RU2575825C2 - Use of glycyrrhetinic acid, glycyrrhizic acid and related compounds for preventing and/or treating pulmonary fibrosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely pulmonology, and aims at preventing or treating a radiation-induced pulmonary disease. Preventing, treating or relieving a radiation-induced pulmonary disease, particularly pulmonary fibrosis are ensured by administering an effective amount of a recovered or substantially pure compound or its salt into an individual receiving a dose of radiation and being in need of the above prevention, treatment or relief. The above compound is specified in a group consisting of glycyrrhetinic acid (GA), glycyrrhizic acid (GLA), an ester, ether and/or amide form of glycyrrhetinic acid (GA), and an ester and/or amide form of glycyrrhizic acid (GLA).

EFFECT: using the group of inventions makes it possible to prevent, treat or relieve a radiation-induced pulmonary disease, particularly pulmonary fibrosis.

18 cl, 44 dwg, 1 tbl, 10 ex

Description

FIELD OF THE INVENTION

In General, the present invention relates to the use of glycyrrhetinic acid, glycyrrhizic acid and related compounds for the prevention and / or treatment of pulmonary fibrosis and lung diseases associated with pulmonary fibrosis.

State of the art

Pulmonary fibrosis (LF) is an asymptomatic developing condition characterized by the gradual replacement of normal parenchyma cells by fibrous (fibrous), connective, matrix macromolecules (e.g., collagen, fibronectins and proteogdicans) on the surface and inside the lungs, usually at the sites of injury or infection. Excessive formation of fibrous tissue as a result of activation and proliferation of fibroblasts destroys the normal structure of the lungs and their function. For example, the accumulation of fibrous tissue leads to a thickening of the alveolar septa, reduces the volume of air space and causes damage to the epithelium or even alveolar collapse. As a result, patients suffering from pulmonary fibrosis experience varying degrees of dyspnea during physical exertion, and in the later stages, orthopnea, cyanosis and respiratory failure ¹ . The median survival for pulmonary fibrosis is about 2–3 years, and approximately 65% of patients die within 5 years of diagnosis.

Radiation therapy (IR), the most common treatment for a tumor or cancer, can cause lung damage and ultimately lead to IR-induced pneumopathy, including pulmonary fibrosis ¹ . The risk of IR-induced pneumopathy is further increased with the simultaneous administration of cytotoxic chemotherapeutic agents. IR-induced pneumopathy not only has a devastating effect on a patient’s quality of life, but can sometimes be even more life-threatening than a primary tumor or cancer ¹ . Thus, the risk of IR-induced pneumopathy, such as pulmonary fibrosis, has become the main factor limiting the dose of radiation, and sometimes it does not even allow the use of radiation therapy.

There is currently no cure for pulmonary fibrosis. While anti-inflammatory or lung-protective agents can alleviate the symptoms of pulmonary fibrosis to some extent or improve a patient’s life, they cannot stop the progression of the disease. Common drugs for pulmonary fibrosis include amifostine, celebrex, and dexamethasone. Although amifostine gives a definite cytoprotective effect from IR-induced lung damage ² , it must be administered to patients 30 minutes before lung damage, and thus it cannot prevent or treat accidental lung damage. There is no information on the effectiveness of celebrex, a widely used anti-inflammatory drug, for treating pneumonitis or pulmonary fibrosis. Dexamethasone is a steroidal anti-inflammatory drug and can cause serious side effects with prolonged use. In addition, the inventors of the present invention have found that dexamethasone can lead to a worsening of pulmonary fibrosis, leading to pulmonary failure or even earlier death compared to untreated control groups. Thus, therapeutic agents with improved efficacy and safety are urgently needed.

Traditional Chinese medicine has been practiced by the Chinese people for 2-3 millennia. It applies to various pathologies, as well as the diagnosis, treatment and prevention of diseases. Chinese medicinal substances are recorded in various pharmacopoeias. One of the classic references for medicinal herbs is Ben Cao Gang Mu, written by Li Shizhen at the end of the 14th century. This book describes about 2,500 herbs and other foods, including animals and minerals.

Licorice vulgaris (Glycyrrhiza glabra) has a long and diverse history of medicinal use in East Asia. Its root is used as a therapeutic agent for the treatment of dermatitis and peptic ulcer (peptic ulcer). Glycyrrhizic acid (also known as Glycyrrhizin) (GLA) is a natural compound that can be isolated from the root of the species Glycyrrhiza, including licorice (Glycyrrhiza glabra). GLA is widely used as a flavoring agent in the United States and Europe, and has been approved by the State Administration for Drug and Food Quality Control of China for the treatment of chronic hepatitis and cirrhosis. Glycyrrhetinic acid (GA), a pentacyclic triterpenoid derivative that forms the functional fragment of glycyrrhizic acid (GLA), has been used to treat inflammation, peptic ulcer disease and infection. However, it was not previously noted that GA, GLA and its related compounds play any role in the treatment of pulmonary fibrosis.

SUMMARY OF THE INVENTION

The present invention relates to new uses of glycyrrhetinic acid (GA), glycyrrhizic acid (GLA) and related compounds for the prevention and / or treatment of pulmonary fibrosis and lung diseases associated with pulmonary fibrosis. The method of the invention comprises administering to a patient in need of such treatment an effective amount of one or more of the compounds and compositions of the present invention. The therapeutic uses of prodrugs, metabolites, derivatives (for example, acids, esters, ethers and amides) as well as salts of glycyrrhetinic (GA) and glycyrrhizic (GLA) acids are also described.

In one embodiment, the compounds and pharmaceutical compositions of the present invention can be used to treat or ameliorate pulmonary fibrosis, including, but not limited to, radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis.

The present invention also relates to therapeutic or pharmaceutical compositions comprising the compounds of the present invention in a form that can be combined with a pharmaceutically acceptable carrier. In preferred embodiments of the invention, the compositions are formulated for delivery to the lungs. The present invention also relates to food additives and dietary products or beverage compositions containing the compounds of the invention.

Brief Description of the Figures

Figure 1 illustrates the occurrence of inflammatory reactions resulting from irradiation. (A) - (E) represent the change in the levels of inflammatory mediators (IM) in mice exposed to IR (radiation) at a dose of 0, 5 or 9 Gy.

Figure 2 shows the fluctuation in IM levels in C57BL / 6 and C3H / NeH mice with brain damage caused by IR (irradiation) (6 hours and 1, 2, 4, and 10 days after irradiation at a dose of 9 Gy).

Figure 3 illustrates the inhibitory effects of glycyrrhetinic acid (GA) on radiation-induced (IR) inflammation during the acute phase of IR-induced pneumonitis.

Figure 4 shows that GA reduces inflammatory cell infiltration and plasma exudation in the interstitial space during the acute phase of IR-induced pneumonitis (2.5 days after confirmation of thoracic radiation at a dose of 15 Gy).

Figure 5 shows that GA lowers plasma levels of SP-D and ILα during the acute phase of IR-induced pneumonitis.

6 shows that GA lowers the levels of inflammatory mediators during the subacute phase of IR-induced pneumonitis (17 days after IR).

FIG. 7 shows that GA lowers the number of neutrophils and the activity of myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF) during the subacute phase of IR-induced pneumonitis (17 days after IR at a dose of 15 Gy).

Fig. 8 shows that GA attenuates the subacute phase of IR-induced pneumonitis (17 days after confirmation of IR at a dose of 15 Gy).

Figure 9 shows that GA inhibits inflammatory reactions during the subacute phase of IR-induced pneumonitis. GA was administered to mice 24 hours after IR at a dose of 15 Gy.

Figure 10 shows that GA reduces the respiratory rate in mice with pulmonary fibrosis caused by IR. (A) shows a decrease in respiratory rate 7 months after IR at a dose of 15 Gy. (B) shows a decrease in respiratory rate 5 months after IR at a dose of 18 Gy.

11 depicts a lung of a mouse by cone beam computed tomography (CBCT).

12 shows CBCT images of lungs of mice.

13 shows an increase in lung density as a result of IR. The lung density of the mouse of the normal control group (not exposed to IR) is represented by the dashed line. The lung density of the mouse one month after thoracic IR at a dose of 15 Gy is shown by solid lines.

14 shows that GA lowers lung density in mice with IR-induced pulmonary fibrosis 7.5 months after IR at a dose of 15 Gy.

Fig. 15 shows the effects of GA on lung compliance 6.5 months after IR at a dose of 15 Gy.

16 shows that GA attenuates pulmonary fibrosis caused by IR.

17 shows that GA attenuates pulmonary fibrosis caused by IR.

Fig. 18 shows that GA lowers collagen deposition in mice with pulmonary fibrosis caused by IR.

19 shows that GA increases the body weight of mice with pulmonary fibrosis caused by IR.

Figure 20 shows that glycyrrhizic acid (GLA) attenuates pneumonitis in cancer patients who received radiation therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new uses of glycyrrhetinic acid (GA), glycyrrhizic acid (GLA) and related compounds for the prevention and / or treatment of pulmonary fibrosis, in particular pulmonary fibrosis caused by radiation. The therapeutic uses of prodrugs, metabolites, derivatives (e.g. acids, esters and ethers) and glycyrrhetinic acid salts (GA) and glycyrrhizic acid (GLA) are also described.

In one embodiment, the method of the invention comprises administering to a subject in need of such treatment an effective amount of the compounds and compositions of the present invention. The present invention can also be used to treat lung diseases associated with pulmonary fibrosis.

The present invention is also intended for therapeutic or pharmaceutical compositions containing a compound of the invention in a form that can be combined with a pharmaceutically acceptable carrier. In preferred embodiments, the compositions of the invention are in a form suitable for delivery to the lungs.

Connections

The present invention relates to therapeutic applications of glycyrrhetinic acid (GA), glycyrrhizic acid (GLA) and related compounds. The therapeutic uses of prodrugs, metabolites, derivatives (e.g. acids, esters and ethers) and glycyrrhetinic acid salts (GA) and glycyrrhizic acid (GLA) are also described.

GA and GLA have been found to effectively suppress pneumonia, relieve symptoms of lung damage, improve lung function, and reverse the progressive deposition of fibrous tissue in the lungs. In particular, GA attenuates pulmonary inflammatory responses and significantly reduces levels of lung-specific inflammatory mediators (e.g., IL-1, PF-4, SP-D, IL1α, TNFα, lymphotactin, p-selectin, 1-selectin, sTNF-R1 and VACAM-1) during the acute and subacute stages of pneumonitis. In addition, GA helps maintain the integrity of the alveolar-capillary barrier, as evidenced by a decrease in plasma exudation and inflammatory cell infiltration in subjects treated with GA. Additionally, GA significantly alleviates symptoms of pulmonary fibrosis, improves lung tissue morphology, and suppresses excess collagen deposition in the lungs.

In one embodiment, the present invention relates to glycyrrhetinic acid (GA) (MV: 470.68) having the following structure (Structure A):

In another embodiment, the present invention relates to glycyrrhizic acid (GLA) (MB: 822.93), a triterpenoid saponin glycyrrhetinic acid glycoside having the following structure (Structure B):

Glycyrrhizic acid can be isolated from the licorice root (Glycyrrhiza glabrd) or other types of licorice (Glycyrrhiza).

In some embodiments, the present invention provides ester, ester, and / or amide forms of glycyrrhetinic acid (GA) represented by the following structure (Structure C):

,

,

Where

X represents any group forming an ether or ester bond with a hydroxyl radical; and

Y represents any group that forms an ester or amide bond with a carboxyl group.

In some embodiments, the present invention provides ester and / or amide forms of glycyrrhizic acid (GLA) represented by the following structure (Structure D):

where Y 'represents any group forming an ester or amide bond with a carboxyl group.

In some embodiments, X may be alkyl substituted with alkyl (e.g., haloalkyl and hydroxyalkyl), alkenyl substituted with alkenyl, —COOH, acyl, alkylcarbonyl, benzyl, cyclic alkyl, or cyclic alkenyl.

In some embodiments, X may be an organic or inorganic acid group including, but not limited to, acetic acid, carboxylic acid, aspartic acid, formic acid, citric acid, benzoic acid, hippuric acid, malic acid, mucic acid, phosphoric acid, sulfuric acid, gluconic acid, maleic acid, succinic acid, tartaric acid and lactic acid.

In some embodiments, X may be a carbohydrate group in which the monosaccharide, disaccharide, oligosaccharide or its derivative loses —H in the hydroxyl group and, accordingly, forms a radical. Suitable carbohydrate groups can be obtained, for example, from glucose, fructose and sucrose.

In some embodiments, Y and Y ′ may independently be —NH ₂ , alkylamino, or alkoxy.

“Alkyl” means a linear saturated monovalent radical containing from one to sixteen carbon atoms, or a branched saturated monovalent radical containing from three to sixteen carbon atoms. Alkyl may include hydrocarbon radicals containing from one to four or from one to three carbon atoms, which may be linear. Examples include methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl and the like. “Alkenyl” means a linear or branched C ₂ -C ₁₆ hydrocarbon radical containing one or more carbon-carbon double bonds. Examples include propylene, buten-1-yl, isobutenyl, penten-1-yl, 2,2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, hepten-1-yl, octen- 1-yl and the like. “Acyl” means the radical —C (O) R, in which R is hydrogen, alkyl or cycloalkyl, or heterocycloalkyl. Examples include formyl, acetyl, ethylcarbonyl and the like.

“Carboxyl” means the radical —CO (O) H.

“Carbalkoxy” means the radical —C (O) R, in which R is, for example, hydrogen, alkyl or cycloalkyl, heterocycloalkyl, halogen or haloalkyl. “Halogen” means fluoro, chloro, bromo or iodo, for example bromo and chloro. “Haloalkyl” means alkyl substituted with one or more identical or different halogen atoms, for example —CH ₂ Cl, —CH ₂ Br, —CF ₃ , —CH ₂ CH ₂ Cl, —CH ₂ CCl ₃ and the like.

“Amino” means the radical —NH ₂ .

“Alkylamino” means the radical —NHR or —NR ₂ in which each R independently represents an alkyl group. Examples include methylamino, (1-methylethyl) amino, dimethylamino, methylethylamino, di (1-methylethyl) amino, and the like.

“Hydroxy” means the radical —OH.

“Hydroxyalkyl” means an alkyl radical, as defined herein, substituted with one or more, preferably one, two, or three hydroxyl groups. Illustrative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1- (hydroxymethyl) -2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl , 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2- (hydroxymethyl) -3-hydroxypropyl, preferably 2-hydroxyethyl, 2,3-dihydroxypropyl and 1- (hydroxymethyl) -2- hydroxyethyl.

“Alkoxy” means the radical —OR _a , where R _a represents an alkyl group or a substituted alkyl group.

Examples of alkoxy groups include methoxy, ethoxy, propoxy and the like.

In one embodiment, the present invention relates to acetoxolone (C ₃₂ H ₄₈ O ₅ , CAS No.6277-14-1), an acetyl derivative of glycyrrhizic acid.

The present invention also relates to salt forms of GA, GLA and related compounds, including (but not limited to) ammonium salts, sodium salts and potassium salts.

The present invention also relates to the use of prodrugs and metabolites of the compounds. The term "prodrug", as used in the present invention, refers to the metabolic precursor of the compounds of the present invention or its pharmaceutically acceptable form. In general, prodrugs include functional derivatives of compounds that may be inactive when administered to a subject, but are readily converted to an active metabolite in vivo.

Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in Design of Prodrugs, edited by H. Bundgaard, Elsevier, 1985. The prodrug of the present invention preferably enhances the desired characteristics of the compounds of the present invention, including solubility, bioavailability and stability, but , not limited only to them. Thus, the compounds used in the methods of the invention can, if desired, be administered in the form of a prodrug. Prodrugs of the compounds used in the present invention can be obtained by modifying the functional groups present in the compound so that the modifying groups are cleaved either during routine manipulation or in vivo to form the basic compound.

The term "metabolite" refers to a pharmacologically active product, including, for example, an active intermediate or end product obtained by in vivo metabolism of a compound of the present invention in a subject. A metabolite can be obtained due, for example, to anabolic and / or catabolic transformations of an administered compound in a subject's body, including, but not limited to, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like.

Metabolites are typically determined by preparing an isotope-labeled (e.g., ¹⁴ C or ³ H) compound of the present invention, administering it parenterally in a detectable dose (e.g., at a dose of more than about 0.5 mg / kg) to an animal, such as a rat , mouse, guinea pig, monkey, or human, waiting enough time for the metabolism to occur (usually from about 30 seconds to 30 hours), and isolating the conversion products of the compound from urine, blood, or other biological samples. These products are easy to isolate, since they are labeled with isotopes (others are isolated by the use of antibodies that can bind epitopes that survive in the metabolism). The structure of the metabolites can be determined in the usual way, for example using MS, LC / MS or NMR analyzes. In general, the analysis of metabolites is carried out in accordance with methods well known to specialists in the field of studies of drug metabolism.

The present invention further relates to isolated enantiomeric compounds. The isolated enantiomeric forms of the compounds of the invention are substantially free of each other (i.e., enantiomeric excess occurs). In other words, the “R” forms of the compounds are substantially free of the “S” forms of the compounds and are thus present in enantiomeric excess relative to the “S” forms. Similarly, the “S” forms of the compounds are substantially free of the “R” forms of the compounds and are thus present in enantiomeric excess relative to the “R” forms. In one embodiment, the isolated enantiomeric compounds are present in at least about 80% enantiomeric excess. In a preferred embodiment, the compounds are present in at least about 90% enantiomeric excess. In a more preferred embodiment, the compounds are present in at least about 95% enantiomeric excess. In an even more preferred embodiment, the compounds are present in at least about 97.5% enantiomeric excess. In a most preferred embodiment, the compounds are present in at least about 99% enantiomeric excess.

In one embodiment, the compounds of the present invention have the same chiral structure as shown in any of the structures AD.

Prevention and / or Treatment of Pulmonary Fibrosis

The present invention relates to methods for preventing and / or treating pulmonary fibrosis, in particular pulmonary fibrosis, caused by radiation. The methods of the present invention can also be used to prevent, treat or ameliorate lung diseases associated with pulmonary fibrosis.

In one embodiment, the method of the invention comprises administering to a subject in need of such treatment an effective amount of the compounds and compositions of the present invention. Preferably, the compounds and compositions of the present invention are prepared in a form suitable for administration to the lungs.

The term "pulmonary fibrosis" or "pulmonary fibrosis", as used in the present invention, refers to the atypical formation or accumulation of fibrous, connective or scar tissue and / or matrix macromolecules (e.g., collagen, fibronectin, proteoglycan) on the surface and / or inside the lungs. Symptoms of pulmonary fibrosis include shortness of breath, dry cough, increased respiratory rate, decreased lung elasticity, increased lung density, chest discomfort, and rapid weight loss. Pulmonary fibrosis does not cover fibrotic conditions that develop in organs other than the lungs, such as fibrotic conditions that develop in the liver.

The term “treatment” or any variation thereof (eg, treat, treatment, therapeutic therapy, etc.) used in the present invention includes amelioration or alleviation of symptoms of a disease or condition, reduction, suppression, deceleration, weakening, or effect on progression , severity and / or scale of the condition, but are not limited to this. In one embodiment, the treatment relates to reducing, suppressing, slowing, weakening, or influencing the progression, severity and / or scale of the atypical formation or accumulation of fibrous, connective or scar tissue and / or matrix macromolecules (e.g., collagen, fibronectin and proteoglycan) on the surface or inside the lungs.

The term “prevention” or any variation thereof (eg, prevent, prevention and prophylaxis, etc.) used in the present invention includes delaying the onset of symptoms, preventing relapse of the disease, increasing the delay between symptomatic episodes, or combinations thereof, but are not limited to by this. Prevention, as the term is used in the present invention, does not require an absolute absence of symptoms.

Pulmonary fibrosis-related lung diseases include complications of pulmonary fibrosis, lung diseases that can develop with pulmonary fibrosis, and lung diseases that result from pulmonary fibrosis. Symptoms and complications of pulmonary diseases include, but are not limited to, hypoxemia, dyspnea, orthopnea, cyanosis, pulmonary hypertension, pulmonary heart, and lung dysfunction. Pulmonary conditions that can develop into pulmonary diseases include (but are not limited to) lung damage (e.g., radiation, chemicals, drug therapy, biological damage or contamination), lung infections (e.g. viral, bacterial, fungal and parasitic infections), interstitial lung disease, lung damage caused by parasitic infections, and pneumonitis.

"Pneumonitis," as the term is used in the present invention, refers to its usual meaning, which is inflammation of the lung tissue.

The term "effective amount" as used in the present invention refers to an amount that is able to prevent, ameliorate or treat pulmonary fibrosis. For example, an effective amount is an amount capable of alleviating one or more symptoms of pulmonary fibrosis. In some embodiments, an effective amount provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, A 90% or 100% reduction in respiratory rate, a decrease in lung density, an increase in body weight and / or an increase in lung tissue elasticity compared to subjects with pulmonary fibrosis not receiving treatment.

In a specific embodiment, an effective amount provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, A 90% or 100% reduction in the atypical accumulation of fibrous tissues (eg, collagen, fibronectin and proteoglycans) in the lungs compared to subjects with non-treated pulmonary fibrosis. For example, an effective amount provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in collagen, fibronectin, proteoglycan, and / or hydroxyproline content in the lungs compared to subjects with non-treated pulmonary fibrosis. In another example, an effective amount provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or a 100% reduction in profibrotic mediators, such as TGFp in lung tissue, compared to subjects with non-treated pulmonary fibrosis.

In addition, since pulmonary fibrosis in many cases arises from inflammatory reactions in response to pulmonary damage or infection, an effective amount can reduce the level of one or more pulmonary inflammatory mediators in lung tissue. Illustrative examples of pulmonary inflammatory mediators include, but are not limited to, SP-D, IL1α, TNFα, IL6, PF4, P-selectin, L-selectin, VCAM-1, lymphotactin and prostaglandin E (PGE). In some embodiments, an effective amount provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, A 90% or 100% reduction in the levels of one or more inflammatory mediators compared to subjects with non-treated pulmonary fibrosis.

The term “subject,” as used herein, describes an organism, including mammals, such as primates, which may be treated with the compositions of the present invention. Types of mammals that may benefit from the described methods of treatment include monkeys, chimpanzees, orangutans, humans, monkeys; and domestic animals such as dogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs and hamsters, but are not limited to them.

The expression “subject in need of such treatment” as used in the present invention refers to a subject who is at risk, has symptoms, or is diagnosed with pulmonary fibrosis and / or pulmonary disease associated with pulmonary fibrosis. In one embodiment, the present invention includes the diagnosis of pulmonary fibrosis in a subject, during which the compounds and compositions of the present invention are administered to a subject who has a diagnosis or symptoms of pulmonary fibrosis.

Identification of subjects who have pulmonary fibrosis is within the knowledge and abilities of a qualified specialist in this field of knowledge. For example, a qualified physician in this area can, using physical tests, such as a pulmonary function test and test exercises, easily notice visible symptoms of pulmonary fibrosis. In addition, a combination of medical methods such as chest x-ray, high-resolution computed tomography (CT) and a surgical lung biopsy can be used to determine pathological changes in lung tissue caused by pulmonary fibrosis.

In another embodiment, the compounds and compositions of the present invention are administered to a subject who does not have visible symptoms of pulmonary fibrosis, but for which a predisposition to the development of pulmonary fibrosis has been established (hereinafter, such a patient is referred to as “patient at risk”). For example, “high-risk patients” include subjects who have had lung damage (eg, radiation, chemicals, drug therapy, biological damage and contamination), pulmonary infection (eg, viral, bacterial, fungal and parasitic infections), as well as diseases like pneumonitis and interstitial lung disease. In one embodiment, prior to administration of the compounds and compositions of the present invention, a patient is evaluated to determine the risk of developing pulmonary fibrosis. In yet another embodiment, the subject is a cancer patient who has received or is receiving radiation therapy.

In one embodiment, the compounds and pharmaceutical compositions of the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis, including, but not limited to, radiation-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, and cystic pulmonary fibrosis.

In one embodiment, the present invention can be used to prevent, treat, or ameliorate lung damage caused by radiation and / or radiation pneumopathy, including pneumonitis and pulmonary fibrosis. In one embodiment, the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis caused by tumor radiation therapy or cancer treatment. In another embodiment, the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis resulting from accidental lung damage caused by radiation or nuclear incidents.

In one embodiment, the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis caused by thoracic radiation. In some embodiments, the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis caused by irradiation in doses of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 120, or 150 Gy. In addition, the present invention can be used to prevent, treat or ameliorate pulmonary fibrosis caused by radiation in doses of at least 0.1, 0.3, 0.5, 0.7, 0.9, 1.0, 1, 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3.0, 3.2, 3.5 or 4.0 Gy per day.

In one embodiment, the compounds and pharmaceutical compositions of the present invention can be used to prevent, treat, or ameliorate pulmonary fibrosis caused by radiation (including radiation therapy), contaminants, toxins, trauma, smoking, autoimmune diseases such as rheumatoid arthritis , drug therapy (e.g., amiodarone, bleomycin, busulfan, methotrexate and nitrofurantoin), asbestos and infection (e.g., viral, bacterial, fungal and steam itarnoy infection).

In one embodiment, the compounds and pharmaceutical compositions of the present invention can be used to prevent, treat, or alleviate a disease that develops into pulmonary fibrosis, including interstitial lung disease, acute and / or chronic pneumonitis, chronic obstructive pulmonary disease (COPD), asthma, silicosis lung damage and pneumonia.

In one embodiment, the compounds and pharmaceutical compositions of the present invention can be used to prevent, treat, or ameliorate fibrotic diseases or conditions that develop in the skin, heart, intestines, and / or retroperitoneal space. In one embodiment, the present invention excludes the treatment of liver fibrosis.

Although in experimental models of the present invention, pulmonary fibrosis was induced by irradiation of the chest, one skilled in the art will recognize that the therapeutic benefits of the present invention extend beyond IR-induced pulmonary fibrosis.

In yet another embodiment, the present invention does not cover the treatment of inflammation of tissues other than lung tissue. In one embodiment, the present invention does not cover the treatment of inflammatory conditions of the liver and skin. In another embodiment, the present invention does not include the treatment of one or more inflammatory conditions and diseases, including hepatitis, cirrhosis, hypertension, and non-pulmonary edema. In another embodiment, the present invention does not cover the treatment of viral (e.g., influenza A, herpes simplex virus, SARS and / or HIV), bacterial (e.g., staphylococcus) or fungal infections that do not develop into pulmonary fibrosis. In another embodiment, the present invention does not cover the treatment of dermatitis, peptic ulcer or rheumatoid arthritis. In another embodiment, the present invention does not cover the treatment of respiratory diseases that do not lead to pulmonary fibrosis.

Therapeutic compositions and formulations

The present invention also relates to therapeutic or pharmaceutical compositions comprising a compound of the invention in a form that can be combined with a pharmaceutically acceptable carrier. In this regard, the compound of the invention may be isolated or substantially pure. The present invention also includes nutritional supplements and diet food or beverage compositions containing a compound of the invention.

The term “carrier” refers to a diluent, adjuvant, excipient or delivery vehicle with which the compound of the invention is administered. Such pharmaceutical carriers may be sterile fluids, such as water and oils, including petroleum oils, such as mineral oil, vegetable oils, such as peanut oil, soybean oil and sesame oil, animal oils or synthetic oils. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially in the case of injectable solutions. Particularly preferred pharmaceutical carriers for treating or alleviating inflammatory processes in the central nervous system are carriers that can cross the blood-brain barrier. In accordance with the use in the present invention, the carriers do not include natural plant materials as it exists in nature.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol and the like. The therapeutic composition, if desired, may also contain small amounts of moisturizing or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, capsules, powders, sustained release formulations and the like. The composition may be formulated using conventional binders and carriers, such as triglycerides. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin Such compositions comprise a therapeutically effective amount of the therapeutic composition together with a suitable amount of carrier to obtain a form suitable for administration to a patient. The composition should correspond to the method of administration.

In preferred embodiments, the compositions are prepared in a form adapted for delivery to the lungs. For example, upon inhalation delivery, the liquid pharmaceutical composition can be lyophilized prior to use, where the lyophilized composition is ground to obtain a finely divided dry powder consisting of particles with sizes within the above desired range. On the other hand, for example, spray drying can be used to obtain a dry powder form of a liquid pharmaceutical composition, and this process is carried out under conditions that result in a substantially amorphous finely divided dry powder consisting of particles with sizes within the desired range. For methods for preparing dry powder forms of pharmaceutical compositions, see, for example, WO 96/32149, WO 97/41833, WO 98/29096 and US patents. No. 59976574, 5985248, 6001336 and 6875749, which are incorporated herein by reference. In addition, a dry powder form of a pharmaceutical composition can be prepared and used as an aqueous or non-aqueous solution or suspension in metered-dose inhalers.

In addition, a pharmaceutically effective amount of a dry powder form of the composition can be prepared in the form of an aerosol or other preparation suitable for pulmonary inhalation. The amount of dry powder form of the composition placed in the delivery device should be sufficient to ensure the delivery of a pharmaceutically effective amount of the composition to the subject upon inhalation. The delivery device provides the introduction in one or more fractional doses due to pulmonary inhalation of a pharmaceutically effective amount of the composition into the patient's lungs. The aerosol propellant may be any conventional material used for this purpose.

When used in the context of pharmaceutical compositions suitable for delivery to the lungs, these terms have the following meanings. By "aqueous" is meant a composition obtained with water, containing water or dissolved in water, including mixtures, where water is the predominant substance in the mixture. By "non-aqueous" is meant a composition obtained with, containing or dissolved in a substance other than water and a mixture in which water is not the predominant substance. By “solution” is meant a homogeneous preparation of two or more substances, which may be solids, liquids, gases, or various combinations thereof. By "suspension" is meant a mixture of substances such that one or more insoluble substances are uniformly dispersed in another predominant substance.

For the purposes of the present invention, the terms “solid” and “dry powder” are used interchangeably where pharmaceutical compositions are concerned. By “solid” or “dry powder” form of a pharmaceutical composition is meant a composition that has been dried to a fine powder with a moisture content below about 10% by weight, typically below about 5% by weight and preferably below about 3% by weight. The specified dry powder form of the composition consists of particles containing the peptides of the present invention. Preferably, the average particle diameter is less than about 90.0 microns, more preferably less than about 70.0 microns, more preferably less than about 50.0 microns, even more preferably less than about 30.0 microns, more preferably less than about 10 , 0 microns, more preferably less than about 7.0 microns, even more preferably in the range of 0.1 to 5.0 microns, most preferably in the range of about 1.0 to about 5.0 microns.

To reduce the adhesion of the dry powder on the walls of the delivery device from which the aerosol is distributed, a surfactant can be added to the pharmaceutical composition. Suitable surfactants for this intended use include, but are not limited to sorbitan trioleate, soya lecithin, and oleic acid. Devices suitable for inhaled delivery of a dry powder form of a non-aqueous suspension composition are commercially available. Examples of such devices include a Ventolin dose-controlled inhaler (Glaxo Inc., Research Triangle Park, N.C.), and an Intal inhaler (Fisons, Corp., Bedford, Mass.). See also aerosol delivery devices disclosed in US Pat. Nos. 5,522,378; 5,775,320; 5,934,272; and 5960792, which are incorporated herein by reference.

In yet another embodiment of the invention, the pharmaceutical composition may be delivered using a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; and Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment of the invention, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105). In yet another embodiment, a controlled release system can be placed in close proximity to the target of the composition, namely the lung, so only a fraction of the usual systemic dose is required (see, for example, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).

In one embodiment of the invention, the composition is prepared in accordance with conventional procedures in the form of a pharmaceutical composition adapted for topical injection into humans. Typically, compositions for topical injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are supplied either separately or mixed in unit dosage form, for example, in the form of a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the amount of active substance. In the case where the composition is administered by injection, ampoules with sterile water for injection or saline can be supplied so that the ingredients can be mixed before administration.

The therapeutic or pharmaceutical compositions of the invention may be in the form of a neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as groups derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and salts formed with free carboxyl groups, such as obtained from sodium, potassium, ammonium , calcium, iron hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The present invention also provides for the modification of the compound so that it is more stable after administration to the subject, that is, so that after administration of the compound, it has a longer period of effectiveness compared to the unmodified compound. Such modifications are well known to those skilled in the art, for example microencapsulation, etc.

The invention also relates to a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients with a pharmaceutical composition of the invention, for example, a compound, a carrier.

The compounds of the present invention can also be prepared in accordance with traditional Chinese medical practice. The composition and dosage of the composition that are effective in treating a particular disease, condition or disorder will be determined by the nature of the disease, condition or disorder using standard clinical methods.

In traditional Chinese medicine, prescription amounts can be easily converted into any form of a drug suitable for administration to humans or animals. Suitable forms include, for example, tinctures, decoctions, and dry extracts. They can be taken orally, injected into a vein or mucous membranes. The active ingredient may also be placed in capsules, powders, pallets, troches, suppositories, oral solutions, pasteurized gastrointestinal suspension injections, small or large injections, frozen powder injections, pasteurized powder injections and the like. All of the above methods are known to specialists in this field of technology, are described in books and are widely used by specialists in the field of herbal medicine.

Tinctures are prepared by suspending herbs in an alcoholic solution, such as, for example, wine or liquor. After a suspension period, a liquid (alcohol solution) can be taken, for example, two or three times a day, one teaspoon each time.

Broth is a common form of herbal preparation. The broth is traditionally prepared in a clay pot, but can also be prepared in glass, enameled containers or stainless steel containers. The composition can be soaked for some time in water, and then brought to a boil and cooked over low heat until the amount of water is reduced, for example, by half.

The extract is a concentrated preparation of the necessary ingredients of medicinal herbs. Typically, the main ingredients are extracted from herbs by suspending the herbs in an appropriate selected solvent, typically in water, ethanol / water, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether or other organic solvents. The extraction process can be additionally equipped with means of maceration, percolation, re-percolation, countercurrent extraction, turbo extraction or supercritical carbon dioxide (temperature / pressure) extraction. After filtration, in order to get rid of plant debris, the extraction solution can be further evaporated and thus concentrated to obtain a soft extract (extractum spissum) and / or, ultimately, the dried extract (extracum siccum) by spray drying, drying in a vacuum oven fluidized bed drying or lyophilization. The soft extract or dried extract can be further dissolved in a suitable liquid to the desired concentration for administration or processing into a form, such as dragees, capsules, injections, etc.

Routes of Introduction

The compounds and compositions of the present invention can be administered to a subject to be treated using standard routes, including oral, inhaled or parenteral administration, including intravenous, subcutaneous, local, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraocular, intraocular, intraocular transracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal nuyu injection, infusion and electroporation, as well as co-administration as a component of any medical device or object that is to be placed (temporarily or permanently) in that subject.

In preferred embodiments, the compounds and compositions of this invention are administered by any route suitable for pulmonary delivery. Pulmonary administration requires the release of a biologically active substance from a delivery vehicle into the subject's oral cavity during inspiration. For the purposes of the present invention, pharmaceutical compositions can be administered by inhalation of an aerosol or other suitable preparation, which is obtained from an aqueous or non-aqueous solution or a suspension form or a solid or dry powder form of a pharmaceutical composition, depending on the delivery device used. Pulmonary inhalation results in the application of an inhaled composition to the lung alveoli of the subject. After precipitation, the compounds or compositions can be absorbed, passively or actively, through the layers of the alveolar epithelium and capillary epithelium into the blood.

The amount of a therapeutic or pharmaceutical composition of the invention that is effective in treating a particular disease, condition or disorder will depend on the route of administration and the severity of the disease, condition or disorder, and should be determined in accordance with the clinical experience of the attending physician and the circumstances for each patient. Typically, the dose is in the range of about 0.001 mg / kg to about 3 g / kg. For example, suitable unit doses may be from about 0.01 to about 3 g, from about 0.01 to about 1 g, from about 0.01 up to about 500 mg, from about 0.01 to about 400 mg, from about 0.01 to about 300 mg, from about 0.01 to about 200 mg, from about 0.01 to about 100 mg, from about 0.01 up to about 50 mg; from about 0.01 to about 30 mg; from about 0.01 to about 20 mg; from about 0.01 to about 10 mg; from about 0.01 to about 5 mg; from about 0.01 up to about 3 mg; from about 0.01 to about 1 mg; or from about 0.01 to about 0.5 mg. Such a unit dose may be administered more than once a day, for example, two or three times a day.

The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending, for example, on the type of condition and the subject to be treated. Typically, the therapeutic composition contains from about 5% to about 95% of the active ingredient (w / w). More specifically, the therapeutic composition comprises from about 20% (w / w) to about 80%, or from about 30% to about 70% of the active ingredient (w / w).

After the patient has improved, a maintenance dose is administered if necessary. Subsequently, the dose or frequency of administration, or both, can be reduced, depending on the symptoms, to a level at which improvement is maintained. When symptoms are relieved to the desired level, treatment should be discontinued. Patients may, however, require periodic treatment on a long-term basis in the event of any recurrent symptoms of the disease.

In addition, in vitro assays may optionally be used to identify optimal dose ranges. The exact dose to be used in the composition will also depend on the route of administration and the severity of the disease, condition or disorder, and should be determined in accordance with the clinical experience of the attending physician and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained from test systems in in vitro or animal models.

The following are examples that illustrate the techniques for implementing the present invention. These examples should not be construed as limiting the invention.

Example 1 - the induction of inflammatory reactions through IR

Briefly, fibrosis-prone C57BL / 6 mice were exposed to IR (radiation) at a dose of 0, 5, or 9 Gy. As shown in FIG. 1, IR induces a first stage of an inflammatory reaction 6 hours after exposure, a second stage 4-10 days after exposure, and a third stage 180 days after exposure (IL2 only) (FIG. 1, first rectangle : normal group control). Figure 2 shows an increase in IM levels in C57BL / 6 and C3H / NeH mice with IR-induced brain damage (MP) 6 hours, 1, 2, 4, and 10 days after exposure at 9 Gy. This means that IR causes inflammatory reactions, and the levels of inflammatory mediators can be used to monitor IR-induced lung damage.

Example 2 - modulation of inflammatory reactions during the acute phase of pneumonitis caused by IR

This example shows that GA inhibits the growth of inflammatory reactions during the acute phase of IR-induced pneumonitis, which occur within 48 hours after exposure to mice by IR (radiation). Briefly, C57BL / 6 mice predisposed to fibrosis received a single dose of thoracic IR at 15 Gy with a dose rate of 1.9 Gy / min. Six hours after exposure to IR, mice were divided into the following treatment groups: 1) normal (received a dose of 0 Gy, without treatment); 2) processed by the carrier; 3) GA-treated (30-40 mg / kg / qod (i.e. every other day) (the GA dosage used in this case was obtained based on the effective human dose (3-4 mg / kg), which, as a rule, 6-10 times lower than in mice), 4) treated with dexamethasone (Positive control group 1) (Dex, 3 mg / kg, im or p / o, every other day); 5) treated with amifostine (Positive control group 2) (Ami, 200 mg / kg / intravenously, 0.5 hours before irradiation); and 6) treated with celebrex (Positive control group 3) (30 mg / kg, oral, every other day) .

Mice were sacrificed 2.5 days after exposure to IR and fresh lung tissue homogenate was prepared using lysis buffer mixed with a mixture of protenase inhibitor. After the protein concentration was adjusted to 1 mg / ml, fresh lung homogenate was added to the ELISA plates at a concentration of 100 μl / well. Inflammatory mediator levels were measured by ELISA.

As shown in FIG. 3, exposure to IR causes an increase in inflammatory mediator (IM) levels, including IL1α, TNFα, PF4, and VCAM-1. GA effectively reduced IM levels compared to vehicle treated mice (FIG. 2, P <0.05). The degree of inhibition of GA was comparable to the degree of inhibition of celebrex, dexamethasone and amifostine.

Example 3 - Suppression of plasma exudation and inflammatory cell infiltration during the acute phase of IR-induced pneumonitis

This example shows that GA inhibits IR-induced plasma exudation and inflammatory cell infiltration during the acute phase of IR-induced pneumonitis. Briefly, fibrosis-prone C57BL / 6 mice received one dose of thoracic IR at 15 Gy with a dose rate of 1.9 Gy / min and were treated as described in Example 2. Mice were sacrificed 2.5 days after exposure to IR and pulmonary tissues were collected and analyzed for morphological changes.

As shown in FIG. 4, carrier-treated lungs were filled with infiltrated inflammatory cells and plasma exudates compared to normal non-IR-exposed lungs. GA significantly inhibited plasma exudation and inflammatory cell infiltration within the interstitial space. Celebrex, Dexamethasone and Amifostin also produced a similar treatment effect.

Example 4 - reduction in plasma levels of SP-D and IL1a during the acute phase of IR-induced pneumonitis

This example shows that GA reduced plasma levels of SP-D and IL1α .. IR damages the epithelium of the lungs and endothelial cells and causes dysfunction of tight junctions, increased cell permeability and loss of integrity of the alveolar-capillary barrier. As a result, SP-D and IL1α abnormally enter the bloodstream from inflamed lung tissue, resulting in elevated plasma levels of SP-D and IL1α.

In this example, mouse fibrosis-prone C57BL / 6 lines received a single dose of thoracic IR of 15 Gy with a dose rate of 1.9 Gy / min and were processed as described in Example 2. Mice were killed 2.5 days after exposure to IR and measured plasma levels of SP-D and IL1α. 5A shows that exposure to IR leads to a dose-dependent increase in plasma SP-D levels. In particular, plasma SP-D did not increase when exposed to IR at 5 Gy, but increased at 10 Gy and 15 Gy. As shown in FIGS. 5A-B, GA effectively reduced plasma SP-D levels (P <0.05). Furthermore, FIG. 5C shows that GA effectively reduced plasma IL1α levels (P <0.05).

Example 5 - the effect of GA on the subacute phase of IR-induced pneumonitis

This example shows that GA effectively modulates the subacute phase of IR-induced pneumonitis, which develops approximately 2-4 weeks after exposure to IR in mice. Briefly, mouse fibrosis-prone C57BL / 6 lines received thoracic IR and were treated as described in Example 2. Mice were sacrificed 17 days after exposure to IR and lung tissue was harvested. Bronchoalveolar lavage fluid (BALF) analyzes were performed.

The results showed that GA reduced the levels of lymphotactin and p-selectin, two key inflammatory mediators involved in the pulmonary immune response and innate immunity (FIGS. 6A and B, P <0.05). In addition, GA decreased the level of neutrophils penetrating the fluid of bronchoalveolar lavage (Fig. 7A, P <0.05). GA also decreased myeloperoxidase (MPO) activity in (BALF) (Fig. 7B, P <0.05). Figa-C also show the suppression of neutrophil infiltration in the interstitial (intercellular) fluid (figa-C).

In addition, it was noted that the growth of inflammatory mediators during the subacute phase is not dose dependent. IR exposure at a dose of 5 Gy can cause inflammatory reactions. The authors of the present invention also noted that exposure to IR with a dose higher than 5 Gy can cause pulmonary fibrosis. The larger the dose, the faster it can affect the development of fibrosis. The inventors of the present invention found that mice treated with IR at a dose of 7.5 Gy developed pulmonary fibrosis and died of pulmonary failure 1.5 years after exposure to IR.

The introduction of GA 24 hours after exposure to IR can effectively block the growth of inflammatory reactions. Figure 9 shows that GA significantly reduced the levels of key inflammatory mediators, including IL1α, TNFα, lymphotactin and p-selectin, in mice exposed to thoracic IR at doses of 5, 10 and 15 Gy, respectively.

Example 6 - GA-attenuating effect on IR-induced pulmonary fibrosis

This example shows that GA improves lung function and attenuates IR-induced pulmonary fibrosis. Mice exposed to thoracic IR developed pulmonary fibrosis 6 months after exposure to IR (irradiation) at 18 Gy, 8 months after exposure to IR at 15 Gy and 10 months after exposure to IR at 12.5 Gy. In order to evaluate the effectiveness of GA in relation to pulmonary fibrosis and lung function, respiratory rate, lung density, and lung compliance were determined in mice.

To assess the improvement in respiratory function of mice exposed to thoracic IR at a dose of 15 Gy, GA (30-40 mg / kg) q.o.d (every other day) or twice a week for 3 months was treated. The respiration rate of mice was measured using a Harvard rodent breathing apparatus connected to a plethysmograph under pressure. As shown in FIGS. 10A-B, exposure to IR significantly increased the respiration rate of mice, and GA treatment caused an effective decrease in respiration rate (P <0.05).

To assess the effect of GA on lung density, cone-beam CT (CBCT) of mouse lungs was performed. CBCT provides a complete 3D 650 × 650 × 428 scan (i.e., 428 sections) of the lungs with an isotropic resolution of 270 μm (central section shown in FIG. 11) for 30 seconds. The scanner provides a CT density sensitivity of ~ 5 HU ³ , and thus changes in lung density can be distinguished.

CBCT data were further analyzed using MATLAB software. The lungs were automatically segmented (FIG. 12A) and a 3D image was created for each mouse lung (FIG. 12B). To reduce the effect of cardiac movements on the average lung density, lung boundaries were not used for analysis. A histogram of voxel intensity (lung tissue density) was created for each mouse (FIG. 13).

Table 1 shows the mean and standard deviation (SD) of lung density. Exposure to IR (irradiation) at 18 Gy gradually increased lung density from a normal density of -454 + 60 HU (Hounsfield scale) up to -364 + 45 HU per year. As shown in FIG. 14, GA significantly reduced the abnormal increase in lung density (P <0.05). The decrease in lung density due to GA is more powerful than with Celebrex.

Table 1 Comparison of lung density in mice in different treatment groups Group Medium Density (HU) Standard Deviation (HU) Normal control -454 60 1 month after exposure to IR at 15 Gy -420 56 1 year after exposure to IR at 18 Gy -364 45 1 year after exposure to IR at 18 Gy with GA treatment -458 88

To assess the effect of GA on the extensibility of the lungs, mice were anesthetized and their tracheas were surgically removed and dissected. A 1 cm 18-gauge stainless steel tube was inserted into the trachea and secured with surgical sutures. The respiration rate and inspiratory volume of the mice were measured using a Harvard rodent breathing apparatus. The average respiratory rate was 150 breaths per minute, and the inspiratory volume was adjusted according to weight (0.01 ml per gram of body weight). Then the mice were placed in a plethysmograph for pressure-volume measurements. As shown in FIG. 15, GA treatment significantly and generally effectively increased lung extensibility, reduced by exposure to IR (P <0.05).

Example 7 - the weakening of fibrotic conditions

This example shows that GA reverses the pathological alteration of lung tissue, improves lung morphology, and suppresses collagen deposition in IR-induced fibrosis. Briefly, mice treated with GA (40 mg / kg / qod (every other day)) for 3 months were sacrificed 7 months after exposure to IR (irradiation) at a dose of 15 Gy, and lung tissue samples were stained using a hematoxylin stain and eosin (H&E). As shown in FIG. 16, carrier-treated lungs showed impaired normal lung structure, including thickening of the alveolar septa and increased levels of fibroblasts. In comparison, GA-treated lungs showed an almost normal morphology (Fig. 16).

To assess how GA reduces abnormal collagen deposition in the lungs, a lung tissue sample was stained with trichromic blue collagen-binding dye. On Fig shows that GA reduces collagen deposition caused by exposure to IR, and improves lung morphology.

Reducing collagen deposition was further analyzed by measuring levels of hydroxyproline, a key component of collagen. Fig. 18A shows that GA decreased lung hydroxyproline levels compared to IR + control (pure vehicle) (P <0.05). Figv shows that GA reduced the level of TGFβ, pro-fibrous factor.

Example 8 - low toxicity

This example shows that long-term use of GA causes few side effects, based on observations of the activity, coat morphology, and body weight (MT) of the treated mice. In particular, 7.5 months after exposure to IR at a dose of 15 Gy, carrier-treated mice lost 6-7 grams of body weight, while less severe MT loss was observed in GA- or Celebrex-treated mice (FIG. 19). No mouse died during GA treatment.

Example 9 - the effect of GA on the endocrine system

The chemical structure of GA is similar to that of cortisol. Thus, this example explores the effect of long-term use of GA on the formation of cortisol and the occurrence of side effects. Plasma levels of ACTH (adrenocorticotropic hormone) - the hormone that releases cortisol - were measured by ELISA 6, 7, and 10 weeks after GA treatment (40 mg / kg, qod (every other day), orally). Results (data not shown) showed that although GA decreased ACTH levels by 60% at week 6, ACTH levels quickly returned to normal after 7–10 weeks. In comparison, dexamethasone treatment caused a 10–50-fold decrease in ACTH levels over the course of all 7–10 weeks.

In addition, plasma growth hormone (GH) levels were measured by ELISA 6, 7, and 10 weeks after GA treatment. The results showed that both GA and dexamethasone increased plasma GH levels. It has been noted that exposure to IR atypically reduces growth hormone levels and, therefore, GA-induced increase in GH level is useful for patient recovery ⁴ .

Example 10 — Effect of GLA Treatment on IR-Induced Pneumonitis

In collaboration with radiation oncologists at the First Affiliated Hospital at the Fujian Medical School in China, GLA tablets (obtained by Minophagen Co. from Japan and available in pharmacies in China) were administered to a patient who received a second course of IR-treatment for lung cancer. During the first course of IR treatment, the patient developed severe IR-induced pneumonitis three months after treatment with IR and symptoms of lung damage, including fever, cough and shortness of breath, were revealed. To control the progression of pneumonitis, she received dexamethasone treatment. Due to the resumption of tumor growth 9 months after her first IR treatment, she underwent a second course of IR treatment with the warning that IR could worsen pneumonitis.

In order to reduce IR-induced lung damage, GLA was administered at a dose of 150 mg per day (GA equivalent of about 85 mg per day), starting from the date of the 2nd IR-treatment for 3 months. The result was extremely amazing. The patient, whose pneumonitis was expected to worsen, did not have any symptoms of pneumonitis or fibrosis during the course of GLA treatment. Comparison of CT results taken after the 1st and 2nd courses of IR treatment also showed that GLA significantly reduced the lung density in the patient (Fig. 20).

It should be understood that the examples and embodiments of the present invention described herein are for illustrative purposes only, and that various modifications or changes will be apparent to those skilled in the art in light of the present disclosure. Such modifications and changes should be included in the concept and disclosure of this application and should fall within the scope of the attached claims.

All references, including publications, patent applications, and patents cited herein, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and its contents set forth herein completely.

In the present invention, the term in the singular also covers the plural, unless explicitly stated otherwise and if it is not contrary to the context.

The use of value ranges herein is intended only as a method of abbreviation of text. It should be understood that such ranges imply an individual reference to each individual value included in this range, unless explicitly stated otherwise, and each such individual value is considered to be included in the description, as if it were individually presented in this document.

All methods described in the present invention can be performed in any suitable order, unless otherwise indicated herein and if this is not contrary to the context.

All examples or illustrative terms (eg, “such as”) provided herein are intended only to better clarify the present invention and are not intended to limit the scope of the invention unless expressly indicated otherwise. No term in the description should be construed as indicating that any element is crucial to the practice of the present invention, unless explicitly stated.

The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having” or “including” with reference to any element or elements, also includes that embodiment of the invention in which the terms “consists of "," consists essentially of "or" essentially only contains "this particular element or elements, unless explicitly stated otherwise and if this is not contrary to the context (for example, a composition described in the present invention as containing a specific element, according to it is also understood as a composition consisting of this element, unless otherwise indicated and if this does not contradict the context).

Information sources

1. Tsoutsou, P.G. & Koukourakis, M.I. Radiation pneumonitis and fibrosis: mechanisms underlying its pathogenesis and implications for future research. Int J Radiat Oncol Biol Phys 66, 1281-1293 (2006).

2. Choi, N.C. Radioprotective effect of amifostine in radiation pneumonitis. Semin Oncol 30, 10-17 (2003).

3. Ning, R., Tang, X., Conover, D. & Yu, R. Flat panel detector-based cone beam computed tomography with a circle-plus-two-arcs data acquisition orbit: preliminary phantom study. Medical Physics 30, 1694-1705 (2003).

4. Vazquez et al., Protective effect of enriched diet plus growth hormone administration on radiation-induced intestinal injury and on its evolutionary pattern in the rat, Digestive Diseases and Sciences, 44 (11) 2350-2058 (1999).

Claims (18)

1. A method of preventing, treating, or attenuating radiation-induced pulmonary fibrosis, comprising administering to a subject receiving a radiation dose and in need of such prevention, attenuation or treatment, an effective amount of an isolated or substantially pure compound or salt thereof, wherein said compound is selected from the group consisting of of:
(A) glycyrrhetinic acid (GA);
(B) glycyrrhizic acid (GLA);
(C) ester, ester and / or amide forms of glycyrrhetinic acid (GA) represented by the following structure (Structure C):

;
and
(D) ester and / or amide forms of glycyrrhizic acid (GLA) represented by the following structure (Structure D):

;
Where
X represents any group forming an ether or ester bond with a hydroxyl radical; and
Y and Y 'each independently represents any group forming an ester or amide bond with a carboxyl group. 2. The method of claim 1, wherein the subject is a human. 3. The method of claim 1, wherein the compound is glycyrrhetinic acid (GA) or a salt thereof. 4. The method of claim 1, wherein the compound is glycyrrhizic acid (GLA) or a salt thereof. 5. The method according to p. 1, in which X is selected from the group consisting of
a) alkyl, substituted alkyl, alkenyl, substituted alkenyl, —COOH, acyl, alkylcarbonyl, benzyl, cyclic alkyl, and cyclic alkenyl;
b) an acid group selected from the group consisting of acetic acid, carboxylic acid, aspartic acid, formic acid, citric acid, benzoic acid, hippuric acid, malic acid, mucic acid, phosphoric acid, sulfuric acid, gluconic acid, maleic acid succinic acid, tartaric acid and lactic acid; and
c) a carbohydrate group. 6. The method of claim 1, wherein each of Y and Y ′ is —NH ₂ , alkylamino, or alkoxy. 7. The method of claim 1, wherein the subject receives radiation therapy for treating cancer. 8. The method of claim 1, wherein the subject is randomly exposed. 9. The method according to p. 1, in which the level of the inflammatory mediator in the lung tissue of a subject is reduced, wherein said inflammatory mediator is selected from the group consisting of SP-D, IL1α, TNFα, IL6, PF4, P-selectin, L-selectin, VCAM -1, lymphotactin and prostaglandin E (PGE). 10. The method according to p. 1, in which the content of collagen, fibronectin, proteoglycan and / or hydroxyproline in the lung tissue of a subject is reduced. 11. The method of claim 1, wherein the level of TGFβ in the lung tissue of the subject is reduced. 12. The method of claim 1, wherein the respiratory rate of the subject is reduced, the density of the lung of the subject is reduced, and / or the compliance of the lung tissue of the subject is increased. 13. The method of claim 1, wherein the compound is administered before, during, or after the subject receives a radiation dose. 14. The method of claim 1, wherein the subject receives a radiation dose of at least 1 Gy. 15. A method of preventing, treating, or attenuating radiation-induced lung damage, comprising administering to a subject who is receiving a radiation dose and in need of such prevention, attenuation or treatment, an effective amount of an isolated or substantially pure compound or its salt, wherein said compound is selected from the group consisting of:
(A) glycyrrhetinic acid (GA);
(B) glycyrrhizic acid (GLA);
(C) ester, ester and / or amide forms of glycyrrhetinic acid (GA) represented by the following structure (Structure C):

; and
(D) ester and / or amide forms of glycyrrhizic acid (GLA) represented by the following structure (Structure D):

;
Where
X represents any group forming an ether or ester bond with a hydroxyl radical; and
Y and Y 'each independently represents any group forming an ester or amide bond with a carboxyl group. 16. The method of claim 15, wherein the compound is glycyrrhetinic acid (GA) or a salt thereof. 17. The method of claim 15, wherein the compound is glycyrrhizic acid (GLA) or a salt thereof. 18. The method of claim 15, wherein the subject receives a radiation dose of at least 1 Gy.

Images