GINSENOSIDE M1 AS A MODULATOR OF ANGIOTENSIN REGULATING ENZYMES AND ITS USE FOR TREATING DISEASES OR CONDITIONS INCLUDING SYMPTOMS CAUSED BY CORONAVIRUS

Abstract

It relates to a new use of ginsenoside M1 as a modulator of angiotensin regulating enzymes and treatment for infection caused by coronavirus using ginsenoside M1.

Claims

1-13. (canceled)

14. A method for treating a disease or condition associated with abnormal balance of angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) or accumulation of angiotensin II in a subject in need, comprising: administering to the subject an amount of ginsenoside M1 effective to treat the subject.

15. The method of claim 14, wherein ginsenoside M1 is administered in an amount (a) effective in inhibiting ACE, (b) effective in activating ACE2 or (c) effective in inhibiting ACE and activating ACE2, in the subject.

16. The method of claim 14, wherein ginsenoside M1 is administered in an amount effective in degrading angiotensin II and preventing accumulation of angiotensin II in the subject.

17. The method of claim 14, wherein the disease or condition includes damages in organs or tissues in the subject.

18. The method of claim 17, wherein the damages includes damages in one or more selected from the group consisting of lung, gastrointestinal tract, spleen, lymph nodes, heart, kidney, bladder, liver, gallbladder, adrenal glands and testis.

19. The method of claim 17, wherein the damages are caused by infection of coronavirus.

20. The method of claim 19, wherein the coronavirus is one or more selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (2019-nCoV).

21. The method of claim 14, further comprising: administering the ginsenoside M1 in combination with one or more additional therapeutic methods or agents useful for degrading angiotensin II and preventing accumulation of angiotensin II.

22. The method of claim 21, wherein said one or more additional therapeutic agents include ACE2.

23. The method of claim 14, further comprising: administering the ginsenoside M1 in combination with one or more additional therapeutic agents selected from the group consisting of corticosteroids, non-steriodal anti-inflammatory drugs (NSAIDs), cytotoxic drugs, immunosuppressants, and vasodilators.

24. The method of claim 14, further comprising: administering the ginsenoside M1 in combination with one or more additional therapeutic agents selected from the group consisting of an antibiotic, interferon and an anti-viral agent.

25-28. (canceled)

29. A method for treating pulmonary injuries in a subject in need thereof, comprising: administering to the subject an amount of ginsenoside M1 effective to treat the subject.

30. The method of claim 29, wherein the pulmonary injuries include one or more selected from the group consisting of alveolitis, lymphocyte infiltration and fibrosis in the lung.

31. The method of claim 29, further comprising: administering the ginsenoside M1 in combination with one or more additional therapeutic methods or agents useful for degrading angiotensin II and preventing accumulation of angiotensin II.

32. The method of claim 31, wherein said one or more additional therapeutic agents include angiotensin-converting enzyme 2 (ACE2).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] For the purpose of illustrating the invention, there are shown in the drawings embodiments. It should be understood, however, that the invention is not limited to the preferred embodiments shown. In the drawings:

[0029] FIG. 1: Angiotensin levels in the renal tissue in rats. Ang II levels in renal tissue (upper panel). Ang-(1-7) levels in renal tissue (lower panel). The same superscript letters indicate no significant difference between groups (P>0.05); significant difference existed between groups that do not have the same superscript letter (P<0.05).

[0030] FIG. 2: Levels of the angiotensin-converting enzymes in the renal tissue in rats. Quantitative results of IHC staining were presented as IOD/area and were proportional to the levels of ACE (upper panel) and ACE2 (lower panel). The same superscript letters indicate no significant difference between groups (P>0.05); significant difference existed between groups that do not have the same superscript letter (P<0.05).

[0031] FIG. 3: Angiotensins levels in the renal tissue in mice (infusion model). Ang II (upper panel) and Ang-(1-7) (lower panel) levels in the renal tissue. The same superscript letters indicate no significant difference between groups (P>0.05); significant difference existed between groups that do not have the same superscript letter (P<0.05).

[0032] FIG. 4: Levels of the ACE in the renal tissue in mice. Quantitative results of IHC staining were presented as IOD/area and were proportional to the levels of ACE (upper panel) and ACE2 (lower panel). The same superscript letters indicate no significant difference between groups (P>0.05); significant difference existed between groups that do not have the same superscript letter (P<0.05).

[0033] FIG. 5: The local lung renin-angiotensin system. ACE2 and M1 improved histological changes in bleomycin-induced lung fibrosis. In different groups, recombinant mouse ACE2 or ACE2 together with M1 and bleomycin were administered on day 0. Szapiel's scoring was obtained to quantify the degree of alveolitis (upper panel) and fibrosis (lower panel) and inhibitory potency of ACE2 and ACE2 together with M1. Data represent as means±SEM. *p<0.05 when compared with the Saline group, #p<0.05 when compared with BLM.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0035] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0036] The term “comprise” or “comprising” is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”

[0037] Ginsenoside M1, also named Compound K (CK), 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, is one of saponin metabolites known in the art. The chemical structure of ginsenoside M1 is as follows:

##STR00001##

[0038] Ginsenoside M1 is known as one metabolite of protopanaxadiol-type ginsenosides via the gypenoside pathway by human gut bacteria. Ginsenoside M1 can be found in blood or urine after intake. Ginsenoside M1 may be prepared from ginseng plants through fungi fermentation by methods known in the art, such as Taiwan Patent Application No. 094116005 (1280982) and U.S. Pat. No. 7,932,057, the entire content of which is incorporated herein by reference. In certain embodiments, the ginseng plants for preparing the ginsenoside M1 include Araliaceae family, Panax genus, e.g. P. ginseng and P. pseudo-ginseng (also named Sanqi). In general, the method of preparation of ginsenoside M1 includes the steps of (a) providing powder of ginseng plant materials (e.g. leaves or stems); (b) providing a fungus for fermenting the ginseng plant materials, wherein the fermentation temperature is ranged from 20-50° C., the fermentation humidity is ranged from 70-100%, the pH value is ranged from 4.0-6.0, and the fermentation period is ranged from 5-15 days; (c) extracting and collecting the fermentation products; and (d) isolating 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol from the fermentation products.

[0039] When ginsenoside M1 is described as “isolated” or “purified” in the present invention, it should be understood as not absolutely isolated or purified, but relatively isolated or purified. For example, purified ginsenoside M1 refers to one that is more purified compared to its naturally existing form. In one embodiment, a preparation comprising purified ginsenoside M1 may comprise ginsenoside M1 in an amount of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100% (w/w) of the total preparation. It should be understood that when a certain number was used herein to show a ratio or dosage, said number generally includes that within the range of 10% more and less, or more specifically, the scope of 5% more and less than the number.

[0040] The term “individual” or “subject” used herein includes human and non-human animals such as companion animals (such as dogs, cats and the like), farm animals (such as cows, sheep, pigs, horses and the like), or laboratory animals (such as rats, mice, guinea pigs and the like).

[0041] The term “treating” as used herein refers to the application or administration of a composition including one or more active agents to a subject afflicted with a disorder, a symptom or conditions of the disorder, a progression of the disorder or at risk of developing the disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms or conditions of the disorder, the disabilities induced by the disorder, or the onset or progression of the disorder.

[0042] The term “effective amount” used herein refers to the amount of an active ingredient to confer a desired therapeutic effect in a treated subject. In some embodiments, an effective amount as used herein can be an amount effective in downregulating ACE, upregulating ACE2, degrading angiotensin II, and/or preventing accumulation of angiotensin II. In some embodiments, an effective amount as used herein can be an amount effective in alleviating or reducing damages in organs or tissues caused by accumulation of angiotensin II, for example, pulmonary injuries e.g. alveolitis, lymphocyte infiltration and/or fibrosis in the lung.

[0043] The therapeutically effective amount may change depending on various reasons, such as administration route and frequency, body weight and species of the individual receiving said pharmaceutical, and purpose of administration. Persons skilled in the art may determine the dosage in each case based on the disclosure herein, established methods, and their own experience. For example, in certain embodiments, the oral dosage of ginsenoside M1 used in the present invention is 10 to 1,000 mg/kg daily. In some examples, the oral dosage of ginsenoside M1 used in the present invention is 100 to 300 mg/kg daily, 50 to 150 mg/kg daily, 25 to 100 mg/kg daily, 10 to 50 mg/kg daily, or 5 to 30 mg/kg daily. In addition, in some embodiments of the invention, ginsenoside M1 is administered periodically for a certain period of time, for example, daily administration for at least 15 days, one month or two months or longer.

[0044] In one embodiment, a therapeutically effective amount of the active ingredient may be formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition of an appropriate form for the purpose of delivery and absorption. Depending on the mode of administration, the pharmaceutical composition of the present invention preferably comprises about 0.1% by weight to about 100% by weight of the active ingredient, wherein the percentage by weight is calculated based on the weight of the whole composition.

[0045] As used herein, “pharmaceutically acceptable” means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize said active ingredient and is safe to the individual receiving the treatment. Said carrier may be a diluent, vehicle, excipient, or matrix to the active ingredient. Some examples of appropriate excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, Arabic gum, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, sterilized water, syrup, and methylcellulose. The composition may additionally comprise lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents. The composition of the present invention can provide the effect of rapid, continued, or delayed release of the active ingredient after administration to the patient.

[0046] According to the present invention, the form of said composition may be tablets, pills, powder, lozenges, packets, troches, elixers, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, and packaged powder.

[0047] The composition of the present invention may be delivered via any physiologically acceptable route, such as oral, parenteral (such as intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. Regarding parenteral administration, it is preferably used in the form of a sterile water solution, which may comprise other substances, such as salts or glucose sufficient to make the solution isotonic to blood. The water solution may be appropriately buffered (preferably with a pH value of 3 to 9) as needed. Preparation of an appropriate parenteral composition under sterile conditions may be accomplished with standard pharmacological techniques well known to persons skilled in the art, and no extra creative labor is required.

[0048] According to the present invention, ginsenoside M1 may be used as an active ingredient for treating a disease or condition associated with abnormal balance of ACE and ACE2 or accumulation of angiotensin II in the subject. In some embodiments, such disease or condition includes damages in organs or tissues in the subject such as those in lung, gastrointestinal tract, spleen, lymph nodes, heart, kidney, bladder, liver, gallbladder, adrenal glands and/or testis. In certain examples, the damages are caused by infection of coronavirus, including but not limited to SARS-CoV, MERS-CoV and 2019-nCoV.

[0049] In some embodiments, ginsenoside M1 or compositions comprising ginsenoside M1 as the active ingredient may be used in combination with existing therapeutic methods or medicaments, for example, those for treating a disease or condition associated with abnormal balance of ACE and ACE2 or accumulation of angiotensin II as described herein. In one example, ginsenoside M1 is administrated with ACE2. Accordingly, a particular combination of ginsenoside M1 with ACE2 is administrated to a subject in need as described herein.

[0050] In some embodiments, ginsenoside M1 or compositions comprising ginsenoside M1 as the active ingredient may be used in combination with corticosteroids (such as prednisolone), non-steriodal anti-inflammatory drugs (NSAIDs), cytotoxic drugs (such as cyclophosphamide, chlorambucil, and azathioprine), immunosuppressants (such as cyclosporine and Mycophenolate Mofetil), and vasodilators (such as angiotensin-converting-enzyme inhibitors (ACE inhibitors)).

[0051] In one particular embodiment, ginsenoside M1 is administrated in combination with ACE2 for use treatment of pulmonary injuries, for example, alveolitis, lymphocyte infiltration and/or fibrosis in the lung.

[0052] In some embodiments, ginsenoside M1 is administered in combination with an antibiotic, interferon and an anti-viral agent.

[0053] In one embodiment, the medicament or therapeutic method used in combination may be used simultaneously (parallel) or sequentially. When medicaments are used in combination, the medicaments may be mixed in the same formula or put in different formulas separately, such as separate capsules, pills, tablets, and injections.

[0054] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

[0055] Ginsenoside M1, also named Compound K (CK), 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (named LCHK168 below), was prepared by methods known in the art, such as those described in Taiwan Patent Application No. 094116005 (I280982) and U.S. Pat. No. 7,932,057.

[0056] According to previous research, the spike protein (S protein) on coronavirus will bind to ACE2, and the amount of ACE2 will affect the health of the lungs and kidneys (ACE2 Expression in Kidney and Testis May Cause Kidney and Testis Damage After 2019-nCoV Infection https://doi.org/10.1101/2020.02. 12.20022418, Posted Feb. 13, 2020; and Angiotensin-Converting Enzyme 2 Attenuates Bleomycin-Induced Lung Fibrosis in Mice, Cellular Physiology and Biochemistry, 2015; 36: 697-711). This study uses ginsenoside M1 to regulate the content of ACE2 to maintain a healthy body.

[0057] Angiotensin II (Ang II)-mediated renal injury represents a major pathogenetic mechanism in most chronic kidney diseases. We found that ginsenoside M1 attenuated Ang II expression by upregulating angiotensin-converting enzyme 2 (ACE2) in the renal tissue. We confirmed this finding in an exogenous Ang II-infused mice model of renal injury, and the results showed consistent in two models. Local renin-angiotensin system (RAS) activation has been shown to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). It has been reported that ACE2 could inhibit RAS-mediated epithelial injury and fibrogenesis and ACE2 deficiency could aggravate acute and chronic lung injury.

[0058] 1. Material and Methods

[0059] 1.1 Animals and Treatments.

[0060] 1.1.1 Rat (Renal Damages)

[0061] A total of 20 Wistar-Kyoto rats (WKY) and 20 SHR (male, 16˜17 weeks old) animals were grouped into four: WKY group (8 WKY, orally administered 0.5% CMC-Na); SHR group (8 SHR, orally administered 0.5% CMC-Na); WKY+Ginsenoside M1 group (8 WKY, orally administered 20 mg/kgGinsenoside M1); SHR +Ginsenoside M1 group (8 SHR, orally administered 20 mg/kg Ginsenoside M1). Ginsenoside M1 or placebo administration was carried out once daily for 42 days is was followed by animal sacrifice and blood and renal tissue sample collection. The renal tissue specimens underwent fixation with 4% formalin (histopathology) or were snap-frozen with liquid nitrogen and kept at −80° C. for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA).

[0062] 1.1.2 Mice (Infusion Model) (Renal Damages)

[0063] A total of twenty C57BL/6 mice (male, 10 weeks old) were maintained with rodent chow and water at will. Subcutaneous implantation of a 1002 osmotic minipump was performed at the dorsum of the neck for Ang II (1.5 mg/kg) or normal saline infusion animals were assigned to four groups: Saline group (5 mice, infused with normal saline and orally administered 0.5% CMC-Na); Ang II group (5 mice, infused with Ang II and orally administered 0.5% CMC-Na); Saline+Ginsenoside M1 group (5 mice, infused with normal saline and orally administered 20 mg.Math.kg−1.Math.d−1 Ginsenoside M1); Ang II +Ginsenoside M1 group (5 mice, infused with Ang II and orally administered 20 mg/kg Ginsenoside M1). Ginsenoside M1 or placebo administration was performed daily for 14 days, followed by animal sacrifice and blood and renal tissue sample collection. The renal tissue specimens underwent fixation with 4% formalin (histopathology) or were snap-frozen with liquid nitrogen and kept at −80° C. (RT-qPCR and ELISA).

[0064] 1.1.3 Mice (Pulmonary Damages)

[0065] The animals were randomly divided into 5 groups as: (1) Saline group: 0.9% saline (200 μL) injection via the tail vein; (2) ACE2 group: a single intraperitoneal injection of ACE2 (0.2 mg/kg); (3) BLM group: a single injection of bleomycin at 150 mg/kg via the tail vein; and (4) BLM+ACE2 group: an injection of bleomycin at 150 mg/kg via the tail vein, followed by an intraperitoneal injection of ACE2 at 0.2 mg/kg. (5) BLM+ACE2+M1 group: an injection of bleomycin at 150 mg/kg via the tail vein, followed by an intraperitoneal injection of 0.2 mg/kg ACE2 and 60mg/kg M1. After administration, ten animals randomly selected from each group were euthanized on days 7, 14 and 28 by cervical dislocation.

[0066] 1.2 Histopathological Assessment.

[0067] Renal tissue specimens underwent fixation with 4% formalin, paraffin embedding, sectioning at 4 μm, and staining with hematoxylin and eosin (H&E) and Masson trichrome stain, respectively. Then, a Nikon E100 light microscope was employed for analysis.

[0068] 1.3 Immunohistochemistry (IHC)

[0069] ACE and ACE2 primary antibodies were produced by Bioss Antibodies. Peroxidase-linked goat anti-rabbit secondary antibodies and the DAB and two-step rabbit IHC kits were provided by ZSGB-BIO. IHC was carried out as proposed by ZSGB-BIO.

[0070] 1.4 Ang II and Ang-(1-7) Level Assessment in the Renal Tissue

[0071] The supernatants of renal specimens were prepared as follows. A total of 100 mg of the renal tissue was homogenized in 900 μL of ice-cold normal saline and submitted to centrifugation (1000 g, 4° C. for 15 min). The resulting supernatants were kept at −80° C. until analysis with Ang II and Ang-(1-7) ELISA kits, respectively, in accordance with the manufacturer's protocols.

[0072] 2. Results

[0073] 2.1 Ginsenoside M1 Reduces Ang II Levels in the Renal Tissue in SHR

[0074] There are two main ACE which regulate the levels of Ang II in vivo, including ACE and ACE2. ACE transforms angiotensin I into Ang II, and ACE2 transforms Ang II into Ang-(1-7). So upregulation of ACE induces Ang II increase, while ACE2 upregulation attenuates Ang II increase. Spontaneously hypertensive rat (SHR) is an animal model of genetic hypertension. According to ELISA data (FIG. 1), Ang II levels in the renal tissue of the SHR group were significantly higher than those of WKY and WKY+Ginsenoside M1 groups. Treatment with Ginsenoside M1 significantly reduced Ang II levels in the renal tissue in SHR, while showing limited effects in the WKY group. Ang-(1-7) amounts in the SHR group were significantly higher than those of the WKY and WKY+Ginsenoside M1 groups, with the SHR+Ginsenoside M1 showing even significantly higher values compared with the SHR group. IHC results (FIG. 2) showed that ACE levels were markedly elevated in the two SHR groups compared with the two WKY groups. ACE2 amounts in the renal tissue were compensatorily upregulated in the SHR group, compared with the WKY and WKY+Ginsenoside M1 groups. Meanwhile, Ginsenoside M1 treatment could further upregulate ACE2 in the renal tissue in SHR. is was the main mechanism that Ginsenoside M1 treatment downregulated Ang II and upregulated Ang-(1-7) in the kidneys of SHR. Meanwhile, Ginsenoside M1 had no significant effect on ACE expression in either WKY or SHR.

[0075] 2.2 Ginsenoside M1 Reduces Ang II Levels in the Renal Tissue in Mice Infused Ang II

[0076] According to ELISA data (FIG. 3), Ang II infusion significantly raised Ang II levels in the renal tissue in mice, and Ginsenoside M1 treatment could attenuate this increase significantly, although Ang II levels in the Ang II+Ginsenoside M1 group were still significantly higher than those of the two groups infused saline. Meanwhile, Ang-(1-7) levels in the Ang II group were significantly higher than those of the Saline and Saline+Ginsenoside M1 groups; the Ang II+Ginsenoside M1 group showed a significant increase compared with the Ang II group. IHC data (FIG. 4) showed no marked differences in various group pairs in ACE expression. ACE2 expression levels in the Ang II +Ginsenoside M1 group were markedly elevated compared with those of the other three groups, which might be the main mechanism for degrading exogenous angiotensin.

[0077] 2.3 Treatment of Alveolitis and Fibrosis by M1 and ACE2

[0078] As shown in FIG. 5, a single injection of bleomycin at the dose of 150 mg/kg via the tail vein in mice induced mild to moderate alveolitis on day 7. Administration of bleomycin is a well-characterized model of pulmonary alveolitis or fibrosis in mice. The fibrotic changes in the lungs were determined using the Szapiel scoring method, higher score indicating greater disease condition. Lungs exhibited focal distribution of alveolitis that was predominantly subpleural and perivascular, in which the alveolar septa were mildly thickened with edema and inflammatory cell infiltration; no apparent fibrosis was observed. At the same time, administration of ACE2 did not significantly alter the pathological changes on day 7. On day 14, the BLM group showed disease progression. Lungs exhibited moderate to severe alveolitis and moderate fibrosis with patchy inflammatory consolidation and collagen deposition, while the administration of ACE2 significantly attenuated both alveolitis and fibrosis. On day 28, in the BLM group, alveolitis spontaneously resolved; however, derangement of the alveolar architecture continued, and fibrosis persisted, characterized by diffuse, dense, thick collagen bundles and fibroblast foci. ACE2 noticeably reflected a long-term therapeutic effect on day 28, with sections in the BLM+ACE2 group presenting only mild to moderate alveolitis, with sections in the BLM+ACE2+M1 group presenting only mild alveolitis and significant attenuation of fibrosis. No signs of alveolitis or fibrosis were found in the Saline group or the ACE2 group among the different time points.

REFERENCES

[0079] 1. Journal of Molecular Medicine/October 2006, Volume 84, Issue 10, pp 814-820| Cite as/Lessons from SARS: control of acute lung failure by the SARS receptor ACE2. Pharmacology & Therapeutics/Volume 128, Issue 1, October 2010, Pages 119-128/Trilogy of ACE2: A peptidase in the renin—angiotensin system, a SARS receptor, and a partner for amino acid transporters.

[0080] 2. Published in final edited form as: Annu Rev Virol. 2016 Sep. 29; 3(1): 237-261. doi:10.1146/annurev-virology-110615-042301./Structure, Function, and Evolution of Coronavirus Spike Proteins/Fang Li Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minn. 55455

[0081] 3. Antioxidant Treatment and Alcoholism/Camila S. Silva PhD, . . . Helio Vannucchi MD, PhD, in Molecular Aspects of Alcohol and Nutrition, 2016

[0082] 4. Beta-Glucosidase From Penicillium/Gustavo Molina, . . . Glàucia M. Pastore, in New and Future Developments in Microbial Biotechnology and Bioengineering, 2018

[0083] 5. Ginseng and Gastrointestinal Protection*/Min-Hyun Kim, Hyeyoung Kim, in Gastrointestinal Tissue, 2017