Use of LCN2 inhibitor in treatment of neuron wax-like lipidosis in infants

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of an LCN2 inhibitor in treatment of neuron wax-like lipidosis of infants. According to the invention, the LCN2 inhibitor is used, so that overexpression of glial fibrillary acidic protein in astrocytes and hypertrophy and excessive proliferation of the astrocytes are remarkably inhibited. When the LCN2 inhibitor is applied to a mouse of which the PPT1 gene is knocked out, overexpression of colloidal fibrillary acidic protein in brain tissues of the mouse is remarkably inhibited. Experiments show that the LCN2 inhibitor has the potential of treating hypertrophy of astrocytes and / or hyperproliferative diseases of astrocytes, and particularly has the potential of treating neuron wax-like lipid deposition of infants. For the LCN2 inhibitor, the purpose of intervention treatment can be achieved through oral administration without a special administration mode, the advantages of being safe, simple and convenient are achieved, a new direction is brought to treatment of INCL diseases, and the development prospect is good.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of LCN2 inhibitors in treating infantile neuronal ceroid lipidosis. Background Art

[0002] Infantile Neuronal Ceroid Lipofuscinosis (INCL) is a rare hereditary neurodegenerative disease in children. INCL usually develops in childhood and is pathologically manifested as the deposition of yellow autofluorescent lipid pigments in nerve cells and other cells, leading to the loss of nerve cells mainly in the cerebral cortex and retina. Clinical manifestations include progressive dementia, refractory epileptic seizures, and vision loss. As the disease progresses, affected children gradually lose basic motor skills such as sitting and walking, usually need to use a wheelchair, and have a short lifespan, often not surviving their teens. Therefore, delaying the course of the disease is crucial to the treatment of INCL.

[0003] Currently, the only drug approved for the treatment of INCL (especially type 2, also known as CLN2) is Cerliponase α (trade name Brineura), a drug that requires intraventricular administration and was approved by the FDA in 2017 to relieve symptoms such as loss of walking ability in pediatric patients with CLN2 aged 3 years and older. In addition, other treatments are under development, including stem cell transplantation, enzyme replacement therapy, gene therapy, and immunotherapy. These treatments offer hope for the future treatment of INCL patients. However, these methods involve complex treatments, and their widespread use is limited. Further development of new treatments is still necessary.

[0004] Finding new targets or pathways to treat INCL is a feasible approach. Lipocalin-2 (LCN2) is a secreted glycoprotein and a lipophilic small molecule transporter. It plays an important role in inducing apoptosis of hematopoietic cells, regulating inflammation, metabolic homeostasis, and the occurrence and development of tumors. According to current research, LCN2 expression levels are abnormally elevated in a variety of cancers and is considered to be a promising target. LCN2 inhibitors have shown potential application value in the treatment of a variety of cancers, especially in the treatment of inflammatory breast cancer, brain injury, and bile duct cancer.

[0005] Therefore, it is promising to explore LCN2 targets to treat ICNL. However, in the prior art, there is no report on methods based on LCN2 targets for the treatment of ICNL, and the art is unable to establish methods for the treatment of ICNL based on LCN2 inhibitors through existing theoretical guidance. This has created a major obstacle to the use of LCN2 inhibitors to delay the course of ICNL. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a use of an LCN2 inhibitor in the intervention treatment of INCL disease, aiming to establish a safe and simple treatment method for INCL based on the LCN2 target.

[0007] The present invention provides use of an LCN2 inhibitor in preparing a medicament for treating diseases of neural astrocyte hypertrophy and / or excessive proliferation.

[0008] Preferably, the disease of neuroastrocyte hypertrophy and / or excessive proliferation is infantile neuronal ceroid lipidosis.

[0009] Preferably, the neuronal ceroid lipidosis of infancy is neuronal ceroid lipidosis of infancy in mice.

[0010] Preferably, the dosage of the LCN2 inhibitor is 5-20 mg / kg; and / or, when the mice are less than 3 months old, the drug is administered once a day, and when they are 3 months old, the drug is administered once every two days until they are 6 months old; when the mice are more than 3 months old, the drug is administered once a day for at least 7 days.

[0011] Preferably, the structural formula of the LCN2 inhibitor is:

[0012]

[0013] Wherein, R1, R2, and R3 are independently selected from halogen, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, C1-C 10 Alkoxy;

[0014] m is selected from 0, 1, 2, 3, 4, 5;

[0015] n is selected from 0, 1, 2, 3;

[0016] k is selected from 0, 1, 2, 3.

[0017] Preferably, the R1 is C1-C 10 and / or, m is 1, n is 0, and k is 0.

[0018] Preferably, the compound represented by formula I includes the following compounds:

[0019]

[0020] Preferably, the LCN2 inhibitor is selected from ZINC 00640089 and ZINC 00784494.

[0021] The present invention also provides a pharmaceutical composition, which is prepared by taking the LCN2 inhibitor as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0022] Preferably, the structural formula of the LCN2 inhibitor is as follows:

[0023]

[0024] Wherein, R1, R2, and R3 are independently selected from halogen, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, C1-C 10 Alkoxy;

[0025] m is selected from 0, 1, 2, 3, 4, 5;

[0026] n is selected from 0, 1, 2, 3;

[0027] k is selected from 0, 1, 2, 3.

[0028] The present invention uses LCN2 inhibitors to significantly inhibit the overexpression of glial fibrillary acid protein (GFAP) in astrocytes, the hypertrophy and overproliferation of astrocytes. When the LCN2 inhibitor is applied to PPT1-KO mice, the overexpression of GFAP protein in the brain tissue of PPT1-KO mice is significantly inhibited. This shows that LCN2 inhibitors have the potential to treat INCL and can be used to prepare drugs for treating INCL. For LCN2 inhibitors, no special administration method is required, and the purpose of intervention treatment can be achieved by oral administration, which has the advantages of safety and simplicity, and brings a new direction for the treatment of INCL diseases. At the same time, LCN2 inhibitors can also be used to prepare drugs for treating diseases related to hypertrophy or overproliferation of astrocytes, and have a good development prospect.

[0029] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0030] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0031] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C 1～4 The term "alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0032] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 member atoms, such as 1 to 4 member atoms. The alkyl group can be straight or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl. The alkyl group may also be part of other groups, such as C1-C6 alkoxy.

[0033] "Alkoxy" refers to an alkyl group attached to the site of attachment through an oxygen atom, for example, methoxy refers to -OCH3.

[0034] "Ether" refers to a saturated alkane chain having the specified number of carbon atoms containing at least one oxygen atom.

[0035] The "halogen" refers to at least one of fluorine, chlorine, bromine and iodine.

[0036] "Haloalkyl" means that the hydrogen atoms in the alkyl group may be replaced by one or more halogen atoms. 1～4 The halogenalkyl group refers to an alkyl group containing 1 to 4 carbon atoms in which hydrogen atoms are substituted by one or more halogen atoms.

[0037] "LCN2 inhibitor" refers to any substance that has the effect of inhibiting the activity of LCN2. These substances can be small molecule compounds, polypeptides or antibodies, including molecules that can inhibit the activity of LCN2 reported in the prior art, such as ZINC 00640089, ZINC 00784494, etc.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The graph shows the results of the LCN2 inhibitor concentration and astrocyte survival rate;

[0041] Figure 2 is the gel electrophoresis diagram of GFAP expression in astrocytes;

[0042] Figure 3 This is the enhanced chemiluminescence (ECL) detection result of GFAP expression in astrocytes.

[0043] Figure 4 This is a microscope image of astrocytes;

[0044] Figure 5 This is the pixel ratio map corresponding to the microscope image of astrocytes;

[0045] Figure 6 is a graph showing the proliferation of astrocytes over time;

[0046] Figure 7 This is the gel electrophoresis diagram of GFAP expression in mouse brain tissue;

[0047] Figure 8 This is the ECL detection result of GFAP expression in mouse brain tissue. DETAILED DESCRIPTION

[0048] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0049] INCL model animals are PPT1 knockout mice (PPT1-KO), which were donated by Professor Anil B. Mukherjee of the National Institutes of Health. The construction method is based on the following literature: Bouchelion A, Zhang Z, Li Y, Qian H, Mukherjee AB. Mice homozygous for c.451C>T mutation in Cln1 gene recapitulate INCL phenotype. Ann Clin Transl Neurol.

[0050] 2014;1(12):1006-1023.doi:10.1002 / acn3.144.

[0051] Example 1 Use of LCN2 inhibitors in interventional treatment of INCL

[0052] This embodiment provides a use of an LCN2 inhibitor in the intervention treatment of INCL.

[0053] 1. Reagents

[0054] LCN2 inhibitor: ZINC 00640089, catalog number HY-Q4570, structural formula is as follows:

[0055]

[0056] LCN2 inhibitor solution: Dissolve 5 mg of ZINC 00640089 compound in 2.7 mL of DMSO to prepare a stock solution with a concentration of 5 mM. Before use, take 67.5 μL of the stock solution and dissolve it in 5 mL of sterile water.

[0057] 2. LCN2 inhibitors for interventional treatment of INCL

[0058] The mice were accurately weighed and the LCN2 inhibitor solution was orally administered to PPT1-KO mice at a dose of 5 mg / kg. The administration was started once a day from 2 months of age, and then once every 2 days after 3 months of age, and continued until 6 months of age.

[0059] In other embodiments, if the mouse is over 3 months old, the drug can be orally administered once a day for one week.

[0060] The technical solution of the present invention is further illustrated by experiments below.

[0061] Experimental Example 1 Effects of LCN2 inhibitor on astrocytes in PPT1-KO mice

[0062] 1. Experimental Methods

[0063] 1. Acquisition of astrocytes

[0064] Wild-type mice (WT) and PPT1-KO newborn mice were sterilized with 75% alcohol and killed by decapitation. The brains were taken and placed in cold D-Hanks balanced salt solution to remove the meninges and large blood vessels. The cerebral cortices on both sides were separated and cut into pieces. Then, 0.25% trypsin was added to digest in a 37°C water bath for 15 minutes. After that, the digestion was terminated with DME / F12 medium containing 13% fetal bovine serum (FBS) and the tissue was gently blown. After the tissue was completely separated, it was filtered through a 75μm mesh, the filtrate was collected, and the filtrate was centrifuged at 500rcf for 10 minutes. The precipitate was collected and resuspended with DME / F12 medium containing 13% FBS to adjust the cell density to 1.5×10 6 / ml, planted at 75cm 2 The cells were cultured in culture flasks (the culture medium was changed every 3 days). On the 9th day, the culture flasks were placed on a constant temperature rotary shaker at 37°C, 240 rpm, and shaken for 18 h. The suspended cells were removed and washed 3 times with D-Hanks solution to obtain purified WT and PPT1-KO astrocytes, respectively.

[0065] 2. Effect of LCN2 inhibitors on astrocyte IC 50 Determination of

[0066] PPT1-KO astrocytes were taken and prepared into a single cell suspension in DME / F12 medium containing 13% FBS. 1x10 4 Cells were inoculated into 96-well plates, with a volume of 100 μL per well. After 3 days of culture, the culture medium was removed. Then, LCN2 inhibitors were prepared into solutions with concentrations of 0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, 1 mM, and 2 mM, and 100 μL was added to astrocytes. After 48 hours of treatment, 10 μL of CCK8 reagent was added to each well and reacted for 4 hours. Finally, a wavelength of 450 nm was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the results were recorded.

[0067] The inhibitor concentration was converted into logarithmic form, and the logarithmic inhibitor concentration was used as the X-axis, and the reduced survival rate of astrocytes compared with the control group (0 μM) was used as the Y-axis. The X value corresponding to the Y value when the survival rate was reduced by 50% was calculated according to the linear formula, and then the corresponding inhibitor concentration was calculated. The result is IC 50 value.

[0068] 3. Determination of the effect of LCN2 inhibitor on protein expression in astrocytes

[0069] Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is mainly present in astrocytes of the central nervous system and is a marker for astrocyte activation. The present invention measures the expression of GFAP in mouse astrocytes, and the measurement method is as follows:

[0070] WT and PPT1-KO astrocytes were taken separately, and astrocytes were treated with LCN2 inhibitor (5μM) for 48h. The cells were collected and lysed in cell lysis buffer on ice. After lysis, centrifuged at 12000rpm and 4℃ for 10 minutes, and then the protein supernatant was taken. The protein concentration was determined by Bicinchoninic Acid Assay (BCA) method, and samples were prepared. After separation in 12% SDS-PAGE gel, it was transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in skim milk for 90 minutes. After blocking, the membrane was washed in 1xTBST buffer and incubated with the primary antibody of GFAP at 4℃ overnight. After washing 3 times with 1xTBST buffer, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1h, and finally the membrane was detected by enhanced chemiluminescence (ECL) kit, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

[0071] 4. Determination of the effect of LCN2 inhibitor on astrocyte size

[0072] WT and PPT1-KO astrocytes were taken separately, plated on coverslips coated with poly-D-lysine (PDL), and cultured for 24 hours. When the cells were in good condition, the cells were treated with LCN2 inhibitor (5 μM) for 4 days, and then fixed with 4% paraformaldehyde at room temperature for 20 minutes, and the slides were washed 3 times with 1xPBS. Then, the cells were treated with 0.1% Triton X-100 at 4°C for 5 minutes, and the slides were washed 3 times with 1xPBS. The cells were blocked with 3% BSA at room temperature for 30 minutes, and the blocking solution was aspirated. Without washing, 50 μL of diluted GFAP primary antibody was added to each slide and placed in a wet box, incubated overnight at 4°C, and the slides were washed 3 times with 1xPBS on the next day. The excess liquid on the slides was aspirated, and 50 μL of diluted Alexa Fluor Plus The secondary antibody labeled with 488 fluorescence was incubated at room temperature in a wet box for 1 h, and then the slides were washed 3 times with 1xPBS and sealed with a sealing solution containing an anti-fluorescence quencher. Finally, the images were observed and collected under a fluorescence microscope, and the pixel ratio of the cells in the fixed-size image was calculated for quantitative analysis.

[0073] 5. Determination of the effect of LCN2 inhibitor on astrocyte proliferation

[0074] WT and PPT1-KO astrocytes were taken and prepared into single cell suspension in DME / F12 medium containing 13% FBS, with 1x10 per well. 4 The cells were inoculated into a 96-well plate with a volume of 100 μL per well and cultured for 3 days. The cells were then treated with an LCN2 inhibitor (5 μmol) at different time points. 10 μl of CCK8 reagent was added to each well at the corresponding time point for reaction for 4 h. The wavelength of 450 nm was then selected, and the light absorbance of each well was measured on an enzyme-linked immunosorbent assay monitor. The results were recorded to obtain the OD values ​​of the cells at different time points after inhibitor treatment for quantitative analysis.

[0075] 2. Experimental Results

[0076] 1. Relationship between LCN2 inhibitor concentration and astrocyte survival rate

[0077] The experimental results of LCN2 inhibitor concentration and astrocyte survival rate are as follows Figure 1 As shown in the figure, as the concentration of the inhibitor increases, the survival rate of astrocytes decreases. The results show that LCN2 inhibitors significantly inhibit the proliferation of astrocytes, and the IC value of LCN2 inhibitors on the survival of astrocytes is 50 The value is 18.22μM.

[0078] 2. Effects of LCN2 inhibitors on protein expression in astrocytes

[0079] Electrophoresis experiment and corresponding ECL results are shown in Figure 2 , 3 As shown: Compared with WT astrocytes, GFAP protein expression is excessive in PPT1-KO astrocytes. After LCN2 inhibitor treatment of WT and PPT1-KO astrocytes, GFAP protein expression is reduced. Among them, LCN2 inhibitor has a greater effect on the expression of GFAP protein in PPT1-KO astrocytes. The results show that LCN2 inhibitor significantly inhibits the overexpression of GFAP protein in PPT1-KO astrocytes.

[0080] 3. Effect of LCN2 inhibitor on astrocyte size

[0081] The results of the astrocyte size experiment are as follows Figure 4 , 5 As shown: Compared with WT astrocytes, PPT1-KO astrocytes occupy a larger proportion of the pixels in the image, and after adding the inhibitor, the proportion of the pixels in the image occupied by PPT1-KO astrocytes significantly decreased. The results show that LCN2 inhibitors significantly inhibit the hypertrophy of PPT1-KO astrocytes.

[0082] 4. Effects of LCN2 inhibitors on astrocyte proliferation

[0083] The results of astrocyte proliferation assay were as follows Figure 6 As shown: LCN2 inhibitors had no significant effect on WT astrocytes. Compared with WT astrocytes, PPT1-KO astrocytes showed an overproliferation trend. When treated with LCN2 inhibitors, the proliferation behavior of PPT1-KO astrocytes was significantly inhibited. The results showed that LCN2 inhibitors could significantly inhibit the overproliferation of PPT1-KO astrocytes.

[0084] These results indicate that the IC value of LCN2 inhibitors on astrocyte viability is 50 The value was 18.22 μM, and LCN2 inhibitors could significantly inhibit the overexpression of GFAP protein, hypertrophy and overproliferation of PPT1-KO astrocytes. Therefore, LCN2 inhibitors have the potential to treat INCL.

[0085] Experimental Example 2 Application of LCN2 inhibitor in intervention treatment of PPT1-KO mice

[0086] 1. Experimental Methods

[0087] Four-month-old PPT1-KO mice were orally administered with LCN2 inhibitor solution once a day at a dose of 5 mg / kg. One week later, fresh brain tissues were obtained from WT mice, PPT1-KO mice, and PPT1-KO mice that had been orally treated with LCN2 inhibitors, and the brain tissues were lysed on ice using RIPA tissue lysis buffer. After lysis, the cells were centrifuged at 12000 rpm at 4°C for 10 minutes, and then the supernatant protein was taken, the protein concentration was determined by the BCA method, and the samples were prepared. After that, they were separated in a 12% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked in skim milk for 90 minutes. After blocking, the membrane was washed in 1xTBST and incubated with the primary antibody for GFAP overnight at 4°C. After that, it was washed 3 times with 1xTBST, and the membrane was incubated with the HRP-labeled secondary antibody at room temperature for 1 hour. Finally, the membrane was detected by ECL kit, and GAPDH was used as an internal reference.

[0088] 2. Experimental Results

[0089] The electrophoresis experiment and the corresponding ECL signal results are shown in Figure 7 , 8 As shown: GFAP was overexpressed in PPT1-KO mice compared with GFAP expression in WT mice. When LCN2 inhibitors were orally administered, GFAP expression in PPT1-KO mice was significantly reduced. The results showed that LCN2 inhibitors significantly inhibited the overexpression of GFAP protein in the brain tissue of PPT1-KO mice and could be used to intervene in the treatment of INCL.

[0090] Through the above embodiments and experimental examples, it can be seen that the present invention uses LCN2 inhibitors to significantly inhibit the overexpression of GFAP in astrocytes, the hypertrophy of astrocytes and the excessive proliferation of astrocytes. When the LCN2 inhibitor is applied to PPT1-KO mice, the overexpression of GFAP protein in the brain tissue of PPT1-KO mice is significantly inhibited. This shows that LCN2 inhibitors have the potential to treat INCL and can be used to prepare drugs for the treatment of INCL. For LCN2 inhibitors, no special administration method is required, and the purpose of intervention treatment can be achieved orally, which has the advantages of safety and simplicity, and brings a new direction for the treatment of INCL diseases. At the same time, LCN2 inhibitors can also be used to prepare drugs for the treatment of diseases related to hypertrophy or excessive proliferation of astrocytes, and have a good development prospect.

Claims

1. Use of LCN2 inhibitors in the preparation of drugs for treating diseases caused by hypertrophy and / or excessive proliferation of neural astrocytes.

2. The use according to claim 1, characterized in that: The disease of neuroastrocyte hypertrophy and / or excessive proliferation is infantile neuronal ceroid lipidosis.

3. The use according to claim 2, characterized in that: The neuronal ceroid lipidosis of infancy is neuronal ceroid lipidosis of infancy in mice.

4. The use according to claim 3, characterized in that: The dosage of the LCN2 inhibitor is 5-20 mg / kg; and / or, when the mice are less than 3 months old, the drug is administered once a day, and when they are 3 months old, the drug is administered once every two days until they are 6 months old; when the mice are more than 3 months old, the drug is administered once a day for at least 7 days.

5. The use according to claim 1, characterized in that: The structural formula of the LCN2 inhibitor is: Wherein, R1, R2, and R3 are independently selected from halogen, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, C1-C 10 Alkoxy; m is selected from 0, 1, 2, 3, 4, 5; n is selected from 0, 1, 2, 3; k is selected from 0, 1, 2, 3.

6. The use according to claim 5, characterized in that: The R1 is C1-C 10 and / or, m is 1, n is 0, and k is 0.

7. The use according to claim 6, characterized in that: The compounds shown in formula I include the following compounds:

8. The use according to claim 1, characterized in that: The LCN2 inhibitor is selected from ZINC 00640089 and ZINC00784494.

9. A pharmaceutical composition, characterized in that: The invention is prepared by taking the LCN2 inhibitor as the active ingredient and adding pharmaceutically acceptable auxiliary materials or supplementary ingredients.

10. The pharmaceutical composition according to claim 9, characterized in that: The structural formula of the LCN2 inhibitor is as follows: Wherein, R1, R2, and R3 are independently selected from halogen, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, C1-C 10 Alkoxy; m is selected from 0, 1, 2, 3, 4, 5; n is selected from 0, 1, 2, 3; k is selected from 0, 1, 2, 3.

Images