Pharmaceutical composition for reducing cytotoxicity and use thereof
By combining rapamycin with chloroquine derivatives, the cytotoxicity problem of rapamycin was solved, enabling the drug to be used alone and effectively inhibiting HSV-2, thus expanding the therapeutic window of rapamycin.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2026-04-02
AI Technical Summary
Existing rapamycin compositions have significant cytotoxicity issues, limiting their application scope and therapeutic window, and they lack the activity of monotherapy when used in combination with other drugs.
Rapamycin or its derivatives are combined with chloroquine or its derivatives as the sole active ingredients to prepare a pharmaceutical composition for reducing cytotoxicity and inhibiting herpes simplex virus (HSV), wherein the chloroquine derivatives can reverse the autophagy-promoting effect of rapamycin to autophagy inhibition, thus forming a synergistic effect.
It significantly reduced the cytotoxicity of rapamycin, expanding its scope of use, and had a significant inhibitory effect on HSV-2, realizing the drug's use alone and synergistic effects.
Smart Images
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Abstract
Description
Pharmaceutical composition for reducing cytotoxicity and its use TECHNICAL FIELD
[0001] The present application belongs to the technical field of medicine, and in particular, the present application provides a pharmaceutical composition for reducing cytotoxicity and / or anti-herpes simplex virus and its use. BACKGROUND
[0002] Rapamycin (Sirolimus) is a macrolide antibiotic with anti-inflammatory and bactericidal activity, mainly used for immunosuppression. For example, rapamycin can be used to treat rheumatoid arthritis, systemic lupus erythematosus and other autoimmune diseases. Rapamycin has broad-spectrum antibacterial activity and can effectively inhibit the infection of various pathogenic bacteria, including gram-positive bacteria, gram-negative bacteria and mycobacteria. Therefore, it is also used to treat infectious diseases caused by these pathogens, such as pneumonia, skin infection, pulmonary infection, etc. Rapamycin also has certain potential in the treatment of neurological diseases. Studies have shown that it can delay the degeneration of nerves and has certain therapeutic effect on neurological diseases such as cerebral malaria. In addition, rapamycin is also believed to have a role in preventing complications of diabetes-related heart disease. In the treatment of tuberous sclerosis, rapamycin also plays an important role. It can delay the progression of the disease and has a certain preventive effect on the disease. For patients with tuberous sclerosis complicated with epilepsy, subependymal giant cell astrocytoma, angioleiomyolipoma, lymphatic smooth muscle tumor, etc., the therapeutic effect of rapamycin is particularly significant. In addition, rapamycin has also been studied for the treatment of Alzheimer's disease, improvement of vascular stenosis, etc. In recent years, rapamycin has also been developed into an anticancer drug, such as advanced renal cell carcinoma. However, due to its relatively large toxic side effects, the effect and application range of rapamycin are limited to a certain extent.
[0003] Herpes simplex virus (HSV) is divided into two serotypes, HSV-1 and HSV-2, and the DNA of the two types of viruses has 50% homology, with type-specific antigens and type-specific antigens. HSV infection is extremely common in the population, and can cause a variety of diseases, such as gingivostomatitis, keratoconjunctivitis, encephalitis, and genital infection and infection in newborns, and the common clinical manifestations are local herpes of mucosa or skin. In addition, HSV has also been reported to be associated with lip cancer, vulvar cancer and cervical cancer, etc.
[0004] The existing combination of rapamycin and chloroquine derivatives is usually applied in combination with other drugs or therapeutic agents, not as a single drug. For example, our granted Chinese patents 201110098959.X and 201110100357.3 provide sensitizing agents for cancer treatment, which need to be applied in combination with cancer chemotherapy drugs or radiation to enhance the therapeutic effect on cancer or tumor; the drug provided by Chinese patent 201780087985.8 further needs to be applied in combination with hormones.
[0005] However, surprisingly, we found that the combination of rapamycin and chloroquine derivatives can be applied alone (i.e. they are the only active ingredients), i.e. effectively, even synergistically, inhibit HSV, and in particular, we also found that the use of chloroquine derivatives can significantly reduce the cytotoxicity of rapamycin, thereby expanding the scope of use and treatment window of rapamycin. SUMMARY
[0006] The technical problem to be solved by the present application is to provide a new pharmaceutical composition, which can be used to reduce the cytotoxicity caused by rapamycin and its derivatives. Moreover, the pharmaceutical composition of the present application can also be used as the only active ingredient to inhibit HSV, such as HSV-2. In addition, the present application also provides the corresponding use.
[0007] Specifically, in a first aspect, the present application provides a pharmaceutical composition for reducing the cytotoxicity caused by rapamycin and its derivatives, which consists of rapamycin or its derivatives and chloroquine or its derivatives.
[0008] The pharmaceutical composition of the first aspect of the present application can be used to inhibit HSV, preferably to inhibit HSV-2.
[0009] Preferably, the pharmaceutical composition of the first aspect of the present application is applied as the only active ingredient, i.e. alone, without being applied in combination with other active pharmaceutical ingredients.
[0010] In addition, the present application provides a pharmaceutical preparation, which comprises the pharmaceutical composition of the first aspect of the present application and a pharmaceutically acceptable excipient.
[0011] In a second aspect, the present application provides the use of rapamycin or its derivatives and chloroquine or its derivatives as the only active ingredient in the preparation of a drug for inhibiting HSV.
[0012] In a third aspect, the present application provides the use of chloroquine or its derivatives in the preparation of a pharmaceutical composition for reducing the cytotoxicity caused by rapamycin and its derivatives.
[0013] Preferably in the third aspect of the present application, the pharmaceutical composition is the pharmaceutical composition of the first aspect of the present application.
[0014] Preferably in the third aspect of the present application, the pharmaceutical composition is for use in inhibiting HSV, such as HSV-2.
[0015] Preferably in the present application, the rapamycin or derivative thereof is rapamycin.
[0016] Preferably in the present application, the chloroquine or derivative thereof is hydroxychloroquine sulfate.
[0017] Preferably in the present application, the chloroquine or derivative thereof reverses the cell autophagy promoting effect of the rapamycin or derivative thereof to an autophagy inhibiting effect.
[0018] Preferably in the present application, the pharmaceutical composition is for use in inhibiting autophagy.
[0019] Preferably in the present application, the weight ratio of the chloroquine or derivative thereof and the rapamycin or derivative thereof is 0.1 to 1000: 0.1 to 100, preferably 1 to 500: 0.1 to 50, more preferably 1 to 300: 0.5 to 30.
[0020] Preferably in the present application, the molar ratio of the chloroquine or derivative thereof and the rapamycin or derivative thereof is 0.1 to 1000: 0.1 to 100, preferably 1 to 500: 0.1 to 50, more preferably 1 to 300: 0.5 to 30, for example 3: 1. DETAILED DESCRIPTION
[0021] In this text, the terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise defined. For better understanding of the present application, the definitions and explanations of relevant terms are provided below.
[0022] The pharmaceutical composition of the first aspect of the present application consists of rapamycin or derivative thereof and chloroquine or derivative thereof, and is for use in reducing the cytotoxicity caused by rapamycin and its derivatives. The pharmaceutical composition of the first aspect of the present application only contains rapamycin or derivative thereof and chloroquine or derivative thereof, and does not contain other active ingredients, i.e. rapamycin or derivative thereof and chloroquine or derivative thereof are the only active ingredients of the pharmaceutical composition of the first aspect of the present application.
[0023] As used herein, "rapamycin derivative" refers to a derivative of rapamycin that has been chemically or biologically modified or substituted, but still retains the properties of rapamycin or has properties similar to rapamycin, and can include ester, ether, hydrazone or hydroxylamine derivatives of rapamycin, such as everlimus, temsirolimus, tacrolimus, deforolimus, biolimus or (42S)-42-deoxy-42-(1 hydrogen-tetrazol-1-yl)-rapamycin (zotaroliumus). Rapamycin or its derivatives can cause cytotoxicity, such as promoting autophagy. In one embodiment of the present application, the rapamycin or its derivative is rapamycin.
[0024] As used herein, "quinoline derivative" can refer to chloroquine in addition to derivatives of chloroquine that have been further modified or substituted, or analogs of chloroquine. The derivatives of chloroquine that have been further modified or substituted can be those that have the chemical structure of chloroquine, but one or more hydrogens or functional groups thereon have been modified or substituted with one or more substituents. In particular, the substituents can be halogen, C 1-10 alkyl, -OC 1-10 alkyl, hydroxyl, C 6-10 aryl, heteroaryl, heterocycloalkyl, alkylheterocycloalkyl, heteroalkyl or alkylheteroalkyl. For example, the quinoline derivative can be selected from, but not limited to, the group consisting of hydroxychloroquine, primaquine, amoproquine, amodiaquine, cycloquine, sontoquine, quinacrine, tebuquine and bis-pyroquine. The inventors have found that chloroquine or its derivatives can reverse the autophagy-promoting effect of rapamycin or its derivatives into an autophagy-inhibiting effect, and the pharmaceutical composition of the present application as a whole exhibits an inhibitory effect on autophagy and thus can be used to inhibit autophagy. In one embodiment of the present application, the chloroquine or its derivative is hydroxychloroquine sulfate.
[0025] The pharmaceutical preparation of the present application includes the pharmaceutical composition of the first aspect of the present application, and pharmaceutically acceptable excipients.
[0026] As used herein, "pharmaceutically acceptable excipient" refers to a carrier, diluent, and / or vehicle that is compatible with pharmacological and / or physiological action of the subject and the active ingredient (e.g., an antibody) and is well-tolerated by the subject. Pharmaceutically acceptable excipients include, but are not limited to, pH adjusting agents (e.g., phosphate buffer), surfactants (e.g., cationic, anionic, or non-ionic surfactants such as Tween-80), adjuvants, ion strength enhancers (e.g., salts such as sodium chloride).
[0027] The pharmaceutical composition of the first aspect of the present application can be used for anti-HSV. In one embodiment of the present application, the pharmaceutical composition of the first aspect of the present application can be used for anti-HSV-2. That is, rapamycin or a derivative thereof and chloroquine or a derivative thereof can be used for the manufacture of a medicament for inhibiting HSV.
[0028] In a second aspect, the present application provides the use of rapamycin or a derivative thereof and chloroquine or a derivative thereof as the sole active ingredient for the manufacture of a medicament for inhibiting HSV. Accordingly, the present application provides a method for inhibiting HSV, which comprises administering to a subject in need thereof an effective amount of rapamycin or a derivative thereof and an effective amount of chloroquine or a derivative thereof, wherein rapamycin or a derivative thereof and chloroquine or a derivative thereof are used as the sole active ingredient.
[0029] As used herein, "effective amount" means an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a viral infection) means an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., a viral infection); an effective amount for treating a disease (e.g., a viral infection) means an amount sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the state of the individual's immune system, the individual's general condition (e.g., age, body weight, and sex), the mode of administration of the drug, and other therapies being administered to the individual, etc. In the present context, the individual can be a human or an experimental animal, preferably a human.
[0030] In a third aspect, the present application provides the use of chloroquine or a derivative thereof for the manufacture of a pharmaceutical composition for reducing cytotoxicity caused by rapamycin and its derivatives. Accordingly, the present application provides a method for reducing cytotoxicity caused by rapamycin and its derivatives, which comprises administering to an individual to whom rapamycin and its derivatives are administered or will be administered an effective amount of chloroquine or a derivative thereof.
[0031] Preferably in the third aspect of the present application, the pharmaceutical composition is the pharmaceutical composition of the first aspect of the present application. That is, an effective amount of rapamycin or a derivative thereof and an effective amount of chloroquine or a derivative thereof are administered to a subject in need thereof, wherein the rapamycin or a derivative thereof and the chloroquine or a derivative thereof are the only active ingredients.
[0032] Also preferably in the third aspect of the present application, the pharmaceutical composition is used against HSV, such as HSV-2. That is, a subject in need thereof is infected with HSV, such as HSV-2.
[0033] Preferably in the present application, the weight ratio of chloroquine or a derivative thereof to rapamycin or a derivative thereof is 0.1 to 1000: 0.1 to 100, preferably 1 to 500: 0.1 to 50, more preferably 1 to 300: 0.5 to 30.
[0034] Also preferably in the present application, the molar ratio of chloroquine or a derivative thereof to rapamycin or a derivative thereof is 0.1 to 1000: 0.1 to 100, preferably 1 to 500: 0.1 to 50, more preferably 1 to 300: 0.5 to 30, for example 3: 1.
[0035] The present application is advantageous in that it provides a new pharmaceutical composition which can reduce the cytotoxicity of rapamycin and its derivatives and can also be used against HSV.
[0036] For the purpose of facilitating understanding, the present application will be described in detail below with specific examples. It should be particularly noted that these descriptions are merely exemplary and do not limit the scope of the present application. Many variations and modifications of the present application will be apparent to those skilled in the art in light of the discussion of the present specification. DETAILED DESCRIPTION
[0037] The present application will be further illustrated by the following examples. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art and commercially available common instruments and reagents, which can be referred to the manufacturer's instructions of the corresponding instruments and reagents, etc.
[0038] Example 1 Cytotoxicity test of hydroxychloroquine sulfate and rapamycin alone and in combination
[0039] (1) Cytopathic effect (CPE) test
[0040] Vero cells (purchased from ATCC, Cat. No. CCL-81) were seeded into 96-well cell culture plates at a certain cell density (10,000 cells / well) and cultured overnight in a 5% CO2, 37 ℃ incubator. The next day, the test compounds hydroxychloroquine sulfate (H) and rapamycin (R) were respectively 2-fold serially diluted into 7 concentration points, and then the two compounds were combined respectively into 7 different concentrations for orthogonal array design (see Table 1), and the compounds were added to the 96-well plates, and each combination was tested in triplicate. The final concentration of DMSO in the cell culture medium was 0.5%. The cells were cultured in a 37 ℃, 5% CO2 incubator for 5 days. Cell Counting Kit-8 reagent was used to detect cell viability, and the raw data were used for compound cytotoxicity calculation.
[0041] Table 1 Compound arrangement chart-cell cytotoxicity test.
[0042] [Amended according to Rule 26 13.10.2025]
[0043] Note: a0;b0 is the cell control (cell control), MC is the solvent control (medium control)
[0044] [Amended according to Rule 26 13.10.2025]
[0045] (2) Experimental results
[0046] The results of the CPE experiment of the two drugs in combination are shown in Table 2, wherein column 9 is the cell viability when R is used alone, and columns 2-8 are the cell viability when H and R are used in combination. The results show that when the concentration of drug H is between 7.5 and 30 μM and the concentration of R is between 0.156 and 10 μM, the cytotoxicity of H and R used in combination is lower than that of drug R used alone.
[0047] Table 2 Cytotoxicity test results of two drugs in combination
[0048] [Amended according to Rule 26 13.10.2025]
[0049] Example 2 HSV-2 inhibition test of hydroxychloroquine sulfate and rapamycin used alone and in combination
[0050] (1) qPCR experiment method of two drugs in combination:
[0051] Vero cells (purchased from ATCC, Cat. No. CCL-81) were seeded at 10,000 cells per well in 96-well cell culture plates and incubated overnight in a 37 °C incubator with 5% CO2. The next day, test compounds H and R were 2-fold serially diluted to 7 concentration points, respectively, and then the two compounds were combined to perform orthogonal array design at 7 different concentrations (Table 3), and the compound was added to the 96-well plate, and three replicates were tested for each combination. Subsequently, diluted virus (Herpes Simplex Virus Type II HSV-2, MS strain, purchased from ATCC, Cat. No. CCL-81) was added at MOI = 0.1. The final concentration of DMSO in the cell culture medium was 0.5%. The cells were incubated at 37 °C in a 5% CO2incubator for 1 day. DNA in the cells was extracted using QuickExtract™ DNA Extraction Solution, and the viral DNA was quantified by qPCR. The viral DNA copy number was used for the analysis of the in vitro joint efficacy of H and R. The test data were processed using MacSynegy software to analyze the in vitro joint efficacy of H and R on Herpes Simplex Virus Type II.
[0052] Table 3 Compound arrangement chart - antiviral activity test
[0053] [Amended in accordance with Rule 26 13.10.2025]
[0054] Note: a0;b0is the virus control group (Virus control), CC: cell control group (cell control)
[0055] [Amended in accordance with Rule 26 13.10.2025]
[0056] (2) Experimental results
[0057] The qPCR experiment of the two drugs in combination is shown in Table 4, wherein column 9 is the average EC50 value of R alone, column 10 is the average EC50 value of H alone, and columns 2-8 in the middle are the average EC50 values of H and R in combination.
[0058] Table 4 Results of the inhibition of HSV-2 by the combination of drugs H and R
[0059] [Amended in accordance with Rule 26 13.10.2025]
[0060] From the results, R alone has no inhibitory effect on HSV-2 virus, and in combination with the cytotoxicity shown in Table 2, R alone can even increase viral activity due to its activation of autophagy; after R is combined with H, not only does it show that R alone can reduce toxicity (Table 2), but it also improves viral inhibition (Table 4), indicating that after R is combined with H, the original autophagy activation unexpectedly turns to autophagy inhibition, and significantly improves the autophagy inhibition effect of H; moreover, when the concentration of drug R is 10 μM and the concentration of H is between 3.750 μM and 30 μM, the combination of H and R has a synergistic effect on the inhibition of HSV-2.
Claims
1. A pharmaceutical composition for reducing the cytotoxicity caused by rapamycin and its derivatives, consisting of rapamycin or its derivatives and chloroquine or its derivatives; preferably wherein, Rapamycin or a derivative thereof is rapamycin, and / or chloroquine or a derivative thereof is hydroxychloroquine sulfate.
2. The pharmaceutical composition of claim 1 for use against HSV, such as HSV-2.
3. The pharmaceutical composition of claim 1 or 2 as the total active ingredient.
4. The pharmaceutical composition of claim 1, wherein, Chloroquine or a derivative thereof reverses the cell autophagy promoting effect of rapamycin or a derivative thereof to an autophagy inhibiting effect.
5. The pharmaceutical composition of claim 1, wherein, The weight ratio of chloroquine or a derivative thereof and rapamycin or a derivative thereof is 0.1-1000:0.1-100, preferably 1-500:0.1-50, more preferably 1-300:0.5-30.
6. A pharmaceutical preparation comprising the pharmaceutical composition of any one of claims 1-5, and pharmaceutically acceptable excipients.
7. Use of rapamycin or a derivative thereof and chloroquine or a derivative thereof as the total active ingredient for the manufacture of a medicament for use against HSV.
8. Use of chloroquine or a derivative thereof for the manufacture of a pharmaceutical composition for reducing the cytotoxicity caused by rapamycin and derivatives thereof.
9. The use of claim 8, wherein, The pharmaceutical composition is the pharmaceutical composition of any one of claims 1-5, preferably wherein the pharmaceutical composition is for use against HSV, such as HSV-2.
10. The use according to claim 8 or 9, wherein, The pharmaceutical composition is for use in inhibiting autophagy. The pharmaceutical composition is for use in inhibiting autophagy.