dextran sulfate, or a pharmaceutically acceptable salt thereof, is capable of reducing undesired fibrosis formation in a subject, and in particular reducing or inhibiting such fibrosis formation in ischemic tissue, in particular ischemic cardiac tissue.
Fibrotic diseases include a wide spectrum of medical conditions potentially affecting different organs and tissue in a subject's body. These medical conditions are characterized by elevated expression of genes encoding matrix proteins and the resulting fibrosis disrupts the normal architecture of the affected organ or tissue, ultimately leading to its dysfunction or failure.
The embodiments are capable or treating, inhibiting or preventing cardiac fibrosis in terms of treating, inhibiting or preventing adverse effects of fibrosis diseases by reducing the amount of formed fibrosis in the heart or cardiac tissue. Thus, dextran sulfate of the embodiments is capable of treating, inhibiting or preventing deleterious or injurious cardiac fibrosis.
Accordingly, an aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, formulated for systemic administration to a subject for use in treating, inhibiting or preventing cardiac fibrosis in the subject.
It was highly surprising that systemically delivered dextran sulfate could significantly reduce deleterious fibrosis formation in the light of U.S. Pat. No. 5,605,938. This patent document discloses that dextran sulfate administered locally could inhibit fibrosis and scar formation in connection with, among others, implantation of various implants. It was speculated therein that the negative charges of the dextran sulfate polymer were involved in inhibiting invasions of various cells at the implantation site. In order for such negative charges to have the cell-invasion-inhibiting effect dextran sulfate needs to be administered locally at the desired site. Accordingly, the implant was advantageously coated with dextran sulfate in U.S. Pat. No. 5,605,938.
The experimental data as presented herein shows that dextran sulfate of the embodiments can be systemically administered, i.e. not locally at the target site, and still exert its desired anti-fibrotic effect.
Dextran sulfate, or the pharmaceutically acceptable salt thereof, is formulated for systemic administration to the subject. In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is formulated for parenteral administration as an example of systemic administration to achieve a systemic effect in the subject.
Examples of parenteral administration routes include intravenous (i.v.) administration, intra-arterial administration, intra-muscular administration, intracerebral administration, intracerebroventricular administration, intrathecal administration and subcutaneous (s.c.) administration.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is preferably formulated for intravenous (i.v.) or subcutaneous (s.c.) administration to the subject. Accordingly, i.v. and s.c. administration are preferred examples of systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof.
Orally delivered dextran sulfate is known to induce colitis and intestinal fibrosis in mice, rats, hamsters and guinea pigs. Accordingly, systemic administration as used herein preferably excludes oral administration of dextran sulfate, or the pharmaceutically acceptable salt thereof. In a particular embodiment, systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, is a systemic administration other than oral administration, preferably other than enteral administration.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is formulated as an aqueous injection solution, preferably as an aqueous i.v. or s.c. injection solution. Thus, dextran sulfate, or the pharmaceutically acceptable salt thereof, of the embodiments is preferably formulated as an aqueous injection solution with a selected solvent or excipient. The solvent is advantageously an aqueous solvent and in particular a buffer solution. A non-limiting example of such a buffer solution is a citric acid buffer, such as citric acid monohydrate (CAM) buffer, or a phosphate buffer. For instance, dextran sulfate of the embodiments can be dissolved in saline, such as 0.9% NaCl saline, and then optionally buffered with 75 mM CAM and adjusting the pH to about 5.9 using sodium hydroxide. Also non-buffered solutions are possible, including aqueous injection solutions, such as saline, i.e. NaCl (aq). Furthermore, other buffer systems than CAM and phosphate buffers could be used if a buffered solution are desired.
Dextran sulfate is preferably a so-called low molecular weight dextran sulfate.
In the following, reference to (average) molecular weight and sulfur content of dextran sulfate applies also to any pharmaceutically acceptable salt of dextran sulfate. Hence, the pharmaceutically acceptable salt of dextran sulfate preferably has the average molecular weight and sulfur content as discussed in the following embodiments.
Dextran sulfate is a sulfated polysaccharide and in particular a sulfated glucan, i.e. polysaccharide made of many glucose molecules. Average molecular weight as defined herein indicates that individual sulfated polysaccharides may have a molecular weight different from this average molecular weight but that the average molecular weight represents the mean molecular weight of the sulfated polysaccharides. This further implies that there will be a natural distribution of molecular weights around this average molecular weight for a dextran sulfate sample.
Average molecular weight (Mw) of dextran sulfate is typically determined using indirect methods such as gel exclusion/penetration chromatography, light scattering or viscosity. Determination of average molecular weight using such indirect methods will depend on a number of factors, including choice of column and eluent, flow rate, calibration procedures, etc.
Average molecular weight
typical for methods sensitive to molecular size rather than numerical value, e.g. light scattering and size exclusion chromatography (SEC) methods. If a normal distribution is assumed, then a same weight on each side of Mw, i.e. the total weight of dextran sulfate molecules in the sample having a molecular weight below Mw is equal to the total weight of dextran sulfate molecules in the sample having a molecular weight above Mw.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, preferably has an average molecular weight equal to or below 40000 Da, more preferably equal to or below 20000 Da and in particular equal to or below 10000 Da.
Dextran sulfate of a molecular weight exceeding 10000 Da generally has a lower effect vs. toxicity profile as compared to dextran sulfate having a lower average molecular weight. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10000 Da) as compared to dextran sulfate molecules having an average molecular weight within the preferred range. As a consequence, such larger dextran sulfate molecules are less appropriate in clinical uses when the dextran sulfate is to be systemically administered to subjects in vivo.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average molecular weight within a range of 2000 and 10000 Da. In another embodiment, the average molecular weight is within a range of 2500 and 10000 Da. In a particular preferred embodiment, the average molecular weight is within a range of 3000 to 10000 Da.
In an optional, but preferred embodiment, less than 40% of the dextran sulfate molecules have a molecular weight below 3000 Da, preferably less than 35%, such as less than 30% or less than 25% of the dextran sulfate molecules have a molecular weight below 3000 Da. In addition, or alternatively, less than 20% of the dextran sulfate molecules have a molecular weight above 10000 Da, preferably less than 15%, such as less than 10% or less than 5% of the dextran sulfate molecules have a molecular weight above 10000 Da. Thus, in a particular embodiment, the dextran sulfate has a substantially narrow molecular weight distribution around the average molecular weight.
In a particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically acceptable salt thereof, is within a range of 3500 and 9500 Da, such as within a range of 3500 and 8000 Da.
In another particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically acceptable salt thereof, is within a range of 4500 and 7500 Da.
In a further particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically acceptable salt thereof, is within a range of 4500 and 5500 Da.
Thus, in a currently preferred embodiment the average molecular weight of dextran sulfate, or the pharmaceutically acceptable salt thereof, is preferably approximately 5000 Da or at least substantially close to 5000 Da, such as 5000±500 Da, for instance 5000±400 Da, preferably 5000±300 Da or 5000±200 Da, such as 5000±100 Da. Hence, in an embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically acceptable salt thereof, is 4.5 kDa, 4.6 kDa, 4.7 kDa, 4.8 kDa, 4.9 kDa, 5.0 kDa, 5.1 kDa, 5.2 kDa, 5.3 kDa, 5.4 kDa or 5.5 kDa.
In a particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically salt thereof as presented above is average Mw, and preferably determined by gel exclusion/penetration chromatography, size exclusion chromatography, light scattering or viscosity-based methods.
In a particular embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, consists, on average, of about or slightly above 5 glucose units and has an average sulfate number per glucose unit of at least 2.0, such as of at least 2.5.
Dextran sulfate is a polyanionic derivate of dextran and contains sulfur. The average sulfur content for dextran sulfate of the embodiments is preferably 15 to 20% and more preferably approximately 17%, generally corresponding to about or at least two sulfate groups per glucosyl residue. In a particular embodiment, the sulfur content of dextran sulfate is preferably equal to or at least close to the maximum possible degree of sulfur content of the corresponding dextran molecules.
In a particular embodiment, dextran sulfate of the embodiments has a number average molecular weight (Mn) as measured by nuclear magnetic resonance (NMR) spectroscopy within an interval of 1850 and 2000 Da.
In another particular embodiment, dextran sulfate of the embodiments has on average 5.1 glucose units and an average sulfate number per glucose unit of 2.6 to 2.7, typically resulting in a number average molecular weight (Mr) as measured by nuclear magnetic resonance (NMR) spectroscopy within an interval of 1850 and 2000 Da.
Number average molecular weight
typically derived by end group assays, e.g. NMR spectroscopy or chromatography. If a normal distribution is assumed, then a same number of dextran sulfate molecules can be found on each side of Mn, i.e. the number of dextran sulfate molecules in the sample having a molecular weight below Mn is equal to the number of dextran sulfate molecules in the sample having a molecular weight above Mn.
A dextran sulfate, or pharmaceutically salt thereof, that can be used according to the embodiments is described in WO 2016/076780.
The dextran sulfate according to the embodiments can be provided as a pharmaceutically acceptable salt of dextran sulfate. Such pharmaceutically acceptable salts include e.g. a sodium or potassium salt of dextran sulfate.
Suitable dose ranges for the dextran sulfate, or the pharmaceutically acceptable salt, of the embodiments may vary according to the size and weight of the subject, the condition for which the subject is treated, and other considerations. In particular for human subjects, a possible dosage range could be from 1 μg/kg to 150 mg/kg of body weight, preferably from 10 μg/kg to 100 mg/kg of body weight.
In preferred embodiments, dextran sulfate, or the pharmaceutically acceptable salt thereof, is formulated to be systemically administered at a dosage in a range from 0.05 to 50 mg/kg of body weight of the subject, preferably from 0.05 or 0.1 to 40 mg/kg of body weight of the subject, and more preferably from 0.05 or 0.1 to 30 mg/kg, or 0.1 to 25 mg/kg or from 0.1 to 15 mg/kg or 0.1 to 10 mg/kg body weight of the subject.
Systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, of the embodiments is preferably initiated as soon as possible after occurrence of an event or condition that may otherwise cause fibrosis and in particular deleterious fibrosis in the subject. For instance, fibrosis often occur following an injury or other condition causing ischemia or a cardiovascular disease in the subject. In such a case, systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, is preferably performed as soon as possible following detection or diagnosis of the injury or other condition causing ischemia or a cardiovascular disease.
Systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, does not necessarily have to be limited to treatment of a present medical condition but could alternatively, or in addition, be used for prophylaxis. In other words, dextran sulfate of the embodiments could be systemically administered to a subject that will undergo a medical procedure, such as surgery, that may cause or induce cardiac fibrosis.
Dextran sulfate, or the pharmaceutically acceptable salt thereof, of the embodiments can be systemically administered at a single administration occasion, such as in the form of a single bolus injection. This bolus dose can be injected quite quickly to the patient but is advantageously infused over time so that the dextran sulfate solution is infused over a few minutes of time to the patient, such as during 5 to 10 minutes or more.
Alternatively, dextran sulfate, or the pharmaceutically acceptable salt thereof, of the embodiments can be systemically administered at multiple, i.e. at least two, occasions during a treatment period. Thus, dextran sulfate of the embodiments could be systemically administered once or at multiple times per day, once or at multiple times per week, once or at multiple times per month as illustrative examples.
In a particular embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is formulated for systemic administration at multiple times, such as 2-5 times, preferably 3 times, a week for multiple consecutive weeks, such as at least 2-5 consecutive, preferably at least 3 consecutive weeks.
In an embodiment, systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, could be initiated as soon as possible following an event or condition causing deleterious fibrosis in the subject, such as soon as possible following an ischemic event or heart infarct as mentioned above. Alternatively, the systemic administration could be initiated at a time period following the event or condition causing deleterious fibrosis. A reason for such a delay in systemic administration is that the fibrosis process generally takes a period of time following a fibrosis causing event or condition. For instance, systemic administration of dextran sulfate, or the pharmaceutically acceptable salt thereof, could be initiated within the first week or from one week following an event or condition causing deleterious fibrosis in the subject.
In an embodiment, the subject is a mammalian subject, preferably a primate, and more preferably human subject. Although the embodiments are in particular directed towards treating, inhibiting or preventing cardiac fibrosis in human subjects, the embodiments may also, or alternatively, be used in veterinary applications. Non-limiting example of animal subjects include primate, cat, dog, pig, horse, mouse, rat.
In an embodiment, the subject is suffering from a disease, condition or disorder causing cardiac fibrosis and in particular detrimental, deleterious or injuries fibrosis. Such detrimental, deleterious or injurious fibrosis causes disruption of the normal architecture of the affected organ or tissue, ultimately leading to its dysfunction and failure. This means that detrimental, deleterious or injurious fibrosis is a pathological state or pathological fibrosis of excess deposition of fibrous tissue that will have a negative and detrimental effect on the organ or tissue where the fibrosis takes place.
Accordingly, in an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is for use in treating, inhibiting or preventing pathological fibrosis causing excess deposition of fibrous tissue in the heart or cardiac tissue of the subject causing dysfunction of the organ or tissue.
In a particular embodiment, the disease, condition or disorder causing fibrosis is selected from a group consisting of endomyocardial fibrosis, fibrosis following myocardial infarction or atrial fibrosis.
Dextran sulfate, or the pharmaceutically acceptable salt thereof, is thereby used to treat, inhibit or prevent the fibrosis component of any of the above mentioned diseases, conditions or disorders. Accordingly, dextran sulfate, or the pharmaceutically acceptable salt thereof, does not necessarily treat, inhibit or prevent the disease, condition or disorder per se but reduces the fibrosis process and thereby the amount of fibrotic tissue resulting from the disease, condition or disorder.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, formulated for systemic administration is used to treat, inhibit or prevent cardiac fibrosis in a subject suffering from cardiac fibrosis.
The pathological accumulation of extracellular matrix (fibrous connective tissue) is a key contributor to cardiac heart failure (CHF) in both diabetic and non-diabetic patients, resulting in progressive stiffening of the ventricular walls and loss of contractility of the heart. Heart failure is a global health problem, appearing most commonly in patients with previous myocardial infarction (MI). Cardiac remodeling, due to fibrosis, seen in both the infarcted and non-infarcted myocardium is recognized to be a major determinant of the development of impaired ventricular function, leading to a poor prognosis.
Accordingly, fibrosis may occur in heart (cardiac fibrosis) in the form of, for instance, endomyocardial fibrosis, fibrosis following myocardial infarction or atrial fibrosis. Fibrosis often occurs following ischemia at a site in the heart muscle, which may have severe and negative consequences in terms of heart wall stiffening, loss of contractility and cardiac remodeling.
In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is for use in treating, inhibiting or preventing cardiac fibrosis in a subject having suffered from myocardial infarction or another ischemic condition in the heart, e.g. myocardial ischemia.
In a particular embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is for use in treating, inhibiting or preventing interstitial fibrosis in an infarct area of a heart of the subject.
Experimental data as presented herein show that dextran sulfate has an anti-fibrosis effect in a myocardial infarction model. Dextran sulfate resulted in significantly less cardiac fibrosis and in particular significantly less interstitial fibrosis in the infarct area of the test subjects.
Fibrosis may also occur in connection with transplantation of the heart or cardiac tissue in a subject. Dextran sulfate, or the pharmaceutically acceptable salt thereof, may accordingly be systemically administered to a subject that will be subject to or has recently been subject to heart or cardiac tissue transplantation. Non-limiting examples of such transplanted cardiac tissue includes heart valves, etc.
Dextran sulfate, or the pharmaceutically acceptable salt thereof, may also be systemically administered to a subject in connection with implantation of a medical device, such as pacemaker, stent, prosthesis, in or in connection with the heart of the subject. Thus, pathological cardiac fibrosis may also occur following implantation of medical devices.
Another aspect of the embodiments relates to use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament formulated for systemic administration for treatment, inhibition or prevention of cardiac fibrosis in a subject.
A further aspect of the embodiments relates to a method of treating, inhibiting or preventing cardiac fibrosis in a subject. The method comprises systemically administering dextran sulfate, or a pharmaceutically acceptable salt thereof, to the subject.
Evaluation of Anti-Fibrosis Effect of Dextran Sulfate in a Myocardial Infarction Model
The present study assessed the effect of dextran sulfate in inhibiting or reducing fibrosis in a rat model of myocardial infarction (MI).
Materials and Methods
The myocardial infarction model in rat involved ligations of the left coronary artery permanently with an intramural stitch. The surgery caused obstruction of the blood flow and subsequently to severe ischemic damage and cardiac walls infarct.
In total 120 female SD rats having an average body weight of 178 g at study initiation (Day 0) were obtained from Harlan Laboratories, Israel. Animals were fed ad libitum a commercial rodent diet (Teklad Certified Global 18% Protein Diet). Animals had free access to autoclaved and acidified drinking water (pH between 2.5 and 3.5) obtained from the municipality supply. Animals were housed under standard laboratory conditions, air conditioned and filtered (HEPA F6/6) with adequate fresh air supply (minimum air changes/hour). Animals were kept in a climate controlled environment with a temperatures range of 20-24° C. and RH range of 30-70% with 12 hours light and 12 hours dark cycle.
Dextran sulfate with an average molecular weight within a range of 5-7 kDa was dissolved in 0.9% NaCl (saline) (Teva Pharmaceutical Industries Ltd) to be injected subcutaneous at doses of 15 mg/kg or 3 mg/kg.
On the day of surgery the animals were anesthetized with a combination of 90 mg/kg ketamine and 10 mg/kg xylazine, and animals were intubated and mechanically ventilated. In order to induce MI, under anesthesia, the rat chest was opened by left thoracotomy, the pericardium was removed and the proximal left coronary artery was permanently occluded with an intramural stitch (Circulation 2008, 117: 1388-1396). Two hours post-surgery, each animal in all treated groups were injected s.c. with dextran sulfate or saline vehicle according to Table 1.
On day 36 after MI induction, the rats were sacrificed by CO2 inhalation and the hearts were harvested and fixed in buffered formalin solution. Routine paraffin embedding was performed using standard histological procedures.
Masson's Trichrome Staining was used for fibrosis evaluation. The hearts were sectioned transversely into five sections that were imbedded in paraffin. Five paraffin sections at 5 μm were performed on a Lika microtome. All sections were stained according to standard Masson's trichrome protocol. The collagen fibers were stained blue, the nuclei were stained black and the background was stained red. The sections were visualized in a computer-imaging system and infarct size was marked and calculated using the ImageJ program. For each animal, five serial sections including one containing the ligature were analyzed and the mean value of all sections for each heart was treated as one value for statistical analysis.
Statistical analysis was performed by two ways ANOVA for repeated measures, followed by Bonferroni post-hoc test.
Results of Cardiac Fibrosis Analysis
Interstitial fibrosis in the marginal area of the infarct was estimated manually in heart sections stained with Masson's Trichrome Staining. The area of interstitial fibrosis was calculated as the percent of the total area of the left ventricle. For each rat five cross-sections were analyzed. The extent of interstitial fibrosis in the margin zone of the infarct is shown in
The extent of interstitial fibrosis was significantly lower in the dextran sulfate treated group 2M (11.9±0.9%) as compared to the control vehicle treated group 1M (22.7±1.9%). The extent of interstitial fibrosis was also lower in the other dextran sulfate treated groups 3M-5M, although the difference was not significant.
The effect of dextran sulfate seems to be primarily during the time fibrosis is developing following MI. Accordingly, treatment group 2M comprising treatment with dextran sulfate during 3 weeks significantly decreased fibrosis while the effect in the other groups (3M-5M) with shorter treatment was less pronounced. This seems to be in agreement with the pathogenesis of fibrosis in the rat model of myocardial infarction (American Journal of Pathology 1995, 147(2): 325-338). This article discloses that the fibrosis process in infarcted cardiac tissue is regulated differently than fibrosis in dermal wounds and in non-infarcted areas of the heart.
The results thereby revealed that dextran sulfate treatment significantly decreased fibrosis at five weeks post-infarction compared to the vehicle control treated group. Dextran sulfate is thereby capable of decreasing the fibrogenesis following myocardial infarction.
The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1551050-6 | Jul 2015 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SE2016/050720 | 7/15/2016 | WO | 00 |
