Use of a trpm11-specific small molecule inhibitor ml-si3

Abstract

The application provides application of a TRPML1 specific small molecule inhibitor ML-SI3 in preparation of a medicine for treating or preventing nephritis. The application has the following beneficial effects: the TRPML1 specific small molecule inhibitor ML-SI3 can significantly reduce the serum anti-dsDNA antibody titer level of MRL / lpr lupus mice, reduce the urine albumin / creatinine ratio level, reduce the serum urea nitrogen level, reduce the swelling degree of the spleen and axillary lymph nodes, improve the kidney pathological changes, has a comprehensive protective effect on nephritis of MRL / lpr lupus mice, and provides application of a TRPML1 specific small molecule inhibitor ML-SI3 in preparation of a medicine for treating or preventing nephritis, provides a new medicine for treating lupus nephritis, and has good market value and clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a TRPML1-specific small molecule inhibitor, ML-SI3. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple systems. The kidneys are among the most commonly affected organs. Thanks to advances in treatment, the 5-year survival rate for SLE patients has reached 94%, and the 10-year survival rate has reached 89%. However, kidney inflammation caused by SLE remains a leading cause of death. Even with aggressive treatment using glucocorticoids, hydroxychloroquine, and immunosuppressants, nephritis can be relieved and kidney function can recover. Nevertheless, a significant proportion of lupus nephritis patients eventually develop end-stage renal disease, leading to death. Therefore, actively seeking other effective drugs for lupus nephritis remains essential. Summary of the Invention

[0003] This application provides an application of ML-SI3, a TRPML1-specific small molecule inhibitor, which aims to address the problem of kidney inflammation caused by lupus erythematosus in existing technologies.

[0004] This application provides the use of ML-SI3, a TRPML1-specific small molecule inhibitor, in the preparation of drugs for the treatment or prevention of nephritis.

[0005] According to some embodiments of the application described in this application, the nephritis is lupus nephritis.

[0006] According to some embodiments of the application described in this application, the structural formula of the TRPML1-specific small molecule inhibitor ML-SI3 is shown below:

[0007]

[0008] According to some embodiments of the application described in this application, in the preparation of drugs for treating or preventing nephritis, the effective dose of the TRPML1-specific small molecule inhibitor ML-SI3 is 2-5 mg / kg, such as 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, etc.

[0009] According to some embodiments of the application described in this application, in the preparation of drugs for treating or preventing nephritis, the effective dose of the TRPML1-specific small molecule inhibitor ML-SI3 is 3 mg / kg.

[0010] A second aspect of this application provides a medicament for treating lupus nephritis, comprising a pharmaceutically active ingredient and pharmaceutically acceptable excipients, wherein the pharmaceutically active ingredient comprises a TRPML1-specific small molecule inhibitor, ML-SI3.

[0011] According to some embodiments of the drug for treating lupus nephritis described in this application, the content of the active pharmaceutical ingredient in the drug is 2-5 mg / kg, such as 2 mg / kg, 2.8 mg / kg, 3.2 mg / kg, 3.5 mg / kg, 4.1 mg / kg, 4.6 mg / kg, 5.0 mg / kg, etc.

[0012] According to some embodiments of the medicament for treating lupus nephritis described in this application, the content of the active pharmaceutical ingredient in the medicament is 3 mg / kg.

[0013] According to some embodiments of the medicament for treating lupus nephritis described in this application, the dosage form of the medicament includes injections, tablets, capsules, granules, aerosols, or oral liquids.

[0014] The beneficial effects of this application include:

[0015] The TRPML1-specific small molecule inhibitor ML-SI3 described in this application can significantly reduce the serum anti-dsDNA antibody titer in MRL / lpr lupus mice; reduce the urinary albumin / creatinine ratio; reduce serum urea nitrogen levels; alleviate the swelling of the spleen and axillary lymph nodes; improve kidney pathological changes; and provide comprehensive protection against nephritis in MRL / lpr lupus mice.

[0016] This application provides the use of ML-SI3, a TRPML1-specific small molecule inhibitor, in the preparation of drugs for the treatment or prevention of nephritis, providing a new drug for the treatment of lupus nephritis, with good market value and clinical application prospects. Attached Figure Description

[0017] Figure 1 A schematic diagram illustrating the preparation and drug administration timeline of MRL / lpr lupus nephritis model mice;

[0018] Figure 2 Figure showing the effect of ML-SI3 administration on serum anti-dsDNA antibody titers in MRL / lpr lupus mice;

[0019] Figure 3 Figure showing the effect of ML-SI3 administration on the urinary albumin / creatinine ratio in MRL / lpr lupus mice;

[0020] Figure 4 Figure showing the effect of ML-SI3 administration on serum urea nitrogen levels in MRL / lpr lupus mice;

[0021] Figure 5 Figure showing the effect of ML-SI3 administration on the degree of spleen and axillary lymph node swelling in MRL / lpr lupus mice;

[0022] Figure 6 The image shows the results of H&E and PAS staining to assess the pathological changes in mouse kidneys. Detailed Implementation

[0023] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0024] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0025] This application provides a novel use of the TRPML1-specific small molecule inhibitor ML-SI3 in the preparation of a drug for treating lupus nephritis. Specifically, when the inhibitor is used to prepare a drug for treating lupus nephritis, it can significantly reduce the serum anti-dsDNA antibody titer in MRL / lpr lupus mice; reduce urinary protein levels; reduce serum urea nitrogen levels; alleviate splenomegaly and lymph node swelling; and improve renal pathological changes, thus providing comprehensive protection against lupus nephritis in MRL / lpr mice.

[0026] In the treatment of lupus nephritis, the effective dose of the TRPML1-specific small molecule inhibitor ML-SI3 is 2-5 mg / kg, preferably 3 mg / kg.

[0027] Studies have shown that the lysosomal cation channel TRPML1 is a target for regulating cation release from lysosomes. TRPML1 expression is significantly upregulated in the kidney tissue of MRL / lpr lupus mice, and the expressed TRPML1 channel participates in the pathological process of lupus nephritis by releasing cations from lysosomes. By using an inhibitor to reduce TRPML1 channel activity, the fusion barrier between lysosomes and autophagosomes in renal podocytes can be relieved, thereby alleviating podocyte autophagy and restoring normal podocyte autophagy. This restored podocyte autophagy can improve podocyte function in the kidneys of MRL / lpr mice, thus reducing the degree of kidney damage in lupus mice and providing protection against lupus nephritis in MRL / lpr mice. The following is ML-SI3, a specific small molecule inhibitor of TRPML1 in this application (its chemical structure is shown below). Figure 1 This study validated the therapeutic effect of MRL / lpr mice on lupus nephritis.

[0028] 1. Test Methods

[0029] Adult female MRL / lpr mice were used as experimental animals. Sixteen 8-10 week old female lupus-susceptible MRL / lpr mice were selected and randomly divided into two groups: the ML-SI3 group and the Vehicle group. Eight 8-10 week old adult female MRL / Mpj mice were selected as a normal control group. All mice were raised under normal conditions, and their body weight was recorded weekly starting at 11 weeks of age. At 12 weeks of age, ML-SI3 mice were intraperitoneally injected with ML-SI3 (0.1 ml per 10 g body weight, a concentration of 0.3 mg / ml), while Vehicle mice were injected with saline (0.1 ml per 10 g body weight) for 6 consecutive weeks. At 18 weeks of age, the effects of ML-SI3 on ​​LN mice were analyzed—serum anti-dsDNA antibody titer levels were analyzed; urinary albumin / creatinine ratio was measured; serum urea nitrogen levels were measured; the length of axillary lymph nodes and spleen was measured; and kidney sections were stained with PAS or H&E to evaluate the degree of kidney lesions. MRL / lpr mice were pathologically confirmed to have nephritis. The MRL / Mpj mice were the normal control group (without nephritis phenotype, raised under normal conditions). Modeling and ML-SI3 intervention methods are as follows: Figure 1 As shown:

[0030] Vehicle group: MRL / lpr mice were injected intraperitoneally with saline.

[0031] ML-SI3 group: MRL / lpr mice were administered ML-SI3 intraperitoneally.

[0032] MRL / Mpj group: This group consists of normal control mice without the nephritis phenotype.

[0033] Figure 1 Here is a diagram of the ML-SI3 intervention model in MRL / lpr lupus mice.

[0034] 2. Effect of ML-SI3 administration on serum anti-dsDNA antibody titers in MRL / lpr lupus mice

[0035] 2.1 Measurement method:

[0036] Mice were harvested at 18 weeks of age. Mice were anesthetized, and their whiskers were trimmed with ophthalmic scissors. With the mice head down, their necks were gently compressed to cause their eyeballs to protrude, and the eyeballs were removed with curved forceps to collect venous blood. Immediately after blood collection, the blood was transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature (25°C) for 30 minutes. The blood sample was centrifuged at 14000g for 15 minutes, and the separated serum was pipetted and transferred to a clean centrifuge tube. The serum sample could be centrifuged again at 14000g for 2 minutes to separate any remaining red blood cells. The separated serum was transferred to a clean centrifuge tube, and the level of anti-dsDNA antibodies in the serum was analyzed. All data were statistically analyzed and plotted using Graphpad 7.0 software.

[0037] The specific process is as follows: All steps are carried out at room temperature (25℃). Before each addition of reagent to start incubation, the plate should be gently tapped to mix the contents of the well.

[0038] (a) Sample addition: Dilute mouse serum samples at a ratio of 1:10000. Then add 100 μL each of the sample dilution buffer (blank control), standard, and diluted sample provided by the mouse anti-double-stranded DNA IgG ELISA kit to the designated wells. Gently tap the plate to mix the liquid and incubate at room temperature (25°C) for 60 min. Discard the liquid in the wells, add 200 μL / well of working wash buffer, incubate at room temperature (25°C) for 30 s, then discard. Repeat the washing process 3 times.

[0039] (b) Antibody incubation: Add 100 μL of diluted HRP anti-mouse IgG to each reaction well, incubate at room temperature (25°C) for 30 min, and then repeat the washing steps above for 5 washes.

[0040] (c) Adding substrate for color development: Add 100 μL of TMB substrate to each reaction well. The liquid in the well will start to turn blue. Incubate at room temperature (25°C) in the dark for 15 min.

[0041] (d) Termination of reaction: Add 100 μL of termination solution to each well, gently tap to mix, the enzyme reaction will stop, and the liquid in the well will turn yellow.

[0042] (e) Absorbance readings: Within 30 minutes of adding the stop reaction solution, read the OD values ​​of each well at single wavelengths of 450 nm and 630 nm using a microplate reader. Plot a standard curve and calculate the sample concentration. The results are as follows: Figure 2 As shown.

[0043] from Figure 2 As can be seen, ML-SI3 administration significantly reduced the serum anti-dsDNA antibody titer level in MRL / lpr lupus mice. MRL / Mpj mice were normal control mice.

[0044] 3. Effect of ML-SI3 administration on the urinary albumin / creatinine ratio in MRL / lpr lupus mice

[0045] 3.1 Measurement Method

[0046] Sixteen 8-10 week old female lupus-susceptible MRL / lpr mice were selected as experimental animals and randomly divided into two groups: the ML-SI3 group and the Vehicle group. Eight 8-10 week old adult female MRL / Mpj mice were selected as a normal control group. All mice were raised under normal conditions. Starting at 11 weeks of age, mouse weight was recorded weekly for 7 weeks. At 12 weeks of age, ML-SI3 mice were intraperitoneally injected with ML-SI3 (0.1 ml per 10 g body weight, concentration 0.3 mg / ml), while Vehicle mice were intraperitoneally injected with physiological saline (0.1 ml per 10 g body weight) for 6 consecutive weeks. At 18 weeks of age, 12-hour urine samples were collected from the mice in metabolic cages, and the urinary albumin / creatinine ratio was measured. All data were statistically analyzed and plotted using Graphpad 7.0 software.

[0047] The method for measuring urinary albumin is as follows (urinary albumin reagent kit):

[0048] a. Reagent preparation

[0049] b. Sample preparation: Add 25 μL of mouse urine sample to the desired wells in a 96-well white plate. Adjust the volume to 50 μL / well with albumin diluent.

[0050] c. Preparation of Standard Dilution: Dilute the 2 mg / mL BSA Standard II / BSA to 0.5 mg / mL by adding 25 μL of the BSA Standard II / BSA provided in the albumin-creatinine ratio kit to 75 μL of albumin diluent. Add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of 0.5 mg / mL BSA Standard II / BSA to the wells of a 96-well plate. Adjust the total volume of each well to 50 μL with albumin diluent to generate 0 μg, 1 μg, 2 μg, 3 μg, 4 μg, and 5 μg BSA Standard II / BSA per well.

[0051] d. Albumin Assay Procedure: Prepare a total of 50 μL of mixture for each well. Prepare the reaction mixture by mixing sufficient reagents. The reaction mixture consists of 46 μL of albumin assay buffer II and 4 μL of albumin probe; the background mixture consists of only 50 μL of albumin assay buffer II. Mix thoroughly before use. Add 50 μL of the reaction mixture to each well containing BSA standard II / BSA standard (sample); add 50 μL of the background mixture to each well containing the sample background control.

[0052] e. Measurement: Incubate at 25°C in the dark for 30 minutes. Measure fluorescence in endpoint mode (excitation / emission = 600 / 630 nm).

[0053] The method for measuring urine creatinine is as follows (urine creatinine reagent kit):

[0054] a. Reagent preparation

[0055] b. Sample Preparation: Add 25 μL of mouse urine sample to the required wells in a 96-well clear plate. Adjust the volume to 50 μL / well using assay buffer XXXIV / creatinine assay buffer. Standard Dilution Preparation: Dilute the 10 μg / mL creatinine standard to 0.1 μg / μL by adding 10 μL of the standard to 990 μL of ddH₂O. Add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of 0.1 μg / μL creatinine standard to the wells of the 96-well plate. Adjust the total volume of each well to 50 μL using assay buffer XXXIV / creatinine assay buffer to generate 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg, and 1 μg creatinine standards per well.

[0056] c. Creatinine Assay Procedure: Prepare a reaction mixture by mixing sufficient reagents. For each well, prepare a total of 50 μL of mixture. The reaction mixture consists of 42 μL of assay buffer XXXIV / creatinine assay buffer, 2 μL of creatine kinase, 2 μL of creatinine kinase, 2 μL of creatinine kinase mixture / creatinine kinase mixture, and 2 μL of LOxiRed probe / creatinine probe. The background mixture consists of 44 μL of assay buffer XXXIV / creatinine assay buffer, 2 μL of creatine kinase, 2 μL of creatine kinase mixture / creatinine kinase mixture, and 2 μL of LOxiRed probe / creatinine probe, mixed thoroughly before use. Add 50 μL of the reaction mixture to each well containing a creatinine standard (sample); add 50 μL of the background mixture to each well containing a sample background control.

[0057] e. Urine creatinine determination: Incubate the plate at 37°C for 60 minutes, avoiding light exposure. Measure the absorbance (OD570nm) in endpoint mode. Results are as follows: Figure 3 As shown.

[0058] from Figure 3 As can be seen, compared with the control group (Vehicle), ML-SI3 administration significantly reduced the urinary albumin / creatinine level in MRL / lpr lupus mice. MRL / Mpj mice represent the urinary albumin / creatinine ratio of normal control mice.

[0059] 4. Effect of ML-SI3 administration on serum urea nitrogen levels in MRL / lpr lupus mice

[0060] 4.1 Measurement Method

[0061] Sixteen 8-10 week old female lupus-susceptible MRL / lpr mice were randomly divided into two groups: the ML-SI3 group and the Vehicle group. Eight 8-10 week old adult female MRL / Mpj mice served as a normal control group. All mice were raised under normal conditions. At 12 weeks of age, the ML-SI3 group mice were intraperitoneally injected with ML-SI3 (0.1 ml per 10 g body weight, 0.3 mg / ml), while the Vehicle group mice were intraperitoneally injected with saline (0.1 ml per 10 g body weight) for 6 consecutive weeks. At 18 weeks of age, the mice were harvested. The mice were anesthetized, and their whiskers were trimmed on both sides. The mice were positioned head down, and their necks were gently compressed to cause their eyeballs to protrude. The eyeballs were then removed with curved forceps to collect venous blood. Immediately after blood collection, the blood was placed in a 1.5 mL centrifuge tube and allowed to stand at room temperature (25°C) for 30 minutes. Centrifuge blood samples at 14000g for 15 minutes, aspirate the separated serum using a pipette, and transfer it to a clean centrifuge tube. The serum sample can be centrifuged again at 14000g for 3 minutes to separate any remaining red blood cells. Transfer the separated serum to a clean centrifuge tube and analyze the blood urea nitrogen level in the serum. All data were statistically analyzed and plotted using Graphpad 7.0 software.

[0062] The method for measuring blood urea nitrogen levels is as follows:

[0063] a. Equilibrate the aluminum foil bag at room temperature (25°C) for 20 minutes, remove the required strips, seal the remaining strips in a self-sealing bag, and store them dry at 4°C.

[0064] b. Set up standard wells and sample wells, and add 50 μL of standard solution of different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1, 3, 5, 10, 15, 20, 25 mmol / L) from the urea nitrogen (BUN) test kit to each standard well.

[0065] c. Add 10 μL of the sample solution to be tested to the test sample well, followed by 40 μL of sample diluent. Do not add any sample to the blank well.

[0066] d. Except for the blank wells, add HRP-labeled detection antibody to the standard wells and the sample wells at a rate of 100 μL / well, then seal the reaction wells with sealing film and incubate at 37°C in a water bath or incubator for 1 hour.

[0067] e. Discard the liquid in the well, drain the remaining liquid on absorbent paper, fill with washing buffer, let stand for 1 minute, shake off the washing buffer, drain on absorbent paper, and repeat the washing process 5 times.

[0068] f. Add 50 μL of substrates A and B from the kit to each well and incubate at 37°C in the dark for 15 minutes.

[0069] g. Add 50 μL of the stop solution from the kit to each well, set the detection wavelength of the microplate reader to 450 nm, and measure the OD value of each well within 15 minutes. The results are as follows: Figure 4 As shown.

[0070] from Figure 4 As can be seen, compared with the control group (Vehicle), ML-SI3 administration significantly reduced the blood urea nitrogen level in MRL / lpr lupus mice. MRL / Mpj mice represent the normal control mice in terms of blood urea nitrogen level.

[0071] 5. Effects of ML-SI3 administration on the degree of spleen and axillary lymph node swelling in MRL / lpr lupus mice

[0072] 5.1 Measurement Method

[0073] Sixteen female lupus-susceptible MRL / lpr mice aged 8-10 weeks were randomly divided into two groups: an ML-SI3 group and a Vehicle group. Eight adult female MRL / Mpj mice aged 8-10 weeks served as a normal control group. Mice were raised under normal conditions. At 12 weeks of age, the ML-SI3 group mice were intraperitoneally injected with ML-SI3 (0.1 ml per 10 g body weight, 0.3 mg / ml), while the Vehicle group mice were intraperitoneally injected with physiological saline (0.1 ml per 10 g body weight) for 6 consecutive weeks. At 18 weeks of age, the mice were anesthetized, and the left ventricle was perfused with PBS buffer. The spleen and axillary lymph nodes were collected, and their sizes were measured and compared between the control group and the ML-SI3 group. All data were statistically analyzed and plotted using Graphpad 7.0 software. The results are shown below. Figure 5 As shown.

[0074] from Figure 5 As can be seen, compared with the control group (Saline), ML-SI3 administration significantly reduced the swelling of the spleen and axillary lymph nodes in MRL / lpr lupus mice. The spleen and axillary lymph node sizes in MRL / Mpj mice are those of normal control mice. The scale bar is in centimeters.

[0075] 6. Effects of ML-SI3 administration on renal pathological changes in MRL / lpr lupus mice

[0076] 6.1 Measurement Method

[0077] Sixteen 8-10 week old female lupus-susceptible MRL / lpr mice were randomly divided into two groups: the ML-SI3 group and the Vehicle group. Eight 8-10 week old adult female MRL / Mpj mice served as a normal control group. Mice were raised under normal conditions. At 12 weeks of age, the ML-SI3 group mice were intraperitoneally injected with ML-SI3 (0.1 ml per 10 g body weight, 0.3 mg / ml), while the Vehicle group mice were intraperitoneally injected with physiological saline (0.1 ml per 10 g body weight) for 6 consecutive weeks. At 18 weeks of age, the mice were anesthetized, and the left ventricle was perfused with PBS buffer. Intact mouse kidneys were collected for HE and PAS staining.

[0078] HE staining: Kidney tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and paraffin sections were prepared from the renal cortex. The prepared paraffin sections were dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin, differentiated with hydrochloric acid and ethanol, stained with eosin, then dehydrated with graded ethanol, cleared with xylene, and finally mounted with neutral resin. Changes in glomerular inflammatory cell infiltration, capillary lesions, and glomerular cell proliferation were observed under a light microscope to assess the degree of LN renal lesions.

[0079] PAS staining: Paraffin sections of renal cortex tissue fixed in 4% paraformaldehyde were dewaxed with xylene and then stained with PAS. PAS staining, also known as glycogen staining, is a routine staining method in pathology. This staining solution can visualize not only glycogen but also neutral mucous substances, certain acidic substances, amyloid substances, and the basement membrane. Under a light microscope, glomerular cell proliferation, basement membrane thickening, and changes in the mesangial area can be observed. It is also one of the methods for assessing the pathological changes in nephropathy (LN). The staining results are as follows: Figure 6 As shown.

[0080] from Figure 6 As can be seen, compared with the control group, ML-SI3 administration significantly reduced the renal pathological changes in MRL / lpr lupus mice. MRL / Mpj mice are normal control mice, and the results of HE and PAS staining are shown. The scale bar is 20 micrometers.

[0081] The TRPML1-specific small molecule inhibitor ML-SI3 described in this application can significantly reduce the serum anti-dsDNA antibody titer in MRL / lpr lupus mice; reduce the urinary albumin / creatinine ratio; reduce urea nitrogen levels; alleviate the swelling of the spleen and axillary lymph nodes; improve kidney pathological changes; and provide comprehensive protection against lupus nephritis in MRL / lpr mice.

[0082] The above experimental results demonstrate that ML-SI3 small molecule inhibitors can comprehensively improve the progression of lupus nephritis in mouse models. This invention provides a new drug for the treatment of lupus nephritis, with good market value and clinical application prospects.

[0083] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made to the above embodiments by those skilled in the art are within the protection scope of the present invention.

Claims

1. The application of a TRPML1-specific small molecule inhibitor, ML-SI3, in the preparation of drugs for the treatment or prevention of lupus nephritis.

2. The application according to claim 1, characterized in that, The structural formula of the TRPML1-specific small molecule inhibitor ML-SI3 is shown in the figure below: 。 3. The application according to claim 1, characterized in that, In the preparation of drugs for the treatment or prevention of lupus nephritis, the effective dose of the TRPML1-specific small molecule inhibitor ML-SI3 is 2-5 mg / kg.

4. The application according to claim 1, characterized in that, In the preparation of drugs for the treatment or prevention of lupus nephritis, the effective dose of the TRPML1-specific small molecule inhibitor ML-SI3 is 3 mg / kg.

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