CN116173042A - Application of ursodeoxycholic acid in preparing medicine for preventing and treating porcine viral diarrhea - Google Patents
Application of ursodeoxycholic acid in preparing medicine for preventing and treating porcine viral diarrhea Download PDFInfo
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- ursodeoxycholic acid
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- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 title claims abstract description 56
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 title claims abstract description 56
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- GHCZAUBVMUEKKP-NHIHLBCISA-N 2-[[(4R)-4-[(3R,5S,7S,10S,13R,17R)-3,7-Dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)C1C2C2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)CC1 GHCZAUBVMUEKKP-NHIHLBCISA-N 0.000 claims description 4
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses application of ursodeoxycholic acid in preparing a medicine for preventing and treating porcine viral diarrhea, which belongs to the technical field of biological medicines, and has remarkable effect of resisting porcine epidemic diarrhea virus and remarkable inhibition effect on proliferation of porcine epidemic diarrhea virus particles; ursodeoxycholic acid can reduce the expression of intracellular pro-inflammatory factor interleukin-1 beta (IL-1 beta) and up-regulate the expression of anti-inflammatory factor interleukin-10 (IL-10), thereby exhibiting remarkable anti-inflammatory activity. In conclusion, ursodeoxycholic acid can inhibit the proliferation of virus particles and regulate the expression level of anti-inflammatory factors by inhibiting the replication of porcine epidemic diarrhea virus in host cells, thereby having the prevention and treatment effect on porcine epidemic diarrhea. Ursodeoxycholic acid has obvious effect, low cost, convenient storage, safety, reliability and no toxic or side effect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of ursodeoxycholic acid in preparation of a medicine for preventing and treating porcine viral diarrhea.
Background
Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute intestinal infectious disease of a highly-contagious pig caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) infection, pigs at all day-old stages are susceptible, clinical symptoms are mainly watery diarrhea, vomiting and serious dehydration, and the mortality rate of the porcine epidemic diarrhea is as high as 80-100 percent for suckling pigs. PEDV is a classical Coronavirus (coronavir) and is a enveloped, non-segmented, single-stranded positive-strand RNA virus. Because the genome and replication characteristics of the virus cause easy mutation of epidemic strains, but the current vaccine strains are not matched with the epidemic strains, the current vaccine strains cannot have good cross immunity protection effect, and a large number of outbreaks of porcine epidemic diarrhea are caused, so that the virus becomes an industrial bottleneck for restricting the development of pig industry. Therefore, new PEDV prevention and control technologies based on non-vaccine strategies need to be established.
The antiviral drug can well replace and supplement the problem of poor vaccine protection effect in clinic. In recent years, due to rapid development of middle veterinarian and pharmaceutical engineering technologies, a large number of natural and synthetic medicines are reported successively, but most medicines have single target points on virus antigens or unclear active ingredients of the medicines, and coronaviruses are extremely easy to generate variation, so that clinical application of the medicines is severely restricted; in addition, while a large number of antiviral drugs are effective in inhibiting infection and replication of the virus, they have serious side effects. Thus, there is a need to develop new, safe, efficient and inexpensive anti-PEDV drugs as non-vaccine prevention and control strategies.
Ursodeoxycholic acid (UrsodeoxycholicAcid, UDCA) is chemically named as 3 alpha, 7 beta-dihydroxyl-5 beta-cholestane-24-acid, is a natural secondary bile acid, widely exists in bile of human and animals, has important clinical significance, is approved by FDA, and has good clinical application foundation worldwide. At present, the ursodeoxycholic acid is mainly used for treating liver and gall system diseases including primary cholangitis, primary biliary cirrhosis and dissolved cholesterol calculus clinically, has an immunoregulatory effect, and is an important medicament for clinically protecting liver and resisting inflammation. In addition, recent studies indicate that UDCA can effectively prevent new coronavirus infection by antagonizing farnesyl ester X receptor (FXR) and then shutting down angiotensin converting enzyme 2 (ACE 2), and is likely to be an important antiviral drug against new crowns. However, ursodeoxycholic acid has not been used in anti-swine coronavirus drug studies, and whether it has anti-PEDV activity is unclear. Therefore, the invention provides application of ursodeoxycholic acid in preparing a medicine for preventing and treating porcine viral diarrhea.
Disclosure of Invention
The invention provides application of ursodeoxycholic acid in preparing a medicine for preventing and treating porcine viral diarrhea, and an in vitro cell test shows that the ursodeoxycholic acid (UDCA) is used as an application prospect for potentially preventing and controlling porcine epidemic diarrhea, so that the technical problems that most medicines have single target points on virus antigens, clinical application of the medicines is limited due to virus variation, and serious side effect reaction is easy to occur in antiviral medicines are effectively solved.
The invention provides application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as a unique active ingredient in preparing a medicament for preventing and treating viral diarrhea, which is characterized in that the virus is porcine epidemic diarrhea virus.
Preferably, the control is inhibition of diarrhea virus.
Preferably, the ursodeoxycholic acid pharmaceutically acceptable salt is at least one selected from the alkali metal salt or ammonium salt of glycoursodeoxycholic acid and the alkali metal salt or ammonium salt of tauroursodeoxycholic acid.
The invention also provides application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as the only active ingredient in preparing a medicament for resisting replication of porcine epidemic diarrhea virus.
The invention also provides application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt serving as the sole active ingredient in preparing a medicament for preventing and treating porcine epidemic diarrhea virus particle proliferation.
The invention also provides application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as the only active ingredient in preparing a medicament for inhibiting intestinal inflammatory response induced by porcine epidemic diarrhea virus infection.
Preferably, the intestinal inflammatory response comprises an increase in the amount of IL-1. Beta. Expression and/or a decrease in the amount of IL-10 expression.
The invention also provides application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as the only active ingredient in preparing a medicament for preventing and treating diseases caused by porcine epidemic diarrhea virus infection, which is characterized in that the diseases comprise enteritis, diarrhea, vomiting or dehydration.
Preferably, the pharmaceutical formulation is a liquid formulation, including an aqueous solution, a non-aqueous solution or a suspension.
Preferably, the content of ursodeoxycholic acid in the liquid preparation is 50-250 mug/mL.
The ursodeoxycholic acid or the ursodeoxycholic acid combined bile acid or the pharmaceutically acceptable salt of the ursodeoxycholic acid is at least one selected from glycoursodeoxycholic acid, tauroursodeoxycholic acid, ursodeoxycholic acid, alkali metal salt or ammonium salt of glycoursodeoxycholic acid and alkali metal salt or ammonium salt of tauroursodeoxycholic acid; the sodium salt of tauroursodeoxycholic acid is preferred.
Compared with the prior art, the invention has the beneficial effects that:
the application of ursodeoxycholic acid provided by the invention in preparing a medicine for preventing and treating porcine viral diarrhea is found by the research of the invention: ursodeoxycholic acid has remarkable effect of resisting porcine epidemic diarrhea virus and remarkable inhibiting effect on proliferation of porcine epidemic diarrhea virus particles; ursodeoxycholic acid can reduce the expression of intracellular pro-inflammatory factor interleukin-1 beta (IL-1 beta) and up-regulate the expression of anti-inflammatory factor interleukin-10 (IL-10), thereby exhibiting remarkable anti-inflammatory activity. In conclusion, ursodeoxycholic acid can inhibit the proliferation of virus particles and regulate the expression level of anti-inflammatory factors by inhibiting the replication of porcine epidemic diarrhea virus in host cells, thereby having the prevention and treatment effect on porcine epidemic diarrhea. Ursodeoxycholic acid has obvious effect, low cost, convenient storage, safety, reliability and no toxic or side effect.
Drawings
FIG. 1 shows the detection of toxicity of UDCA to Vero E6 cells by MTT method of the present invention, with the abscissa indicating the concentration of UDCA or blank control and the ordinate indicating the cell viability;
FIG. 2 shows the fluorescent quantitative PCR assay of the invention for inhibiting replication of PEDV virus by UDCA on Vero E6 cells, with the concentration of UDCA on the abscissa or a blank control and the relative mRNA level of the N gene of PEDV on the ordinate;
FIG. 3 shows the relative levels of mRNA of inflammatory cytokine interleukin-1β (IL-1β) after inhibition of PEDV infection by UDCA on Vero E6 cells by fluorescent quantitative PCR assay of the present invention;
FIG. 4 shows the relative levels of mRNA of inflammatory cytokine interleukin-10 (IL-10) after inhibition of PEDV infection by UDCA on Vero E6 cells as determined by fluorescent quantitative PCR according to the present invention;
FIG. 5 is a TCID of the invention 50 Detection of proliferation of UDCA inhibiting PEDV Virus particles on Vero E6 cells, the abscissa indicates the different time points after cell infection, and the ordinate indicates the viral TCID in whole cell culture 50 。
In the figures, the differences between the different treatment groups are statistically significant, P <0.05, P <0.01, and P <0.001.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to the specific examples and the accompanying drawings, but the examples are not intended to be limiting. The following test methods and detection methods, if not specified, are conventional methods; the reagents and starting materials, unless otherwise specified, are commercially available.
Experimental materials
Medicament and reagent
Ursodeoxycholic acid (Ursodeoxycholic Acid) is obtained from Allatin Corp., china, and has molecular formula C 24 H 40 O 4 The molecular weight is 392.57g/moL, and the purity is more than or equal to 99 percent. The product is dissolved in DMSO to prepare 10g/L mother liquor, and the mother liquor is diluted to corresponding action concentration in proportion for cell test.
The MTT assay kit was purchased from shanghai bi yun biotechnology limited;
fluorescent quantitative PCR (Real-time PCR) SYBR Green MasterMix kit was purchased from Nanjinouzan medical science and technology Co., ltd;
primers were ordered from the biological engineering Co., ltd.
Virus: PEDV LW/L strain (GenBank: MK 392335.1) was stored at-80℃by the laboratory;
and (3) cells: african green monkey kidney cells (Vero E6) were maintained in liquid nitrogen by the present laboratory;
serum: serum was purchased from Gibco, usa.
Example 1
Cytotoxicity assay of UDCA on Vero E6 cells:
vero E6 cells were cultured in 96-well plates in MEM medium containing 10% fetal bovine serum to a cell density of about 70%. UDCA was diluted separately to: 50 mu g/mL, 100 mu g/mL, 250 mu g/mL and 500 mu g/mL, 100 mu L of UDCA treated cells with different concentrations are respectively added into each hole after the complete culture medium is removed, 3 groups of the cells are repeatedly arranged at each concentration, the supernatant is removed after 48 hours of treatment, 90 mu L of fresh culture solution and 10 mu L of MTT solution are sequentially added, the culture is continued for 4 hours, and 110 mu L of UDCA treated cells are added into each hole after the supernatant is removed; the absorbance of each well was measured at a wavelength of 450nm using a multifunctional microplate reader, and the results are shown in FIG. 1.
As can be seen from FIG. 1, when the concentration of UDCA was 0 to 100. Mu.g/mL, the effect of UDCA on Vero E6 cell activity was not significant, and when the concentration was 100. Mu.g/mL, the growth of cells was promoted to some extent, indicating that there was no significant cytotoxicity (P > 0.05) in this concentration range. Thus, three concentrations of 50, 100, 250. Mu.g/mL were selected for the subsequent antiviral assay.
Example 2
Fluorescent quantitative PCR detection of replication of UDCA in Vero E6 cells against porcine epidemic diarrhea virus:
UDCA is dissolved in MEM culture medium containing 10% of fetal bovine serum to prepare three concentrations of 50 mug/mL, 100 mug/mL and 250 mug/mL for pretreatment of cells; a MEM culture solution containing pancreatin at a concentration of 10. Mu.g/mL was prepared as a virus infection solution, and UDCA was dissolved in the virus infection solution to obtain a virus infection solution containing UDCA at each concentration.
In a 24-well plate, vero E6 cells were grown in MEM medium containing 10% fetal bovine serum to a cell confluency of 80%, the medium was removed, the experimental group was pretreated with cell culture solutions of different concentrations of UDCA for 1h, respectively, the control group was added with MEM complete medium containing an equal amount of DMSO solvent, after the medium was removed, the cells were washed 3 times with PBS, and replaced with virus-infected solutions containing each concentration of UDCA (the concentration of UDCA in each well was the same as that in pretreatment) or with blank virus-infected solutions (negative control), followed by inoculation of PEDV LW/L strain at a multiplicity of infection of MOI=0.1, followed by exposure to 5% CO at 37 ℃ 2 After culturing for 12 hours in an incubator, cell samples are collected, total RNA of the cells is extracted by a Trizol method, and then the change of mRNA expression level of the N gene of PEDV is detected by fluorescent quantitative PCR.
The relative quantitative detection of the N gene of PEDV was carried out by the 2-delta Ct method, and the result is shown in FIG. 2, when the UDCA concentration is 50 mug/mL or above, the inhibition effect on PEDV replication is remarkable, and the inhibition effect shows dose dependency. When UDCA was 250. Mu.g/mL, N gene expression of PEDV was almost completely inhibited, indicating that UDCA had a significant anti-PEDV viral effect.
Example 3
Fluorescent quantitative PCR assay for UDCA expression of inflammatory cytokines after Vero E6 cells inhibit PEDV infection:
in 24-well plate, vero E6 cells are grown in MEM culture medium containing 10% of fetal calf serum until cell confluence reaches 80%, the culture medium is removed, cell culture solutions of different concentrations of UDCA are added into experimental group to respectively pretreat cells for 1h, and the cells are added into control group to contain the sameAfter removing the medium, PBS was used to wash the cells 3 times, and the cells were replaced with a virus-infected solution (the concentration of UDCA in each well was the same as that in pretreatment) or a blank virus-infected solution (negative control), and then the strain PEDV LW/L was inoculated at a multiplicity of infection MOI=1, and then placed at 37℃in 5% CO 2 After culturing for 8 hours in an incubator, cell samples are obtained, total RNA of cells is extracted by adopting a Trizol method, and then mRNA expression level changes of interleukin-1 beta (IL-1 beta) and interleukin-10 (IL-10) in the cells are detected by adopting fluorescent quantitative PCR.
The relative quantification of interleukin-1 beta (IL-1 beta) and interleukin-10 (IL-10) of pro-inflammatory cytokines was performed in this assay using the 2-DeltaCt method, and the results are shown in FIGS. 3-4, where when UDCA concentration was 100 μg/mL and above, the expression of the intracellular pro-inflammatory factor interleukin-1 beta (IL-1 beta) was reduced while the expression of the anti-inflammatory factor interleukin-10 (IL-10) was upregulated, and the effect exhibited a dose-dependent, indicating that UDCA had significant anti-inflammatory activity after PEDV infection.
Example 4
TCID50 assay UDCA inhibited proliferation of PEDV virions on Vero E6 cells:
vero E6 cells were grown in 12 well plates in MEM medium with 10% foetal calf serum to a cell confluency of 80%, the medium was removed, the experimental group was pre-treated with cell culture solutions of different concentrations of UDCA, respectively, for 1h, the control group was added with MEM complete medium with equivalent amounts of DMSO solvent, after the medium was removed, the cells were washed 3 times with PBS, replaced with virus-infected solutions containing each concentration of UDCA (the UDCA concentration in each well was identical to that at the time of pre-treatment) or with blank virus-infected solutions (negative control), then PEDV LW/L strain was inoculated at a multiplicity of infection MOI=0.01, and then whole cell cultures were harvested after continued culture in a 5% CO2 incubator at 37℃for 12, 24, 48 h. Determination of viral titres referring to the Reed-Muench method, cells were plated in 96-well plates at 1X 105/well, cultured until cell confluency reached more than 90%, the culture broth was aspirated and washed 3 times with PBS. And (3) respectively adding the whole cell cultures to be tested (dilution factor 10-1-10-10) with the dilution ratio into the corresponding holes, counting the culture holes with cytopathy after 48 hours after infection, and calculating the corresponding virus titer.
As a result, as shown in FIG. 5, when the UDCA concentration was 100. Mu.g/mL, the effect of inhibiting the proliferation of PEDV virus particles was remarkable, and the effect of inhibiting was most remarkable at the early stage of virus infection. The results indicate that UDCA can significantly inhibit proliferation of PEDV LW/L strain virions at the cellular level.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. The application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as the only active ingredient in preparing a medicament for preventing and treating viral diarrhea is characterized in that the virus is porcine epidemic diarrhea virus.
2. The use according to claim 1, wherein the control is inhibition of diarrhea virus.
3. The use according to claim 1, wherein the pharmaceutically acceptable salt of ursodeoxycholic acid is selected from at least one of an alkali metal or ammonium salt of glycoursodeoxycholic acid, an alkali metal or ammonium salt of tauroursodeoxycholic acid.
4. The use of ursodeoxycholic acid or ursodeoxycholic acid conjugated bile acid or a pharmaceutically acceptable salt thereof as the sole active ingredient in the manufacture of a medicament against replication of porcine epidemic diarrhea virus.
5. The application of ursodeoxycholic acid or ursodeoxycholic acid combined bile acid or ursodeoxycholic acid pharmaceutically acceptable salt as the only active ingredient in preparing the medicine for preventing and treating porcine epidemic diarrhea virus particle proliferation.
6. Use of ursodeoxycholic acid or ursodeoxycholic acid conjugated bile acid or a pharmaceutically acceptable salt thereof as the sole active ingredient in the manufacture of a medicament for inhibiting the intestinal inflammatory response induced by porcine epidemic diarrhea virus infection.
7. The use according to claim 6, wherein the intestinal inflammatory response comprises an increase in the amount of IL-1 β expression and/or a decrease in the amount of IL-10 expression.
8. Use of ursodeoxycholic acid or ursodeoxycholic acid conjugated bile acid or a pharmaceutically acceptable salt thereof as sole active ingredient in the manufacture of a medicament for the prevention and treatment of a disease caused by porcine epidemic diarrhea virus infection, characterized in that the disease comprises enteritis, diarrhea, vomiting or dehydration.
9. The use according to any one of claims 1 to 8, wherein the formulation of the medicament is a liquid formulation, including an aqueous solution, a non-aqueous solution or a suspension.
10. The use according to claim 9, wherein the ursodeoxycholic acid content of the liquid formulation is 50-250 μg/mL.
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