USE OF KAURANE COMPOUNDS IN PREPARATION OF DRUG FOR PREVENTION AND TREATMENT OF SEPSIS AND MULTIPLE ORGAN DAMAGE

Abstract

The invention discloses the medicinal use of kaurene compound in the prevention and treatment of sepsis, systemic inflammatory response syndrome (SIRS) and multiple organ failure caused by sepsis, including acute pulmonary failure, acute heart failure and renal failure.

Claims

1. The use of Kaurene compounds or its pharmaceutically acceptable salts thereof in a pharmaceutical preparation for preventing and treating sepsis or systemic inflammatory response syndrome (SIRS) and the multi-organ damage or failure caused by it.

2. The use of claim 1, wherein the disease of sepsis was induced by pathogens includes bacteria, viruses or fungi, and pathogen-associated molecular patterns (PAMPs) related to the abovementioned pathogens, include LPS, lipoproteins, glycoprotein, lipopeptides, nucleic acids, etc.

3. The use of claim 1, wherein the systemic inflammatory response syndrome (SIRS) is caused by non-pathogenic infections, including trauma, burns, myocardial infarction and heart failure, cerebral infarction, and inflammatory bowel disease, etc.

4. The use of claim 1, wherein the sepsis or systemic inflammatory response syndrome (SIRS) is characterized by the overproduction of systemic cytokines or cytokine storm.

5. The use of claim 1, wherein the multi-organ damage or failure is lung damage or failure, heart damage or failure, circulatory failure, liver damage or failure, kidney damage or failure, spleen damage or failure, or the combination of one or several of the above situation.

6. The use of claim 5, wherein the lung damage or failure is characterized by acute pulmonary depression, acute respiratory distress syndrome, or acute pulmonary failure.

7. The use of claim 5, wherein the heart damage or failure is characterized by cardiac dysfunction, heart failure or arrhythmia caused by sepsis or systemic inflammatory response syndrome (SIRS).

8. The use of claim 1, wherein the multi-organ damage or failure is characterized by organ fibrosis and remodeling caused by sepsis.

9. The use of claim 1, wherein the mechanism of action of the prevention and treatment involves inhibition of cytokine production or inhibition of cytokine storm. Cytokines include IFN-γ, TNF , IL-1β, IL-1, IL-6, IL-12, IL-13, IL-10, IL-23, IL-17 and IL-6, etc.

10. The use of claim 1, wherein the mechanism of action involves the inhibition and regulation of the activation and proliferation of macrophages.

11. The use of claim 1, wherein the mechanism of action involves the inhibition of activation and proliferation of inflammatory cells including leukocytes, neutrophils, monocytes and lymphocytes.

12. The use of claim 1, wherein the said compounds are represented by structural formula (I). Compounds of structural formula (I) may have one or more asymmetric centers, and may exist as different stereoisomers. ##STR00004## wherein ii. R1: Hydrogen, hydroxyl, or alkoxy. iii. R2: Carboxyl, carboxylate, acyl halides, aldehyde, hydroxymethyl, and ester, acrylamide, acyl, or ether groups that can form carboxyl. which can generate carboxyl group. iv. R3, R4, R5, R6, R8: Oxygen, hydroxyl, hydroxymethyl, and ester or alkoxymethyl  groups that hydrolyze to hydroxymethyl. v. R7: Methyl, hydroxyl, and ester or alkoxymethyl groups that hydrolyze to hydroxymethyl. that can be hydrolyzed to hydroxymethyl. vi. R9: Methylene or oxygen.

13. The compound described in claim 12 is characterized by the fact that the structural formula (I) compound described therein is the compound shown in structural formula (II). The compound may have multiple asymmetric centers and have different stereoisomers or diastereoisomers. The absolute configurations of positions 8 and 13 are either (8R, 13S) or (8S, 13R). ##STR00005## wherein vii. R2: Carboxylate, carboxylate, aldehyde group, hydroxymethyl, methyl ester, acyl methyl, acyl halide. viii. R7: Methyl, hydroxymethyl or methyl ether. ix. R9: Methylene or oxygen.

14. The compound described in claim 12 is characterized by the fact that the structural formula (I) compound described therein is the compound shown in structural formula (A). ##STR00006##

15. The compound described in claim 12 is characterized by the fact that the structural formula (I) compound described therein is the compound shown in structural formula (B). ##STR00007##

16. The use of claim 1, wherein said medicament are tablets, capsules, granules, suppositories, ointments, patches, injections, buccal tablets, chewables, and controlled release agents via oral, parenteral or implanted channels.

17. The use of claim 1, wherein said medicament is characterized by could be inhalant nebulizer, metered dose inhalant or dry powder inhalant via pulmonary or nasal delivery.

18. The use of claim 1, wherein said medicament is characterized by delivery to the patient in need through muscle, vein, abdominal cavity, interventional catheter and ventilator, using a standard medicinal liquid injection or infusion or other suitable dosage form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is the survival curve of each group of mice after intraperitoneal injection of different doses of LPS in Example 1 of the present invention.

[0031] FIG. 2 is the effect of intraperitoneal injection of different doses of compound A on the mortality of LPS-induced septic Balb/C mice in Example 2 of the present invention.

[0032] FIG. 3 is the effect of Compound A on cardiac function of LPS-induced mice in Example 4 of the present invention.

[0033] FIG. 4 is the effect of compound A on plasma CK and LDH of LPS-induced septic mice in Example 5 of the present invention.

[0034] FIG. 5 is the effect of compound A on pulmonary function after compound A administration by whole body plethysmography recording (Penh, EF50, TV) in Example 6 of the present invention.

[0035] FIG. 6 is the effect of compound A on LPS-induced liver dysfunction in Example 7 of the present invention

[0036] FIG. 7 is the effect of compound A on LPS-induced renal dysfunction in Example 8 of the present invention

[0037] FIG. 8 is the effect of compound A on M1/M2 primary peritoneal macrophages of LPS induced septic mice in Example 9 of the present invention.

EXAMPLES

[0038] The methods and examples of the present invention are provided in detail as follows.

[0039] Detail Methods

[0040] To further illustrate the technologies used to achieve the objects of the present invention, detailed methods, techniques, procedures and special features regarding in determining and identifying the pharmaceutical and therapeutic uses of the kaurene compounds in the present invention are described below. Examples provide experimental methods and results which are utilized for supporting the invention, and for validating the animal models used in the present invention. Proper control and statistical analysis are used in all the experiments in this invention. The following examples are provided to illustrate, not limit, the invention. The methods and techniques utilized to screen and to determine the therapeutic use of some kaurene compounds of formula (I). The therapeutic use of other compounds of formula (I) can also be determined in the same way.

[0041] The examples provided in the present invention are used to support the experimental methods and results of the present invention and to validate the animal models used in the present invention. Appropriate controls and statistical tests were used in all experiments of the present invention. The following examples are provided to illustrate, not limit, the invention. The methods and techniques utilized to screen and to determine the therapeutic use of some kaurene compounds of formula (I). The therapeutic use of other compounds of formula (I) can also be determined in the same way.

Experimental Materials

[0042] Experimental animal: Adult male Balb/c mice, body weight 20 g±5 g, 6-8 weeks old. Mice are housed in acrylic cages with food and water ad libitum under an environmentally controlled condition.
Chemical reagent: Compound A (ent-17-norkaurane-16-oxo-18-oic acid, molecular formula, C20H40O3, molecular weight: 318.5) is obtained from stevioside through acid hydrolysis and crystallization purification. The sodium salt of compound A can be obtained by adding NaOH or other sodium-containing bases; the purity of the sodium salt of compound A measured by high performance liquid chromatography is greater than 99%.
Administration of test compound: intravenous or intraperitoneal injection or oral. Dosage; Compound A (or its sodium salt), 5 mg/kg to 60 mg/kg.

Statistical Analysis

[0043] Differences between groups were compared by ANOVA (one-way ANOVA) and Fisher's test. P values for all tests were two-tailed, and P<0.05 was considered statistically significant.

EXAMPLE 1

[0044] In this case, a mouse model of sepsis with intraperitoneal injection of LPS was established.

[0045] Forty-eight Balb/c mice (6-8 weeks, ♂) were randomly divided into 6 groups: the Control group, 10 mg/kg LPS group, 15 mg/kg LPS group, 20 mg/kg LPS group, 25 mg/kg LPS group. and 30 mg/kg LPS group, 8 mice per group. Mice were given intraperitoneal injection of LPS to establish a mouse model of sepsis. Different doses of LPS were given respectively, and the Control group was given the same volume of 0.9% saline to observe the changes in the survival rate of mice.

[0046] In this study, five different doses of 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg and 30 mg/kg were selected. In FIG. 1, 10 mg/kg is the median lethal dose, and 20-30 mg/kg is the lethal dose. To investigate the effect of Compound A on the survival rate of septic mice, we chose 20 mg/kg LPS to induce acute sepsis model. In the follow-up experiments, we chose intraperitoneal injection of 20 mg/kg LPS for 6 h as the lethal dose and modeling time of the mouse sepsis model.

EXAMPLE 2

[0047] This example mainly observes the effect of intraperitoneal injection of different doses of Compound A on the mortality of LPS-septic Balb/C mice.

[0048] To observe the effect of intraperitoneal injection of different doses of compound A on the mortality of LPS-induced sepsis mice.

[0049] The mice were randomly divided into 6 groups: [0050] (1) Control group: intraperitoneal injection of physiological saline (solvent) (0.1 ml/10 g) once a day for three consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection of physiological saline (0.2 ml/10 g); [0051] (2) LPS group: intraperitoneal injection of saline (0.1 ml/10 g), 1 time/d for 3 consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection of LPS (20 mg/kg, 0.1 ml/10 g). [0052] (3) Compound A (5 mg/kg)+LPS group: intraperitoneal injection of compound A (5 mg/kg, 0.1 ml/10 g), 1 time/d for 3 consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection LPS (20 mg/kg, 0.1 ml/10 g). [0053] (4) Compound A (10 mg/kg)+LPS group: intraperitoneal injection of compound A (10 mg/kg, 0.1 ml/10 g), 1 time/d for 3 consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection LPS (20 mg/kg, 0.1 ml/10 g). [0054] (5) Compound A (20 mg/kg)+LPS group: intraperitoneal injection of compound A (20 mg/kg, 0.1 ml/10 g), 1 time/d for 3 consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection LPS (20 mg/kg, 0.1 ml/10 g). [0055] (6) Compound A (60 mg/kg)+LPS group: intraperitoneal injection of compound A (60 mg/kg, 0.1 ml/10 g), 1 time/d for 3 consecutive days, 1 hour after intraperitoneal injection on the third day, intraperitoneal injection LPS (20 mg/kg, 0.1 ml/10 g).

[0056] After intraperitoneal injection of LPS, the survival of mice in each group was observed every 12 h for six consecutive days.

[0057] As shown in FIG. 2, after intraperitoneal injection of 20 mg/kg LPS, all mice died within 24 hours, and 6 hours after intraperitoneal injection of LPS, all mice in the 5 mg/kg compound A group died, and there was no significant difference with the LPS group (p>0.05), while 10 mg/kg and 20 mg/kg of Compound A improved the survival rate of mice by 52% and 67%, respectively, and there was a significant difference between the two groups compared with the Control group (P<0.01), no death observed in the Control group at each time period. Based on the previously reports, the dose of 5 mg/kg for adults is an acceptable and safe dose, and the maximum blood concentration reaches 20 μM, which is equivalent to 45 mg/kg in mice. Accordingly, 10 mg/kg STV-Na was employed for further studies.

EXAMPLE 3

[0058] This example mainly observes the general behavior of experimental mice in each group.

[0059] The fur color, consciousness, physical activity, diet, etc. of the mice were observed, and the heart, liver, spleen, lung and kidney of the mice were weighed 6 hours after intraperitoneal injection of LPS. In the Control group, there was no obvious abnormality in behavior, spirit, eating and fur color; After intraperitoneal injection of LPS, the mice in the other five groups gradually showed fatigue, lethargy, decreased appetite, fear of cold and unresponsiveness.

EXAMPLE 4

[0060] This example mainly illustrates the effect of compound A in improving cardiac dysfunction in septic mice.

[0061] Six hours after intraperitoneal injection of LPS, mice in each group were weighed and anesthetized, and the cardiac function of the mice was detected by Vevo2100 imaging system. The mice were put into the recumbent position and fixed on a constant temperature heating plate under heart rate of 400-500 times per minute. The limbs were fixed on the four metal poles with tape. The ultrasound probe was placed in the left thoracic region of the mouse, and the short axis of the parasternal left ventricle was obtained by 2D ultrasound. The left ventricular motion was recorded at the papillary muscle level by M-ultrasound, and heart rate (HR), left ventricular systolic diameter (LVID: FS), left ventricular diastolic diameter (LVID: d), left ventricular systolic diameter (LVID: FS), left ventricular diastolic diameter (LVID: Fd), and left ventricular diastolic forearm thickness (LVPWF: FdF) were measured. Using Vevo2100 small animal ultrasound system software to process and analyze the acquired images. Determination of mouse plasma myocardial injury indexes (LDH and CK)

[0062] Ejection Fractions (EF), refers to the percentage of cardiac stroke volume in ventricular end-diastolic volume, which is an indicator of myocardial contractility. As shown in FIG. 3A, the echocardiography results showed that the ejection fraction of the LPS-induced septic mice was significantly lower than that of the Control group (p<0.01), indicating that intraperitoneal injection of LPS resulted in decreased cardiac contractility and decreased cardiac function. However, after compound A administration, the EF was significantly improved (p<0.05) compared with the LPS group, while the effect of dexamethasone was not significant. The left ventricular short-axis shortening rate (fractional shortening, FS) refers to the ratio of the inner diameter of the ventricle at the end systole to the inner diameter of the ventricular end diastole, and also reflects the systolic function of the heart (FS=(left ventricular end-diastolic diameter (LVDd)−Left ventricular end-systolic diameter (LVDs))/left ventricular end-diastolic diameter×100%). As shown in FIG. 3B, the FS of the LPS-induced septic mice was significantly lower than that of the Control group (p<0.01), while the FS of the compound A group was significantly higher than that of the LPS group (p<0.05), indicating that the cardiac systolic function of LPS-induced septic mice decreased, whereas that of the Dex group was not significant.

[0063] In conclusion, intraperitoneal injection of LPS can decrease the left ventricular ejection fraction and fractional shortening, and induce a certain degree of cardiac dysfunction. After treated with compound A, the cardiac function was improved, which could alleviate the cardiac dysfunction caused by LPS.

EXAMPLE 5

[0064] This example mainly illustrates the effect of compound A in improving cardiac dysfunction in sepsis.

[0065] Serum lactate dehydrogenase (LDH) and creatine kinase (CK) were determined according to the istructions provided by Nanjing Jiancheng Biotechnology Co., Ltd.

[0066] Lactate dehydrogenase (LDH) activity in plasma (FIG. 4) LDH is an important glycolytic enzyme in human energy metabolism. When myocardial injury such as viral and rheumatic myocardial inflammation occurs, the level of plasma LDH increases, so it can be used as an effective indicator for the diagnosis of myocardial injury. As shown in FIG. 4A, the plasma LDH level of the LPS-induced septic mice was significantly higher than that of the Control group (P<0.01). After treated with compound A and Dex, the LDH levels in the plasma showed a downward trend, which was statistically significant when compared with the LPS group (p<0.01). Taken together, these data indicate that Compound A and Dex could ameliorate the myocardial injury of mice with LPS-induced sepsis.

[0067] Creatine kinase (CK) activity in plasma (FIG. 4B): In general, myocardial tissue CK is present in cardiomyocytes, and when blood levels of CK are elevated, it usually indicates or is developing myocardial damage. As shown in FIG. 4B, the plasma CK level in the LPS group was significantly higher than that in the control group (P<0.01). After compound A and Dex administration, the level of serum CK was significantly decreased with statistical significance (p<0.01) when compared with the LPS group, indicating that Compound A and Dex can alleviate LPS-induced myocardial injury in mice and achieve therapeutic effect.

EXAMPLE 6

[0068] This example illustrates the effect of compound A in improving lung function in septic mice.

[0069] The lung function of each group of mice was measured at 6 h. Mice were placed in a closed chamber of a whole body plethysmograph (BUXCO, USA), and the plethysmographic chamber was connected to a sensor. As the animal breathes, the rise and fall of the chest changes the volume inside the chamber, and pressure transducers and amplifiers convert this volume change into an electrical signal, After processing, the respiration curve is displayed on screen, and the graphics are processed by relevant software to calculate the tidal volume (TV), the expiratory flow at 50% tidal volume (50% tidal volume expiratory flow, EF50), airway responsiveness (Penh), etc.

[0070] In this experiment, we used Buxco's pulmonary function testing system to measure and record the 50% tidal expiratory volume, tidal volume, and changes in airway responsiveness in each group of mice by whole body plethysmography to assess lung function. This method offers a real-time lung function of mice under non-invasive conditions. Penh is a pulmonary function parameter related to airway resistance and intrapleural pressure, and Penh is used as an index to evaluate airway response. It can be seen from FIG. 5 that 6 hours after LPS injection, the Penh of the LPS group was significantly higher than that of the Control group (p<0.01), and the EF50 and TV values were significantly lower than those of the Control group (p<0.01), indicating that the LPS-induced lung injury mice exhibited obvious airway hyperresponsiveness. However, EF50 and TV were significantly higher than those of the LPS group (p<0.01), indicating that compound A can ameliorate LPS-induced lung injury.

EXAMPLE 7

[0071] This example illustrates the role of compound A in sepsis-induced liver damage.

[0072] The prepared plasma of each group was taken to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD) in plasma by a microplate reader according to the manufacture's instruction.

[0073] In order to observe the effect of compound A on the liver function of LPS-induced systemic inflammatory response in mice, we detected the changes of plasma ALT, ALT and SOD in mice, respectively. As shown in FIG. 6, the changes of ALT, ALT and SOD in plasma of mice were significantly decreased after compound A and Dex treatment when compared with the Control group (P<0.01), indicating that compound A can ameliorate liver function disorder of LPSinduced septic mice.

EXAMPLE 8

[0074] This example illustrates the effect of Compound A in improving renal impairment in sepsis.

[0075] The prepared plasma of each group was taken to determine the levels of urea nitrogen (BUN) and creatinine (Crea) in plasma by a microplate reader following the manufacture's instruction.

[0076] In order to observe the effect of compound A on the renal function of LPS-induced systemic inflammatory response in mice, we detected the changes of plasma BUN and Crea in mice, respectively. As shown in FIG. 7, after Compound A and Dex administration, the changes of BUN in the plasma of mice were significantly decreased when compared with the Control group (P<0.01). However, Crea level in plasma did not change significantly after Dex treatment, indicating that compound A can ameliorate renal function disorder of LPS-induced septic mice.

EXAMPLE 9

[0077] This example illustrates the effect of compound A on macrophage polarization. [0078] (1) Grouping of experimental animals: 6-8 week old Balb/c male mice were randomly divided into 4 groups after one week of adaptive feeding, 10 mice in each group, namely the Control group, the LPS group, the compound A group and the dexamethasone group. The LPS group, compound A group and dexamethasone group were given intraperitoneally injection of a 20 mg/kg of LPS. Samples are collected six hours after LPS injection. [0079] (2) Extraction of primary peritoneal macrophages: Six hours after LPS injection, mice were sacrificed and the primary peritoneal macrophages were extracted. [0080] (3) Flow cytometry for M1 and M2 macrophages: After blocked with MACS for 20 min on ice, cells are centrifuged at 1000 rpm, 4° C. for 5 min, and then discard the supernatant, add 0.2 μl of PE-anti-mouse F4/80 antibody and BV421-anti-CD11c antibody to the cell suspension, incubate on ice for 30 min in the dark, wash once with PBS, fix with 50 μl fixative solution on ice for 10 min, 50 μl of 1× permeabilization solution, 1000 rpm, centrifuge at 4° C. for 5 min, add 100 μl of 1× permeabilization solution to rupture the membrane, centrifuging, add 0.2 μl of FITC-anti-mouse CD206 antibody and incubate on ice for 30 min in the dark, Add 100 μl of permeabilization fluid and centrifuging, add 1 ml PBS to wash once, centrifuging, collecting sediment, resuspend in 200 μl PBS, FACSCelesta flow cytometry assay, FlowJo 7.6.1 software analyzes M1 (F4/80+CD11c+ CD206−) and M2 (F4/80+CD11c− CD206+).

[0081] In vivo, mouse peritoneal macrophages were used as the research object. Three days after the preventive administration of compound A, 20 mg/kg LPS was injected intraperitoneally for stimulation, and the mice were sacrificed 6 hours later to collect primary peritoneal macrophages for flow cytometry. As shown in FIG. 8, it was found that the M1 and M2 peritoneal macrophages were significantly reduced in the compound A and dexamethasone (the positive drug) group when compared with the LPS group. These indicate that compound A can regulate the M1/M2 macrophages ratio in LPS-induced septic mice thus maintaining the balance of M1/M2 macrophages in vivo.

[0082] The above-mentioned examples are only preferred embodiments of the present invention, but do not limit the invention in any way. It is obvious that all the modifications or rearrangements to these examples may be made by any technical persons skilled in art according to what have been disclosed by the invention. These modifications and rearrangements shall be included within the same scope of the invention.