Patent Application
20230321185
The Sequence Listing XML that is contained in the file named “azam2.xml”, which is 1921 bytes (as measured in Microsoft Windows®) and was created on Jul. 4, 2023, is filed herewith by electronic submission, and is incorporated by reference herein.
Please replace any previously submitted sequence listing with the attached sequence listing.
The present invention relates to a method for treatment of Type-1 diabetes, and more particularly, the present invention relates to the use of a combination of magainin peptides and human growth hormone in the treatment of Type-1 diabetes.
Diabetes is a general term for heterogeneous metabolic disorders with chronic hyperglycemia as the main outcome due to disorder in insulin secretion or malfunction or both. According to the International Diabetes Federation (IDF) report, the number of diabetic patients from 138 countries was reported to be 463 million in 2019 (with an 8.3% outbreak). In addition, it is estimated that by 2030 and 2045, this number will rise to 578 million and 700 million, respectively.
Treatment of diabetes is currently a universal challenge due to the growing number of diabetic patients and high medication costs. In both Type-1 diabetes mellitus (T1DM) and Type-2 diabetes mellitus (T2DM), the two main types of diabetes, the progressive loss of functional β-cell mass and imbalanced blood glucose level, are the main common criteria. Therefore, the primary goal in the treatment of affected individuals is based on compensation of the endogenous insulin pool size via expanding the functional β-cell population, especially in T1DM.
At a glance, the pathophysiology of T1DM besides the effective role of environmental factors is related to evoking the immune system against β-cell antigens and beginning proinflammatory responses. β-cell antigens are presented to the immune system by antigen-presenting cells (APCs), then inefficient regulation of immune reactions induces chronic immunological responses that can result in β-cell destruction. The dendritic cells (DCs) then uptake the released antigens from destructed β-cells and present them to T cells. β-cells death through physiological or virus-directed mechanisms triggers the release of antigens and begins more immune responses against other β-cells. Dendritic cells (DCs) usually absorb these antigens and then present them to T-cells and induce autoreactive T-cells. Only when thymic negative selection does not recognize these autoreactive T cells, an auto-immune response is possible.
Activated autoreactive T cells by DCs stimulate autoreactive B and cytotoxic T cells. Finally, the effective mechanism of beta cell degradation requires the cooperation of DCs, T cells, B cells, natural killer (NK) cells, and macrophages. Based on the considerable number of destructed β-cells in T1DM, compensation of the functional β-cells population looks more promising treatment option.
Toward this goal, the maturation of various cell sources capable of differentiation, de-differentiation, and trans-differentiation have been evaluated. Many transcription factors have been identified that contribute to the differentiation of pancreatic epithelium, specification of endocrine progenitors, functional specialization of the α cells, and inducing of β and δ cells like Pdx1(7), Ngn3 (8), Arx, and Pax4. Among these transcription factors, the Arx and Pax4 have received more attention due to their roles in the final differentiation of β-cells. For instance, the ectopic expression of Pax4 can age-independently induce the continuous replacement of the embryonic glucagon-producing cells and their conversion into β-like cells. Furthermore, it has been shown that the expression of Pax4 in the mouse α-cells can result in the neogenesis or conversion of α-cells into functional β-like cells. Studies have also shown that Arx inactivation in pancreatic glucagon-positive cells can be transformed into β-like cells. It is interesting to note that, however, the function of Arx and Pax4 in the maturation process of endocrine cell lines have an inhibitory effect on each other's expression. Consequently, any therapeutic agent used to compensate for the loss of beta cells is expected to result in similar changes in these transcription factors with similar trends.
Among the reported antidiabetic agents under investigation, antimicrobial peptides have received extensive attention in the last decade. Magainin-AM2, as an orthologue of Magainin-2 from amphibians, is a cationic, amphipathic, α-helical antimicrobial peptide with 23 amino acids with the lowest hemolytic activity. The proposed mechanisms for the antidiabetic activity of Magainin-AM2 include cell membrane depolarization, increasing intracellular calcium content, and enhancing the release of GLP-1 from GLUTag cells followed by insulin-release from the treated cells. Although magainin has been frequently studied to treat T2DM models, there are no reports of using magainin in beta cell regeneration in T1DM models.
Thus, a need is appreciated for novel methods for the treatment of type-1 diabetes.
The following presents a simplified summary of one or more embodiments of the present invention in order to provide a basic understanding of such embodiments. This summary is not an extensive overview of all contemplated embodiments and is intended to neither identify key or critical elements of all embodiments nor delineate the scope of any or all embodiments. Its sole purpose is to present some concepts of one or more embodiments in a simplified form as a prelude to the more detailed description that is presented later.
The principal object of the present invention is therefore directed to novel methods for the treatment of type-1 diabetes.
It is another object of the present invention to provide secondary and complementary treatment for Type-1 diabetes in patients that have received pancreatic Graft
In one aspect, disclosed is a method for treatment of type-1 diabetes in patients in need thereof using a synergistic combination of Magainin and growth hormone for beta cell regeneration.
The accompanying figures, which are incorporated herein, form part of the specification and illustrate embodiments of the present invention. Together with the description, the figures further explain the principles of the present invention and to enable a person skilled in the relevant arts to make and use the invention.
Subject matter will now be described more fully hereinafter with reference to the accompanying drawings, which form a part hereof, and which show, by way of illustration, specific exemplary embodiments. Subject matter may, however, be embodied in a variety of different forms and, therefore, covered or claimed subject matter is intended to be construed as not being limited to any exemplary embodiments set forth herein; exemplary embodiments are provided merely to be illustrative. Likewise, a reasonably broad scope for claimed or covered subject matter is intended. Among other things, for example, the subject matter may be embodied as methods, devices, components, or systems. The following detailed description is, therefore, not intended to be taken in a limiting sense.
The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. Likewise, the term “embodiments of the present invention” does not require that all embodiments of the invention include the discussed feature, advantage, or mode of operation.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprise”, “comprising,”, “includes” and/or “including”, when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
The following detailed description includes the best currently contemplated mode or modes of carrying out exemplary embodiments of the invention. The description is not to be taken in a limiting sense but is made merely for the purpose of illustrating the general principles of the invention, since the scope of the invention will be best defined by the allowed claims of any resulting patent.
Disclosed is a method for treatment of Type-1 diabetes in patients in need thereof. Disclosed is a method for treating Type-1 diabetes in patients in need thereof using a synergistic combination of Magainin and growth hormone. More specifically Magainin peptide is Magainin-II (23 aa) of SEQ ID No. 1. Magainin II is a cationic, amphipathic, a-helical antimicrobial peptide with twenty-three amino acids, and both the N-Terminus and C-terminus are free i.e., free acid and free amine. The method includes the steps of administering a combination of Magainin-II (SEQ ID No. 1) and growth hormone (SEQ ID No. 2) in a patient in need thereof. It is understood, however, that any terminal amine-derivative or terminal acid derivative including salts of carboxylic acid are within the scope of the present invention.
Treatments with the synergistic combinations of Magainin and hGH improve the results of Fasting blood sugar (FBS) and glucose tolerance test (GTT).
Chemicals: Streptozotocin, glucose, sodium dihydrogen phosphate, and tris sodium hydroxide were purchased from Sigma. Citric acid, sodium citrate, potassium tartrate sodium, sodium deoxycholate, acrylamide, methanol, ethanol and isopropanol were obtained from MERK, also from LKB, N,N′-Methylene base acrylamide, sodium dodecyl sulfate, bromophenol blue, Triton-X, TEMED, sodium chloride, and Dithiothreitol were prepared from SERVA.
Peptide Magainin II (GIGKFLHSAKKFGKAFVGEIMNS): It was synthesized by the Canadian Biomatik Company. The purification of this peptide (>95% purity) was performed by reverse phase chromatography using the Cromasil C18 column and the accuracy of this peptide was confirmed by mass spectrometry.
Human Growth Hormone (hGH): It was produced by EXIR company under the brand name of Exitropin 4 IU (1.3 mg) or (Somatropin), and their vials were purchased from a pharmacy in lyophilized powder form.
Laboratory animals: Male Balb/c mice, weighing about 25-30 g, were purchased from Razi institute (Karaj, Iran) and kept under standard conditions (an air-conditioned room (23±2° C.) with a 12 h light: 12 h dark cycle (light: 08:00-20:00 h)) and standard rodent diet in the animal house of the Institute of Biochemistry and Biophysics, University of Tehran. Also, all the processes of animal experiments were performed based on the national animal ethics committee guidelines.
Antibodies: mouse anti-p-ERK 1/2 antibody sc-81492 (1/1000), goat anti-PAX4 antibody (ab101721) (1/1000), rabbit anti-STAT5a antibody (ab30648) (1/1000), rabbit anti-GAPDH antibody (ab181602) (1/1000), rabbit anti-insulin antibody (ab63820) (1/500), mouse anti-glucagon antibody sc-514592 (1/500), mouse anti-vimentin antibody sc-6260 (1/500), rabbit recombinant anti-Ki67 (ab197547) (1/500), rat anti-CD3 antibody (ab11089) (1/500), mouse anti-CD19 antibody (sc-373897) (1/500). All secondary antibodies were utilized in at a 1/1000 concentration, including goat anti-rat IgG H&L (ab6840), goat anti-rabbit IgG H&L (ab6717), goat anti-rabbit IgG H&L (ab72465), goat anti-mouse IgG H&L (ab6785), goat anti-mouse IgG H&L (ab6787).
In a 14-day period, diabetes type 1 was induced in a group of mice via injecting multiple low doses of Streptozotocin (STZ). Also, the other group of mice received an equal volume of citrate buffer (pH 4.5) intraperitoneally (I.P.) in the same procedure that was called the normal group.
Each of the normal (N) (n=20) and diabetic (D) (n=20) groups was divided into four subgroups. The first group (N1 and D1) (n=5) received 6.7 mg/kg intraperitoneally growth hormone for two weeks. The second group (N2 and D2) (n=5) administrated 0.185 mg/kg intraperitoneally magainin II for approximately four weeks. The third group (N3 and D3) (n=5) first received 0.185 mg/kg of magainin II for four weeks, and then 6.7 mg/kg of growth hormone for two weeks. Finally, the fourth group (N4 and D4) (n=5) received only an equal volume of physiological saline instead of these treatments.
FBS measurements before the treatment and weekly during the treatment, and glucose tolerance test (GTT) at the end of the treatment were performed. The pancreatic tissue was quickly removed and placed in a cold phosphate buffer saline (PBS). Some of the extracted tissues for additional tests were stored in a freezer (−80° C.) and some were immersed in 4% paraformaldehyde (PFA) for immunohistochemical staining.
All protocols especially animal maintenance and manipulation were conducted according to the guidelines of the animal ethics committee of the University of Tehran that were audited and accepted by this committee. Male Balb/c mice, weighing about 25-30 g, were purchased from Razi institute (Karaj, Iran) and housed in the animal laboratory of the institute of Biochemistry and Biophysics and maintained at 23° C.±2° C. with a 12-hour (8:00 to 20:00) light-dark cycle.
Diabetes type 1 was induced in a group of mice via injecting multiple low doses of Streptozotocin (STZ) (40 mg/kg, intraperitoneal) for five consecutive days. The STZ dissolved in the 50 mM sodium citrate buffer (pH 4.5) to a final concentration of 4 mg/ml, immediately prior to injection. Nine days after the last STZ injection, all the mice have their FBS (fasting blood sugar) over 200 mg/dl were considered as type 1 diabetic mice group.
FBS levels were measured after fasting mice for 6 h (7:00-13:00) with an Accu-Chek glucometer via tail vein. Also in the glucose tolerance test, six hours after the mice were deprived of food (7:00-13:00), the first FBS was measured and after that mouse administrated glucose (2 g/kg bodyweight, intraperitoneally), and then at the indicated time points post-injection, blood glucose levels were measured.
Fixed tissues in 4% paraformaldehyde, after embedding in paraffin, were cut in 6-μm sections, and the sections were applied to slides. For hematoxylin-eosin staining tissues respectively pass this process; rehydration incubation in hematoxylin (2.5 min), rinsing in water, dipping in 0.5% HCl/70% ethanol (v/v), washing in water, and again after immersion in 0.2% NaHCO3, rinsing in water, dipping 20 secs in 0.1% eosin, washing briefly in water and finally dehydration and mounting.
Fixed tissue in 4% paraformaldehyde for 30 min at 4° C., embedded in paraffin and applied to slides. The prepared 6-μm sections were assayed as described previously. The following primary antibodies were used in these assays: rabbit anti-insulin antibody (ab63820) (1/500), mouse anti-glucagon antibody sc-514592 (1/500), mouse anti-vimentin antibody sc-6260 (1/500), rabbit recombinant anti-Ki67 (ab197547) (1/500), rat anti-CD3 antibody (ab11089) (1/500), mouse anti-CD19 antibody (sc-373897) (1/500). All secondary antibodies were utilized in at a 1/1000 concentration, including goat anti-rat IgG H&L (ab6840), goat anti-rabbit IgG H&L (ab6717), goat anti-rabbit IgG H&L (ab72465), goat anti-mouse IgG H&L (ab6785), goat anti-mouse IgG H&L (ab6787).
Proteins were extracted in RIPA buffer which are containing Tris-HCl buffer (100 mM, pH 7.5), ethylene diamine tetra acetic acid (EDTA, 10 mM), sodium pyrophosphate (10 mM), sodium fluoride (0.1 mM), sodium orthovanadate (10 mM), phenylmethylsulphonyl fluoride (PMSF, 2 mM), and aprotinin (10 mg/ml). The pancreas extracts were vortexed for 30 min frequently at 4° C. The homogenates were centrifuged at 12,000 rpm for 20 min at 4° C. The supernatants were stored in an −80° C. freezer. Total protein concentration was determined by the Lowry method.
De-freeze sample was added to loading buffer and dithiothreitol 0.5 M solution, then heated for 5 min at 95° C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and then protein transfer to a polyvinylidene difluoride (PVDF) blotting membrane was performed. Transferred PVDF membrane was nonspecifically blocked with fat free milk and after overnight incubation at 4° C. with primary antibody, (HRP)-conjugated secondary antibody was added and incubated for 120 min at room temperature. Finally, a result image was developed based on Western Blot-ECL (electrochemiluminescence) Development Protocol.
All values are depicted as mean±SEM and considered significant if p<0.05. The arbitrary optical density unit was acquired using ImageJ software (version 1.46). Data were analyzed using GraphPad Prism software and the results of groups were compared with the ANOVA test.
The fasting blood sugars of mice in all diabetic and normal groups were measured before the treatment and weekly during the treatments. At the end of the treatments, the percentage of FBS changes were calculated. The average percentage of FBS alterations in each group was then determined. Finally, the average result of each group was compared and analyzed with other groups by prism software.
As shown in
Also, the approximate improvement rate of fasting blood sugar for treated diabetic groups in comparison with untreated control diabetic group, demonstrated 57.43%, 97.13% and 98.83% improvement in GH (D1), magainin (D2) and magainin then GH (D3) treated diabetic mice, respectively.
As evident from
GH and magainin cumulative effect on P-ERK level as an apoptosis reducer and pdx1 (differentiated beta cell factor) enhancer. Increased ERK and PDX1 expression could be survival for islet β-cells, the maintenance of islet cell mass, the cell proliferation and differentiation, and also the survival and pro-survival role for islet β-cells in tolerating gluco- or lipo-toxicity.
In this study, as shown in
In this study as shown in
GH and Magainin Cumulative Effect on mTOR Pathway can Induce Anabolic Process of Beta Cell Regeneration:
The activity of mTOR pathway was appraised by evaluating the changes in ribosomal P-S6 expression. As a downstream factor of the mTOR pathway, results of pancreatic P-S6 expression in all treated diabetic and normal mice were compared with their control groups. Therefore, changes in P-S6 expression proportionally reflects the activity of mTOR pathway. According to the
Based on previous studies, stimulating the expression of Pax4 is essential for the a-to-β cell transdifferentiating strategy. Consequently, according to increased PAX4 expression in our treated groups, we have expected transdifferentiated or regenerated functional beta cells, especially in mice that have consecutively received magainin and then growth hormone, (N3, D3) and have significantly increased PAX4 expression.
The hematoxylin and eosin staining clearly demonstrated the effective role of magainin and growth hormone in islet size and average of cell numbers per islet. In hematoxylin and eosin staining, the islets number, and also cell numbers per islet in all sections were counted and the results were reported as the average number of cells per island for each group. Then, a comparative analysis was done for all treated normal and diabetic mice with their control groups.
As shown in
Magainin and growth hormone have a synergistic effect in beta cell mass compensation in STZ-induced diabetic mice. GH via growth hormone receptor (GHR) can affect glucose homeostasis, metabolism, growth, differentiation, and apoptosis in mammals. Also, in pancreatic beta cells, GH is effective in the cell cycle regulation, growth, and function of Langerhans Islands beta cells. On the other side, based on studies, magainin via enhancing GLP-1 release can induce insulin releasing and sensitivity and suppression of glucagon secretion in type 2 and obesity diabetic animal models. Also, Magainin-AM2 enhances insulin-releasing from mouse beta cell line via depolarization of cell membrane and enhancing intracellular calcium level. In this study, magainin and GH have been used to induce beta cell regeneration in type1 diabetes and almost all of the pancreatic beta cells have been eliminated by STZ.
The examination of magainin, GH, and consequently magainin then GH treated mice demonstrated effective beta cell regeneration in type 1 diabetic mice. As shown in
In this study, Ki67 and vimentin were respectively used as a marker of cell division and differentiation of epithelial cells to mesenchymal stem cells in immunohistochemical staining of pancreatic tissue sections. Ki67 is actually an antigen that is found in the nucleus at all active stages of cell division (G1, S, G2 and mitosis); and the highest level of ki67 expression is related to the S phase of the cell cycle. However, this protein is not seen in the G0 phase (differentiation or cessation of the cell cycle) from the cell cycle. On the other hand, vimentin is a type of cytoskeletal protein that is specifically expressed in mesenchymal cells. This factor is a marker of differentiation of epithelial cells into mesenchymal stem cells (EMT) that these mesenchymal stem cells have the ability to differentiate into different types of cells.
In
Due to the activation of multiple pathways in this treatment, the expression comparison of Ki67 and vimentin in pancreatic tissue sections is helpful to realize that the compensation of beta cells in these treatments depend on cell proliferation or regeneration of cells.
All comparative results confirm that consecutive treatment with magainin and growth hormone has a greater effect on increasing proliferation as well as increasing differentiation from mesenchymal cell lines in comparison with three control groups. For example, in
The effect of magainin and growth hormone in the treatment of type 1 diabetes suggests it as a secondary treatment for transplant recipients
Although the pathophysiology of T1DM is related to environmental factors, evoking the immune system against β-cell antigens, and beginning proinflammatory responses, the effective mechanism of beta cell degradation requires the collective cooperation of DCs, T, B and natural killer (NK) cells and also macrophages. CD3 and CD19 are known as T and B lymphocyte surface antigens, respectively. So increased expression of these two factors indicates an increase in the distribution of immune T and B cells.
Referring to
Here, disclosed is an unsuspected role of magainin in beta cell regeneration that was confirmed based on hematoxylin and eosin staining that clearly demonstrated regeneration of islet cells and reconstruction of islets in type 1 diabetic mice and also a more effective role of consecutive treatment with magainin and growth hormone in islet size and the average of cell numbers per islet in both normal and diabetic mice. Improved FBS and GTT results in treated diabetic mice, demonstrated that regenerated cells including functional beta cells that magainin can induce their insulin secretion more effectively.
Furthermore, IHC staining analysis approved insulin+ cell regeneration increased up to approximately 5-fold, although glucagon+ cell enhanced significantly, the ratio of insulin positive cell versus glucagon positive cell preserved in a normal range. Also on one side, based on increased PAX4, we expected more alpha to beta cell trans-differentiation and increased cell differentiation that resulted in differentiated beta cells. On another side, according to the semi-quantitative investigation performed on IHC staining and counting analysis, co-expression of insulin and glucagon in D3 was found to be significantly increase which could be evidence of enhancement in differentiation and trans-differentiation.
Due to the evaluation results of the activation of multiple pathways in this study, the effects of these treatments have been simplified in
In addition, growth hormone also acts by increasing the amount of ERK1,2 and (p-S6) AKT and STAT5 and also increases intracellular calcium through the prolactin receptor (PRLR), and consequently increasing insulin secretion and inhibitory effects on the apoptotic pathway.
Furthermore, Pdx1 is an important transcription factor in differentiating progenitor cell lines into pancreatic endocrine cells such as beta cells. Based on a study, the increase in Pdx1 was associated with an increase in ERK. In other words, Pdx1, as a downstream gene regulated by ERK, had increased directly or indirectly under the influence of ERK1,2. Also as another transcription factor PAX4 acts as a determining factor in the differentiation of endocrine cells into beta and delta cells. under the influence of PAX4, beta and delta cells differentiate, or some alpha cells can trans-differentiate into beta cells. In general, magainin and growth hormone can compensate beta cells by the de-differentiation pathway, directly and indirectly, inhibiting apoptosis, inducing alpha cell trans-differentiation into beta cell and also progenitor cell differentiation into beta cell.
Regarding these approximately successful results probably the disclosed treatment can be also effective in pancreas implant cases, in which there are some normal cells to accelerate regeneration more effectively. As another suggests, the disclosed treatment strategies can be adjusted based on personal medicine to get better remedies.
In certain implementations, the disclosed growth hormone can be Somatropin which is a purified polypeptide of recombinant human growth hormone, for example commercially available Exitropin®. The dose of Somatropin in combination with Magainin II can be commercially determined, for example, the dose of growth hormone can be about 4 IU i.e., 1.3 mg.
In certain implementations, the effective dose based on previous research and IC50 results were determined: The normal (N, n=20) and diabetic (D, n=20) groups were each divided into four subgroups. The first group (N1 and D1, n=5) received (IP) 6.7 mg/kg/day of growth hormone for 14 days. The second group (N2 and D2, n=5) were daily administrated 0.185 mg/kg magainin II (IP) for approximately 28 days. The third group (N3 and D3, n=5) first received 0.185 mg/kg magainin II for 28 days and then 6.7 mg/kg growth hormone for 14 days. Finally, the fourth group (N4 and D4, n=5) received only an equal volume of physiological saline. In this study administration of solved lyophilized powder has done intraperitoneally (IP), but in the developing phase drug delivery based on personalized medicine can multiply effectiveness. Thus, it is understood that all modes of drug administration including parenteral and oral are within the scope of the present invention. Also, different dosage forms, such as tablets, capsules, transdermal patches, and the like are within the scope of the present invention. Also, due to the possibility of insulin hypersensitivity, the third group first received 0.185 mg/kg magainin II for 28 days and then 6.7 mg/kg growth hormone for 14 days. Combined administration of Magainin and GH was found to have a synergistic effect, more preferably, the consecutive administration of Magainin followed by GH was found to be more effective to treat type 1 diabetes. The treatment with magainin peptide and growth hormone can be used as a primary treatment due to significantly induce regeneration of beta cells in type 1 diabetes. Treatment with magainin peptide and growth hormone can significantly reduce T and B cells, so can prevent beta cell destruction during the treatment and as secondary treatment in pancreas transplantation to accelerate cell proliferation and differentiation and reduce immune responses.
According to the treatment strategy, treatment with magainin peptide and growth hormone in diabetic and normal mice (groups N3 and D3 in this study) and a significant decrease in CD3+ and CD19+ cells in these groups, also due to a temporary increase in the mTOR pathway (temporary immunosuppressant), this treatment can be used as an adjunctive or secondary treatment in pancreatic transplantation of type 1 diabetes, kidney transplantation and other transplants in order to increase transplantation efficiency and reduce the level of immune responses in transplant rejection.
Considering the action mechanism of magainin and growth hormone, the growth factors including GLP-1, IGF, EGF and etc., can show similar effects with magainin. According to the treatment strategy, treatment with magainin peptide and growth hormone in diabetic and normal mice (groups N3 and D3 in this study) has a synergistic effect on signaling pathways, which is confirmed by a significant increase in P-ERK and P-S6. It can pave the way for the widespread use of this treatment in other diseases, disorders and transplants (In the case of diseases or complications due to decreased function of the mTOR pathway, such as sarcopenia, aging, or other diseases with a similar mechanism, this treatment can be used, with the effect that this pathway has on glucose, fat, brain function and it has muscle as well as muscle mass, it can reduce complications or improve function).
While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above-described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed.
This application claims priority to the U.S. provisional patent application Ser. No. 63/280,623 filed on 18 Nov. 2021, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63280623 | Nov 2021 | US |
