WO2018223763A1

WO2018223763A1 - Pharmaceutical composition for treating liver injury

Info

Publication number: WO2018223763A1
Application number: PCT/CN2018/082301
Authority: WO
Inventors: 万晓春; 陈倩; 张青梅; 夏蒙
Original assignee: 深圳市中科艾深医药有限公司
Priority date: 2017-06-06
Filing date: 2018-04-09
Publication date: 2018-12-13
Also published as: CN108992671B; CN108992671A

Abstract

Provided are a pharmaceutical composition and the use of the same in the preparation of a drug for treating liver injury. The pharmaceutical composition comprises a tumour necrosis factor antagonist and a tumour necrosis factor-related apoptosis-inducing ligand antagonist.

Description

[Name of invention established by ISA according to Rule 37.2] Pharmaceutical composition for treating liver damage

Technical field

The present invention relates to pharmaceutical compositions and their use in the treatment of liver damage.

Background technique

When the liver is seriously damaged by a variety of factors, the liver cells die in a short period of time, leading to serious disorders or decompensation of their functions, and then a group of clinical manifestations with coagulation disorders and jaundice, hepatic encephalopathy, ascites, etc. Syndrome, called liver failure. The general onset is rapid, the condition is heavy, the progress is fast, the treatment is difficult, the complications are high, the mortality rate is high, the medical expenses are expensive, and the prognosis is poor. If liver failure is not treated in time, the mortality rate is as high as 70%. When liver failure develops into severe coma, 90% of patients will die. Therefore, liver failure has a "nine deaths and one lifetime", and the treatment of liver failure is still a world problem.

Endotoxin is the outer layer of the cell wall of Gram-negative bacteria, and its main chemical component is lipopolysaccharide (LPS). Under normal physiological conditions, there are a large number of Gram-negative bacteria in the human intestine and produce LPS. The intestinal mucosa has a strict shielding function, which can block most pathogenic bacteria from entering the human body, but there will still be a small amount of bacteria and endotoxin absorbed. When these small amounts of bacteria and endotoxes reach the liver, they are phagocytized and degraded by macrophages in the liver, so they are not harmful to the human body. However, when the liver is damaged, bacteria in the blood or lesions produce and release a large amount of LPS, the liver's ability to clear endotoxin is reduced, the permeability of the intestinal mucosa is increased, the intestinal barrier is impaired, and a large amount of endotoxin is taken into the blood circulation. Causes the rapid increase of blood LPS concentration, forming intestinal endotoxemia. The retention of these endotoxins in venous blood activates Kupffer cells to secrete large amounts of pro-inflammatory cytokines and various inflammatory mediators, resulting in a multiplicity of sensitivity to endotoxin. The production of endotoxemia can aggravate liver damage and can lead to various complications. Patients with chronic liver disease are often associated with intestinal endotoxemia, the incidence of which is in various types of liver disease: acute severe hepatitis 59% to 100%, fulminant hepatic failure 100%, chronic hepatitis 20% to 50%, cirrhosis 15% to 92%.

D-galactosamine (D-GalN) is an amino sugar that is only metabolized in hepatocytes and can specifically deplete UTP in the liver, thereby inhibiting the corresponding nucleic acids, proteins, etc. The synthesis of substances, the structure and function of cell membranes are altered by impaired membrane protein synthesis, which ultimately leads to damage or even death of liver cells. LPS alone can cause systemic inflammatory response, while LPS combined with D-GaIN can specifically trigger fulminant damage and failure of the liver without affecting other organs.

At present, the treatment of liver failure still lacks special effects drugs and means. The general medical treatment is poor, and the clinical mortality rate is high. Artificial liver treatment as a supportive treatment method cannot completely reverse the liver failure caused by large necrosis of liver cells. Liver transplantation is the only effective treatment, but liver transplantation is far from universal due to its high price, shortage of donor liver and technical factors. Need to develop new mechanisms of action, improve treatment and survival rates of therapeutic drugs.

At present, the basic treatment principle of endotoxin-induced liver failure is: mainly to reduce inflammatory factors and alleviate complications.

Therapeutic drugs mainly include:

(1) N-acetylcysteine (NAC): NAC is a precursor of intracellular reduced glutathione (GSH), which has hepatoprotective effects and is used to treat livers caused by various causes. Cell damage. NAC can directly scavenge free radicals, increase the body's ability to resist oxidative stress, and reduce the production of inflammatory cytokines, chemokines and adhesion molecules. In addition, NAC can also regulate the body's immune status and apoptosis program. It is used for early treatment of liver failure on the basis of comprehensive treatment to reduce bilirubin and increase prothrombin activity.

(2) chlorotin (reduced glutathione for injection, GSH): GSH is a naturally synthesized peptide in human cytoplasm composed of glutamic acid, cysteine and glycine, containing sulfhydryl (-SH) Participate in the body's tricarboxylic acid cycle and sugar metabolism. It activates SH enzymes in the body and promotes the metabolism of carbohydrates, fats and proteins. Reduced glutathione can also promote the formation of low-toxic compounds that are easily metabolized by combining thiol groups with free radicals in the body. Therefore, it can attenuate some exogenous toxic substances and protect liver synthesis, detoxification, inactivation, etc. Features.

(3) Microecological regulators, also known as microecological preparations: are ecological preparations made from normal microorganisms or their promoting substances that are beneficial to the host, which can effectively adjust the intestinal disorders and restore the intestinal micro-ecological environment and inhibit G-bacteria excessively proliferate, significantly reducing serum endotoxin levels. At the cytokine level, microecological modulators can reduce TNFα, IL-6 levels, increase IL-10, IL-2 levels, inhibit the production of inflammatory mediators and reduce the damage of immune cells to hepatocytes, thereby improving chronic heavyness. Clinical symptoms and liver function in patients with hepatitis. Micro-ecological regulators can be divided into three categories: the first is a live bacterial preparation (probiotics), the representative drug is Peficon; the second is a dominant population growth-promoting substance (prebiotic), representing a drug for lactulose; The three types are a combined preparation of living bacteria and probiotics, called synbiotics.

Therefore, finding more effective and safe drugs is of great significance for the treatment of endotoxin-induced liver failure.

Tumor Necrosis Factor α (TNF-α) is the main cytokine of inflammation and the earliest and most important endogenous medium in inflammatory response. Studies have shown that LPS can activate a variety of cells, including peripheral blood mononuclear cells, to induce the production of various endogenous active substances such as TNFs, IFNs, ILs and CSFs. TNF-α binds to Tumor Necrosis Factor Receptor (TNFR) on the target cell membrane to achieve its biological functions such as cytotoxicity, antiviral and immunomodulation. TNFR is present on the surface of a variety of normal cells and tumor cells, and has two subtypes of TNFR1 and TNFR2. Since the intracellular domain of TNFR1 contains a death domain, it plays a major role in cytolytic activity. When TNF-α binds to the extracellular domain of TNFR2 on the target cell membrane, NF-κB is activated by signaling, which initiates and amplifies a series of cytokine expressions involved in the inflammatory response, resulting in an uncontrolled inflammatory response. TNF-α also has functions such as binding to a receptor to induce apoptosis and activation of a JNK signaling pathway. Therefore, TNF-α-mediated NF-κB signaling can be blocked by early inhibition of TNF-α activity to alleviate cytokine overexpression, regulate uncontrolled inflammatory response, and achieve the purpose of protecting organs.

The currently marketed TNF antagonists mainly include a fusion protein of soluble tumor necrosis factor receptor II (sTNFRII) and an Fc segment of IgG (sTNFRII-Fc, etanercept) and a monoclonal antibody against TNF (such as a chimeric type). TNF antibody - Infliximab infliximab and humanized TNF antibody - Adalimumab adalimumab) two major categories. They can alleviate and prevent the clinical and imaging progress of rheumatoid arthritis, significantly reduce the symptoms, improve function and improve the quality of life of patients with rheumatoid arthritis; can significantly improve the arthritis symptoms of patients with psoriatic arthritis, Slow down the progress of imaging. It can reduce the disease activity of patients with ankylosing spondylitis, delay the progress of imaging and improve the quality of life. The trade names of sTNFRII-Fc fusion proteins from different manufacturers are: Yisaipu, Qiangke, Enli. Etanercept, a homodimer, is secreted and expressed by Chinese hamster ovary cells (CHO). With the dimerization of Fc, Etanercept's ability to antagonize TNF is 50- of the corresponding sTNFR II monomer. 1000 times, it competitively binds to TNF-α in the blood, blocks its binding to the TNF receptor on the cell surface, and reduces its activity. The drug is currently used for treatment: 1) moderate to severe active rheumatoid arthritis; 2) moderate to severe plaque psoriasis in adults 18 years of age and older; 3) active ankylosing spondylitis. Currently, no TNF antagonists have been approved for the treatment of liver injury diseases.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) binds to death receptor 5 (DR5) on the cell membrane surface to induce apoptosis. Many bacterial infections have been found to trigger or inhibit apoptosis, and there have been reports of TRAIL involvement in bacterial-induced apoptosis. Soluble DR5 (soluble DR5, sDR5) is a soluble form of DR5 that does not contain a transmembrane region and binds to TRAIL ligands, but does not transmit apoptotic signals to cells, blocking TRAIL-DR5-mediated apoptosis. sDR5 is a human body's own protein, which has the advantages of low toxicity and no immunogenicity. At present, there is no drug for the treatment of liver diseases for this target at home or abroad.

Summary of the invention

In order to solve the above problems, the present invention provides a pharmaceutical composition and use of the pharmaceutical composition for the preparation of a medicament for treating and preventing liver damage.

In particular, one aspect of the invention provides a pharmaceutical composition comprising a tumor necrosis factor antagonist and a tumor necrosis factor-related apoptosis inducing ligand antagonist.

In some embodiments, the tumor necrosis factor antagonist is selected from the group consisting of one or more of an Fc fusion protein of soluble tumor necrosis factor receptor II, a monoclonal antibody against TNF; more preferably a sTNFR-Fc fusion protein.

In some specific embodiments, the tumor necrosis factor-related apoptosis inducing ligand antagonist is one or more selected from the group consisting of an Fc fusion protein of a death receptor, and a monoclonal antibody against a tumor necrosis factor-related apoptosis inducing ligand. A combination thereof is preferably one or more of SEQ ID No. 1-4.

Still another aspect of the present invention provides the use of the aforementioned pharmaceutical composition for the preparation of a medicament for treating liver damage.

In some embodiments, wherein the liver injury is selected from the group consisting of acute liver failure or acute liver injury, subacute liver failure, chronic acute liver failure, chronic liver failure or chronic liver injury, liver damage with endotoxemia, and liver failure Use of the drug.

According to still another aspect of the present invention, a medicament for treating liver damage comprising a tumor necrosis factor antagonist and a tumor necrosis factor-related apoptosis-inducing ligand antagonist, wherein the tumor necrosis factor receptor antagonist is associated with tumor necrosis factor Apoptosis-inducing ligand antagonists are mixed or separated.

The invention uses a tumor necrosis factor antagonist and a tumor necrosis factor-related apoptosis-inducing ligand antagonist in combination, which can effectively inhibit inflammation and cell death, improve survival rate, and play a significant synergistic therapeutic effect.

Tumor necrosis factor-related apoptosis-inducing ligand antagonists and tumor necrosis factor antagonists can neutralize TRAIL and TNF-α by binding to TRAIL and TNF-α, respectively, and the inventors have unexpectedly discovered that simultaneous blocking of both pathways can be Significantly improve the survival rate, reduce the pathological damage of the liver, reduce the level of inflammatory factors, thereby effectively improving the condition and having a synergistic effect. The invention adopts the active component in the form of a fusion protein, and the sDR5-Fc combined with the sTNFR-Fc fusion protein proves that the two pathways of TRAIL and TNF have synergistic relationship and can be used as a medicine for treating liver failure, and the mechanism of action is clear and novel, and the effect is unique. It has remarkable curative effect, high safety and great potential for development.

The general terms used herein are defined to have the following meanings:

The terms "comprising" and "including", as used herein, are intended to have an open and non-limiting meaning.

The term "pharmaceutical composition" as defined herein refers to a kit of fixed combinations, non-fixed combinations or components for combined administration in one unit dosage form; the term "fixed combination" means that the active ingredient is a single active ingredient. Or the dosage form is administered to the subject simultaneously. The term "non-fixed combination" means that the active ingredient is administered to the subject sequentially with separate active ingredients, either simultaneously or without specific time constraints, wherein such administration results in both active ingredients in the subject achieving therapeutically effective levels. The tumor necrosis factor antagonist and the tumor necrosis factor-related apoptosis inducing ligand antagonist may be administered simultaneously, independently, or separately during the time interval in which the combination shows an assisting effect.

As used herein, the term "synergistic effect" refers to two active ingredients that produce a simple additive effect that is greater than the effect of each drug alone (eg, increases the survival rate of a subject with liver failure). According to the calculation formula of synergy factor (SF) (Mario Cortina-Borja, A David Smith, Onofre Combarros, Donald J Lehmann. The synergy factor: a statistic to measure interactions in complex diseases. BMC Res Notes. 2009; 2: 105.), it can be calculated that the synergistic factor of sDR5-Fc and sTNFR-Fc in the treatment of endotoxin-induced liver failure is 100% / (25% * 66.67%) = 5.997 > 1, indicating that sDR5-Fc and sTNFR-Fc are obvious Synergistic treatment effect.

The sDR5-Fc fusion protein of the present invention is selected from the sDR5-Fc fusion protein disclosed in the patent application CN201610067931.2 or PCT/CN2016/089650. Specifically, it is selected from the sDR5-Fc fusion protein having the following amino acid sequences of SEQ ID NOS. 1 to 4.

SEQ ID NO. 1:

SEQ ID NO. 2:

SEQ ID No. 3:

SEQ ID NO. 4:

Beneficial effect

1. The present inventors have provided new ideas for the development of therapeutic drugs for liver damage.

2. The tumor necrosis factor antagonist of the present invention in combination with a tumor necrosis factor-related apoptosis-inducing ligand antagonist blocks apoptosis induced by the TRAIL/DR5 pathway and blocks TNF-a/TNFRII pathway-mediated inflammation and cells Apoptosis reduces the death and liver pathological changes caused by endotoxin-induced acute liver failure, thereby achieving the function of protecting liver function and improving survival rate.

DRAWINGS

Figure 1 is a graph showing the survival rate of mice with endotoxin-induced acute liver failure, in which sDR5-Fc combined with sTNFR-Fc administration significantly increased survival rate in mice compared to sDR5-Fc or sTNFR-Fc alone. P < 0.05. The synergistic factor was 100%/(25%*66.67%)=5.997>1, indicating that sDR5-Fc and sTNFR-Fc have significant synergistic therapeutic effects.

Figure 2 is a graph showing the results of serum transaminase levels in mice in which sDR5-Fc in combination with sTNFR-Fc significantly reduced serum transaminase levels in endotoxin-induced acute liver failure mice. At 6h, the level of liver injury in the sDR5-Fc treatment group was similar to that in the saline group, while the sTNFR-Fc treatment group and the combination therapy group significantly inhibited or delayed the occurrence of liver injury, and the transaminase level was significantly lower (P<0.05). ). At 24h, the mice in the sDR5-Fc treatment group and the saline group had died a lot, and the sTNFR-Fc treatment group showed obvious liver damage, while the combination therapy group still significantly inhibited the occurrence of liver injury, and the transaminase level was obvious. Low (P<0.05).

Figure 3 is a graph showing the results of serum inflammatory factor levels in mice in which sDR5-Fc in combination with sTNFR-Fc significantly reduced the level of serum inflammatory factors in endotoxin-induced acute liver failure mice. At 6 h after modeling, the levels of IL-6 in the sDR5-Fc group were similar to those in the saline group, while the sTNFR-Fc treatment group and the combination therapy group significantly reduced IL-6 levels (P<0.05). Compared with the saline group, the sDR5-Fc group, the sTNFR-Fc treatment group and the combination treatment group can significantly increase the level of the anti-inflammatory factor IL-10, thereby down-regulating the inflammatory response and antagonizing the inflammatory mediator. At 24h and 48h, the level of IL-10 in the sTNFR-Fc-treated group and the combination therapy group continued to increase, and the IL-6 level in the combination therapy group was significantly lower than that in the sTNFR-Fc-treated group (P<0.05). As can be seen from the figure, sDR5-Fc can not only block apoptosis by binding to TRAIL, but also has an obvious anti-inflammatory effect, and can synergize with sTNFR-Fc.

Figure 4 is a graph showing the pathological damage of liver in mice, in which A is a saline group, and hepatocytes die in a large area; B is a sDR5-Fc group, and the number of hepatocyte death is less than that of a saline group; C is a sTNFR-Fc group, Hepatocytes with normal morphology were observed; D was sDR5-Fc+sTNFR-Fc group, the liver structure remained good, and most of the liver cells were normal. It can be seen from the figure that sDR5-Fc combined with sTNFR-Fc can significantly reduce liver pathological damage in mice with endotoxin-induced acute liver failure.

Fig. 5 is a graph showing the results of the number of TRAIL-positive infiltrating lymphocytes in the liver of mice, wherein A is a sTNFR-Fc group, and a large amount of infiltrating TRAIL-positive lymphocytes around the blood vessels is observed; B is a sDR5-Fc+sTNFR-Fc group, see There are few infiltrating lymphocytes around the blood vessels, and fewer TRAIL-positive lymphocytes. Thus, sDR5-Fc in combination with sTNFR-Fc significantly reduced the number of TRAIL-positive infiltrating lymphocytes in the liver of mice with endotoxin-induced acute liver failure.

detailed description

The invention is further illustrated by the following examples, but the scope of protection of the invention is not limited thereto.

The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. The various reagents and samples used in the examples are commercially available unless otherwise stated.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning The terms used in the description of the present invention are for the purpose of describing the specific embodiments and are not intended to limit the invention. The term "and/or" used in the present invention includes any and all combinations of one or more of the associated listed items.

Example 1 Human sDR5-Fc antibody fusion protein in combination with sTNFR-Fc antibody fusion protein is capable of treating endotoxin-induced acute liver failure in mice.

Fourteen C57BL/6 mice were randomly divided into 4 groups (1 to 4 groups), 3 to 4 mice in each group. Each mouse was intraperitoneally administered with 800 mg/kg D-galactose (D-GaIN) and 50 ug/kg. Bacterial lipopolysaccharide (LPS) with an injection volume of 10 ml/kg. Before the model was established for 1 h, each group of mice was given: (1 group) intravenous injection of 10 ml/kg of normal saline, (2 groups) intravenous injection of 16.1 mg/kg sDR5-Fc protein, and (3 groups) intraperitoneal injection of 50 mg/kg recombinant Human type II tumor necrosis factor receptor-antibody fusion protein (Shanghai CITIC Guojian Pharmaceutical Co., Ltd., Sinopharm S20050058, Yisaipu, sTNFR-Fc), (4 groups) intravenous injection of 16.1 mg/kg sDR5-Fc protein (SEQ ID NO. 4) and intraperitoneal injection of 50 mg/kg sTNFR-Fc. Blood was collected from the submandibular vein at 6h, 24h and 48h after model establishment. Serum was separated and serum transaminase levels and inflammatory cytokine levels were measured. The mice were sacrificed 48 h after model establishment, and some livers were fixed in 4% PFA, embedded in paraffin, sectioned, HE stained, and TRAIL immunohistochemical staining. It can be seen that the combination of sDR5-Fc and sTNFR-Fc can significantly improve the survival rate of mice, reduce the serum transaminase level of mice, reduce the pathological damage of liver in mice, reduce the expression level of inflammatory factors, and simultaneously infiltrate lymphocytes positive for TRAIL. The number is significantly reduced.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A pharmaceutical composition comprising a tumor necrosis factor antagonist and a tumor necrosis factor-related apoptosis inducing ligand antagonist.
The pharmaceutical composition according to claim 1, wherein the tumor necrosis factor antagonist is selected from the group consisting of one or more of an Fc fusion protein of soluble tumor necrosis factor receptor II and a monoclonal antibody against TNF-α; Preferred is a combination of one or more of the sTNFR-Fc fusion proteins.
The pharmaceutical composition according to claim 1, wherein the tumor necrosis factor-related apoptosis inducing ligand antagonist is selected from the group consisting of an Fc fusion protein of a death receptor and a monoclonal antibody against a tumor necrosis factor-related apoptosis inducing ligand. A combination of one or more of them is preferably one or more of SEQ ID No. 1-4.
Use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for treating liver damage.
The use according to claim 4, wherein the liver injury is selected from the group consisting of acute liver failure or acute liver injury, subacute liver failure, chronic acute liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin Use in induced liver damage, liver damage with endotoxemia, and liver failure.
A medicament for treating liver injury, comprising a tumor necrosis factor antagonist and a tumor necrosis factor-related apoptosis-inducing ligand antagonist, wherein the tumor necrosis factor antagonist and the tumor necrosis factor-related apoptosis-inducing ligand antagonist are mixed or Separate placement.
The medicament according to claim 6, which is a parenteral preparation, preferably an injection.