CN113304160A - Novel medical application of digoxin in cholestasis - Google Patents
Novel medical application of digoxin in cholestasis Download PDFInfo
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- CN113304160A CN113304160A CN202110568864.3A CN202110568864A CN113304160A CN 113304160 A CN113304160 A CN 113304160A CN 202110568864 A CN202110568864 A CN 202110568864A CN 113304160 A CN113304160 A CN 113304160A
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- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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Abstract
The invention relates to a new medical application of digoxin in cholestasis, in particular to an application of digoxin in preparing a medicament for preventing and/or treating cholestatic diseases, and particularly relates to an application in preparing a medicament for preventing and/or treating intrahepatic cholestasis, intrahepatic cholestasis in pregnancy, hepatic fibrosis caused by cholestasis, liver cirrhosis and other diseases in oral contraceptives and postmenopausal hormone treatment crowds.
Description
Technical Field
The invention relates to an application of Digoxin (Digoxin) in improving cholestasis, belonging to the technical field of medicines.
Background
Intrahepatic cholestasis (IHC) is a common underlying pathology for many liver diseases and can progress to liver fibrosis, cirrhosis, and even liver cancer without effective intervention. Estrogens are a high risk factor for inducing IHC in sensitive pregnant women, oral contraceptives or postmenopausal hormone-treated people. In the case of Intrahepatic Cholestasis (ICP) during pregnancy, ICP is a unique type of estrogen-induced IHC, a severe liver disease with a high prevalence of the middle and late pregnancy (0.2-2%), primarily characterized by skin itching in pregnant women, with elevated bile acids and/or liver enzymes, and ICP subsides spontaneously after delivery, but is prone to relapse in a more severe form (30-70%) upon re-pregnancy or oral contraceptive. ICP has serious influence on the fetus, can cause premature birth (20-60 percent), bradycardia of the fetus, intrauterine asphyxia (less than or equal to 44 percent) and the like, and often troubles clinicians and pregnant women. Ursodeoxycholic acid is a clinically preferred medicament for treating ICP, can improve symptoms and biochemical indexes of pregnant women, but the Lancet journal reports that the ursodeoxycholic acid cannot improve the poor outcome of ICP on perinatal infants in 2019. The pathogenesis of cholestasis is complex, and effective treatment targets and medicines have not yet made breakthrough progress.
Disclosure of Invention
In recent years, changes in immune function during the course of cholestasis have attracted increasing attention. IL-17 is up-regulated in ICP and is closely related to cholestasis inflammation, NKT cells are predominantly gathered in liver, and IL-17 secreting iNKT17 cells are probably involved in cholestasis in 2007, but no clear research is available. The 2019 Nature journal reports that bile acid metabolites directly influence transcriptional activity of a transcription factor ROR gamma t of Th17 cells, and suggests that the bile acid metabolites may directly influence iNKT17 cells. Furthermore, targeting IL-17 and ROR γ t improved cholestasis in mice, suggesting that iNKT17 cells are potential targets for cholestasis. iNKT cells express high levels of CXCR3, CXCR3 and its ligands are the primary receptors that activate, promote lymphocyte differentiation and recruit into the inflamed liver, playing a key role in cholestasis. Therefore, iNKT17 cells are presumed to regulate cholestasis via the CXCR3 signaling pathway.
Digoxin (CAS number: 20830-75-5), a secondary cardiac glycoside compound isolated from Digitalis lanata Ehrh. leaves of Digitalis lanata, was identified as the first specific inhibitor that binds to ROR γ t and inhibits its activity, and is effective in delaying the onset of autoimmune diseases and reducing the severity in mice, but its use for the treatment of cholestasis has not been reported clinically and the molecular mechanism is unclear.
The invention discovers a medicament capable of improving cholestasis and inhibiting the expression of ROR gamma t and CXCR3 of iNKT17 cells through a cell co-culture model and an in vivo animal model.
The invention provides application of digoxin in preparation of a medicament for preventing and/or treating cholestatic diseases.
The chemical structural formula of digoxin is as follows:
further, the digoxin can be used as a medicine for relieving cholestasis diseases, wherein the cholestasis comprises intrahepatic cholestasis in oral contraceptives and postmenopausal hormone treatment populations, intrahepatic cholestasis in pregnancy, hepatic fibrosis caused by cholestasis and liver cirrhosis diseases.
In another aspect, a pharmaceutical composition comprising a therapeutically effective amount of digoxin is used for preparing a medicament for preventing and/or treating cholestatic diseases.
In another aspect, a pharmaceutical preparation comprising a therapeutically effective amount of digoxin and a pharmaceutically acceptable carrier, adjuvant or vehicle, for use in the preparation of a medicament for the prevention and/or treatment of cholestatic diseases.
In the present invention, the above-mentioned compounds and pharmaceutically acceptable salts thereof, and solvates of these compounds (collectively referred to herein as "therapeutic agents") can be administered to mammals either alone or, preferably, in combination with pharmaceutically acceptable carriers or diluents in accordance with standard pharmaceutical practice. The mode of administration can be by various routes, including oral, parenteral, or topical administration. Parenteral administration as used herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.
The invention is proved by experiments that: digoxin can significantly improve estrogen-induced cholestasis in vitro and in vivo models, and inhibit expression of iNKT17 cell ROR gamma t and CXCR 3.
In the experimental process, the dosage of the digoxin is as follows: cells 97.5nM, C57BL/6 mice were injected intraperitoneally with 20. mu.g/mouse.
The results of the experiments in the cells show that: successful induction of cholestasis by estrogen (17 α -ethinylestradiol, EE, a synthetic estrogen derivative widely used in oral contraceptives and hormone replacement therapy) was successful in an in vitro model of co-culture of the mouse liver parenchymal cell line (AML-12) and the iNKT cell line with iNKT17 cell characteristics (dn32.d3), up-regulation of the protein expression of the transcription factor ROR γ t of iNKT17 cells, and promotion of the up-regulation of the protein expression of the pro-inflammatory chemokine receptor CXCR 3. After the digoxin with the concentration of 97.5nM is administered for 15 hours, the elevation of biochemical indexes ALT and AST can be obviously improved, and ROR gamma t and CXCR3 protein expression of iNKT17 cells can be inhibited.
Experimental results in mice show that: in a model of using EE to induce intrahepatic cholestasis on a C57BL/6J mouse, digoxin (20 mug/mouse) can obviously inhibit the rise of ALP and TBA which are biochemical indicators of cholestasis, and inhibit the mRNA expression of Ror gamma t and Cxcl9/10-Cxcr3 which are up-regulated by EE.
The invention has the advantages that:
(1) the invention discovers for the first time that digoxin has the effect of improving intrahepatic cholestasis induced by estrogen, and has no obvious toxicity under the effective dose.
(2) The invention discovers for the first time that digoxin inhibits the expression of iNKT17 cell transcription factor ROR gamma t and chemokine receptor CXCR3, and plays a role in resisting inflammation and cholestasis.
Drawings
FIG. 1 is a CCK-8 method for examining the Effect of Estrogen (EE) or Digoxin (DGX) alone on the proliferation viability of DN32.D3 cells or AML-12 cells;
FIG. 2 is a graph showing the effect of Digoxin (DGX) on the biochemical markers ALT and AST in the estrogen (EE) -induced co-culture system of AML-12 cells and DN32.D3 cells. Wherein*P<0.05，**P<0.01，***P<Control group at 0.001vs. control,#P<0.05，##P<ee single-given group (Graphpad 7.0, Tukey's test) 0.01 vs;
FIG. 3 is a graph showing the effect of Digoxin (DGX) on protein expression of DN32.D3 cell transcription factor ROR γ t in estrogen (EE) -induced co-culture systems of AML-12 cells and DN32.D3 cells. Wherein***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) 0.001 vs;
fig. 4 is a graph examining the effect of Digoxin (DGX) on estrogen (EE) -induced protein expression of CXCR3 on the surface of dn32.d3 cells in AML-12 cells and dn32.d3 cells co-culture system. Wherein*P<0.05，**P<0.01，***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) 0.001 vs;
FIG. 5 is a graph showing the effect of Digoxin (DGX) on the biochemical indicators ALP, TBA and AST of estrogen (EE) -induced C57BL/6J mouse serum. Wherein**P<0.01，***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) 0.001 vs;
FIG. 6 is a graph showing the effect of Digoxin (DGX) on estrogen (EE) induction of mRNA expression of Ror γ t and Cxcl9/10-Cxcr3 in the liver of C57BL/6J mice. Wherein*P<0.05，**P<0.01，***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) at 0.001vs.
Detailed Description
To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.
Drugs and reagents: the digoxin (CAS: 208030-75-5) used in this experiment was purchased from American APEXBIO, the estrogen (CAS: 57-63-6) was purchased from Chiese chemical industry development Limited, and the other reagents were commercially available analytical grade reagents.
Cell proliferation activity assay: DN32.D3 cells or AML-12 cells were treated with either estrogen (EE, 1.56-1000. mu.M) or digoxin (DGX, 48.7-390nM) alone for 24 hours and the CCK-8 method determined the effect on cell proliferation viability.
Determination of biochemical indices in cell supernatants or mouse sera: treatment of AML-12 cells with digoxin (DGX, 97.5nM) and Estrogen (EE, 6.25. mu.M) and DN32.D3 cells Co-culture systems for 15, 19 or 24 hours, or C57BL/6J mice were given digoxin (intraperitoneal injection, 20. mu.g/mouse) and Estrogen (subcutaneous injection, 10mg/kg) for 3, 5 or 7 days, and cell supernatants or sera were assayed for ALP, ALT, AST viability and TBA concentration according to commercial kit protocol.
Expression assay of the transcription factor ROR γ t: co-culture systems of AML-12 cells and dn32.d3 cells were treated with digoxin (DGX, 97.5nM) and estrogen (EE, 6.25 μ M) for 15, 19, or 24 hours, or C57BL/6J mice were given digoxin (i.p., 20 μ g/mouse) and estrogen (s.c., 10mg/kg) for 3, 5, or 7 days, protein expression of ROR γ t in dn32.d3 cells in co-culture systems was detected by flow cytometry, and mRNA expression of ROR γ t in mouse liver was detected by RT-PCR.
Expression assay of chemokine CXCL9/10 and its receptor CXCR 3: treating AML-12 cells and DN32.D3 cells by using digoxin (DGX, 97.5nM) and estrogen (EE, 6.25 mu M) for 15, 19 or 24 hours, or feeding digoxin (intraperitoneal injection, 20 mu g/mouse) and estrogen (subcutaneous injection, 10mg/kg) to C57BL/6J mice for 3, 5 or 7 days, detecting protein expression of CXCR3 on the surface of the DN32.D3 cells in the coculture system by using a flow cytometry method, and detecting mRNA expression of Cxcl9/10-Cxcr3 in liver of the C57BL/6J mice by using an RT-PCR method.
Example 1
Cell proliferation activity assay: DN32.D3 cells or AML12 cells were treated with estrogen (EE, 1.56-1000. mu.M) or digoxin (DGX, 48.7-390nM) for 24 hours and cell proliferation viability was determined by the CCK-8 method. As shown in FIG. 1, it can be seen that estrogen 6.25. mu.M and digoxin 97.5nM were not significantly toxic to DN32.D3 cells or AML-12 cells.
Example 2
And (3) determination of biochemical indexes: co-culture of AML-12 cells and DN32.D3 cells at 15, 19 or 24 hours after treatment with digoxin (DGX, 97.5nM) and estrogen (EE, 6.25. mu.M) according to commercial reagentsCassette instructions biochemical markers ALT and AST were measured in the cell supernatants. The results of the tests are shown in figure 2,*P<0.05，**P<0.01，***P<control group at 0.001vs. control,#P<0.05，##P<EE Single-given group (Graphpad 7.0, Tukey's test) at 0.01vs. It can be seen that digoxin inhibits estrogen (6.25 μ M) induced elevation of the cellular supernatant biochemical markers ALT and AST by inhibiting ROR γ t at doses as low as 97.5nM, 15 hours after administration.
Example 3
Protein expression assay for the transcription factor ROR γ t: co-culture systems of AML-12 cells and DN32.D3 cells were treated with digoxin (DGX, 97.5nM) and estrogen (EE, 6.25. mu.M) for 15, 19 or 24 hours, and protein expression of the DN32.D3 cell transcription factor ROR gamma t in the co-culture systems was detected by flow cytometry. The results of the test are shown in FIG. 3, wherein***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) at 0.001vs. The results show that 97.5nM digoxin can significantly inhibit estrogen-induced up-regulated ROR gamma t protein expression and play a role in inhibiting inflammation.
Example 4
Protein expression assay for chemokine receptor CXCR 3: co-culture systems of AML-12 cells and DN32.D3 cells were treated with digoxin (DGX, 97.5nM) and estrogen (EE, 6.25. mu.M) for 15, 19 or 24 hours, and protein expression of CXCR3 on the surface of DN32.D3 cells in the co-culture systems was examined by flow cytometry. The test results are shown in FIG. 4, in which*P<0.05，**P<0.01，***P<Control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) at 0.001vs. As can be seen from the results, 97.5nM digoxin can significantly inhibit estrogen-induced up-regulated protein expression of CXCR3 by inhibiting ROR γ t, CXCR3 may be downstream of ROR γ t, digoxin further inhibits the recruitment and activation of iNKT cells.
Example 5
And (3) determination of biochemical indexes: c57BL/6J mice were given digoxin (20. mu.g/mouse) and estrogen (10mg/kg) for 3, 5, or 7 days, and serum was assayed for ALP and AST viability according to the instructions of the commercial kit protocolAnd TBA content. The results of the test are shown in figure 5,**P<0.01，***P<control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) at 0.001vs. As can be seen from the results, 20. mu.g of digoxin significantly reduced the estrogen-induced biochemical indicators ALP and TBA, and AST was not significantly different from the control group. Thus, digoxin can reduce cholestatic liver injury in mice by inhibiting ROR γ t.
Example 6
mRNA expression assay for Ror γ t and Cxcl9/10-Cxcr 3: c57BL/6J mice were given digoxin (20. mu.g/mouse) and estrogen (10mg/kg) for 3, 5 or 7 days, and mRNA expression of mouse liver Ror. gamma.t and Cxcl9/10-Cxcr3 were examined by RT-PCR. The results of the tests are shown in figure 6,*P<0.05，**P<0.01，***P<control group at 0.001vs. control,###P<ee single-given group (Graphpad 7.0, Tukey's test) at 0.001vs. As can be seen from the results, 20. mu.g digoxin significantly reduced estrogen-induced mRNA expression of Ror γ t and Cxcl9/10-Cxcr 3. Consistent with in vitro experimental results, CXCR3 may be downstream of ROR γ t, digoxin further inhibits the recruitment and activation of iNKT cells, possibly being the reason it reduces cholestatic liver injury.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (9)
1. Use of digoxin in the manufacture of a medicament for the prevention and/or treatment of cholestatic diseases.
2. The use according to claim 1, wherein the digoxin has the following chemical formula:
3. the use of claim 1 or 2, wherein the cholestatic disorder comprises oral contraceptives, intrahepatic cholestasis in post-menopausal hormone-treated people, intrahepatic cholestasis in pregnancy, liver fibrosis due to cholestasis, liver cirrhosis disease.
4. A pharmaceutical composition comprising a therapeutically effective amount of digoxin.
5. Use of the pharmaceutical composition of claim 4 for the preparation of a medicament for the prevention and/or treatment of cholestatic diseases.
6. The use of claim 5, wherein said cholestatic disorder comprises oral contraceptives, intrahepatic cholestasis in postmenopausal hormone-treated populations, intrahepatic cholestasis in pregnancy, liver fibrosis due to cholestasis, and liver cirrhosis disorders.
7. A pharmaceutical formulation comprising a therapeutically effective amount of digoxin and a pharmaceutically acceptable carrier, adjuvant or vehicle.
8. Use of a pharmaceutical formulation according to claim 7 for the preparation of a medicament for the prevention and/or treatment of cholestatic diseases.
9. The use of claim 8, wherein said cholestatic disorder comprises oral contraceptives, intrahepatic cholestasis in postmenopausal hormone-treated populations, intrahepatic cholestasis in pregnancy, liver fibrosis due to cholestasis, and liver cirrhosis disorders.
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Non-Patent Citations (3)
| Title |
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| CHUANWANG等: "The effect of 17α-ethynylestradiol induced intrahepatic cholestasis of pregnancy on placental P-glycoprotein in mice: Implications in the individualized transplacental digoxin treatment for fetal heart failure", 《PLACENTA》 * |
| K. E. ANDERSEN等: "The Effect on Serum Enzymes of Intramuscular Injections of Digoxin, Bumetanide, Pentazocine and Isotonic Sodium Chloride", 《ACTA MED. SCAND.》 * |
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