Compositions for protecting islet transplantation

Abstract

The present invention relates to a composition having a protective effect during islet transplantation, and more particularly, to a composition containing a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof and capable of providing a protective effect against oxidative stress, inflammation, etc. during islet transplantation.

Description

Technical Field

[0001] The present invention relates to a composition having a protective effect during islet transplantation. More specifically, the present invention relates to a composition for protecting islet transplantation, which comprises a compound of formula 1 or a pharmaceutically acceptable salt thereof, and can provide protection against oxidative stress and inflammation during islet transplantation:

[0002] [Formula 1]

[0003]

[0004] wherein n, X, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are as defined herein. Background Art

[0005] Type 1 diabetes or long-term type 2 diabetes, which has been increasing rapidly in recent years, is highly dependent on insulin due to the destruction of pancreatic β-cells, and has a high incidence of life-threatening hypoglycemia and diabetic complications. Islet transplantation refers to a surgery in which the pancreas donated by a brain-dead person is biochemically processed to isolate pure islets and then administered to the recipient. The purpose of the surgery is to allow diabetic patients to normally control blood glucose without administering insulin. The transplanted islets secrete insulin, allowing the recipient to live a normal life without administering insulin.

[0006] Allogeneic islet transplantation has always been a promising strategy for eliminating severe hypoglycemia and achieving normal blood glucose in type 1 diabetic patients with impaired hypoglycemia awareness. However, the substantial loss of transplanted islet mass during the peri-transplant period remains a major obstacle to widespread clinical use.

[0007] During the peri-transplant period, hypoxia / reoxygenation-induced injury and the release of damage-associated molecular patterns (DAMPs) such as high-mobility group protein 1 (HMGB1) have increasingly been accepted as potent activators of innate immunity and the production of pro-inflammatory cytokines. In contrast to apoptosis, which is a non-inflammatory and even anti-inflammatory mode of cell death, necroptosis or necrosis is highly immunogenic. Both the release of DAMPs from necrotic cells and the intracellular activation of the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome during necroptosis are at least partially responsible for this immunogenicity.

[0008] The production of mitochondrial reactive oxygen species (ROS) that induces the opening of the mitochondrial permeability transition pore (mPTP) is one of the key factors promoting necroptosis and necrosis. In transplanted islets, the role of mitochondrial ROS in necroptosis and necrosis may be crucial because β-cells are highly sensitive to oxidative stress due to the low expression of antioxidant enzyme genes such as catalase, superoxide dismutase, and glutathione peroxidase. Therefore, oxidative stress generated during various processes of islet transplantation and hypoxia / reoxygenation-induced injury may play an important role in significant β-cell damage and substantial loss of transplanted islets. In this context, various antioxidants have been used in a few studies to provide possible protection against oxidative damage to islets, resulting in enhanced islet survival rate and insulin secretion in vitro and improved islet transplantation procedures. However, the clinical translation of these methods requires stronger efficacy and the availability of clinical-grade materials. In addition, in previous studies, the production of mitochondrial ROS was not a specific target, and these studies mainly focused on reducing apoptosis. Summary of the Invention

[0009] Technical Problem

[0010] Therefore, the technical problem of the present invention is to provide a composition that can effectively protect islets during islet transplantation.

[0011] Technical Solution

[0012] To achieve the above object, the present invention provides a composition for protecting islet transplantation, which comprises a compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[0013] [Formula 1]

[0014]

[0015] Wherein

[0016] n is 0 or 1;

[0017] X is C or N, provided that when X is N, n is 0, and when X is C, n is 1;

[0018] R 1 is hydrogen or C1-C6 alkyl;

[0019] R 2 is phenyl or pyridine;

[0020] R 3 is hydrogen, halogen, or C1-C6 alkyl;

[0021] R 4is hydrogen, a halogen, 2-carboxy-pyrrolidin-1-yl, pyrrolidin-1-yl, 4-acetamido-1,3-thiazolin-2-yl, -CH2-(1,1-dioxo-thiomorpholin-4-yl) or -CH2-(2-oxo-piperazin-4-yl);

[0022] R 5 is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl; and

[0023] R 6 is -D-W-R 7 wherein D is cyclopentyl, cyclohexyl, pyrrolidine, tetrahydropyran, tetrahydrofuran or piperidine; W is a direct bond, -SO2-, -CO- or -C(O)O-; and R 7 is hydrogen, hydroxy or C1-C6 alkyl.

[0024] In one embodiment according to the present invention, the compound of formula 1 may be (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine of formula 2:

[0025]

[0026] In another embodiment according to the present invention, the pharmaceutically acceptable salts may include acid addition salts formed from the following acids: inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid; organic 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 and salicylic acid; or sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid, which form non-toxic acid addition salts including pharmaceutically acceptable anions. In addition, the pharmaceutically acceptable salts may include pharmaceutically acceptable base addition salts, such as salts with alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium and magnesium; salts with amino acids such as lysine, arginine and citrulline; and organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, but not limited thereto. The compounds of 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 formula 1 without separate explanation.

[0027] Islet transplantation is a surgery that separates only insulin-producing islet cells from the pancreas and transplants them into the recipient, and is a representative example of cell therapy rather than solid organ transplantation.

[0028] However, β-cells are vulnerable to oxidative stress due to their weak antioxidant defense system. During islet isolation, various factors including hypothermia, mechanical stress, and exposure to collagenase lead to the generation of oxidative stress and ultimately trigger cell death. Apoptosis, autophagy, necrosis, and necroptosis occur in islets under low availability of oxygen and nutrients during islet transplantation and angiogenesis. Although apoptosis generally does not involve an inflammatory response in contrast to necrosis, primary apoptosis caused by hypoxia in islets may induce immune activation, presenting uncleaved caspase-9 and caspase-3 rather than their cleaved forms. This suggests that the major cell death process is towards necrosis that induces the release of damage-associated molecular patterns (DAMPs) rather than secondary apoptosis. DAMPs are molecules released from the intracellular space such as HMGB1, dsDNA, and uric acid, which are mainly recognized by Toll-like receptors (TLRs) involved in the innate immune system. Since DAMPs are released as a result of cell death caused by necrosis and necroptosis during hypoxia and reperfusion injury and lead to an inflammatory cascade in islet transplantation failure, inhibiting necrosis and necroptosis rather than apoptosis or autophagy may be a preferred strategy to reduce DAMP release and related immune responses during islet transplantation.

[0029] HMGB1 is secreted by damaged or necrotic cells during cell death and acts as a DAMP signal and a pro-inflammatory cytokine, and is associated with the pathogenesis of various inflammatory diseases. It is also known that several pro-inflammatory cytokines such as IL-1β, IFN-γ, and TNF-α are responsible for inducing β-cell dysfunction and apoptosis through a series of intracellular signaling pathways including ROS formation, JNK activation, and NF-κB translocation. NF-κB is a transcription factor that controls NADPH oxidase, leading to the generation of oxidative stress. The synergistic action of pro-inflammatory cytokines induces the expression of inducible nitric oxide synthase (iNOS) and the blockade of iNOS activity, or the enhancement of manganese superoxide dismutase (MnSOD) expression in insulin-producing cell lines under in vitro conditions attenuates cytokine-induced NF-κB activation.

[0030] Due to safety regulations, to avoid the use of animal products, isolated human islets are typically cultured under serum deprivation culture conditions prior to clinical allogeneic intraportal islet transplantation. During islet culture under serum deprivation conditions, the islets are further exposed to diverse stresses in the culture medium, including deprivation of nutrients, various pro-inflammatory cytokines, enzymes from the exocrine pancreas, and deprivation of sufficient oxygenation, and these stresses are harmful to β-cells, inducing the accumulation of excessive ROS. In addition, it is believed that the toxicity of islet amyloid polypeptide (IAPP) is the cause of the progressive decrease in the amount of β-cells and islet fibrosis in type 2 diabetes, and serum deprivation culture under normoglycemic conditions evokes the accumulation of toxic hIAPP oligomers. Inhibition of the IL-1 / IL-1 receptor axis can attenuate the inflammatory response and functional damage of islets caused by serum deprivation culture, but IL-1 blockade cannot attenuate the accumulation of toxic oligomers during serum deprivation culture.

[0031] When processed while isolating the islets, the compound of Formula 1 according to the present invention can protect the islets by reducing oxidative stress and the amount of reactive oxygen species (ROS).

[0032] Furthermore, when processed during serum deprivation culture of isolated islets, the compound of Formula 1 according to the present invention reduces the expression of c-jun N-terminal kinase, HMGB1 (high mobility group protein-1), and pro-inflammatory cytokines (such as interleukin-1β, interleukin-6, and tumor necrosis factor (TNF)-α) due to its strong mitochondrial reactive oxygen species (ROS) scavenging activity, and reduces the accumulation of amyloid and toxic IAPP (islet amyloid polypeptide) oligomers.

[0033] When processed during islet isolation or serum deprivation culture of isolated islets, the compound of Formula 1 according to the present invention effectively protects the islets through the mechanism of action described above, thereby significantly increasing the diabetes reversal rate after islet transplantation.

[0034] In another embodiment according to the present invention, if desired, the composition of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, the term "carrier" refers to a material that facilitates the administration of a compound in a cell or tissue, and there is no particular limitation thereto.

[0035] Advantageous Effects

[0036] The composition of the present invention can significantly increase the success rate of islet transplantation by providing excellent protection to islet cells. Brief Description of the Drawings

[0037] Figure 1 Shows compound 1 on isolated hIAPP during isolation + / -Evaluation results of the effects on the in vitro survival rate, function, and pro-inflammatory cytokine expression of mouse islets.

[0038] Figure 2 The evaluation results showing the effects of Compound 1 on the in vitro survival rate, function, and pro-inflammatory cytokine expression of isolated C57BL / 6 mouse islets during isolation are presented.

[0039] Figure 3 The evaluation results showing the effects of Compound 1 on hIAPP + / - during transplantation in vivo of mouse islets are presented.

[0040] Figure 4 The evaluation results showing the effects of Compound 1 on the cell viability of RINm5F cells and hIAPP + / - mouse islets and the ROS therein during serum deprivation culture are presented.

[0041] Figure 5 The evaluation results showing the effects of Compound 1 on the cell viability of non-human primate (NHP) islets and the ROS therein during serum deprivation culture are presented.

[0042] Figure 6 The evaluation results showing the effects of Compound 1 on isolated hIAPP + / - during in vitro survival rate, function, pro-inflammatory cytokine expression, and oligomer accumulation of mouse islets are presented.

[0043] Figure 7 The evaluation results showing the effects of Compound 1 on hIAPP + / - during transplantation in vivo of mouse islets are presented. Detailed Description of the Invention

[0044] Hereinafter, the present invention will be described in more detail by way of Preparation Examples and Examples. However, these Examples are merely illustrative, and the scope of the present invention is not limited thereto.

[0045] I. Experimental Materials and Methods

[0046] 1. Compounds

[0047] (Tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxido-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine (hereinafter referred to as "Compound 1") was prepared according to the method described in Example 36 of International Publication No. WO2009 / 025478A1.

[0048] 2. Experimental animals and cell lines

[0049] Islets were isolated from heterozygous human islet amyloid polypeptide transgenic (hIAPP + / - ) FVB / N mice (Jackson Laboratory, Bar Harbor, ME, USA), C58BL / 6 mice (Orientbio, Sungnam, Korea), and cynomolgus monkeys (Macaca fascicularis; Orientbio, Sungnam, Korea). For additional in vitro studies, rat insulinoma cells (RINm5F cells) were treated with or without compound 1 during culture under tert-butyl hydroperoxide (tBHP) exposure or serum deprivation 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 fully humidified 5% CO2 atmosphere. All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Samsung Biomedical Research Institute.

[0050] 3. Islet isolation

[0051] Mouse islets were isolated from 10- to 12-week-old hIAPP + / - and C57BL / 6 mice as previously described. Briefly, 0.8 mg / mL collagenase P (Roche, Indianapolis, IN, USA) in Hank's balanced salt solution (HBSS, Sigma-Aldrich, St. Louis, MO, USA) with or without 20 μM compound 1 was injected into the common bile duct for mouse pancreas digestion. Islets were purified from the digested pancreas using a Ficoll (Biochrom, Berlin, Germany) gradient and washed several times with 1×HBSS. The isolated islets were cultured floating freely in 10 mL RPMI 1640 (Gibco, Grand Island, NY, USA). Before in vitro and in vivo studies, the medium was supplemented with 10% heat-inactivated FBS (fetal bovine serum) and incubated at 37 °C and 5% CO2.

[0052] 4. Islet isolation from non - human primates

[0053] The dissected non-human primate (NHP) pancreas was injected intraductally with cold Liberase MTF C / T solution (4 mL / g pancreas; Roche). Digestion and isolation were performed using the Ricordi automated isolation technique as previously described. Before in vitro and in vivo studies, all isolated NHP islets were cultured in CMRL medium supplemented with 10% heat-inactivated porcine serum at 37 °C and 5% CO2 incubation.

[0054] 5. Serum - deprivation ex vivo culture of isolated islets

[0055] Purified hIAPP + / - FVB / N and C57BL / 6J mouse islets were suspended in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 100 IU / mL penicillin and 100 μg / mL streptomycin and cultured at 37 °C in a fully humidified 5% CO2 atmosphere. Purified NHP islets were suspended in CMRL1066 medium (Corning Life Sciences, Tewksbury, MA; catalog number 99-663-CV) containing 100 IU / mL penicillin and 100 μg / mL streptomycin and cultured at 37 °C in a fully humidified 5% CO2 atmosphere. During the ex vivo culture of murine islets, for the use of hIAPP + / - Three experimental groups were designated for experiments with FVB / N and C57BL / 6J mouse islets: medium supplemented with 10% FBS (Tissue Culture Biologicals, Los Alamitos, CA, USA; catalog number 101; FBS group), medium supplemented with 0.625% bovine serum albumin (Qbiogene, Carlsbad, CA, USA; catalog number BSA003; BSA group), and medium supplemented with 0, 0.1, 1, 10, and 20 μM compound 1 plus 0.625% BSA (BSA + compound 1 group). In experiments using NHP islets, the medium was supplemented with 10% FBS (FBS group) and 0 - 20 μM compound 1 plus 0.625% human serum albumin (HSA; Greencross, Yongin, Korea) to determine the optimal concentration of compound 1 for treatment during serum deprivation culture. In addition, the medium was supplemented with 10% FBS (FBS group), 20 μM compound 1 plus 10% FBS (FBS + compound 1 group), 0.625% human serum albumin (HSA; Greencross, Yongin, Korea; HSA group), or 20 μM compound 1 plus 0.625% HSA (HSA + compound 1 group).

[0056] 6. Assessment of islet viability by Alamar Blue assay

[0057] The viability of islets was evaluated using Alamar blue staining according to the manufacturer's protocol (Invitrogen, Grand Island, NY, USA). Briefly, from hIAPP + / -Mouse isolated islets were cultured in 24-well plates at a density of 100 islet equivalents (IE) per well in RPMI 1640 containing 10% FBS and 0.625% BSA with or without Compound 1. After 1 day and 3 days, 10× Alamar Blue solution was added directly to each well, and the islets were incubated at 37 °C for 4 hours in the dark. 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 the values were normalized against the blank.

[0058] 7. Acridine Orange / Propidium Iodide (AO / PI) assay

[0059] The viability of islets after isolation and culture was evaluated using dual fluorescence in 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 using a fluorescence microscope (Nikon ECLIPSE 80i, Tokyo, Japan), and the areas of live and dead cells were quantified using NIS-Element AR 3.0 (Nikon). Islet viability (%) was calculated as live islet cells / total islet cells × 100.

[0060] 8. ATP assay

[0061] The ATP content of islets was determined by luminescence. Samples were mixed with 200 μL of a commercially available lyophilized ATP monitoring reagent containing firefly luciferase and luciferin (ATP Bioluminescence Assay Kit CLS II, Roche Diagnostics) reconstituted first in imidazole buffer (100 mM, pH 7.75). The emitted light was measured in a luminometer (LKB 1250 luminometer). The adenine nucleotide content in the samples was determined after correcting for the control (no islets or cells) and calculated by reference to an ATP standard treated in the same manner as the samples.

[0062] 9. Fluorescence - based detection of intracellular free radicals and lipid peroxidation

[0063] hIAPP was determined immediately after islet isolation and after ex vivo culture of isolated islets + / -Oxidative stress levels in mouse islets. Oxidative stress in islets was quantified using a fluorescence-based intracellular ROS assay and by estimating the concentration of malondialdehyde (MDA), a byproduct of lipid peroxidation. The formation of total ROS, superoxide ions, and nitric oxide was determined using a reactive oxygen species detection kit (ENZ-51011, Enzo Life Sciences, Farmingdale, New York, USA). The ROS content was measured by the level of DHR123 fluorescence. The kit was used according to the manufacturer's protocol.

[0064] 10. Glucose - stimulated insulin secretion (GSIS) assay

[0065] After manual picking and washing with PBS, islets were seeded at a density of 10 islets per well in 12-mm diameter insert wells (Merck Millipore, Billerica, MA, USA) and incubated with 60 mg / dL glucose in Krebs-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) at 37 °C for 90 min. After washing, islets were incubated with 300 mg / dL glucose-KRB for 1 h and then continued to be incubated with 60 mg / dL glucose-KRB for 1 h. Insulin released from mouse islets into the supernatant was measured by ELISA (ALPCO, Salem, NH, USA) and a multiplex kit (Merck Millipore), respectively.

[0066] 11. RNA isolation and cDNA synthesis

[0067] The harvested islets were treated with 500 μL of Trizol (Life Technologies, Grand Island, NY, USA), and then 100 μL of chloroform was added. After incubation at 4 °C for 5 min, the mixture was centrifuged at 12,000 rpm for 15 min, and 250 μL of isopropanol was added to precipitate total RNA. The RNA pellet was washed with 75% ethanol, and the RNA was eluted from the pellet with RNase-free water (WelGene, Daegu, South Korea). RNA purity was evaluated by NanoDrop (Thermo Scientific, Wilmington, DE, USA) and agarose gel electrophoresis. The purity of the RNA was in the range of 1.9 to 2.0 as measured by the ratio of optical density (OD) at 260 / 230 and 260 / 280. Using SuperScript TMThe II reverse transcription system (Life Technologies) was used to reverse transcribe total RNA according to the manufacturer's protocol to quantify gene expression.

[0068] 12. Real - time quantitative reverse transcription (qRT) - PCR

[0069] Real-time qRT-PCR was performed using gene-specific primer pairs (Table 1). The amplified PCR products were normalized to the β-actin PCR products amplified from the same sample. The PCR products were separated on a 1% agarose gel, and images were obtained using a GelDocTM XR instrument (Bio-Rad, Hercules, CA, USA). To quantify the gene expression levels, 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.

[0070] [Table 1]

[0071]

[0072] IL-1β: Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α;

[0073] HMGB1: High mobility group box 1

[0074] 13.hIAPP + / - In vivo islet function of pancreatic islets

[0075] The marginal mass kidney subcapsular islet transplantation model was used to evaluate the in vivo islet function of hIAPP + / - in mice. To induce diabetes, 180 mg / kg streptozotocin (STZ, Sigma-Aldrich) was administered to 8- to 10-week-old hIAPP - / - FVB / N mice. Mice were considered diabetic when two consecutive blood glucose level readings were higher than 300 mg / dL. Equal amounts (400 IEQ per recipient) of islets isolated from hIAPP + / - mice were allotted to the following two groups: medium supplemented with 0.625% BSA (BSA group) and 0.625% BSA plus compound 1 (BSA + compound 1 group) to evaluate the combined effects of compound 1 on islet mass and function during in vitro culture. After 72 hours of culture, the islets were transplanted into diabetic hIAPP - / -In the subcapsular space of the mouse kidney. Using the Ricordi algorithm, the number of IEs was calculated by classifying islets according to diameter. After islet transplantation, non-fasting blood glucose levels were measured three times a week for the first 2 weeks and twice a week for the last 2 weeks.

[0076] 14. Histological analysis

[0077] Four weeks after transplantation, the transplantation site on the mouse kidney was removed and embedded in paraffin. Immunohistochemical staining is briefly explained below. After deparaffinization, 4-μm tissue sections were stained with polyclonal guinea pig anti-insulin (1:1000, A0546, DAKO, Denmark), glucagon (1:500, ab92517, abcam, United Kingdom), and amyloid (1:2500, #44–344, Invitrogen) antibodies. The stained slides were observed using an Olympus BX40 optical microscope (Olympus, Japan) with objectives of ×10 / 22 numerical aperture and ×40 / 0.75 numerical aperture. Photographic images were acquired using a digital camera (Olympus DP50) and analyzed using Image-Pro Plus 5.1 software.

[0078] For the quantification of DAB-labeled β-cells, all slides were imaged at 10× on a Vectra 3.0 automated quantitative pathology imaging system and analyzed using inForm software (both from Perkin-Elmer, Waltham, MA, USA). The transplanted kidney tissue sections were scanned to obtain images, and then, to set the transplantation tissue segmentation, a large number of representative regions of the transplanted islet cells and regions to be excluded from the analysis, such as kidney tissue, were examined through the instance learning interface. For β-cell segmentation, a spectral library was used to identify hematoxylin-stained cell nuclei and DAB-stained cytoplasm (β-cells) within the transplanted tissue. Using these training sets, an algorithm for identifying only DAB-stained cells in the transplanted tissue was verified. All captured 10× images were analyzed, and the DAB-stained transplanted β-cells were counted.

[0079] 15. Assessment of hIAPP oligomer accumulation

[0080] Accumulation of hIAPP oligomers was determined using hIAPP stained with A11 Ab (anti-oligomer antibody, Invitrogen) and insulin antibody (Dako) + / -Evaluated using mouse pancreatic islet sections. The islets were blocked with 10% normal donkey serum and incubated with rabbit anti-oligomer primary antibody (1:100). The islets were washed and incubated with Cy3-conjugated anti-rabbit secondary antibody for 1 hour. After washing, the sections were stained with guinea pig anti-insulin primary antibody (1:500; Dako), and then with Alexa Fluor 488 anti-guinea pig secondary antibody (1:200; Jackson ImmunoResearch Laboratories). After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI), the sections were mounted. To quantify the accumulation of hIAPP oligomers in each islet, the proportion of A11-stained cells among total DAPI+ islet cells was determined using confocal microscopy.

[0081] 16. Assessment of amyloid accumulation

[0082] Sections of islet grafts under the renal capsule were examined for islet amyloid after staining with 0.5% thioflavin S (Sigma), and insulin immunostaining was performed using guinea pig anti-insulin antibody (1:500; Dako, Carpenteria, CA) followed by Cy3-conjugated anti-guinea pig secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) to examine β-cells. The sections were mounted after DAPI staining. To quantify amyloid accumulation in each section, the percentage of thioflavin S-stained cells (green) among the total islet graft area (green + red + blue parts) was determined using confocal microscopy. The original magnification of the figure was 100×.

[0083] 17. Statistical analysis

[0084] Where appropriate, the results were expressed as mean ± standard deviation (SD) or median and interquartile range. Where appropriate, continuous variables were compared using Student's t-test, one-way analysis of variance (ANOVA), or Mann–Whitney U test. Longitudinal data were analyzed using Graphpad Prism 5 (GraphPad Software, La Jolla, CA, USA) by two-way ANOVA and Bonferroni post hoc test. A p-value < 0.05 was accepted as statistically significant for comparison.

[0085] II. Results

[0086] 1. Protective effects against oxidative stress and pro - inflammatory responses during islet isolation

[0087] To evaluate the effect of compound 1 during islet isolation, hIAPP was isolated by collagenase with and without supplementation of 20 μM compound 1+ / - Islets isolated from FVB / N mice. The relative cell viability evaluated by the Alamar Blue assay was significantly increased in the compound 1-treated group compared with the untreated group ( Figure 1 A). In addition, significantly elevated ATP levels were presented during islet isolation in the compound 1-treated group compared with the compound 1-untreated group ( Figure 1 B). In addition, the ROS content and oxidative stress levels were significantly reduced in the islets treated with compound 1 compared with the control islets not treated with compound 1 ( Figure 1 C and 1D). The stimulation index presenting in vitro islet function obtained in the glucose-stimulated insulin secretion (GSIS) assay showed comparable values between the two groups ( Figure 1 E). Next, we also examined the transcriptional expression of c-Jun N-terminal kinase and high-mobility group box protein-1 (HMGB1) and pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the medium after isolation with or without supplementation of compound 1. These transcriptional expression levels were significantly reduced in the compound 1-treated group compared with the untreated group ( Figure 1 F). These results were replicated in islets from wild-type C57BL / 6 mice ( Figure 2 ).

[0088] 2. Effects on improving post - transplantation outcomes

[0089] To examine the in vivo effect of supplementing compound 1 during islet isolation, marginal pieces of islets isolated with or without supplementation of compound 1 were transplanted into the subrenal capsular space of streptozotocin (STZ)-induced diabetic hIAPP - / - FVB / N mice ( Figure 3 A). Compared with the untreated group, the glucose levels were significantly lower after transplantation in the compound 1-treated group, and the removal of the transplanted kidney in the compound 1-treated group confirmed the re-acquisition of hyperglycemia ( Figure 3 B). Compared with the control group, the compound 1 group showed better post-transplantation results in terms of the diabetes reversal rate and glucose levels during the intraperitoneal glucose tolerance test (IPGTT) ( Figure 3 C, 3D, and 3E). Compared with the control group, the compound 1 group presented a larger proportion of insulin-positive areas in the transplanted tissue ( Figure 3 F).

[0090] 3. Protection of hIAPP transgenic mice and NHP islets during serum deprivation culture + / - ​

[0091] We examined the effect of supplementation with compound 1 on tert-butyl hydroperoxide (tBHP) exposure or islet culture under serum deprivation conditions. Although RINm5F cells exposed to tBHP showed LDH release and ROS formation, cytotoxicity and ROS levels were significantly reduced after supplementation with compound 1 ( Figure 4 A and 4B). The relative cell viability of rat insulinoma cells (RINm5F cells) and islets from hIAPP + / - FVB / N mice was significantly improved in a dose-dependent manner from 0.1 μM to 20 μM against serum deprivation-induced cytotoxicity ( Figure 4 C and 4D). In RINm5F cells and islets from hIAPP + / - FVB / N mice, supplementation of the culture medium with compound 1 under serum deprivation culture conditions also reduced serum deprivation-induced ROS in a dose-dependent manner from 0.1 μM to 20 μM ( Figure 4 E and 4F). Similar results were observed in NHP islets ( Figure 5 A, 5B and 5C).

[0092] Figure 6 Figure A shows the cell viability of hIAPP + / - FVB / N mouse islets during serum deprivation culture with or without supplementation of compound 1 evaluated by AO / PI staining. Compared with FBS-treated islets, islets cultured with BSA for 72 h experienced more cell death, showing that the positive rate of red PI-positive cells and impaired cell viability were significantly improved by supplementation with compound 1 ( Figure 6 A and 6B). Compared with the BSA group, the transcript levels of TNF-α, IL-1β, IL-6, c-JUN and HMGB1 were significantly attenuated 1 day and 3 days after ex vivo culture in the BSA + compound 1 group ( Figure 6 C and 6D). To evaluate the function of islets, in islets from hIAPP + / - mice, a significantly increased stimulation index was observed when compound 1 was added to the culture medium under serum deprivation culture conditions ( Figure 6 E). After culturing for 72 h in media with and without compound 1 under serum deprivation culture conditions, compared with the BSA group, the accumulation ratio of hIAPP + / - oligomers in hIAPP islets was significantly reduced in the BSA + compound 1 group ( Figure 6 F).

[0093] 4. Improvement of post - transplantation blood glucose outcomes and amyloid accumulation in transplanted islets

[0094] We investigated whether supplementation of the culture medium with compound 1 during serum deprivation culture improved post-transplantation outcomes in an in vivo mouse model. Islets from hIAPP+ / - Edge pieces of islets from FVB / N mice were cultured for 72 hours with and without compound 1 supplementation and transplanted into STZ-induced diabetic hIAPP - / - under the renal subcapsule of FVB / N mice ( Figure 7 A). The compound 1 group showed significantly better post-transplantation outcomes, including post-transplant glucose levels, diabetes reversal rate, and glucose levels and glucose AUC during IPGTT ( Figure 7 B, 7C, 7D, and 7E). In the compound 1 group, the proportion of insulin-positive area in the transplanted tissue was greater than that in the control group ( Figure 7 F). In addition, compared with the control group, the proportion of amyloid accumulation in islet graft sections from the compound 1 group was significantly reduced ( Figure 7 G).

[0095] III. Discussion

[0096] Supplementation with compound 1 during islet isolation led to an increase in in vitro islet cell viability and attenuation of oxidative stress, reactive oxygen species (ROS) content, and the expression of c-Jun, HMGB1, and pro-inflammatory cytokines. In addition, in vivo studies confirmed improved post-transplantation outcomes by treating with compound 1 during islet isolation.

[0097] In addition, compound 1 provided protection against serum deprivation-induced impairment of in vitro islet viability. More importantly, supplementation of the culture medium with compound 1 during serum deprivation culture significantly reduced the accumulation of amyloid and toxic IAPP oligomers and improved in vivo islet graft function.

[0098] From these results, it can be confirmed that supplementation with compound 1 during islet isolation and serum deprivation culture during islet transplantation has beneficial effects.

Claims

1. Use of (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxido-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine of formula 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for increasing the cell viability of islets during islet isolation or during serum-deprived culture of isolated islets, wherein the islets are treated with the compound of formula 2 during islet isolation or during serum-deprived culture of isolated islets: [Formula 2] 2. The use according to claim 1, wherein the compound of formula 2 reduces the accumulation of amyloid and toxic IAPP (islet amyloid polypeptide) oligomers.

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