Use of fingolimod for the preparation of a medicament for the treatment of hepatolenticular degeneration

Abstract

The application discloses application of fingolimod in preparation of prevention, improvement, treatment or adjuvant treatment of hepatolenticular degeneration, and relates to the field of medicine. Through in-vivo experiment research, it is found that fingolimod (FTY720) can improve the liver phenotype of a hepatolenticular degeneration mouse by blocking sphingosine-1-phosphate (S1P) signal transmission, and the drug use scheme of FTY720 in improving the liver symptoms of the mouse can be used for treating liver-type hepatolenticular degeneration; and the treatment potential of FTY720 on hepatolenticular degeneration is confirmed, thereby providing a new selection for treatment of hepatolenticular degeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of fingolimod in the preparation of a drug for treating hepatolenticular degeneration. BACKGROUND

[0002] Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is a copper metabolism disorder autosomal recessive genetic disease 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 cause copper excretion disorder in liver cells, and excessive copper is deposited in liver, brain, kidney, cornea and other tissues and organs, and then cause complex phenotypes such as liver damage and neuropsychiatric abnormalities. Since HLD has insidious onset, patients at the initial diagnosis often have multiple clinical manifestations, among which the liver is one of the most commonly involved organs in HLD, and can show various forms such as asymptomatic, acute hepatitis, acute liver failure, chronic hepatitis, and cirrhosis.

[0003] Currently, the treatment of HLD mainly depends on low-copper diet, drug therapy and liver transplantation. It is currently believed that all newly diagnosed patients with hepatolenticular degeneration should start lifelong drug therapy. On the one hand, existing drug therapy is not effective for all patients, and is faced with problems such as low drug therapy compliance and adverse reactions. On the other hand, the development of liver transplantation is often limited by the shortage of donor organs, the high cost and the need for lifelong immunosuppressive therapy. Therefore, the development of new drugs for HLD helps to explore more effective treatment methods, improve the quality of life of patients, and delay the progression of the disease.

[0004] As early as 2007, Lang PA et al. found that ceramide mediated hepatocyte death caused by copper overload through in vivo and in vitro models, and clarified that ceramide has a core function in the process of metabolic diseases and acute poisoning caused by heavy metals. Another study on the detection of serum sphingolipid spectrum in HLD patients also found that the levels of various sphingolipid components in the serum of HLD patients changed significantly. These collectively suggest that sphingolipid metabolism disorder plays 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, chemical formula C19H33NO2.HCl, is a synthetic sphingosine analogue modified from a polystictin extracted from Cordyceps sinensis (Ascomycota Cordyceps), which has a wide range of biological activities. In 2010, the U.S. Food and Drug Administration (FDA) approved FTY720 as a first-line treatment for relapsing multiple sclerosis. In 2019, it was approved for listing in China. In addition to relapsing multiple sclerosis, based on the regulatory function of FTY720 on sphingolipid biosynthesis, FTY720 has been found to play a therapeutic role in experimental models of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, spastic paraplegia, and amyotrophic lateral sclerosis caused by sphingolipid disorders.

[0006] Currently, the treatment methods for HLD include low-copper diet, drug therapy, and liver transplantation. Among them, drug therapy is divided into two categories: one is to increase the excretion of urinary copper (copper chelator), including D-penicillamine, sodium dimercapto-propane sulfonate, dimercaptosuccinic acid, and trientine; the other is to reduce copper absorption, including zinc agent and ammonium tetrathiomolybdate. The main purpose of 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 defect of hepatolenticular degeneration. However, the current diagnosis and treatment programs still have the following shortcomings.

[0007] (1) The existing drug treatment methods mainly aim to improve the copper overload state to prevent further damage to the liver, but there is a lack of specific drug treatment methods for liver damage caused by copper overload.

[0008] (2) Copper chelators stimulate the transfer of copper from accumulated liver to blood, and then excreted through urine. During treatment, as the level of non-copper albumin-bound copper in the blood increases, it may accumulate in the brain by crossing the blood-brain barrier, leading to worsening of neurological symptoms in patients.

[0009] (3) Liver transplantation surgery has high risk, and it is difficult to find donors, there is a risk of rejection of transplanted organs, and the cost is high. SUMMARY

[0010] The application provides application of fingolimod hydrochloride (FTY720) in preparation of a medicine for treating liver damage of hepatolenticular degeneration, and finds that FTY720 can improve the liver phenotype of a hepatolenticular degeneration mouse by blocking sphingosine-1-phosphate (S1P) signal transduction through in-vivo experiment research, the use of FTY720 in improving the liver symptoms of an Atp7b gene knockout mouse can be used for treating liver-type hepatolenticular degeneration, and the treatment potential of FTY720 on hepatolenticular degeneration is proved, and a new selection is provided for the treatment of hepatolenticular degeneration.

[0011] To achieve the above object, the application provides application of fingolimod in preparation of a medicine for preventing, improving, treating or assisting in treating hepatolenticular degeneration.

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

[0013] Preferably, the fingolimod improves hepatolenticular degeneration by blocking sphingosine-1-phosphate signal transduction.

[0014] The application also provides a medicine, and an effective component of the medicine is fingolimod, and the function of the medicine is preventing, improving, treating or assisting in treating hepatolenticular degeneration.

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

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

[0017] Preferably, the administration mode is injection.

[0018] Preferably, the medicine powder is dissolved in 0.5% dimethyl sulfoxide and injected.

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

[0020] The application has the following beneficial technical effects:

[0021] (1) The FTY720 is innovatively used in the treatment of hepatolenticular degeneration.

[0022] (2) In the animal experiment, the FTY720 significantly down-regulates the inflammation phenotype of the liver of the Atp7b gene knockout mouse, and shows excellent treatment effect on the liver damage related to hepatolenticular degeneration. As a potential medicine for treating hepatolenticular degeneration, the FTY720 can help patients to obtain more medicine treatment opportunities, reduce the liver transplantation rate, and reduce the disease burden of patients.

[0023] (3) FTY720 is not only suitable for adult patients, but also the only approved drug for the treatment of multiple sclerosis in children aged 10 years and older, which reflects its wide applicability.

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

[0025] The concept, specific structure and technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a representative picture of hematoxylin-eosin staining of the histological improvement of FTY720 on the liver of liver-wilson disease mice,

[0027] Among them: the liver cell nucleus of the control group mice is enlarged, the liver cells are deformed and swollen with balloon-like degeneration, and there is extensive inflammatory cell infiltration in the lobule; the histological morphology of the experimental group mice is significantly improved, and the liver lobule structure is restored to normal;

[0028] Figure 2 is the effect of FTY720 on the plasma liver enzyme level of liver-wilson disease mice,

[0029] Among them: compared with the control group mice, the plasma glutathione transaminase (ALT) and glutathione transaminase (AST) levels of the experimental group mice are significantly decreased, and the liver damage is reduced;

[0030] Figure 3 is the effect of FTY720 on the liver fibrosis of liver-wilson disease mice,

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

[0032] Figure 4 is the effect of FTY720 on the neutrophil infiltration in the liver of liver-wilson disease mice,

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

[0034] Figure 5 Effect of FTY720 on liver macrophage infiltration in liver of mouse model of hepatolenticular degeneration,

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

[0036] Figure 6 Effect of FTY720 on expression level of liver inflammation and fibrosis related genes in mouse model of hepatolenticular degeneration,

[0037] wherein, Figure 6 A-F is the mRNA expression level of liver inflammation related genes, Figure 6 G-J 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 mice is significantly lower than that in the control group, indicating that the degree of liver damage in the experimental group mice is significantly improved). DETAILED DESCRIPTION

[0038] The following reference specification drawings introduce a plurality of preferred embodiments of the present application, so that the technical content thereof is more clear and convenient to understand. The present application can be embodied in many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned herein.

[0039] The technical scheme of the present application is as follows: by giving the Atp7b gene knockout mouse model of hepatolenticular degeneration mouse model Fingolimod (FTY720) treatment, the therapeutic effect of FTY720 on the liver damage index of Atp7b gene knockout mouse is evaluated. The administration route is intraperitoneal injection, and the treatment cycle is one month. The drug belongs to an effective dose of FTY720 and pharmaceutically acceptable adjuvant, specifically, the FTY720 drug powder is dissolved in 0.5% dimethyl sulfoxide, and the administration concentration is 1 mg / kg.

[0040] The experimental research by drug treatment shows that FTY20 can effectively reduce the plasma glutathione transaminase and glutathione transaminase level of mouse model of hepatolenticular degeneration. FTY720 can completely reverse the histological lesions of liver tissue of mouse model of hepatolenticular degeneration, significantly improve the degree of liver cell death, liver macrophage and neutrophil infiltration, and effectively reduce the expression of inflammation factor and chemokine related genes in liver tissue of mouse. FTY720 can significantly alleviate the degree of liver fibrosis of mouse model of hepatolenticular degeneration, and inhibit the expression of liver fibrosis related genes.

[0041] The specific content of the present application is illustrated below with specific examples:

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

[0043] 1. Method:

[0044] 1.1 Tissue fixation and embedding:

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

[0046] (2) The specimen is dehydrated in different concentrations of alcohol gradient (30%, 50%, 70%, 80%, 90%, 95%, 100%), changing every 30 minutes;

[0047] (3) The specimen is placed in a mixture of anhydrous ethanol and xylene (1:1) for 30 minutes, and then in 100% xylene, observing the tissue block at all times until it is transparent;

[0048] (4) The specimen is immersed in pre-melted paraffin liquid twice, each time for 3 hours;

[0049] (5) The melted paraffin liquid is poured into the embedding box, and the tissue block is placed in the 4°C refrigerator or water for condensation. After marking the direction of the tissue block, it is stored at 4°C for standby;

[0050] (6) The embedded tissue is cut into slices with a thickness of 4-6 μm;

[0051] 1.2 Staining:

[0052] (1) The slide is placed in a 55-60°C oven for 30 minutes, and then immersed in xylene for 5 times of deparaffinization, each time for 5-10 minutes;

[0053] (2) The slide is hydrated in different concentrations of ethanol (from high to low, 100%, 95%, 80%, 70%, each for 5 minutes), and then rinsed with single distilled water for 5 minutes;

[0054] (3) The slice is stained with hematoxylin dye for 5-10 minutes, rinsed with tap water for 3-5 minutes, then placed in 1% hydrochloric acid alcohol for differentiation for 5-30 seconds, rinsed with tap water for 1-3 minutes, and finally placed in 0.5% eosin alcohol dye for staining for 1 minute, and rinsed with single distilled water for 1 minute;

[0055] (4) The neutral resin is mounted, and the microscope is observed and photographed.

[0056] 2. Results: As Figure 1As shown, the liver cells of the untreated hepatolenticular degeneration mice were swollen and degenerated, the liver lobule structure was disordered, and balloon-like degenerative hepatocytes and extensive inflammatory cell infiltration were observed. The liver cell morphology and lobule structure of the FTY720-treated experimental mice were restored to normal, and the inflammatory cell infiltration was reduced. This indicates that FTY720 treatment can significantly improve the pathological damage of the liver of the hepatolenticular degeneration mice.

[0057] Example 2: Detection of plasma liver enzyme levels

[0058] 1. Method: The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the mouse liver were determined according to the instructions of the kit. The kit was obtained from Shanghai Shen Suyoufu Medical Diagnostics Limited Company, and the detection steps were as follows:

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

[0060] (2) The 96-well plate was removed, 50 μL of working solution R2 was added to each well, and incubated in a 37°C incubator for 5 minutes;

[0061] (3) The enzyme marker was preheated to 37°C, and then the 96-well plate with complete reaction was placed in the enzyme marker, and the absorbance at 340 nm was detected for a total of 20 cycles;

[0062] (4) Content calculation: the absorbance values of the first 5 and last 6 cycles were discarded, the absorbance difference between the 6th cycle and the 14th cycle was recorded as ΔOD, the time difference was recorded as ΔT, and the final concentration was calculated according to the following formula: concentration (U / L) = ΔOD / ΔT x 60 x 207.5 / 6.22 / 7.5 / 0.8 x 1000.

[0063] 2. Results: As shown, the plasma ALT and AST levels of the FTY720-treated mice were significantly lower than those of the control group, indicating that FTY720 treatment can significantly alleviate the liver damage of the hepatolenticular degeneration mice. Figure 2

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

[0065] 1. Method:

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

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

[0068] ​(2) Hydrating: The slides were hydrated in different concentrations of ethanol (from high to low, 100%, 95%, 80%, 70%, each for 5 minutes), and then rinsed with single distilled water for 5 minutes;

[0069] (3) Drop staining the sections with Sirius red staining solution for 1 hour, and then rinsing with running water to remove the floating color, and then restaining the cell nucleus with hematoxylin staining solution for 10 minutes, and then rinsing with running water to remove the floating color;

[0070] (4) Neutral resin mounting, and then observing under a microscope and taking photos, and then quantifying the photos by using ImageJ software.

[0071] 2. Results: As shown in Figure 3 A, the liver of the control mice without treatment showed extensive collagen fiber deposition, while the degree of fibrosis was significantly reduced after FTY720 treatment. Quantification of the Sirius red positive area showed that the positive area of the experimental group was significantly reduced (B), indicating that FTY720 treatment can significantly improve the liver fibrosis of the mouse with hepatolenticular degeneration. Figure 3

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

[0073] 1. Method:

[0074] The tissue fixation and embedding method was the same as that in Example 1, and the staining method was as follows:

[0075] (1) Baking the slides: The slides were placed in an oven at 55-60°C for 30 minutes;

[0076] (2) De-waxing: The slides were immersed in xylene for de-waxing 5 times, each for 5-10 minutes;

[0077] (3) Hydrating: The slides were hydrated in different concentrations of ethanol (from high to low, 100%, 95%, 80%, 70%, each for 5 minutes), and then rinsed with single distilled water for 5 minutes;

[0078] (4) Antigen repair: The slides were immersed in 10 mM citrate buffer (PH = 6.0), and then heated to 90-98°C by using a microwave oven, and incubated for 15 minutes;

[0079] (5) Inactivation: The 30% H2O2 solution was diluted 10 times by using single distilled water, and the sections were incubated in the dark for 30 minutes, and then washed with single distilled water for 3 times;

[0080] (6) Blocking: 5% BSA was used for blocking at room temperature for 20 minutes;

[0081] (7) Incubating the primary antibody: The primary antibody solution (antibody dilution ratio 1:400) was added dropwise to the slides, and then incubated at 4°C overnight;

[0082] ​(8) Incubate secondary antibody: Wash the slide with PBS for 3 times, 5 minutes each time, after absorbing the liquid, add secondary antibody solution (antibody dilution ratio 1:200) to the slide, incubate at room temperature for 30 minutes;

[0083] (9) Color development: Wash the slide with PBS for 3 times, 5 minutes each time, after absorbing the liquid, add DAB color developing solution to the slide, observe in real time under a microscope (reaction time about 5 minutes), use single distilled water to wash and stop color development;

[0084] (10) Hematoxylin staining: use hematoxylin staining solution to restain the cell nucleus for 5 minutes, and then wash with tap water for 1 minute;

[0085] (11) Dehydration: immerse the specimen in alcohol gradient of different concentrations (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, and then place it in 100% xylene, observe the tissue block at all times until it becomes transparent;

[0087] (13) Mounting: mount with neutral resin, observe under a microscope and take photos, and use ImageJ software to quantify the photos.

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

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

[0090] 1、Method:

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

[0092] (2) The target gene was detected using specific primers, and the internal reference GAPDH primers were:

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

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

[0095] The primer sequence for amplifying Tnfα was:

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

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

[0098] The primer sequence for amplifying I11β was:

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

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

[0101] The primer sequence for amplifying I16 was:

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

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

[0104] The primer sequence for amplifying Ccl2 was:

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

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

[0107] The primer sequence for amplifying Ccl3 is:

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

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

[0110] The primer sequence for amplifying Cxcl2 is:

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

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

[0113] The primer sequence for amplifying Tgfβ is:

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

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

[0116] The primer sequence for amplifying αSMA is:

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

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

[0119] The primer sequence for amplifying Col1α1 is:

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

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

[0122] The primer sequence for amplifying Col3α1 is:

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

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

[0125] The PCR reaction condition is: 95℃ pre-denaturation for 30s, 95℃ for 10s, 60℃ for 30s, 40 cycles.

[0126] 2、Results: As shown in Figs. Figure 6 A-F, the mRNA levels of inflammatory factors Tnfα, Il1β, Il6 and chemotactic factors Ccl2, Ccl3, Cxcl2 in the liver of the experimental mice were significantly lower than those of the control group. Meanwhile, the mRNA levels of fibrosis-related genes Tgfβ, αSMA, Col1α1, Col3α1 in the liver of the experimental mice were also significantly lower than those of the control group. It is indicated that FTY720 treatment can significantly improve the degree of liver inflammation and fibrosis in the liver of the mouse with hepatolenticular degeneration.

[0127] In summary, the above examples show the application of FTY720 in the treatment of liver damage in hepatolenticular degeneration. The combination of FTY720 and copper chelating agents can provide new drug treatment opportunities for patients who do not respond well to copper chelating agents, and reduce the occurrence of decompensated liver disease and death.

[0128] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make modifications and changes to the embodiments without creative effort based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. The use of fingolimod hydrochloride in the preparation of drugs for the prevention or treatment of liver injury caused by Wilson's disease.

2. The application according to claim 1, characterized in that, The chemical formula of the fingolimod hydrochloride is C19H33NO2.HCl.

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

4. The application according to claim 1, characterized in that, The drug is administered by injection.

5. The application according to claim 4, characterized in that, The drug powder is dissolved in 0.5% dimethyl sulfoxide and administered by injection.

6. The application according to claim 5, characterized in that, The dosage concentration is 1 mg / kg.

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