Pharmaceutical composition containing thiamine derivative for prevention or treatment of sepsis

Abstract

The present disclosure is a pharmaceutical composition for the prevention or treatment of sepsis containing a thiamine derivative or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition of the present disclosure can be used for the prevention, alleviation, or treatment of sepsis by containing fursultiamine or allithiamine to inhibit the expressions of CD40, CD86, and TNF.

Claims

1. A method for alleviating or treating sepsis, the method comprising: administering to a subject a composition containing a thiamine derivative represented by chemical formula 1 or a pharmaceutically acceptable salt thereof: ##STR00015## wherein R is selected from the group consisting of ##STR00016##

2. The method of claim 1, wherein the thiamine derivative is at least one selected from the group consisting of fursultiamine and allithiamine.

3. The method of claim 2, wherein the pharmaceutically effective amount of fursultiamine contained in the composition is 5-150 mg/kg according to the body weight of the subject to be administered.

4. The method of claim 2, wherein the pharmaceutically effective amount of allithiamine contained in the composition is 5-150 mg/kg according to the body weight of the subject to be administered.

5. The method of claim 1, wherein a causative pathogen of the sepsis is at least one selected from the group consisting of streptococci, staphylococci, Escherichia coli, pneumococcus, Pseudomonas aeruginosa, fungi, and Klebsiella modified pseudomonas aeruginosa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic diagram of the whole experimental schedule according to the present disclosure.

(2) FIG. 2 shows a graph showing the survival rate over time after LPS injection in control mice intraperitoneally injected with lipopolysaccharide (LPS) and test group mice administered LPS along with fursultiamine.

(3) FIG. 3 shows a graph showing the survival rate over time in control mice intraperitoneally injected with LPS, test group mice administered LPS along with allithiamine, and test group mice administered allithiamine 3 hours after LPS injection.

(4) FIG. 4 shows graphs comparing CD40 expression levels in mice administered different concentrations of thiamine derivatives along with LPS.

(5) FIG. 5 shows graphs comparing IL-1 expression levels in mice administered different concentrations of thiamine derivatives along with LPS.

(6) FIG. 6A shows graphs comparing CD40 expression levels in mice administered different concentrations of thiamine derivatives along with LPS.

(7) FIG. 6B shows graphs comparing CD86 expression levels in mice administered different concentrations of thiamine derivatives along with LPS.

(8) FIGS. 7A and 7B show graphs depicting the inhibition of TNF or IL-1 expression by allithiamine and fursultiamine according to an experimental example of the present disclosure.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

(9) Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

Experimental Example 1: Measurement of Humane Endpoint of LPS-Induced Murine Sepsis Model

(10) A dose of 50 mg/kg of lipopolysaccharide (LPS) corresponds to the lethal dose for 50 percent kill that kills half of the population within 24 hours. Mice were subjected to intraperitoneal injection of 50 mg/kg LPS in 1PBS for a vehicle control, and when the mice showed the signs of the moribund state, such as impaired motility, labored breathing, or inability to maintain an upright position, the mice were sacrificed by CO.sub.2 euthanasia, and the point was recorded as a humane endpoint. (The signs of the moribund state: impaired mobility, inability to maintain upright position, prolonged lack of activity and labored breathing)

(11) All animal studies were performed according to protocols approved by Kyungpook National University (permit No. 2019-0003) and under recommendations for the proper use and care of the specific pathogen-free housing facility at Kyungpook University.

Experimental Example 2: Measurement of Survival Rate of Murine Sepsis Model

(12) 2-1. Fursultiamine Treatment on Murine Sepsis Model

(13) Six- to eight-week old C57BL/6J male mice were grouped, each group containing seven mice. The whole experimental schedule is shown FIG. 1. The mice were fed fursultiamine 24 hours and 30 minutes before intraperitoneal injection of 40 mg/kg LPS, and then again administered fursultiamine 6 hours after the intraperitoneal injection, and then the survival rates of the mice were monitored and the results are shown in FIG. 2 and Table 1.

(14) TABLE-US-00001 TABLE 1 Percent survival (%) Days 0 d 1 d 2 d 3 d 4 d 5 d 6 d 7 d Fursultiamine 100 mg/kg 100 100 100 100 100 100 100 100 V.C 100 57.1% 14.2% 0 0 0 0 0 (V.C. designating control mice not fed fursultiamine)

(15) As can be identified in FIG. 2 and Table 1, the mice fed 100 mg/kg fursultiamine were not dead despite intraperitoneal injection of 40 mg/kg LPS, whereas the mice not fed fursultiamine were all dead three days after LPS injection.

(16) 2-2. Allithiamine Treatment on Murine Sepsis Model

(17) Six- to eight-week old C57BL/6J male mice were grouped, each group containing six mice. The mice were intravenously (I.V.) administered 5 mg/kg allithiamine immediately before intraperitoneal injection of 50 mg/kg LPS, and then the survival rates were monitored (allithiamine 5 mg/kg co-treatment). In addition, the mice were intravenously administered 5 mg/kg allithiamine three hours after LPS injection, and then the survival rates were monitored (allithiamine 5 mg/kg 3 h-delayed co-treatment), and the results are shown in FIG. 3 and Table 2.

(18) TABLE-US-00002 TABLE 2 Percent survival (%) Hours 0 h 20 h 24 h 28 h 32 h 36 h 40 h 44 h 48 h 52 h 56 h 60 h Allithiamine 5 mg/kg 100 100 83.3 83.3 83.3 66.7 66.7 66.7 66.7 66.7 66.7 66.7 co-treatment Allithiamine 5 mg/kg 100 100 100 83.3 83.3 83.3 83.3 66.7 66.7 66.7 66.7 66.7 3 h-delayed treatment V.C 100 100 83.3 33.3 16.7 0 0 0 0 0 0 0

(19) As can be identified in FIG. 3 and Table 2, when the mice were administered allithiamine before LPS injection, 66.7% of the mice were live even 60 hours after LPS injection, whereas all the mice of the control (V.C.) injected with LPS alone were dead. Considering that 66.7% of the mice were live even 60 hours after LPS injection even when administered allithiamine 3 hours after LPS injection, even the administration of allithiamine after LPS injection were identified to improve the survival rates.

Experimental Example 3: TNF, CD40, and CD86 Expression Inhibition by Thiamine Derivatives

(20) 3-1. Inhibition of TNF Expression

(21) Bone marrow was extracted from the legs of six- to eight-week old C57BL/6J male mice, and subjected to red blood cell (RBC) lysis, followed by differentiation into dendritic cells in RPMI 1640 medium (10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) along with 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) for six days. The dendritic cells at 110.sup.6 cells/mL were treated with benfotiamine and fursultiamine at different concentrations of 0, 1, 10, and 100 and allithiamine at different concentrations of 0, 1, and 10 M.

(22) Thereafter, the dendritic cells were activated with 100 ng/mL LPS for 18 hours. TNF was assessed in supernatants using the enzyme-linked immunospecific assay (ELISA), and the results are shown in FIG. 4 and Table 3.

(23) TABLE-US-00003 TABLE 3 TNF (pg/mL) Benfotiamine 0 M* 0 M 1 M 10 M 50 M 100 M 100 M* 93.68 5,248.97 4,909.05 4,614.42 4,806.97 4,696.43 84.82 Fursultiamine 0 M* 0 M 1 M 10 M 50 M 100 M 100 M* 93.68 5,248.97 4,954.64 3,600.63 3,003.47 1,628.26 38.37 Allithiamine 0 M* 0 M 1 M 10 M 10 M* 93.68 5,248.97 4,654.84 3,919.75 78.51 (*Dendritic cells are not stimulated by 100 ng/mL LPS)

(24) As can be seen from FIG. 4 and Table 3, benfotiamine could not inhibit the TNF expression increased by LPS, whereas fursultiamine significantly inhibited the TNF expression to levels of about 68.60%, about 57.22%, and about 31.02% at the concentrations of 10 M, 50, and 100 M, respectively, and allithiamine significantly inhibited the TNF expression to levels of about 88.68% and about 74.68% at the concentrations of 1 M, and 10 M, respectively.

(25) 3-2. Inhibition of IL-1 Expression

(26) The dendritic cells were subjected to pro-IL1b priming by the treatment with 500 ng/mL LPS for 3 hours, and then treated with benfotiamine at 0, 10, 50, or 100 and fursultiamine at 0, 10, 50, or 100 M, for 15 minutes. Then, pro-IL1b was converted into IL-1 through pro-IL1b cleavage, followed by 5 mM ATP treatment for inducing inflammasome formation. After 30 minutes, IL-1 was assessed in supernatants, and the results are shown in FIG. 5 and Table 4.

(27) TABLE-US-00004 TABLE 4 IL-1 (pg/mL) Benfotiamine 0 M.sup.ab 0 M.sup.b 0 M 10 M 50 M 100 M 11.0950 37.0371 931.5479 912.2951 914.9398 914.2512 Fursultiamine 0 M.sup.ab 0 M.sup.b 0 M 10 M 50 M 100 M 11.0950 37.0371 931.5479 878.0075 799.4891 592.4987 (.sup.aDendritic cells are not stimulated by 100 ng/mL LPS, .sup.bNo ATP treatment)

(28) As can be seen from FIG. 5 and Table 4, benfotiamine could not inhibit the IL-1 expression increased by LPS, whereas fursultiamine significantly inhibited the IL-1 expression to levels of about 85.8237% and about 63.6037% at the concentrations of 50 M and 100 M, respectively.

(29) 3-3. Inhibition of CD40 and CD86 Expressions

(30) The dendritic cells were measured for CD40 and CD86 in CD11c+ cells by flow cytometry, and the results are shown in FIGS. 6A and 6B and Tables 5 and 6.

(31) TABLE-US-00005 TABLE 5 CD40 (Mean fluorescence intensity; MFI) Benfotiamine 0 M* 0 M 1 M 10 M 100 M 100 M* 251 2,004 2,134 1,975 1,803 248 Fursultiamine 0 M* 0 M 1 M 10 M 100 M 100 M* 251 2,004 1,992 2,026 1,245 253 Allithiamine 0 M* 0 M 1 M 10 M 10 M* 251 2,004 1,998 982 253 (*Dendritic cells are not stimulated by 100 ng/mL LPS.)

(32) As can be seen from FIG. 6A and Table 5, benfotiamine could not inhibit the CD40 expression increased by LPS, whereas fursultiamine significantly inhibited the CD40 expression to a level of about 62% at the concentration of 100 M and allithiamine significantly inhibited the CD40 expression to a level of about 49% at the concentration of 10 M.

(33) TABLE-US-00006 TABLE 6 CD86 (Mean fluorescence intensity; MFI) Benfotiamine 0 M* 0 M 1 M 10 M 50 M 100 M 100 M* 382 2,653 2,734 2,198 2,448 430 382 Fursultiamine 0 M* 0 M 1 M 10 M 50 M 100 M 100 M* 382 2,653 2,684 2,864 1,275 351 382 Allithiamine 0 M* 0 M 1 M 10 M 10 M* 382 2,653 2,184 1,704 304 (*Dendritic cells are not stimulated by 100 ng/mL LPS.)

(34) As can be seen from FIG. 6B and Table 6, benfotiamine could not inhibit the CD86 expression increased by LPS, whereas fursultiamine significantly inhibited the CD86 expression to a level of about 13% at the concentration of 100 M and allithiamine significantly inhibited the CD86 expression to a level of about 64% at the concentration of 10 M.

(35) 3-4. Inhibition of TNF Expression (In Vivo)

(36) The inhibition of TNF expression in vivo was investigated by the administration of 5 mg/kg allithiamine along with 50 mg/kg LPS, and the results are shown in FIG. 7A and Table 7.

(37) TABLE-US-00007 TABLE 7 Adminis- LPS (50 mg/kg) + + tration Allithiamine + + or not (5 mg/kg) TNF (pg/mL) 79.561 82.442 4,484.206 2,084.883

(38) As can be seen from FIG. 7A and Table 7, allithiamine reduced the TNF expression, which had been increased to 4,484.206 pg/mL by LPS, to 2,084.883 pg/mL. Allithiamine significantly inhibited the TNF expression considering a reduction of about 46.494%.

(39) The inhibition of IL-1 expression was investigated by the administration of 100 mg/kg fursultiamine, instead of allithiamine, along with LPS, and the results are shown in FIG. 7B and Table 8.

(40) TABLE-US-00008 TABLE 8 IL-1 (pg/mL) Hours 0 h 1 h 3 h 6 h 12 h 18 h LPS only (50 mg/kg) 5.147 11.290 103.144 338.739 815.804 577.802 Fursultiamine (50 mg/kg) 4.483 6.115 96.715 186.088 495.824 171.451

(41) As can be seen from FIG. 7B and Table 8, the administration of fursultiamine reduced the IL-1 expression to a level of about 54.936%, from 338.739 pg/mL to 186.088 pg/mL, for 6 hours, relative the control without administration, in the mice with IL-1 expression increased by LPS. Especially, considering that fursultiamine, relative to the control, reduced the IL-1 expression to a level of about 60.777%, from 815.804 pg/mL to 495.824 pg/mL, for 12 hours, and to a level of about 29.673%, from 577.802 pg/mL to 171.451 pg/mL, for 18 hours, it was identified that fursultiamine significantly inhibited the IL-1 expression.