Treatment method for allograft rejection reaction

Abstract

The present disclosure generally relates to methods of inhibiting immune responses involved in transplant rejection, such as immune responses and allograft rejection. In particular, the present invention relates to the use of certain enzyme inhibitors, such as heparanase inhibitors, that can be used to treat transplant rejection and / or extend the survival of transplanted tissues or organs, particularly allografted tissues or organs.

Description

Technical Field

[0001]

[0001] The present disclosure generally relates to methods of inhibiting immune responses involved in transplant rejection responses such as immune responses and allograft rejection responses. In particular, the present invention relates to the use of specific enzyme inhibitors for treating allograft rejection responses and / or prolonging the survival of allografted tissues or organs.

Background Art

[0002]

[0002] Any reference to prior art methods, devices, or documents should not be construed as constituting evidence or admission that they form part of, or form, common general knowledge.

[0003]

[0003] A preferred treatment for end-stage organ failure is organ transplantation. Transplantation of various organs such as kidneys, livers, hearts, lungs, and cardiopulmonary organs is a potential treatment option for patients presenting with organ failure. However, organ dysfunction due to recipient compatibility, donor shortage, and immune rejection responses all pose significant constraints to long-term success.

[0004]

[0004] Despite significant progress in the understanding of transplantation immunology, organ rejection remains the most significant obstacle to the success of short - and long - term organ transplantation. The following are important facts regarding the immune response in allograft rejection. First, recipient macrophages recognize foreign antigens in the allograft. Macrophages release cytokines and chemokines, which activate vascular endothelial cells and T cells, thereby promoting the adhesion of alloreactive cells and their invasion through the vascular basement membrane of the allograft. T cells proliferate and activate B cells, which are responsible for the production of alloantibodies. Both T cells and alloantibodies recognize foreignness conferred by antigens derived from the recipient's major histocompatibility complex (MHC), and both damage the allograft. When T / B cell lines are controlled by immunosuppressive agents, there are other types of immune cells (e.g., eosinophils, macrophages, mast cells, etc.) that can damage the allograft. Thus, the primary immune rejection reaction is adaptive and specific, mobilizing T cells and B cells, while the secondary immune rejection reaction uses macrophages and other similar cell types that are present innately. This damage is an allograft rejection reaction that can be acute or chronic, or both.

[0005]

[0005] Typically, a transplantation immunosuppressive agent (or immunosuppressant) is a drug that reduces the recipient's ability to reject a graft, as defined by its ability to extend the survival period of an allograft (compared to an untreated control group) and the immunological mechanisms that promote the reduction of allograft rejection.

[0006] [

[0006] ]The immune response involved in allograft rejection, particularly organ allograft rejection, is known to be a very special immune response different from other immune responses, such as autoimmune responses (including immune responses related to insulin-dependent diabetes) and immune responses caused by ischemia-reperfusion injury. Autoimmune rejection differs from allograft rejection in the following aspects: Autoimmune rejection is specific to autoantigens (recognized as self) and not specific to allogeneic antigens recognized as non-self. Autoimmune rejection may disappear naturally (e.g., polymyalgia rheumatica, rheumatoid arthritis), while allograft rejection does not disappear naturally. Autoimmune rejection tends to be weaker than allograft rejection that can mobilize other cell types when the T cell-mediated rejection is controlled by immunosuppressants. Furthermore, the mechanisms of autoimmune rejection and allograft rejection are different. Allograft rejection is a rejection reaction against foreign (non-self) antigens introduced in allotransplantation, while autoimmune rejection results from the breakdown of the autoimmune regulatory mechanism configured to accept autoantigens. Therefore, the assumption that autoimmune rejection is equivalent to allograft rejection is incorrect (Patent Document 1)(1).

[0007] [

[0007] ]Reperfusion injury, also known as ischemia-reperfusion injury (IRI) or reoxygenation injury, is tissue damage caused when blood supply to tissues is restored after a period of ischemia or oxygen deficiency (anoxia or hypoxia). In the context of transplantation, ischemia-reperfusion injury (IRI) differs from allograft rejection in the following aspects: IRI is an injury to the organ (or tissue) caused by oxygen deficiency, insufficient blood supply, reperfusion, and resulting toxic products (reactive oxygen species) when the organ (or tissue) is removed from the donor, transplanted into the recipient, and reperfused. In contrast, allograft rejection is a host reaction against foreign antigens in allotransplantation. Furthermore, the recipient's reaction to IRI mediated by innate immune cells (neutrophils, eosinophils, macrophages) can regress without immunosuppression, while allograft rejection is usually mediated by T cells and antibodies and can never regress without immunosuppression. Therefore, the assumption that IRI is equivalent to allograft rejection is also incorrect.

[0008]

[0008] The heparanase inhibitor, roneparstat, has been shown to suppress ischemia-reperfusion injury (Non-Patent Document 1)(2). However, the reduction of IRI by heparanase inhibitors (Non-Patent Documents 1 to 3)(2, 3, 4) should not be confused with the treatment of allograft rejection.

[0009]

[0009] In Patent Document 2 (The Australian National University, September 15, 2011)(5), the authors claim that the survival period of allogeneic pancreatic islets in mice is extended by the heparan sulfate mimetic, PI88 (Examples 7 and Figure 15). It is stated that this mimetic acts by restoring the heparan sulfate content of islets with destructive insulitis, compared with control mice treated with saline, which shows a significant decrease in islet heparan sulfate in the presence of destructive insulitis. However, there is no control group for IRI consisting of mice with syngeneic transplanted pancreatic islets treated with the same heparanase inhibitor (PI88). Without this control, the improvement in the survival rate of allogeneic pancreatic islets is more consistent with a control for IRI rather than a control for allograft rejection. In this regard, the disclosure of Patent Document 2 assumes that heparan sulfate (or a heparan sulfate mimetic) inhibits oxidative damage, which is characteristic of IRI.

[0010]

[0010] In Patent Document 3 (The Australian National University, April 24, 2008) (6), the authors claim that the heparan sulfate mimetic PI88 inhibits the rejection of allogeneic pancreatic islets in mice by preventing the degradation of heparan sulfate in the pancreatic islet BM / ECM (basement membrane / extracellular matrix) by heparanase (Examples 8 and Figure 10). Therefore, this study is related to the preservation of transplanted allogeneic pancreatic islet cells and not to the long-term survival of allogeneic pancreatic islets. Also, similar to the disclosure of Patent Document 2, there is no control for IRI in Patent Document 3. Therefore, the disclosure in Patent Document 3 that there is less destruction of allogeneic pancreatic islets may be due to the effect of the heparan sulfate mimetic only on IRI (as mentioned later in Patent Document 2). Furthermore, although Patent Document 3 discloses a long list of heparanase inhibitors, there is nothing in Patent Document 3 to support the use of these structurally different inhibitors in the treatment of allograft rejection. Importantly, allogeneic pancreatic islets are not primarily angiogenic grafts but secondarily angiogenic grafts. Secondarily angiogenic grafts are cell grafts without vascular anastomosis, but are usually placed into or under the capsule of another organ using a catheter inserted into the recipient's vein. In contrast, primarily angiogenic grafts are organs that are connected to the recipient's blood supply by surgical arterial and venous anastomoses (connections) to obtain immediate blood supply. Therefore, considering the high risk of immediate antibody-mediated rejection in primarily angiogenic grafts, there is no basis to conclude that factors effective for secondarily angiogenic grafts are necessarily effective for primarily angiogenic grafts.

[0011]

[0011] Heparanase is a complex multifunctional enzyme. It mediates, for example, proliferative diseases, autoimmune diseases, psoriasis, macular degeneration, and diabetes. Heparanase is highly expressed in various cancers, and its increased expression is associated with metastasis and increased tumor size, so it has been mainly considered as a promising target for cancer treatment for nearly 20 years (Non-Patent Document 4) (7).

[0012]

[0012] Recent studies have shown that heparanase is involved not only in cancer but also in various pathological conditions such as diabetes, osteonecrosis, liver fibrosis, amyloidosis, Alzheimer's disease, etc., and in the infection and spread of a number of viruses (7).

[0013]

[0013] Heparanase has been shown to stimulate the release of pro-inflammatory cytokines [interleukins IL-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α] from peripheral blood mononuclear cells, but its role in transplant rejection is, at present, unclear, if any.

[0014]

[0014] In previous studies on secondarily angiogenic grafts using heparin derivatives, the role of heparanase inhibitors has not been established (Non-Patent Documents 5, 6) (8, 9). This is due to the fact that none of the agents were specific inhibitors of heparanase, the absence of a control for IRI, and the fact that the prolongation could be explained by other mechanisms. For example, heparin can act by inhibiting tumor necrosis factor α (Non-Patent Document 7) (10) and can act as an anticoagulant. In Non-Patent Document 5 (8) and Non-Patent Document 6 (9), since there was no control for IRI in the mouse skin allograft model, the prolongation of the survival period of the transplanted skin may have been affected by the suppression of IRI. Furthermore, secondarily angiogenic allogeneic skin grafts exhibit different behavior from primarily angiogenic grafts (these differences are described above). These data do not establish that heparanase inhibitors control the rejection of allogeneic skin grafts, nor do they establish by inference that they control the rejection of primarily angiogenic grafts.

[0015]

[0015] Despite the availability of clinical immunosuppressive therapies for transplant rejection, there remains a need for more effective treatments for transplant rejection. For example, there is still a need for treatments with low toxicity and the ability to provide long-term immunosuppression after transplantation.

[0016]

[0016] The objective of the present invention is to overcome or improve at least one of the drawbacks of the prior art, or to provide a useful alternative means.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0018]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0019]

[0017] Surprisingly, it has been found that specific inhibition of heparanase activity prolongs the survival period of allografts in the transplantation recipient. Also unexpectedly, inhibition of heparanase activity has been found to be useful as a means for preventing and treating transplant rejection and / or maintaining the integrity of allografts.

[0020]

[0018] Also unexpectedly, it has been discovered that systemic administration of a heparanase inhibitor reduces the expression level of heparanase in lymphocytes isolated from transplanted organs. Surprisingly, this decrease in heparanase expression level was not observed in lymphocytes isolated from the peripheral blood of animals that received a transplant administered with a heparanase inhibitor. Heparanase is known to be a complex multifunctional enzyme involved in many systems and processes in the body. Therefore, the results presented here suggest that treatment of transplant rejection with a heparanase inhibitor is unlikely to have a significant impact on a wide range of biological systems and processes in which heparanase may play a role, other than immune allograft rejection at the transplant site. That is, it can be said that treatment of transplant rejection by administration of a heparanase inhibitor is unlikely to be associated with, for example, serious side effects or toxicity.

[0021]

[0019] Thus, in one aspect, the present invention relates to a method for preventing or treating transplant rejection, said method comprising administering a heparanase inhibitor to a subject in need thereof, said heparanase inhibitor preventing or treating transplant rejection by inhibiting heparanase activity.

[0022]

[0020] In certain embodiments, the method of the invention comprises treating transplant rejection, said rejection being primarily the rejection of a vasculated organ transplant. Preferably, the method of the invention treats or prevents transplant rejection in, for example, but not limited to, heart, heart-lung, kidney, liver, pancreas, stomach or intestine transplants. Thus, the method of the invention excludes the treatment of grafts that secondarily become vascularized (such as islet beta cell transplants).

[0023]

[0021] In one embodiment, the method of the invention relates to the prevention or treatment of allograft rejection.

[0022] In certain embodiments, the heparanase inhibitor used in the method of the invention is, for example, an acid derivative of benzoxazole, benzothiazole or benzimidazole as described in WO 2004 / 046122, the content of which is incorporated by reference. In a further embodiment, the heparanase inhibitor is a benzoxazol-5-ylacetic acid derivative. In yet another embodiment, the heparanase inhibitor is of the formula:

[0024]

Chemical formula

[0025] OGT2115 having the following formula or a functional derivative or functional analog thereof.

[0023] OGT2115 is a benzoxazol-5-ylacetic acid derivative also known as 2-[4-[[3-(4-bromophenyl)-1-oxo-2-propenyl]amino]-3-fluorophenyl]-5-benzoxazoleacetic acid and is a cell-permeable heparanase inhibitor.

[0026] One of ordinary skill in the art will understand that OGT2115 is commercially available, for example, from MCE Med Chem Express and R&D Systems. Further production methods of heparanase inhibitors of acid derivatives of benzoxazole, benzothiazole and benzimidazole, including OGT2115, are described, for example, in International Publication No. WO 2004 / 046122, the content of which is incorporated by reference.

[0027]

[0025] One of ordinary skill in the art will appreciate that various heparanase inhibitors are suitable for use in the methods of the present invention. For example, quinazoline compounds as described in International Publication Nos. WO 2018 / 107200 and WO 2018 / 107201 (the contents of which are incorporated by reference). Such heparanase inhibitors include compounds of general formula A

[0028]

Chemical formula

[0029] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 3 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 4 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2Selected from phenyl, O-phenyl; or R 1 and R 2 or R 2 and R 3 or R 3 and R 4 together form C 1~3 alkylenedioxy; R 1 R 2 R 3 and R 4 at least one of which is not H, L 1 is NHC 1~4 alkyl-NHC(O)-, NHC 1~4 alkyl-NHSO 2 -, azetidinyl-NHC(O)-, and azetidinyl-NHSO 2 - selected from; R 5 is C 3~9 cycloalkyl, aryl optionally substituted with one or two RX groups, C 6~10 aryl, heteroaryl optionally substituted with one or two RX groups, C 2~9 heteroaryl, heterocycloalkyl optionally substituted with one or two RX groups, C 2~5 heterocycloalkyl, alkyl C 1~4 heterocycloalkyl selected from; L 2 is C 1~4 alkyl, azetidinyl-C(O)-, C 1~4 alkyl-NHC(O)-, C 1~4 alkyl-NHSO 2 -, -C(O)-, SO 2 - selected from, or L 2 does not exist; 6 R is H, C 2~6 alkyl, guanidinyl, NHC(NH)NH(C 1~3 alkyl), ureido, NHC(O)NH(C 1~3 alkyl), aryl optionally substituted with one or two RX groups, C 6~10 aryl, aryl optionally substituted with one or two RX groups, C 1~9 aryl, aryl optionally substituted with one or two RX groups, C1~9 heteroaryl, C optionally substituted with 1 or 2 RX groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 RX groups 3~9 selected from cycloalkyl; R 7 is H or C 1~6 alkyl; L 1 is NHC 1~4 alkyl-NHSO 2 - when R 5 is not phenyl substituted with 1 methyl group, tert-butyl group or phenyl group; each RX is, independently, hydroxyl, halo, nitro, NR’R” (R’ and R” are, independently, H and C 1~3 alkyl selected), C 1~4 alkyl, C 3~9 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C(O)C 1~3 alkyl, C(O)OC 1~4 alkyl, C(O)NHRY, C optionally substituted with 1 or 2 RY groups 6~10 aryl, C optionally substituted with 1 or 2 RY groups 2~9 heteroaryl, C 1~4 alkyl-(C 2~9 heteroaryl), C optionally substituted with 1 or 2 C 1~4 alkyl groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 C 1~4 alkyl groups 1~4 alkyl-(C 2~5 heterocycloalkyl), C optionally substituted with 1 or 2 C 1~4 alkyl groups to form C(O)-C 2~9 heteroaryl; 1 or 2 C 1~4 alkyl groups, or haloC 1~4 heteroaryl optionally substituted with alkyl groups; or two adjacent RX groups together form C 2~9 alkylenedioxy and are selected from those; 1~3 RY is H, hydroxyl, halo, C RYは、H、ヒドロキシル、ハロ、C1~4 Alkyl, halo C 1~4 Alkyl, C 1~4 Selected from alkoxy.

[0030]

[0026] The heparanase inhibitor is also a compound of general formula I

[0031]

Chemical formula

[0032] Or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein X is S or O; R 1 Is H, hydroxyl, halo, C 1~6 Alkyl, C 1~4 Alkoxy, O-CH 2 Selected from phenyl, O-phenyl; R 2 Is H, hydroxyl, halo, C 1~6 Alkyl, C 1~4 Alkoxy, O-CH 2 Selected from phenyl, O-phenyl; R 3 Is H, hydroxyl, halo, C 1~6 Alkyl, C 1~4 Alkoxy, O-CH 2 Selected from phenyl, O-phenyl; R 4 Is H, hydroxyl, halo, C 1~6 Alkyl, C 1~4 Alkoxy, O-CH 2 Selected from phenyl, O-phenyl; or Or R 1 And R 2 Or R 2 And R 3 Or R 3 And R 4 Together form C 1~3 Alkylenedioxy; R 5 Is H, C 1~6 Alkyl, C 1~3 Alkyl C(O)OC1~4 Alkyl and C 1~3 Alkyl C 6~10 Selected from aryl, optionally and independently halo C 1~3 Alkyl and halo C 1~3 May be substituted with one or two groups selected from alkoxy; L is C 1~6 Alkyl, azetidinyl, C 1~6 Alkyl - indolyl, NH, C 1~6 Alkyl - NI - IC(O)O, azetidinyl - C(O)-, C 1~6 Alkyl - NHC(O)-indolyl, C 1~6 Alkyl - NHSO 2 Selected from - or does not exist; R 6 Is H, halo, hydroxyl, d. 6 Alkyl, C 1~6 Alkenyl, C 1~6 Alkynyl, one or two R x Groups optionally substituted C 6~10 Aryl, one or two R x Groups optionally substituted C 1~9 Heteroaryl, one or two R x Groups optionally substituted C 2~5 Heterocycloalkyl, one or two R x Groups optionally substituted C(O)-(heterocycloalkyl), one or two R x Groups optionally substituted C(O)(C 2~5 Heterocycloalkyl), C(O)NHR Y Selected from - or does not exist; Each R x Is independently hydroxyl, halo, nitro, NR’R”, C 1~4 Alkyl, C 3~6 Cycloalkyl, halo C 1~4 Alkyl, C 1~4 Alkoxy, one or two R Y Groups optionally substituted C 6~10 Aryl, C 1~9 Heteroaryl, C 1~4 Alkyl-(C 1~9 Heteroaryl), C(O)OC 1~4Alkyl, C(O)NHR Y , 1 or 2 C 1~4 heterocycloalkyl optionally substituted with C 2~5 alkyl groups, 1 or 2 C 1~4 alkyl groups, C(O)-(heterocycloalkyl) optionally substituted with C x or two adjacent R 1~3 groups together form C R Y is selected from H, hydroxyl, halo, C 1~4 alkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C 1~4 alkylheterocycloalkyl, C(O)-(C 1~4 alkylheterocycloalkyl), C 1~4 alkylNR’R”;; R’ and R” are independently selected from H, C 1~4 alkyl, C 1~4 alkylheterocycloalkyl; R 7 is H, C 1~4 alkyl and C 1~6 alkylC 1~9 heteroaryl.

[0033]

[0027] The heparanase inhibitor also includes a compound of general formula II

[0034]

Chemical formula

[0035] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein X is S or O; R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl, and is selected from; R 3 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl, and is selected from; R 4 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl, or is selected from; or R 1 and R 2 or R 2 and R 3 or R 3 and R 4 together form C 1~3 alkylenedioxy; R 5’ is H, C 1~6 alkyl, C 1~3 alkyl C Λ OC 1~4 alkyl and C 1~3 alkyl C 6~10 aryl, optionally independently substituted with one or two groups selected from halo C 1~3 alkyl and halo C 1~3 alkoxy; L is C 1~6 alkyl, azetidinyl, C 1~6 alkyl-indolyl, NH, C 1~6 alkyl-NI-IC(O)O, azetidinyl-C(O)-, C 1~6 alkyl-NHC(O)-indolyl, C 1~6 alkyl-NHSO 2 - and is selected from, or is absent; R 6 is H, halo, hydroxyl, C 1~6 alkyl, C 1~6 alkenyl, C 1~6 alkynyl, one or two R x groups and is optionally substituted with C6~10 Aryl, 1 or 2 Rs x O optionally substituted with a group 1~9 Heteroaryl, 1 or 2 Rs x C optionally substituted with a group 2~5 Heterocycloalkyl, 1 or 2 Rs x C(O)-(heterocycloalkyl) optionally substituted with a group, 1 or 2 Rs x C(O)(C optionally substituted with a group 2~5 heterocycloalkyl), C(O)NHR Y is selected from or absent; Each R x is independently hydroxyl, halo, nitro, NR’R”, C 1~4 alkyl, C 3~6 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, 1 or 2 Rs Y C optionally substituted with a group 6~10 aryl, C 1~9 heteroaryl, C 1~4 alkyl-id-heteroaryl), 0(0)00 1~4 alkyl, C(O)NHR Y , 1 or 2 Cs 1~4 C optionally substituted with an alkyl group 2~5 heterocycloalkyl, 1 or 2 Cs 1~4 C(O)-(heterocycloalkyl) optionally substituted with an alkyl group, or two adjacent Rs x groups together form C 1~3 alkylenedioxy and is selected from those forming; R Y is H, hydroxyl, halo, C 1~4 alkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C 1~4 alkylheterocycloalkyl, C(O)-(C 1~4 alkylheterocycloalkyl), C 1~4 alkylNR’R” and is selected from; R’ and R” are independently H, C 1~4 alkyl, C 1~4selected from alkylheterocycloalkyl; R 7 is H, C 1~4 alkyl and C 1~6 alkylC 1~9 selected from heteroaryl.

[0036]

[0028] The heparanase inhibitor is also a compound of general formula III

[0037]

Chemical formula

[0038] wherein the group R 1 、R 2 、R 3 、and R 4 are as defined in formulas (I) and (II); R A 、R B 、R C and R D are independently H, OH, C 1~3 alkyl, OO 1~3 alkyl, C(O)-(N - heterocycloalkyl) (e.g., C(O) morpholinyl), C(O) piperazinyl optionally substituted with 1 or 2 C 1~3 alkyl groups); OH, halo, C 1~3 alkyl, OC 1~3 alkyl - substituted N - heteroaryl (e.g., 3 - pyridyl, 4 - pyridyl, 2,1,3 - benzoxadiazolyl, pyrazolyl) optionally substituted with 1 or 2 groups selected from; OH, halo, C 1~3 alkyl, OC 1~3 alkyl - substituted phenyl, CiOJNHC 1~3 alkylKNiC 1~3 alkyl^], C(O)(heterocycloalkyl) (e.g., morpholinyl, piperazinyl, piperidinyl) optionally substituted with 1 or 2 C 1~3 alkyl groups; R E is H, C 1~3It is alkyl or C(O)-heterocycloalkyl (e.g., C(O)-(N-morpholino)).

[0039]

[0029] One skilled in the art will be able to easily understand the method for producing the heparanase inhibitors of Formula A, Formula I, Formula II and Formula III from the disclosures of International Publication No. 2018 / 107200 and International Publication No. 2018 / 107201 incorporated by reference.

[0040]

[0030] In another embodiment, the present invention relates to the use of monoclonal or polyclonal antibodies that target heparanase in allografts during the induction of treatment or during rejection or chronic allograft rejection.

[0041]

[0031] In a further embodiment, the present invention relates to a method for preventing or treating allograft rejection, said method comprising the step of administering a heparanase inhibitor to a subject in need thereof, said heparanase inhibitor reducing the level of heparanase activity, thereby preventing or treating transplant rejection.

[0042]

[0032] In a further embodiment, the present invention relates to a method for preventing or treating transplant rejection, said method comprising the step of administering a heparanase inhibitor to a subject in need thereof, said heparanase inhibitor reducing the expression level of heparanase in lymphocytes, thereby preventing or treating allograft rejection. Preferably, the reduction in the expression level of heparanase occurs in lymphocytes at the allograft site, such as the organ allograft site.

[0043]

[0033] In a particularly preferred embodiment, the present invention relates to a method for preventing or treating transplant rejection, the method comprising the step of administering a heparanase inhibitor to a subject in need thereof, wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the transplant site, for example, in the transplanted organ, and does not reduce the expression level of heparanase in lymphocytes in the peripheral blood of the subject. In one embodiment, the heparanase inhibitor administered to the subject to reduce the heparanase expression level in lymphocytes at the transplant site is selected from OGT2115 or castanospermine. The inventors have shown that both OGT2115 and castanospermine selectively reduce the expression level of heparanase in lymphocytes at transplant sites such as the transplanted organ site.

[0044]

[0034] Those skilled in the art will understand from the present invention that any heparanase inhibitor capable of reducing the heparanase expression level is suitable for the method of the present invention. Those skilled in the art will also understand that it is routine work to identify a heparanase inhibitor capable of reducing the expression level in lymphocytes.

[0045]

[0035] In certain embodiments, the method of the present invention comprises administering the heparanase inhibitor in combination with one or more immunosuppressive agents. Suitable immunosuppressive agents are well known to those skilled in the relevant art. Preferably, the one or more immunosuppressive agents may be selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analogs of leflunomide; anti-CTLA4 antibodies, anti-CTLA4 Ig fusions, anti-B lymphocyte stimulator antibodies, anti-CD80 antibodies, etanercept, infliximab, anti-T cell antibodies, anti-CD3 antibodies, OKT3, anti-CD4 antibodies, anti-il-2 receptor antibodies, prednisone or its derivatives, anti-CD52 monoclonal antibodies; anti-CD20 monoclonal antibodies; belatacept; eculizumab; and intravenous immunoglobulin.

[0046]

[0036] In certain embodiments, the immunosuppressant is cyclosporine A or tacrolimus, which are routinely used for maintenance immunosuppressive therapy in organ transplant patients.

[0037] In further embodiments, the method of the present invention comprises administering a heparanase inhibitor in combination with one or more anti-inflammatory agents. Suitable anti-inflammatory agents will be well known to those skilled in the relevant art. Preferably, the one or more immunosuppressants may be selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

[0047]

[0038] In further embodiments, the present invention relates to a method for preventing or treating allograft rejection, said method comprising administering to a subject in need thereof an OGT2115 of formula

[0048]

Chemical formula

[0049] or a functional derivative or analog thereof, said OGT2115 inhibiting heparanase activity and thereby preventing or treating transplant rejection.

[0050]

[0039] In another embodiment, the present invention relates to a method for preventing or treating allograft rejection, said method comprising administering to a subject in need thereof an OGT2115 of formula

[0051]

Chemical formula

[0052] or a functional derivative or analog thereof in combination with cyclosporine A or tacrolimus, said OGT2115 inhibiting heparanase and thereby preventing or treating transplant rejection.

[0053]

[0040] In another embodiment, in the method of the present invention, the heparanase inhibitor is administered to the subject in a therapeutically effective amount. In another embodiment, in the method of the present invention, the heparanase inhibitor is administered to the subject at a dose of 1 mg / kg to 50 mg / kg. Specifically, the heparanase inhibitor is administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg. In a further embodiment, the heparanase inhibitor is administered at a dose of 2.0 mg / kg to 10 mg / kg. Specifically, this includes 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, or 10 mg / kg.

[0054]

[0041] In the method of the present invention, the heparanase inhibitor is administered to the subject daily, for example, once a day or twice a day, at any of the doses described in the above paragraph

[0040] . Alternatively, the heparanase inhibitor is administered weekly, for example, once a week.

[0055]

[0042] When the heparanase inhibitor of the present invention is administered in combination with one or more immunosuppressive agents, the immunosuppressive agent is administered in a therapeutically effective amount.

[0043] In certain embodiments, the OGT2115 of formula

[0056]

Chemical formula

[0057] or its functional derivatives or analogs are administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0058]

[0044] In a further embodiment, the formula

[0059]

Chemical formula

[0060] OGT2115 is administered at a dose of 2.0 mg / kg to 10 mg / kg. Specifically, this includes 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, or 10 mg / kg.

[0061]

[0045] In one embodiment of the method of the present invention, OGT2115 is administered to a subject daily, for example, once a day, at any of the doses described in the above paragraph

[0043] . Alternatively, the heparanase inhibitor is administered weekly, for example, once a week.

[0062]

[0046] When OGT2115 is administered in combination with a therapeutically effective amount of cyclosporine A or tacrolimus.

[0047] In a further embodiment, the present invention relates to a method of preventing or treating allograft rejection, said method comprising the step of administering a quinazoline compound to a subject in need thereof.

[0063]

[0048] In another embodiment, the present invention relates to a method of preventing or treating allograft rejection, said method comprising the step of administering to a subject in need thereof a compound of general formula A

[0064]

Chemical formula

[0065] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein, R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4Alkoxy, O-CH 2 selected from phenyl, O-phenyl; R 3 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 selected from phenyl, O-phenyl; R 4 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 selected from phenyl, O-phenyl; or R 1 and R 2 , or R 2 and R 3 , or R 3 and R 4 together form C 1~3 alkylenedioxy; R 1 , R 2 , R 3 and R 4 at least one of which is not H, L 1 is NHC 1~4 alkyl-NHC(O)-, NHC 1~4 alkyl-NHSO 2 -, azetidinyl-NHC(O)-, and azetidinyl-NHSO 2 -; selected from R 5 is C 3~9 cycloalkyl, C optionally substituted with one or two RX groups 6~10 aryl, C optionally substituted with one or two RX groups 2~9 heteroaryl, C optionally substituted with one or two RX groups 2~5 heterocycloalkyl, C optionally substituted with one or two RX groups 1~4 alkyl C 2~5 selected from heterocycloalkyl; L 2 is C 1~4 alkyl, azetidinyl-C(O)-, C 1~4 alkyl-NHC(O)-, C 1~4 alkyl-NHSO2 -, -C(O)-, SO 2 selected from, or L 2 does not exist; R 6 is H, C 2~6 alkyl, guanidinyl, NHC(NH)NH(C 1~3 alkyl), ureido, NHC(O)NH(C 1~3 alkyl), C optionally substituted with 1 or 2 RX groups 6~10 aryl, C optionally substituted with 1 or 2 RX groups 1~9 heteroaryl, C optionally substituted with 1 or 2 RX groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 RX groups 3~9 selected from cycloalkyl; R 7 is H or C 1~6 alkyl; L 1 is NHC 1~4 alkyl-NHSO 2 -, when R 5 is not phenyl substituted with 1 methyl, tert-butyl or phenyl group; Each RX is independently hydroxyl, halo, nitro, NR’R” (R’ and R” are independently selected from H and C 1~3 alkyl), C 1~4 alkyl, C 3~9 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C(O)C 1~3 alkyl, C(O)OC 1~4 alkyl, C(O)NHRY, C optionally substituted with 1 or 2 RY groups 6~10 aryl, C optionally substituted with 1 or 2 RY groups 2~9 heteroaryl, C 1~4 alkyl-(C 2~9 heteroaryl), C optionally substituted with 1 or 2 C 1~4 alkyl groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 C 1~4 alkyl groups 1~4 alkyl-(C 2~5(heterocycloalkyl), 1 or 2 C 1~4 C(O)-C optionally substituted with an alkyl group 2~9 heteroaryl; 1 or 2 C 1~4 alkyl group, or halo C 1~4 SO2-C optionally substituted with an alkyl group 2~9 heteroaryl; or two adjacent RX groups together form C 1~3 selected from those forming alkylenedioxy; RY is H, hydroxyl, halo, C 1~4 alkyl, halo C 1~4 alkyl, C 1~4 selected from alkoxy, a compound of formula A or a salt, hydrate, solvate, tautomer or stereoisomer thereof inhibits heparanase, thereby preventing or treating transplant rejection.

[0066]

[0049] In another embodiment, the present invention relates to a method of preventing or treating allograft rejection, said method comprising administering to a subject in need thereof a compound of general formula I

[0067]

Chemical formula

[0068] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein X is S or O; R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 3 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH2 selected from phenyl and O-phenyl; R 4 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 selected from phenyl and O-phenyl; or R 1 and R 2 or R 2 and R 3 or R 3 and R 4 together form C 1~3 alkylenedioxy; R 5 is H, C 1~6 alkyl, C 1~3 alkylC(O)OC 1~4 alkyl and C 1~3 alkylC 6~10 selected from aryl, optionally independently substituted with one or two groups selected from haloC 1~3 alkyl and haloC 1~3 alkoxy; L is C 1~6 alkyl, azetidinyl, C 1~6 alkyl-indolyl, NH, C 1~6 alkyl-NI-IC(O)O, azetidinyl-C(O)-, C 1~6 alkyl-NHC(O)-indolyl, C 1~6 alkyl-NHSO 2 - or absent; R 6 is H, halo, hydroxyl, d. 6 alkyl, C 1~6 alkenyl, C 1~6 alkynyl, C optionally substituted with one or two R x groups, C 6~10 aryl optionally substituted with one or two R x groups, C 1~9 heteroaryl optionally substituted with one or two R x groups, C 2~5 heterocycloalkyl optionally substituted with one or two R xC(O)-(heterocycloalkyl) optionally substituted with a group, 1 or 2 Rs x C(O)(C optionally substituted with a group 2~5 (heterocycloalkyl), C(O)NHR Y selected from, or absent; Each R x is independently hydroxyl, halo, nitro, NR’R”, C 1~4 alkyl, C 3~6 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C optionally substituted with 1 or 2 R Y groups 6~10 aryl, C 1~9 heteroaryl, C 1~4 alkyl-(C 1~9 (heteroaryl), C(O)OC 1~4 alkyl, C(O)NHR Y , C optionally substituted with 1 or 2 C 1~4 alkyl groups 2~5 (heterocycloalkyl), C(O)-(heterocycloalkyl) optionally substituted with 1 or 2 C 1~4 alkyl groups, or 2 adjacent Rs x groups together form C 1~3 alkylenedioxy; selected from those which R Y is H, hydroxyl, halo, C 1~4 alkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C 1~4 alkylheterocycloalkyl, C(O)-(C 1~4 (alkylheterocycloalkyl), C 1~4 alkylNR’R”; selected from R’ and R” are independently H, C 1~4 alkyl, C 1~4 (alkylheterocycloalkyl); selected from R 7 is H, C 1~4 alkyl and C 1~6 alkylC 1~9 (heteroaryl).

[0069]

[0050] In an alternative embodiment, the present invention relates to a method of preventing or treating allograft rejection, the method comprising administering to a subject in need thereof a compound of general formula II

[0070]

Chemical formula

[0071] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein X is S or O; R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 3 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 4 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; or or R 1 and R 2 or R 2 and R 3 or R 3 and R 4 together form C 1~3 alkylenedioxy; R 5’ is H, C 1~6 alkyl, C 1~3 alkyl C Λ OC 1~4 alkyl and C1~3 Alkyl C 6~10 selected from aryl, and optionally independently halo C 1~3 alkyl and halo C 1~3 substituted with one or two groups selected from alkoxy; L is C 1~6 alkyl, azetidinyl, C 1~6 alkyl - indolyl, NH, C 1~6 alkyl - NI - IC(O)O, azetidinyl - C(O)-, C 1~6 alkyl - NHC(O)- indolyl, C 1~6 alkyl - NHSO 2 - selected from, or absent; R 6 is H, halo, hydroxyl, C 1~6 alkyl, C 1~6 alkenyl, C 1~6 alkynyl, one or two R x groups optionally substituted C 6~10 aryl, one or two R x groups optionally substituted O 1~9 heteroaryl, one or two R x groups optionally substituted C 2~5 heterocycloalkyl, one or two R x groups optionally substituted C(O)-(heterocycloalkyl), one or two R x groups optionally substituted C(O)(C 2~5 heterocycloalkyl), C(O)NHR Y selected from, or absent; each R x is independently hydroxyl, halo, nitro, NR’R”, C 1~4 alkyl, C 3~6 cycloalkyl, halo C 1~4 alkyl, C 1~4 alkoxy, one or two R Y groups optionally substituted C 6~10 aryl, C 1~9 heteroaryl, C 1~4 alkyl - id - g heteroaryl), 0(0)00 1~4 alkyl, C(O)NHR Y , one or two C1~4 C optionally substituted with an alkyl group 2~5 heterocycloalkyl, 1 or 2 C 1~4 C(O)-(heterocycloalkyl) optionally substituted with an alkyl group, or 2 adjacent R x groups together form C 1~3 selected from those forming alkylenedioxy; R Y is H, hydroxyl, halo, C 1~4 alkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C 1~4 alkylheterocycloalkyl, C(O)-(C 1~4 alkylheterocycloalkyl), C 1~4 selected from alkylNR’R”; R’ and R” are independently H, C 1~4 alkyl, C 1~4 selected from alkylheterocycloalkyl; R 7 is H, C 1~4 alkyl and C 1~6 alkylC 1~9 selected from heteroaryl.

[0072]

[0051] In a further embodiment, the present invention relates to a method of preventing or treating allograft rejection, said method comprising administering to a subject in need thereof a compound of general formula III

[0073]

Chemical formula

[0074] wherein the groups R 1 , R 2 , R 3 , and R 4 are as defined in formulas (I) and (II); R A , R B , R C and R D are independently H, OH, C 1~3 alkyl, OO1~3 Alkyl, C(O)-(N-heterocycloalkyl) (e.g., C(O) morpholinyl), one or two C 1~3 C(O) piperazinyl optionally substituted with alkyl groups); OH, halo, C 1~3 Alkyl, OC 1~3 N-heteroaryl (e.g., 3-pyridyl, 4-pyridyl, 2,1,3-benzoxadiazolyl, pyrazolyl) optionally substituted with one or two groups selected from alkyl; OH, halo, C 1~3 Alkyl, OC 1~3 Phenyl optionally substituted with one or two groups selected from alkyl, CiOJNHC 1~3 Alkyl KNiC 1~3 Alkyl ^], one or two C 1~3 Selected from C(O)(heterocycloalkyl) (e.g., morpholinyl, piperazinyl, piperidinyl) optionally substituted with alkyl groups; R E is H, C 1~3 Alkyl, or C(O)-heterocycloalkyl (e.g., C(O)-(N-morpholino)).

[0075]

[0052] Preferably, in embodiments of the method of the present invention, prevention or treatment of transplant rejection includes extending the survival period of the graft.

[0053] In a further aspect, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of transplant rejection, wherein the heparanase inhibitor inhibits heparanase activity, thereby preventing or treating transplant rejection.

[0076]

[0054] In yet another aspect, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of allograft rejection, wherein the heparanase inhibitor reduces the expression level of heparanase, thereby preventing or treating transplant rejection.

[0077]

[0055] In one embodiment, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for preventing or treating allograft rejection, wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the transplantation site, such as the transplanted organ or tissue, and does not reduce the expression level of heparanase in lymphocytes in the peripheral blood of the subject.

[0078]

[0056] In a preferred embodiment, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for preventing or treating transplant rejection, wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the transplantation site, such as the transplanted organ or tissue, and does not reduce the expression level of heparanase in lymphocytes in the peripheral blood of the subject.

[0079]

[0057] In one embodiment of the present invention, the heparanase inhibitor is selected from OGT2115 or castanospermine. Those skilled in the art will understand that both OGT2115 and castanospermine are commercially available compounds.

[0080]

[0058] In one embodiment, by the use of the present invention, rejection of a primarily vascularized transplanted organ (i.e., excluding grafts that are secondarily vascularized) is prevented or treated. Preferably, the use of the present invention treats or prevents transplant rejection, for example, but not limited to, in the transplantation of the heart, heart-lung, kidney, lung, liver, pancreas, stomach or intestine.

[0081]

[0059] In one embodiment, the use of the present invention relates to the prevention or treatment of allograft rejection.

[0060] In a particular embodiment, the present invention relates to the use of a benzoxazol-5-ylacetic acid derivative in the manufacture of a medicament for preventing or treating transplant rejection, wherein the benzoxazol-5-ylacetic acid derivative inhibits heparanase activity, thereby preventing or treating transplant rejection.

[0082]

[0061] In certain embodiments, the present invention relates to the use of OGT2115 having the formula

[0083]

Chemical Formula

[0084] or a functional derivative or functional analog thereof in the manufacture of a medicament for preventing or treating transplant rejection, wherein said OGT2115 inhibits heparanase activity and thereby prevents or treats transplant rejection.

[0085]

[0062] In an alternative embodiment, the present invention relates to the use of a quinazoline compound in the manufacture of a medicament for preventing or treating transplant rejection, wherein said quinazoline compound inhibits heparanase activity and thereby prevents or treats transplant rejection.

[0086]

[0063] In certain embodiments, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for preventing or treating transplant rejection, wherein said heparanase inhibitor inhibits heparanase activity and thereby prevents or treats transplant rejection, and said heparanase inhibitor is a compound of general formula A

[0087]

Chemical Formula

[0088] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein in the formula R 1 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 2 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 3is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 4 is selected from H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; or R 1 and R 2 , or R 2 and R 3 , or R 3 and R 4 together form C 1~3 alkylenedioxy; R 1 , R 2 , R 3 and R 4 at least one of which is not H, L 1 is NHC 1~4 alkyl-NHC(O)-, NHC 1~4 alkyl-NHSO 2 -, azetidinyl-NHC(O)-, and azetidinyl-NHSO 2 -; R 5 is C 3~9 cycloalkyl, C optionally substituted with one or two RX groups 6~10 aryl, C optionally substituted with one or two RX groups 2~9 heteroaryl, C optionally substituted with one or two RX groups 2~5 heterocycloalkyl, C optionally substituted with one or two RX groups 1~4 alkyl C 2~5 heterocycloalkyl; L 2 is C 1~4 alkyl, azetidinyl-C(O)-, C 1~4 alkyl-NHC(O)-, C 1~4 alkyl-NHSO 2 -,-C(O)-, SO 2 -; or L 2does not exist; R 6 is H, C 2~6 alkyl, guanidinyl, NHC(NH)NH(C 1~3 alkyl), ureido, NHC(O)NH(C 1~3 alkyl), C optionally substituted with one or two RX groups 6~10 aryl, C optionally substituted with one or two RX groups 1~9 heteroaryl, C optionally substituted with one or two RX groups 2~5 heterocycloalkyl, C optionally substituted with one or two RX groups 3~9 selected from cycloalkyl; R 7 is H or C 1~6 alkyl; L 1 is NHC 1~4 alkyl-NHSO 2 -, R 5 is not phenyl substituted with one methyl group, tert-butyl group or phenyl group; Each RX is independently hydroxyl, halo, nitro, NR’R” (R’ and R” are independently selected from H and C 1~3 alkyl), C 1~4 alkyl, C 3~9 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C(O)C 1~3 alkyl, C(O)OC 1~4 alkyl, C(O)NHRY, C optionally substituted with one or two RY groups 6~10 aryl, C optionally substituted with one or two RY groups 2~9 heteroaryl, C 1~4 alkyl-(C 2~9 heteroaryl), C optionally substituted with one or two C 1~4 alkyl groups 2~5 heterocycloalkyl, C optionally substituted with one or two C 1~4 alkyl 1~4 alkyl-(C 2~5 heterocycloalkyl), C optionally substituted with one or two C 1~4 alkyl groups and optionally substituted with C(O)-C2~9 heteroaryl; 1 or 2 C 1~4 alkyl group, or halo C 1~4 SO2-C optionally substituted with an alkyl group 2~9 heteroaryl; or two adjacent RX groups together form C 1~3 selected from those forming alkylenedioxy; RY is H, hydroxyl, halo, C 1~4 alkyl, halo C 1~4 alkyl, C 1~4 selected from alkoxy.

[0089]

[0064] In a further embodiment, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of transplant rejection, wherein the heparanase inhibitor inhibits heparanase activity, thereby preventing or treating transplant rejection, and the heparanase inhibitor is, for example, a compound of general formula I, formula II or formula III described in paragraphs

[0049] to

[0051] above.

[0090]

[0066] In another embodiment, the use of the present invention includes the use of a heparanase inhibitor suitable for administration in combination with one or more immunosuppressive agents. Suitable immunosuppressive agents will be well known to those skilled in the relevant art. Preferably, the one or more immunosuppressive agents are selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analogs of leflunomide; anti-CTLA4 antibodies, anti-CTLA4 Ig fusions, anti-B lymphocyte stimulator antibodies, anti-CD80 antibodies, etanercept, infliximab, anti-T cell antibodies, anti-CD3 antibodies, OKT3, anti-CD4 antibodies, anti-il-2 receptor antibodies, prednisolone or its derivatives, anti-CD52 monoclonal antibodies; anti-CD20 monoclonal antibodies; belatacept; eculizumab; and intravenous immunoglobulin.

[0091]

[0066] In a preferred embodiment, the immunosuppressive agent is cyclosporine A or tacrolimus.

[0067] In a further embodiment, the use of the present invention includes the use of a heparanase inhibitor suitable for administration in combination with one or more anti-inflammatory agents. Suitable anti-inflammatory agents are well known to those skilled in the relevant art. Preferably, the one or more immunosuppressive agents may be selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

[0092]

[0068] In another embodiment, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of transplant rejection, wherein the heparanase inhibitor has the formula

[0093]

Chemical formula

[0094] and is OGT2115 or a functional derivative or functional analog thereof, and the OGT2115 inhibits heparanase, thereby preventing or treating transplant rejection.

[0069] In yet another embodiment, the present invention relates to the use of a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of transplant rejection, wherein the heparanase inhibitor has the formula

[0095]

Chemical formula

[0096] and is OGT2115 or a functional derivative or functional analog thereof, and the OGT2115 inhibits heparanase activity, thereby preventing or treating transplant rejection and being suitable for administration in combination with cyclosporin A or tacrolimus.

[0097]

[0070] Preferably, in the embodiments relating to the use of the present invention, the prevention or treatment of transplant rejection includes extending the survival period of the graft.

[0071] In yet another aspect, the present invention relates to a heparanase inhibitor for use in preventing or treating transplant rejection, wherein the heparanase inhibitor inhibits heparanase activity and thereby prevents or treats transplant rejection.

[0098]

[0072] In one embodiment, with respect to a heparanase inhibitor for use in preventing or treating transplant rejection, the rejection is primarily the rejection of a vasculogenic transplanted organ. Preferably, the prevention or treatment is, for example, but not limited to, treating or preventing transplant rejection in the transplantation of a heart, heart-lung, kidney, lung, liver, pancreas, stomach or intestine.

[0099]

[0073] In a further embodiment, the transplant rejection is an allograft rejection.

[0074] In yet another embodiment, the heparanase inhibitor of the present invention is a benzoxazole-5-yl acetic acid derivative. In a particular embodiment, the heparanase inhibitor is OGT2115 having the formula

[0100]

Chemical formula

[0101] or a functional derivative or functional analog thereof.

[0075] In a further embodiment, the heparanase inhibitor used in the method of the present invention is a quinazoline compound. For example, a compound of general formula A

[0102]

Chemical formula

[0103] or a salt, hydrate, solvate, tautomer or stereoisomer thereof, wherein R 1 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2Selected from phenyl, O-phenyl; R 2 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 3 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; R 4 is H, hydroxyl, halo, C 1~6 alkyl, C 1~4 alkoxy, O-CH 2 phenyl, O-phenyl; or R 1 and R 2 or R 2 and R 3 or R 3 and R 4 together form C 1~3 alkylenedioxy; R 1 R 2 R 3 and R 4 at least one of which is not H, L 1 is NHC 1~4 alkyl-NHC(O)-, NHC 1~4 alkyl-NHSO 2 -, azetidinyl-NHC(O)-, and azetidinyl-NHSO 2 -; selected from R 5 is C 3~9 cycloalkyl, C optionally substituted with 1 or 2 RX groups 6~10 aryl, C optionally substituted with 1 or 2 RX groups 2~9 heteroaryl, C optionally substituted with 1 or 2 RX groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 RX groups 1~4 alkyl C 2~5 selected from heterocycloalkyl; L2 is C 1~4 alkyl, azetidinyl-C(O)-, C 1~4 alkyl-NHC(O)-, C 1~4 alkyl-NHSO 2 -, -C(O)-, SO 2 selected from, or L 2 is absent; R 6 is H, C 2~6 alkyl, guanidinyl, NHC(NH)NH(C 1~3 alkyl), ureido, NHC(O)NH(C 1~3 alkyl), C optionally substituted with 1 or 2 RX groups 6~10 aryl, C optionally substituted with 1 or 2 RX groups 1~9 heteroaryl, C optionally substituted with 1 or 2 RX groups 2~5 heterocycloalkyl, C optionally substituted with 1 or 2 RX groups 3~9 selected from cycloalkyl; R 7 is H or C 1~6 alkyl; L 1 when is NHC 1~4 alkyl-NHSO 2 -, R 5 is not phenyl substituted with 1 methyl group, tert-butyl group or phenyl group; each RX is independently hydroxyl, halo, nitro, NR’R” (R’ and R” are independently selected from H and C 1~3 alkyl), C 1~4 alkyl, C 3~9 cycloalkyl, haloC 1~4 alkyl, C 1~4 alkoxy, C(O)C 1~3 alkyl, C(O)OC 1~4 alkyl, C(O)NHRY, C optionally substituted with 1 or 2 RY groups 6~10 aryl, C optionally substituted with 1 or 2 RY groups 2~9 heteroaryl, C 1~4 alkyl-(C 2~9 heteroaryl), 1 or 2 C 1~4C optionally substituted with an alkyl group 2~5 heterocycloalkyl, 1 or 2 C 1~4 C optionally substituted with an alkyl group 1~4 alkyl-(C 2~5 heterocycloalkyl), 1 or 2 C 1~4 C(O)-C optionally substituted with an alkyl group 2~9 heteroaryl; 1 or 2 C 1~4 alkyl group, or halo C 1~4 SO2-C optionally substituted with an alkyl group 2~9 heteroaryl; or two adjacent RX groups together form C 1~3 selected from those forming alkylenedioxy; RY is H, hydroxyl, halo, C 1~4 alkyl, halo C 1~4 alkyl, C 1~4 selected from alkoxy.

[0104]

[0076] In a further embodiment, the present invention relates to a heparanase inhibitor in the manufacture of a medicament for the prevention or treatment of transplant rejection, the heparanase inhibitor inhibits heparanase activity, thereby preventing or treating transplant rejection, and the heparanase inhibitor is a compound of general formula I, formula II or formula III as described in paragraphs

[0049] to

[0051] above, for example.

[0105]

[0077] In a further embodiment, the heparanase inhibitor of the present invention is administered in combination with one or more immunosuppressive agents. For example, the immunosuppressive agent is selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analogs of leflunomide; anti-CTLA4 antibodies, anti-CTLA4 Ig fusions, anti-B lymphocyte stimulator antibodies, anti-CD80 antibodies, etanercept, infliximab, anti-T cell antibodies, anti-CD3 antibodies, OKT3, anti-CD4 antibodies, anti-il-2 receptor antibodies, prednisone or its derivatives, anti-CD52 monoclonal antibodies; anti-CD20 monoclonal antibodies; belatacept; eculizumab; and intravenous immunoglobulin, but is not limited thereto.

[0106]

[0078] In a particular embodiment, the heparanase inhibitor of the present invention is administered in combination with cyclosporine A or tacrolimus.

[0079] In a further embodiment, the heparanase inhibitor of the present invention is administered in combination with one or more anti-inflammatory agents. For example, the anti-inflammatory agent is selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone, but is not limited thereto.

[0107]

[0080] In yet another embodiment, the heparanase inhibitor of the present invention prolongs the survival period of the graft.

[0081] In a further embodiment, the heparanase inhibitor is administered at a dose of 1 mg / kg to 50 mg / kg. Specifically, the heparanase inhibitor is administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg. In a further embodiment, the heparanase inhibitor is administered at a dose of 2.0 mg / kg to 10 mg / kg. Specifically, this includes 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, or 10 mg / kg.

[0108]

[0082] The heparanase inhibitor can be administered to the subject daily, for example, once a day, at any of the above doses. Alternatively, the heparanase inhibitor is administered weekly, for example, once a week.

[0083] In a further aspect, the present invention relates to a heparanase inhibitor for use in preventing or treating transplant rejection, wherein the heparanase inhibitor inhibits heparanase activity, thereby preventing or treating transplant rejection.

[0109]

[0084] It will be well-known to those skilled in the art that the heparanase inhibitor of the present invention can be administered by any suitable route of administration, such as parenteral administration including, but not limited to, subcutaneous administration, intravenous administration, intradermal administration, intramuscular administration, and intraperitoneal administration. Those skilled in the art will also understand that the heparanase inhibitor of the present invention can be administered by enteral administration such as oral or rectal administration.

[0110]

[0085] The present invention is not limited to the use of OGT2115, and it will be apparent to those skilled in the art that other specific heparanase inhibitors, such as those described above, are also suitable for the methods and uses of the present invention for treating transplant rejection reactions, including, but not limited to, prolonging the survival period of transplanted organs in allotransplantation.

[0111]

[0086] In particular, it is routine for those skilled in the art to identify heparanase inhibitors suitable for the methods and uses of the present invention. Suitable assays for determining heparanase inhibitory activity are known in the art, for example, the in vitro assay described in Rivara et al. (2016) Future Med Chem, 8(6):647-680. For example, this method involves contacting a preparation containing heparanase and a heparanase substrate (e.g., heparan sulfate or fondaparinux) with a test compound and detecting the amount of the complete substrate compared to a reference level of the complete substrate in the absence of the test compound, or detecting the modulation of the activity of a downstream target of the complete heparanase substrate. Detection of the amount or modulation of the complete substrate can be achieved using techniques including, but not limited to, ELISA, cell-based ELISA, inhibition ELISA, Western blot, RIA, immunoprecipitation, immunostaining, solid-phase labeled substrate assays such as solid-phase radioactive or fluorescent labeling or biotinylated substrates, ultrafiltration assays, proximity assays such as HTRF and scintillation proximity assays, fluorescence assays using fluorescent substrate-heparanase substrate conjugates such as fluorescein or rhodamine, colorimetric assays, and fluorescence immunoassays, all of which are well-known to those skilled in the art.

[0112]

[0087] One of ordinary skill in the art can readily prepare derivatives or analogs of OGT2115, or compounds of Formula A, Formula I, Formula II, or Formula III, for example, using well-known chemical synthesis methods, and can test for heparanase inhibitory activity using an assay such as that described in the above paragraph

[0086] .

[0113]

[0088] Other additional aspects and features of the present disclosure will become apparent upon reading the following detailed description of embodiments intended to illustrate, and not limit, the present disclosure.

Brief Description of the Drawings

[0114]

[0089] The illustrated embodiments of the disclosed subject matter are best understood by reference to the figures and tables, in which like parts are indicated by like numbers throughout. The following description is for purposes of illustration only and merely exemplifies certain selected embodiments of devices, systems, and processes consistent with the disclosed subject matter claimed herein.

Figure 1

[0090] FIG. 1 shows the distribution of the survival times (in days) of allogeneic rat hearts. The black dots within the box-and-whisker plot correspond to the raw data points in Table 2. Each black dot represents the survival of one rat that received an allogeneic heart transplant.

Figure 2A

[0091] FIG. 2 graphically depicts the percent cDNA present in each experimental group in the 2A) kidney samples and 2B) blood samples described in Example 2.

Figure 2B

Modes for Carrying Out the Invention

[0115] Detailed Description

[0092] Preferred features, embodiments, and variations of the present invention can be understood from the following detailed description that provides sufficient information for one of ordinary skill in the art to practice the present invention. The detailed description should not be regarded as limiting the scope of the foregoing summary of the present invention in any way.

[0116] Definition

[0093] In the context of the present invention, the terms "comprising" and the like are to be construed in an inclusive sense, i.e., "including but not limited to", rather than in an exclusive sense.

[0117]

[0094] Throughout the specification and the claims (if any), unless the context requires otherwise, the terms "substantially" or "about" are not to be construed as being limited to the values within the ranges defined by those terms. Thus, unless otherwise specified, the numerical parameters set forth in the following specification and the appended claims (if any) are approximations, which may vary depending on the desired properties sought to be obtained by the present invention. At the very least, each numerical parameter should be construed in light of the number of significant digits and the normal rounding conventions. The term "about" is understood to refer to a range of + / - 10%, for example, + / - 5% or + / - 1% or + / - 0.1%.

[0118]

[0095] The terms "a", "the", "said" and similar referents used in the context of describing the present invention (particularly in the context of the following claims) are to be construed as including both the singular and the plural forms, unless otherwise specifically stated herein or clearly inconsistent with the context.

[0119]

[0096] All methods described herein can be performed in any suitable order, unless otherwise specifically stated herein or clearly inconsistent with the context.

[0097] The use of any examples or exemplary language provided herein (e.g., "such as") is intended merely to better illustrate the invention and is not intended to limit the scope of the claimed invention. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention, unless otherwise specifically stated herein or clearly inconsistent with the context.

[0120] In the context of the present invention, as far as tissues and organs are concerned, the term "transplantation" may include "autotransplantation", "allotransplantation" or "xenotransplantation". These terms are further defined as follows.

[0121] In the context of the present invention, the term "autotransplantation" is defined as the transplantation of cells, tissues, or organs between sites within the same individual or to an identical twin. In the context of the present invention, the term "allotransplantation" is defined as the transplantation of an organ or tissue from a donor to a genetically non-identical individual of the same species.

[0122] In the context of the present invention, the term "xenotransplantation" is defined as the transplantation of an organ or tissue between two different species. In the transplantation context defined above, the term "primarily vascularized organ transplant" (PVT) refers to an organ transplant that is primarily vascularized, and includes, but is not limited to, heart, heart-lung, kidney, lung, and liver grafts. A primarily vascularized graft is an organ that is connected to the recipient's blood supply by surgical arterial and venous anastomoses (connections) to obtain immediate blood supply.

[0123]

[0103] In the context of transplantation as defined above, the term "secondarily vascularized transplant" (SVT) refers to transplant tissue that becomes secondarily vascularized, including, but not limited to, pancreatic islet cells, skin, or corneal transplants. A secondarily vascularized transplant is a transplant of cells or tissue without vascular anastomosis, but is usually placed into or under the capsule of another organ using a catheter inserted into the recipient's vein. For an SVT to survive, it is necessary to grow a network of blood vessels that connect to the recipient's blood supply. This can take several days to several weeks. PVT is subject to immediate antibody attack by the recipient's alloantibodies that can be generated by pregnancy, blood transfusions prior to transplantation, or previous allografts. Such attacks usually cause loss of the allograft, which often occurs on the operating table. Since an SVT does not have a surgical blood supply and the blood vessels need to grow slowly, it is rarely subject to this attack. PVT acquires function earlier than SVT. SVT usually has a relatively longer ischemic time (the time from harvest from the donor to transplantation / revascularization into the transplant recipient) compared to PVT. For example, pancreatic islets are removed from the pancreas by chemical digestion of a cadaveric donor pancreas, perfused, washed, stored at 4°C, and transplanted after a functional test (at least 24 hours). In contrast, in the case of a living kidney transplant, the warm ischemic time is 2 - 3 minutes and the cold ischemic time is about 1 - 2 hours. Therefore, in studies on the survival of pancreatic islet cells, control of ischemia-reperfusion injury is necessary.

[0124]

[0104] For example, in an allograft of skin tissue, since secondary vascularization occurs, the possibility of an immediate vascular rejection reaction by alloantibodies is reduced. In contrast, an allograft heart is primarily vascularized and is immediately subject to attack by alloantibodies.

[0125]

[0105] As used herein, the term "inhibitor" refers to an agent that reduces, inhibits, or impairs at least one function or biological activity of a target molecule. As used herein, the term "heparanase inhibitor" refers to an agent that reduces, inhibits, or impairs at least one function or biological activity of heparanase. The term "heparanase inhibitor" includes agents that reduce the biological activity of heparanase. The term "heparanase inhibitor" also includes agents that inhibit or reduce heparanase activity by reducing the level of heparanase, such as the expression level of heparanase. One of ordinary skill in the art will understand, for example, that a decrease in the expression level of heparanase results in a decrease in heparanase activity.

[0126]

[0107] In the context of the present invention, the properties of a heparanase inhibitor include, for example, the ability to treat allograft rejection by prolonging the survival period of an allograft in vivo.

[0127]

[0108] The term "transplant site" refers to the site of a transplanted tissue or organ. The term may also refer to the transplanted tissue or organ. In this regard, the phrase "lymphocytes at the transplant site" refers to lymphocytes within and in the vicinity of a transplanted tissue or organ.

[0128]

[0109] In the context of the present invention, phrases such as "a heparanase inhibitor reduces the expression level of heparanase" mean a decrease in the expression level of the heparanase gene, and are measured, for example, as a decrease in heparanase mRNA or the corresponding cDNA in a subject treated with a heparanase inhibitor compared to the expression level of the heparanase gene in a subject not treated with a heparanase inhibitor.

[0129]

[0110] The term "subject" includes any human or non-human mammal. Thus, in addition to being useful for the treatment of humans, the compounds of the present invention may also be useful for veterinary treatment of mammals, including but not limited to pet animals and livestock such as dogs, cats, horses, cows, sheep, and pigs. In a preferred embodiment, the subject is a human.

[0130]

[0111] The term "therapeutically effective amount" refers to an amount of a drug sufficient to produce a desired therapeutic or pharmacological effect in a subject being treated. This term is synonymous with the amount of a drug that achieves the goal of improving the severity and / or frequency of occurrence of a disease over the course of treatment with each drug itself, while avoiding or minimizing, to the extent possible, adverse side effects, including those adverse side effects that are normally associated with other forms of treatment, and is intended to identify that amount. It will be routine for those of ordinary skill in the art to determine a therapeutically effective amount using information known in the art and ordinary methods.

[0131]

[0112] The compounds used in the method of the present invention described herein can be administered as a formulation containing a pharmaceutically effective amount of the compound in combination with one or more pharmaceutically acceptable excipients, including carriers, vehicles, and diluents. As used herein, the term "excipient" refers to a substance that is not itself a therapeutic agent but is used as a diluent, adjuvant, or vehicle for delivering a therapeutic agent to a subject, or for improving handling or storage characteristics, or for enabling or facilitating the formation of solid dosage forms such as tablets, capsules, or solutions or suspensions suitable for oral, parenteral, intradermal, or subcutaneous administration, or for adding to a pharmaceutical composition. Excipients include, by way of example and not limitation, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, glidants, stabilizers. Acceptable excipients include, but are not limited to, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose materials (such as cellulose esters of alkanoic acids and cellulose alkyl esters), low melting waxes, cocoa butter or powder, polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, and other pharmaceutically acceptable materials. Examples of excipients and their use are described in Remington’s Pharmaceutical Sciences, 20th Edition (Lippincott Williams & Wilkins, 2000). The selection of excipients depends largely on factors such as the route of administration, the effect on the solubility and stability of the excipient, and the nature of the dosage form.

[0132]

[0113] The term "pharmaceutically acceptable salts" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, etc., and that have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19.

[0133]

[0114] The compounds and pharmaceutical compositions used in the methods of the present invention can be formulated for oral, injection, rectal, parenteral, subcutaneous, intravenous, topical, intravitreal or intramuscular delivery. Non-limiting examples of specific formulation types include tablets, capsules, caplets, powders, granules, injections, ampoules, vials, ready-to-use (RTU) solutions or suspensions, lyophilized materials, suppositories and implants. Solid formulations such as tablets and capsules can contain any number of suitable pharmaceutically acceptable excipients or carriers described herein. The compounds of the present invention can also be formulated for sustained delivery.

[0134]

[0115] Tablets and capsules for oral administration may be in unit dosage form and may contain conventional excipients such as binders (e.g., acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone), fillers (e.g., lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine), tablet lubricants (e.g., magnesium stearate, talc, polyethylene glycol, or silica), disintegrants (e.g., potato starch), or acceptable wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated according to methods well known in the ordinary pharmaceutical practice.

[0135] For parenteral administration, which includes intravenous, intramuscular, subcutaneous, or intraperitoneal administration, fluid unit dosage forms can be prepared by combining the compound with a sterile vehicle (usually a sterile aqueous solution that is preferably isotonic with the recipient's blood). Depending on the vehicle and concentration used, the compound can be suspended or dissolved in the vehicle or other suitable solvent. When preparing a solution, the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Advantageously, agents such as local anesthetics, preservatives, buffers, etc. can be dissolved in the vehicle. To enhance stability, after filling the composition into vials, it can be frozen and the water removed under vacuum. The dried lyophilized powder can then be sealed in the vial and supplied with a vial of water for injection or other suitable liquid for reconstitution prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended rather than dissolved in the vehicle and sterilization by filtration cannot be achieved. The compound can be sterilized by exposure to ethylene oxide prior to suspension in the sterile vehicle. A surfactant or wetting agent may be included in the composition to facilitate uniform distribution of the compound.

[0136]

[0117] In certain embodiments, the compounds used in the methods of the invention are formulated as injectable solutions, suspensions, or emulsions.

[0118] The term "pharmaceutical carrier, diluent or excipient" includes, but is not limited to, any physiologically buffered (i.e., about pH 7.0 to 7.4) medium containing a suitable water-soluble organic carrier, conventional solvents, dispersion media, fillers, solid carriers, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. Suitable water-soluble organic carriers include, but are not limited to, physiological saline, dextrose, corn oil, dimethyl sulfoxide, and gelatin capsules. Other conventional additives include binders such as lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, cellulose derivatives such as hydroxypropylmethylcellulose, disintegrants such as acacia, gelatin, sodium carboxymethylcellulose, and lubricants such as talc and magnesium stearate.

[0137]

[0119] The terms "therapeutically effective amount" or "pharmacologically effective amount" or "effective amount" refer to an amount of a drug sufficient to produce a desired therapeutic or pharmacological effect in a subject being treated. These terms are synonymous with, and are intended to identify, the amount of each drug that will achieve the goal of improving the severity and / or frequency of occurrence of a disease over the course of treatment with each drug itself, while avoiding or minimizing, if possible, adverse side effects, including those that are normally associated with other forms of treatment. One of ordinary skill in the art can determine the effective dosage using information and methods known in the art.

[0138]

[0120] It is also known to those of ordinary skill in the art that there are further differences between secondarily vascularized grafts and primarily vascularized grafts. For example, cell transport to allografts, basement membranes, etc., and the possibility that these differences may affect the way rejection reactions occur and the methods for treating rejection reactions.

[0139]

[0121] In the context of the present invention, insofar as it relates to administering compositions in combination with each other, the term "in combination" means that the compositions are administered together, for example simultaneously, or sequentially in any order, over a period of time during which the two compositions act together to result in the treatment of transplant rejection.

[0140]

[0122] In the context of the present invention, the term "functional derivative or functional analog" means, with respect to a particular compound, a compound that is structurally similar to that particular compound and exhibits the same functional properties as that particular compound. For example, functional derivatives or functional analogs of OGT2115 include compounds that are benzoxazol-5-ylacetic acid derivatives, and salts, hydrates, solvates, tautomers or stereoisomers of such related compounds that are capable of inhibiting heparanase activity.

[0141]

[0123] Any embodiment of the present invention is intended for illustration only and is not intended to limit the present invention. Thus, it should be understood that various other changes and modifications can be made to any of the described embodiments without departing from the spirit and scope of the present invention.

[0142]

[0124] As used herein, "treatment" of allograft rejection means the achievement of one or more of the following: (a) reducing / shortening the severity and / or duration of the rejection, (b) stopping the rejection, (c) limiting or preventing the manifestation of symptoms characteristic of the rejection being treated, (d) suppressing the worsening of symptoms characteristic of the rejection being treated, (e) limiting or preventing the recurrence of rejection in a patient who has previously shown signs of rejection, and (f) limiting or preventing the recurrence of symptoms in a patient who has previously shown symptoms of rejection. The term "treatment" does not necessarily mean that the subject is treated until complete recovery.

[0143]

[0125] As used herein, "prevention" of allograft rejection means preventing allograft rejection in a subject. As used herein, "maintaining the integrity of" or "maintaining" an allograft in a subject means keeping the allograft substantially healthy and functional and substantially preserving the biological structure of the allograft.

[0144] As used herein, the expressions "for administration" and "intended to be administered" have the same meaning as "prepared for administration". In other words, the description that an active compound is "for administration" must be understood to mean that the active compound is formulated and compounded in a dosage such that it can exert a therapeutic effect.

[0145] Preferred embodiments of the present invention are further described by the following non-limiting examples with reference to the accompanying drawings. Examples Materials and Methods Inbred rat strains PVG (RT1c) (donor) and DA (RT1a) (recipient) used for heart and kidney transplantation were bred under standard conditions. All procedures were approved and monitored by the Animal Care and Ethics Committee of Australia.

[0146] Rat heterotopic heart transplantation

[0130] Heterotopic heart transplantation was performed using standard techniques (10). Heart function was evaluated daily by abdominal palpation. The endpoint of transplanted heart survival was defined as the last day of palpable heartbeat. The breeding of all rats in this study complied with the Animal Research Act 1985 (New South Wales, Australia). This protocol was designed to minimize animal pain and discomfort. Before the experiment, the animals were acclimatized to the laboratory environment (22 °C, 12-hour light-dark cycle, 50% humidity, free access to food and water) for at least 1 week. Intragastric forced oral administration was performed on conscious animals using a curved gastric tube needle (gauge 16, 100 mm suitable for animals weighing 250 - 300 g). All transplanted rats were administered postoperative analgesics (carprofen 4 mg / kg, subcutaneous injection every 12 - 24 hours). Before tissue collection, the animals were euthanized by approved carbon dioxide asphyxiation and the heart was stopped.

[0147] Rat kidney transplantation

[0131] Orthotopic left kidney transplantation was performed using standard techniques (11) well-known to those skilled in the art, with an average anastomosis time of 37 ± 4 minutes. The harvested kidneys were perfused with 2 ml of Custodiol HTK solution (Chemie GMBH, Alsdorf-Hainline, Germany) at 40 °C. Transplanted rats were administered analgesics (carprofen 5 mg / kg, subcutaneous injection every 12 - 24 hours) and prophylactic antibiotics (enrofloxacin 5 mg / kg, subcutaneous injection every 12 hours) for 3 days. The transplanted kidneys and blood were collected on the 6th day, immediately after euthanasia by asphyxiation. The kidneys and blood were used for the measurement of gene expression in graft lymphocytes and peripheral blood mononuclear cells. 2 Collected immediately after euthanasia by asphyxiation. The kidneys and blood were used for the measurement of gene expression in graft lymphocytes and peripheral blood mononuclear cells.

[0148] Study design

[0132] Research on the survival rate of allogeneic hearts was conducted in six groups: syngeneic grafts (controls for ischemia-reperfusion injury); untreated (rejected) allogeneic grafts; dimethyl sulfoxide (DMSO) (controls for the vehicle of OGT2115); and allogeneic grafts treated with three doses of OGT2115 (a heparinase inhibitor): 2.5 mg / kg, 5 mg / kg, and 10 mg / kg. Research on gene expression was conducted in five groups: syngeneic grafts (controls for ischemia-reperfusion injury), untreated (rejected) allogeneic grafts, allogeneic grafts treated with OGT2115 (a heparinase inhibitor), and allogeneic grafts treated with castanospermine. In these studies, OGT2115 was administered at 10 mg / kg. Research on the gene expression of heparinase was conducted using lymphocytes derived from transplanted kidney tissue and peripheral blood on day 6 after transplantation. This time frame included the period when the rejection reaction was maximal on day 6.

[0149] Drug administration

[0133] The heparinase inhibitor (OGT2115) was suspended in dimethyl sulfoxide and saline and then administered subcutaneously at 2.5 mg / kg or 5 mg / kg or 10 mg / kg. Castanospermine was administered by an Alzet osmotic pump (Alza Corporation, Palo Alto, USA) at an immunosuppressive dose of 150 mg / kg per day. The OGT2115 treatment was started on day 1 after surgery, and the castanospermine treatment was started on the day of transplantation.

[0150] Cell preparation

[0134] Briefly, kidney tissue was minced and digested with 200 units / ml of collagenase type 4 (Worthington Biochemical Corp, Lakewood, NJ, USA). The tissue suspension was passed through a sieve and then through a 70-μm nylon cell strainer (Falcon Corning Inc, Corning, NY, USA). Lymphocyte cells were prepared from peripheral blood using standard methods. Next, 100-μl aliquots (1×10 6 cells) of the cell suspensions obtained for the kidney and blood were made.

[0151] Sample

[0135] Cell suspensions of tissues and blood (1×10 6 cells) were suspended in RNAlater (trademark) stabilization solution (Sigma, St. Louis, Missouri, USA) and frozen at -80°C.

[0152] RNA Isolation

[0136] The cell suspension was thawed before use. 5 ml of cold PBS was added, the cells were resuspended and then centrifuged at 6000 g for 10 minutes. The supernatant was removed, 600 μl of RLT Plus buffer (Qiagen, Hilden, Germany) was added, the sample was resuspended and then vortexed to ensure complete homogenization of the sample.

[0153]

[0137] RNA was manually extracted using the RNeasy Plus Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The RNA was eluted with 30 μl of RNase-free water. The quality of the RNA was measured using a Nanodrop 2000 (ThermoFisher Scientific, Massachusetts, USA) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Massachusetts, USA), and the RNA was stored at -80°C. The conversion of RNA to cDNA was performed using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad, Hercules, California, USA) according to the manufacturer's instructions. The cDNA samples were stored at -20°C.

[0154] Droplet Digital PCR (ddPCR)

[0138] Custom ddPCR assays were designed using the IDT PrimerQuest™ tool and PrimeTime qPCR assays (IDT, Singapore). Probes were tagged with either the reporter dye 5’-FAM™ or 5’-HEX™ and the fluorescence quencher 3’lowaBlack™. ddPCR assays were resuspended in IDTE and diluted to a working concentration of 900 nM primer / 250 nM probe. The sequences of the primers and probes for each assay are described in the following paragraph. A 25 μl reaction mixture containing ddPCR SuperMix for Probes (without dUTP) (BioRad, Hercules, CA, USA), hypoxanthine-guanine phosphoribosyl transferase reference control (HPRT-1), and heparanase target probe (HPSE) (IDT, Singapore) was prepared. cDNA and water were made for each sample and added to a 96-well plate ready for ddPCR droplet generation.

[0155]

[0139] ddPCR was performed using a QX200AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA). After droplet generation, the samples were placed on a thermocycler under the following conditions.

[0156]

Table 1

[0157]

[0140] The samples were left on the cycler at 4°C for at least 4 hours. The droplets were then read on a QX200 droplet reader and analyzed using QuantaSoft software (Bio-Rad, Hercules, CA, USA). The results were expressed using the fractional abundance ratio parameter of cDNA.

[0158] HPSE primer / probe assay

[0159]

Table 2

[0160] The above HPSE primer 1 sequence is SEQ ID NO: 1. The above HPSE primer 2 sequence is SEQ ID NO: 2. The above HPSE probe sequence is SEQ ID NO: 3 having a FAM phosphor and 3IAbkFQ and ZEN fluorescence quencher. Those skilled in the art will understand that these are necessary to enable accurate reading of the dyes.

[0161] HPRT1 primer / probe assay

[0162] [Table 3]

[0163] The above HPRT1 primer 1 sequence is SEQ ID NO: 4. The above HPRT1 primer 2 sequence is SEQ ID NO: 5. The above HPRT1 probe sequence is SEQ ID NO: 6 having a HEX phosphor and 3IAbkFQ and ZEN fluorescence quencher. Those skilled in the art will understand that these are necessary to enable accurate reading of the dyes.

[0164] Example 1

[0141] An experiment was conducted to compare the survival rates of allogeneic transplanted hearts in six groups of inbred rats. Among these groups, three of groups 4, 5, and 6 were treated with a specific heparinase inhibitor OGT2115. The groups are shown in Table 1 as follows.

[0165] [Table 4]

[0166]

[0142] The data of the first group of rats were not included in these analyses, and the five groups were targeted. All 11 rats transplanted with syngeneic grafts survived for 100 days until euthanasia.

[0167]

[0143] There are two main sets of null hypotheses. 1. M2 = M4; M2 = M5; M2 = M6 2. M3 = M4; M3 = M5; M3 = M6

[0144] And two secondary null hypotheses: 3. M2 = (M4 + M5 + M6) / 3 4. M3 = (M4 + M5 + M6) / 3

[0145] These tests were evaluated using the Wilcoxon rank-sum test. The mean, median, minimum, maximum, first and third quartiles, and the estimated difference in the location parameter of each group are shown in the 95% confidence interval. The difference in the location parameter is the median of the difference between the sample of the first group and the sample of the second group.

[0168]

[0146] This study was powered to detect the alternative hypothesis that samples of 11 rats each in groups 2, 3, and 6 provide 80% power to reject the null hypothesis at a 5% type I error rate, and that the survival time in group 6 is 20% longer compared to groups 2 and 3.

[0169]

Table 5

[0170]

[0147] Table 2 summarizes the allogeneic heart survival periods of the five rat groups in days. A plot of the distribution of the survival periods of each group is shown in Figure 1. The results shown in Table 2 and Figure 1 show a clear tendency for the administration of OGT2115 to extend the survival period of heart allografts. Specifically, the mean and median survival rates of heart allografts increased in the low-dose groups (2.5 mg / Kg and 5 mg / Kg), and significantly increased in the highest-dose group (10 mg / Kg).

[0171] Conclusion

[0148] As a result of the experimental treatment (use of heparinase inhibitor), the distribution of the survival days of allografts was significantly longer compared to the untreated control group or the DMSO control group. The highest dose group (Group 6: 10 mg / Kg) showed the largest difference compared to the control group (an estimated increase in survival period of 2 days compared to untreated PVG-DA and 3 days compared to DMSO). Also, when compared to both control groups, there was an obvious tendency for the average and median values of the transplantation survival rate to increase in the low dose groups.

[0172] Example 2

[0149] The heparinase expression levels in lymphocytes extracted from transplanted kidneys of inbred rats on the 6th day after transplantation were measured. The heparinase expression levels were also measured in peripheral blood lymphocytes isolated from the same rats. The heparinase levels were measured by isolating mRNA and converting it to cDNA using reverse transcriptase. The cDNA was measured using the parameter of fractional abundance ratio. The experimental design consisted of four treatment groups: a castanospermine (Cast) treatment group, an OGT2115 treatment group, an isograft treatment group, and an untreated control group. There were 3 rats in each experimental group, and each rat received a maximum of 3 observations. A total of 21 observations were made in the kidney group (Table 3). A total of 24 observations were made in the blood group. The experiments were performed in duplicate in the two assays, and these results were averaged as part of data preparation.

[0173]

Table 6

[0174]

[0151] Figure 2A graphically shows the abundance ratio of cDNA in lymphocytes extracted from transplanted kidneys of inbred rats, and Figure 2B shows the abundance ratio of cDNA in peripheral blood lymphocytes isolated from the same rats.

[0175] Statistical methods The comparisons shown in Table 4 were estimated using a linear mixed effects regression model, with separate models estimated for the relative proportions of kidney lymphocytes and DNA in the blood. The models included a fixed categorical effect for the experimental group (untreated as the reference group) and a random subject-specific intercept to account for repeated measures from up to three assays per rat. The mean of duplicate measurements within each rat / assay was calculated prior to analysis. The residuals from this model were assumed to be from a normal distribution and were evaluated using a normal quantile plot. Also, the variance of these residuals was initially assumed to be constant (evaluated by examining a plot of standardized residuals against fitted values). This assumption was later relaxed to allow for changes in variance within treatment groups. The estimated values of the comparisons are presented with 95% confidence intervals, along with the degrees of freedom estimated using the Satterthwaite approximation. All analyses were performed in the R programming environment (R version 4.2.0), and the linear mixed model was estimated using the Ime function from the "nlme" package.

[0176] Results

[0153] The mean difference in the percentage of heparanase expression levels (heparanase cDNA levels) of lymphocytes isolated from transplanted kidneys is shown in detail in Table 4. Table 4 shows a clear difference in heparanase levels between the heparanase inhibitor OGT2115 group and the untreated group. Specifically, the percent difference in the heparanase expression level of lymphocytes isolated from grafts from OGT2115 (10 mg / kg) compared to untreated transplanted rats was -14.5%, indicating that the heparanase expression level in the OGT2115 group was 14.5% lower than that in the untreated group. Furthermore, the heparanase expression level of lymphocytes isolated from grafts treated with castanospermine was -31% different compared to untreated transplanted rats. Therefore, in the castanospermine-treated group, the heparanase expression level decreased by 31% compared to the untreated group. Therefore, in rats treated with the heparanase inhibitor OGT2115, the heparanase expression level in graft lymphocytes was clearly decreased compared to the untreated group. Without wishing to be bound by theory, the results in Table 4 indicate that systemic administration of a heparanase inhibitor specifically decreased the heparanase expression level in graft lymphocytes involved in allograft immune rejection reactions.

[0177]

Table 7

[0178]

[0154] To determine whether systemic administration of a heparanase inhibitor results in the expected general decrease in heparanase in all lymphocytes, the heparanase expression levels of lymphocytes isolated from the peripheral blood of treated and untreated transplanted rats were measured. These results are shown in Table 5. Surprisingly, the level of heparanase in peripheral blood lymphocytes isolated from rats treated with a heparanase inhibitor was not decreased compared to untreated rats.

[0179]

Table 8

[0180] Conclusion

[0155] The results of Example 2 clearly show that the systemic administration of a heparinase inhibitor decreased the expression level of heparinase in isolated lymphocytes of the graft. However, this decrease in the heparinase expression level was not observed in lymphocytes isolated from the peripheral blood of rats that received transplants treated with a heparinase inhibitor. Therefore, this result indicates that the systemic administration of a heparinase inhibitor to a transplant recipient decreased the expression of heparinase in a targeted manner at the immune rejection site.

[0181]

[0156] Heparinase is known as a complex multifunctional enzyme involved in many systems and processes in the body. In Example 1, we showed that the administration of a heparinase inhibitor prolonged the survival period of allografts. The results of Example 2 revealed an unexpected and surprising advantage that the treatment of transplant rejection with a heparinase inhibitor is particularly targeted at decreasing the heparinase expression level at the transplant site. Therefore, it is considered that the treatment of transplant rejection with a heparinase inhibitor is less likely to affect a wide range of biological systems and processes in which heparinase may play a role, other than the immune rejection reaction at the transplant site. Thus, for example, the targeted treatment of transplant rejection by the administration of a heparinase inhibitor is considered to be beneficial as a treatment for transplant rejection because it is less likely to cause serious side effects.

[0182] Example 3 An experiment was conducted to examine the effect of castanospermine treatment on rats that received kidney allografts. Specifically, the inventor understood that the integrity of the cell membrane, extracellular matrix, and vascular endothelial basement membrane depends on heparan sulfate proteoglycan (HSPG). The inventor hypothesized that when HSPG is degraded by heparanase, the invasion of alloreactive cells into the allograft becomes possible. As a result, it is considered that the heparan sulfate moiety, including inflammatory cytokines and chemokines, is released from HSPG, activating alloreactive cells and causing rejection. Therefore, it was interesting to examine the ability of castanospermine to maintain the integrity of allograft organs by stopping the degradation of the HSPG entity.

[0183] Groups and statistical analysis

[0158] The groups used in this study are shown in Tables 6 and 7. Comparisons were made between the syngraft control treatment group (i.e., the control for ischemia-reperfusion injury), the untreated allograft group, and the castanospermine-treated allograft group. The primary comparison was between the untreated group and the castanospermine-treated group. The secondary comparisons were between the untreated group and the syngraft group, and between the castanospermine-treated group and the syngraft group.

[0184]

[0159] Multiple post hoc comparisons were made for each data source, so there was a possibility of a type I error. To address this, multiple methods were used: 1. Raw p-values using the comparison of the keyword-Roger approximation to the degrees of freedom and the Bonferroni correction threshold; 2. p-values adjusted using the Benjamin-Hochberg method to control the false discovery rate (FDR) at 5%; 3. The Bayes factor that measures how much evidence the data provides to support (or refute) the null hypothesis. The Bayes factor can be interpreted using the scale of Kass and Raftery (Journal of the American Statistical Association Vol. 90, No. 430: 773-795). In this analysis, values from 1 to 3.2 are considered irrelevant, from 3.2 to 10 are considered substantial, from 10 to 100 are considered strong, and values exceeding 100 are considered decisive.

[0185]

[0160] Within each group of hypotheses related to each primary and secondary objective, and for each data source individually, multiple testing adjustments were performed. A normal prior distribution (N(0,1000)) was used for the regression parameters, and a Cauchy (Cauchy(0,25)) was used for the residual variance. Samples from the posterior distribution were obtained using the No-U-Turn sampler with the Rstan package (Stan Development Team, https: / / mc-stan.org / ). The basic assumptions regarding the normality and heteroscedasticity of the residuals were evaluated graphically. There were no violations of normality, and the assumption of linearity was corrected by logarithmic transformation of the dose concentration. When there was evidence of unequal variance, robust heteroscedasticity-consistent regression standard errors were used when appropriate. All analyses were performed in R version 3.4.3 and SAS v9.4 (The SAS Institute, Cary, North Carolina).

[0186]

[0161] The H scores (tissue scores, outcome indicators) of the three groups on day 6 were compared using a linear mixed regression model incorporating fixed effects for the treatment group. The untreated group was set as the reference. The random effect for each rat modeled the dependence due to repeated measurements. The subject number was used as a random effect that enabled random intercepts and dependent data. Robust standard errors were used to adjust for heteroscedasticity of the residuals.

[0187] Digital image analysis

[0162] Digital images of the whole slide were acquired at 20× magnification using Aperio AT2 (Leica Biosystems, Wetzlar, Germany) and analyzed using HALO v2.0 software (Indica laboratory, Albuquerque, NM, USA). After selecting the tissue area, the “Area Quantification V1.0” algorithm was used to measure the percentage of the total pixels with weak, moderate, and strong staining intensities. The quantification of the histological score was calculated as H-score = (1 × weak%) + (2 × moderate%) + (3 × strong%) for each slide, and the range was 0–300 (30). Four to six sections were scored at various levels of the whole tissue for each sample, and the mean value was obtained.

[0188] ELISA assay for serum heparan sulfate

[0163] Serum samples separated from whole blood were aliquoted and stored at -80 °C. Then, a commercially available ELISA assay kit for heparan sulfate (CSB-E09585h) was used according to the manufacturer's recommended protocol (Cusabio, Wuhan, China). Briefly, samples were first incubated with the antibody for 30 min, and then the horseradish peroxidase-conjugated solution was added for 30 min. The substrate was added for 20 min in the dark to stop the reaction. All incubations were performed at 37 °C. The absorbance at 450 nm was immediately measured using a Versa Max microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the results were calculated in ng / ml according to the internal standard curve.

[0189] Treatment

[0164] Castanospermine was administered at a dose of 150 mg / kg per day by an Alzet osmotic pump (Alza Corporation, Palo Alto, USA).

[0190] Results

[0165] The results surprisingly show that castanospermine effectively blocks the degradation of HSPG in both allografts and sera of allogeneic rats. This is because there is no significant difference in the heparan sulfate (HS) concentration between castanospermine-treated rats and syngraft controls. Furthermore, in both situations, the untreated (rejected) level of HS was significantly higher than that of syngrafts and significantly higher than that of castanospermine-treated rats. Syngraft controls are controls for ischemia-reperfusion injury (IRI). Therefore, the dataset presented here excludes the influence of IRI and is limited to the influence of rejection reactions by the presence or absence of anti-rejection treatment (castanospermine).

[0191]

[0166] Table 6 shows the H scores for the comparison among three groups. The three treatment groups were studied on day 6 after transplantation. The results presented are the mean and 95% CI for each treatment group, the mean and 95% CI for the differences between them, the p-value of the differences, and the Bayes factors for the alternative hypothesis (BF 10 ) and the null hypothesis (BF 01 ). The quantification of the H score was calculated as H score = (1 × weak %) + (2 × moderate %) + (3 × strong %) for each slide, resulting in scores in the range of 0 - 300. Four to six slides per kidney sample per rat were scored (taken at different levels of the tissue), and the mean for that rat was obtained. Bayes factors estimate the degree to which the data provide evidence in support of (or against) the null hypothesis.

[0192]

[0167] Table 7 shows the comparison of serum heparan sulfate concentrations by treatment group and by study period (combinations of day 2, day 4, and day 6 after transplantation). The results presented are the ratios of the mean (estimated values) and 95% CI for the comparison of each group for the combinations of day 2, day 4, and day 6.

[0193] Discussion

[0168] The discovery that castanospermine effectively blocks the degradation of HSPG in allografts and serum is unexpected and has great significance for the treatment and prevention of allograft rejection. The use of syngeneic graft controls eliminates the effects of ischemia-reperfusion injury. This data means that, despite allografts being exposed to allograft rejection that destroys them, including their structure, the heparanase inhibitor castanospermine has the effect of maintaining the integrity of allografts. Therefore, the use of heparanase inhibitors has applications in induction, acute rejection, and maintenance therapy in allotransplantation.

[0194]

Table 9

[0195]

Table 10

[0196] Examples of preferred claims:

[0169] A method for preventing or treating allograft rejection, said method comprising the step of administering a heparanase inhibitor to a subject in need thereof, said heparanase inhibitor preventing or treating allograft rejection by reducing the level of heparanase activity.

[0197]

[0170] A method for maintaining the integrity of an allograft, said method comprising the step of administering a heparanase inhibitor to a subject in need thereof, said heparanase inhibitor maintaining the integrity of the allograft organ by reducing the level of heparanase activity.

[0198]

[0171] The method of

[0169] or

[0170] , wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the allograft site and does not reduce the expression level of heparanase in lymphocytes in the peripheral blood of the subject.

[0199] The method according to any one of

[0169] to

[0171] , wherein the allograft is a transplanted organ that has been primarily vascularized. The method according to any one of

[0169] to

[0172] , wherein the allograft is a graft of the heart, kidney, lung, heart-lung, liver, pancreas, stomach or intestine.

[0200] The method according to any one of

[0169] to

[0173] , wherein the heparinase inhibitor is a benzoxazole-5-yl acetic acid derivative.

[0175] The heparinase inhibitor is of the formula

[0201]

Chemical formula

[0202] The method according to any one of

[0169] to

[0174] , which is OGT2115 having the following formula, or a functional derivative or functional analog thereof. The method according to any one of

[0169] to

[0173] , wherein the heparinase inhibitor is castanospermine.

[0203] The method according to any one of claims

[0169] to

[0176] , wherein the heparinase inhibitor is administered in combination with one or more immunosuppressive agents. The method of

[0177] , wherein the immunosuppressive agent is selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analog of leflunomide; anti-CTLA4 antibody, anti-CTLA4 Ig fusion, anti-B lymphocyte stimulator antibody, anti-CD80 antibody, etanercept, infliximab, anti-T cell antibody, anti-CD3 antibody, OKT3, anti-CD4 antibody, anti-IL-2 receptor antibody, prednisolone or its derivative, anti-CD52 monoclonal antibody; anti-CD20 monoclonal antibody; belatacept; eculizumab; and intravenous immunoglobulin.

[0204] The method according to

[0178] , wherein the immunosuppressant is cyclosporine A or tacrolimus. The method according to any one of

[0169] to

[0176] , wherein the heparanase inhibitor is administered in combination with one or more anti-inflammatory drugs.

[0205] The method according to

[0180] , wherein the anti-inflammatory drug is selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

[0206] The method according to any one of

[0169] to

[0181] , which prolongs the survival period of the allograft. The method according to any one of

[0169] to

[0182] , wherein the heparanase inhibitor is administered to the subject at a dose of 2.5 mg / kg to 10 mg / kg.

[0207] The method according to any one of

[0169] to

[0183] , wherein the heparanase inhibitor is administered to the subject once a day. The method according to any one of

[0169] to

[0184] , wherein the heparanase inhibitor is administered to the subject once a week.

[0208] The method according to any one of

[0170] to

[0185] , wherein the integrity of the transplant is maintained by substantially preventing the degradation of heparan sulfate proteoglycan (HSPG) by heparanase.

[0209] Use of a heparanase inhibitor in the preparation of a medicament for the prevention or treatment of allograft rejection, wherein the heparanase inhibitor prevents or treats allograft rejection by reducing the level of heparanase activity.

[0210] Use of a heparanase inhibitor in the preparation of a medicament for maintaining the integrity of an allograft, wherein the heparanase inhibitor maintains the integrity of the transplant by reducing the level of heparanase activity.

[0211] Use according to

[0187] or

[0188] , wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the allograft site and does not reduce the expression level of heparanase in lymphocytes in peripheral blood.

[0212] Use according to any one of

[0187] to

[0189] , wherein the allograft is a transplant organ that is primarily angiogenic. Use according to any one of claims

[0187] to

[0190] , wherein the allograft is a transplant of the heart, kidney, lung, heart-lung, liver, pancreas, stomach or intestine.

[0213] Use according to any one of claims

[0187] to

[0191] , wherein the heparanase inhibitor is a benzoxazole-5-ylacetic acid derivative.

[0193] The heparanase inhibitor is of the formula

[0214]

Chemical

[0215] Use according to any one of claims

[0187] to

[0192] , which is OGT2115 having the formula or a functional derivative or functional analog thereof. Use according to any one of

[0187] to

[0191] , wherein the heparanase inhibitor is castanospermine.

[0216] Use according to any one of

[0187] to

[0194] , wherein the heparanase inhibitor is formulated for administration in combination with one or more immunosuppressive agents.

[0196] The use according to

[0195] , wherein the immunosuppressant is selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analogs of leflunomide; anti-CTLA4 antibodies, anti-CTLA4 Ig fusions, anti-B lymphocyte stimulator antibodies, anti-CD80 antibodies, etanercept, infliximab, anti-T cell antibodies, anti-CD3 antibodies, OKT3, anti-CD4 antibodies, anti-il-2 receptor antibodies, prednisone or its derivatives, anti-CD52 monoclonal antibodies; anti-CD20 monoclonal antibodies; belatacept; eculizumab; and intravenous immunoglobulin.

[0217]

[0197] The use according to

[0196] , wherein the immunosuppressant is cyclosporin A or tacrolimus.

[0198] The use according to any one of claims

[0187] to

[0194] , wherein the heparanase inhibitor is formulated for administration in combination with one or more anti-inflammatory agents.

[0218]

[0199] The use according to

[0198] , wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

[0219]

[0200] The use according to any one of claims

[0187] to

[0199] , wherein the use prolongs the survival period of the graft.

[0201] The use according to any one of claims

[0188] to

[0200] , wherein the integrity of the transplant is maintained by substantially preventing the degradation of heparan sulfate proteoglycan (HSPG) by heparanase.

[0220] References

[0221]

Table 11

Claims

**Claim 1** A method for preventing or treating allograft rejection reactions, said method comprising the step of administering a heparinase inhibitor to a subject in need thereof, said heparinase inhibitor preventing or treating allograft rejection reactions by reducing the level of heparinase activity. **Claim 2** A method for maintaining the integrity of an allograft, said method comprising the step of administering a heparinase inhibitor to a subject in need thereof, said heparinase inhibitor maintaining the integrity of an allograft by reducing the level of heparinase activity. **Claim 3** The method according to claim 1 or claim 2, wherein the heparinase inhibitor reduces the expression level of heparinase in lymphocytes at the allograft site, and does not reduce the expression level of heparinase in lymphocytes in the peripheral blood of the subject. **Claim 4** The method according to any one of claims 1 to 3, wherein the allograft is a transplant organ that has been primarily vascularized. **Claim 5** The method according to any one of claims 1 to 3, wherein the allograft is a transplant of the heart, kidney, lung, heart-lung, liver, pancreas, stomach or intestine. **Claim 6** The method according to any one of claims 1 to 5, wherein the heparinase inhibitor is a benzoxazole-5-yl acetic acid derivative. **Claim 7** The heparinase inhibitor has the formula 【Chemical 1】 The method according to any one of claims 1 to 6, which is OGT2115 or a functional derivative or functional analog thereof. **Claim 8** The method according to any one of claims 1 to 5, wherein the heparinase inhibitor is castanospermine. **Claim 9** The method according to any one of claims 1 to 8, wherein the heparinase inhibitor is administered in combination with one or more immunosuppressive agents. **Claim 10** The method according to claim 9, wherein the immunosuppressant is selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analogs of leflunomide; anti-CTLA4 antibodies, anti-CTLA4 Ig fusions, anti-B lymphocyte stimulator antibodies, anti-CD80 antibodies, etanercept, infliximab, anti-T cell antibodies, anti-CD3 antibodies, OKT3, anti-CD4 antibodies, anti-IL-2 receptor antibodies, prednisolone or its derivatives, anti-CD52 monoclonal antibodies; anti-CD20 monoclonal antibodies; belatacept; eculizumab; and intravenous immunoglobulin.

11. The method according to claim 9, wherein the immunosuppressant is cyclosporine A or tacrolimus.

12. The method according to any one of claims 1 to 8, wherein the heparanase inhibitor is administered in combination with one or more anti-inflammatory drugs.

13. The method according to claim 12, wherein the anti-inflammatory drug is selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

14. The method according to any one of claims 1 to 13, which prolongs the survival period of the allograft.

15. The method according to any one of claims 1 to 14, wherein the heparanase inhibitor is administered to the subject at a dose of 2.5 mg / kg to 10 mg / kg.

16. The method according to any one of claims 1 to 14, wherein the heparanase inhibitor is administered to the subject once a day.

17. The method according to any one of claims 1 to 14, wherein the heparanase inhibitor is administered to the subject once a week.

18. The method according to any one of claims 2 to 14, wherein the integrity of the allograft is maintained by substantially preventing the degradation of heparan sulfate proteoglycan (HSPG) by heparanase.

19. Use of a heparanase inhibitor in the preparation of a medicament for the prevention or treatment of allograft rejection, wherein the heparanase inhibitor prevents or treats transplant rejection by reducing the level of heparanase activity.

20. Use of a heparanase inhibitor in the preparation of a medicament for maintaining the integrity of an allograft, wherein the heparanase inhibitor maintains the integrity of the allograft by reducing the level of heparanase activity.

21. The use according to claim 19 or 20, wherein the heparanase inhibitor reduces the expression level of heparanase in lymphocytes at the allograft site, and does not reduce the expression level of heparanase in lymphocytes in peripheral blood.

22. The use according to any one of claims 19 to 21, wherein the allograft is a transplant organ that is primarily angiogenic.

23. The use according to any one of claims 19 to 22, wherein the allograft is a transplant of the heart, kidney, lung, heart-lung, liver, pancreas, stomach or intestine.

24. The use according to any one of claims 19 to 23, wherein the heparanase inhibitor is a benzoxazole-5-yl acetic acid derivative.

25. The heparanase inhibitor is [Chemical 2] OGT2115 having the formula or a functional derivative or functional analog thereof, and the use according to any one of claims 19 to 24.

26. The use according to any one of claims 19 to 23, wherein the heparanase inhibitor is castanospermine.

27. The use according to any one of claims 19 to 26, wherein the heparanase inhibitor is formulated for administration in combination with one or more immunosuppressive agents.

28. The immunosuppressive agent is selected from the group consisting of methotrexate, mizoribine, cyclosporine, aerosolized cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, sirolimus and other mTOR inhibitors, deoxyspergualin, leflunomide, malononitrile amide analog of leflunomide; anti-CTLA4 antibody, anti-CTLA4 Ig fusion, anti-B lymphocyte stimulatory antibody, anti-CD80 antibody, etanercept, infliximab, anti-T cell antibody, anti-CD3 antibody, OKT3, anti-CD4 antibody, anti-IL-2 receptor antibody, prednisolone or its derivatives, anti-CD52 monoclonal antibody; anti-CD20 monoclonal antibody; belatacept; eculizumab; and intravenous immunoglobulin, and the use according to claim 27.

29. The use according to claim 28, wherein the immunosuppressive agent is cyclosporin A or tacrolimus.

30. The use according to any one of claims 19 to 26, wherein the heparanase inhibitor is formulated for administration in combination with one or more anti-inflammatory agents.

31. The use according to claim 30, wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinolone, fluocinonide, prednisone, prednisolone and methylprednisolone.

32. The use according to any one of claims 19 to 31, wherein the use prolongs the survival period of the graft.

33. The use according to any one of claims 20 to 32, wherein the integrity of the allograft is maintained by substantially preventing the degradation of heparan sulfate proteoglycan (HSPG) by heparanase.

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