Composition for protecting pancreatic islet transplantation

Abstract

The present invention relates to a composition having a protective effect during pancreatic islet transplantation, and more specifically to a composition comprising a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, which can provide a protective effect against oxidative stress, inflammation, etc. during pancreatic islet transplantation.

Description

[Technical field]

[0001] The present invention relates to a composition having a protective effect during pancreatic islet transplantation, more specifically, a composition represented by the formula (1) [ka] (In the formula, n, X, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 has the same meaning as defined herein.) or a pharma- ceutically acceptable salt thereof, and is capable of providing a protective effect against oxidative stress, inflammation, and the like during pancreatic islet transplantation. [Background technology]

[0002] Type 1 diabetes, or the long-term type 2 diabetes that has been rapidly increasing in recent years, is highly insulin-dependent due to pancreatic β-cell destruction, and has a high incidence of life-threatening hypoglycemia and diabetic complications. Pancreatic islet transplantation refers to a procedure in which a donated pancreas from a brain-dead donor is biochemically processed to isolate pure islets, which are then administered to the recipient. The purpose of the procedure is to enable diabetic patients to normally control their blood sugar without the administration of insulin. The transplanted islets secrete insulin, allowing the recipient to live a normal life without the administration of insulin.

[0003] Allogeneic islet transplantation has been a promising strategy to eliminate severe hypoglycemia and achieve normoglycemia in type 1 diabetic patients with impaired hypoglycemic awareness, but the substantial loss of transplanted islet mass during the peri-transplant period remains a significant hurdle for widespread clinical use.

[0004] During the peri- and post-transplantation period, hypoxia / reoxygenation-induced damage and release of damage-associated molecular patterns (DAMPs), such as HMGB1, are increasingly recognized as potent activators of innate immunity and proinflammatory cytokine production. In contrast to apoptosis, a non-inflammatory or even anti-inflammatory mode of cell death, necroptosis or necrosis is highly immunogenic. At least in part, both DAMPs released from necrotic cells and the cell-intrinsic activation of the nucleotide-binding oligomerization (NOD)-like receptor protein 3 (NLRP3) inflammasome during the necrotic process are responsible for such immunogenicity.

[0005] The generation of mitochondrial reactive oxygen species (ROS), which induces the opening of the mitochondrial permeability transition pore (mPTP), is one of the main players promoting the necroptosis and necrosis process. The role of mitochondrial ROS in the necroptosis and necrosis of transplanted islets may be important, since β-cells are known to be highly susceptible to oxidative stress due to the low expression of antioxidant enzyme genes such as catalase, superoxide dismutase, and glutathione peroxidase. Therefore, the oxidative stress and hypoxia / reoxygenation-induced damage generated during various islet transplantation processes may play a key role in the severe β-cell damage and the main loss of transplanted islets. In this context, various antioxidants have been used in several studies, with their possible impact on the protection of islets against oxidative damage, enhancing islet viability and insulin secretion in vitro, and improving the islet transplantation procedure. However, the possibility of clinical application of these approaches may require stronger efficacy and availability of clinical grade substances. Furthermore, mitochondrial ROS production has not been a specific target in previous studies, which mainly focused on reducing apoptosis. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, a technical object of the present invention is to provide a composition that can efficiently protect pancreatic islets during pancreatic islet transplantation. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention provides a compound represented by the following formula (1) as an active ingredient: [ka] [wherein n is 0 or 1; X is C or N, provided that when X is N, n is 0, and when X is C, n is 1; R 1 is hydrogen or C1-C6 alkyl; R 2 is phenyl or pyridine; R 3 is hydrogen, halogen or C1-C6 alkyl; R 4 is hydrogen, halogen, 2-carboxy-pyrrolidin-1-yl, pyrrolidin-1-yl, 4-acetic acid-1,3-thiazolin-2-yl, -CH2-(1,1-dioxo-thiomorpholin-4-yl) or -CH2-(2-oxopiperazin-4-yl); R 5 is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl; R 6 -DWR 7 where D is cyclopentyl, cyclohexyl, pyrrolidine, tetrahydropyran, tetrahydrofuran or piperidine, and W is a direct bond, -SO2-, -CO-, -C(O)O-; R 7 is hydrogen, hydroxy, or C1-C6 alkyl. or a pharma- ceutically acceptable salt thereof.

[0008] In one embodiment of the present invention, the compound represented by formula (1) is represented by the following formula (2): [ka] The compound is (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine, represented by the formula:

[0009] In another embodiment according to the invention, the pharmaceutically acceptable salts include those formed with acids which form non-toxic acid addition salts containing pharmaceutically acceptable anions, e.g., inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the like; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, and the like; sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. In addition, the pharma- ceutically acceptable salts include, but are not limited to, pharma-ceutically acceptable base addition salts, such as alkali metal or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc.; amino acid salts such as lysine, arginine, guanidine, etc.; organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, triethylamine, etc. The compounds represented by the formula (1) according to the present invention can be converted into their salts by conventional methods, and the preparation of the salts can be easily carried out by those skilled in the art based on the structure of the formula (1) without further explanation.

[0010] Pancreatic islet transplantation is a surgical procedure in which only islet cells that produce insulin are extracted from the pancreas and transplanted into a recipient, and is a typical example of cell therapy rather than solid organ therapy.

[0011] However, β-cells are vulnerable to oxidative stress due to their weak antioxidant defense system. During the isolation of islets, various factors such as hypothermia, mechanical stress and exposure to collagenase contribute to the generation of oxidative stress, which ultimately leads to cell death. During the process of islet transplantation and revascularization, the lack of oxygen and nutrients leads to the occurrence of apoptosis, autophagy, necrosis and necroptosis in islets. Apoptosis, in contrast to necrosis, is not usually accompanied by an inflammatory response, but primary apoptosis of islets due to hypoxia may induce immune activation that provides uncleaved caspase-9 and caspase-3, rather than the cleaved forms. This makes the primary cell death process towards necrosis, which induces the release of damage-associated molecular patterns (DAMPs), rather than secondary apoptosis. DAMPs are molecules such as HMGB1, dsDNA and uric acid, which are released from the intracellular space and are mainly recognized by Toll-like receptors (TLRs), which are mainly involved in the innate immune system. Because DAMPs are released during ischemia and reperfusion injury as a result of cell death by necrosis and necroptosis and contribute to the inflammatory cascade in islet transplant failure, inhibiting necrosis and necroptosis rather than apoptosis or autophagy may be a preferred strategy to reduce the immune response associated with DAMP release during islet transplantation.

[0012] HMGB1 is secreted from damaged or necrotic cells during cell death, functions as a DAMP signal and proinflammatory cytokine, and is associated with the pathogenesis of various inflammatory diseases. Several proinflammatory cytokines, such as interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, are also known to induce β-cell dysfunction and apoptosis through a series of intracellular signaling pathways, including ROS formation and JNK activation, and translocation of NF-kB, a transcription factor that controls NADPH oxidase, leading to the generation of oxidative stress. The concerted action of proinflammatory cytokines induces the expression of inducible nitric oxide synthase (iNOS), and blocking iNOS activity or increasing the expression of manganese peroxide dismutase (MnSOD) attenuated cytokine-induced NF-kB activation under in vitro conditions in insulin-producing cell lines.

[0013] To avoid the use of animal products for safety regulations, isolated human islets are generally cultured under serum-starved culture conditions before clinical allogeneic islet transplantation. During islet culture under serum-starved conditions, islets are further exposed to various stresses from the medium, nutrient deprivation, various proinflammatory cytokines, enzymes from the exocrine pancreas, and lack of sufficient oxygen supply. Such stresses are harmful to β-cells and induce the accumulation of excessive ROS. In addition, the toxicity of islet amyloid polypeptide (IAPP) is thought to be the cause of the gradual loss of β-cell mass and islet fibrosis in type 2 diabetes, and it was thought that serum-starved culture under euglycemic conditions induces the accumulation of toxic hIAPP oligomers. Although inhibition of the IL-1 / IL-1 receptor axis can attenuate the inflammatory response and functional impairment of islets caused by serum-starved culture, IL-1 blockade cannot attenuate the accumulation of toxic oligomers during serum-starved culture.

[0014] The compound of formula (1) of the present invention, when administered during islet isolation, can protect islets by reducing oxidative stress and the amount of reactive oxygen species (ROS).

[0015] Furthermore, when the compound represented by formula (1) of the present invention is treated with isolated pancreatic islets during serum-starved culture, it can reduce the expression of c-jun N-terminal kinase, HMGB1 and proinflammatory cytokines (e.g., interleukin-1β, interleukin-6 and tumor necrosis factor (TNF)-α), and reduce the accumulation of amyloid and toxic IAPP (islet amyloid polypeptide) oligomers due to its potent mitochondrial reactive oxygen species (ROS) scavenging activity.

[0016] The compound represented by formula (1) of the present invention, when administered during isolation of pancreatic islets or during serum-starved culture of isolated pancreatic islets, can effectively protect the pancreatic islets through the above-mentioned mechanism of action, thereby dramatically improving the recovery rate of diabetes after islet transplantation.

[0017] In another embodiment of the present invention, the composition of the present invention may further comprise a pharma- ceutically acceptable carrier, if necessary. As used herein, the term "carrier" refers to a substance that facilitates administration of a compound to a cell or tissue, and is not particularly limited. Effect of the Invention

[0018] The composition of the present invention can provide excellent protective effect on pancreatic islet cells, thereby significantly improving the success rate of pancreatic islet transplantation. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of evaluating the effect of Compound 1 on in vitro viability, function and pro-inflammatory cytokine expression of isolated hIAPP+ / − mouse islets during isolation. [Diagram 2] FIG. 1 shows the results of evaluating the effect of Compound 1 on in vitro viability, function and pro-inflammatory cytokine expression of isolated C57BL / 6 mouse islets during isolation. [Diagram 3]FIG. 1 shows the results of evaluating the effect of compound (1) during isolation on the in vivo outcome after transplantation of hIAPP+ / − mouse islets. [Figure 4] FIG. 1 shows the results of evaluating the effect of Compound 1 on cell viability and ROS in serum-starved culture of RINm5F cells and hIAPP+ / − mouse islets. [Diagram 5] FIG. 1 shows the results of evaluating the effect of Compound 1 on cell viability and ROS in non-human primate (NHP) islets during serum-starved culture. [Figure 6] FIG. 1 shows the results of evaluating the effect of Compound 1 on the in vitro viability, function, proinflammatory cytokine expression, and oligomer accumulation of isolated hIAPP+ / − mouse islets during serum-starved culture. [Figure 7] FIG. 1 shows the results of evaluating the effect of Compound 1 in serum-starved culture on in vivo outcomes following transplantation of hIAPP+ / − mouse islets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention will be described in more detail with reference to the following Preparation Examples and Examples, however, these Examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0021] I. Experimental Materials and Methods 1.Compound (Tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine (hereinafter referred to as 'Compound 1') was prepared according to the method disclosed in Example 36 of International Publication WO2009 / 025478.

[0022] 2. Experimental animals and cell lines Heterozygous human islet amyloid polypeptide (hIAPP) gene transfer + / -Pancreatic islets were isolated from FVB / N mice (Jackson Laboratory, Bar Harbor, ME, USA), C58BL / 6 mice (Orientbio, Korea), and cynomolgus monkeys (Orientbio, Korea). For further in vitro studies, rat insulinoma cells (RINm5F cells) were treated with or without compound 1 during tert-butyl hydroperoxide (tBHP) exposure or culture under serum-starved conditions. RINm5F cells were suspended in DMEM medium (Gibco, Grand Island, NY, USA) containing 100 IU / mL penicillin and 100 μg / mL streptomycin and cultured at 37°C in a well-humidified 5% CO2 atmosphere. All experimental protocols in this study were approved by the Institutional Animal Care and Use Ethics Committee (IACUC) of the Samsung Life Science Institute.

[0023] 3. Islet Isolation Mouse islets were cultured at 10–12 weeks of age using hIAPP ELISA kits, as previously described. + / - Collagenase was isolated from C57BL / 6 and C57BL / 6 mice. Briefly, mouse pancreases were digested by injecting 0.8 mg / mL collagenase in HBSS buffer solution (Sigma-Aldrich, St. Louis, MO, USA) with or without 20 μM compound 1 through the common bile duct. Islets were purified from the digested mouse pancreases using a Ficoll (Biochrom, Berlin, Germany) gradient and washed several times with 1xHBSS. The isolated islets were cultured in suspension in 10 mL of RPMI1640 (Gibco, Grand Island, NY, USA). Prior to ex vivo and in vivo studies, the medium was supplemented with 10% heat-inactivated FBS (fetal bovine serum) at 37°C and 5% CO2.

[0024] 4. Non-human Primate Islet Isolation Dissected non-human primate (NHP) pancreases were intraductally injected with cold Liberase MTFC / T solution (4 mL / g pancreas; Roche). Digestion and isolation were performed using the Ricordi automated isolation technique described previously. All isolated NHP islets were cultured in CMRL medium supplemented with 10% heat-inactivated porcine serum at 37°C and 5% CO2 prior to in vitro and in vivo studies.

[0025] 5. Serum-starved Ex Vivo Culture of Isolated Islets Purified hIAPP + / - FVB / N and C57BL / 6J mice are suspended in RPMI1640 (Gibco, Grand Island, NY, USA) containing 100 IU / mL penicillin and 100 μg / mL streptomycin and cultured at 37°C in a well-humidified 5% CO2 atmosphere. Purified NHP islets are suspended in CMRL1066 medium (Corning Life Sciences, Tewksbury, MA, USA; Catalog No. 99-663-CV) containing 100 IU / mL penicillin and 100 μg / mL streptomycin and cultured at 37°C in a well-humidified 5% CO2 atmosphere. During ex vivo culture of mouse islets, hIAPP + / -Three experimental groups were designated for experiments using FVB / N and C57BL / 6J mouse islets: medium supplemented with 10% FBS (Tissue Culture Biologicals, Los Alamitos, CA, USA; Catalog No. 101) (FBS group), medium supplemented with 0.625% BSA (Qbiogene, Carlsbad, CA, USA; Catalog No. BSA003) (BSA group), and medium supplemented with 0, 0.1, 1, 10, and 20 μM compound 1 + 0.625% BSA (BSA + compound 1 group). For experiments using NHP islets, the medium was supplemented with 10% FBS (FBS group) and 0-20 μM compound 1 + 0.625% human serum albumin (HSA; Greencross, Yongin, Korea) to determine the optimal concentration of compound 1 for treatment during serum-starved culture. In addition, the medium was supplemented with 10% FBS (FBS group), 20 μM compound 1 + 10% FBS (FBS + compound 1 group), 0.625% human serum albumin (HSA; Greencross, Yongin, Korea; HSA group), or 20 μM compound 1 + 0.625% HSA (HSA + compound 1 group).

[0026] 6.Assessment of islet viability by Alamar Blue assay Islet viability was assessed using Alamar Blue staining according to the manufacturer's protocol (Invitrogen, Grand Island, NY, USA). + / - Pancreatic islets isolated from mice were cultured in 24-well plates at a density of 100 islet equivalents (IEs) per well in RPMI 1640 containing 10% FBS and 0.625% BSA with or without compound 1. After 1 and 3 days, 10x AlamarBlue solution was added directly to each well and the islets were cultured for 4 hours at 37°C protected from direct light. The fluorescence intensity of each well was measured at 570 / 585 nm (excitation / emission) using a GloMax-Multi Plus Detection system (Promega, Fitchburg, WI, USA) and values ​​were normalized to a blank.

[0027] 7. Acridine Orange / Propidium Iodide (AO / PI) Assay Islet viability after isolation and culture was assessed using dual fluorescence with acridine orange (0.67 μmol / L) and propidium iodide (75 μmol / L) (AO / PI) staining to visualize live and dead islet cells simultaneously. Fluorescence imaging was performed with a fluorescence microscope (Nikon ECLIPSE 80i, Tokyo, Japan), and live and dead areas were quantified using NIS-Element AR3.0 (Nikon). Islet viability (%) was calculated as live islet cells / total islet cells × 100.

[0028] 8. ATP Assay The ATP content of islets was analyzed by luminometry. Samples were first mixed with 200 μL of a commercial lyophilized ATP monitoring reagent (ATP Bioluminescence Assay KIT CLS II, Roche Diagnostics) containing firefly luciferase and luciferin, reconstituted in imidazole buffer (100 mM, pH 7.75). The emitted light was measured with a luminometer (LKB 1250 luminometer). The adenine nucleotide content in the samples was determined after correction for a control group (without islets or cells) and calculated with reference to an ATP standard treated in the same way as the samples.

[0029] 9. Fluorescence-based Intracellular Free Radical Detection and Lipid Peroxidation hIAPP + / -Oxidative stress levels in mouse islets were measured immediately after islet isolation and after ex vivo culture of isolated islets. Oxidative stress in islets was quantified by estimating the concentration of malondialdehyde (MDA), a by-product of lipid peroxidation, using a fluorescence-based intracellular ROS detection method. Total ROS, superoxide ions, and nitric oxide formation were determined using a ROS detection kit (ENZ-51011) (Enzo Life Sciences, Farmingdale, New York, USA). ROS content was measured by DHR123 fluorescence levels. The kit was applied according to the manufacturer's protocol.

[0030] 10. Glucose-stimulated Insulin Secretion (GSIS) Assay After hand-picking and washing with PBS, islets were seeded into 12-mm insert wells (Merck Millipore, Billerica, MA, USA) at 10 islets per well and pre-incubated for 90 min at 37°C in Kreb's-Ringer buffer (KRB: 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, and 0.2% BSA) supplemented with 60 mg / dL glucose. After washing, islets were incubated with 300 mg / dL glucose-KRB for 1 h, followed by incubation with 60 mg / dL glucose-KRB for an additional 1 h. Insulin release into the supernatant by mouse islets was measured by ELISA (ALPCO, Salem, NH, USA) and multiplex kit (Merck Millipore), respectively.

[0031] 11. RNA isolation and cDNA synthesis The collected islets were treated with 500 μL of Trizol (Life Technologies, Grand Island, NY, USA), followed by the addition of 100 μL of chloroform. After incubation at 4 °C for 5 min, the mixture was centrifuged at 12,000 rpm for 15 min, and 250 μL of isopropyl alcohol was added to precipitate the total RNA. The RNA pellet was washed with 75% ethanol, and the RNA was eluted from the pellet using RNase-free water (WelGene, Daegu, South Korea). RNA purity was assessed by NanoDrop (Thermo Scientific, Wilmington, DE, USA) and agarose gel electrophoresis. The purity of the RNA ranged from 1.9 to 2.0, based on the ratios of 260 / 230 and 260 / 280 optical density (OD). Total RNA was reverse transcribed for quantifying gene expression using the SuperScript® II Reverse Transcription System (Life Technologies) according to the manufacturer's protocol.

[0032] 12. Real-time quantitative reverse transcription (qRT)-PCR Real-time qRT-PCR was performed using gene-specific primer pairs (Table 1). Amplified PCR products were normalized to β-actin PCR products amplified from the same sample. PCR products were separated on 1% agarose gels and images were obtained using a Gel DocTM XR instrument (Bio-Rad, Hercules, CA, USA). To quantify the expression levels of genes, real-time PCR was performed using a SYBR premix kit (Takara Bio Inc., Tokyo, Japan) and an ABI Prism 7000 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. [Table 1]

[0033] 13.hIAPP + / - In vivo islet function of pancreatic islets Using a marginal mass renal subcapsular islet transplantation model, we investigated the effect of hMAPP + / - We assessed in vivo islet function in mice. To induce diabetes, 180 mg / kg streptozotocin (STZ, Sigma-Aldrich) was administered to 8–10 week-old hIAPP mice. - / - FVB / N mice were administered 100 mg / dL of DMSO. Mice were considered diabetic if their blood glucose values ​​exceeded 300 mg / dL on two consecutive occasions. To evaluate the combined effects of Compound 1 on islet mass and function in ex vivo culture, hIAPP + / - Equal amounts of islets isolated from mice (400 IEQ islets per recipient) were assigned to two groups: 0.625% BSA (BSA group) and 0.625% BSA + Compound 1 (BSA + Compound 1 group). After 72 hours of culture, the islets were incubated with diabetic hIAPP - / - The islets were transplanted into the renal subscapular space of mice. The IE number was calculated by classifying the islets according to their diameter using the Ricordi algorithm. After islet transplantation, non-fasting plasma glucose levels were measured three times a week for the first two weeks and twice a week for the last two weeks.

[0034] 14.Histological analysis Four weeks after transplantation, the transplanted kidneys of the mice were removed and embedded in paraffin. The immunohistochemical staining was briefly described below. After deparaffinization, 4-μm tissue sections were stained with polyclonal guinea pig anti-insulin antibody (1:1000, A0546, DAKO, Denmark), glucagon (1:500, ab92517, abcam, United Kingdom), and amyloid (1:2500, #44-344, Invitrogen). The stained slides were observed using an Olympus BX40 optical microscope (Olympus, Japan) equipped with ×10 / 22 numerical aperture and ×40 / 0.75 numerical aperture objective lens. Photographic images were collected with a digital camera (Olympus DP50) and analyzed using Image-Pro Plus5.1 software.

[0035] For quantification of DAB-labeled β-cells, all slides were imaged at 10X on a Vectra 3.0 automated quantitative pathology imaging system and analyzed using inForm software (all Perkin-Elmer, Waltham, MA, USA). Transplanted kidney tissue sections were scanned for image acquisition, and to set up transplanted tissue segmentation, the number of representative areas of transplanted islet cells and areas to be excluded from analysis such as kidney tissue were examined by a learn-by-example interface. For β-cell segmentation, a spectral library was used to identify hematoxylin-stained nuclei and DAB-stained cytoplasm (β-cells) within the transplanted tissue. Using these training sets, the algorithm was validated to identify only DAB-stained cells within the transplanted tissue. All captured 10X images were analyzed and DAB-stained transplanted β-cells were counted.

[0036] 15. Assessment of hIAPP oligomer accumulation Accumulation of h11APP oligomers was measured using hIAPP stained with A11Ab (anti-oligomer antibody, Invitrogen) and insulin antibody (Dako). + / - Sections of mouse islets were used to assess hIAPP oligomer accumulation. Islets were blocked with 10% normal donkey serum and incubated with primary rabbit anti-oligomer (1:100). Islets were washed and incubated with secondary Cy3-conjugated anti-rabbit antibody for 1 h. After washing, sections were stained with guinea pig anti-insulin primary antibody (1:500; Dako) followed by Alexa Fluor 488 anti-guinea pig secondary antibody (1:200; Jackson Immuno Research Laboratories). Mounting was performed after counterstaining with 4',6-diamidino-2-phenylindole (DAPI). To quantify accumulation of hIAPP oligomers per islet, the percentage of A11 stained cells among total DAPI+ islet cells was measured using confocal microscopy.

[0037] 16. Assessment of amyloid accumulation Sections of islet grafts under the kidney capsule were examined for islet amyloid after 0.5% Thioflavin S staining (Sigma) and for β-cells using insulin immunostaining with a guinea pig anti-insulin antibody (1:500; Dako, Carpenteria, CA) and a Cy3-conjugated anti-guinea pig secondary antibody (1:200; Jackson Immuno Research Laboratories, West Grove, PA). Sections were mounted after staining with DAPI. To quantify amyloid accumulation per section, the percentage of Thioflavin S stained cells (green) in the entire islet graft site (green + red + blue areas) was measured using a confocal microscope. The original magnification of the panels was ×100.

[0038] 17. Statistical analysis Results were expressed as mean ± standard deviation (SD) or median and interquartile range, as appropriate. Continuous variables were compared using Student's t test, one-way analysis of variance (ANOVA), or Mann-Whitney U test, as appropriate. Longitudinal data were analyzed by two-way ANOVA with Bonferroni post hoc tests using Graphpad Prism5 (Graph Pad Software, La Jolla, CA, USA). p values ​​<0.05 were accepted as statistically significant for comparisons.

[0039] II. Results 1. Protective effect against oxidative stress and inflammatory responses during islet isolation To evaluate the effect of Compound 1 during islet isolation, hIAPP + / -Pancreatic islets from FVB / N mice were isolated by collagenase with and without supplementation of 20 μM compound 1. The relative ex vivo cell viability assessed by Alamar Blue analysis was significantly increased in the compound 1-treated group compared to the untreated group (Figure 1A). In addition, compound 1 treatment during islet isolation showed significantly elevated levels of ATP compared to the compound 1-untreated group (Figure 1B). Also, the ROS content and oxidative stress levels of islets treated with compound 1 during islet isolation were significantly decreased compared to the compound 1-untreated control islets (Figure 1C and D). The stimulation index obtained in the glucose-stimulated insulin secretion (GSIS) assay, which indicates in vitro islet function, showed similar values ​​between the two groups (Figure 1E). Next, we investigated the transcriptional expression of c-Jun N-terminal kinase and HMGB1, and proinflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the culture medium after isolation with and without compound 1 supplementation. These transcript expression levels were significantly decreased in the compound 1-treated group compared to the untreated group (Figure 1F). These results were reproduced in wild-type C57BL / 6 mouse islets (Figure 2).

[0040] 2. Improved outcomes after islet transplantation To investigate the effect of Compound 1 supplementation during in vivo islet isolation, we compared the marginal mass of isolated islets with or without Compound 1 supplementation in streptozotocin (STZ)-induced diabetic hIAPP - / - The compound 1 group was transplanted into the renal subscapular space of FVB / N mice (Fig. 3A). The glucose level after transplantation in the compound 1-treated group was significantly lower than that in the non-treated group, and removal of the transplanted kidney confirmed the recovery of hyperglycemia in the compound 1-treated group (Fig. 3B). The compound 1 group showed better post-transplant results in terms of diabetes reversal rate and glucose level during intraperitoneal glucose tolerance test (IPGTT) compared with the control group (Fig. 3C, D, and E). The compound 1 group showed a larger proportion of insulin-positive area in the transplanted tissue compared with the control group (Fig. 3F).

[0041] 3. hIAPP in Serum-starved Culture + / -Protection of transgenic mice and NHP islets The effects of compound 1 supplementation on tert-butyl hydroperoxide (tBHP) exposure or pancreatic islet culture under serum-starved conditions were examined. Although RINm5F cells exposed to tBHP exhibited LDH release and ROS formation, the cytotoxicity and ROS content were significantly reduced after compound 1 supplementation (Figure 4, A and B). Rat insulinoma cells (RINm5F cells) and hIAPP + / - The phase-dependent cell viability of pancreatic islets from FVB / N mice significantly increased serum deprivation-induced cytotoxicity in a dose-dependent manner from 0.1 μM to 20 μM (Figure 4C and D). Supplementation of the medium with compound 1 in serum-starved culture conditions dose-dependently increased hINAPP + / - Serum deprivation-induced ROS was also reduced in RINm5F cells and islets from FVB / N mice (Fig. 4E and F). Similar results were observed in NHP islets (Fig. 5A, B, and C).

[0042] FIG. 6A shows the upregulation of hIAPP expression during serum-starved culture with or without supplementation of Compound 1 as assessed by AO / PI staining. + / - Cell viability of FVB / N mouse islets is shown. Compared with FBS-treated islets, islets cultured with BSA for 72 h experienced more cell death, seen in red PI-positive cells, and showed a decrease in cell viability that was significantly improved by supplementation with compound 1 (Figure 6A and B). The transcription levels of TNF-α, IL-1β, IL-6, c-JUN, and HMGB1 after 1 and 3 days of ex vivo culture were significantly decreased in the BSA + compound 1 group than in the BSA group (Figure 6C and D). To evaluate the function of the islets, the addition of compound 1 to the medium in serum-starved culture conditions significantly increased the expression of hIAPP + / - A significant increase in the stimulation index was observed in mouse-derived islets (Figure 6E). After 72 hours of culture under serum-starved culture conditions in medium with or without Compound 1, the percentage of hIAPP oligomer accumulation in hIAPP+ / - islets was significantly decreased in the BSA + Compound 1 group compared to the BSA group (Figure 6F).

[0043] 4. Improvement of post-transplant glycemic outcomes and amyloid accumulation in transplanted islets We investigated whether supplementation of medium with Compound 1 during serum-starved culture would improve post-transplant outcomes in an in vivo mouse model of hIAPP. + / - Marginal mass of pancreatic islets from FVB / N mice were cultured with or without compound 1 supplementation for 72 h and were analyzed for STZ-induced diabetic hIAPP - / - The compound 1 group was transplanted under the renal capsule of FVB / N mice (A in FIG. 7). The compound 1 group showed significantly better post-transplant outcomes, including post-transplant glucose levels, diabetes reversal rate, and glucose levels and AUC of glucose during IPGTT (B, C, D, and E in FIG. 7). The insulin-positive area of ​​the transplanted tissue was larger in the compound 1 group than in the control group (F in FIG. 7). In addition, the rate of amyloid accumulation was significantly reduced in the islet grafts of the compound 1 group compared with the control group (G in FIG. 7).

[0044] III. Review Supplementation of compound 1 during islet isolation increased islet cell viability in vitro and reduced oxidative stress, reactive oxygen species (ROS) content, and expression of c-Jun, HMGB1, and proinflammatory cytokines. In vivo studies also confirmed that treatment with compound 1 during islet isolation improved post-transplant outcomes.

[0045] Furthermore, Compound 1 protected against serum deprivation-induced impairment of in vitro islet viability, and more importantly, supplementation of the medium with Compound 1 during serum-starved culture significantly reduced the accumulation of amyloid and toxic IAPP oligomers and improved in vivo islet graft function.

[0046] These results confirmed the beneficial effects of supplementation with Compound 1 of the present invention during islet isolation as well as serum-starved culture during the islet transplantation process.

Claims

1. The active ingredient is represented by the following formula (1): 【Chemistry 1】 [Wherein, n is 0 or 1; X is C and n is 1; R 1 is hydrogen or C 1 -C 6 is alkyl; R 2 is phenyl; R 3 is hydrogen or C 1 -C 6 is alkyl; R 4 is -CH 2 -(1,1-dioxo-thiomorpholin-4-yl) or -CH 2 -(2-oxopiperazin-4-yl); R 5 is hydrogen, C 1 -C 6 Alkyl or C 3 -C 6 is cycloalkyl; R 6 is tetrahydropyran or tetrahydrofuran. or a pharma- ceutically acceptable salt thereof, A composition for protecting islets from transplantation, for protecting islets by reducing oxidative stress and the amount of reactive oxygen species (ROS).

2. The compound represented by the formula (1) is represented by the following formula (2): 【Chemistry 2】 2. The composition according to claim 1, wherein the compound is (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine represented by the formula:

3. The composition according to claim 1, characterized in that the compound represented by formula (1) is administered during isolation of pancreatic islets.

4. 2. The composition according to claim 1, characterized in that the compound represented by formula (1) is administered to isolated pancreatic islets during serum-deprived culture.

5. The composition according to claim 3, characterized in that the compound represented by formula (1) reduces the expression of c-jun N-terminal kinase, HMGB1 (high mobility group box-1) and proinflammatory cytokines.

6. The composition of claim 5, wherein the proinflammatory cytokines are interleukin-1β, interleukin-6 and tumor necrosis factor (TNF)-α.

7. The composition according to claim 4, characterized in that the compound represented by formula (1) reduces the accumulation of amyloid and toxic IAPP (islet amyloid polypeptide) oligomers.

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