WO2009091134A2

WO2009091134A2 - Pharmaceutical composition for sepsis and septic shock

Info

Publication number: WO2009091134A2
Application number: PCT/KR2008/007757
Authority: WO
Inventors: Sun Mee Lee; Chan Ho Lee
Original assignee: Sungkyunkwan University Foundation For Corporate Collaboration
Priority date: 2007-12-28
Filing date: 2008-12-29
Publication date: 2009-07-23
Also published as: KR20090071871A; WO2009091134A3

Abstract

The composition of the present invention is a pharmaceutical composition for the treatment of sepsis and septic shock, and inhibits multiple organ dysfunction syndrome that is associated with sepsis-induced organ injury, leading to reduction in mortality rate. Therefore, the pharmaceutical composition can be effectively used for the treatment of sepsis and septic shock.

Description

[DESCRIPTION]

[invention Title]

PHARMACEUTICAL COMPOSITION FOR SEPSIS AND SEPTIC SHOCK

[Technical Field]

The present invention relates to a therapeutic agent for sepsis and septic shock.

[Background Art] Sepsis is known as a major cause of morbidity and mortality in intensive care units worldwide, and it is estimated that more than 750, 000 cases of severe sepsis occur annually in the United States . Although no official statistics are available, very high incidence rates have been reported in Korea. However, there is only one FDA-approved therapeutic agent for sepsis, and a shortage of effective therapy and drugs currently available.

Since sepsis development is characterized by the overactivated inflammatory system at initial stage, many efforts have been made to inhibit the inflammatory mediators, such as TNF-α, interleukin-β, interleukin-1, and interleukin-8, for the treatment of sepsis. However, survival rates of the patients have not been improved yet.

On the other hand, chlorogenic acid is one of polyphenolic compounds widely distributed in plants and an important factor in plant metabolism.

Chlorogenic acid is a compound that is a protector against lipid peroxidation, an inhibitor of cholesterol biosynthesis, and a potent anti-oxidant carcinogenic inhibitor. In addition, chlorogenic acid delays the release of glucose into the bloodstream after a meal, prevents cardiovascular diseases, increases the levels of glycogen and glucose-β-phosphate in the liver, and reduces the blood glucose level. Other studies have reported that chlorogenic acid improves mineral pool distribution, decreases some plasma and liver lipids, and improves glucose tolerance.

However, there are no reports of therapeutic activity of chlorogenic acid in sepsis and septic shock.

[Disclosure] [Technical Solution] It is an object of the present invention to provide a pharmaceutical composition for the treatment of sepsis and septic shock.

[Advantageous Effects] As described above, the pharmaceutical composition for the treatment of sepsis and septic shock comprising one or more selected from the compound of Formula 1 of the present invention and pharmaceutically acceptable salts thereof as an active ingredient is expected to provide great therapeutic benefits to patients, notwithstanding the lack of effective therapy and drugs currently available. In addition, being derived from a natural source, the therapeutic agent is advantageous over chemical compounds in that it possesses various therapeutic actions and generates fewer side effects.

[Description of Drawings]

FIGs. 1 to 5 are graphs showing the changes in survival rate of sepsis mouse model according to administration of the pharmaceutical composition of one embodiment of the present invention;

FIG. 6 is a graph showing the changes in alanine aminotransferase concentration during 48 hrs after administration of the pharmaceutical composition to examine the inhibitory effect of the pharmaceutical composition of one embodiment of the present invention on liver injury;

FIG. 7 is a graph showing the changes in aspartate aminotransferase concentration during 48 hrs after administration of the pharmaceutical composition to examine the inhibitory effect of the pharmaceutical composition of one embodiment of the present invention on liver injury;

FIG. 8 is a graph showing the changes in blood urea nitrogen concentration during 48 hrs after administration of the pharmaceutical composition to examine the inhibitory effect of the pharmaceutical composition of one embodiment of the present invention on renal injury;

FIG. 9 is a graph showing the changes in creatinine concentration during 48 hrs after administration of the pharmaceutical composition to examine the inhibitory effect of the pharmaceutical composition of one embodiment of the present invention on renal injury; FIG. 10 is a graph showing the changes in lactate dehydrogenase concentration during 48 hrs after administration of the pharmaceutical composition to examine the inhibitory effect of the pharmaceutical composition of one embodiment of the present invention on heart injury; FIGs .11 to 19 are graphs showing the cytokine and chemokine expression levels in sepsis mouse model according to administration of the pharmaceutical composition of one embodiment of the present invention; and

FIGs. 20 to 27 are graphs showing the TLR (Toll-like receptor) mRNA expression levels in the liver, kidney, lung and heart of sepsis mouse model according to administration of the pharmaceutical composition of one embodiment of the present invention .

[Best Mode]

The present invention pertains to a pharmaceutical composition for the treatment of sepsis and septic shock, comprising the compound of Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient.

[Formula l]

wherein Ri, R₂ and R₃ are each independently selected from hydrogen and a residue of the following Formula 2, and at least one of Ri, R₂ and R₃ is the residue of the following Formula 2, [Formula 2]

wherein R₄ is any one selected fromhydrogen and alkyl having 1 to 6 carbon atoms.

Further, the pharmaceutical composition of the present invention is characterized in that it inhibits sepsis-induced organ injury.

Further, the pharmaceutical composition of the present invention is characterized in that it inhibits sepsis-induced injury to one or more organs of liver, kidney, and heart. Furthermore, the pharmaceutical composition of the present invention is characterized in that it further includes typical carriers, excipients or diluents.

The compound of Formula 1 of the present invention is commercially available or prepared by the known method from the leaves and fruits of dicotyledonous plants, such as Rubiaceae, Rubiaceae, and Solanaceae.

Examples of the compound of Formula 1 of the present invention include those structurally similar to chlorogenic acid, specifically 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3, 4-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid, 4 , 5-dicaffeoylquinic acid, 3-ferulylquinic acid, 4-ferulylquinic acid, 5-ferulylquinic acid, and 3-ferulyl-4-caffeoylquinic acid, preferably a compound having hydrogen in R₄ of Formula 2, and more preferably chlorogenic acid.

The compound of Formula 1 of the present invention may be used in the form of a salt to improve its water solubility and increase its physiological function. Pharmacologically accepted salt is preferably used as the above described salt. As basic substances used for producing the above described salt, it is possible to use hydroxides of alkali metals such as lithium hydroxide, sodium hydroxide, and potassium hydroxide; hydroxides of alkaline earth metals such as magnesium hydroxide and calcium hydroxide; inorganic bases such as ammonium hydroxide; basic amino acids such as arginine, lysine, histidine, and ornithine; organic bases such as monoethanolamine, diethanolamine, and triethanolamine, and among these, hydroxides of alkali metals or alkaline earth metals are preferable. In the present invention, the above described salt may be prepared and then added to a composition comprising other components, or alternatively, the compound of Formula 1 and the salt-forming component may be separately added to the above described composition to form therein the above described salt.

[Mode for Invention] Hereinafter, the present invention will be described in detail with reference to the following Examples, Preparation Examples and Experimental Examples. However, these Examples are for the illustrative purposes only, and the invention is not intended to be limited by these Examples.

The pharmaceutical composition comprising one or more ingredients selected from the compound of Formula 1 of the present invention and pharmaceutically acceptable salts thereof as an active ingredient may be used by formulating it into oral formulations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and aerosol, external preparation, suppository or sterilized solution for injection according to the typical method.

Carriers, excipients and diluents which can be included in the pharmaceutical composition are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

In case of formulating, diluents or excipients such as filler, extender, bonding agent, wetting agent, disintegrating agent, and surfactant may be used. Solid preparation for oral administration comprises tablet, pill, powder, granule, capsule, etc . and is preparedbymixing one ormore excipients, forexample, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above extract. Also, besides simple excipients, lubricant such as magnesium stearate and talc may be used. Solution for oral administration comprises suspension, internal solution, emulsion, syrup, etc., and besides simple diluents such as water and liquid paraffin, various excipients, for example, wetting agent, sweetening agent, odorant, preservative or the like may be contained therein. Formulation for parenteral administration includes sterilized water solution, non-aqueous solution, suspension, emulsion, lyophilization preparation, or suppository. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyloleate may be used as non-aqueous solution and suspension. Witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin or the like may be used as suppository base.

The preferable dosage of the pharmaceutical composition of the present invention is different depending on condition and weight of patient, severity of disease, forms of medicine, and route and period of administration, but may be appropriately selected by a skilled person in the art. However, to have desirable effect, it is preferable to administer the pharmaceutical composition of the present invention in 0.01 ~ 1000 mg/kg, preferably 0.1 ~ 1000 mg/kg, and more preferably 1 ~ 1000 mg/kg a day, based on chlorogenic acid. The administration may be made once or several times a day. The scope of the present invention should not be limited by the above dosage in any manner . The extract of the present invention can be administered through various routes to mammals of rat, mouse, livestock, human, etc. The administration can be made through all routes, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

Hereinbelow, the methods of formulating the pharmaceutical composition of the present invention will be described with reference to Preparation Examples. The pharmaceutical composition of the present invention may be administered in the form of the following formulations. However, these Preparation Examples are for the illustrative purposes only, and the invention is not intended to be limited by these Examples.

Preparation Example 1. Preparation of injectable formulation

3-Caffeoylquinic acid 500 mg Sterile distilled water proper amount

Sterile distilled water was added to a total volume of 25 ml. According to a typical procedure, the above ingredients were mixed well, filled in a brown bottle, and sterilized to prepare an injectable formulation.

Preparation Example 2. Preparation of pill

3, 4-Dicaffeoylquinic acid 25 mg Corn starch 100 mg

Binder proper amount

The above ingredients were mixed, and pills were prepared to have the same size as a phoenix tree seed or a large pill according to a typical procedure.

Preparation Example 3. Preparation of tablet 3-Ferulylquinic acid 25 mg

Lactose 100 mg

Starch 100 mg Magnesium Stearate proper amount

The above ingredients were mixed, and tabletted to prepare a tablet according to a typical procedure.

Preparation Example 4. Preparation of capsule 3-Caffeoylquinic acid 25 mg

Lactose 50 mg

Starch 50 mg Talc 2 mg

Magnesium Stearate proper amount

The above ingredients were mixed, and filled into a gelatin capsule according to a typical procedure to give a capsule agent .

It was found that the pharmaceutical composition of the present invention significantly reduced mortality rate after sepsis in a murine cecal ligation and puncture (hereinbelow, referred as CLP) model, and inhibits injury to the major organs, such as liver, kidney, and heart, so as to prevent multiple organ dysfunction syndrome (hereinbelow, referredasMODS) , which will be described in detail with reference to Experimental Examples and Drawings .

Experimental Example 1. Evaluation for survival rate of sepsis

1. Changes in the survival rate were examined in CLP-induced sepsis mouse model according to administration of the inj ectable formulation of Preparation Example 1. 2. After anesthesia with an intraperitoneal injection, sepsis was induced in mouse by CLP. That is, a ventral midline incision was made, and the cecum was exposed and ligated with a silk suture just distal to the ileocecal valve. The cecum was then punctured twice with a needle. The punctured cecum was squeezed to expel a small amount of fecal material, and then returned to the abdominal cavity. The abdominal incision was then closed, and the animals received normal saline subcutaneously. After induction of sepsis by the above procedure, the injectable formulation of Preparation Example

1 or a vehicle was administered into the mouse via tail vein.

3. Test material : the injectable formulation of Preparation Example 1; instead of chlorogenic acid, 4-caffeoylquinic acid,

5-caffeoylquinic acid, 3, 5-dicaffeoylquinic acid, or

3-ferulylquinic acid was used to prepare an injectable formulation in Preparation Example 1; and a vehicle excluding the active ingredient was used as a control group. 4. Test result:

(1) The survival rate was 60% at 10 days after administrations of chlorogenic acid to sepsis-induced mice at a dosage of 10, 20, 30 and 40 mg/kg (FIG. 1) .

(2) The survival rate was 40% at 10 days after administrations of 4-caffeoylquinic acid to sepsis-inducedmice at a dosage of 10 and 20 mg/kg (FIG. 2) .

(3) Each survival rate was 50% and 60% at 10 days after administrations of 5-caffeoylquinic acid to sepsis-inducedmice at a dosage of 10 and 20 mg/kg (FIG. 3) . (4) The survival rate was 60% at 10 days after administrations of 3, 5-dicaffeoylquinic acid to sepsis-induced mice at a dosage of 10 and 20 mg/kg (FIG. 4) .

(5) Each survival rate was 50% and 60% at 10 days after administrations of 3-ferulylquinic acid to sepsis-inducedmice at a dosage of 10 and 20 mg/kg, which were remarkably improved as compared to that (20%) of non-treated group (FIG. 5) .

Accordingly, it can be seen that the pharmaceutical composition of the present invention significantly reduced the mortality rate of sepsis.

Experimental Example 2. Evaluation of efficacy on liver function

1. Liver injury during sepsis was examined, and the levels of alanine aminotransferase (hereinbelow, referred as ALT) and aspartate aminotransferase (hereinbelow, referred as AST) were measured in order to assess changes in liver injury according to administration of the injectable formulation of Preparation Example 1.

2. At the time points of 1, 3, 6, 12, 24 and 48 hr after CLP, blood was collected, and serum was isolated therefrom. The ALT andAST levels were determined in the isolated serumto examine injury to the liver, shown in FIGs. 2 and 3. A control group is a mouse that were not administered with the injectable formulation of Preparation Example 1 after induction of CLP sepsis, and Sham is an operative control.

3. Test result: the results are shown in FIGs. 6 and 7, and the injectable formulation of Preparation Example 1 remarkably inhibits the increase of the ALT and AST levels, which indicates the degree of liver injury during sepsis.

Experimental Example 3. Evaluation of efficacy on renal function

1. Renal injury during sepsis was examined, and the levels of blood urea nitrogen (hereinbelow, referred as BUN) and creatinine (hereinbelow, referred as CRE) were measured in order to assess changes in renal injury according to administration of the injectable formulation of Preparation Example 1.

2. At the time points of 1, 3, 6, 12, 24 and 48 hr after CLP, blood was collected, and serum was isolated therefrom. The

BUN and CRE levels were determined in the isolated serumto examine injury to the kidney.

3. Test result: the results are shown in FIGs. 8 and 9, and the injectable formulation of Preparation Example 1 remarkably inhibits the increase of the BUN and CRE levels, which indicates the degree of renal injury during sepsis.

Experimental Example 4. Evaluation of efficacy on heart function 1. Heart injury during sepsis was examined, and the level of lactate dehydrogenase (hereinbelow, referred as LDH) was measured in order to assess changes in heart injury according to administration of the injectable formulation of Preparation Example 1. 2. At the time points of 1, 3, 6, 12, 24 and 48 hr after CLP, blood was collected, and serum was isolated therefrom. The LDH level was determined in the isolated serum to examine injury to the heart.

3. Test result: the result is shown in FIG. 10, and the injectable formulation of Preparation Example 1 remarkably inhibits the increase of the LDH level, which indicates the degree of heart injury during sepsis. Experimental Example 5. Measurement of cytokines and chemokines in mouse sepsis model

1. Concentrations of TNF-α, IFN-γ, IL-lβ, IL-2, IL-6, IL-IO, IL-12, MCP-I (monocyte chemotactic protein-1) (BD Bioscience, San Diego, CA), MIP-2 (macrophage inflammatory protein-2) (R&D Systems, Inc., Minneapolis, MN) and HMGBl (Shino-Test Corp., Japan) were measured using an ELISA kit in accordance with the manufacturer's instructions. 2. At the time points of 1, 3, 6, 12, 18, 24 and 48 hr after

CLP, blood was collected from the CLP-inducedmouse sepsismodel, and serum was isolated therefrom. The expression levels of cytokines and chemokines were determined in the isolated serum.

3. Test result: the results are shown in FIGs. 11 to 19. The increased levels of TNF-α, IL-lβ, and IL-6 after CLP were inhibitedby the administration of chlorogenic acid, whereas the increased levels of IFN-γ, IL-2 and IL-12 after CLP were increased by the administration of chlorogenic acid. In addition, chlorogenic acid did not affect the level of Th2 cytokine, IL-10. The increased level of MIP-2 was significantly inhibited by chlorogenic acid at 6 hr after CLP, and the increased level of MCP-I was significantly increased by chlorogenic acid at 3 hr after CLP.

Experimental Example 6. Examination of TLRmBHAandprotein expressions in mouse sepsis model

1. Total RNA extraction Total RNA isolation was performed using the method of

Chomczynski and Sacchi (1987) . At the time points of 1, 3, 6,

12 and 24 hr after CLP or CLP and chlorogenic acid (20 mg/kg) administration, the mice were anesthetized, and heart, lung, liver, and kidney were excised therefrom. About 100 mg of each tissue was homogenized in 1 ml of TRIZOL^® reagent (GibcoBRL,

NY, USA) , andthen200μ£ of chloroformwas added thereto, followed by centrifugation. The aqueous phase was put in a microtube, and RNAwas precipitatedwith400mlof isopropanol . The obtained RNA pellet was washed with 75% ethanol, centrifuged, and dried under vacuum. Thereafter, RNA was dissolved in DEPC

(diethylpyrocarbonate) -treated deionized water, and stored at

-70 ^°C. Total RNA concentration was determined by absorbance at 260 nm.

2. RT-PCR

Reverse transcription was performed using total RNA, oligo dT-adaptor primer and AMV reverse transcriptase to synthesize cDNA. PCR amplification was performed with cDNA sample diluted in 10-μJt reaction solution. A GeneAmp^® 2700 thermocycler (Perkin-Elmer, Inc., Waltham, MA, USA) was used for PCR amplification consisting of an initial denaturation at 94 ^°C for 5 min, and annealing at 72 ^°C for 7 min. The PCR cycling conditions (denaturation, annealing, extension) were as follows : 30 cycles of 94 ^°C for 30 sec, 5β^°C for 30 sec, and 72 ^°C for 30 sec for TLR4 ; 25 cycles of 94 ^°C for 30 sec, 64 ^°C for 30 sec, and 72 ^°C for 30 sec for TLR2; 26 cycles of 94 ^°C for 30 sec, 54 ^°C for 30 sec, and 72^°Cfor 60 sec for β-actin. TLR4 forward primer was 5'-AGTGGGTCAAGGAACAGAAGCAG-S', and reverse primer 5'-CTTTACCAGCTCATTTCTCACCC-S' . TLR2 forward primer was 5'-TGGAGACGCCAGCTCTGGCTCA-S', and reverse primer 5'-CAGCTTAAAGGGCGGGTCAGAG-S' .

3. Western blot analysis

At the time point of 6 hr after CLP, the liver tissue sample was homogenized in 1 ml of lysis buffer (150 mM NaCl, 1% Triton X-IOO, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate,

50 mM Tris, pH 8.0), and incubated in ice for 60 min, followed by sonication for 30 sec. Then, the sample was centrifuged at

10, 000 rpm for 10 min, and protein extract was obtained at -70 ^°C

The protein from each sample was mixed with 2X sodium dodecyl sulphate sample buffer, and heated at 95 ^°C for 5 min, followed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane for 2 hrs, and then incubated with the primary antibody against TLR4 (1: 1000, Santacruz, USA) at 4 ^°C overnight. The membrane was washed with TBS-O.05% Tween 20 three times, and incubated with the secondary antibody, peroxidase-conjugated goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch, Baltimore, USA) at room temperature for 1 hr. The signals were quantified by densitometry and computer image analysis.

4. Results

The CLP group showed increased expression levels of TLR2 and TLR4 mRNA at all time points, as compared to the sham-operated group (FIGs. 20 to 27) .

In the liver, the increased expression levels of TLR2 and TLR4 mRNA after CLP reached a peak at the time point of 6 hr, and gradually decreased by the time point of 24 hr. The TLR2 expression level was significantly reduced by administration of chlorogenic acid during 3 to 24 hrs after CLP. In addition, chlorogenic acid remarkably inhibited the increase of TLR4 mRNA expression level at the time points of 3, 6, and 24 hr after CLP.

In the kidney, the expression levels of TLR2 and TLR4 mRNA after CLP reached a peak at the time point of 6 hr, and gradually decreased by the time point of 24 hr . Chlorogenic acid remarkably inhibited the TLR2 mRNA expression level at the time points of 3 to 12 hrs after CLP, as well as TLR4 mRNA expression level.

In the lung, the TLR2 mRNA expression level was significantly increased at the time points of 1 and 3 hr after

CLP, and maintained at a constant level at the time points of

6, 12, and 24 hr after CLP, as compared to the sham group. The TLR4 mRNA expression level after CLP reached a peak at the time point of 6 hr, and gradually decreased at the time points of 12 and 24 hr. Chlorogenic acid remarkably inhibited the increased expression level of TLR2 mRNA at the time point of 3 hr, and did not reduce the TLR4 mRNA expression level. In the heart, the TLR2 mRNA expression level was significantly increased at the time point of 6 hr after CLP, and maintained at a constant level at the time points of 6, 12, and 24 hr after CLP, as compared to the sham group. The TLR4 mRNA expression level after CLP was significantly increased at all time points, as compared to the sham group. Chlorogenic acid remarkably inhibited the TLR2 mRNA expression level at the time points of 3 to 24 hrs after CLP, and the TLR4 mRNA expression level at the time points of 6 to 24 hrs after CLP.

On the other hand, the pharmaceutical composition of the present invention may be formulated with or used in combination with anti-inflammatory agents, antipyretic analgesic agents, anticoagulants, antibiotics, antimicrobial agents, and anti-allergic agents.

[Indus-trial Applicability]

The pharmaceutical composition for the treatment of sepsis and septic shock comprising one ormore selected fromthe compound of Formula 1 of the present invention and pharmaceutically acceptable salts thereof as an active ingredient is expected to provide great therapeutic benefits to patients, notwithstanding the lackof effective therapy and drugs currently available. In addition, being derived from a natural source, the therapeutic agent is advantageous over chemical compounds in that it possesses various therapeutic actions and generates fewer side effects.

Claims

[CLAIMS]

[Claim l]

A pharmaceutical composition for the treatment of sepsis and septic shock, comprising the compound of the following Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient:

[ Formula 1 ]

wherein R₁, R₂ and R₃ are each independently selected from hydrogen and a residue of the following Formula 2, and at least one of Ri, R₂ and R₃ is the residue of the following Formula 2,

[Formula 2]

wherein R₄ is any one selected fromhydrogen and alkyl having 1 to β carbon atoms. [Claim 2]

The pharmaceutical composition for the treatment of sepsis and septic shock according to claim 1, wherein R₄ is hydrogen.

[Claim 3] The pharmaceutical composition for the treatment of sepsis and septic shock according to claim 1 or 2, wherein R₂ and R₃ are hydrogens.

[Claim 4]

The pharmaceutical composition for the treatment of sepsis and septic shock according to claim 1 or 2 , wherein the composition inhibits sepsis-induced organ injury.

[Claim 5]

The pharmaceutical composition for the treatment of sepsis and septic shock according to claim 4, wherein the organ is one or more selected from liver, kidney, and heart.

[Claim β]

The pharmaceutical composition for the treatment of sepsis and septic shock according to claim 1 or 2, further comprising a carrier, excipient, or diluent that is typically used in the production of pharmaceutical composition.