Pharmaceutical composition containing homoharringtonine and its application in preparing anti-HIV drug

Abstract

The present invention discloses a pharmaceutical composition containing homoharringtonine and its application in the preparation of anti-HIV viral drugs. The pharmaceutical composition is composed of 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 zidovudine, lamivudine, and abacavir, and the non-nucleoside reverse transcriptase inhibitor is selected from one of the compounds nevirapine, efavirenz, and rilpivirine. The pharmaceutical composition has a significant synergistic effect in treating HIV, improves the efficacy of the drug, reduces the dosage of a single drug, reduces toxic side effects, and can significantly inhibit the production of viruses in combination, producing a very significant antiviral effect, and improving the current status of the treatment of HIV-1, especially middle and late stage AIDS.

Description

Technical Field

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

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

[0003] Although highly active antiretroviral therapy (HARRT) is currently a viable option for HIV treatment, it can still lead to the development of viral resistance, which can lead to treatment failure. Simply put, drug resistance occurs when a virus mutates under the influence of drugs, rendering previously effective drugs less effective or even completely ineffective. There are two types of HIV drug resistance. One is primary drug resistance, also known as transmitted drug resistance (TDR), which occurs before treatment begins. This is often caused by HIV infection with a resistant strain. The other is secondary drug resistance, also known as acquired drug resistance (ADR), which is currently the main cause of HIV drug resistance. The main factors contributing to secondary drug resistance include: HIV is a highly variable virus. During viral replication, genetic mutations can alter drug targets, leading to drug resistance; patients failing to follow their doctor's orders, adjusting drug dosages without authorization, or stopping medications, can also lead to the development of drug resistance; and when multiple drugs are used simultaneously, drug interactions can reduce antiviral efficacy, accelerating the development of drug resistance. HIV can eventually develop cross-resistance to different drugs within the same class, or even to drugs from different classes. For HIV patients, drug resistance not only means treatment failure, but can also lead to rapid deterioration of the disease due to viral mutations, threatening their lives.

[0004] Therefore, finding an effective and proactive treatment for HIV is crucial both in clinical practice and in basic research. Currently, HIV treatments are small molecules developed to target viral proteins. The virus itself is prone to mutations in its genome, leading to drug resistance. Therefore, finding a combination drug regimen or formulation 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 their use in preparing anti-HIV drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The first aspect of the present invention provides a pharmaceutical composition containing homoharringtonine, which is composed 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.

[0008] In some embodiments of the present invention, the molar ratio of homoharringtonine to nucleoside reverse transcriptase inhibitor in the pharmaceutical composition is no more than 1:3. In some embodiments, the molar ratio of homoharringtonine to nucleoside reverse transcriptase inhibitor is 1:4; in some embodiments, 1:5000; in some embodiments, 1:7500; in some embodiments, 1:10000; and in some embodiments, 1:15000.

[0009] 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.

[0010] The structure of homoharringtonine is as follows:

[0011]

[0012] The structural formulas of the nucleoside reverse transcriptase inhibitors zidovudine, lamivudine, and abacavir are as follows:

[0013]

[0014] The structural formulas of the non-nucleoside reverse transcriptase inhibitors Nevirapine, Efavirenz, and Rilpivirine are as follows:

[0015]

[0016] In some embodiments of the present invention, the homoharringtonine is replaced by a homoharringtonine derivative.

[0017] In some embodiments of the present invention, the homoharringtonine derivatives include harringtonine, deoxyharringtonine, and isoharringtonine.

[0018] In some embodiments of the present invention, the nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors are hydrates, analogs, derivatives and organic or inorganic salts of the selected compounds.

[0019] 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.

[0020] 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.

[0021] In some embodiments of the present invention, the medicament comprises the pharmaceutical composition or pharmaceutically acceptable carrier described in the first aspect.

[0022] In some embodiments of the present invention, the pharmaceutical dosage forms include clinically acceptable preparations: tablets, capsules, pills, controlled-release preparations, sustained-release preparations, granules, and injections.

[0023] 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 a tablet or capsule. Alternatively, HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be formulated into separate preparations and administered separately. Alternatively, the components of the above-mentioned composition can be formulated into a controlled-release preparation that first releases one component of the composition and then releases the other component of the composition. The patient only needs to take the controlled-release composition preparation.

[0024] The HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor in the composition can be administered simultaneously or in any order. For example, HHT and the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be administered to the patient simultaneously; HHT can be administered 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 first, followed by HHT. HHT and existing nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor formulations can also be combined, or the nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor can be formulated into a controlled-release formulation and administered in combination with an HHT injection.

[0025] 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.

[0026] 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.

[0027] The dosage of the pharmaceutical composition of the present invention is adjusted according to the subject of administration, route of administration or form of pharmaceutical preparation, but it is premised on ensuring that the pharmaceutical composition can reach an effective blood concentration in the body.

[0028] The present invention also provides a use of the pharmaceutical composition described in the first aspect in preparing an inhibitor for inhibiting HIV virus production in vitro.

[0029] 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.

[0030] Beneficial effects of the present invention

[0031] Compared to existing technologies, the present invention has the following advantages: It provides a pharmaceutical composition in which HTT achieves an antiviral effect by inhibiting programmed ribosomal frameshifting. Nucleoside reverse transcriptase inhibitors compete with natural deoxynucleoside triphosphates for binding to HIV reverse transcriptase (RT), inhibiting RT and hindering proviral synthesis. Non-nucleoside reverse transcriptase inhibitors bind to the hydrophobic region near the active site of reverse transcriptase, disrupting the structure of the catalytic site and thereby inhibiting viral synthesis. Combinations of HTT with either nucleoside reverse transcriptase inhibitors or non-nucleoside reverse transcriptase inhibitors exhibit significant synergistic effects in the treatment of HIV, improving drug efficacy, reducing the dosage of individual drugs, and minimizing toxic side effects.

[0032] 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-to-late stage AIDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The 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;

[0034] 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

[0035] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0036] The numerical ranges in this application are approximate, so unless otherwise stated, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if a component, physical or other property (such as molecular weight, melt index, etc.) is recorded as 100 to 1000, it means that all individual values, such as 100, 101, 102, etc., and all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc., are clearly listed. For ranges containing values ​​less than 1 or containing fractions 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 ranges containing single digits 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.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0038] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0039] 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.

[0040] Example 1

[0041] The laboratory constructed pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase and transformed 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 using a multifunctional microplate reader. The results are as follows: Figure 1 shown.

[0042] 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.

[0043] HHT's antiviral mechanism is to inhibit programmed ribosomal frameshifting, thereby preventing the production of the Gag-Pol fusion protein and the cleavage of the Pol protein into reverse transcriptase, integrase, and protease. Compared to the single inhibitory effects of currently available small molecule compounds, HHT simultaneously depletes multiple viral proteins, achieving an unparalleled effect.

[0044] Example 2

[0045] In this example, we investigated the efficacy of a pharmaceutical combination of HHT and the nucleoside reverse transcriptase inhibitors zidovudine, lamivudine, and abacavir in inhibiting HIV production.

[0046] The research scheme and concentration design are shown in Table 1.

[0047] Table 1 HHT and zidovudine, lamivudine, abacavir drug combination inhibition scheme design

[0048]

[0049] 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.

[0050] When no drug is added, the p24 level detected in the supernatant is defined as a viral inhibition rate of 0. After drug treatment, the rate of decrease in the p24 value is the drug's viral inhibition rate. Whether two drugs have a synergistic effect is determined by calculating the q value using King's formula for the combined drug effect: The q value for the combined drug effect is calculated as: viral inhibition of the AB combination / (viral inhibition of drug A + viral inhibition of drug B - viral inhibition of drug A × viral inhibition of drug B). When q > 1, the two drugs have a synergistic effect. When q = 1, the two drugs are additive. When q < 1, the two drugs have an antagonistic effect. The results of Schemes 1 to 24 are shown in Table 2.

[0051] Table 2 HHT and zidovudine, lamivudine, abacavir drug combination inhibition regimen test results

[0052]

[0053] The results in Table 2 show that the calculated q values ​​for HHT combined with zidovudine, lamivudine, or abacavir were all greater than 1, indicating significant synergistic effects between the two. In particular, the q value for HHT combined with zidovudine reached over 1.8, indicating a strong synergistic effect.

[0054] Example 3

[0055] 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 production.

[0056] The research scheme and concentration design are shown in Table 3.

[0057] Table 3 HHT and nevirapine, efavirenz, rilpivirine drug combination inhibition scheme design

[0058]

[0059] The specific research process and the calculation and judgment methods of the q value of the combined drug effect are the same as those in Example 2.

[0060] The results of the above schemes 1 to 24 are shown in Table 4.

[0061] Table 4 HHT and zidovudine, lamivudine, abacavir drug combination inhibition regimen test results

[0062]

[0063] The results in Table 4 show that the calculated q values ​​for HHT combined with nevirapine, efavirenz, and rilpivirine are all greater than 1, indicating significant synergistic effects between the two drugs. In particular, the q value for HHT combined with rilpivirine can reach greater than 2, indicating a strong synergistic effect between the two drugs.

[0064] Example 4

[0065] To investigate the synergistic effect of HHT with nucleoside and non-nucleoside reverse transcriptase inhibitors in inhibiting HIV in animals, we compared the use of DMSO, HHT, zidovudine, and rilpivirine alone, as well as the combined use of HHT with zidovudine and HHT with rilpivirine. The study regimens and concentration designs are shown in Table 5.

[0066] Table 5 Animal experiment research plan design

[0067]

[0068] 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, DMSO, HHT, zidovudine, and rilpivirine were injected into the tail vein of the mice alone or in combination according to the scheme in Table 5. 24 hours after drug injection, the content of HIV virus particles in the mouse blood was detected by p24 ELISA. The results are shown in the figure below. Figure 2 shown.

[0069] The results showed that when HHT was combined with the nucleoside transcriptase inhibitor zidovudine, the q value was 1.48, compared to when either was used alone, indicating that the combination also exhibited a significant synergistic effect in mice. Similarly, when HHT was combined with the non-nucleoside reverse transcriptase inhibitor rilpivirine, the q value was 1.51, also indicating that the combination also had a significant synergistic effect in mice.

[0070] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. Use of a pharmaceutical composition containing homoharringtonine in the preparation of an anti-HIV drug, characterized in that: The pharmaceutical composition consists of homoharringtonine and a nucleoside reverse transcriptase inhibitor or a non-nucleoside reverse transcriptase inhibitor. The nucleoside reverse transcriptase inhibitor is the compound zidovudine, and the non-nucleoside reverse transcriptase inhibitor is the compound rilpivirine.

2. The use according to claim 1, characterized in that: The nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor are organic or inorganic salts of selected compounds.

3. The use according to claim 1, characterized in that: The anti-HIV virus drug comprises the pharmaceutical composition or the pharmaceutical composition and a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The homoharringtonine, nucleoside reverse transcriptase inhibitor or non-nucleoside reverse transcriptase inhibitor in the anti-HIV drug are prepared simultaneously, or prepared separately and used in combination.

5. The use according to claim 4, characterized in that: The anti-HIV drug includes clinically acceptable preparations: tablets, capsules, pills, controlled-release preparations, sustained-release preparations, granules and injections.

6. Use of a pharmaceutical composition containing homoharringtonine in the preparation of an in vitro HIV virus inhibitor, characterized in that: The pharmaceutical composition consists of homoharringtonine and a nucleoside reverse transcriptase inhibitor or a non-nucleoside reverse transcriptase inhibitor. The nucleoside reverse transcriptase inhibitor is the compound zidovudine, and the non-nucleoside reverse transcriptase inhibitor is the compound rilpivirine.

7. The use according to claim 6, characterized in that: In the above-mentioned use, homoharringtonine 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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