Use of ritonavir in preparation of drug for treating psychological disorder caused by traumatic event

Abstract

Disclosed in the present application is the use of ritonavir in the preparation of a drug for treating a psychological disorder caused by a traumatic event. In the present application, it is found that intraperitoneal injection of ritonavir to fear memory extinction resistant model mice (AtLAS- / - mice) and extinction resistant (EXT-R) mice distinguished from common C57BL6 mice can significantly relieve the rigidity of the model mice and EXT-R mice. It is further confirmed by means of immunoblotting and co-immunoprecipitation methods that ritonavir can significantly block the binding of Syn2a to Syngr-3 and promote the formation of a trimer of VAMP2, Syntaxin and SNAP25 to promote and restore the normal release of presynaptic membrane vesicles in neurons, so as to restore the rigidity of the extinction resistant model mice and EXT-R mice, and treat a psychological disorder, such as post traumatic stress disorder (PTSD), caused by a traumatic event.

Description

Use of ritonavir in preparing medicine for treating psychological disorders caused by traumatic events

[0001] Cross-reference to related applications

[0002] This patent application claims priority to the Chinese patent application filed on June 15, 2023, with application number 2023107141989 and invention name “Application of ritonavir or its derivatives in the preparation of drugs for the treatment of mental disorders”. The full text of the above application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to the use of ritonavir in preparing a medicament for treating psychological disorders caused by traumatic events. Background Art

[0004] Post-traumatic stress disorder (PTSD) is a delayed and persistent mental disorder caused by experiencing, witnessing, or encountering one or more events involving the actual death of oneself or others, the threat of death, severe injury, or a threat to physical integrity. It is a common mental disorder caused by exposure to extreme traumatic events, involving multiple brain circuits mediating stress and fear responses.

[0005] Pavlovian fear conditioning and extinction, known as extinction learning, is a widely accepted tool that allows healthy individuals to return to baseline emotional reactivity upon re-exposure to traumatic memories. Numerous preclinical studies have shown that impaired fear extinction mechanisms following traumatic stress play a significant role in the development and maintenance of post-traumatic stress disorder (PTSD). Clinically, exposure therapy, based on extinction learning mechanisms, is widely used to treat PTSD. However, this treatment is only effective for some individuals, with some efficacy, ineffectiveness, or even unavailable for others. Combining medication with psychotherapy has become a major research focus, with medications significantly relieving symptoms and enhancing psychotherapy. Currently, SSRIs, such as sertraline, paroxetine, and fluoxetine, are commonly used. However, these medications can have significant side effects, including severe gastrointestinal reactions, nausea, vomiting, and diarrhea, as well as neurological side effects such as drowsiness, dizziness, and nightmares. In some cases, they can also cause sexual dysfunction.

[0006] Therefore, there is an urgent need in this field to find new methods for treating post-traumatic stress disorder.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a pharmaceutical use of ritonavir.

[0009] Another object of the present invention is to provide a method for treating post-traumatic stress disorder.

[0010] In order to solve the above technical problems, the first aspect of the present invention provides the use of ritonavir for:

[0011] (i) Treating psychological disorders caused by traumatic events;

[0012] (ii) preparing a medicament for treating a psychological disorder resulting from a traumatic event;

[0013] (iii) promoting the release of vesicles from the presynaptic membrane of neurons;

[0014] (iv) inhibiting the formation of the Syn2a-Syngr-3 complex; and / or

[0015] (v) Promotes the formation of a trimeric complex of vesicle-associated membrane protein 2 (VAMP2)-synaptotagmin (Syntaxin)-25kD synaptosome-associated protein (SNAP25).

[0016] In some preferred embodiments, the psychological disorder caused by the traumatic event is selected from at least one of the following: post-traumatic stress disorder, post-traumatic anxiety disorder and post-traumatic depression.

[0017] In some preferred embodiments, the use of ritonavir is for:

[0018] (i) treatment of post-traumatic stress disorder; and / or

[0019] (ii) preparing a medicament for treating post-traumatic stress disorder.

[0020] In some preferred embodiments, the ritonavir prevents and / or treats psychological disorders caused by traumatic events by inhibiting the formation of the Syn2a-Syngr-3 complex.

[0021] In some preferred embodiments, the ritonavir prevents and / or treats psychological disorders caused by traumatic events by promoting the formation of the VAMP2-Syntaxin-SNAP25 trimer complex.

[0022] In some preferred embodiments, the ritonavir prevents and / or treats psychological disorders caused by traumatic events by promoting the release of presynaptic vesicles from neurons.

[0023] In some preferred embodiments, the ritonavir promotes the release of presynaptic vesicles from neurons by promoting the formation of a VAMP2-Syntaxin-SNAP25 trimer complex.

[0024] A second aspect of the present invention provides a method for treating post-traumatic stress disorder, comprising the steps of:

[0025] A therapeutically effective amount of ritonavir is administered to the subject.

[0026] In some preferred embodiments, ritonavir is administered to the subject at a dose of 0.1-100 mg / kg.

[0027] In some preferred embodiments, ritonavir is administered to the subject orally or by injection.

[0028] In some preferred embodiments, ritonavir is prepared into tablets, capsules or injections and administered to the subject.

[0029] Compared with the prior art, the present invention has at least the following advantages:

[0030] The present invention unexpectedly discovered a model mouse (AtLAS) that is difficult to extinct fear memory. - / - Intraperitoneal injection of ritonavir into traumatic mice (EXT-R) and EXT-R mice significantly restored the rigidity rate of both mice. Further immunoblotting and immunoprecipitation confirmed that ritonavir could significantly block the binding of Syn2a and Syngr-3, promote the formation of VAMP2, Syntaxin and SNAP25 trimers, and promote the release of presynaptic vesicles from neurons to normal, thereby restoring the rigidity rate of EXT-R mice and achieving the treatment of psychological disorders caused by traumatic events, such as post-traumatic stress disorder.

[0031] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0033] FIG1 is a diagram of co-immunoprecipitation of the binding of Syn2a and Syngr3 disrupted in HEK 293T cells according to an embodiment of the present invention;

[0034] FIG2 is a schematic diagram of a fear memory extinction behavioral experiment process according to an embodiment of the present invention;

[0035] FIG3 is a graph showing changes in the freezing rate of mice during fear memory extinction according to an embodiment of the present invention;

[0036] FIG4 is a statistical diagram of the immunoblotting results of the total amount of Syn2a / b protein according to an embodiment of the present invention;

[0037] FIG5 is a statistical diagram of immunoblotting results after immunoprecipitation of Syn2a and Syngr3 proteins according to an embodiment of the present invention;

[0038] FIG6 is a diagram of the immunoprecipitation of VAMP2, Syntaxin and SNAP25 proteins according to an embodiment of the present invention;

[0039] FIG7 is a diagram showing changes in spontaneous excitatory postsynaptic currents in the brain of mice according to an embodiment of the present invention;

[0040] FIG8 is an electron microscopic image of a presynaptic membrane vesicle according to an embodiment of the present invention;

[0041] FIG9 is a statistical diagram of the number of vesicles in the presynaptic membrane according to an embodiment of the present invention;

[0042] FIG10 is a schematic diagram of a behavioral experiment process for extinction-resistant mice (EXT-R) according to an embodiment of the present invention;

[0043] 11 is a graph showing changes in the freezing rate of fear memory extinction-resistant mice (EXT-R) according to an embodiment of the present invention;

[0044] FIG12 is a diagram showing changes in spontaneous excitatory postsynaptic currents in the brain region of extinction-resistant mice (EXT-R) according to an embodiment of the present invention. DETAILED DESCRIPTION

[0045] Existing drugs have limited therapeutic effects on post-traumatic stress disorder and have significant side effects. The present inventors have found in their research that ritonavir (RTV), which is used to treat adult HIV-1 infection, can effectively block the biological effects of the binding of Synapsin IIa (Syn2a) and Synaptogyrin-3 (Syngr-3), restoring the release of presynaptic vesicles from neurons to a certain extent, thereby reducing the severity of post-traumatic stress disorder in the AtLAS model mouse model. - / - mice, a mouse model of difficult-to-extinction fear memory) and the fear memory of difficult-to-extinction mice, and therefore is an effective drug for preventing / treating post-traumatic stress disorder.

[0046] Uses of compounds

[0047] The present invention relates to a novel use of ritonavir for: (i) treating psychological disorders caused by traumatic events; (ii) preparing a medicament for treating psychological disorders caused by traumatic events; (iii) promoting the release of presynaptic vesicles from neurons; (iv) inhibiting the formation of the Syn2a-Syngr-3 complex; and / or (v) promoting the formation of the vesicle-associated membrane protein 2 (VAMP2)-synaptotagmin (Syntaxin)-25kD synaptosome-associated protein (SNAP25) trimeric complex.

[0048] In an embodiment of the present invention, ritonavir prevents and / or treats psychological disorders caused by traumatic events by affecting the process of neuronal exocytosis. Specifically, during neuronal exocytosis, the body rapidly secretes signaling molecules such as neurotransmitters or hormones by forming a soluble trimeric complex of SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP, Synaptobrevin). Ritonavir can promote the formation of the VAMP2-Syntaxin-SNAP25 trimeric complex, thereby promoting the release of vesicles from the presynaptic membrane of neurons / restoring the release of vesicles from the presynaptic membrane of neurons to normal, thereby preventing and / or treating psychological disorders caused by traumatic events.

[0049] In an embodiment of the present invention, ritonavir can reduce the stiffness rate of model mice (AtLAS- / - mice) with difficulty in extinction of fear memory, preferably reducing the stiffness rate of model mice with difficulty in extinction of fear memory by at least 10%, preferably at least 15%, more preferably at least 20%, more preferably at least 25%.

[0050] In an embodiment of the present invention, ritonavir can reduce the freezing rate of mice with difficulty in extinction of fear memory (EXT-R), preferably reducing the freezing rate of EXT-R mice by at least 10%, preferably at least 15%, more preferably at least 20%, more preferably at least 25%.

[0051] In an embodiment of the present invention, ritonavir prevents and / or treats psychological disorders caused by traumatic events by inhibiting the formation of the Syn2a-Syngr-3 complex.

[0052] In a preferred embodiment of the present invention, ritonavir is used for: (i) treating post-traumatic stress disorder; and / or (ii) preparing a medicament for treating post-traumatic stress disorder.

[0053] As used in the present invention, the term "ritonavir (RTV)" is a commercially available oral inhibitor of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) aspartic protease, which is used to treat HIV-1 infection in adults. The chemical name of ritonavir is N-[(2S,3S,5R)-3-hydroxy-5-[[(2S)-3-methyl-2-[[methyl-[(2-isopropyl-1,3-thiazol-4-yl)methyl]carbamoyl]amino]butyryl]amino]-1,6-diphenyl-hexan-2-yl]carbamic acid 5-thiazolylmethyl ester, CAS No. 155213-67-5, and the structural formula is shown in Formula I below.

[0054] As used in the present invention, the term "psychological disorders caused by traumatic events" refers to all individual psychological disorders caused by sudden, threatening or catastrophic life events, including anxiety disorders, depression, post-traumatic stress disorder and other diseases. The aforementioned anxiety disorders and depression are directly caused by traumatic events and are also called post-traumatic anxiety disorder and post-traumatic depression. As used in the present invention, the term "posttraumatic stress disorder (PTSD)" refers to an individual's delayed and long-lasting psychological stress response caused by sudden, threatening or catastrophic life events.

[0055] Treatment

[0056] The present invention also relates to a method for treating a psychological disorder caused by a traumatic event, the method comprising the steps of:

[0057] A therapeutically effective amount of ritonavir is administered to the subject.

[0058] As used herein, the term "subject" is defined herein to include animals, such as mammals, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the subject is a human.

[0059] As used herein, the term "treating" refers to the eradication or amelioration of a disease or condition, or one or more symptoms associated with the disease or condition. In one embodiment, the symptom is known to those skilled in the art to be associated with the disease or condition to be treated. In specific embodiments, the term refers to minimizing the spread or worsening of the disease or condition by administering one or more preventive or therapeutic agents to a subject suffering from the disease or condition. In some embodiments, the term refers to administering a compound of the present invention, with or without other additional active agents, after the onset of symptoms of a particular disease.

[0060] As used in the present invention, the term "therapeutically effective amount" refers to the amount of a compound that is sufficient to provide a therapeutic effect in the treatment or control of a disease or disorder, or is sufficient to delay or minimize one or more symptoms associated with the disease or disorder. The therapeutically effective amount of a compound refers to the amount of a therapeutic agent that can provide a therapeutic effect in the treatment or control of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. In some embodiments of the invention, the therapeutically effective amount is 0.1-100 mg / kg (mouse), preferably 0.1-10 mg / kg, preferably 1-10 mg / kg, for example 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg.

[0061] In a preferred embodiment of the present invention, ritonavir is administered to the subject at a dose of 0.1-100 mg / kg.

[0062] In the present invention, the mode of administering ritonavir to the study subject is not limited, and can be administered with single dose or divided dose by oral, parenteral or local route.For example, it can be administered orally several times a day from continuous (intravenous drip), and can include oral, local, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which can include a penetration enhancer), buccal, sublingual and suppository administration, and other administration routes.In a preferred embodiment of the present invention, ritonavir is administered to the study subject by oral or injection mode.

[0063] In the present invention, ritonavir can be prepared into any dosage form commonly used in the art, preferably tablets, capsules, injections, etc., more preferably capsules.

[0064] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0065] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0066] In the following examples, the experimental subjects were male C57 / BL mice (WT mice, purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd.) and AtLAS- / - mice, 8 weeks old, SPF grade, weighing 20-22 grams, and raised in a conventional environment.

[0067] Example 1

[0068] In this example, it was demonstrated that ritonavir disrupts the binding of Syn2a and Syngr-3 in HEK 293T cells.

[0069] HEK 293T cells were co-transfected with plasmids expressing EGFP-Syn2a and HA-Syngr3. Forty-eight hours after transfection, cells were treated with 0, 1, or 2.5 μM ritonavir for 12 hours, and then cellular proteins were harvested. Overexpressed Synapsin 2a (Syn2a), a fusion protein of Synapsin 2a and EGFP, was precipitated with anti-EGFP antibodies and detected with anti-HA antibodies. The interaction between Syngr3 and Syn2a was demonstrated by a black blot on a NC membrane. After treatment with 2.5 μM ritonavir, the anti-HA antibody barely detected the corresponding protein on the NC membrane, indicating that 2.5 μM ritonavir disrupted the interaction between Syn2a and Syngr3. In contrast, HA protein was clearly detected in the 0 μM control, demonstrating that 2.5 μM ritonavir disrupted the binding of Syn2a and Syngr3. The results are shown in Figure 1.

[0070] Example 2

[0071] In this example, a behavioral test of fear memory extinction in mice confirmed that ritonavir reduces the freezing rate of fear memory extinction in mice.

[0072] Six normal C57 mice and six AtLAS- / - mice were randomly selected and intraperitoneally injected with PBS solution. Another six normal C57 mice and seven AtLAS- / - mice were intraperitoneally injected with 5 mg / kg ritonavir solution for four consecutive injections (Figure 2). Fear memory testing subsequently showed that the freezing rate of fear memory extinction in normal C57 mice injected with 5 mg / kg ritonavir solution did not significantly change. However, in mice with difficult fear memory extinction (AtLAS- / - mice), the freezing rate of fear memory extinction was significantly reduced after administration of 5 mg / kg ritonavir solution (see Figure 3 for details).

[0073] Example 3

[0074] In this example, the expression of Syn2a / b on the membrane of the mouse mPFC brain region was quantified by immunoblotting.

[0075] After the fear memory extinction behavioral experiment of Example 2 was completed, the mice were killed by cervical dislocation and decapitation, the brain tissue was quickly removed and placed in 0-4°C PBS, the bilateral mPFC was quickly isolated, the total protein was extracted according to conventional steps, and the protein content was determined for later use. The Syn2a / b content of the ritonavir drug group and the control group mice was detected respectively, and GAPDH was used as the internal reference band to prove the consistency of the protein loading amount. By comparison, it was found that the protein content of Syn2a / b did not change significantly in the mice injected with 5 mg / kg ritonavir solution compared with the control group mice (Figure 4).

[0076] Example 4

[0077] In this example, immunoprecipitation was used to find that ritonavir could block the binding of Syn2a and Syngr-3 in the mouse mPFC brain region.

[0078] After collecting the proteins from Example 3, tissue proteins were precipitated with anti-Syn2a antibodies, and the precipitated proteins were detected with anti-Syngr3 antibodies. The binding of Syn2a and Syngr3 was indicated by a black blot on the NC membrane. Compared to the control group, mice injected with a 5 mg / kg ritonavir solution showed a significant decrease in the amount of the corresponding protein detected on the NC membrane using anti-Syngr3 antibodies, suggesting that ritonavir can partially block the interaction between Syn2a and Syngr3 in vivo (Figure 5).

[0079] Example 5

[0080] In this example, it was found through co-immunoprecipitation that ritonavir can improve the release of presynaptic vesicles.

[0081] After collecting the proteins in Example 3, tissue proteins were precipitated with anti-VAMP2 antibodies, and then anti-Syntaxin and SNAP25 antibodies were used to detect the precipitated proteins. The black blot on the NC membrane indicated the binding of VAMP2 to Syntaxin and SNAP25. Compared with the control group, the amount of corresponding proteins detected on the NC membrane by anti-Syntaxin and SNAP25 antibodies in mice injected with 5 mg / kg ritonavir solution was significantly increased, indicating that ritonavir can enhance the expression of AtLAS. - / - Release of vesicles from mouse presynaptic membranes (Figure 6).

[0082] Example 6

[0083] In this example, spontaneous postsynaptic potentials (sEPSCs) in mouse brain slices were detected by electrophysiological experiments, confirming that ritonavir can increase the frequency of sEPSCs.

[0084] Mice were anesthetized and sacrificed by cervical dislocation and decapitation. Brain tissue was quickly removed and placed in chilled artificial cerebrospinal fluid (aCSF). Brain regions were separated according to experimental requirements and sliced ​​into 300 μm-thick slices using a vibratome. The slices were incubated at room temperature for 1 hour. Subsequently, 2.5 μM ritonavir solution or PBS solution was administered in the prepared solution for 30 minutes. Appropriate stimulating and recording electrode positions were selected to record spontaneous excitatory aftercurrents (sEPSCs) in the mPFC. Three to six cells were recorded per slice.

[0085] The experiment found that after treatment with 2.5 μM ritonavir solution, the frequency of sEPSC was significantly increased compared with the control group, indicating that the presynaptic transmission of neurons was enhanced ( Figure 7 ).

[0086] Example 7

[0087] In this example, the distribution of synaptic vesicles in the presynaptic membrane region and the number of synaptic vesicles anchored to the presynaptic membrane were photographed by electron microscopy, and it was found that the number of vesicles anchored to the presynaptic membrane also increased significantly after ritonavir treatment.

[0088] After anesthesia, mice were sacrificed by cervical dislocation and decapitation, and brain tissue was quickly removed for electron microscopy. Electron microscopy revealed that ritonavir-treated AtLAS- / - mice showed significantly increased presynaptic vesicle accumulation at the presynaptic membrane compared to control AtLAS- / - mice, as well as a significant increase in the number of vesicles anchored to the presynaptic membrane (Figures 8 and 9).

[0089] Example 8

[0090] In this example, a behavioral test of fear memory extinction in mice confirmed that ritonavir reduced the freezing rate of fear memory extinction in EXT-R mice.

[0091] First, we performed a classic fear memory extinction behavioral test on 70 normal C57BL6 mice. By calculating the freezing rate of each mouse, we screened out 12 mice with difficulty in extinction (EXT-R). Six mice with difficulty in extinction were randomly selected and intraperitoneally injected with PBS and six mice with difficulty in extinction were intraperitoneally injected with ritonavir solution for four consecutive injections (Figure 10). After that, fear memory was tested for four consecutive days, namely EXT3, 4, 5, and 6. The results showed that the group given 5 mg / kg ritonavir solution showed lower freezing rates in EXT4, 5, and 6 compared to the PBS group (see Figure 11 for details).

[0092] Example 9

[0093] In this example, spontaneous postsynaptic potentials (sEPSCs) in mouse brain slices were detected by electrophysiological experiments, confirming that ritonavir can increase the sEPSC frequency in EXT-R mouse brain slices.

[0094] EXT-R mice were anesthetized and sacrificed by cervical dislocation and decapitation. Brain tissue was quickly removed and placed in chilled artificial cerebrospinal fluid (aCSF). Brain regions were separated according to experimental requirements and sliced ​​into 300 μm thick slices using a vibratome. The slices were then incubated at room temperature for 1 hour. Subsequently, 2.5 μM ritonavir solution or PBS solution was administered in the prepared solution for 30 minutes. Appropriate stimulating and recording electrode positions were selected to record spontaneous excitatory aftercurrents (sEPSCs) in the mPFC. Three to six cells were recorded per slice.

[0095] The experiment found that after treatment with 2.5 μM ritonavir solution, the frequency of sEPSC increased significantly compared with the control group, indicating that the presynaptic transmission of neurons was enhanced ( FIG. 12 ).

[0096] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. The use of ritonavir, characterized in that: Used for: (i) Treating psychological disorders caused by traumatic events; (ii) preparing a medicament for treating psychological disorders caused by traumatic events; (iii) promote the release of vesicles from the presynaptic membrane of neurons; (iv) inhibiting the formation of the Syn2a-Syngr-3 complex; and / or (v) Promote the formation of VAMP2-Syntaxin-SNAP25 trimeric complex.

2. The use according to claim 1, characterized in that The psychological disorder caused by the traumatic event is selected from at least one of the following: post-traumatic stress disorder, post-traumatic anxiety disorder and post-traumatic depression.

3. The use according to claim 1, characterized in that The psychological disorder caused by the traumatic event is post-traumatic stress disorder.

4. The use according to claim 1, characterized in that The ritonavir prevents and / or treats psychological disorders caused by traumatic events by inhibiting the formation of Syn2a-Syngr-3 complex.

5. The use according to claim 1, characterized in that: The ritonavir prevents and / or treats psychological disorders caused by traumatic events by promoting the formation of a VAMP2-Syntaxin-SNAP25 trimer complex.

6. The use according to claim 1, characterized in that: The ritonavir prevents and / or treats psychological disorders caused by traumatic events by promoting the release of presynaptic vesicles of neurons.

7. A method for treating post-traumatic stress disorder, characterized in that: The method comprises the step of administering a therapeutically effective amount of ritonavir to a subject.

8. The method according to claim 7, characterized in that Ritonavir was administered to the subject at a dose of 0.1-100 mg / kg.

9. The method according to claim 7, characterized in that: Ritonavir is administered to the subject orally or by injection.

10. The method according to claim 7, characterized in that Ritonavir is formulated into tablets, capsules or injections and administered to the subject.

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