Application of fingolimod in preparation of medicine for treating hepatolenticular degeneration

Abstract

The invention discloses application of fingolimod in preparation of drugs for prevention, improvement, treatment or adjuvant treatment of hepatolenticular degeneration, and relates to the field of medicine and pharmacology. In-vivo experimental research finds that fingolimod (FTY720) can improve the liver phenotype of a mouse with hepatolenticular degeneration by blocking sphingosine-1-phosphoric acid (S1P) signal transduction, and the medication scheme of FTY720 in the aspect of improving the liver symptoms of the mouse can be used for treating liver-type hepatolenticular degeneration; the treatment potential of FTY720 on hepatolenticular degeneration is proved, and a new choice is provided for treatment of hepatolenticular degeneration.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an application of fingolimod in preparing a drug for treating Wilson's disease. Background Art

[0002] Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an autosomal recessive genetic disease of copper metabolism disorder caused by Atp7b gene mutation. The pathogenic gene Atp7b is located on the long arm of chromosome 13, contains 20 introns and 21 exons, and encodes a P-type copper transport ATPase. Atp7b gene mutation can lead to impaired bile excretion of copper in hepatocytes, and excessive copper is successively deposited in tissues and organs such as the liver, brain, kidneys, and cornea, thereby causing complex phenotypes such as liver damage and neuropsychiatric abnormalities. Due to the insidious onset of HLD, patients at the first diagnosis often have multiple clinical manifestations, among which the liver is one of the most commonly affected organs of HLD. Clinically, it can manifest in various forms such as asymptomatic, acute hepatitis, acute liver failure, chronic hepatitis, and cirrhosis.

[0003] Currently, the treatment of HLD mainly relies on a low-copper diet, drug therapy, and liver transplantation. It is currently believed that all patients newly diagnosed with Wilson's disease should begin lifelong drug treatment. However, on the one hand, existing drug treatments are not effective for all patients and face problems such as low drug compliance and adverse reactions. On the other hand, liver transplantation is often limited by the shortage of donor organs, high costs, and the need for lifelong immunosuppressive therapy. Therefore, the development of new drugs for HLD will help explore more effective treatments, improve patients' quality of life, and delay the progression of the disease.

[0004] As early as 2007, Lang PA et al. found that ceramide mediated copper overload-induced liver cell death through in vitro and in vivo models, and clarified that ceramide has a core function in metabolic diseases and acute poisoning caused by heavy metals. Another test on the serum sphingolipid profile of HLD patients also found that the levels of sphingolipid components in the serum of HLD patients changed significantly. These together suggest that sphingolipid metabolism disorders play an important role in the occurrence and development of HLD.

[0005] FTY720 (2-amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydro-chloride), with the trade name Fingolimod and the chemical formula C19H33NO2.HCl, is a sphingosine analogue synthesized by modifying myriocin extracted from Cordyceps sinensis (Ascomycetes), and has a wide range of biological activities. In 2010, the US Food and Drug Administration (FDA) approved FTY720 as a first-line treatment for relapsing multiple sclerosis. It was approved for marketing in my country in 2019. In addition to relapsing multiple sclerosis, based on FTY720's regulatory function on sphingolipid biosynthesis, FTY720 has also been found to play a therapeutic role in experimental models of neurodegenerative diseases caused by sphingolipid disorders, such as Alzheimer's disease, Parkinson's disease, spastic paraplegia, and amyotrophic lateral sclerosis.

[0006] Current treatments for HLD include a low-copper diet, drug therapy, and liver transplantation. Drug therapy is divided into two categories: one is drugs that increase urinary copper excretion (copper chelators), including D-penicillamine, sodium dimercaptopropanesulfonate, dimercaptosuccinic acid, and trientine; the other is drugs that reduce copper absorption, including zinc agents and ammonium tetrathiomolybdate. The main purpose of a low-copper diet and drug therapy is to reduce copper accumulation in the body and reverse copper overload in the body. Liver transplantation can completely correct the underlying genetic defects of Wilson's disease. However, the above-mentioned diagnosis and treatment schemes still have the following shortcomings.

[0007] (1) Existing drug treatments are mainly aimed at improving copper overload to prevent further damage to the liver, but there is a lack of specific drug treatments for liver damage already caused by copper overload.

[0008] (2) Copper chelators stimulate the transfer of copper from the liver to the blood, where it is then excreted through the urine. During treatment, as the level of non-ceruloplasmin-bound copper in the blood increases, it may cross the blood-brain barrier and accumulate in the brain, leading to worsening of the patient's neurological symptoms.

[0009] (3) Liver transplantation surgery is risky, donors are difficult to find, there is a risk of transplanted organ rejection, and the cost is high. Summary of the invention

[0010] The present invention provides an application of fingolimod hydrochloride (FTY720) in the preparation of a drug for treating hepatolenticular degeneration liver injury. Through in vivo experimental research, it is found that FTY720 can improve the liver phenotype of hepatolenticular degeneration mice by blocking sphingosine-1-phosphate (S1P) signal transduction. The medication scheme of FTY720 in improving the liver symptoms of Atp7b gene knockout mice can be used to treat hepatic hepatolenticular degeneration; the therapeutic potential of FTY720 on hepatolenticular degeneration is confirmed, and a new option for the treatment of hepatolenticular degeneration is provided.

[0011] To achieve the above objectives, the present invention provides the use of fingolimod in the preparation of a method for preventing, improving, treating or assisting in the treatment of Wilson's disease.

[0012] Preferably, the chemical formula of fingolimod is C19H33N02.HCl.

[0013] Preferably, the fingolimod improves Wilson's disease by blocking sphingosine-1-phosphate signaling.

[0014] Another aspect of the present invention provides a drug, the active ingredient of which is fingolimod, and the function of the drug is to prevent, improve, treat or assist in the treatment of Wilson's disease.

[0015] Preferably, the chemical formula of fingolimod is C19H33N02.HCl.

[0016] Preferably, the drug is prepared from an effective dose of fingolimod and pharmaceutically acceptable excipients.

[0017] Preferably, the administration method is injection.

[0018] Preferably, the drug powder is dissolved in 0.5% dimethyl sulfoxide for injection.

[0019] Preferably, the administration concentration is 1 mg / kg.

[0020] Beneficial technical effects of the present invention:

[0021] (1) Innovative application of FTY720 in the treatment of Wilson's disease;

[0022] (2) In animal experiments, FTY720 significantly downregulated the inflammatory phenotype of the liver of Atp7b knockout mice, showing excellent therapeutic effects on liver damage associated with hepatolenticular degeneration. As a potential drug for the treatment of hepatolenticular degeneration, it may provide patients with more drug treatment opportunities, reduce the rate of liver transplantation, and reduce the disease burden of patients.

[0023] (3) FTY720 is not only suitable for adult patients, but is also the only disease-modifying treatment for multiple sclerosis approved for children aged 10 years and above, reflecting its wide applicability.

[0024] (4) In existing clinical use, although the use of FTY720 is associated with some adverse reactions, overall it is considered to be a relatively safe and well-tolerated drug.

[0025] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a representative picture of hematoxylin-eosin staining showing the improvement of liver histology in mice with Wilson's disease by FTY720.

[0027] The mice in the control group had enlarged liver cell nuclei, deformed and swollen liver cells with ballooning, and extensive inflammatory cell infiltration in the lobules. The mice in the experimental group had significantly improved histological morphology, and the liver lobule structure returned to normal.

[0028] Figure 2 The effect of FTY720 on plasma liver enzyme levels in mice with Wilson's disease.

[0029] Among them: compared with the control group mice, the plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the experimental group mice were significantly decreased, and liver damage was alleviated;

[0030] Figure 3 The effect of FTY720 on liver fibrosis in mice with Wilson's disease.

[0031] in, Figure 3 A is Sirius red staining of liver tissue. The red-stained part represents collagen fibers (it can be seen that the collagen deposition in the liver of the experimental group of mice is significantly less than that in the control group). Figure 3 B is the quantitative result of the Sirius red staining positive area (it can be seen that the Sirius red positive area of ​​the liver of the experimental group of mice is significantly less than that of the control group);

[0032] Figure 4 The effect of FTY720 on neutrophil infiltration in the liver of mice with Wilson's disease.

[0033] in, Figure 4 A is the MPO immunohistochemical staining of liver tissue. The brown dots represent the labeled neutrophils (it can be seen that the degree of neutrophil infiltration in the liver of the experimental group of mice is significantly less than that in the control group). Figure 4 B is the quantitative result of the MPO staining positive area (it can be seen that the content of neutrophils in the liver of mice in the experimental group is significantly less than that in the control group);

[0034] Figure 5 The effect of FTY720 on macrophage infiltration in the liver of mice with Wilson's disease.

[0035] in, Figure 5 A is F4 / 80 immunohistochemical staining of liver tissue. The brown dots represent the labeled macrophages (it can be seen that the infiltration of macrophages in the liver of the experimental group of mice is significantly less than that in the control group). Figure 5 B is the quantitative result of the F4 / 80 staining positive area (it can be seen that the content of macrophages in the liver of the experimental group of mice is significantly less than that in the control group);

[0036] Figure 6 The effect of FTY720 on the expression levels of genes related to liver inflammation and fibrosis in mice with hepatolenticular degeneration.

[0037] in, Figure 6 AF is the mRNA expression level of liver inflammation-related genes, Figure 6 GJ is the mRNA expression level of liver fibrosis-related genes (it can be seen that the expression level of liver inflammation and fibrosis-related genes in the experimental group of mice was significantly lower than that in the control group, indicating that the degree of liver damage in the experimental group of mice was significantly improved). DETAILED DESCRIPTION

[0038] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0039] The technical scheme of the present invention is as follows: by administering fingolimod (FTY720) to Atp7b knockout mice in a hepatolenticular degeneration mouse model, the therapeutic effect of FTY720 on liver damage indicators of Atp7b knockout mice is evaluated. The administration route is intraperitoneal injection, and the treatment cycle is one month. The drug is made of an effective dose of FTY720 and pharmaceutically permitted excipients. Specifically, the present invention dissolves FTY720 drug powder in 0.5% dimethyl sulfoxide, and the administration concentration is 1 mg / kg.

[0040] Experimental studies on drug administration have shown that FTY20 can effectively reduce the plasma alanine aminotransferase and aspartate aminotransferase levels in mice with hepatolenticular degeneration. FTY720 can completely reverse the histological lesions of the liver in mice with hepatolenticular degeneration, significantly improve hepatocyte death, hepatic macrophage and neutrophil infiltration, and effectively reduce the expression of inflammatory factors and chemokine-related genes in mouse liver tissue. FTY720 can significantly alleviate the degree of liver fibrosis in mice with hepatolenticular degeneration and inhibit the expression of genes related to liver fibrosis.

[0041] Below, specific embodiments are used to illustrate the specific content of the present invention:

[0042] Example 1 Hematoxylin-eosin staining of mouse liver tissue

[0043] 1. Methods:

[0044] 1.1 Tissue fixation and embedding:

[0045] (1) After the mice were anesthetized and killed, a piece of liver tissue was taken from the right lobe of the liver and fixed with 4% paraformaldehyde at room temperature for 24 hours;

[0046] (2) Immerse the specimen in different concentrations of alcohol for gradient dehydration (30%, 50%, 70%, 80%, 90%, 95%, 100%), changing it every 30 minutes;

[0047] (3) Place the specimen in a mixture of anhydrous ethanol and xylene (1:1) for 30 minutes, then place it in 100% xylene, and observe the tissue block until it becomes transparent;

[0048] (4) Immerse the specimen in pre-melted paraffin twice, each time for 3 hours;

[0049] (5) Pour the melted paraffin into the embedding box, then put the tissue block in a 4°C refrigerator or water to condense, mark the direction of the tissue block and store it at 4°C for later use;

[0050] (6) The embedded tissue was sliced ​​at a thickness of 4 to 6 μm;

[0051] 1.2 Dyeing:

[0052] (1) Place the slide in a 55-60°C oven for 30 minutes, then dewax the slide in xylene for 5 times, each time for 5-10 minutes;

[0053] (2) Hydrate the slides in different concentrations of ethanol (from high to low, 100%, 95%, 80%, 70%, 5 minutes each), and then rinse with distilled water for 5 minutes;

[0054] (3) Place the sections in hematoxylin stain for 5 to 10 minutes, rinse with tap water for 3 to 5 minutes, then place in 1% hydrochloric acid alcohol for 5 to 30 seconds, rinse with tap water for 1 to 3 minutes, and finally place in 0.5% eosin alcohol stain for 1 minute, and rinse with distilled water for 1 minute;

[0055] (4) Seal the slide with neutral resin, observe under a microscope and take photos.

[0056] 2. Results: Figure 1As shown in the figure, the hepatocytes of the untreated hepatolenticular degeneration mice were swollen and degenerated, the liver lobule structure was disordered, balloon-like hepatocytes were visible, and there was extensive inflammatory cell infiltration in the lobules. The hepatocyte morphology and lobule structure of the experimental group of mice treated with FTY720 returned to normal, and the inflammatory cell infiltration was reduced. This shows that FTY720 treatment can significantly improve the liver pathological damage of hepatolenticular degeneration mice.

[0057] Example 2 Detection of plasma liver enzyme levels

[0058] 1. Method: The liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice were measured according to the kit instructions. The kits used were purchased from Shanghai Shensuo Youfu Medical Diagnostic Products Co., Ltd. The detection steps are as follows:

[0059] (1) Add 7.5 μL of the plasma sample to be tested to each well of a 96-well plate, then add 150 μL of working solution R1 and incubate in a 37°C incubator for 5 minutes;

[0060] (2) Take out the 96-well plate, add 50 μL of working solution R2 to each well, and incubate in a 37°C incubator for 5 minutes;

[0061] (3) Preheat the microplate reader to 37°C, then place the 96-well plate with complete reaction into the microplate reader and detect the absorbance at 340 nm for a total of 20 cycles;

[0062] (4) Calculation of content: Discard the absorbance values ​​of the first 5 and last 6 cycles, take the absorbance difference between the 6th cycle and the 14th cycle as ΔOD, and the time difference as ΔT, and calculate the final concentration according to the following formula: concentration (U / L) = ΔOD / ΔT × 60 × 207.5 / 6.22 / 7.5 / 0.8 × 1000.

[0063] 2. Results: Figure 2 As shown, the plasma ALT and AST levels of the treated FTY720 mice were significantly lower than those of the control group, indicating that FTY720 treatment can significantly alleviate liver damage in mice with Wilson's disease.

[0064] Example 3 Sirius red staining of mouse liver tissue

[0065] 1. Methods:

[0066] The tissue fixation and embedding methods are the same as in Example 1, and the staining method is as follows:

[0067] (1) Place the slide in a 55-60°C oven for 30 minutes, then dewax the slide in xylene for 5 times, each time for 5-10 minutes;

[0068] (2) Hydrate the slides in different concentrations of ethanol (from high to low, 100%, 95%, 80%, 70%, 5 minutes each), and then rinse with distilled water for 5 minutes;

[0069] (3) Use Sirius red staining solution to stain the sections for 1 hour, rinse with running water to remove the floating color, then use hematoxylin staining solution to counterstain the cell nuclei for 10 minutes, and rinse with running water to remove the floating color;

[0070] (4) Seal the slides with neutral resin, observe and take photos under a microscope, and quantify the photos using ImageJ software.

[0071] 2. Results: Figure 3 As shown in A, extensive collagen fiber deposition was observed in the liver of the untreated control group mice, while the degree of fibrosis was significantly reduced after FTY720 treatment. The Sirius red positive area was quantified, and the results showed that the positive area in the experimental group was significantly reduced ( Figure 3 B). This indicates that FTY720 treatment can significantly improve liver fibrosis in mice with Wilson's disease.

[0072] Example 4 Immunohistochemical staining of F4 / 80 and MPO in mouse liver tissue

[0073] 1. Methods:

[0074] The tissue fixation and embedding methods are the same as in Example 1, and the staining method is as follows:

[0075] (1) Baking: Place the slide in a 55-60°C oven for 30 minutes;

[0076] (2) Dewaxing: Immerse the slides in xylene for dewaxing 5 times, 5 to 10 minutes each time;

[0077] (3) Hydration: Place the slides in different concentrations of ethanol for hydration (from high to low, 100%, 95%, 80%, 70%, 5 minutes each), and then rinse with distilled water for 5 minutes;

[0078] (4) Antigen retrieval: immerse the slide in 10 mM citrate buffer (PH = 6.0), heat to 90°C-98°C in a microwave oven, and incubate for 15 min;

[0079] (5) Inactivation: dilute the 30% H2O2 solution 10-fold with distilled water, incubate the sections in the dark for 30 min, and then wash three times with distilled water;

[0080] (6) Blocking: Block with 5% BSA at room temperature for 20 minutes;

[0081] (7) Incubation with primary antibody: add primary antibody solution (antibody dilution 1:400) onto the slide and incubate overnight at 4°C;

[0082] (8) Incubation with secondary antibody: Wash the slides three times with PBS, 5 minutes each time, absorb the liquid and add secondary antibody solution (antibody dilution multiple 1:200) to the slides, and incubate at room temperature for 30 minutes;

[0083] (9) Color development: Wash the slides with PBS three times, 5 minutes each time, and add DAB color development solution to the slides after absorbing the liquid. Observe in real time under a microscope (reaction time is about 5 minutes). Wash with distilled water to stop color development.

[0084] (10) Hematoxylin staining: Counterstain the cell nucleus with hematoxylin stain for 5 minutes and rinse with tap water for 1 minute;

[0085] (11) Dehydration: Immerse the specimen in different concentrations of alcohol for gradient dehydration (75%, 85%, 90%, 95%, 100% for 1 hour each);

[0086] (12) Transparency: Place the specimen in a mixture of anhydrous ethanol and xylene (1:1) for 30 minutes, then place it in 100% xylene, and observe the tissue block at all times until it becomes transparent;

[0087] (13) Sealing: Seal the slides with neutral resin, observe and take photos under a microscope, and quantify the photos using ImageJ software.

[0088] 2. Results: As shown in the figure, the positive signal of MPO staining in the liver of mice in the experimental group treated with FTY720 was significantly less than that in the control group ( Figure 4 A), the quantitative results of the positive area showed that the MPO positive area of ​​the experimental group mice was significantly reduced, indicating that FTY720 treatment can reduce the degree of neutrophil infiltration in mice with Wilson's disease. Figure 5 As shown in A, the positive signal of F4 / 80 in the experimental group mice was significantly reduced, and the quantitative results showed that the F4 / 80 positive area in the experimental group mice was significantly less than that in the control group, indicating that FTY720 treatment can reduce the degree of macrophage infiltration in mice with Wilson's disease. The results of immunohistochemical staining showed that FTY720 can significantly alleviate the inflammatory damage of the liver in mice with Wilson's disease.

[0089] Example 5 RT-PCR detection of the expression of genes related to inflammation and fibrosis in mouse liver

[0090] 1. Methods:

[0091] (1) According to the kit instructions, use Total RNA was extracted from mouse liver tissue using Super Total RNA Extraction Kit (Promega, Shanghai), and then reverse transcribed using HiScript III RT SuperMixfor qPCR (+gDNA wiper) (Vazyme, Nanjing) to generate cDNA. cDNA was used for real-time PCR reaction.

[0092] (2) Use specific primers to detect the target gene. The internal reference GAPDH primer is:

[0093] P1: 5'-CATCACTGCCACCCAGAAGACTG-3',

[0094] P2: 5'-ATGCCAGTGAGCTTCCCGTTCAG-3';

[0095] The primer sequences used to amplify Tnfα are:

[0096] P3: 5'-GGTGCCTATGTCTCAGCCTCTT-3',

[0097] P4: 5'-GCCATAGAACTGATGAGAGGGAG-3';

[0098] The primer sequences used to amplify I11β are:

[0099] P5: 5'-TGGACCTTCCAGGATGAGGACA-3',

[0100] P6: 5'-GTTCATCTCGGAGCCTGTAGTG-3';

[0101] The primer sequences used to amplify I16 are:

[0102] P7: 5'-TACCACTTCACAAGTCGGAGGC-3',

[0103] P8: 5'-CTGCAAGTGCATCATCGTTGTTC-3';

[0104] The primer sequences used to amplify Ccl2 are:

[0105] P9: 5'-GCTACAAGAGGATCACCAGCAG-3',

[0106] P10: 5'-GTCTGGACCCATTCCTTCTTGG-3';

[0107] The primer sequences used to amplify Ccl3 are:

[0108] P11: 5'-ACTGCCTGCTGCTTCTCCTACA-3',

[0109] P12: 5'-ATGACACCTGGCTGGGAGCAAA-3';

[0110] The primer sequences used to amplify Cxcl2 are:

[0111] P13: 5'-CATCCAGAGCTTGAGTGTGACG-3',

[0112] P14: 5'-GGCTTCAGGGTCAAGGCAAACT-3';

[0113] The primer sequences used to amplify Tgfβ are:

[0114] P15: 5'-TGATACGCCTGAGTGGCTGTCT-3',

[0115] P16: 5'-CACAAGAGCAGTGAGCGCTGAA-3';

[0116] The primer sequences used to amplify αSMA are:

[0117] P17: 5'-TGCTGACAGAGGCACCACTGAA-3',

[0118] P18: 5'-CAGTTGTACGTCCAGAGGCATAG-3';

[0119] The primer sequences used to amplify Col1α1 are:

[0120] P19: 5'-CCTCAGGGTATTGCTGGACAAC-3',

[0121] P20: 5'-CAGAAGGACCTTGTTTGCCAGG-3';

[0122] The primer sequences used to amplify Col3α1 are:

[0123] P21: 5'-GACCAAAAGGTGATGCTGGACAG-3',

[0124] P22: 5'-CAAGACCTCGTGCTCCAGTTAG-3'.

[0125] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s, for 40 cycles.

[0126] 2. Results: Figure 6 As shown in A to F, the mRNA levels of inflammatory factors Tnfα, Il1β, Il6 and chemokines Ccl2, Ccl3, and Cxcl2 in the experimental group were significantly lower than those in the control group. At the same time, the mRNA levels of fibrosis-related genes Tgfβ, αSMA, Col1α1, and Col3α1 in the liver tissue of the experimental group were also significantly reduced compared with those in the control group. This indicates that FTY720 treatment can significantly improve the degree of liver inflammation and fibrosis in mice with hepatolenticular degeneration.

[0127] The above examples show that FTY720 is useful in treating liver damage caused by Wilson's disease. The combination of FTY720 and copper chelators can provide new drug treatment opportunities for patients who do not respond well to copper chelators, thereby reducing the occurrence of decompensated liver disease and death.

[0128] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of fingolimod in the preparation of a drug for preventing, improving, treating or assisting in the treatment of Wilson's disease.

2. The use according to claim 1, characterized in that: The chemical formula of fingolimod is C19H33NO2.HCl.

3. The use according to claim 1 or 2, characterized in that: Fingolimod improves Wilson's disease by blocking sphingosine-1-phosphate signaling.

4. A drug, characterized in that The active ingredient is fingolimod, and the function of the drug is to prevent, improve, treat or assist in the treatment of Wilson's disease.

5. The drug according to claim 4, characterized in that The chemical formula of fingolimod is C19H33NO2.HCl.

6. The drug according to claim 4, characterized in that The medicine is prepared from an effective dose of fingolimod and pharmaceutically permitted auxiliary materials.

7. The drug according to claim 6, characterized in that The administration method is injection.

8. The drug according to claim 7, characterized in that The drug powder was dissolved in 0.5% dimethyl sulfoxide for injection.

9. The drug according to claim 8, characterized in that The dosage concentration was 1 mg / kg.

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