Vidarabine monophosphate liposome freeze-dried powder injection as well as preparation method and application thereof

Abstract

The invention discloses vidarabine monophosphate lipidosome freeze-dried powder injection and a preparation method and application thereof, and belongs to the technical field of medicine, and the lipidosome is composed of vidarabine monophosphate, phospholipid, cholesterol, pegylated phospholipid and a freeze-drying protective agent (such as trehalose). By optimizing a formula and a preparation process, the liposome disclosed by the invention has excellent placement stability and freeze-drying stability, and can still keep good encapsulation efficiency and particle size distribution after long-term storage. By introducing polyethylene glycol (PEG), the retention time of liposome in systemic circulation is remarkably prolonged, and passive targeting enrichment of liver and spleen is reduced, so that the toxicity of immune organs is reduced, and the accumulation of drugs at focus parts is facilitated. The liposome has an excellent slow release characteristic, can improve the antiviral ability and cell entry ability of drugs and reduce toxic and side effects, and is suitable for treating viral infectious diseases, such as hepatitis B, herpes and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a lyophilized powder injection of adenosine monophosphate liposomes, its preparation method, and its application. Background Technology

[0002] Aabinofuranosyl adenine monophosphate (Ara-AMP), chemically named 9-(β-D-arabinofuranose)-adenine-5'-monophosphate, is a phosphorylated adenosine monophosphate synthesized from vidarabine and phosphoryl chloride. It is a glycoside compound formed from adenine and arabinose monophosphate and is a broad-spectrum antiviral drug. It appears as a white or off-white crystalline powder, soluble in water but sparingly soluble in methanol, ethanol, and ether. It possesses multiple antiviral mechanisms of action: firstly, it significantly inhibits the biological activity of viral nucleotide reductase, thereby blocking the supply of deoxynucleotide precursors required for viral DNA synthesis; secondly, this metabolite effectively inhibits the function of viral DNA terminal deoxynucleotidyl transferase. This dual inhibition not only leads to the misincorporation of Ara-AMP into the viral DNA chain but also promotes its specific binding to the 3'-OH end of the DNA chain, ultimately forming an irreversible chain termination effect and completely blocking the replication process of the viral genome. It is commonly used clinically to treat viral infections such as hepatitis B and herpes. However, its short half-life (about 3.5 hours) requires frequent administration, and its lack of targeted therapy leads to significant toxic side effects.

[0003] Currently, only one dosage form of vidarabine monophosphate (MPS) is available on the market: injectable MPS. There is no literature on its liposomal formulation. Its short half-life makes it easily degraded into vidarabine hypoxanthine (with lower activity) by adenosine deaminase in vivo, requiring frequent administration to maintain efficacy. Its inhibitory effect on DNA polymerase is not limited to viruses but acts non-selectively throughout the body, resulting in hematologic toxicity such as anemia and neutropenia, as well as damage to the skin and its appendages, gastrointestinal damage, and systemic injury. Severe cases can cause anaphylactic shock. However, there is a lack of experience and reports both domestically and internationally regarding reducing the toxic side effects of injectable MPS and improving its formulation. Due to these toxic side effects, the number of cases treated with MPS in clinical practice is currently limited. However, with the increasing frequency of use of drugs such as acyclovir and tenofovir, the virus targets and mutates against the activation mechanism before the drug takes effect in the body, leading to drug resistance. As a result, vidarabine monophosphate has reappeared in clinical applications. Vidarabine monophosphate can effectively avoid viral targeting mutations due to its unique mechanism of action that does not require activation. Therefore, research on strategies to enhance the efficacy and reduce the toxicity of injectable vidarabine monophosphate is urgently needed.

[0004] Publication No. CN105232571A discloses a method for preparing a low-dose adenosine monophosphate (AMP) pharmaceutical composition powder for injection. However, the AMP instructions do not specify a pediatric dosage. In clinical applications, medication is usually administered according to adult dosages based on weight, sometimes even exceeding the recommended dose. Administering excessive doses to children significantly increases the incidence of adverse drug reactions and raises medical costs. This low-dose AMP pharmaceutical composition can effectively treat herpesvirus or cytomegalovirus infections in children, avoiding the toxic side effects and increased medical costs associated with high or ultra-high dose administration. However, this formulation relies on AMP synergistically with other drugs for clinical treatment. In this patent, AMP only plays an adjunctive therapeutic role. While it addresses the issue of toxic side effects, it does not resolve the issue of dosing frequency, and the efficacy of AMP is not directly demonstrated. Publication No. CN1562068A discloses a arabinophosphate monophosphate aqueous injection and its preparation method. The preparation method is simple, using only activated carbon treatment and autoclaving, resulting in a short production cycle and ease of industrial production. However, the formulation only contains arabinophosphate monophosphate and water for injection, leading to low formulation stability, easy drug degradation, and difficulty in ensuring quality. Publication No. CN103202804A discloses a arabinophosphate monophosphate aqueous injection and its preparation method. Although this method adds pH adjusters such as sodium hydroxide or phosphate buffer to the formulation in addition to arabinophosphate monophosphate and water for injection to improve stability, it still has limitations in transportation and storage, as well as sustained-release and targeted delivery. Furthermore, it fails to address the issues of toxic side effects and dosing frequency. Publication number CN116270498A discloses an injectable adenosine monophosphate powder for injection and its preparation method. The powder for injection is convenient to use, highly stable, and easy to transport and store. The formulation includes adenosine monophosphate, sodium chloride, mannitol, and water for injection. The preparation process uses a repeated freeze-drying method, which is relatively complex, has a long production cycle, and still has limitations in terms of sustained release and targeted delivery. Overall, current domestic and international improvements to injectable adenosine monophosphate focus on the stability and convenience of the formulation, without addressing improvements in sustained release or reducing toxic side effects.

[0005] Liposomes are lipid bilayer vesicles composed of phospholipids and cholesterol. They are researched and applied in numerous fields, including cancer, eye diseases, viral infections, gene delivery, vaccines, anesthesia and analgesia, and imaging diagnostics. More than 20 liposomal formulations have been approved for marketing worldwide, such as cytarabine liposome (DepoCyte®), daunorubicin liposome (DaunoSome®), and doxorubicin liposome (Doxil®). This structure, similar to a natural cell membrane, allows them to overcome cellular and tissue uptake barriers, improving drug distribution in tissues and organs throughout the body. This enables encapsulated drugs to be effectively delivered to the target site, minimizing systemic toxicity. Simultaneously, internal encapsulation prevents enzymatic degradation and rapid plasma clearance, prolonging circulation time, improving sustained-release effects and bioavailability, thereby enhancing clinical therapeutic efficacy.

[0006] As a nanomedicine delivery system, liposomes are susceptible to oxidation, hydrolysis, aggregation, or fusion due to their phospholipid bilayer structure, leading to drug leakage and decreased stability, especially under liquid storage conditions (such as aqueous media). To improve the long-term stability of adenosine monophosphate liposomes, freeze-drying (lyophilization) can be used to convert the liposomes into a solid powder by removing moisture, thus extending their shelf life. There are two main hypotheses regarding the protection mechanism of freeze-drying. One is the water substitution model, which proposes that hydrogen bonds are formed between the polar part (hydroxyl group) of sugar molecules and the polar head of phospholipid molecules. The presence of these hydrogen bonds can maintain the spatial position of the polar head of phospholipid molecules while reducing the van der Waals forces between acyl chains, thereby lowering the phase transition temperature of the plasma membrane and preventing membrane fusion and aggregation. The other is the glass transition model, which proposes that sugar solutions are concentrated into a stable glassy state during freezing. This amorphous glassy state has high viscosity and low fluidity, which can encapsulate liposomes. Through osmotic and volume effects, it can inhibit the fusion and aggregation of liposomes, protecting the lipid membrane from damage by ice crystals. At the same time, the glassy state of sugar can also inhibit conformational changes related to the phase transition of liposomes.

[0007] Given that there is currently no literature on the research of liposome formulations of adenosine monophosphate, and that existing dosage forms of adenosine monophosphate (such as aqueous injections and powder injections) have problems such as poor stability, large toxic side effects, and multiple administration times, this invention applies liposomes to adenosine monophosphate injection and performs freeze-drying treatment to develop a lyophilized powder injection of adenosine monophosphate liposomes with sustained-release and targeted properties.

[0008] The lyophilized powder injection of adenosine monophosphate liposome developed in this invention has high stability and sustained-release effect, reduces the number of dosings, improves drug targeting, and can significantly enhance antiviral ability while reducing systemic toxic side effects. Summary of the Invention

[0009] The purpose of this invention is to provide a lyophilized liposome powder injection containing adenosine monophosphate, its preparation method, and its application. The lyophilized liposome powder injection of this invention significantly improves the stability, safety, and sustained-release performance of the product by optimizing the formulation and preparation process. It also has the advantages of simple preparation process and suitability for industrial production.

[0010] Specifically, the present invention has the following outstanding features:

[0011] (1) Using specific components and ratios, the encapsulation efficiency and particle size of lyophilized liposome injections are kept stable under long-term storage conditions;

[0012] (2) PEG surface modification prolongs the body circulation time and significantly reduces toxicity;

[0013] (3) It has excellent sustained-release properties, which can reduce the frequency of clinical administration;

[0014] (4) It has better antiviral effects;

[0015] (5) The preparation process is simple and controllable, with low equipment requirements, and is easy to achieve industrial production.

[0016] The objective of this invention can be achieved through the following technical solutions:

[0017] A lyophilized powder injection of adenosine monophosphate liposomes, comprising, by weight:

[0018] One part of adenosine monophosphate;

[0019] Phospholipids 2-10 parts;

[0020] 0.5–3.0 parts of membrane flow modifier;

[0021] 0.02–0.80 parts of surface modification agent;

[0022] 5-40 parts of freeze-drying protectant.

[0023] Furthermore, the phospholipid is selected from one or more of soybean phospholipids, hydrogenated soybean phospholipids, dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC).

[0024] Furthermore, the phospholipid is distearylphosphatidylcholine (DSPC).

[0025] Furthermore, the membrane fluidity regulator is selected from one or more of cholesterol, polysorbate, and phytosterols.

[0026] Furthermore, the membrane fluidity regulator is cholesterol.

[0027] Furthermore, the surface modifier is selected from one or more of distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), polysialic acid, polyhydroxyethyl starch, and hyaluronic acid.

[0028] Furthermore, the surface modifier is distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0029] Furthermore, the freeze-drying protectant is selected from one or more of sucrose, trehalose, glucose, lactose, and mannitol.

[0030] Furthermore, the freeze-drying protectant is trehalose.

[0031] As a further aspect of the present invention, the preparation method of the above-mentioned lyophilized adenosine monophosphate liposome injection includes the following steps:

[0032] (1) Dissolve phospholipids, membrane flow modifiers and surface modifiers in an organic solvent to form an organic phase;

[0033] (2) Dissolve adenosine monophosphate in a buffer salt solution to form an aqueous phase;

[0034] (3) The organic phase obtained in step (1) is mixed with the aqueous phase obtained in step (2), and emulsified by high-speed shearing for 5 to 20 minutes. The organic solvent is then removed by rotary evaporation, and the mixture is hydrated at 55 to 70°C for 30 to 120 minutes. The mixture is then extruded or homogenized to make the particle size uniform. After removing the free drug, a freeze-drying protectant is added, and the mixture is then filtered to remove bacteria and freeze-dried to obtain adenosine monophosphate liposome freeze-dried powder injection.

[0035] Furthermore, the organic solvent mentioned in step (1) is selected from one or more of dichloromethane, trichloromethane, tetrahydrofuran, ethanol, methanol, acetone or ethyl acetate.

[0036] Furthermore, the organic solvent mentioned in step (1) is chloroform.

[0037] Furthermore, the buffer salt solution mentioned in step (2) is a borate buffer, phosphate buffer, citrate buffer, or glycine buffer.

[0038] Furthermore, the buffer salt solution mentioned in step (2) is a phosphate buffer solution with a pH value of 7.4 to 9.6.

[0039] Furthermore, the volume ratio of the buffer salt solution to the organic solvent in step (3) is 1:2 to 5.

[0040] As a further aspect of the present invention, the above-mentioned lyophilized adenosine monophosphate liposome powder for injection is used in the preparation of drugs for treating viral infections.

[0041] The beneficial effects of this invention are:

[0042] (1) Stability: After 180 days of accelerated testing, the liposome particle size change rate was <5%, and the encapsulation efficiency retention rate was >95%;

[0043] (2) Safety: Cytotoxicity test and in vivo animal safety test showed that its safety was significantly better than that of ordinary preparations. The acute toxicity test showed that the intravenous LD50 was 2557±64mg / kg, which was more than twice as good as ordinary adenosine monophosphate injection (1200mg / kg).

[0044] (3) Sustained-release properties: In vitro release test and in vivo pharmacokinetic test in animals showed that it has a significant sustained-release effect. Compared with solution, liposomes can significantly prolong the in vivo residence time of the drug (the prolongation time can be up to 16.5 times) and reduce the drug clearance rate (the clearance rate is reduced by 25.2 times).

[0045] (4) Good antiviral effect: Experiments on the inhibition of viral surface antigens showed that the inhibition rate of the virus was significantly higher than that of the solution.

[0046] (5) Quality controllable: The residual amount of organic solvents meets the ICH Q3C limit standard for injections.

[0047] The preparation process of this invention is simple, the product quality is stable, it is suitable for large-scale industrial production, and it has good prospects for clinical application. Attached Figure Description

[0048] The present invention will now be further described with reference to the accompanying drawings.

[0049] Figure 1 This is a transmission electron microscopy (TEM) image of the liposome suspension after the removal of free drug in Example 1 of the present invention.

[0050] Figure 2 This is a scanning electron microscope (SEM) characterization image of the lyophilized adenosine monophosphate liposome injection in Example 1 of this invention.

[0051] Figure 3 This is the in vitro release curve of lyophilized adenosine monophosphate liposome powder for injection;

[0052] Figure 4 This is the in vivo pharmacokinetic curve of lyophilized adenosine monophosphate liposome for injection;

[0053] Figure 5 This is a graph showing the ability of lyophilized adenosine monophosphate liposomes to inhibit HBV antigen expression in cells. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, unless otherwise specified, the raw materials, reagents, or devices used in the following embodiments can be obtained from conventional commercial channels or by existing known methods.

[0055] Example 1

[0056] Preparation of lyophilized adenosine monophosphate liposome powder for injection:

[0057] Table 1. Formulation of lyophilized adenosine monophosphate liposome for injection, Example 1

[0058]

[0059] Preparation process:

[0060] (1) Weigh out the prescribed amounts of DSPC, cholesterol and DSPE-PEG2000 and dissolve them in chloroform to form the organic phase for later use;

[0061] (2) Weigh out the prescribed amount of adenosine monophosphate and dissolve it in a phosphate buffer solution to form an aqueous phase;

[0062] (3) Mix the organic phase obtained in step (1) and the aqueous phase obtained in step (2) at a volume ratio of 4:1, and emulsify under high-speed shear for 5 minutes;

[0063] (4) Remove organic solvent by rotary evaporation, and hydrate at 65°C for 30 minutes to obtain liposome initial suspension;

[0064] (5) Place the initial liposome suspension obtained in step (4) above into a high-pressure homogenizer to homogenize the particle size, then remove the free drug by tangential flow chromatography, and then add the prescribed amount of trehalose to obtain the liposome suspension.

[0065] (6) The liposome suspension obtained in (5) was filtered and sterilized, and then freeze-dried to obtain adenosine monophosphate liposome lyophilized powder for injection.

[0066] (7) The lyophilized powder of adenosine monophosphate liposome obtained in (6) is dispensed under aseptic conditions.

[0067] Example 2

[0068] Preparation of lyophilized adenosine monophosphate liposome powder for injection:

[0069] Table 2. Formulation of lyophilized adenosine monophosphate liposome for injection in Example 2

[0070]

[0071] The preparation process is the same as in Example 1 above, and finally, a lyophilized powder injection of adenosine monophosphate liposomes is prepared. The obtained lyophilized powder injection of adenosine monophosphate liposomes is dispensed under sterile conditions.

[0072] Example 3

[0073] Preparation of lyophilized adenosine monophosphate liposome powder for injection:

[0074] Table 3. Formulation of lyophilized adenosine monophosphate liposome for injection in Example 3

[0075]

[0076] The preparation process is the same as in Example 1 above, and finally, a lyophilized powder injection of adenosine monophosphate liposomes is prepared. The obtained lyophilized powder injection of adenosine monophosphate liposomes is dispensed under sterile conditions.

[0077] Example 4

[0078] Preparation of lyophilized adenosine monophosphate liposome powder for injection:

[0079] Table 4. Formulation of lyophilized adenosine monophosphate liposome for injection, Example 4

[0080]

[0081] Preparation process:

[0082] (1) Weigh out the prescribed amounts of DSPC, cholesterol and DSPE-PEG2000 and dissolve them in chloroform to form the organic phase for later use;

[0083] (2) Weigh out the prescribed amount of adenosine monophosphate and dissolve it in a phosphate buffer solution to form an aqueous phase;

[0084] (3) Mix the organic phase obtained in step (1) and the aqueous phase obtained in step (2) at a volume ratio of 4:1, and emulsify under high-speed shear for 5 minutes;

[0085] (4) Remove organic solvent by rotary evaporation, and hydrate at 60°C for 30 minutes to obtain liposome initial suspension;

[0086] (5) The liposome initial suspension obtained in step (4) above is placed in a liposome extruder and extruded 5 times with a 200 nm polycarbonate membrane and 3 times with a 100 nm polycarbonate membrane. After uniform particle size, the free drug is removed by tangential flow chromatography. Then, the prescribed amount of trehalose is added to obtain the liposome suspension.

[0087] (6) The liposome suspension obtained in (5) was filtered and sterilized, and then freeze-dried to obtain adenosine monophosphate liposome lyophilized powder for injection.

[0088] (7) The lyophilized powder of adenosine monophosphate liposome obtained in (6) is dispensed under aseptic conditions.

[0089] Example 5

[0090] Preparation of lyophilized adenosine monophosphate liposome powder for injection:

[0091] Table 5. Formulation of lyophilized adenosine monophosphate liposome for injection, Example 5

[0092]

[0093] Preparation process:

[0094] (1) Weigh out the prescribed amounts of DSPC, cholesterol and DSPE-PEG2000 and dissolve them in chloroform to form the organic phase for later use;

[0095] (2) Weigh out the prescribed amount of adenosine monophosphate and dissolve it in a phosphate buffer solution to form an aqueous phase;

[0096] (3) Mix the organic phase obtained in step (1) and the aqueous phase obtained in step (2) at a volume ratio of 4:1, and emulsify under high-speed shear for 5 minutes;

[0097] (4) Remove organic solvent by rotary evaporation, and hydrate at 65°C for 60 minutes to obtain liposome initial suspension;

[0098] (5) The liposome initial suspension obtained in step (4) above is placed in a liposome extruder and extruded 5 times with a 200 nm polycarbonate membrane and 3 times with a 100 nm polycarbonate membrane. After uniform particle size, the free drug is removed by tangential flow chromatography. Then, the prescribed amount of trehalose is added to obtain the liposome suspension.

[0099] (6) The liposome suspension obtained in (5) was filtered and sterilized, and then freeze-dried to obtain adenosine monophosphate liposome lyophilized powder for injection.

[0100] (7) The lyophilized powder of adenosine monophosphate liposome obtained in (6) is dispensed under aseptic conditions.

[0101] Test Example 1

[0102] The encapsulation efficiency of liposomes in the initial liposome suspensions before removal of free drug in Examples 1 to 5 was determined:

[0103] Take appropriate amounts of the initial liposome suspensions from Examples 1-5, add methanol to demulsify, and determine the content by high-performance liquid chromatography (HPLC), recording it as m1. Then, take appropriate amounts of the initial liposome suspensions from Examples 1-5 into ultrafiltration centrifuge tubes, centrifuge at 4000 rpm for 5 min, take the lower layer liquid, dilute to volume, and determine the content by HPLC, recording it as m2. The encapsulation efficiency is calculated using the following formula:

[0104] Encapsulation efficiency (%) = (1 - m2 / m1) × 100%;

[0105] Table 6 Encapsulation efficiency test results

[0106]

[0107] Test Example 2

[0108] The encapsulation efficiency of liposomes in the liposome suspensions after removal of free drug in Examples 1 to 5 was determined:

[0109] Take appropriate amounts of the liposome suspensions from Examples 1-5, add methanol to demulsify, and determine the content by high-performance liquid chromatography (HPLC), recording it as m1. Then, take appropriate amounts of the liposome suspensions from Examples 1-5 into ultrafiltration centrifuge tubes, centrifuge at 4000 rpm for 5 min, take the lower layer liquid, dilute to volume, and determine the content by HPLC, recording it as m2. The encapsulation efficiency is calculated using the following formula:

[0110] Encapsulation efficiency (%) = (1 - m2 / m1) × 100%;

[0111] Table 7 Encapsulation efficiency test results

[0112]

[0113] The test results of Test Example 1 and Test Example 2 show that the encapsulation efficiency of the adenosine monophosphate liposomes prepared by the present invention is high and meets the industrial production standards.

[0114] Test Example 3

[0115] The drug loading of the lyophilized adenosine monophosphate liposome injections in Examples 1 to 5 was determined:

[0116] Drug loading (%) = drug mass / total mass of lyophilized adenosine monophosphate liposome powder for injection;

[0117] Table 8 Results of Drug Loading Measurement

[0118]

[0119] Test Example 4

[0120] The particle size of liposomes in the liposome suspensions after removal of free drug in Examples 1 to 5 was determined:

[0121] Under normal temperature conditions, the liposome suspensions from Examples 1 to 5 were diluted to a suitable concentration, and the average particle size and polydispersity index of the liposomes were determined by dynamic light scattering (DLS).

[0122] Table 9 Results of average particle size and particle size dispersion index determination

[0123]

[0124] The test results of Test Examples 3 and 4 show that, in addition, the liposome suspension after removal of free drug in Example 1 was characterized by structure (transmission electron microscopy), and the characterization results are as follows. Figure 1 As shown, the structure of the lyophilized adenosine monophosphate liposome injection finally prepared in Example 1 was characterized (scanning electron microscopy), and the characterization results are as follows. Figure 2 As shown above, the liposomes of adenosine monophosphate prepared by this invention have a particle size of about 100 nm, are uniformly distributed, and are suitable for intravenous administration.

[0125] Test Example 5

[0126] The stability of the lyophilized powder of adenosine monophosphate liposomes for injection in Examples 1 to 5 was determined:

[0127] The lyophilized powder of adenosine monophosphate liposome prepared in Examples 1 to 5 was placed at 40℃±2℃ and 75%±5%RH for 6 months for accelerated testing.

[0128] Table 10 Accelerated Test Results

[0129]

[0130] The results above show that the liposomes prepared by this invention exhibit no significant changes in appearance or encapsulation efficiency under accelerated testing conditions, and demonstrate good reconstitution properties. This solves the problem of poor storage stability in ordinary solutions.

[0131] Test Example 6

[0132] The in vitro release performance of the lyophilized adenosine monophosphate liposome injection from Example 1 was determined:

[0133] Accurately pipette 2g of the lyophilized adenosine monophosphate liposome injection from Example 1 into a pretreated dialysis bag (8000-14000 Da), seal tightly, and place in a brown release vial containing 10mL of release medium. Perform triplicate dispensing, placing each vial in a 37°C water bath shaker. Take 2mL of release medium at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours, and simultaneously add an equal volume of release medium at the same temperature. Determine the adenosine monophosphate content in the release medium using high-performance liquid chromatography (HPLC). The results are shown in the appendix. Figure 3 The results showed that the lyophilized liposome monophosphate (LPD) liposome injection prepared in Example 1 had excellent sustained-release properties. Under the three pH values, the drug was released more under acidic conditions. Fitting the release curves revealed that the in vitro release curves of the LPD liposome injection at pH 7.4 and pH 6.8 best matched the first-order release curves, indicating that the release was primarily driven by the concentration gradient. The in vitro release curve of the LPD liposome injection at pH 4.5 better matched the Weibull model, presumably because the phospholipids underwent some hydrolysis at the more acidic pH 4.5 environment, leading to increased membrane permeability and facilitating drug release. In the more neutral environments of pH 6.8 and pH 7.4, the lipid membrane structure was stable, and the oil-water partition coefficient of LPD was around -0.22, resulting in poor membrane permeability. Therefore, the drug could be stably encapsulated in the aqueous cavities within the liposomes.

[0134] Based on the above characteristics, the lyophilized powder of adenosine monophosphate liposomes of the present invention can remain stable in human plasma at a pH of approximately 7.4 for a relatively long period after intravenous administration, significantly prolonging the drug's blood circulation time. Furthermore, when the human body is infected with a virus, the site of viral infection typically experiences a strong inflammatory response, prompting the infiltration of numerous immune cells such as neutrophils and macrophages. This infiltration, through anaerobic glycolysis, produces lactic acid and other substances, leading to a more acidic microenvironment at the infection site. This induces a large release of the drug encapsulated in the liposomes, achieving specific accumulation of the drug at the lesion site and solving the problems of sustained release and targeting inherent in conventional solutions.

[0135] Test Example 7

[0136] In vivo pharmacokinetic study of the lyophilized liposome arabinophosphate powder for injection in Example 1:

[0137] Twelve SD rats, weighing 200±20g, were randomly divided into three groups: a free adenosine monophosphate (ADP) group and an ADP liposome lyophilized powder injection group, with six rats in each group. The dosage was 30mg / kg. Plasma samples were collected at 30min, 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h after administration. The collected plasma samples were collected in heparin-coated centrifuge tubes, centrifuged at 4000rpm for 10min, and the supernatant plasma was collected and stored at -80℃. The results are attached. Figure 4 .

[0138] The results showed that the area under the curve (AUC0-t) of the lyophilized liposomes for injection of adenosine monophosphate was 25 times that of the solution, indicating that liposomes significantly increased the bioavailability of adenosine monophosphate in vivo. The mean residence time (MRT0-t) of the liposomes was 16.5 times that of the solution group, while the plasma clearance (CL) of the drug solution group was 25.2 times that of the liposomes. This indicates that encapsulating adenosine monophosphate in liposomes can greatly prolong its retention time in the bloodstream. The longer circulation time and slower plasma elimination rate of liposomes may be due to their good stability in the blood and the slow release of the drug.

[0139] Test Example 8

[0140] The ability of the lyophilized liposome arabinophosphate powder prepared in Example 1 to inhibit HBV antigen expression in cells was determined:

[0141] HepG2.2.15 cells in the logarithmic growth phase were harvested and treated with 1×10⁻⁶ cells. 5 Cells were seeded at a density of [number] cells / mL in 24-well plates, 1 mL of culture medium was added, and the plates were incubated for 24 hours. After cell attachment, the old culture medium was removed, and 1 mL of drug-containing medium at different concentration gradients and lyophilized medium containing adenosine monophosphate (APD) liposome injection powder were added to each well. Each group was in triplicate, and the drug-containing medium was replaced every 4 days, for a total of 2 times. The old culture medium was collected after each replacement and stored at -20°C for later use. HBV surface antigen (HBsAg) and e antigen (HBeAg) were detected using ELISA. Results are attached. Figure 5 .

[0142] The three concentrations of free drug and vidarabine monophosphate liposome lyophilized powder selected in this experiment all showed inhibitory effects on HBsAg and HBeAg. The inhibition rate of vidarabine monophosphate liposome lyophilized powder on both drugs was significantly higher than that of the free drug. This may be because vidarabine monophosphate liposome lyophilized powder is better taken up by cells, resulting in a higher effective intracellular drug concentration. Furthermore, free vidarabine monophosphate is rapidly metabolized into inactive vidarabine hypoxanthine by cellular secreted nucleotide metabolic enzymes, while vidarabine monophosphate liposome lyophilized powder protects the drug from enzymatic degradation, improving its intracellular stability. The inhibition rates against HBsAg and HBeAg showed time- and concentration-dependent effects, increasing with both treatment time and concentration. The inhibition rate reached its maximum at a drug concentration of 40 μg / mL, and was significantly higher than that of the ordinary solution.

[0143] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0144] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A lyophilized powder injection of adenosine monophosphate liposomes, characterized in that, Its composition, by weight, includes: One part of adenosine monophosphate; Phospholipids 2-10 parts; 0.5–3.0 parts of membrane flow modifier; 0.02–0.80 parts of surface modification agent; 5-40 parts of freeze-drying protectant.

2. The lyophilized powder injection of adenosine monophosphate liposomes according to claim 1, characterized in that, The phospholipid is selected from one or more of soybean phospholipids, hydrogenated soybean phospholipids, dipalmitoyl phosphatidylcholine, and distearate phosphatidylcholine.

3. The lyophilized powder injection of adenosine monophosphate liposomes according to claim 1, characterized in that, The membrane fluidity regulator is selected from one or more of cholesterol, polysorbate, and phytosterols.

4. The lyophilized powder injection of adenosine monophosphate liposomes according to claim 1, characterized in that, The surface modifier is selected from one or more of distearylphosphatidylethanolamine-polyethylene glycol, polysialic acid, polyhydroxyethyl starch, and hyaluronic acid.

5. The lyophilized powder injection of adenosine monophosphate liposomes according to claim 1, characterized in that, The freeze-drying protectant is selected from one or more of sucrose, trehalose, glucose, lactose, and mannitol.

6. A method for preparing a lyophilized powder injection of adenosine monophosphate liposomes according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Dissolve phospholipids, membrane flow modifiers and surface modifiers in an organic solvent to form an organic phase; (2) Dissolve adenosine monophosphate in a buffer salt solution to form an aqueous phase; (3) The organic phase obtained in step (1) is mixed with the aqueous phase obtained in step (2), and emulsified by high-speed shearing for 5 to 20 minutes. The organic solvent is then removed by rotary evaporation, and the mixture is hydrated at 55 to 70°C for 30 to 120 minutes. The mixture is then extruded or homogenized to make the particle size uniform. After removing the free drug, a freeze-drying protectant is added, and the mixture is then filtered to remove bacteria and freeze-dried to obtain adenosine monophosphate liposome freeze-dried powder injection.

7. The method for preparing a lyophilized powder injection of adenosine monophosphate liposomes according to claim 6, characterized in that, The organic solvent mentioned in step (1) is selected from one or more of dichloromethane, trichloromethane, tetrahydrofuran, ethanol, methanol, acetone or ethyl acetate.

8. The method for preparing a lyophilized powder injection of adenosine monophosphate liposomes according to claim 6, characterized in that, The buffer salt solution mentioned in step (2) is borate buffer, phosphate buffer, citrate buffer or glycine buffer.

9. The method for preparing a lyophilized powder injection of adenosine monophosphate liposomes according to claim 6, characterized in that, The volume ratio of the buffer salt solution to the organic solvent in step (3) is 1:2 to 5.

10. The application of a lyophilized powder injection of adenosine monophosphate liposome according to any one of claims 1 to 5, characterized in that, The application of the lyophilized adenosine monophosphate liposome powder for injection in the preparation of drugs for treating viral infections.

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