Treatment of necrotizing enterocolitis and symptoms thereof

Abstract

Methods of treating necrotizing enterocolitis (NEC) in a subject in need thereof are disclosed herein. Particularly, the methods include administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APE1 / Ref-1) inhibitor such as APX3330, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

Description

TREATMENT OF NECROTIZING ENTEROCOLITIS AND SYMPTOMS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority and the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 617,923, filed on January 5, 2024, which is incorporated by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0002] The present disclosure relates generally to the methods of treating necrotizing enterocolitis (NEC). Particularly, it has been found herein that by blocking the APE1 pathway, through the administration of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor (e.g., APX3330), transcription factors (TFs) involved in inflammation of the gastrointestinal tract are regulated, thereby alleviating the acute inflammatory cascade and oxidative stress within the gut of subjects suffering from necrotizing enterocolitis (NEC).

[0003] Necrotizing enterocolitis (NEC) is a devastating intestinal disease that affects premature or very low birth weight infants. NEC is characterized by destructive and progressive intestinal injury. Symptoms may include poor feeding, bloating, decreased activity, blood in the stool, vomiting of bile, bowel death, multiorgan failure, and even death.

[0004] The exact cause of NEC is unclear and there are no pharmacological therapeutic treatment options. The injury seen in NEC is thought to be caused by the inability to combat oxidative injury due to their premature state, allowing for the proliferation of toxins and exacerbation of injury. However, several risk factors have been identified. Consistently described risk factors include formula feeding, intestinal dysbiosis, low birth weight, and prematurity. Maternal factors such as chorioamnionitis, cocaine abuse, in utero growth restriction, intrahepatic cholestasis during pregnancy, increased body mass index, lack of prenatal steroids, mode of delivery, placental abruption, preeclampsia, and smoking have not been consistentlyimplicated with the development of NEC. Other risk factors potentially implicated include congenital heart disease, birth asphyxia, exchange transfusion, and prelabor rupture of membranes. The underlying mechanism is believed to involve a combination of poor blood flow and infection of the intestines. Infection can lead to both pro-inflammatory and oxidative stress induced conditions. Diagnosis is based on symptoms and confirmed with medical imaging.

[0005] Additionally, markers of oxidative stress in cord blood are predictive of developing NEC, which suggests that disturbances in the redox balance may precede disease onset. After birth, there is a dramatic shift in oxygenation from the intra to the extrauterine environment. At later stages of gestation, antioxidant defenses are strengthened prior to birth. Given that 90% of NEC cases occur in preterm infants, prevailing hypotheses surrounding oxidative stress in NEC include an inability to successfully modulate free radical scavenging, which can be accentuated by O2 supplementation, and formula feeding, which lacks the antioxidant properties of breast milk. Notwithstanding, inflammation is a critical aspect in the pathophysiology of NEC. Lipopolysaccharide (LPS) has been shown to increase O2 production via NOS uncoupling in rat models of NEC, which implicates oxidative stress in mediating later stages of the disease.

[0006] Prevention includes the use of breast milk and probiotics. Treatment includes bowel rest, orogastric tube, intravenous fluids, and intravenous antibiotics. Surgery is required in those who have free air in the abdomen. A number of other supportive measures may also be required. Complications may include short-gut syndrome, intestinal strictures, or developmental delay.

[0007] About 7% of those who are bom prematurely develop NEC; however, the odds of an infant developing this illness is directly related to the intensive care unit they are placed in. Onset is typically in the first four weeks of life. Among those affected, about 25% die. Furthermore, "surgical NEC" survivors are still at risk for possible long-term complications, such as narrowing of the intestines or short bowel syndrome and neurodevelopmental disability.

[0008] Treatment options are limited and continued research into targeted therapies is needed. Accordingly, the present disclosure provides insight into the inflammatory pathway that is present in NEC. APEl / Refl works to inhibit the inflammatory cascade in the gut and allows appropriate intestinal restitution. It is believed herein that blocking APEl / ref-1 redox signaling function will lead to the quieting of over-activated TF function and a subsequent dampening of inflammatory molecules back to normal levels. This APEl / Ref-1 redox inhibition with specific APX molecules such as APX3330 and subsequent related molecules also allows for the stimulation of the oxidative DNA repair component of APEl / Ref-1 to repair subsequent DNA damage. It has also been shown that oxidative DNA damage, e.g. 8oxoG, within the promoter regions of genes impacts gene expression such that repair of this lesion via APE1 repair activity will allow for correct transcription of genes required for normal cellular function and not dysfunctional gene activity.

[0009] APEl / Ref-1 (APE1) is a multifunctional protein found throughout the body known for its role in deoxyribonucleic acid (DNA) repair and transcription factor activation. Blocking the redox function of APE1 will not only block the activation of important TFs such as NFkB, STAT3, AP-1 and others w hich regulate inflammatory cytokines and proteins, but will lead to an increase in the APE1 DNA repair activity on oxidative DNA damage and subsequent protection and correction of cellular balance. It has also been shown that oxidative DNA damage, e.g. 8oxoG, within the promoter regions of genes impacts gene expression such that repair of this lesion via APE1 repair activity will allow for correct transcription of genes required for normal cellular function and not dysfunctional gene activity.BRIEF DESCRIPTION

[0010] The present disclosure relates generally to methods of treating NEC in subjects in need thereof. Particularly, it has been found herein that blocking APE1, through the administration of APX3330 (and / or analogs thereof), leads to inability of APE1 to interact with and reduce the oxidized TF targets, thereby reducing their activation of inflammatory signaling molecules. This involves a direct interaction of the APX3330 and analogs with the APE1 protein and causes unfolding of the proteinrendering it inactive for the redox signaling function. This also causes stimulation of the APE1 oxidative DNA repair function which affords protection of the cellular DNA from damage.

[0011] Further, oxidative stress is increasingly recognized as a central player in a range of gastrointestinal (GI) disorders. Apurinic / Apyrimidinic Endonuclease 1 / Redox Factor-1 (APEl / Ref-1) is a vital dual functioning protein that acts as an essential regulator of cellular responses to oxidative stress.

[0012] Based on the foregoing, in one aspect, the present disclosure is directed to a method of treating necrotizing enterocolitis (NEC) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0014] FIGS. 1 A-1D depict the effects of treatment in NEC mice with APX3330. Particularly, treatment with APX3330 resulted in significantly improved clinical sickness scores compared to control (FIG. 1 A) and improved median intestinal macroscopic score (FIG. IB). FIG. 1C depicts the change in weight with treatment with APX3330. FIG. ID depicts change in thorax / abdominal flux with treatment with APX33330.

[0015] FIG. 2 depicts baseline expression levels of TNF-alpha, TNFAIP2, IL- 6, and IL-8 in HIEC-6 cells were compared to cells stimulated with 0. 1 ng of TNF- alpha alone and those treated with APX3330 at concentrations of EC50, EC70, and EC80. The average fold change in TNF-alpha expression was 94.65 in the control group without APX3330 compared to 40.27, 33, and 25.52 for cells treated withAPX3330 at concentrations corresponding to EC50, EC 70. and EC80 respectively. The average fold change in TNFAIP2 was 6.87 in the control group without APX3330 compared to 3.54, 3.3, and 2.65 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively. The average fold change in IL-6 expression was 6.97 in the control group without APX3330 compared to 4. 14, 3.38, and 3.49 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively. The average fold change in IL- 8 expression was 22.59 in the control group without APX3330 compared to 15.62, 16.9, 13.43 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively

[0016] FIGS. 3A-3E depict improvement in NEC outcome with intraperitoneal administration of APX3330. FIG. 3A depicts Ref-1 inhibitor APX3330 improved median clinical sickness scores (Control score 2 (IQR 1.5-2.5) vs APX3330 score 0.5 (IQR 0.5-2.5)]. FIG. 3A shows higher median macroscopic intestinal injury scores in the control group compared to the APX3330-treated group [Control score 3 (IQR 3-3); APX3330 score 0 (IQR 0-0); p<0.0001] . FIG. 3C shows higher median microscopic intestinal injury scores in the control group compared to the APX3330- treated group [Control score 2.25 (IQR 0.88-3); APX3330 score 0 (IQR 0-1); pO.0001]. FIG. 3D shows no significant difference in WBC between control and APX3330 groups [Control 2.43 (IQR 1.74-4.18); APX3330 3.38 (IQR 0.09-4.6); p>0.99], FIG. 3E shows no significant difference in weight loss between the control and APX3330-treated group [Control 0.05 (IQR 0.01-0.18); APX3330 0.08 (IQR 0.05-0.3); p=0.5],DETAILED DESCRIPTION

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.

[0018] The present disclosure relates generally to methods of treating necrotizing enterocolitis (NEC) in a subject in need thereof. Particularly, it has been found herein that blocking APE1, through the administration of APX3330 (and / or analogs thereof), leads to inability7of APE1 to interact and reduce the oxidized TF targets, thereby reducing their activation of inflammatory signaling molecules. This involves a direct interaction of the APX3330 and analogs with the APE1 protein and causes unfolding of the protein rendering it inactive for the redox signaling function. This also causes a stimulation of the APE1 oxidative DNA repair function which affords protection of the cellular DNA from damage. Further, as APEl / Ref-1 is a dual functioning protein that acts as an essential regulator of cellular responses to oxidative stress, which stress plays an important role in pathophysiological mechanisms involved in gut inflammation, blocking APE1 through administration of APX3330 further reduces oxidative stress, thereby further reducing inflammation and other symptoms of NEC.

[0019] APEl / Ref-1 acts as a dual functioning molecule containing a redox active domain and a DNA repair domain (Kelley et al., 2012). APEl / Ref-1 redox active domain regulates cellular stress responses, angiogenesis, inflammation, and proliferation. In oxidative stress, levels of NO and cellular differentiation are controlled by APEl / Ref-1, by subsiding proapoptotic Tumour necrosis factor-a (TNF-a) signaling via pro-survival signaling.

[0020] In suitable embodiments, the present disclosure includes administering to a subject in need thereof an effective amount of an APE1 inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, the APE1 inhibitor capable of interacting with the APE1 protein such to cause unfolding of the APE1 protein in the amino terminal portion of APE1, inhibiting the ability of APE1 to interact with other proteins in the neurons or to perform its redox signaling function. In suitable embodiments, the present disclosure includes use of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof in a medicament for treating necrotizing enterocolitis (NEC) in a subject in need thereof. More particularly, APE1 inhibitors used in the present disclosure have anti-inflammatory effects, blocking the ability of APEl / Ref-1 to convert NF-KB and AP-1 from an oxidized state to reduced state, thereby altering their transcriptional activity. These inhibitors have been shown to suppress the production of pro-inflammatory cytokines and inflammatory mediators in murine macrophages. This results in the inability of NF-KB and AP-1 to bind to their target DNA sequence. Moreover, the inhibition allows direct down regulation of inflammatory cytokine secretion and ROS activation.

[0021] Targeting the specific inhibition of APEl / Ref-1 redox pathways and utilizing the DNA repair domain can lead to a NEC therapy. The inhibition by APX3330 and analogs results in the inability of APE1 to interact and reduce the oxidized TF targets, thereby reducing their activation of inflammatory signaling molecules. This involves a direct interaction of the APX3330 and analogs with the APE1 protein and causing unfolding of the protein rendering it inactive for the redox signaling function. This also causes stimulation of the APE1 oxidative DNA repair function w hich affords protection of the cellular DNA from damage

[0022] Accordingly, in particularly suitable embodiments, the APE1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R2 is an alkyl; R3 and Re are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R4 and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted napthoquinone;X is selected from the group consisting of CH=CR.2 and NCH, wherein R2 is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(RZ)R.2 or NRAORA, wherein each Rzis independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rzand R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

[0023] Particularly suitable APE1 inhibitors include 3-[(5-(2,3-dimethoxy-6- methyl l,4-benzoquinoyl)]-2-nonyl-2-proprionic acid, (hereinafter "E3330" or "3330" or "APX3330"), and / or its analogs (e.g., [(2E)-2-[(3-methoxy-l,4-dioxo-l,4- dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide] (hereinafter "APX2009"), (2E)-2-[(3-methoxy-l,4-dioxo-l,4-dihydronapthalen -2- yl)methylidene]-N,N-dimethylpentanamide] (hereinafter "APX2007"), (2E)-2-[(3- methoxy-1 ,4-dioxo-l ,4-dihydronapthalen -2-yl)methylidene]-N- methoxypentanamide] (hereinafter "APX2014"), (2E)-2-(3-methoxy-l,4-dioxo-l,4- dihydronaphthalen-2-yl)-N,N,2-trimethylprop-2-enamide (hereinafter "APX2032")). Additional suitable analogs are shown below and in Table 1. Further information on APX3330 may be found in Abe et al., U.S. Pat. No. 5,210,239, and information on APX2009 may be found in Kelley et al., J Pharmacol Exp Ther. 2016 Nov, 359(2): 300-309, each incorporated herein by reference to the extent they are consistent herewith.APX2032Table 1 :

[0024] It has herein been found that the administration of APX333O (and / or its analogs) inhibits APE1 protein from interacting with other proteins in the neurons. This interaction inhibition blocks the activation of the transcription factors (TFs) through a reduction-oxidation mechanism. Blocking TF activation results in the lack of their functional activity involving binding to the promoter region of genes involved in inflammation. Further, the inhibition allows for APE1 to be free to perform enhanced DNA repair functions at an oxidized or abasic site in damaged DNA (damaged by inflammatory and other effectors of neuronal pain pathway induction), thereby repairing the DNA and allowing for the proper activity of the genes needed for normal cellular function. Therefore, the mechanism is two-fold; blocking inflammatory TFs from being active as well as enhancing the repair of damaged DNA leading to the proper function cells in the gastrointestinal system.

[0025] Suitable dosages of the APE1 inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, for use in the methods of the present disclosure will depend upon a number of factors including, for example, age and weight of an individual, severity of inflammatory or chronic pain, nature of a composition, route of administration and combinations thereof. Ultimately, a suitable dosage can be readily determined by one skilled in the art such as, for example, a physician, a veterinarian, a scientist, and other medical and research professionals. For example, one skilled in the art can begin with a low dosage that can be increased until reaching the desired treatment outcome or result. Alternatively, one skilled in the art can begin with a high dosage that can be decreased until reaching a minimum dosage needed to achieve the desired treatment outcome or result. Pharmaceutically acceptable salts are known to one skilled in the art and acceptable salts include salts of benzoquinone derivatives with inorganic acids, such as hydrochloride, hydrobromide, sulfate, and phosphate; those with organic acids, such as acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and toluenesulfonate; and those with amino acids, such as arginine, aspartic acid, and glutamic acid. Pharmaceutically acceptable salts are also in the form of metallic salts such as sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg) salts, and thesemetallic salts as well are within the scope of the pharmacologically acceptable salts of the present disclosure.

[0026] In one particularly suitable embodiment, the APEl / Ref-1 inhibitor is APX3330, and the subject is administered from about 1.0 pM to about 50 pM APX333O.

[0027] In another suitable embodiment, the APEl / Ref-1 inhibitor is APX333O and the subject is a human. In an in vivo administration, the APEl / Ref-1 inhibitor is administered to the human patient in concentrations of from about 100 to about 600 mg per day. In one embodiment, the administration is intraperitoneal. In another embodiment, the administration is intravenous.

[0028] In some embodiments, the APE1 inhibitor is administered via a composition that includes the APE1 inhibitor and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be, for example, excipients, vehicles, diluents, and combinations thereof. For example, where the compositions are to be administered orally, they may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intravitreal), drop infusion preparations, or suppositories. These compositions can be prepared by conventional means, and, if desired, the active compound (e.g., APX333O) may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, or combinations thereof.

[0029] It should be understood that the pharmaceutical compositions of the present disclosure can further include additional known therapeutic agents, drugs, modifications of the synthetic compounds into prodrugs, and the like for alleviating, mediating, preventing, and treating the diseases, disorders, and conditions described herein. For example, in one embodiment, the APE1 inhibitor can be administered with one or more of current therapeutic agents and drugs for treating NEC (e.g., 5-aminosalicylic acid (5-ASA), corticosteroids, anti-TNF drugs (e.g., infliximab, certolizumab, adalimumab, and golimumab), vedolizumab, natalizumab, ustekinumab, probiotics, antibiotics, anti-inflammatories (e.g., mesalamine (Asacol HD, Delzicol, others), balsalazide (Colazal) and olsalazine (Dipentum), and the like).

[0030] The pharmaceutical compositions including the APE1 inhibitor and / or pharmaceutical carriers used in the methods of the present disclosure can be administered to a subset of individuals / subjects in need. As used herein, a "subject in need" refers to an individual at risk for or having NEC. As such, in some embodiments, the methods disclosed herein are directed to a subset of the general population such that, in these embodiments, not all of the general population may benefit from the methods. Based on the foregoing, because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified individuals (that is, the subset or subclass of subjects "in need" of assistance in addressing one or more specific conditions noted herein), not all individuals will fall within the subset or subclass of individuals as described herein. In particular, the individual in need is a human. The individual in need can also be, for example, a research animal such as, for example, a non-human primate, a mouse, a rat, a rabbit, a cow, a pig, and other types of research animals known to those skilled in the art.

[0031] Various functions and advantages of these and other embodiments of the present disclosure will be more fully understood from the examples shown below. The examples are intended to illustrate the benefits of the present disclosure, but do not exemplify the full scope of the disclosure.EXAMPLESEXAMPLE 1

[0032] In this Example, cross-sectional slices of neonatal bowel tissue with and without NEC were assessed for APE1 expression from the NEC Biorepository by immunohistochemistry. 2-way ANOVA testing was performed with p-value <0.05considered significant. For the experimental murine model, five-day old pups were separated from their mothers and placed in a satellite house. To induce NEC, pups were gavage fed a hyperosmolar formula containing lipopolysaccharide and stressed with intermittent hypoxia and hypothermia. The control mice group (n=9) underwent experimental NEC alone while the treatment mice group (n=5) also received oral APX3330 (50mg / kg) once daily. On post-natal day 9, the pups were euthanized. Intestinal histologic injury and clinical sickness were scored and analyzed with Mann- Whitney testing with p<0.05 considered significant.

[0033] Results are shown in FIGS. 1 A-1D. Weight gain was similar between both groups (p=0.15). Treatment with APX3330 resulted in significantly improved clinical scores compared to control (APX= 1 (IQR 1-1), Control= 3 (IQR 2-3), p=0.001). The APX3330 group had a median intestinal macroscopic score of 1 (IQR 0-1), while the control group had a score of 2 (IQR 2-3) (p=0.002). Median intestinal histology score was 0 (IQR 0-0.75) for the APX3330 group and 1.75 (1.125-2) for the control group (p=0.0054).

[0034] In conclusion, administration of oral APX3330 improves clinical outcomes and protects the intestine from injury in a murine model of necrotizing enterocolitis.EXAMPLE 2

[0035] In this Example, APX3330 was used in an in vitro human-derived cell injury model to evaluate bimolecular changes and analyze whether APX333O suppresses inflammation in TNF-alpha injured human intestinal epithelial cells (HIEC-6).

[0036] HIEC-6 cells were cultured with media and stimulated with 0.1 ng of TNF-alpha to induce cellular injury. Additionally, a separate group of cells was stimulated with 0.1 ng of TNF-alpha followed by treatment with APX3330 at concentrations corresponding to EC50, EC70, and EC80. RNA was then extracted 6 hours after injury, and the quality and quantity were confirmed via spectrophotometer.Complementary DNA was synthesized from these samples. Using these cDNA samples, real time quantitative polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of TNF-alpha, TNF-alpha induced protein 2 (TNFAIP2), IL-6, and IL-8, markers of inflammation commonly seen upregulated in NEC. Using the delta-delta Ct method, qRT-PCR data was used to quantify relative gene expressions. A Mann- Whitney test was used to compare groups. P <0.05 was considered significant.

[0037] Baseline expression levels of TNF-alpha, TNFAIP2, IL-6, and IL-8 in HIEC-6 cells were compared to cells stimulated with 0.1 ng of TNF-alpha alone and those treated with APX333O at concentrations of EC50, EC70, and EC80. The average fold change in TNF-alpha expression was 94.65 in the control group without APX3330 compared to 40.27, 33, and 25.52 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively (FIG. 2A). The average fold change in IL-6 expression was 6.97 in the control group without APX3330 compared to 4.14, 3.38, and 3.49 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively (FIG. 2B). The average fold change in TNFAIP2 was 6.87 in the control group without APX3330 compared to 3.54, 3.3, and 2.65 for cells treated with APX333O at concentrations corresponding to EC50, EC70, and EC80 respectively (FIG. 2C). The average fold change in IL-8 expression was 22.59 in the control group without APX3330 compared to 15.62, 16.9, 13,43 for cells treated with APX3330 at concentrations corresponding to EC50, EC70, and EC80 respectively (FIG. 2D).

[0038] A significant reduction of TNF-alpha, TNFAIP2, IL-6, and IL-8 expression was seen in HIEC-6 cells stimulated by TNF-alpha when in the presence of increasing concentrations of APX3330. There was a stepwise decrease in the expression level of TNF-alpha, TNFAIP2, IL-6, and IL-8 with increasing APX3330 concentration.

[0039] In this injury model, there was a significant reduction of TNF-alpha, TNFAIP2, IL-6, and IL-8 with increasing concentrations of APX3330. This reduction ininflammation observed in the HIEC-6 cells indicated a dose-dependent anti-inflammatory effect of APX3330.EXAMPLE 3

[0040] In this Example, APX3330 was used to evaluate whether inhibiting Ref-1 with intraperitoneal (IP) APX3330 would improve outcomes in experimental NEC.

[0041] NEC was induced in post-natal day 5 (P5) mice using hyperosmolar lipopolysaccharide formula and intermittent hypoxia and hypothermia. NEC control mice (n=3) underwent experimental NEC with IP vehicle injection while the treatment group (n=3) received IP APX3330 (50mg / kg) twice daily. On post-natal day 9, the pups were euthanized. Clinical sickness and intestinal injury were scored and analyzed by Mann- Whitney, where p<0.05 was considered significant.

[0042] APX3330-treated mice had lower median clinical sickness scores [APX3330 score 0.5 (IQR 0.5-2.5) vs Control score 2 (IQR 1.5-2.5)] and median histological intestinal injury scores [APX3330 score 0 (IQR 0-1) vs Control score 2.25 (IQR 0.88-3); p<0.0001] compared to control.

[0043] These results demonstrate an improvement in NEC outcomes when mice are treated with APX3330, a novel Ref-1 inhibitor, via intraperitoneal administration. Previous data demonstrated improvement in clinical sickness in a murine NEC model with oral administration of APX3330. However, clinical relevance of oral APX3330 is limited since patients with NEC generally do not receive anything by mouth. These results demonstrate that intraperitoneal administration of an inhibitor of APEl / Ref-1 provides an alternative to oral administration of the inhibitor for treating NEC.

Claims

CLAIMSWhat is claimed is:

1. A method of treating necrotizing enterocolitis (NEC) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

2. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R2 is an alkyl; R3 and Re are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R4 and Rs are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted napthoquinone;X is selected from the group consisting of CH=CR2 and NCH, wherein R2 is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting ofN(Rz)R2 orNRAOR , wherein each Rz is independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rz and R2 taken together with the attached nitrogen form an optionally substituted heterocycle;where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

3. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor is selected from an inhibitor set forth in Table 1.

4. The method as set forth in claim 1 , wherein the APEl / Ref-1 inhibitor is selected from the group consisting of 3-[(5-(2,3-dimethoxy-6-methyl l,4-benzoquinoyl)]- 2-nonyl-2-proprionic acid (APX3330), (2E)-2-[(3 -meth oxy-1, 4-dioxo- 1,4- dihydronapthalen -2-yl)methylidene]-N,N-dimethylpentanamide] (APX2007), [(2E)-2- [(3 -methoxy- 1 , 4-dioxo- 1 ,4-dihydronaphthalen-2-yl)methylidene]-N,N- diethylpentanamide] (APX2009), (2E)-2-[(3-methoxy-l,4-dioxo-l,4-dihydronapthalen -2- yl)methylidene]-N-methoxypentanamide] (APX2014), (2E)-2-(3 -methoxy- 1 ,4-dioxo-l ,4- dihydronaphthalen-2-yl)-N,N,2-trimethylprop-2-enamide (APX2032), pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof, and combinations thereof.

5. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor is APX3330 and the subject is administered from about 1.0 pM to about 50 pM APX3330.

6. The method as set forth in claim 1, wherein the subject is a human patient and the APEl / Ref-1 inhibitor is APX3330, and wherein the subject is administered from about 100 to about 600 mg per day.

7. The method as set forth in claim 1, wherein the administration is intraperitoneal.

8. The method as set forth in claim 1, wherein the administration is intravenous.

9. The method as set forth in claim 1 further comprising administering at least one additional therapeutic agent to the subject.

10. The method as set forth in claim 9, wherein the additional therapeutic agent is selected from the group consisting of 5-aminosalicylic acid (5-ASA), corticosteroids, anti-TNF drugs, vedolizumab, natalizumab, ustekinumab, probiotics, antibiotics, antiinflammatories, balsalazide (Colazal), olsalazine (Dipentum), and combinations thereof.11 . Use of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref- 1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof in a medicament for treating necrotizing enterocolitis (NEC) in a subject in need thereof.

12. The use as set forth in claim 11, wherein the APEl / Ref-1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R2 is an alkyl; R3 and Re are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R4 and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted napthoquinone;X is selected from the group consisting of CH=CR2 and NCH, wherein R2 is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(Rz)R.2 or NRAORA, wherein each Rz is independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rz and R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

13. The use as set forth in claim 11, wherein the APEl / Ref-1 inhibitor is selected from an inhibitor set forth in Table 1.

14. The use as set forth in claim 11, wherein the AREl / Ref-1 inhibitor is selected from the group consisting of 3-[(5-(2,3-dimethoxy-6-methyl 1,4-benzoquinoyl)]- 2-nonyl-2-proprionic acid (APX3330), (2E)-2-[(3 -methoxy- 1,4-di oxo- 1,4- dihydronapthalen -2-yl)methylidene]-N,N-dimethylpentanamide] (APX2007), [(2E)-2- [(3 -methoxy- 1 ,4-dioxo- 1 ,4-dihydronaphthalen-2-yl)methylidene]-N,N- diethylpentanamide] (APX2009), (2E)-2-[(3-methoxy-l,4-dioxo-l,4-dihydronapthalen -2- yl)methylidene]-N-methoxypentanamide] (APX2014), (2E)-2-(3 -methoxy- 1,4-di oxo- 1,4- dihydronaphthalen-2-yl)-N,N,2-trimethylprop-2-enamide (APX2032), pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof, and combinations thereof.

15. The use as set forth in any one of claims 11, 12, 13, or 14, wherein the medicament is for intraperitoneal administration.

16. The use as set forth in any one of claims 11, 12, 13, or 14, wherein the medicament is for intravenous administration.

17. The use as set forth in any one of claims 11, 12, 13, 14, 15 or 16, wherein the APEl / Ref-1 inhibitor is APX333O and the subject is administered from about 1.0 pM to about 50 pM APX3330.

18. The use as set forth in any one of claims 11, 12, 13, 14, 15, or 16, wherein the APEl / Ref-1 inhibitor is APX3330 and the subject is a human patient administered from about 10 mg / day to about 600 mg / day.

19. The use as set forth in any one of claims 11, 12, 13, 14, 15, 16, 17, or 18, further comprising use of at least one additional therapeutic agent in combination with the APE 1 / Ref- 1 inhibitor.

20. The use as set forth in claim 19, wherein the additional therapeutic agent is selected from the group consisting of 5-aminosalicylic acid (5-ASA), corticosteroids, azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, anti-TNF drugs vedolizumab, natalizumab, ustekinumab, probiotics, antibiotics, anti-inflammatories, and combinations thereof.

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