WO2014063660A1

WO2014063660A1 - Naringenin and asiatic acid combination treatment of fibrosis

Info

Publication number: WO2014063660A1
Application number: PCT/CN2013/086058
Authority: WO
Inventors: Hui Yao Lan
Original assignee: The Chinese University Of Hong Kong
Priority date: 2012-10-26
Filing date: 2013-10-28
Publication date: 2014-05-01
Also published as: CN104736151B; CN104736151A

Abstract

The present invention resides in the surprising discovery of the previously unrecognized synergistic effects resulted from combined use of narigenin and asiatic acid. New methods and compositions are therefore provided for the treatment or prevention of fibrosis.

Description

NARINGENIN AND ASIATIC ACID

COMBINATION TREATMENT OF FIBROSIS

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/719,107, filed October 26, 2012, the contents of which are incorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Fibrosis is a process that involves the formation of an excess amount of fibrous connective tissue in an organ or at an anatomic location, often in response to an injury, a disease, or other external stimulation. Such excess amount of fibrous tissue tends to disrupt or even replace the normal cellular architecture of the original tissue, severely diminished or completely lost physiological function of the normal tissue or organ can result. Because fibrosis can take place in virtually all organs and can significantly effect a person's health or even cause death due to organ failure, much effort in the ongoing medical research has been devoted to the treatment and prevention of fibrosis.

[0003] Because of the medical prevalence and significance of fibrosis in human health, there remains an urgent need to develop new and effective means for treating and preventing fibrosis. The present invention addresses this and other related needs.

BRIEF SUMMARY OF THE INVENTION

[0004] Naringenin and Asiatic acid individually have been known as being involved in different signaling pathways relevant to the formation of tissue fibrosis. The present inventor made the surprising discovery that, when naringenin and Asiatic acid are administered together, a synergistic effect is achieved in inhibiting fibrosis. Although the role of each of naringenin and Asiatic acid in regulating fibrosis-related signaling was previously known, such synergistic effect of combined use of the two compounds was not previously recognized until now. As such, the present invention provides novel methods and compositions effective for the treatment and prevention of fibrosis.

[0005] In one aspect, the invention provides a new method of inhibiting fibrosis in a tissue. The method includes the step of contacting the tissue with an effective amount of naringenin and Asiatic acid. The tissue may be a part of an organ, such as liver, kidney, or lung. In some embodiments, the contacting step comprises subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration. In some embodiments, the effective amount is about 5 mg/kg to 250 mg/kg body weight of naringenin and 1 mg/kg to 50 mg/kg body weight of Asiatic acid. In some embodiments, naringenin and Asiatic acid are administered at a weight ratio of about 10: 1. In some embodiments, naringenin and Asiatic acid are administered in a single composition. In other embodiments, naringenin and Asiatic acid are administered in two separate compositions. Naringenin and Asiatic acid are administered in any appropriate form, including but not limited to, a solution, a powder, a tablet, or a capsule.

[0006] In another aspect, the invention provides a new composition that comprises (1) an effective amount of naringenin and Asiatic acid and (2) a pharmaceutically acceptable excipient. In some embodiments, naringenin and Asiatic acid are present in the weight ratio of about 10: 1. In some embodiments, the composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration. For example, the composition may be in the form of a solution, a powder, a tablet, or a capsule. [0007] In yet another aspect, the present invention provides a kit for inhibiting fibrosis. The kit contains at least two containers: the first container contains a first composition, which comprises narigenin; and the second container contains a second composition, which comprises Asiatic acid. In some embodiments, the first composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration. In some embodiments, the second composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration. In some embodiments, the kit further includes an instruction manual for administration of the first and second compositions.

[0008] In each of the above described aspects, the synergistic therapeutic effects of narigenin and Asiatic acid are achievable by substituting Asiatic acid with either of its two known analogs, madecassic acid and asiaticoside, in combined use with narigenin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1. Combination of Asiatic Acid and Naringenin further attenuates UUO- induced renal collagen I deposition. A. Immunohistochemistry results indicate treatment of Asiatic Acid or Naringenin alone decreases collagen I deposition in obstructive kidney, combination of Asiatic Acid and Naringenin further inhibits UUO-induced collagen production in protein level. B. Real-time PCR results confirm that combination of Asiatic Acid and Naringenin largely down-regulates Collagen I synthesis in mRNA level in obstructive nephropathy. Data represent mean ± SEM for 6-8 mice. ***P < 0.001 versus sham group; #P < 0.05, ###P < 0.001 versus UUO+DMSO group. AA: Asiatic Acid treatment; Nar: Naringenin treatment; CB: Combined treatment.

[0010] Fig. 2. Combination of Asiatic Acid and Naringenin further decreases UUO- induced a-SMA+ myofibroblasts infiltration. A. Immunohistochemistry results indicate treatment of Asiatic Acid or Naringenin alone decreases a-SMA+ myofibroblasts infiltration in obstructive kidney, combination of Asiatic Acid and Naringenin further decreases the number of infiltrated a -SMA+ myofibroblasts; B. Real-time PCR results demonstrate that combination of Asiatic Acid and Naringenin substantially down-regulates a-SMA in mRNA level in obstructive nephropathy. Data represent meant SEM for 6-8 mice. ***P < 0.001 versus sham group; #P < 0.05, ###P < 0.001 versus UUO+DMSO group. M: Asiatic Acid treatment; Nar: Naringenin treatment; CB: Combined treatment.

[0011] Fig. 3. Combination of Asiatic Acid and Naringenin decreases Smad3 signaling while increasing Smad7 level in obstructive kidney. A. Results of Western blot analysis indicate that combination of Asiatic Acid and Naringenin further decreases UUO-induced Smad3 phosphorylation while antagonizing UUO-induced Smad7 degradation in protein level; B. Real-time PCR results demonstrate that combination of Asiatic Acid and Naringenin down-regulates Smad3 in mRNA level while inducing Smad7 synthesis in obstructive nephropathy. Data represent mean ± SEM for 6-8 mice. *P < 0.05, **P < 0.01 versus sham group; ##P < 0.01 versus UUO+DMSO group. AA: Asiatic Acid treatment; Nar: Naringenin treatment; CB: Combined treatment.

[0012] Fig. 4. Combination of Asiatic Acid and Naringenin further decreases TGF- βΐ-induced fibrotic response in vitro in hepatic stellate cells and kidney tubular epithelial cells. A. Combination of Asiatic Acid and Naringenin further down-regulates Col. I and a-SMA mRNA level in response to TGF-βΙ in hepatic stellate cells; B. Western blot analysis and semi-quantitative data demonstrate that Combination of Asiatic Acid and Naringenin blocks TGF-βΙ -induced Col.I production in kidney tubular epithelial cells. Data represent mean ± SEM for 3-4 independent experiments in vitro. *P < 0.05, **P < 0.01, ***P < 0.001 versus DMSO only group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus TGF- βΙ+DMSO group. AA: Asiatic Acid treatment; Nar: Naringenin treatment; CB: Combined treatment. DEFINITIONS

[0013] The term "inhibiting" or "inhibition," as used herein, refers to any detectable negative or suppressing effect on a target biological or pathological process, such as the onset or progression of fibrosis. Typically, an inhibition is reflected in a decrease of at least 10%, 20%, 30%), 40%o, or 50%> in a feature characteristic of the target process (e.g., the presence or new formation of fibrous connective tissue) when compared to a control.

[0014] The term "fibrosis" describes the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Fibrosis is distinguished from the formation of fibrous tissue as a normal constituent of an organ or tissue. Scarring is confluent fibrosis that obliterates the architecture of the underlying organ or tissue. Fibrosis can take place in virtually all organs and tissue types. Examples of fibrosis include: pulmonary fibrosis (lungs); cirrhosis (liver); endomyocardial fibrosis (heart); mediastinal fibrosis (soft tissue of the mediastinum); myelofibrosis (bone marrow); peritoneal fibrosis (soft tissue of the peritoneum); progressive massive fibrosis (lungs); nephrogenic systemic fibrosis (kidney); Crohn's Disease (intestine); keloid (skin); old myocardial infarction (heart); and

scleroderma/systemic sclerosis (skin, lungs).

[0015] The term "effective amount," as used herein, refers to an amount of a substance that produces therapeutic effects for which the substance is administered. The effects include the prevention, correction, or inhibition of progression of the symptoms of a disease or condition and related complications to any detectable extent. In a case where two or more substances are used for a desired effect, the "effective amount" may be expressed in more than one way. For example, the "effective amount" may be expressed in the total amount of all active ingredients, or expressed in a separate amount for each active ingredient, or expressed in a ratio (e.g., in weight or volume ratio) of one ingredient over another. The exact amount of an "effective amount" will depend on the purpose of the treatment as well as the form and identify of the active substance, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0016] "Naringenin" is a flavanone with the systematic (IUPAC) name 5,7-dihydroxy-2- (4-hydroxyphenyl)chroman-4-one and synonym 4',5,7-trihydroxyflavanone (CAS Number 480-41-1). Its chemical formula is C15H12O5 and molecular weight is 272.257. In nature, naringenin is present most abundantly in grapefruits, oranges and tomato skin. High purity naringenin can be purchased from various commercial venders.

[0017] "Asiatic acid" is derived from an ancient, traditional herbal extract of the plant Centella asiatica, commonly called Gotu Kola. It is also known as dammarolic acid (CAS Number 464-92-6). Its chemical formula (Hill Notation) is C30H48O5, and molecular weight is 488.70. Asiatic acid shares many similarities with its analogs, madecassic acid and asiaticoside. It is available through commercial suppliers such as Sigma-Aldrich.

[0018] As used herein, the term "administration" encompasses any means of delivering a substance, e.g., an agent with therapeutic or prophylactic effects, to a subject, which may include but is not limited to, systemic, regional, and local applications. Examples of

"administration" are injection (such as by subcutaneous, intramuscular, intravenous, or intraperitoneal means), oral ingestion, intake through the nasal cavity or through the eyes or ears, inhalation, transdermal delivery, and anal or virginal deposit, etc.

[0019] The terms "pharmaceutically acceptable excipient" and "physiologically acceptable excipient" may be used interchangeably to refer to an inert substance that is included in the formulation of a composition containing an active ingredient to achieve certain characteristics, such as more desirable pH, solubility, stability, bioavailability, texture, consistency, appearance, flavor/taste, viscosity, etc., but in itself does not negatively impact the intended therapeutic or prophylactic effects of the active ingredient. [0020] The term "tissue," as used herein, refers to an ensemble of cells that are similar in their biological attributes, such as morphology and biological activity, and are from the same origin, such that these cells together carry out a specific function. An "organ" is a collection of different tissues joined in a structural unit to serve a common function.

[0021] The term "about," as used herein, describes a range of plus or minus 10% from a recited value. For example, a value of "about 10" can be any value within the range of 10±1, i.e., between 9 to 11.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0022] Organ (liver, kidney, lung, and heart etc.) fibrosis or scarring is a major cause and final pathway leading to end-stage organ failure. Tissue fibrosis is characterized by the excessive accumulation of myofibroblasts and extracellular matrix. To this date, however, treatment for fibrosis remains largely ineffective and non-specific. Therefore, there is an urgent need for searching and developing anti-fibrotic strategies in order to prevent, halt, or reverse fibrosis. It is generally believed that TGF-p/Smads mediate fibrosis. Findings in the instant study showed that TGF-βΙ mediates tissue scarring positively by its downstream mediator called Smad3, but negatively by Smad7. In a variety of chronic organ diseases, fibrosis occurs with the imbalance of TGF-p/Smads as demonstrated by over-activation of Smad3 but loss of Smad7. Thus, the identification of specific agents to specifically inhibit Smad3 activity while enhancing Smad7 activity, rather than to block the general effects of TGF-β, which can be harmful due to increased inflammation, is the new invention of this application. In this invention, the inventor discovered that a component purified from the citric fruit skins called naringenin acts as a Smad3 inhibitor to block Smad3 -mediated fibrosis, whereas another purified product, Asiatic acid, from a herb has anti-fibrosis activity by upregulating Smad7. The combined use of naringenin and Asiatic acid restores the balance of TGF-p/Smads and produces a synergistic therapeutic effect for the treatment and prevention of fibrosis, such as seen in chronic kidney and liver diseases.

II. Pharmaceutical Compositions and Administration

[0023] The present invention provides pharmaceutical compositions or physiological compositions comprising an effective amount of naringenin and Asiatic acid, which is effective for inhibiting fibrosis in a tissue or organ in both prophylactic and therapeutic applications. Such pharmaceutical or physiological compositions also include one or more pharmaceutically or physiologically acceptable excipients or carriers. Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems.

Suitable formulations for use in the present invention are found in Remington's

Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249: 1527-1533 (1990).

[0024] The pharmaceutical compositions of the present invention can be administered by various routes, e.g., oral, subcutaneous, transdermal, intramuscular, intravenous, intranasal, or intraperitoneal. The preferred routes of administering the pharmaceutical compositions are local delivery to an organ or tissue suffering from or at risk of developing fibrosis {e.g., intraperitoneal injection to an organ) at daily doses of about 0.35 - 17.5g, preferably 2.5-5.5g, of naringenin and about 0.1 - 3.5g, preferably 0.5-1.0g, of Asiatic acid for a 70 kg adult human per day. The appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.

[0025] For preparing pharmaceutical compositions containing naringenin or Asiatic acid, or containing both, one or more inert and pharmaceutically acceptable carriers are used. The pharmaceutical carrier can be either solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material. [0026] In powders, the carrier is generally a finely divided solid that is in a mixture with the finely divided active component, e.g. , naringenin and/or Asiatic acid. In tablets, the active ingredient (naringenin and/or Asiatic acid) is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. [0027] For preparing pharmaceutical compositions in the form of suppositories, a low- melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.

[0028] Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient of naringenin and/or Asiatic acid. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.

[0029] The pharmaceutical compositions can include the formulation of the active ingredient of naringenin and/or Asiatic acid with encapsulating material as a carrier providing a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the active ingredient. In a similar manner, cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. [0030] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration. Sterile water solutions of the active component (e.g. , naringenin and/or Asiatic acid) or sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.

[0031] Sterile solutions can be prepared by dissolving the active component, such as naringenin and/or Asiatic acid, in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably from 5 to 9, and most preferably from 7 to 8.

[0032] The pharmaceutical compositions containing the active ingredient of naringenin and/or Asiatic acid can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a condition involving fibrosis in an amount sufficient to prevent, cure, reverse, or at least partially slow or arrest the symptoms of the fibrosis and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.35g to about 17.5g of naringenin and 0. lg to about 3.5g of Asiatic acid per day for a 70 kg patient, with dosages of from about 2.5g to about 5.5g of naringenin and 0.5g to about l .Og of Asiatic acid per day for a 70 kg patient being more commonly used. [0033] In prophylactic applications, pharmaceutical compositions containing naringenin and/or Asiatic acid are administered to a patient susceptible to or otherwise at risk of developing a disease or condition in which fibrosis is present, in an amount sufficient to delay or prevent the onset of the fibrosis-related symptoms. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts of naringenin and Asiatic acid again depend on the patient's state of health and weight, but generally range from about 0.35g to about 5.5g of naringenin and O. lg to about 3.5g of Asiatic acid for a 70 kg patient per day, more commonly from about 2.5g to about 5.5g of narigenin and 0.5g to about l .Og of Asiatic acid for a 70 kg patient per day. [0034] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of naringenin and/or Asiatic acid sufficient to effectively inhibit fibrosis in the patient, either therapeutically or prophylatically. III. Kits

[0035] The invention also provides kits for inhibiting fibrosis according to the method of the present invention. The kits typically contain two containers: the first container contains a composition that comprises narigenin and the second container contains a composition that comprises Asiatic acid. [0036] Alternatively, the kits may include a container that contains a pharmaceutical composition having an effective amount of naringenin and Asiatic acid (such as a

composition described herein in detail), as well as informational material containing instructions on how to dispense the pharmaceutical composition, including description of the type of patients who may be treated (e.g., patients who are suffering from or are at risk of developing fibrosis), the schedule (e.g., dose and frequency) and route of administration, and the like.

EXAMPLES

[0037] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1 Naringenin/ Asiatic Acid Combination Treatment of Kidney Fibrosis

[0038] Kidney interstitial fibrosis was induced in a mouse model of unilateral ureteral obstruction (UUO) by ligation of the left ureter for seven days. Diseased mice received daily intra-peritoneum injections of Asiatic Acid (AA, 5 mg/kg/BW), or Naringenin (Nar. 50 mg/kg/BW), or the combination of both traditional Chinese herd compounds of AA (5 mg/kg/BW) plus Nar ( 50 mg/kg/BW) for seven days. Kidney fibrosis was evaluated by histology, immunohistochemistry, Western blot, and real-time PCR for mRNA and protein expression of extracellular matrix including collagen I and alpha-smooth muscle actin, and activation of TGF beta/Smad signaling. The in vitro inhibitory effect of Asiatic Acid (20μΜ), Naringenin (50μΜ), or their combination on TGF-βΙ (2ng/ml) stimulated fibrosis response was tested in rat hepatic stellate cells and kidney tubular epithelial cells and examined by real-time PCR and Western blot analysis as described above. Findings showed that combination of AA and Nar was far more effective on suppressing TGF-βΙ -induced fibrotic response when compared with AA or Nar treatment alone, resulting in more than 30% further reduction in collagen I extracellular matrix production. Moreover, it has been demonstrated that Naringenin inhibited Smad3 transcription, while Asiatic Acid suppressed Smad3 phosphorylation by inducing Smad7 gene transcription and protein expression. Thus, the enhanced therapeutic effect by combination of Asiatic acid and Naringenin may be attributed to the sufficient blockade of Smad3 activation by a directly inhibitory effect of Narigenin on Smad3 and also by increasing Smad7 to counter-regulate Smad3 -mediated fibrosis via the negative feed-back loop. Thus, re -balancing the TGF- /Smad signaling may be a key mechanism by which combination of Asiatic acid and Naringenin treatment produces the additive effect on renal fibrosis.

[0039] All patents, patent applications, and other publications, including GenBank

Accession Numbers, cited in this application are incorporated by reference in the entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method for inhibiting fibrosis in an organ, comprising the step of contacting the organ with an effective amount of naringenin and Asiatic acid.

2. The method of claim 1, wherein the organ is liver.

3. The method of claim 1, wherein the organ is kidney.

4. The method of claim 1, wherein the organ is lung.

5. The method of claim 1, wherein the contacting step comprises subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration.

6. The method of claim 1, wherein the effective amount is about 5 mg/kg to 250 mg/kg body weight of naringenin and 1 mg/kg to 50 mg/kg body weight of Asiatic acid.

7. The method of claim 1, wherein naringenin and Asiatic acid are administered at a weight ratio of about 10: 1.

8. The method of claim 1, wherein naringenin and Asiatic acid are administered in a single composition.

9. The method of claim 1, wherein naringenin and Asiatic acid are administered in two separate compositions.

10. The method of claim 1, wherein naringenin and Asiatic acid are administered in the form of a solution, a powder, a tablet, or a capsule.

11. A composition comprising (1) an effective amount of naringenin and Asiatic acid and (2) a pharmaceutically acceptable excipient.

12. The composition of claim 11, wherein naringenin and Asiatic acid are present in the weight ratio of about 10: 1.

13. The composition of claim 11, which is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or oral administration.

14. The composition of claim 11, which is a solution, a powder, a tablet, or a capsule.

15. A kit for inhibiting fibrosis, comprising a first container containing a first composition that comprises narigenin and a second container containing a second composition that comprises Asiatic acid.

16. The kit of claim 15, wherein the first or second composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or oral

administration.

17. The kit of claim 12, further comprising an instruction manual for administration of the first and second compositions.