Pharmaceutical composition containing homoharringtonine and application of pharmaceutical composition in preparation of anti-HIV (Human Immunodeficiency Virus) drugs

Abstract

The invention discloses a medicine composition containing homoharringtonine and application of the medicine composition in preparation of an anti-HIV (human immunodeficiency virus) medicine. The medicine composition is composed of the homoharringtonine and a nucleoside reverse transcriptase inhibitor or a non-nucleoside reverse transcriptase inhibitor, the nucleoside reverse transcriptase inhibitor is selected from one of the compounds of zidovudine, lamivudine and abacavir, and the non-nucleoside reverse transcriptase inhibitor is selected from one of the compounds of nevirapine, efavirenz and rilpivirine. The pharmaceutical composition has a remarkable synergistic effect on treatment of HIV, the curative effect of the medicine is improved, the dosage of a single medicine is reduced, the toxic and side effects are reduced, the generation of viruses can be remarkably inhibited by combined medication, a very remarkable antiviral effect is generated, and the treatment current situation of HIV-1, especially middle and advanced AIDS, is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pharmaceutical composition containing homoharringtonine and application thereof in preparing anti-HIV virus drugs. Background Art

[0002] HIV (human immunodeficiency virus) is a virus that attacks the human immune system, which eventually leads to AIDS (acquired immunodeficiency syndrome). HIV weakens the immune system by destroying and attacking the body's CD4+ T lymphocytes (a key immune cell), making the body more susceptible to other infections. HIV is treated mainly with antiretroviral drugs (ARVs) to control viral replication, boost the patient's immune system, and slow down the damage the virus does to the body. These drugs are divided into different categories, including: Nucleotide reverse transcriptase inhibitors (NRTIs): These drugs inhibit the growth of the virus by preventing the virus from copying its genetic material, RNA, when it infects new cells. Non-nucleotide reverse transcriptase inhibitors (NNRTIs): This class of drugs is also used to block the way the virus replicates, but they do so through a different mechanism. Protease inhibitors (PIs): These drugs prevent the virus from making new viral particles after infection, thereby slowing the progression of the infection. Integrase inhibitors (INSTIs): This class of drugs prevents the virus from replicating by preventing it from integrating its genetic material into the DNA of the host cell. CCR5 antagonists: These drugs slow infection by blocking the virus from entering the CCR5 receptor on CD4 cells.

[0003] At present, although highly active antiretroviral therapy (HAART) is a feasible option for treating HIV, highly active antiretroviral therapy (HARRT) can still lead to viral resistance, thus causing antiviral treatment failure. In simple terms, drug resistance means that the virus gradually mutates under the action of drugs, making the originally effective drugs no longer so effective or even completely ineffective. There are two types of AIDS drug resistance. One is called primary drug resistance, also known as transmitted drug resistance (TDR), which means that drug resistance exists before receiving antiviral drug treatment, mostly because HIV-infected people are infected with drug-resistant strains. The other is called secondary drug resistance, also known as acquired drug resistance (ADR), which is the main cause of HIV virus resistance. The main factors for secondary drug resistance are: HIV is a highly variable virus. During the virus replication process, gene mutations may cause changes in drug targets, thereby generating drug resistance; patients do not follow doctor's advice, adjust drug dosages or stop taking drugs without authorization, which may cause the virus to develop drug resistance; when using multiple drugs at the same time, the interaction between drugs may reduce the antiviral effect, thereby accelerating the development of drug resistance. HIV can eventually develop cross-resistance to different drugs in the same class, or even to drugs in different classes. For AIDS patients, drug resistance not only means treatment failure, but may also lead to rapid deterioration of the disease due to viral mutation, threatening life.

[0004] Therefore, finding an effective treatment for HIV is of great significance both in clinical application and basic research. Currently, HIV treatment drugs are small molecule compounds developed for viral proteins. The virus itself is extremely prone to mutations in the genome, leading to drug resistance. Therefore, finding a combination drug regimen or preparation is a new research direction. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention provides two pharmaceutical compositions containing homoharringtonine (HHT) and applications of the compositions in the preparation of anti-HIV drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a pharmaceutical composition containing homoharringtonine, which consists of homoharringtonine and a nucleoside reverse transcriptase inhibitor or a non-nucleoside reverse transcriptase inhibitor, wherein the nucleoside reverse transcriptase inhibitor is selected from one of the compounds zidovudine, lamivudine, and abacavir, and the non-nucleoside reverse transcriptase inhibitor is selected from one of the compounds nevirapine, efavirenz, and rilpivirine. Any of the above six combination schemes can be used alone to inhibit the production of HIV virus. In some embodiments of the present invention, the pharmaceutical composition is used to inhibit HIV virus in vitro. In some embodiments of the present invention, the pharmaceutical composition is used to inhibit HIV virus in vivo.

[0007] In some embodiments of the present invention, the molar ratio of homoharringtonine to nucleoside reverse transcriptase inhibitors in the pharmaceutical composition is no more than 1:3. In some embodiments, the molar ratio of homoharringtonine to nucleoside reverse transcriptase inhibitors is 1:4; in some embodiments, it is 1:5000; in some embodiments, it is 1:7500; in some embodiments, it is 1:10000; in some embodiments, it is 1:15000.

[0008] In some embodiments of the present invention, the molar ratio of homoharringtonine to the non-nucleoside reverse transcriptase inhibitor in the pharmaceutical composition ranges from (1:10) to (5:1). In some embodiments, the molar ratio of homoharringtonine to the non-nucleoside reverse transcriptase inhibitor is 1:8; in some embodiments, 1:1.25; in some embodiments, 1:2.5; in some embodiments, 1:5; in some embodiments, 1:1; in some embodiments, 2:1; and in some embodiments, 4:1.

[0009] The structure of homoharringtonine is as follows:

[0010] The structural formulas of the nucleoside reverse transcriptase inhibitors zidovudine, lamivudine, and abacavir are as follows:

[0011] The non-nucleoside reverse transcriptase inhibitors Nevirapine, Efavirenz, and Rilpivirine have the following structural formulas:

[0012] In some embodiments of the present invention, the homoharringtonine is replaced by a homoharringtonine derivative.

[0013] In some embodiments of the present invention, the homoharringtonine derivatives include harringtonine, deoxyharringtonine, and isoharringtonine.

[0014] In some embodiments of the present invention, the nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor are hydrates, analogs, derivatives and organic or inorganic salts of the selected compounds.

[0015] The second aspect of the present invention provides a use of the pharmaceutical composition described in the first aspect in the preparation of a drug for treating HIV.

[0016] In some embodiments of the present invention, homoharringtonine and its derivatives, nucleoside reverse transcriptase inhibitors or non-nucleoside reverse transcriptase inhibitors in the pharmaceutical composition are prepared simultaneously, or are prepared separately and used in combination.

[0017] In some embodiments of the present invention, the medicine comprises the pharmaceutical composition or pharmaceutically acceptable carrier described in the first aspect.

[0018] In some embodiments of the present invention, the pharmaceutical dosage form includes clinically acceptable preparations: tablets, capsules, pills, controlled-release preparations, sustained-release preparations, granules, and injections.

[0019] In the pharmaceutical composition, HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be contained in the same pharmaceutical preparation such as tablets or capsules, or HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be made into preparations separately for separate administration, or the components in the above composition can be made into a controlled-release preparation, which first releases one component of the composition and then releases the other component of the composition, and the patient only needs to take the controlled-release composition preparation.

[0020] The HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor in the composition can be used simultaneously or in any order, such as HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be administered to the patient simultaneously; HHT can also be administered to the patient first, followed by the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor, or the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be administered to the patient first, followed by HHT. HHT and existing preparations of nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can also be combined, or the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be made into a controlled release preparation and used in combination with HHT injection.

[0021] In the present invention, the pharmaceutical composition can be prepared into a pharmaceutical preparation suitable for gastrointestinal administration or parenteral administration by conventional methods in the art. The present invention preferably prepares the pharmaceutical composition into a pharmaceutical preparation for gastrointestinal administration, and the preparation form can be conventional tablets, capsules, controlled-release tablets, sustained-release preparations; it can also be pills, granules and injections.

[0022] In the present invention, there is no particular limitation on the preparation method of the pharmaceutical composition. The two components in the pharmaceutical composition can be directly mixed and then prepared into a preparation, or they can be mixed separately and / or with corresponding excipients to prepare preparations and then packaged together according to conventional methods in the art, or they can be mixed separately with corresponding excipients and then mixed to prepare preparations.

[0023] The dosage of the pharmaceutical composition of the present invention is adjusted according to the administration object, administration route or pharmaceutical preparation form, but it is premised on ensuring that the pharmaceutical composition can reach an effective blood drug concentration in the body.

[0024] The present invention also provides a use of the pharmaceutical composition described in the first aspect in the preparation of an inhibitor for inhibiting HIV virus production in vitro.

[0025] When used as an in vitro inhibitor, the HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor in the composition can be added simultaneously or sequentially in any order.

[0026] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a pharmaceutical composition, in which HTT achieves antiviral effect by inhibiting ribosome programmed frameshifting, nucleoside reverse transcriptase inhibitors compete with natural deoxynucleoside triphosphates to bind to HIV reverse transcriptase (RT), inhibit the action of RT, and hinder the synthesis of provirus; non-nucleoside reverse transcriptase inhibitors bind to the hydrophobic region near the active site of reverse transcriptase to cause structural destruction of its catalytic site, thereby inhibiting viral synthesis. The combination of HTT and nucleoside reverse transcriptase inhibitors or non-nucleoside reverse transcriptase inhibitors has a significant synergistic effect in the treatment of HIV, improves the efficacy of the drug, reduces the dosage of a single drug, and reduces toxic side effects.

[0027] Developing the above-mentioned composition into a combined drug regimen or preparation for HIV can significantly inhibit the production of the virus, produce a very significant antiviral effect, and improve the current treatment status of HIV-1, especially mid- and late-stage AIDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The graph shows the change in the Flu / Rlu ratio after pLVX-Rennila Luciferase-(-1PRF)-FireflyLuciferase was transfected into Hela cells and treated with DMSO and HHT in Example 1 of the present invention; Figure 2 The figure shows the comparison of the results of p24 ELISA detection of virus content in mouse blood after treatment under different conditions in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0030] The numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the recorded component, physical or other properties (such as molecular weight, melt index, etc.) are 100 to 1000, it means that all single values ​​are clearly listed, such as 100, 101, 102, etc., and all sub-ranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For a range containing a value less than 1 or containing a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are only specific examples of what is intended, and all possible combinations of numerical values ​​between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0031] 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 the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0032] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0033] In the following examples, pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was constructed by the laboratory itself, and the Rennila Luciferase-(-1PRF)-Firefly Luciferase fragment was constructed into the pLVX-Puro plasmid. The pLVX-Puro plasmid was purchased from TAKARA, and the pNL43-dE plasmid was purchased from Addgene.

[0034] Example 1 The laboratory constructed pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase and transferred it into Hela cells. After 24 hours, 40nM DMSO and 40nM HHT were added to the cells for 6 hours, and the expression levels of Rennila Luciferase and Firefly Luciferase were detected by a multifunctional microplate reader. The results are as follows: Figure 1 shown.

[0035] The results showed that the ratio of Firefly Luciferase to Rennila Luciferase was significantly reduced, indicating that the compound HHT can significantly reduce the efficiency of programmed glycosome frameshifting.

[0036] The antiviral mechanism of HHT is to inhibit programmed ribosomal frameshifting, thereby preventing the production of Gag-Pol fusion protein and the cleavage of Pol protein into reverse transcriptase, integrase and protease. Compared with the single inhibitory effect of currently available small molecule compounds, HHT can make multiple proteins of the virus lack at the same time, which has an effect that other small molecule compounds cannot match.

[0037] Example 2 In this example, we investigated the efficiency of HHT and the nucleoside reverse transcriptase inhibitors zidovudine, lamivudine, and abacavir in inhibiting the production of HIV virus.

[0038] The research scheme and concentration design are shown in Table 1.

[0039] Table 1 HHT and zidovudine, lamivudine, abacavir drug combination inhibition scheme design

[0040] The specific research process is as follows: the pNL43-dE plasmid was first transferred into the cells. After 24 hours, the experiments were carried out according to the design schemes 1 to 24 in Table 1. After 12 hours of drug treatment, the virus content in the supernatant of the cell culture medium was detected by p24 ELISA.

[0041] When no drug is added, the p24 content detected in the supernatant is defined as a virus inhibition rate of 0. After drug treatment, the ratio of the decrease in the p24 value is the drug's inhibition rate on the virus. Whether the two drugs have a synergistic effect, the King's formula for the combined drug effect is used to calculate the q value. The calculation method for the combined drug effect q value is: AB combined drug virus inhibitor / (A drug virus inhibitor + B drug virus inhibitor - A drug virus inhibitor × B drug virus inhibitor). When q>1, it indicates that the two drugs have a synergistic effect when used together. When q=1, it indicates that the two drugs are normally accumulated. When q<1, it indicates that the two drugs have an antagonistic effect when used together. The results of the above schemes 1 to 24 are shown in Table 2.

[0042] Table 2 HHT and zidovudine, lamivudine, abacavir drug combination inhibition scheme test results

[0043] From the results in Table 2, it can be seen that the q values ​​calculated for the combined use of HHT with zidovudine, lamivudine or abacavir are all greater than 1, indicating that the combination of the two has a significant synergistic effect. In particular, the q value of HHT and zidovudine is above 1.8, indicating that the combination of the two has a very strong synergistic effect.

[0044] Example 3 In this example, we investigated the efficacy of HHT in combination with the non-nucleoside reverse transcriptase inhibitors Nevirapine, Efavirenz, and Rilpivirine in inhibiting HIV virus production.

[0045] The research scheme and concentration design are shown in Table 3.

[0046] Table 3 HHT and nevirapine, efavirenz, rilpivirine drug combination inhibition scheme design

[0047] The specific exploration process and the calculation method and judgment method of the q value of the combined drug effect are the same as in Example 2.

[0048] The results of the above-mentioned schemes 1 to 24 are shown in Table 4.

[0049] Table 4 HHT and zidovudine, lamivudine, abacavir drug combination inhibition scheme test results

[0050] From the results in Table 4, it can be seen that the calculated values ​​of HHT combined with Nevirapine, Efavirenz, and Rilpivirine are all greater than 1, indicating that the combination of the two has a significant synergistic effect. In particular, the q value of HHT and Rilpivirine can reach more than 2, indicating that the combination of the two has a very strong synergistic effect.

[0051] Example 4 In order to study the synergistic effect of HHT and nucleoside and non-nucleoside reverse transcriptase inhibitors in inhibiting HIV in animals, we compared DMSO, HHT, zidovudine, and rilpivirine alone, and the combined use of HHT and zidovudine, and HHT and rilpivirine. The research scheme and concentration design are shown in Table 5.

[0052] Table 5 Animal experiment research plan design

[0053] The specific research process is as follows: pNL43-dE plasmid and pCMV-VSV-G plasmid were co-transfected into 293T cells. After 48 hours, the culture medium was collected and the HIV pseudovirus particles in the culture medium were purified by ultracentrifugation. The titer of the purified pseudovirus was determined and 1×10 7 The virus was injected into the mice through the tail vein. 24 hours later, we injected DMSO, HHT, zidovudine, and rilpivirine into the tail vein of the mice alone or in combination according to the scheme in Table 5 above. 24 hours after the drug injection, we detected the content of HIV virus particles in the blood of the mice by p24 ELISA. The results are shown in the figure. Figure 2 shown.

[0054] The results showed that when HHT was used in combination with the nucleoside transcriptase inhibitor zidovudine, the q value was 1.48, compared with the single use, indicating that the combination of the two also showed a significant synergistic effect in mice. Similarly, when HHT was used in combination with the non-nucleoside reverse transcriptase inhibitor rilpivirine, the q value was 1.51, also indicating that the combination of the two also had an extremely significant synergistic effect in mice.

[0055] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A pharmaceutical composition containing homoharringtonine, characterized in that: The pharmaceutical composition consists of homoharringtonine and a nucleoside reverse transcriptase inhibitor or a non-nucleoside reverse transcriptase inhibitor, wherein the nucleoside reverse transcriptase inhibitor is selected from one of the compounds zidovudine, lamivudine and abacavir, and the non-nucleoside reverse transcriptase inhibitor is selected from one of the compounds nevirapine, efavirenz and rilpivirine.

2. The pharmaceutical composition according to claim 1, characterized in that: The homoharringtonine is replaced by a homoharringtonine derivative.

3. The pharmaceutical composition according to claim 2, characterized in that: The homoharringtonine derivatives include harringtonine, deoxyharringtonine and isoharringtonine.

4. The pharmaceutical composition according to claim 1, characterized in that: The nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor are hydrates, analogs, derivatives and organic or inorganic salts of the selected compounds.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of anti-HIV drugs.

6. The use according to claim 5, characterized in that: The medicine comprises the pharmaceutical composition according to any one of claims 1 to 4 or the pharmaceutical composition and a pharmaceutically acceptable carrier.

7. The use according to claim 5, characterized in that: The homoharringtonine and its derivatives, nucleoside reverse transcriptase inhibitors or non-nucleoside reverse transcriptase inhibitors in the medicine are prepared simultaneously, or prepared separately and used in combination.

8. The use according to claim 7, characterized in that: The medicine includes clinically acceptable preparations: tablets, capsules, pills, controlled-release preparations, sustained-release preparations, granules and injections.

9. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of an in vitro inhibitor of HIV virus.

10. The use according to claim 9, characterized in that: In the application, HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor in the pharmaceutical composition can be added simultaneously or sequentially in any order.

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