WO2017066965A1

WO2017066965A1 - Gallic acid-l-leucine conjugate for resolution of inflammation and sepsis

Info

Publication number: WO2017066965A1
Application number: PCT/CN2015/092564
Authority: WO
Inventors: Jianhui Rong
Original assignee: The University Of Hong Kong
Priority date: 2015-10-22
Filing date: 2015-10-22
Publication date: 2017-04-27

Abstract

Compounds used for treatment of inflammation and sepsis, or pharmaceutically acceptable salts or analogs thereof are disclosed.

Description

[Title established by the ISA under Rule 37.2] GALLIC ACID-L-LEUCINE CONJUGATE FOR RESOLUTION OF INFLAMMATION AND SEPSIS

BACKGROUND OF THE INVENTION

Septic shock is a life threatening clinical condition characterized as the systemic inflammatory response syndrome (SIRS) due to bacterial, fungal and viral infection. Upon infection, microbial toxins cause inflammation, tissue damage and subsequently multiorgan failure ⁽ ¹ ⁾ . Previous studies have identified the imbalance in the proinflammatory and anti-inflammatory cytokines, chemokines, antigens, endotoxins, procoagulant, and anticoagulant factors during septic shock ⁽ ² ⁾ . Traditional antimicrobial drugs were designed to correct the imbalance of the inflammatory mediators back to a homeostatic balance and to improve the efficiency of organ perfusion in the microcirculation ⁽ ³ ⁾ . On the other hand, various extracorporeal therapies have also been developed as adjunctive therapeutic strategies ⁽ ² ⁾ . However, clinical outcomes of these therapies may be sometimes disappointing ⁽ ⁴ ⁾ . Thus, effort is still made to develop new strategies for effective resolution of inflammation and sepsis.

Natural products constitute valuable resources for the identification of anti-microbial and anti-inflammatory reagents ⁽ ⁵ ^, ⁶ ⁾ . Gallic acid derivatives exert anti-oxidant, anti-inflammatory, chemopreventive, anticancer and antibacterial properties ⁽ ^7-9 ⁾ . Gallic acid was also evaluated for the treatment of sepsis in a mouse model, but failed to show any protection after 48 hours ⁽ ¹⁰ ⁾ . We have recently isolated gallic acid from the medicinal herb Radix Paeoniae Rubra as one of two active compounds for synergistic induction of leukotriene B4 metalizing enzyme leukotriene B4 12-hydroxydehydrogenase (LTB4DH) ⁽ ¹¹ ⁾ . Induction of LTB4DH represents an endogenous mechanism supporting selective control of proinflammatory lipid mediator LTB4 ⁽ ¹² ⁾ . Bacterial lipopolysaccharide (LPS) is a well-known causative agent of sepsis. Previous studies have shown that LPS reduces L-leucine mediated transport across the rabbit jejunum and thereby reduces L-leucine absorption by about 30％ ⁽ ¹³ ^, ¹⁴ ⁾ . L-leucine is also known for its activity in the regulation of intracellular signaling pathways such as mTORC1 pathway ⁽ ¹⁵ ^, ¹⁶ ⁾ . Upon LPS stimulation, L-leucine becomes restricted in the body so that patients may suffer from dysregulation of autophagy and debilitating skeletal muscle wasting due to the reduced activation of mTORC1 ⁽ ¹⁷ ^, ¹⁸ ⁾ .

BRIEF SUMMARY OF THE INVENTION

Timely resolution of inflammation and sepsis is critical to reduce morbidity and mortality in the infected population. The clinical outcomes of current anti-microbial, anti-inflammatory therapies are frequently disappointing, possibly due to the structure and function of a single chemical compound. This invention provides a new class of chemical compounds, and method for the preparation of the same, for the treatment of inflammation and sepsis.

In one aspect, the present invention provides a compound of Formula (I) :

or a pharmaceutically acceptable salt or analog thereof； wherein X₁, X₂ and X₃ are, independently, H or OH； and wherein R is a methyl group. In some embodiments, the compound of Formula (I) is a gallic acid L-leucine (GAL) conjugate.

In some embodiments, the present invention provides a compound of Formula (II) :

or a pharmaceutically acceptable salt or analog thereof.

In some embodiments, the compound of Formula (I) is provided in pharmaceutical compositions. The pharmaceutical compositions may also comprise a carrier.

In another aspect, the present invention provides a method of treatment for septic shock, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In another aspect, the present invention provides a method of preventing the enlargement of spleen size against LPS-induced endotoxic shock, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In another aspect, the present invention provides a method of attenuating the chemotaxis of macrophages in response to LPS treatment, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In a further aspect, the present invention provides a method of suppressing COX-2 and iNOS expression in response to LPS treatment, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In a further aspect, the present invention provides a method of controlling aberrant production of proinflammatory lipid mediators (such as, for example, PGE2； PGF2α； 13, 14-dh-PGF2α； 12-HETE； 15-HETE； LTB4； and combinations thereof) in response to LPS treatment, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In yet another aspect, the present invention provides a method of suppressing the aberrant production of toxic nitric oxide (NO) in response to LPS treatment, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) .

In another aspect, the present invention provides a compound described herein for use in treatment for septic shock in a subject.

In another aspect, the present invention provides a compound described herein for use in preventing the enlargement of spleen size against LPS-induced endotoxic shock in a subject.

In another aspect, the present invention provides a compound described herein for use in attenuating the chemotaxis of macrophages in response to LPS treatment in a subject.

In a further aspect, the present invention provides a compound described herein for use in suppressing COX-2 and iNOS expression in response to LPS treatment in a subject.

In a further aspect, the present invention provides a compound described herein for use in controlling aberrant production of proinflammatory lipid mediators (such as, for example, PGE2； PGF2α； 13, 14-dh-PGF2α； 12-HETE； 15-HETE； LTB4； and combinations thereof) in response to LPS treatment in a subject.

In yet another aspect, the present invention provides a compound described herein for use in suppressing the aberrant production of toxic nitric oxide (NO) in response to LPS treatment in a subject.

In another aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for treatment for septic shock in a subject.

In another aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for preventing the enlargement of spleen size against LPS-induced endotoxic shock in a subject.

In a further aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for attenuating the chemotaxis of macrophages in response to LPS treatment in a subject.

In a further aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for suppressing COX-2 and iNOS expression in response to LPS treatment in a subject.

In a further aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for controlling aberrant production of proinflammatory lipid mediators (such as, for example, PGE2； PGF2α； 13, 14-dh-PGF2α； 12-HETE； 15-HETE； LTB4； and combinations thereof) in response to LPS treatment in a subject.

In yet another aspect, the present invention provides a use of a compound described herein in manufacture of a medicament for suppressing the aberrant production of toxic nitric oxide (NO) in response to LPS treatment in a subject.

The methods and compositions herein described can be used in connection with pharmaceutical, medical, and veterinary applications, as well as fundamental scientific research and methodologies, as would be identifiable by a skilled person upon reading of the present disclosure. These and other objects, features and advantages of the present invention will become clearer when the drawings as well as the detailed description are taken into consideration.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature of the present invention, reference should be had to the following detailed description taken in connection with the accompanying figures.

Figure 1 shows the general formula of a compound of the present invention.

Figure 2 shows the synthetic scheme for the preparation of a GAL conjugate. The GAL conjugate was synthesized from gallic acid and L-leucine methyl ester through direct formation of amide in one-pot manner. The overall yield is 27％. DMF, NN-dimethyl formamide； BOP, benzo-triazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate； Et₃N, triethylamine.

Figure 3 shows the effect of GAL, L-leucine and gallic acid on cell viability. Mouse macrophage RAW264.7 cells were treated with GAL, L-leucine and gallic acid at various concentrations for 48 h, the cell viability was determined by colorimetric MTT assay. The results were expressed as mean ± SD of three independent experiments.

Figure 4 shows the effect of GAL on LPS-induced endotoxin shock in mice. (A) Experimental protocol for investigating the effect of GAL on LPS-induced endotoxin shock in mice. (B) Effect of GAL on LPS-induced death in mice. Mice were randomly divided into three groups, namely, control, LPS (25 mg/kg) only, and LPS (25 mg/kg) + GAL (5 mg/kg) groups (n＝7/group) . The survival of mice was monitored every 12 h for consecutive 10 days. (C) Effect of GAL on LPS-induced alterations in the size of mouse spleen. After 10 days of drug treatment, mice were sacrificed and the spleens were subsequently collected and weighted. #, p < 0.05 (LPS vs untreated control) ； **, p < 0.01 (GAL + LPS vs LPS alone) .

Figure 5 shows the effect of GAL on LPS-induced expression of COX-2 and iNOS. Following the treatment with GAL and LPS as indicated for 24 h, the proteins were isolated from primary peritoneal macrophages and spleen tissues, and subsequently analyzed by Western blotting using specific antibodies (A) . (B) Quantification of COX-2 and iNOS levels. The protein levels on Western blots were determined by densitometric method. The results were expressed as a percentage of the untreated control (n＝3) . **, p<0.01； ***, p<0.001 (Sample vs LPS alone) .

Figure 6 shows the comparison of GAL with L-leucine and gallic acid in the regulation of COX-2, 5-LOX and iNOS expression in response to LPS stimulation. Following the treatment of LPS-stimulated macrophages with GAL, L-leucine and gallic acid as indicated for 24 h, the proteins were isolated from RAW264.7 cells and primary peritoneal macrophages, and subsequently analyzed by Western blotting using specific antibodies (A) . (B) Quantification of COX-2, 5-LOX and iNOS levels. The protein levels on Western blots were determined by densitometric method. The results were expressed as a percentage of the untreated control (n＝3) . *, p<0.05； **, p<0.01； ***, p<0.001 (Sample vs LPS alone) .

Figure 7 shows the effects of GAL on the generation of pro-inflammatory mediators. (A) Representative chart for illustrating the generation of lipid mediators. (B) LC-MS/MS determination of pro-inflammatory lipid mediators. The lipids were isolated from RAW 264.7 cells after exposure to GAL and LPS as indicated. The individual lipid mediators were quantified by a MRM-based LC-MS/MS method. Results were presented as means ± SD of three independent experiments. **, p<0.01； ***, p<0.001 (Sample vs LPS alone) .

Figure 8 shows the effects of GAL on LPS-induced expression of NO in primary peritoneal macrophages. Following 24 h treatment with LPS and GAL as indicated, the intracellular NO levels were detected and quantified by the production of fluorescence from probe DAF-FM diacetate, Results were presented as means ± SD of three independent experiments. **, p<0.01 (Sample vs LPS alone) .

Figure 9 shows the effect of GAL on the chemotactic potential of macrophages. (A) Transwell assay of chemotactic potential. After 24 h treatment with GAL and LPS as indicated, the conditioned media were collected from primary peritoneal macrophages, and subsequently evaluated by Transwell assay for the chemotactic potential. The conditioned medium was placed in the lower chamber while fresh primary peritoneal macrophages were seeded in the upper chamber. The migrated cells were stained with 0.1％crystal violet and imaged under a microscope. Representative images were shown. (B) Quantification of macrophage chemotaxis. After staining with crystal violet, the membranes were eluted with 30％glacial acetic acid and the absorbance at 570 nm of the cell lysates was measured on a microplate reader. The results were presented as the means ± SD of three separate experiments. **, p < 0.01 (GAL+LPS vs LPS alone) .

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides compounds and related methods for the treatment of inflammation and sepsis.

Several aspects of the invention are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts, steps or events are required to implement a methodology in accordance with the present invention. Many of the techniques and procedures described, or referenced herein, are well understood and commonly employed using conventional methodology by those skilled in the art.

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein.

In one aspect, the present invention provides a compound of Formula (I) :

or a pharmaceutically acceptable salt or analog thereof； wherein X₁ -X₃ are independently OH；and wherein R is a methyl group. In some embodiments, the compound of Formula (I) is a gallic acid L-leucine (GAL) conjugate.

or a pharmaceutically acceptable salt or analog thereof.

The term "carrier" refers to a diluent, adjuvant, excipient, and/or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, lactose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain wetting or emulsifying agents or suspending/diluting agents, or pH buffering agents, or agents for modifying or maintaining the rate of release of the compound. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharine, starch, magnesium stearate, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the compound together with a suitable amount of carrier so as to provide the proper form to the patient based on the mode of administration to be used.

If for intravenous administration, the compositions are packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachette indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.

Moreover, if a packaging material is utilized to package the pharmaceutical composition, it may be biologically inert or lack bioactivity, such as plastic polymers, silicone, etc. and may be processed internally by the subject without affecting the effectiveness of the compound packaged and/or delivered therewith.

As used herein, “treatment” or “treating” refers to arresting or inhibiting, or attempting to arrest or inhibit, the development or progression of a disease or infection and/or causing, or attempting to cause, the reduction, suppression, regression, or remission of a disease and/or a symptom thereof. As would be understood by those skilled in the art, various clinical and scientific methodologies and assays may be used to assess the development or progression of a disease, and similarly, various clinical and scientific methodologies and assays may be used to assess the reduction, regression, or remission of a disease or infection or its symptoms. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease/infection as well as those prone to have the disease/infection or those in whom the disease/infection is to be prevented. In at least one embodiment, the disease/infection being treated is inflammation. In other embodiments, the disease being treated is sepsis.

Administration may be locally (confined to a single cell or tissue) and/or systemically in the subject. It may be desirable to administer the compounds and pharmaceutical compounds of the invention locally to the area in need of treatment, such as areas including inflammation. This method of administration may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery) , by injection, by catheter, or by means of an implant (e.g., a porous membrane) . When administering the compounds, care must be taken to use materials which do not absorb the compounds, thus allowing for effective release, or delayed release if so desired by the materials used.

In some embodiments, the fusion protein or pharmaceutical composition can be delivered in a controlled release system. Such methods may include the use of a pump for administration (e.g., use of an intravenous drip) . In another embodiment, a controlled release system can be placed in the proximity of the therapeutic target (e.g., a tumor) , requiring only a fraction of the dose required if dosed systemically.

Furthermore, it would be understood by those skilled in the art that the therapeutic methods described would not only apply to treatment in a subject, but could be applied to cell cultures, organs, tissues, or individual cells in vivo, ex vivo or in vitro.

As used herein, the term "subject" refers to an animal. Typically, the terms "subject" and "patient" may be used interchangeably herein in reference to a subject. As such, a “subject” includes a human that is being treated for a disease as a patient.

The term “animal, ” includes, but is not limited to, mouse, rat, dog, cat, rabbit, pig, monkey, chimpanzee, and human (i.e., mammals) .

The terms "effective amount" and “therapeutically effective amount, ” used interchangeably, as applied to the compounds and pharmaceutical compositions described herein, mean the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of inflammation or sepsis, for which the composition thereof is being administered. Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the issue being treated and its severity and/or stage of development/progression； the bioavailability and activity of the specific compound or pharmaceutical composition used； the route or method of administration and introduction site on the subject； the rate of clearance of the specific composition and other pharmacokinetic properties； the duration of treatment； inoculation regimen； drugs used in combination or coincident with the specific composition； the age, body weight, sex, diet, physiology and general health of the subject being treated； and like factors well known to one of skill in the relevant scientific art. Some variation in dosage will necessarily occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dosage for an individual patient. The term "pharmaceutically acceptable, " as used herein with regard to pharmaceutical compositions, means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and/or in humans.

Thus, the following non-limiting embodiments are provided:

1. A compound of Formula (I) :

or a pharmaceutically acceptable salt or analog thereof；

wherein X₁ -X₃ are, independently, OH； and

wherein R is a methyl group.

2. A pharmaceutical composition comprising the compound of embodiment 1 and a carrier.

3. A method of treatment of septic shock, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of embodiment 1.

4. A method of preventing the enlargement of spleen size against LPS-induced endotoxic shock, comprising:

5. A method of attenuating the chemotaxis of macrophages in response to LPS treatment, comprising:

6. A method of suppressing COX-2 and iNOS expression in response to LPS treatment, comprising:

7. A method of controlling aberrant production of proinflammatory lipid mediators in response to LPS treatment, comprising:

8. The method of claim 11, wherein the lipid mediators are selected from PGE2； PGF2α； 13, 14-dh-PGF2α； 12-HETE； 15-HETE； LTB4； and combinations thereof.

9. A method of suppressing the aberrant production of toxic NO in response to LPS treatment, comprising:

10. The method of any of embodiments 3-9, wherein the subject is a mammal.

Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 -Synthesis and chemical characterization of gallic acid-L-leucine conjugate

The scheme for the preparation of the gallic acid-L-leucine (GAL) conjugate, as shown in Figure 1, is illustrated in Figure 2. Gallic acid (500 mg) was dissolved in 3 ml of DMF containing 400 μl of Et₃N. 1.33 g of benzo-triazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) in dichloromethane (CH₂Cl₂) and 530 mg of L-leucine methyl ester were sequentially added while stirring on ice. The reaction mixture was stirred at 0 ℃ for 30 min and at room temperature for ～14 h. At the end of reaction, the solvent was removed by rotary evaporator under vacuum. The residues were dissolved in 5 ml of Milli-Q water and extracted with 5 ml of ethyl acetate (EtOAc) . The organic phase was recovered and dried with anhydrous sodium sulfate (Na₂SO₄) . The product GAL was purified by column chromatography on silica gel with chloroform and methanol (CHCl₃ : MeOH ＝ 10 : 1) as eluting solvents to yield a yellow powder (235mg, 27％) . GAL was further characterized by mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy. MS analysis was performed on an ABI/Sciex triple quadrupole 3200 QTRAP mass spectrometer (Framingham, MA, USA) equipped with an TurboV Source operating in positive ionization mode under the control of Analyst v1.4.2 data system (Applied Biosystems/MDS Sciex, Concord, ON, Canada) . ESI-MS (m/z) : 298.2 [M+H] ⁺. NMR spectra were recorded in MeOD: CDCl₃ (50 : 50, v/v) on a Varian Unity plus NMR 400 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) . ¹H NMR (MeOD: CDCl₃, 400 MHz) , δ_H : 8.32 (s, H) , 6.87 (s, 2H) , 4.43 (m, H) , 3.74 (s, 3H) , 1.67 (m, 2H) , 1.53 (m, H) , 0.90 (d, J＝6, 6H) ； ¹³C NMR (MeOD: CDCl₃, 75 MHz) , δ_C: 173.8, 167.2, 145.9, 137.0, 124.6, 107.6, 52.2, 51.3, 40.3, 24.9, 23.4, 21.6.

Example 2 -GAL conjugate was not toxic to mouse macrophage RAW264.7 cells

The cytotoxicity of GAL conjugate was evaluated by a colorimetric assay using 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) . Briefly, following 48 h treatment with GAL conjugate at the concentrations of 0, 10, 25, 50 100, 200 μM, MTT was added to the cell culture of mouse macrophage RAW264.7 cells in a 96-well plate (final concentration of 0.5 mg/ml) . After the incubation at 37 ℃ under 5％CO₂ for 4 h, the supernatant was removed and the resulted formazan crystals in viable cells were solubilized with 150 μl of dimethyl sulfoxide (DMSO) and measured on a Bio-Red microplate reader (Hercules, CA, USA) at 570 nm. The results are shown in Figure 3.

Example 3 -GAL conjugate rescued mice against endotoxin shock and maintained spleen size

The protocols for animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong. Male C57BL/6 (6-8 weeks) mice were randomly divided into three groups, Control, receiving vehicle only； LPS, sequentially receiving vehicle for 3 days and 4 mg/kg LPS； LPS + GAL (10 mg/kg) , sequentially receiving 10 mg/kg GAL for 3 days and 4 mg/kg LPS. In practical, mice were pre-treated with GAL or vehicle via i.p injection for 3 days, and subsequently challenged with LPS via i.p injection at the time of 12 h after the injection of GAL or vehicle on day-3. To examine the effect of GAL on the survival of animals in the LPS-induced model of endotoxin shock, animals were closely examined every 12 hours over the period of 10 consecutive days as previously described ⁽¹⁹⁾ . (Figure 4 (A) ) The number of dead mice was counted. Survival rate was calculated based on the numbers of live and dead animals. (Figure 4 (B) ) . The animals were sacrificed on Day-10 after LPS challenge. The spleens were weighed and examined under a microscope. (Figure 4 (C) ) .

Example 4 -Effect of GAL on LPS-induced expression of COX-2 and iNOS

Male C57BL/6 (6-8 weeks) mice were randomly divided into five groups, Control, receiving vehicle only； LPS, receiving 4 mg/kg； LPS + GAL (i.e., 2.5, 5.0, 10.0 mg/kg) , mice were pre-treated with GAL via i.p injection for 3 days, and subsequently challenged with LPS via i.p injection at the time of 12 h after the injection of GAL on day-3. After LPS stimulation for another 12 hours, the spleens were collected from the animals in each experimental group. (Figure 5 (A) ) . For Western blot analysis, the proteins were extracted from primary peritoneal macrophages (Figure 5 (B) ) and spleen tissues (Figure 5 (C) ) by using ice-cold RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1％NP-40, 1％sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, pH 7.5) . Thirty micrograms of proteins were resolved on 10％SDS-PAGE, and subsequently transferred onto PVDF transfer membrane (EMD Millipore, Billerica, MA, USA) . Following the incubation in 5％non-fatted milk powder in TBST (50 mM Tris-Cl, 150 mMNaCl, and 0.1％Tween-20, pH 7.6) at 4 ℃ for 4 h, the blots were probed with the antibodies specific for COX-2, iNOS and GAPDH (1: 1,000 dilution) at 4 ℃ overnight. The bound antibodies were detected by horseradish peroxidase (HRP) -conjugated secondary antibody (1: 1,000 dilution) at 4 ℃ for 3 h and then visualized by enhanced chemiluminescence (ECL) reaction reagents (GE Healthcare, Uppsala, Sweden) .

Example 5 -Comparison of GAL with L-leucine and gallic acid against LPS-induced expression of COX-2, 5-LOX and iNOS

Primary peritoneal macrophages were freshly isolated male C57BL/6 (6-8 weeks) . For drug treatment, murine macrophage RAW264.7 cells and primary peritoneal macrophages (Figure 6) were treated with LPS (1 μg/mL) in combination with 50 μM of GAL, L-leucine or gallic acid for 24 h, the cellular proteins were prepared in ice-cold RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1％NP-40, 1％sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, pH 7.5) . Thirty micrograms of proteins were resolved on 10％SDS-PAGE, and subsequently transferred onto PVDF transfer membrane (EMD Millipore, Billerica, MA, USA) . Following the incubation in 5％non-fatted milk powder in TBST (50 mM Tris-Cl, 150 mMNaCl, and 0.1％Tween-20, pH 7.6) at 4 ℃ for 4 h, the blots were probed with the antibodies specific for COX-2, iNOS and GAPDH (1: 1,000 dilution) at 4 ℃overnight. The bound antibodies were detected by horseradish peroxidase (HRP) -conjugated secondary antibody (1: 1,000 dilution) at 4 ℃ for 3 h and then visualized by enhanced chemiluminescence (ECL) reaction reagents (GE Healthcare, Uppsala, Sweden) . The protein levels of iNOS, COX-2 and 5-LOX on Western blots were determined by densitometric method.

Example 6 -GAL conjugate inhibited the aberrant production of proinflammatory lipid mediators

Following the treatment with vehicle, LPS, LPS + GAL, the plasma samples were collected. For the isolation of lipids, methanol and water were added to serum to get a mixture of 10％methanol. The samples were kept on ice for 30 min and then centrifuged at 3000 rpm for 5 min. The clear supernatants were transferred into clean 15-ml Falcon tubes, and acidified to pH 3 by gradually adding 1 M hydrogen chloride (HCl) . The lipid mediators were purified on SPE C18 columns (Cayman, Michigan, USA) according to the manufacturer’s instruction. The lipids were eluted with 12 ml of methyl formate. After dried under nitrogen and in the dark (to prevent photo degradation) , the lipid residues were dissolved in 80 μL of ethanol /water (70: 30, v/v) .

Lipids were separated on a Synergi hydro-RP C18 reversed phase column (150 mm ×4.6 mm, 4 m) from Phenomenex (Torrance, CA, USA) . For HPLC separation, an Agilent HLPC 1100 system (Santa Clara, CA, USA) is equipped with a binary pump, a micro vacuum degasser, a column compartment with thermostat control and a temperature controlled HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) . The composition of mobile phase was (A) water with 0.05％ (v/v) formic acid and (B) acetonitrile with 0.05％ (v/v) formic acid. Gradient was set as follows: 0–2.5 min, 40–50％B； 2.5–5.5 min, 50-80％B； 5.5–11.5 min, 80％B； 11.5–12 min, 80-40 ％B, 12–15 min, 40％B. Flow rate was constant at 0.7 ml/min. The column temperature was maintained at 30℃. Sample volume for injection was 20 μL. The elution was monitored by an ABI/Sciex Triple quadrupole mass spectrometer 3200

LC/MS/MS system (Applied Biosystems/MDS Sciex, Concord, ON, Canada) equipped with an ESI-Turbo V source operating in negative ionization mode was used for analysis. The system was controlled by Analyst v1.4.2 data system (Applied Biosystems/MDS Sciex, Concord, ON, Canada) . MS analysis was conducted in electrospray negative mode. The mass spectrometer was operated with an ion spray voltage of -4.5 kV with 325 ℃ drying gas, 40 psi nebulizer gas and 60 psi turbo gas. Multiple-reaction monitoring (MRM) detection was used for the quantitation of all analytes. The collision-induced-dissociation parameters were optimized and MRM pairs were selected by continuous infusion of internal standards at the concentration of 100 nmol/l and at the flow rate of 10 μl/min with Harvard infusion pump (Harvard Apparatus GmbH, March, Germany) . (Figure 7) .

Example 7 -GAL conjugate antagonized the effect of LPS on NO production

Primary peritoneal macrophages were grown in confocal dish. For the detection of intracellular NO production, the cells were pretreated with GAL (0 or 50 μM) for 2 h, and subsequently stimulated with 1 μg/mL LPS for 24 h. At the end of LPS stimulation, the cells were incubated with 5 μM DAF-FM diacetate (4-amino-5-methylamino-2’ , 7’ -difluorofluorescein diacetate) (Life Technologies, NY, USA) at 37 ℃ for 30 min. After the removal of excessive probe, the fluorescence intensity was determined on a Zeiss laser scanning microscope (Carl-Zeiss, Jena, Germany) . The results were expressed as the intracellular NO production percentage compared to that of the cells stimulated with LPS alone (Figure 8) .

Example 8 -GAL conjugate reduced the chemotactic potential of macrophages

For the isolation of primary peritoneal macrophages, male C57BL/6 mice (6-8 weeks old) were treated by i.p injection of 3 ml 3％thioglycollate broth (Fluka, MO, USA) for 3 consecutive days. Peritoneal lavage was carried out using 5 ml of sterile phosphate-buffer saline (PBS) containing 3％FBS. The cells were harvested by centrifugation at 1200 rpm for 8 min, and suspended in DMEM with 10％FBS. After 2 h incubation, the non-adherent cells were removed and the adherent cells were used as primary macrophages ⁽²⁰⁾ .

For the preparation of the conditioned media, primary peritoneal macrophages were treated with 1 mg/ml LPS and 50 μM GAL conjugate, alone or in combination. After 24 h of drug treatment, the cell culture media were recovered as the conditioned media.

Macrophage chemotaxis was measured in a 24-well

plate according to the manufacturer’s instructions ⁽²¹⁾ ⁽²²⁾ . Briefly, the conditioned medium was placed in the lower chamber of the specific Transwells. Freshly isolated primary peritoneal macrophages (1 × 10⁶ cells/ml) were added onto the polycarbonate membrane in the upper chamber. The cells were allowed to migrate through the membrane with a pore size of 5 μm. After 4 h incubation, primary peritoneal macrophages were removed from inside the upper chamber. On the other hand, the cells that migrated through the membrane were fixed by methanol for 20 min, and stained with 0.1％crystal violet for 15min. After the removal of excessive dye, the membranes were carefully placed onto the glass slide, and imaged under a microscopy (Carl Zeiss, Jena, Germany) . (Figure 9A) . After imaging, the membranes were washed with 30％glacial acetic acid for the determination of macrophage migration in a Bio-Rad microplate reader (Hercules, CA, USA) at the wavelength of 570 nm. (Figure 9B) .

Bacterial, fungal and viral infections are common clinical conditions worldwide. Timely resolution of inflammation and sepsis is critical to reduce morbidity and mortality in the infected population. The clinical outcomes of current anti-microbial, anti-inflammatory therapies are frequently disappointing, possibly due to the structure and function of a single chemical compound. This invention provides a method for the preparation of a new class of chemical products such as a GAL conjugate by direct amidation of two chemical compounds gallic acid with L-leucine methyl ester (Figure 2) , and its use in the treatment of inflammation and sepsis. The GAL conjugate significantly reduced LPS-induced death and splenomegaly in mice. In contrast, gallic acid (20 mg/kg body weight) failed to show any protection after 40 h. Mechanistic studies revealed that the GAL conjugate diminished LPS-induced expression of cyclooxygenase-2 (COX-2) , 5-lipoxygenase (5-LOX) and inducible NO synthase (iNOS) in a concentration-dependent manner. This result was confirmed by profiling the intracellular lipids on a LC/MS/MS system and examining the production of toxic NO. In contrast, gallic acid did not show any activity against LPS-induced COX-2 and iNOS expression. Moreover, GAL significantly inhibited LPS-induced chemotactic potential of primary peritoneal macrophages. Collectively, this invention provides a new compound and treatment for the resolution of inflammation and sepsis.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

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Claims

A compound of Formula (I) :

or a pharmaceutically acceptable salt or analog thereof；

wherein X₁ -X₃ are, independently, H or OH； and

wherein R is a methyl group.
A compound of Formula (II) :

or a pharmaceutically acceptable salt or analog thereof.
A pharmaceutical composition comprising the compound of claim 1 or 2 and a carrier.
A method of treatment of septic shock, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
A method of preventing the enlargement of spleen size against LPS-induced endotoxic shock, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
A method of attenuating the chemotaxis of macrophages in response to LPS treatment, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
A method of suppressing COX-2 and iNOS expression in response to LPS treatment, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
A method of controlling aberrant production of proinflammatory lipid mediators in response to LPS treatment, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
The method of claim 8, wherein the lipid mediators are selected from PGE2； PGF2α； 13, 14-dh-PGF2α； 12-HETE； 15-HETE； LTB4； and combinations thereof.
A method of suppressing the aberrant production of toxic NO in response to LPS treatment, comprising:

administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1 or 2.
The method of any of claims 4-10, wherein the subject is a mammal.