HK1253277B - Use of ep4 receptor antagonists for the treatment of nash-associated liver cancer - Google Patents

Description

Use of EP4 receptor antagonists for treating NASH-related liver cancer

Technical Field

The present invention relates to a prostaglandin E2 receptor 4 (EP4) antagonist for treating non-alcoholic steatohepatitis (NASH)-related liver cancer. The method for treating NASH-related liver cancer comprises administering to a human or animal any one of 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl] (hereinafter referred to as "Compound A"), 4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (hereinafter referred to as "Compound B"), or 3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-methylphenyl)sulfonyl]urea (hereinafter referred to as "Compound C"), or a pharmaceutically acceptable salt thereof as an EP4 antagonist (hereinafter referred to as "the compound of the present invention"), or a pharmaceutical composition comprising the compound of the present invention. The treatment method includes administering a single compound of the present invention to a human or animal suffering from NASH-related liver cancer, or administering it in combination with one or more other active agents and/or treatments. The treatment method also includes a therapy that regulates the function of specific immune cells and/or their distribution in the tumor tissue of NASH-related liver cancer by EP4 antagonists, including compounds of the present invention. In addition, the present invention relates to a pharmaceutical composition or kit comprising a compound of the present invention or a pharmaceutically acceptable salt thereof. Hereinafter, "compound of the present invention" includes any one or a combination of compound A, compound B and compound C alone, or a pharmaceutically acceptable salt of any one of these compounds.

Background Art

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States, with an estimated prevalence of 30% to 40% of the adult population. Although it is generally believed that only 5% to 20% of patients with NAFLD meet the histopathological criteria for non-alcoholic steatohepatitis (NASH), this still means that 2% to 5% of the population nationwide is at increased risk of developing cirrhosis. Cirrhosis itself is a sufficient risk factor for liver cancer, including hepatocellular carcinoma (HCC). Obesity may also increase the risk of developing HCC, with meta-analyses of population studies showing a 90% increased risk of HCC in obese patients. This may partly explain the gradual increase in HCC rates in developed countries and the 80% increase in the annual incidence of HCC in the United States over the past two decades (Non-Patent Literature 1: Torres et al., Semin Liver Dis., 2012, 32(1): 30-38).

More worryingly, HCC has been shown to occur in patients with non-cirrhotic NASH. Overall, these data provide strong evidence that the incidence of HCC may be higher in patients with non-cirrhotic NASH than in patients with cirrhotic NASH, but larger prospective studies are needed to obtain incidence rates. The relationship between NASH and liver cancer is well established. Large case studies have demonstrated that elderly people (predominantly male patients) with a specific phenotype of concurrent metabolic syndrome can develop HCC in the absence of a background of cirrhosis. Some of the purportedly complex pathophysiological mechanisms that lead to this condition have been described, but a comprehensive understanding of the interactions and overlap between NASH-promoting and carcinogenic processes is still in progress (Non-Patent Literature 1).

The main causes of hepatitis and HCC can be divided into the following two categories: (1) hepatitis B (HBV) and hepatitis C (HCV) infection, and (2) metabolic causes such as alcohol consumption and NAFLD. In general, chronic viral infection-mediated hepatitis is the most common cause of hepatitis, followed by alcoholic liver disease and NAFLD. Medical treatment for viral infection-mediated hepatitis has been established, and antiviral treatment using interferon α and nucleotide analogs is commonly prescribed. On the other hand, medical treatment for NAFLD, NASH, and diseases related to liver cancer has not yet been established because the disease itself has only recently been recognized.

Obesity has become a global health problem and is known to increase the risk of diabetes, cardiovascular disease, and several types of cancer. Based on epidemiological studies, among obesity-related cancers, a strong relationship between liver cancer and obesity has been confirmed (Non-patent Document 2: Bhaskaran et al., Lancet, 2014, 384: 755-765; Non-patent Document 3: Calle and Kaaks, Nature Reviews Cancer, 2004, 4: 579-591; Non-patent Document 4: Calle et al., New England J. Med., 2003, 348 (17): 1625-1638). The most common risk factor for HCC is long-term infection with HBV or HCV (Non-patent Document 5: El-Serag, New England J. Med., 2011, 365: 1118-1127; Non-patent Document 6: Marengo et al., Annual Review of Medicine, 2016, 67: 103-117). However, obesity-related NAFLD and NASH have recently emerged as risk factors for liver cancer (Non-Patent Document 6; Non-Patent Document 7: Michelotti et al., Nat. Rev. Gastroenterol. Hepatol., 2013, 10: 656-665; and Non-Patent Document 8: Streba et al., World J. Gastroenterology, 2015, 21(14): 4103-4110). Currently, there are no available treatments for NAFLD, NASH, and NASH-related liver cancer. Therefore, there is an urgent need to develop treatments for NASH-related liver cancer.

Prostaglandins are mediators of pain, fever and other symptoms associated with inflammation. Prostaglandin E2 (PGE2) is the main eicosanoid expressed under inflammatory conditions. PGE2 is also involved in various physiological and/or pathological conditions, such as hyperalgesia, uterine contraction, digestive motility, wakefulness, inhibition of gastric acid secretion, blood pressure, platelet function, bone metabolism, angiogenesis and cancer cell growth, invasion and metastasis. Non-patent references disclose the characteristics of prostaglandin receptors, the relationship with treatment and the most commonly used selective agonists and antagonists (see, for example, non-patent literature 9: Konya et al., Pharmacology & Therapeutics, 2013, 138: 485-502; and non-patent literature 10: Yokoyama et al., Pharmacol. Rev., 2013, 65: 1010-1052).

It is reported that PGE2 is highly expressed in the cancerous tissue of various cancers, and has confirmed that PGE2 is relevant to the generation, growth and development of patient's cancer and disease condition.It is generally acknowledged that PGE2 is relevant to the activation of cell proliferation and cell death (apoptosis), and plays an important role in the process of cancer cell proliferation, disease progression and cancer metastasis (see, for example non-patent literature 9; and non-patent literature 10).

There are four kinds of PGE2 receptor subtypes EP1, EP2, EP3 and EP4, and they show different pharmacological properties.EP4 receptor subtype belongs to G protein coupled receptor subfamily, is called as the receptor with seven transmembrane domains.Therefore, EP4 plays an important role in biological events by stimulating the function of cAMP signal mediation.From the angle of pharmacological research, carried out some researches on the compounds with EP4 receptor antagonist activity, and EP4 receptor selectivity antagonist is known (non-patent literature 9).

With respect to the role of the EP4 receptor in cancer, several non-patent documents (e.g., non-patent document 10; and non-patent document 11: Ma et al., Oncoimmunology, 2013, 2(1): e22647) and patent documents (Patent Document 1: U.S. Patent No. 8,921,391B2) have demonstrated the inhibition of growth and/or metastasis of colon cancer, breast cancer, gastric cancer, lung cancer, prostate cancer, and other cancer types in animal tumor models using EP4 receptor antagonists. Some patent documents (e.g., Patent Document 2: WO2015/179615A1 and Patent Document 3: US2015/0004175A1) show that EP4 receptor antagonists have therapeutic efficacy or inhibit EP4 signaling to inhibit tumor growth. In addition, EP4 signaling inhibition in combination with other anticancer therapeutic agents or radiotherapy has shown additional benefits compared to each monotherapy (see Patent Document 2).

The role of EP4 receptor in liver cancer has recently been reported in non-patent literature. PGE2/EP4 receptor signaling activated by PKA/CREB upregulated the expression of c-Myc and led to the promotion of cell growth in HCC cells in vitro (non-patent literature 12: Xia et al., Oncology Reports, 2014, 32: 1521-1530). PGE2 also promotes the accumulation of bone marrow-derived suppressor cells (MDSCs) induced by hepatic stellate cells in vitro and in vivo experiments, which is believed to stimulate the growth of liver cancer (non-patent literature 13: Xu et al., Oncotarget, 2016, 7 (8): 8866-8878). This document suggests that PGE2/EP4 signaling may play a role in liver cancer growth. However, these references did not confirm liver cancer inhibition by PGE2/EP4 signaling inhibition in animals. Inhibition of PGE2/EP4 receptor signaling alone (or in combination with anti-programmed cell death protein 1 (PD-1) antibodies) restored CD8 ⁺ T cell (CTL) functional activity (Non-patent document 14: Chen et al., Nature Medicine, 2015, 21(4):327-334; and Patent document 3). These references indicate that EP4 signaling inhibition mediates the activation of host CTL activity, but there is no direct evidence that EP4 signaling inhibition or EP4 antagonism has an effect on HCC growth and/or metastasis.

U.S. Patent No. 8,921,391B2 (Patent Document 1) demonstrates the antitumor efficacy of Compounds A, B, and/or C in animal tumor models for gastric, lung, prostate, and other cancer types. This patent does not provide any experimental examples related to liver cancer, and liver cancer does not appear in any of the claims. Furthermore, this patent does not disclose treatment for liver cancer associated with nonalcoholic steatohepatitis (NASH) and does not disclose any information related to NASH or NAFLD.

In 2015, a key issue of PGE2/EP4 signal inhibition in the treatment of liver cancer, especially chronic viral infection-related diseases such as HBV and HCV-mediated liver diseases, was reported. In animal models, inhibition of PGE2/EP4 signals significantly induced or activated PD-1 expression in virus-specific CD8 ⁺ T cells (CTLs) (non-patent literature 14). The increase in PD-1 expression on CTLs strongly suggests the suppression of T cell-mediated key immune functions against viral infection. In the case of chronic HBV or HCV-infected liver cancer, the increase in PD-1 expression on CTLs should therefore cause viral amplification and stimulation of tumor development and growth. In recent clinical cancer treatments, the effect of increased PD-1 expression on CTLs on tumor growth and development has been clearly demonstrated by the significant efficacy of their inhibitors (e.g., anti-PD-1 antibodies or immune checkpoint inhibitors). Therefore, this study has attracted widespread attention to the risk of increasing tumor growth (non-patent literature 14) by PGE2/EP4 signal inhibition in the treatment of liver cancer. Therefore, it is expected that PGE2/EP4 signaling inhibition activates PD-1 expression, which will suppress the immune response to viral infection and then promote viral development and HCC progression.

In view of this negative concern about EP4 signaling inhibition in liver cancer treatment, the inventors unexpectedly discovered significant anti-tumor effects and inhibition of PD-1 expression on CD8 ⁺ T cells (CTLs) in NASH-associated liver cancer. This is in contrast to the results of PD-1 expression in a virus-associated liver cancer model of EP4 antagonists in monotherapy in animal models and in combination with another drug. NASH-associated liver cancer has a different etiology from liver cancer associated with viral infection. To date, there is no evidence to support the EP4 mechanism (including EP4 antagonistic activity) in therapies for the treatment of NASH-associated liver cancer. In addition, there is no evidence in the art regarding the efficacy of combination therapy of EP4 receptors with any other therapy in the treatment of NASH-associated liver cancer. Therefore, the use of EP4 antagonists in NASH-associated liver cancer is now unexpected for the prior art.

Reference List

Patent Literature

{Patent Document 1} U.S. Patent No. 8,921,391B2

{Patent Document 2} WO 2015/179615

{Patent Document 3} US 2015/0004175A1

Non-patent literature

{Non-patent document 1} Torres et al., Semin Liver Dis., 2012, 32(1): 30-38

{Non-Patent Document 2} Bhaskaran et al., Lancet, 2014, 384: 755-765

{Non-patent document 3} Calle and Kaaks, Nature Reviews Cancer, 2004, 4: 579-591

{Non-patent document 4} Calle et al., New England J. Med., 2003, 348(17): 1625-1638

{Non-Patent Document 5} El-Serag, New England J. Med., 2011, 365: 1118-1127

{Non-Patent Document 6} Marengo et al., Annual Review of Medicine, 2016, 67: 103-117

{Non-Patent Document 7} Michelotti et al., Nat. Rev. Gastroenterol. Hepatol., 2013, 10: 656-665

{Non-patent document 8} Streba et al., World J. Gastroenterology, 2015, 21(14): 4103-4110

{Non-patent document 9} Konya et al., Pharmacology & Therapeutics, 2013, 138: 485-502

{Non-patent document 10} Yokoyama et al., Pharmacol. Rev., 2013, 65: 1010-1052

{Non-patent document 11} Ma et al., Oncoimmunology, 2013, 2(1):e22647

{Non-patent document 12} Xia et al., Oncology Reports, 2014, 32: 1521-1530

{Non-patent document 13} Xu et al., Oncotarget, 2016, 7(8): 8866-8878

{Non-patent document 14} Chen et al., Nature Medicine, 2015, 21(4): 327-334

{Non-Patent Document 15} Ohtani et al., Cancer Research, 2014, 74: 1885-1889

{Non-Patent Document 16} Fuertes et al., J. Exp. Med., 2011, 208: 2005-2016

{Non-Patent Document 17} Salmon et al., Immunity, 2016, 44: 924-938

{Non-Patent Document 18} Zelenay et al., Cell, 2015, 162: 1257-1270

Summary of the Invention

Technical issues

As mentioned above, obesity has become a global health problem and is known to increase the risk of several types of cancer. Obesity-related liver cancer has different etiological risk factors from HCC mediated by long-term infection with HBV or HCV. According to epidemiological studies, liver cancer has been shown to have a strong relationship with obesity. In addition, obesity-related NAFLD and NASH have recently emerged as risk factors for liver cancer. Therefore, there is an urgent need for precise molecular mechanisms mediating the development of obesity- and/or NASH-related liver cancer and therapeutic agents for these diseases. It is expected that the treatment of chronic HBV- and HCV-infected liver cancer by PGE2/EP4 signaling inhibition will increase liver cancer diagnosis by increasing PD-1 expression on CTLs. Therefore, there are concerns about the use of EP4 signaling inhibition to treat liver cancer, not only chronic virally infected liver cancer, but also alcohol-, NAFLD-, and NASH-related liver cancer.

The present invention aims to provide a method for treating NASH-related liver cancer using an EP4 receptor antagonist alone or in combination with an available therapeutic agent. To achieve this goal, the inventors have found that each of the following three compounds and pharmaceutically acceptable salts thereof significantly reduced the growth and progression of obesity-induced NASH-related liver cancer in a validated mouse model:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C).

Therefore, the present invention is based on the discovery that the compounds of the present invention exhibit selective EP4 antagonist activity in a mouse model and inhibit the growth and progression of obesity-induced NASH-related liver cancer. This is the first discovery of an EP4 antagonist's therapeutic efficacy against obesity-induced NASH-related liver cancer. Furthermore, the inventors discovered that the combination of an EP4 antagonist and an anti-PD-1 antibody for the treatment of NASH-related liver cancer exhibits a more effective synergistic effect than treatment with either agent alone.

Specifically, the present invention is as follows:

[1] A method for treating NASH-related liver cancer, comprising administering a pharmaceutically effective amount of an EP4 antagonist to a human or animal in need thereof.

[2] The method according to [1], further comprising administering the pharmaceutically effective amount of the EP4 antagonist in combination with a second active agent, an anti-tumor therapy, or both.

[3] The method according to [2], wherein the second active agent is an immune checkpoint inhibitor or a PD-1 inhibitor.

[4] The method according to any one of [1] to [3], wherein the EP4 antagonist is at least one compound selected from the group consisting of:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

[5] A method of treating a disease, wherein the method is one or more selected from the group consisting of:

Increase DCs and CD103 ⁺ DCs that can activate anti-tumor immune function in humans or animals in need,

Reducing Foxp3 ⁺ Treg cells that suppress anti-tumor immunity in humans or animals in need thereof,

Increase the proportion of CD8 ⁺ /Treg population in humans or animals in need,

increasing the population of activated CD8 ⁺ T cells (CD69 ⁺ cells) in a human or animal in need thereof, and

reducing PD-1 expression in CD8 ⁺ T cells in a human or animal in need thereof, and

The method comprises administering a pharmaceutically effective amount of an EP4 antagonist to a human or animal in need thereof for treating NASH-related liver cancer.

[6] The method of [5] further comprising administering a pharmaceutically effective amount of the EP4 antagonist in combination with a second active agent, an anti-tumor therapy, or both.

[7] The method according to [5] or [6], wherein the second active agent is an immune checkpoint inhibitor or a PD-1 inhibitor.

[8] The method according to any one of [5] to [7], wherein the EP4 antagonist is at least one compound selected from the group consisting of:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

[9] A pharmaceutical composition for treating NASH-related liver cancer, comprising an EP4 antagonist and a pharmaceutically acceptable additive, diluent or carrier.

[10] The pharmaceutical composition according to [9], further comprising a second active agent and/or an antitumor antibiotic.

[11] The pharmaceutical composition according to [9] or [10], wherein the EP4 antagonist is at least one compound selected from the group consisting of:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

[12] Use of an EP4 antagonist or a pharmaceutically acceptable salt thereof for treating NASH-related liver cancer in humans or animals.

[13] The use according to [12], wherein the EP4 antagonist is used in combination with a second active agent, an anti-tumor therapy, or both.

[14] The use according to [12] or [13], wherein the EP4 antagonist is at least one compound selected from the group consisting of:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

[15] Use of an EP4 antagonist or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating NASH-related liver cancer in humans or animals.

[16] The use according to [15], wherein the EP4 antagonist is used in combination with a second active agent, an anti-tumor therapy, or both.

[17] The use according to [15] or [16], wherein the EP4 antagonist is at least one compound selected from the group consisting of:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

Effects of the Invention

Therefore, the compounds of the present invention can be used in patients in need of treatment for NASH-related liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The EP4 antagonist Compound A inhibits liver tumor development in an obesity-induced NASH-related mouse liver cancer model (Non-Patent Document 15: Ohtani et al., Cancer Research, 2014, 74: 1885-1889).

Figure 1

Figure 1 depicts a timeline of a mouse model developed by treatment with 7,12-dimethylbenz(a)anthracene ("DMBA") and by feeding a high-fat diet (HFD). Compound A was administered daily to mice from 19 to 30 weeks of age and then euthanized ("Eut").

Figure 2

Fig. 2 is the macroscopic photograph of the liver separated from the mouse (left side) treated with vehicle and the mouse (right side) treated with compound A. In this mouse model, the expression of EP4 (but not other PGE2 receptors) in tumor tissue is significantly upregulated. Every day, compound A or vehicle (n=5, every kind) is processed from 19 weeks old to 30 weeks old mice. Arrow indicates hepatocellular carcinoma (HCC).

Figure 3

Figure 3 depicts the average number of liver tumors and relative size distribution (categorized as >6 mm, 2 to 6 mm, ≤2 mm). "NS" indicates not significant. Treatment with Compound A significantly inhibited the number of tumors in the liver compared to vehicle-treated mice. Tumors larger than 6 mm completely disappeared after Compound A treatment.

Figure 4

Figure 4 depicts the mean body weights of the vehicle-treated and compound A-treated groups at 30 weeks of age. Data are presented as mean ± SD. Daily treatment with compound A did not affect the body weight of the mice. Thus, compound A showed significant inhibition of liver cancer development in mice without weight loss.

Figure 5

Figure 5A depicts flow cytometric analysis of immune cells between mice treated with vehicle or mice treated with Compound A. The percentages of total CD11c ^hi MHC class II (MHC II) ^hi , CD11b ⁺ CD11c ^hi MHC II ^hi , or CD103 ⁺ CD11c ^hi MHC II ^hi cells were analyzed by flow cytometry. Data are presented as mean ± SEM.

Figure 5B depicts dot plots of CD11b and CD103 expression in the CD11c ^hi MHC II ^hi cell compartment of livers from vehicle-treated mice (left) and mice treated with Compound A (right). Numbers in the graph represent the percentage of cells in the indicated gates.

Compound A treatment did not change the frequencies of CD11c ^hi MHC class II ^hi dendritic cells (DCs) and CD11b ⁺ DCs. However, compound A treatment increased CD103 ⁺ DCs, a population of DCs essential for anti-tumor immune responses.

Figure 6

Figure 6A depicts the percentages of CD3 ⁺ CD4 ⁺ Foxp3 ⁺ , CD3 ⁺ CD4 ^{+ Foxp3-} , and CD3 ⁺ CD8 ⁺ cells analyzed by flow cytometry. The ratios of CD8 ⁺ T cells to CD4 ⁺ Foxp3 ⁺ Tregs were calculated. Data are presented as mean ± SEM. NS = not significant, *p < 0.05, **p < 0.01.

Figure 6B depicts dot plots of Foxp3 expression in the CD4 ⁺ T cell compartment of livers from vehicle-treated mice (left) and mice treated with Compound A (right). Numbers in the graph represent the percentage of cells in the indicated gates.

Compound A treatment significantly reduced the frequency of CD4 ⁺ Foxp3 ⁺ regulatory T cells (Tregs), but did not reduce the frequency of CD4 ⁺ Foxp3 ^- T cells. In addition, although the ratio of CD8 ⁺ T cells to Tregs increased in Compound A-treated mice, the frequency of CD8 ⁺ T cells did not change.

Figure 7

In FIG. 7A , the percentages of CD69 ⁺ CD8 ⁺ cells were analyzed by flow cytometry (*p<0.05, **p<0.01).

Figure 7B depicts a histogram of CD69 expression on CD8 ⁺ T cells from the livers of mice treated with vehicle (left) and mice treated with Compound A (right). The numbers in the histogram represent the percentage of CD69 ⁺ cells.

The number of CD8 ⁺ T cells expressing the activation marker CD69 was significantly increased in the livers of mice treated with compound A. This result indicates that treatment with compound A activated the function of CD8 ⁺ T cells in tumor tissues.

Figure 8

In FIG. 8A , the percentage of PD-1 ⁺ CD8 ⁺ cells was analyzed by flow cytometry (*p<0.05, **p<0.01).

Figure 8B depicts a histogram of PD-1 expression on CD8 ⁺ T cells from the livers of vehicle-treated mice (left) and mice treated with Compound A (right). The numbers in the histogram represent the percentage of PD-1 ⁺ cells.

Administration of Compound A significantly reduced the number of CD8 ⁺ T cells expressing PD-1, a key inhibitory receptor on T cells in the tumor microenvironment.

DETAILED DESCRIPTION

The compounds of the present invention useful for treating NASH-related liver cancer are:

4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (Compound A),

4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (Compound B), and

3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (Compound C),

or a pharmaceutically acceptable salt thereof.

The compounds of the present invention also include solvates, complexes, polymorphs, prodrugs, isomers and isotopically labeled compounds thereof.

The compounds of the present invention are disclosed in WO2005/021508.

Pharmaceutically acceptable salts include acid addition salts and base salts thereof. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, dibenzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, toluenesulfonate, and trifluoroacetate.

Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts.

For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, Weinheim, Germany, 2002).

Pharmaceutically acceptable salts of the compounds of the present invention can be readily prepared by suitably mixing a solution of the compound of the present invention with an appropriate acid or base. The salt can be precipitated from the solution and collected by filtration, or it can be recovered by evaporating the solvent. The degree of ionization in the salt varies and can range from fully ionized to almost non-ionized.

The compounds of the present invention may exist in both unsolvated and solvated forms.The term "solvate" is used herein to describe a molecular complex comprising a compound of the present invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol.

Included within the scope of the present invention are complexes such as clathrates, drug-host inclusion complexes, wherein, in contrast to the solvates described above, the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of compounds containing two or more organic and/or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of these complexes, see Haleblian, J. Pharm. Sci., 64(8): 1269-1288 (August 1975).

Hereinafter, all references to compounds of the present invention include references to salts, solvates and complexes thereof, as well as solvates and complexes of their salts.

The compounds of the present invention include the compounds of the present invention as defined above, polymorphs, prodrugs and isomers thereof (including optical, geometric and tautomers) as defined below, and isotopically labeled compounds of the present invention.

As stated above, the present invention includes all polymorphs of the compounds of the invention as defined herein.

Also included within the scope of the present invention are so-called "prodrugs" of the compounds of the present invention. Thus, certain derivatives of the compounds of the present invention, which may themselves have little pharmacological activity, can, when administered into or onto the body, be converted, for example by hydrolysis, into compounds having the chemical formula of any of the compounds of the present invention that have the desired activity. Such derivatives are referred to as "prodrugs." Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and "Bioreversible Carriers in Drug Design", Pergamon Press, 1987 (ed. E.B. Roche, American Pharmaceutical Association).

Prodrugs according to the invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the invention with certain moieties known to those skilled in the art as "promoieties", as described in "Design of Prodrugs" by H Bundgaard (Elsevier, 1985).

Some examples of prodrugs according to the present invention include:

(i) When the compound of the present invention contains a carboxylic acid functional group (-COOH), its ester, for example, replaces the hydrogen with a ( _C1 - _C8 ) alkyl group;

(ii) when the compound of the present invention contains an alcohol function (-OH), the ether thereof, for example, replaces the hydrogen with a ( _C1 - _C6 )alkanoyloxymethyl group; and

(iii) When the compound of the invention contains a primary or secondary amino function ( _-NH2 or -NHR, wherein R is not H), its amide, for example, replaces one or both hydrogen atoms with a ( _C1 - _C10 )alkanoyl group.

Other examples of substituents besides the aforementioned examples are known to those skilled in the art and can be found in the above-mentioned references, but are not limited thereto.

Finally, the compounds of the present invention may themselves act as prodrugs of other compounds of the present invention.

Compounds of the present invention containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. When compounds of the present invention contain alkenyl or alkenylene groups, geometric cis/trans (or Z/E) isomers are possible. When compounds contain, for example, ketone or oxime groups or aromatic moieties, tautomerism ("tautomerism") may occur. Thus, a single compound may exhibit more than one type of isomerism.

Included within the scope of the present invention are all stereoisomers, geometric isomers, and tautomeric forms of the compounds of the present invention, including compounds exhibiting more than two types of isomers, and mixtures of one or more thereof. Also included are compounds wherein the counterion is an optically active acid addition salt or base salt, such as D-lactate or L-lysine, or a racemate, such as DL-tartrate or DL-arginine.

Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).

Alternatively, the racemate (or racemic precursor) can be reacted with a suitable optically active compound, such as an alcohol, or, in the case where the compound of the invention contains an acidic or basic moiety, with an acid or base, such as tartaric acid or 1-phenylethylamine. The resulting diastereomeric mixture can be separated by chromatography and/or fractional crystallization, and one or both of the diastereoisomers can be converted to the corresponding pure enantiomer by methods well known to those skilled in the art.

The chiral compounds of the present invention (and chiral precursors thereof) can be obtained in an enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin using a mobile phase consisting of a hydrocarbon (typically heptane or hexane) containing 0 (w/w)% to 50 (w/w)%, typically 2 (w/w)% to 20 (w/w)% of isopropanol, and 0 (w/w)% to 5 (w/w)% of an alkylamine, typically 0.1 (w/w)% of diethylamine. Concentration of the eluate provides an enriched mixture.

Stereoisomeric aggregates can be separated by conventional techniques known to those skilled in the art (see, for example, E. L. Eliel, Stereochemistry of Organic Compounds (Wiley, New York, 1994)).

The present invention includes all pharmaceutically acceptable isotopically labeled compounds of the present invention wherein one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Examples of suitable isotopes for inclusion in the compounds of the present invention include isotopes of hydrogen, such as ² H and ³ H, isotopes of carbon, such as ¹¹ C, ¹³ C, and ¹⁴ C, isotopes of chlorine, such as ³⁶ Cl, isotopes of fluorine, such as ¹⁸ F, isotopes of iodine, such as ¹²³ I and ¹²⁵ I, isotopes of nitrogen, such as ¹³ N and ¹⁵ N, isotopes of oxygen, such as ¹⁵ O, ¹⁷ O, and ¹⁸ O, isotopes of phosphorus, such as ³² P, and isotopes of sulfur, such as ³⁵ S.

Certain isotopically labeled compounds of the present invention, for example those incorporating radioactive isotopes, are useful in drug and/or substrate tissue distribution studies relevant to cancer therapy, including diagnosis, symptom relief, QOL improvement, and prevention. The radioactive isotopes tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready detection.

Substitution with heavier isotopes such as deuterium, ie, ^2H , may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such ^as11C , ^18F , ^15O , ^and13N , can be used in positron emission topography (PET) studies to examine substrate receptor occupancy.

Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and preparations, using an appropriate isotopically labeled reagent in place of the previously non-labeled reagent.

Pharmaceutically acceptable solvates according to the invention include those wherein the solvent of crystallization may be isotopically substituted, for example _D2O , _d6 -acetone, _d6 -DMSO.

The compounds of the present invention intended for pharmaceutical use can be administered as crystalline or amorphous products. They can be obtained, for example, in the form of solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radiofrequency drying can be used for this purpose.

Each compound of the present invention (i.e., compound A, B, or C) can be administered alone or in combination or in combination with one or more other drugs (or as any combination thereof). Typically, they will be administered as a formulation in combination with one or more pharmaceutically acceptable additives. The term "additive" is used herein to describe any ingredient other than the compounds of the present invention. The selection of additives depends largely on various factors, such as the specific mode of administration, the effect of the additive on solubility and stability, and the properties of the dosage form. The compounds of the present invention can be administered alone or with a pharmaceutically acceptable carrier or diluent by any of the above-mentioned routes, and such administration can be performed in single or multiple doses. More specifically, the compounds of the present invention can be administered in various dosage forms, i.e., they can be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, jelly, gels, pastes, lotions, ointments, aqueous suspensions, solutions for injection, elixirs, syrups, etc. Such carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents. In addition, oral pharmaceutical compositions may be suitably sweetened and/or flavored. Generally, the compounds of the present invention are present in such dosage forms at concentration levels of 5% to 95% by weight. For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium hydrogen phosphate, and glycine may be used with various disintegrants such as starch, preferably corn, potato, or tapioca starch, alginic acid, and certain complex silicates, as well as with granulating binders such as polyvinyl pyrrolidone, sucrose, gelatin, and gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are generally very useful for tableting purposes. Similar types of solid compositions may also be used as fillers in gelatin capsules; preferred materials in this regard also include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are required for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents or dyes and, if desired, emulsifying and/or suspending agents and such diluents as water, ethanol, propylene glycol, glycerin and similar combinations thereof.

Thus, the present invention provides compounds of the present invention, solvates thereof, prodrugs thereof, combinations thereof, and combinations with one or more other pharmacologically active agents. Furthermore, the present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable additive, diluent, or carrier, particularly for treating NASH-associated liver cancer. Furthermore, the present invention provides a kit comprising: a first pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof; a second pharmaceutical composition; and a container. Furthermore, the second pharmaceutical composition may comprise a compound of the present invention.

A kit for treating NASH-related liver cancer comprising a compound of the present invention or a pharmaceutically acceptable salt thereof is also an aspect of the present invention. A commercial package comprising a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and written materials related thereto, wherein the written materials state that the compound can or should be used to treat NASH-related liver cancer is also an aspect of the present invention.

Other features and advantages of the present invention will be apparent from the following detailed description and claims. Although specific embodiments of the present invention have been described, various other known or common changes and modifications in the art fall within the present invention and are within the scope of the claims. The present invention also includes equivalents, variations, uses, or modifications within the spirit of the present invention.

The compounds of the present invention are administered in an amount that effectively shrinks cancer, reduces cancer tumor size, reduces cancer metastasis, regulates immune cell function, and/or enhances the effectiveness of cancer treatment. This therapeutically effective amount varies according to the specific compound of the present invention, the specific condition to be treated, the patient's condition, route of administration, formulation, on-site decisions, and other factors. According to the present invention, it is determined by conventional optimization techniques, depending on what is known to those skilled in the art. The compounds of the present invention can be administered to mammals orally, parenterally, or topically. In general, these compounds are most suitably administered to humans in a dosage of 1 mg to 1000 mg, preferably 10 mg to 600 mg, which can be administered in a single dose or divided dose throughout the day. However, depending on the weight and condition of the treated subject, the disease state treated, and the specific route of administration selected, changes are inevitable.

Pharmaceutical compositions may include a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable transport vehicle or carrier.

As used herein, the term "pharmaceutically acceptable transport medium" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. The above-mentioned medium may also contain other active ingredients or inactive ingredients, and target cancer tissues based on the composition.

In light of the present disclosure, the therapeutic efficacy of compounds of the invention can be determined by standard therapeutic procedures in cell cultures or experimental animals, eg, to determine the _ED50 (the dose therapeutically effective in 50% of the population).

The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for humans. The dosage can vary depending on the formulation and route of administration. For any EP4 receptor antagonist (i.e., compound A, B, or C) used in the methods of the present invention, a therapeutically effective dose can be estimated from the cell culture assay. The dosage can be formulated in an animal model to achieve a circulating plasma concentration range that includes the IC ₅₀ measured in cell culture. This information can be used to more accurately determine a useful dose for humans or animals. For example, the level in plasma can be measured by high performance liquid chromatography.

It is well known to those skilled in the art that certain factors may affect the dosage and time required to effectively treat a mammal, including but not limited to the severity of the disease or condition, previous treatment, the general health and/or age of the mammal, and the presence of other diseases. In addition, treatment of a mammal with a therapeutically effective amount of a compound of the present invention may include but is not limited to a single treatment, alternate day treatment, or a series of treatments. The compounds of the present invention can be administered to a mammal orally, parenterally, or topically. In general, these compounds are ideally administered to a human, for example, once daily or in divided portions two to four times daily.

The exact amount of the compound administered to a human patient will be the responsibility of the monitoring physician. However, the dosage used depends on many factors, including the patient's age and sex, the exact condition of the treatment and its severity and route of administration. In the case of oral administration, for example, with respect to the compounds of this invention, the daily dose of mammals (including people) per 1 kg body weight is generally about 0.02 mg to 200 mg, preferably about 0.1 mg to 100 mg, which can be given once a day, or administered in two to four doses per day. More specifically, to people, for example, about 0.02 mg to 20 mg per kg body weight per day, more specifically about 0.2 mg to 12 mg per kg body weight per day. For example, to dogs, about 0.5 mg to 25 mg per kg body weight per day, more specifically about 1 mg to 10 mg per kg body weight per day. For example, to mice, about 1 mg to 100 mg per kg body weight per day, more specifically about 3 mg to 30 mg per kg body weight per day.

The compounds of the present invention can be conveniently administered in the form of a pharmaceutical composition for treating NASH-related liver cancer. Such a composition can be conveniently mixed with one or more pharmaceutically acceptable carriers or excipients in a conventional manner.

Although the compounds of the present invention may be administered as the raw chemical, it is preferable to provide them in the form of a pharmaceutical formulation for administration as a pharmaceutical composition. The formulation comprises the compound together with one or more acceptable carriers or diluents and, optionally, other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical compositions are formulated to conform to the desired route of administration. For example, routes of administration are parenteral (e.g., intravenous, subcutaneous, subcutaneous), oral (e.g., ingestion or inhalation), transdermal (topical), mucosal and rectal, and topical (including transdermal, oral, and sublingual)) administration. Pharmaceutical compositions formulated into solution or suspension forms can be prepared by methods such as those described in Remington's Pharmaceutical Sciences, 18th edition, compiled by Gennaro, Mack Publishing Co., Easton, PA, (1990).

According to the condition and the disease of the patient for example being treated, the most suitable route of administration may be different. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. All methods include the step of combining the compound (i.e., "active ingredient") with the carrier constituting one or more auxiliary components. Generally speaking, the preparation is prepared by combining the active ingredient with a liquid carrier or a finely divided solid carrier or both uniformly and closely, and then, if necessary, the product is shaped into the required preparation.

Formulations of the invention suitable for oral administration may be presented as discrete units, each containing a predetermined amount of the active ingredient, such as capsules, cachets, or tablets (e.g., chewable tablets particularly for pediatric use); as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a pill, electuary, or paste.

Tablet can be prepared by optionally compressing or molding with one or more auxiliary ingredients. Compressed tablets can be prepared by making the active ingredient in a free-flowing form, such as a powder or granule, and optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant, and compressed in a suitable machine. Molded tablets can be prepared by molding a mixture of the powdered active ingredient moistened with an inert liquid diluent in a suitable machine. Tablet can optionally be coated or scored and can be formulated to provide slow or controlled release of the active ingredient therein.

Preparations for parenteral administration include aqueous and non-aqueous sterile injections, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. The preparations can be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, before use. Instant injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the aforementioned types.

Formulations for rectal administration may be presented as a suppository with conventional carriers such as cocoa butter, hard fat or polyethylene glycol.

Formulations for topical administration in the mouth, e.g., buccal or sublingual, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in a flavored basis such as gelatin and glycerin or sucrose and acacia.

The compound of the present invention can also be formulated as a long-acting preparation. Such a long-acting preparation can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the compound of the present invention can be administered with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin or as a slightly soluble derivative, for example, as a slightly soluble salt.

In addition to the ingredients particularly mentioned above, the formulations may include other ingredients conventional in the art having regard to the type of formulation in question, for example, formulations suitable for oral administration may include flavoring agents.

Second active agents that are small molecules can also be used to alleviate the side effects associated with the administration of the compounds of the invention. However, like some macromolecules, when co-administered with the compounds of the invention (e.g., before, after, or simultaneously), it is believed that a synergistic effect can be provided. Examples of small molecule second active agents include, but are not limited to, anticancer agents, antibiotics, immunosuppressants, and steroids.

The present invention also includes combining separate pharmaceutical compositions in kit form. The kit comprises two separate pharmaceutical compositions, a compound of the present invention and a second therapeutic agent as described herein. The kit includes containers for holding the separate compositions, such as separate bottles or separate foil packages, however, the separate compositions may also be contained in a single, unseparated container. Typically, the kit includes instructions for administering the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), administered at different dosage intervals, or when the prescribing physician requires titration of the individual components of the combination.

An example of this test kit is so-called blister pack. Blister pack is well known in the packaging industry and is widely used in the packaging of pharmaceutical unit dosage forms (tablets, capsules, etc.). Blister packs usually comprise a sheet of relatively hard material covered with a foil of preferably transparent plastic material. During the packaging process, a groove is formed in the plastic foil. The groove has the size and shape of the tablet or capsule to be packaged. Next, the tablet or capsule is placed in the groove and the sheet of relatively hard material is sealed with the plastic foil on the face opposite to the direction in which the groove is formed of the foil. As a result, the tablet or capsule is sealed in the groove between the plastic foil and the sheet. Preferably, the strength of the sheet makes it possible for the tablet or capsule to be removed from the blister pack by manually applying pressure on the groove, thereby forming an opening at the recess position of the sheet. The tablet or capsule can then be removed through the opening.

In certain embodiments, provided herein are methods including administering a compound of the present invention in combination with one or more second active agents, and/or administering in combination with radiotherapy and/or surgery. For example, the example of a second active agent includes, for example, another EP4 antagonist, an immune checkpoint inhibitor, PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, adoptive immune cell therapy, cancer vaccines and other immuno-oncology drugs, such as colony stimulating factor 1 receptor (CSF1R), indoleamine 2,3-dioxygenase (IDO) or carcinoembryonic antigen (CEA). In addition, molecularly targeted anticancer drugs and cancer chemotherapeutics are also included as the second active agent. More particularly, the second active agent includes, for example, PD-1 antibodies such as nivolumab, lambrolizumab/pembrolizumab, REGE2810, PD-L1 antibodies such as avelumab, atezolizumab, durvalumab, pembrolizumab, CTLA-4 antibodies such as ipilimumab and tremelimumab, molecular targeted drugs such as anti-HE R2 antibodies, anti-VEGF antibodies, anti-EGFR antibodies, tyrosine kinase inhibitors for EGFR receptors, PDGFR receptors, VEGFR receptor kinases, c-kit and Bcr-Abl, and anti-tumor chemotherapeutics such as alkylating agents, antimetabolites, anti-tumor antibiotics, anti-infective drugs, microtubule inhibitors, hormone therapy agents, platinum drugs, topoisomerase inhibitors, aromatase inhibitors, anti-estrogens, anti-androgens, progesterone, estradiol, LH-RH agonists, and immunotherapy such as adoptive T cell therapy, inherited dendritic cell therapy, adoptive NK cell therapy and cancer vaccine therapy. The administration of the compounds of the present invention and the second active agent to the patient can be carried out simultaneously or sequentially by the same or different routes of administration. The suitability of a specific route of administration for a specific active agent will depend on the active agent itself (e.g., whether it can be orally administered without decomposing before entering the bloodstream) and the disease to be treated. The recommended route of administration of the second active agent is known to those of ordinary skill in the art. Radiotherapy includes all therapies for liver cancer, such as proton beam therapy. Surgery includes all therapies for the treatment of liver cancer.

Definition of terms

" EP4 antagonist " refers to the compound that suppresses or blocks the cell signaling that is triggered by PGE 2 and EP4 receptor interaction.The example of EP4 antagonist includes but is not limited to ER-819762, MK-2894, MF 498, OD-AE3-208, evatanepag, OD-A2E2-227, BGC201531, OD-A3E240, GW 627368 and AH23848 that are listed as E4 receptor antagonists in IUPHAR database.Compound A, B and C and pharmaceutically acceptable salt thereof (compound of the present invention) are also the example of EP4 antagonist.

"Immune checkpoint inhibitors" refer to a type of drug that blocks certain proteins produced by certain types of immune cells, such as T cells and some cancer cells. These proteins help keep the immune response in check and keep T cells from killing cancer cells. When these proteins are blocked, the brake signals on the immune system are released, and T cells are better able to kill cancer cells. Examples of immune checkpoint inhibitors include but are not limited to PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, BTLA inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, B7-1 inhibitors, B7-2 inhibitors, galectin 9 inhibitors and HVEM inhibitors. Immune checkpoint inhibitors can be small molecules, peptides, proteins such as antibodies, nucleic acids, etc.

"PD-1 inhibitors" refer to antibodies or other molecules that inhibit the function of programmed death protein 1 (PD1). Exemplary inhibitors/antibodies include, but are not limited to, antibodies listed in U.S. Patent Nos. 7,029,674, 7,488,802, 7,521,051, 8,008,449, 8,354,509, 8,617,546, and 8,709,417. Specific embodiments of antibodies include MDX-1106/nirolimab, BMS-936558 (Bristol-Myers Squibb), lamblizumab (Merck), MK-3475/pembrolizumab (KEYTRUDA registered trademark, Merck), AMP-224 (GSK), and CT-011 (Cure Tech).

"PD-L1 inhibitor" refers to an antibody or other molecule that inhibits the function of programmed death ligand 1 (PDL1). Exemplary antibodies include, but are not limited to, those listed in U.S. Patent Nos. 8,217,149, 8,383,796, 8,552,154, and 8,617,546. In a specific embodiment, the antibody is MPDL3280A/RG7446 (Roche), BMS-936559 (BMS), MEDI4736 (Astra Zeneca), or MSB0010718C (Merck Serono).

"CTLA4 inhibitor" refers to an antibody or other molecule that inhibits the function of cytotoxic T-lymphocyte antigen 4 (CTLA4). Exemplary inhibitors/antibodies include, but are not limited to, CTLA4 antagonists or the CTLA4 antibodies listed in U.S. Patent Nos. 8,685,394 and 8,709,417. Some embodiments of antibodies include MDX-010 (ipilimumab, Bristol-Myers Squibb) and CP-675,206 (tremelimumab, AstraZeneca). In a specific embodiment, the antibody is ipilimumab or tremelimumab.

"Treatment" refers to alleviating, inhibiting and/or reversing the progression of cancer in a subject in need thereof. The term "treatment" includes any successful marker for treating or improving cancer, including any objective or subjective parameter, such as alleviation of symptoms; relief; reducing or making the subject more tolerant to injury, pathology or illness; delaying or slowing the rate of progression, etc. The measurement of treatment or improvement can be based on the results of, for example, physical examinations, pathological tests and/or diagnostic tests known in the art. Treatment can also refer to reducing the incidence or onset of cancer, or its recurrence (e.g., prolonged remission time), compared to the situation where no measures are taken. The term "treatment" as used herein includes not only shrinking tumor tissue, but also alleviating symptoms, improving quality of life (QOL) and prevention (radiotherapy, postoperative prevention of recurrence, adjuvant chemotherapy, etc.).

"Pharmaceutically effective amount" refers to an amount that is effective in treating cancer as indicated by clinical testing and evaluation, patient observation, and/or other methods. An "effective amount" may further specify an amount that causes a detectable change in biological or chemical activity. For related mechanisms or processes, one skilled in the art can detect and/or further quantify these detectable changes. In addition, an "effective amount" may specify an amount that maintains a desired physiological state, i.e., reduces or prevents a significant decline and/or promotes improvement in a condition. An "effective amount" may further refer to a therapeutically effective amount.

As used herein, the term "pharmaceutically acceptable salts" is consistent with the examples provided above and refers to relatively non-toxic inorganic or organic acid salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in free form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative acid salts include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hexabenzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, and xinafoate. In one embodiment, the pharmaceutically acceptable salt is a hydrochloride/chloride salt.

A "second active agent" is a low molecular weight drug or biologic with pharmacologically effective activity, and includes, but is not limited to, PGE2 signaling inhibitors, such as additional EP4 antagonists, microsomal prostaglandin E synthase (mPGES)-1 inhibitors, COX-2 inhibitors, NSAIDs and immune checkpoint inhibitors, immune cell targeted drugs, molecular targeted anticancer drugs, alkylating agents, antimetabolites, antitumor antibiotics, anti-infective drugs, microtubule inhibitors, hormone therapeutics, platinum drugs, topoisomerase inhibitors, molecular targeted drugs, molecular targeted cancer therapeutics, immunotherapeutics, and the like.

"Anti-tumor therapy" includes, but is not limited to, therapy with anti-tumor vaccine therapy and adoptive immune cell therapy, such as adoptive T cell therapy, adoptive dendritic cell therapy, or adoptive NK cell therapy, and also includes, but is not limited to, cancer radiation therapy and surgical treatment.

"Biological" includes, but is not limited to, pharmacologically active proteins, such as interferon gamma and interleukin 2, as well as pharmacologically active peptides, nucleic acids, and polysaccharides.

“Molecular targeted drugs” include, but are not limited to, anti-HER2 antibodies, anti-VEGF antibodies, anti-EGFR antibodies, and tyrosine kinase inhibitors targeting EGFR receptors, PDGFR receptors, VEGFR receptor kinases, c-kit, and Bcr-Abl.

"Immunotherapy" includes but is not limited to immunomodulatory drugs, adoptive immune cell therapy, anti-tumor vaccine therapy, etc.

"NASH," which stands for nonalcoholic steatohepatitis, is a syndrome that occurs in nonalcoholic patients; it causes liver damage that is histologically indistinguishable from alcoholic hepatitis. It most often develops in patients with at least one of the following risk factors: obesity, dyslipidemia, and glucose intolerance. The pathogenesis is poorly understood but appears to be related to insulin resistance (e.g., as in obesity or metabolic syndrome).

"NASH-associated liver cancer" refers to cancers including hepatocellular carcinoma (HCC) and other cancers that arise in liver-related organs (such as hepatic blood vessels or bile ducts) and/or are associated with NASH. NASH-associated liver cancer differs from hepatitis- or C virus-induced hepatocellular carcinoma in its pathogenesis.

"Metastatic cancer" refers to cancer in which cancer cells from an organ or part of the body have spread (by "metastasis") to another non-adjacent organ or part of the body. Cancer in non-adjacent organs or parts of the body ("secondary tumors" or "metastatic tumors") includes cancer cells that originated in the organ or part of the body from which the cancer or cancer cells have spread. Sites where secondary tumors may occur include, but are not limited to, lymph nodes, lungs, brain, and/or bones.

As used herein, the term "EP4 signal" or "EP4 signaling" means the increase in cAMP and subsequent signaling associated with agonistic stimulation of the EP4 receptor.

Example

Example 1

Compound A inhibited liver cancer growth in a high-fat diet-induced NASH-related mouse liver cancer model.

Animal experiments

C57/BL6 mice were purchased from CLEA Japan Inc. ^{Tlr2 −/−} mice (C57/BL6) were purchased from Oriental Yeast Co. Ltd.

Histological and immunofluorescence analysis

Hematoxylin and eosin staining and immunofluorescence analysis were performed as previously described (Yoshimoto et al., Nature, 2013, 499:97-101).

Immune cell isolation

Immune cells were obtained from mouse livers and subjected to measurement of cytokine production and flow cytometric analysis.

Statistical analysis

Data were analyzed by unpaired t-test with Welch correction (two-sided) or Man-Whitney test (two-sided). P values less than 0.05 were considered significant. "NS" indicates not significant.

Study Results

The mouse model is produced by treating with DMBA, 7,12-dimethylbenz (a) anthracene and feeding a high-fat diet (HFD) (non-patent literature 15). In this mouse model, EP4, rather than the expression of other PGE2 receptors, is significantly increased in tumor tissue, indicating that EP4 can mainly mediate the PGE2 signaling in the NASH-related liver tumor tissues induced by obesity. In this experiment, from 19 to 30 weeks of age, compound A 30 mg/kg QD was administered to mice every day (Fig. 1). As a control, a single group of mice was treated only with vehicle and inactive ingredients. Compared with vehicle-treated mice, the hepatocellular carcinoma (HCC) development in compound A-treated mice was significantly reduced (Fig. 2 and Fig. 3). However, it is worth noting that compound A treatment does not affect body weight (Fig. 4).

The effect of EP4 blocking on the incidence and activation state of immune cells was assessed. Although the frequency of CD11chi MHC class IIhi dendritic cells (DC) and CD11b ⁺ DC cells did not change, the population of CD103 ⁺ DC necessary for anticancer immune response (non-patent literature 16: Fuertes et al., J.Exp.Med., 2011, 208: 2005-2016; Non-patent literature 17: Salmon et al., Immunity, 2016, 44: 924-938; and Non-patent literature 18: Zelenay et al., Cell, 2015, 162: 1257-1270) increased in the Compound A-treated group (Fig. 5). Compound A treatment significantly reduced the frequency of CD4 ⁺ Foxp3 ⁺ regulatory T cells (Treg), rather than the frequency of CD4 ⁺ Foxp ^3- T cells (Fig. 6). In addition, the ratio of CD8 ⁺ T cells to Treg increased in Compound A-treated mice, but the frequency of CD8 ⁺ T cells did not change (Fig. 6). In addition, the number of CD8 ⁺ T cells expressing the activation marker CD69 was significantly increased in the livers of mice treated with Compound A (Figure 7). Conversely, administration of Compound A significantly reduced the number of CD8 ⁺ T cells expressing PD-1, a key inhibitory receptor on T cells in the tumor microenvironment (Figure 8). These results suggest that blocking the EP4 pathway can reactivate anti-tumor immunity in the NASH-associated liver tumor microenvironment.

in conclusion

The EP4 antagonist Compound A significantly inhibited the growth and progression of an obesity-induced NASH-related mouse liver cancer model. Compound A treatment of the mouse model increased the frequency of a DC subtype (CD103 ⁺ ) essential for anti-cancer immune responses and reduced PD-1 expression on cytotoxic CD8 ⁺ T cells in tumor tissue. Compound A treatment also reduced the number of immunosuppressive Treg cells (Foxp3 ⁺ ) in tumor tissue.

Example 2:

In a HFD-induced NASH-associated mouse liver cancer model, the combination of Compound A and PD-1 antibody showed higher anti-tumor efficacy than Compound A alone.

Research Methods

The efficacy of the combination of Compound A and anti-PD-1 antibody in the HFD-induced NASH-related mouse liver cancer model was tested using the same mouse model as in Example 1. Therefore, Example 1 was repeated, except that anti-PD-1 antibody was also administered together with Compound A in the Compound A-treated group.

result

In the HFD-induced NASH-related mouse liver cancer model, the combination therapy of Compound A and PD-1 antibody showed higher anti-tumor efficacy than the mice of Example 1 treated with Compound A.

Example 3:

In a HFD-induced NASH-related mouse liver cancer model, compound B treatment inhibited liver cancer growth.

Research Methods

The same mouse model as in Example 1 was used to test the efficacy of Compound B. Therefore, Example 1 was repeated except that Compound B was used instead of Compound A.

Study Results

In a HFD-induced NASH-associated mouse liver cancer model, Compound B therapy inhibited liver cancer growth. Compound B controls immune cell function in tumor tissue in the same manner as Compound A in Example 1.

in conclusion

EP4 antagonist Compound B inhibited growth and development in a NASH-associated mouse liver cancer model, consistent with the results of Compound A in Example 1. Compound B treatment of the mouse model increased the frequency of a DC subtype (CD103 ⁺ ) essential for anti-cancer immune responses and reduced PD-1 expression on cytotoxic CD8 ⁺ T cells in tumor tissue. Compound B treatment also reduced the number of immunosuppressive Treg cells (Foxp3 ⁺ ) in tumor tissue.

Example 4:

In a HFD-induced NASH-associated mouse liver cancer model, the combination of Compound B and PD-1 antibody showed higher anti-tumor efficacy than Compound B alone.

Research Methods

The efficacy of the combination of Compound B and PD-1 antibody in the HFD-induced NASH-associated mouse liver cancer model was tested using the same mouse model as in Example 3. Therefore, Example 3 was repeated except that PD-1 antibody was also administered together with Compound B in the Compound B-treated group.

result

In the HFD-induced NASH-related mouse liver cancer model, the combination treatment of Compound B and PD-1 antibody showed higher anti-tumor efficacy than the mice treated with Compound B in Example 3.

Example 5:

In the HFD-induced NASH-related liver cancer model in mice, compound C treatment inhibited the growth of liver cancer.

Research Methods

The same mouse model as in Example 1 was used to test the efficacy of Compound C. Therefore, Example 1 was repeated except that Compound C was used instead of Compound A.

Study Results

In an HFD-induced NASH-associated mouse liver cancer model, Compound C therapy inhibited liver cancer growth. Compound C controlled immune cell function in tumor tissue in the same manner as Compound A in Example 1.

in conclusion

EP4 antagonist Compound C inhibited the growth and development of NASH-associated mouse liver cancer models, consistent with the results of Compound A in Example 1 and Compound B in Example 3. Compound C treatment of the mouse model increased the frequency of DC subtypes (CD103 ⁺ ) essential for anti-cancer immune responses and reduced the expression of PD-1 on cytotoxic CD8 ⁺ T cells in tumor tissue. Compound C treatment also reduced the population of immunosuppressive Treg cells (Foxp3 ⁺ ) in tumor tissue.

Example 6:

In a HFD-induced NASH-associated mouse liver cancer model, the combination of compound C and PD-1 antibody showed higher anti-tumor efficacy than compound C alone.

Research Methods

The efficacy of the combination of Compound C and PD-1 antibody in the HFD-induced NASH-related mouse liver cancer model was tested using the same mouse model as in Example 5. Therefore, Example 5 was repeated, except that Compound C was also administered together with PD-1 antibody in the Compound C-treated group.

result

In the HFD-induced NASH-related mouse liver cancer model, the combination treatment of Compound C and PD-1 antibody showed higher anti-tumor efficacy than the mice treated with Compound C in Example 5.

Claims (3)

1. Use of an EP4 antagonist or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating NASH-related hepatocellular carcinoma in humans or animals, wherein said EP4 antagonist is at least one compound selected from the group consisting of: 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]-benzoic acid (compound A), 4-((1S)-1-{[5-chloro-2-(4-fluorophenoxy)benzoyl]amino}ethyl)benzoic acid (compound B), and 3-[2-(4-{2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl}phenyl)ethyl]-1-[(4-toluene)sulfonyl]urea (compound C). 2. The use according to claim 1, wherein the EP4 antagonist is used in combination with a second activator, an antitumor therapy, or both. 3. The use according to claim 2, wherein the second active agent is an immune checkpoint inhibitor or a PD-1 inhibitor.