Use of organic acid lithium amino acid salts in the manufacture of a medicament for the treatment of ovarian cancer

Abstract

The application discloses application of organic acid lithium amino acid salt in preparation of a medicine for treating ovarian cancer and belongs to the technical field of medicines. The organic acid lithium amino acid salt is a salt formed by organic acid lithium and amino acid. The application proves for the first time that the organic acid lithium amino acid salt has a significant inhibiting effect on ovarian cancer cells, can be used for preparing the medicine for treating ovarian cancer and has important clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of organic lithium amino acid salts in the preparation of drugs for treating ovarian cancer. Background Technology

[0002] Ovarian cancer (OC) is one of the three major malignant tumors of the female reproductive system, and its mortality rate remains the highest among gynecological cancers. Studies estimate that in 2022, 39,000 women in China will die from ovarian cancer, with approximately 57,000 new cases, seriously threatening the health and lives of women in the country. Currently, cytoreductive surgery and platinum-paclitaxel-based combination chemotherapy remain the main treatments for ovarian cancer. However, due to the low early diagnosis rate, high recurrence rate, and high drug resistance rate, patients generally have a poor prognosis, with a five-year survival rate of less than 45%. Therefore, finding more effective drugs to alleviate the progression of ovarian cancer and improve chemotherapy resistance has become a hot research topic in the field of ovarian cancer treatment, and is of great significance for improving the prognosis of ovarian cancer patients.

[0003] Lithium, as an important mood stabilizer, is most commonly used to treat neurological disorders such as bipolar disorder. With in-depth research, more and more evidence shows that lithium can affect tumor proliferation in multiple ways. In studies related to ovarian cancer, inorganic lithium salts, represented by lithium chloride, have been shown to reduce the survival rate of ovarian cancer cells and inhibit ovarian cancer growth. However, some studies have found (refer to Liao Jing, Ding Dong, Yang Zongyuan, Xu Qin, Weng Danhui. Effects of lithium chloride on the proliferation and apoptosis of ovarian cancer cells and its mechanism [J]. Chinese Journal of Cancer Prevention and Treatment, 2014, 21(04):241-245.) that lithium chloride only has anti-ovarian cancer cell properties at a supertherapeutic dose concentration of 10 mM. In contrast, the commonly used inorganic lithium salts (lithium chloride and lithium carbonate) in clinical practice have a narrow safety range. Long-term use can lead to blood pH disturbances and damage to kidney and thyroid function. In vivo studies at physiological doses may be limited. According to Chinese invention patent CN114081881A, lithium carbonate has high oral toxicity in mice, and its therapeutic concentration and toxic concentration are relatively close. Therefore, traditional lithium salts have limitations in ovarian cancer research.

[0004] Chinese invention patent CN114081881A discloses a novel organic lithium acid amino acid salt—IsoLiPro (lithium isobutyrate-L-proline conjugate), a novel lithium-ion chelate synthesized by coordination between lithium isobutyrate and proline. It also reveals one application of this type of compound: therapeutic and preventative effects on recurrent episodes of mania and depression in bipolar disorder, effectively alleviating abnormal emotions such as anxiety and depression, reducing suicide risk, and delaying degenerative changes in the central nervous system. However, its preventative and therapeutic effects on cancer remain unknown. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, this invention aims to develop a drug for treating ovarian cancer that is less toxic and has better pharmacokinetics. The inventors unexpectedly discovered that organic lithium acid amino acid salts, including lithium isobutyrate L-proline salt, not only have therapeutic effects on mental and nervous system diseases but also exhibit significant inhibitory effects on ovarian cancer cells, thus completing this invention.

[0006] This invention provides the use of an organic lithium acid amino acid salt in the preparation of a medicament for treating ovarian cancer, wherein the organic lithium acid amino acid salt is a salt formed from the organic lithium acid and an amino acid.

[0007] In some embodiments of the present invention, the organic acid is selected from one of carboxylic acids, sulfonic acids, sulfinic acids, and thiocarboxylic acids, or is a derivative of the corresponding organic acid.

[0008] In some embodiments of the present invention, the carboxylic acid has the molecular formula R-COOH, wherein R is hydrogen, a substituted or unsubstituted C1-C12 hydrocarbon group, aryl or heteroaryl, and the substituted substituent is selected from hydroxyl, carboxyl, amino, thio, guanidine, amide, C6-C8 aryl, C3-C8 heteroaryl, hydroxylated C6-C12 aryl, C3-C6 cycloalkyl, amino-substituted C3-C6 cycloalkyl, and C1-C3 alkylthio.

[0009] In some preferred embodiments of the present invention, the carboxylic acid is selected from one of oxalic acid, butyric acid, isobutyric acid, valeric acid, valproic acid, lactic acid, citric acid, tartaric acid, malic acid, benzoic acid, salicylic acid, and caffeic acid.

[0010] In some embodiments of the present invention, the organic acid derivative is selected from at least one of organometallic acid complexes, covalent organic acid derivatives, and eutectic organic acid compounds.

[0011] In some preferred embodiments of the present invention, the amino acid is one of proline, valine, lysine, taurine, high taurine, or other natural or synthetic amino acids, or a derivative of the corresponding amino acid.

[0012] In some preferred embodiments of the present invention, the amino acid is proline, and its derivatives include chemically modified derivatives of the proline functional group carboxyl group and / or imine site.

[0013] In some preferred embodiments of the present invention, the organic lithium amino acid salt is lithium isobutyrate-L-proline salt. Lithium isobutyrate-L-proline salt, also known as nibulin (IsoLiPro), can significantly downregulate the expression of tau, a neuronal degeneration-related protein, and inhibit neuronal degeneration. Lithium isobutyrate-L-proline salt has therapeutic and preventive effects on recurrent episodes of mania and depression in bipolar disorder, can effectively alleviate abnormal emotions such as anxiety and depression, reduce the risk of suicide, and can delay degenerative changes in the central nervous system.

[0014] In some preferred embodiments of the present invention, the drug for treating ovarian cancer is selected from one of the group consisting of oral preparations, injections, aerosols, suppositories, drops and transdermal patches, and preferably, the drug for treating ovarian cancer is an oral preparation.

[0015] In some embodiments of the present invention, the drug for treating ovarian cancer is administered via the respiratory or digestive system; or subcutaneously; or via mucosa or submucosal administration; or via the eye or ear; or via the rectum; or via the vagina. Administering via the respiratory system refers to intratracheal and intranasal administration; administering via the digestive system refers to oral administration.

[0016] Optionally, when administered via the respiratory system, the drug for treating ovarian cancer is a spray or drops, such as a nasal spray or nasal drops.

[0017] Optionally, when administered via the digestive system, the drug for treating ovarian cancer is in the form of pills, capsules, or chewable gum.

[0018] Optionally, when administered subcutaneously, the drug for treating ovarian cancer is an implant.

[0019] Optionally, when administered rectally, the drug for treating ovarian cancer is a suppository or an infusion.

[0020] Optionally, when administered vaginally, the drug for treating ovarian cancer is a vaginal cream or wash.

[0021] In some preferred embodiments of the present invention, the drug for treating ovarian cancer treats ovarian cancer by inhibiting the proliferation, migration and invasion of ovarian cancer cells, or by increasing the apoptosis rate or necrosis rate of ovarian cancer cells.

[0022] Beneficial effects of the present invention

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention is the first to demonstrate a novel use of organic lithium amino acid salts—for the preparation of drugs to treat ovarian cancer. This provides a drug for treating ovarian cancer with lower toxicity and better pharmacokinetics, and is expected to solve problems such as defective drug distribution in vivo and large toxic side effects in existing technologies, which has important clinical significance. Attached Figure Description

[0025] Figure 1 IsoLiPro was shown to significantly inhibit the proliferation of SKOV3 and OVCAR3 ovarian cancer cells in a time- and dose-dependent manner.

[0026] Figure 2 IsoLiPro demonstrated its ability to significantly inhibit the migration and invasion of SKOV3 ovarian cancer cells.

[0027] Figure 3 IsoLiPro demonstrated its ability to significantly inhibit the migration and invasion of OVCAR3 ovarian cancer cells.

[0028] Figure 4 The study showed that IsoLiPro significantly increased the apoptosis rate of SKOV3 and OVCAR3 ovarian cancer cells in a time- and dose-dependent manner.

[0029] Figure 5 The study demonstrated that IsoLiPro significantly inhibited the relative activity of ovarian cancer organoids. Detailed Implementation

[0030] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0031] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0032] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0033] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0034] Example

[0035] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0037] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0039] Example 1: IsoLiPro significantly inhibited the proliferation of SKOV3 and OVCAR3 ovarian cancer cells.

[0040] 1. Inhibition of the proliferation ability of SKOV3 ovarian cancer cells.

[0041] The SKOV3 cell suspension was diluted to 1×10⁻⁶. 4 / mL, seeded at 200μL per well in a 96-well plate, and cultured at 37℃ for 24 hours. After cell attachment, the culture medium was aspirated, and the medium was replaced with medium containing 0, 10, 20, 30, 40, and 50 mL of SoLiPro and cultured for another 24 hours. Each group was in triplicate. After 24 and 48 hours of culture, 10μL of CCK-8 solution was added to each well. The 96-well plate was incubated in an incubator for 1.5 hours, and the absorbance of each well at 450nm was measured using a microplate reader.

[0042] Cell growth curves were plotted with time on the horizontal axis and absorbance on the vertical axis to observe the effect of IsoLiPro on the proliferation ability of ovarian cancer cells. The survival rate of SKOV3 cells was statistically analyzed in relation to the concentration and duration of IsoLiPro administration.

[0043] The results are as follows Figure 1 As shown in Figure AC, after treating SKOV3 cells with 10 mM IsoLiPro for 24 hours, cell viability was significantly reduced, and the decrease was dose- and time-dependent. The inhibitory effect was most significant under the condition of 50 mM treatment for 48 hours, and the difference was statistically significant.

[0044] 2. Inhibitory effect on the proliferation of OVCAR3 ovarian cancer cells.

[0045] The OVCAR3 cell suspension was diluted to 1×10⁻⁶. 4 / mL, seeded at 200μL per well in a 96-well plate, and cultured at 37℃ for 24 hours. After cell attachment, the culture medium was aspirated, and the medium was replaced with medium containing 0, 10, 20, 30, 40, and 50mM IsoLiPro and cultured for another 24 hours. Each group was in triplicate. After 24 and 48 hours of culture, 10μL of CK-8 solution was added to each well. The 96-well plate was incubated in an incubator for 1.5 hours, and the absorbance of each well at 450nm was measured using a microplate reader.

[0046] Cell growth curves were plotted with time on the horizontal axis and absorbance on the vertical axis to observe the effect of IsoLiPro on the proliferation of ovarian cancer cells OVCAR3. The survival rate of OVCAR3 cells was statistically analyzed in relation to the concentration and duration of IsoLiPro administration.

[0047] The results are as follows Figure 1 As shown in the figure, after 24 hours of treatment with 10 mM IsoLiPro, the cell viability of OVCAR3 cells began to decrease, and the decrease was dose- and time-dependent. The inhibitory effect was most significant under the condition of 48 hours of treatment with 50 mM, and the difference was statistically significant.

[0048] The above results indicate that IsoLiPro affects the proliferation capacity of SKOV3 and OVCAR3 cells in a time- and dose-dependent manner.

[0049] Example 2: IsoLiPro significantly inhibited the migration and invasion abilities of SKOV3 and OVCAR3 ovarian cancer cells.

[0050] 1. Inhibition of the migration ability of SKOV3 and OVCAR3 ovarian cancer cells.

[0051] Prepare a six-well plate. Using a marker pen and ruler, draw evenly spaced horizontal lines on the back of the plate, with a spacing of 0.5-1 cm between lines. Each well should have at least five lines. Then, seed SKOV3 or OVCAR3 cells into the six-well plate. Once the cells have reached 100% confluence, use a 200 μL pipette tip perpendicular to the bottom of the plate to make incisions along the vertical direction of the horizontal lines. After incision, wash with sterile PBS and aspirate any non-adherent cells to make the incision lines clearly visible. Treat the cells with 0 or 10 mM IsoLiPro, respectively, and continue culturing at 37°C in a 5% CO2 incubator. Remove the six-well plate at 0, 24, and 48 hours, observe and measure the width of the incisions under a microscope, and take photographs.

[0052] The mean intercellular distance was calculated using ImageJ software to examine the effect of IsoLiPro on tumor cell migration ability.

[0053] The results are as follows Figure 2 As shown, after treating SKOV3 (A) and OVCAR3 (B) cells with 10 mM IsoLiPro for 24 hours, the cell migration ability was significantly reduced, and the inhibitory effect was more obvious after 48 hours of treatment, with statistically significant differences.

[0054] 2. Inhibition of the invasive ability of SKOV3 and OVCAR3 ovarian cancer cells.

[0055] Prepare Transwell chambers with micropores of 8 μm diameter. Digest SKOV3 or OVCAR3 cells and resuspend them in basal culture medium. Take 8 × 10⁸ cells. 4 Cells were seeded in the upper chamber of a Transwell microarray, and IsoLiPro was added at concentrations of 0, 10, and 30 mM to maintain a total volume of 200 μL. 500 μL of culture medium containing 10% fetal bovine serum was added to the lower chamber, and the cells were incubated at 37°C for 48 hours. Three replicates were performed for each group. After 48 hours, the filter membrane was removed, and cells on the upper chamber surface were wiped clean with a cotton swab. Cells migrating to the lower chamber surface were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS, stained with 0.1% crystal violet for 30 minutes, and washed three times with PBS. The chamber was then inverted, and the number of cells on the lower chamber surface was counted under an inverted microscope to assess the effect of each group on tumor cell migration ability.

[0056] The procedure for detecting cell invasion ability is the same as above, but 50 μL of matrix gel needs to be placed in the upper chamber of the Transwell chamber before adding the cell suspension containing IsoLiPro to the upper chamber. The same counting method is used to detect the effect of each group on the tumor cell invasion ability.

[0057] The results are as follows Figure 3 As shown, after treating SKOV3 (A) and OVCAR3 (B) cells with 10 mM IsoLiPro for 48 hours, the cell migration and invasion abilities were significantly reduced, and the inhibitory effect was more obvious in the 30 mM concentration treatment group, with statistically significant differences.

[0058] Example 3: IsoLiPro significantly increased the apoptosis rate of SKOV3 and OVCAR3 ovarian cancer cells.

[0059] 1. Effect on the apoptosis rate of SKOV3 ovarian cancer cells

[0060] SKOV3 cells were seeded in six-well plates. When confluence reached 80%, the medium was replaced with medium containing 0, 10, 30, and 50 mg / mL MIsoLiPro, and the plates were incubated at 37°C with 5% CO2. At 24 and 48 hours, the plates were removed, and the culture medium from each well was collected into 1.5 mL EP tubes. The cells were then digested and transferred to centrifuge tubes, with the corresponding culture medium added to the centrifuge tubes. After centrifugation, the supernatant was discarded. The cells were washed twice with 4°C PBS and resuspended in 1× Binding Buffer to maintain a concentration of 1×10⁻⁶ cells / mL. 6 At approximately 100 μL / mL, transfer 100 μL of cell suspension to an EP tube, add 5 μL of APCAnnexin V and 5 μL of 7-AAD, mix well, and incubate at room temperature in the dark for 15 minutes. After incubation, add 400 μL of 1×Binding Buffer to each tube, and perform apoptosis analysis using flow cytometry within 1 hour. Analyze the proportion and number of cells in different states (early apoptosis, late apoptosis, and cell death) using FlowJo software.

[0061] The results are as follows Figure 4 As shown in Figure A, after treating SKOV3 cells with 10 mM IsoLiPro for 24 hours, the apoptosis rate significantly increased in a dose-dependent manner, reaching its highest level at 50 mM for 24 hours, with statistically significant differences. Analyzing only the apoptosis rate, the apoptosis rates in each concentration group after 48 hours were not greater than those after 24 hours, showing no time-dependent effect. However, CCK8 assays revealed that SKOV3 cells are highly sensitive to IsoLiPro, and 48 hours of treatment significantly reduced their survival rate. Therefore, the inventors conducted statistical analysis on the necrosis rate in the 48-hour group. The results showed that the necrosis rate of SKOV3 cells after 48 hours of IsoLiPro treatment was significantly higher than that after 24 hours, and in a dose-dependent manner, reaching its highest level at 50 mM for 48 hours, with statistically significant differences.

[0062] 2. Effect on apoptosis rate of OVCAR3 ovarian cancer cells

[0063] OVCAR3 cells were seeded in six-well plates. When confluence reached 80%, the medium was replaced with medium containing 0, 10, 30, and 50 mg / mL MIsoLiPro, and the plates were incubated at 37°C with 5% CO2. At 24 and 48 hours, the plates were removed, and the culture medium from each well was collected into 1.5 mL EP tubes. The cells were then digested and transferred to centrifuge tubes, with the corresponding culture medium added to the centrifuge tubes. After centrifugation, the supernatant was discarded. The cells were washed twice with 4°C PBS and resuspended in 1× Binding Buffer to maintain a concentration of 1×10⁻⁶ cells / mL. 6At approximately 100 μL / mL, transfer 100 μL of cell suspension to an EP tube, add 5 μL of APCAnnexin V and 5 μL of 7-AAD, mix well, and incubate at room temperature in the dark for 15 minutes. After incubation, add 400 μL of 1×Binding Buffer to each tube, and perform apoptosis analysis using flow cytometry within 1 hour. Analyze the proportion and number of cells in different states (early apoptosis, late apoptosis, and cell death) using FlowJo software.

[0064] The results are as follows Figure 4 As shown in Figure B, it can be seen that after treating OVCAR3 cells with 10mM IsoLiPro for 24h, the apoptosis rate began to increase in a dose- and time-dependent manner, and the apoptosis rate reached its highest level under the condition of 50mM treatment for 48h, with statistically significant differences.

[0065] Example 4: IsoLiPro significantly inhibited the relative activity of ovarian cancer organoids.

[0066] The inventors collected tumor tissue from 11 ovarian cancer patients, assessed the tissue condition of each case, and removed non-epithelial components. The tissue blocks were then minced, digested, filtered, and primary ovarian cancer cells were collected, with the cell concentration controlled at 4 × 10⁻⁶. 5 Wash cells twice with a concentration of 1 / mL, centrifuge, aspirate the supernatant, and resuspend the cells in 200 μL of OrganoGel matrix gel, maintaining a concentration of approximately 1 × 10⁶ cells / mL per 50 μL OrganoGel matrix gel. 5 Prepare a preheated 24-well plate. Immediately drop approximately 10 μL of the mixture of primary cells and OrganoGel onto the bottom of each well, placing 5 drops per well. Incubate the plate at 37°C with 5% CO2 for 20 minutes to allow the OrganoGel to solidify. Then, add 500 μL of organoid complete culture medium per well and incubate for 7-10 days. Once the primary organoids have formed (10,000 cells per 40 μL of culture), isolate and passage them to prepare for drug sensitivity testing. After passaged organoids were formed, the 24-well plates were removed and the complete organoid culture medium was aspirated. Organoid culture medium containing 0, 10, 20, 30, 40, and 50 mM IsoLiPro was added and cultured for another 24 hours. The activity of ovarian cancer organoid cells was observed and the area under the IsoLiPro response curve (AUC) was evaluated to explore the relationship between IsoLiPro dose and organoid activity.

[0067] The results are shown in Table 1 and Figure 5 As shown:

[0068] Table 1. Inhibition of relative activity of IsoLiPro on ovarian cancer organoids

[0069]

[0070] It was observed that after 24 hours of treatment with IsoLiPro, the relative activity of ovarian cancer organoids decreased significantly in a dose-dependent manner. Sample LC202211001 was the most sensitive to IsoLiPro, with an IC50 of 0.0071 M; sample LC202209001 was the least sensitive, and its IC50 could not be calculated. The remaining organoid samples showed varying degrees of inhibition after IsoLiPro treatment.

[0071] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of lithium isobutyrate L-proline salt in the preparation of a medicament for the treatment of ovarian cancer.

2. Use according to claim 1, characterized in that, The medicament for the treatment of ovarian cancer is selected from the group consisting of oral preparations, injections, aerosols, suppositories, and transdermal patches.

3. Use according to claim 2, characterized in that, The medicament for the treatment of ovarian cancer is administered through the respiratory system or the digestive system; or is administered subcutaneously; or is administered through the nasal mucosa or submucosa; or is administered rectally; or is administered vaginally.

4. Use according to claim 1, characterized in that, The medicament for the treatment of ovarian cancer treats ovarian cancer by inhibiting the proliferation, migration, and invasion of ovarian cancer cells, or by increasing the rate of apoptosis or necrosis of ovarian cancer cells.

Images