A non-deposition solubilizing micelle system for intramuscular injection of ionizable lipid adjuvant DMPK / toxicological evaluation and its construction method

Abstract

This invention provides a deposition-free solubilizing micelle system and its construction method for DMPK / toxicological evaluation of intramuscularly injected ionizable lipid excipients, belonging to the field of non-clinical evaluation technology for new drugs. This invention formulates a solubilizing agent by combining an ionizable cationic lipid, a C1-C2 alcohol, polyoxyethylene-15-hydroxystearate, and an isotonic carrier; wherein the concentration of the ionizable cationic lipid is 0.1-50 mg / mL, the volume concentration of the C1-C2 alcohol is 1.0%-1.5%, and the volume concentration of the polyoxyethylene-15-hydroxystearate is 10%-15%. The solubilizing agent is completely dissolved during the preparation stage, with a hemolysis rate ≤ 5%; furthermore, the residual time at the administration site after intramuscular injection is short, allowing the ionizable cationic lipid to enter the in vivo circulation and obtain reliable TK / PK parameters, completely solving the deposit problem after intramuscular injection of ionizable cationic lipids in the prior art. In addition, the solubilizing agent of this invention does not require LNP assembly, which is beneficial for rapid batch preparation.

Description

Technical Field

[0001] This invention relates to the field of non-clinical evaluation technology for new drugs, specifically to an intramuscular injection non-deposition solubilizing micelle system used as a novel nucleic acid delivery excipient or candidate molecule for pharmacokinetic (DMPK), toxicokinetic (TK), and GLP toxicological evaluation in animals. Background Technology

[0002] In the non-clinical evaluation stage of new drugs, ionizable cationic lipids (such as SM-102, DLin-MC3-DMA, ALC-0315 and the company's self-developed ester-degradable ionizable lipids, with pKa generally between 5.5 and 7.5) are core excipients or candidate molecules for nucleic acid drug delivery systems. They need to undergo systematic pharmacokinetic (DMPK), toxicokinetic (TK), and GLP toxicological evaluations in animals to support their registration and filing as new excipients (such as CDE excipient registration / DMF data) or IND application.

[0003] Currently, for non-clinical evaluation of intramuscular administration of ionizable cationic lipids, existing technologies mainly employ the following methods:

[0004] 1. Direct intramuscular injection of free lipids (pure alcohol or alcohol-water dilution system):

[0005] Ionizable cationic lipids are dissolved in organic solvents such as anhydrous ethanol or DMSO and then injected directly into the intramuscular cavity, or simply diluted with physiological saline / glucose injection solution before injection. This is the simplest and most commonly used method, with low operating costs and no need for complex formulation processes. However, ionizable cationic lipids are highly hydrophobic molecules (logP is usually > 8) and are almost insoluble in water. When injected intramuscularly with a pure ethanol or simple alcohol-water dilution system, the organic solvent rapidly diffuses and is diluted in the tissue fluid, causing lipid molecules to precipitate from the solution and form micron- to millimeter-sized lipid aggregates / droplets at the injection site. These deposits can remain at the injection site for weeks to months (experiments have shown that the residual time at the injection site after intramuscular injection of free SM-102 is > 4-6 weeks), resulting in: lipids being unable to enter the bloodstream or lymphatic system, extremely low systemic exposure, and inability to obtain modelable TK / PK parameters (AUC, Cmax, t). 1 / 2 (e.g., MRT, etc.); the local residual curve shows an infinite tail, making it impossible to calculate the clearance kinetics and affecting the derivation of NOAEL; local deposition may induce foreign body granuloma / chronic inflammation, interfering with the conclusions of toxicological evaluation.

[0006] 2. Lipid nanoparticle (LNP) encapsulation system:

[0007] Lipid nanoparticles of approximately 80-100 nm are assembled from ionizable cationic lipids, phospholipids (such as DSPC), cholesterol, and PEGylated lipids (such as DMG-PEG2000) in a specific molar ratio (e.g., 50:10:38.5:1.5) for encapsulating mRNA / siRNA before intramuscular injection. This is the classic technical route used in currently marketed mRNA vaccines (such as Spikevax® and Comirnaty®). Although the LNP system can stably encapsulate ionizable lipids in nanoparticles, its design purpose is for therapeutic delivery (protection of nucleic acids, targeted delivery, and endosome escape), rather than for non-clinical evaluation. LNP formulations are complex (four types of lipids + nucleic acids), and the preparation process is cumbersome, making them unsuitable for DMPK tracking of a single ionizable lipid excipient. Components such as PEG lipids and cholesterol in LNPs can generate their own PK signals in vivo, interfering with the determination of the clearance pathway of the target ionizable lipid itself. After intramuscular injection, LNPs still exhibit a certain degree of local retention (due to particle size and interstitial space limitations), failing to fundamentally solve the depot problem. For novel ionizable lipids that are still in the early stages of development (with undetermined structures and not yet assembled into LNPs), LNP encapsulation schemes are not applicable.

[0008] 3. Nonionic surfactant solubilization systems (such as Solutol HS15 / polyoxyethylene alkyl esters):

[0009] Solutol HS15 (polyoxyethylene-15-hydroxystearate, Kolliphor HS15), a nonionic surfactant listed in the European Pharmacopoeia / USP / NF, has been widely used in the solubilization of hydrophobic drugs (such as paclitaxel, tacrolimus, and fat-soluble vitamins) in injectable formulations. Its basic principle is to encapsulate the hydrophobic drug within a hydrophobic core by forming micelles, thereby improving the apparent solubility of the drug. Typical applications include paclitaxel micelle injections and concentrated cyclosporine A solutions. Although Solutol HS15 has been used to solubilize various hydrophobic drugs, its current applications are mainly concentrated in the field of clinical therapeutic injectables. Existing Solutol solubilization systems have not been optimized for the specific requirement of "rapid clearance of ionizable cationic lipids after intramuscular injection to support DMPK modeling." Existing systems typically only focus on single-point solubility without considering phase stability under tissue fluid dilution conditions after intramuscular injection (i.e., "whether precipitation / phase separation occurs after dilution"). The existing system lacks systematic optimization of parameters such as ethanol concentration, surfactant concentration, and aqueous carrier ratio for the characteristics of ionizable lipids. Direct application can lead to problems such as hemolysis or incomplete dissolution, phase separation after dilution (resulting in turbidity / oil droplets / "melting" phenomena), and unnecessary local irritation.

[0010] In summary, the existing technology lacks a solubilizing formulation system specifically designed for intramuscular injection of ionizable cationic lipids in DMPK / toxicological evaluation scenarios. This system needs to simultaneously meet the following mutually restrictive conditions:

[0011] Complete dissolution: Ionizable cationic lipids must be completely dissolved during the preparation stage, leaving no undissolved particles;

[0012] Dilution stability: When diluted with tissue fluid after intramuscular injection, it does not undergo phase separation / precipitation / "melting", avoiding secondary deposition;

[0013] Low hemolytic activity: The final concentration of ethanol and other excipients in the system does not cause unacceptable hemolytic reactions;

[0014] Rapid clearance: The short residual time at the administration site after intramuscular injection (≤ 24 h) allows ionizable lipids to enter the systemic circulation and obtain modelable TK / PK parameters;

[0015] Locally tolerable: It does not introduce additional local irritation or tissue damage, and does not affect the conclusions of the toxicological evaluation. Summary of the Invention

[0016] To address the shortcomings of the existing technologies, this invention provides a deposition-free solubilizing micelle system for intramuscular injection of ionizable lipid excipients DMPK / toxicological evaluation and its construction method.

[0017] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0018] In a first aspect, the present invention provides a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids, comprising the following components:

[0019] Ionizable cationic lipids, C1-C2 alcohols, polyoxyethylene-15-hydroxystearate (Solutol HS15), and isotonic water-carriers;

[0020] Furthermore, the solubilizing agent satisfies the following conditions:

[0021] (1) The volume concentration of the C1-C2 alcohol in the final state system is 1.0%-1.5% v / v;

[0022] (2) The volume concentration of the polyoxyethylene-15-hydroxystearate in the final system is 10%-15% v / v;

[0023] (3) The concentration of the ionizable cationic lipid in the final state system is 0.1-50 mg / mL;

[0024] (4) The solubilizing agent is filtered through a 0.22 μm filter membrane during the preparation process.

[0025] Optionally, the C1-C2 alcohol is anhydrous ethanol.

[0026] Optionally, the volume concentration of the anhydrous ethanol in the final system is 1.2% v / v.

[0027] Optionally, the volume concentration of the polyoxyethylene-15-hydroxystearate in the final system is 12% v / v.

[0028] Optionally, the ionizable cationic lipid is an ionizable cationic lipid with a degradable ester bond structure and a pKa value of 5.5 to 7.5, such as SM-102, DLin-MC3-DMA, ALC-0315 or similar structures thereof.

[0029] Optionally, the isotonic water-bearing carrier is selected from at least one of 5% glucose injection, 0.9% sodium chloride injection, and Ringer's solution.

[0030] In some embodiments, the degree of hemolysis of the solubilizing agent is within the range specified in USP. <797> The hemolysis test grade is no higher than "micro-positive", that is, the hemolysis rate is ≤ 5%, and the residual amount of ionizable cationic lipids at the administration site after intramuscular injection is less than 30% of the initial dose at 24 hours and less than 10% of the initial dose at 168 hours.

[0031] Secondly, the present invention provides a method for preparing the solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids, comprising the following steps:

[0032] Ionizable cationic lipids are dissolved in C1-C2 alcohols to form an ester-alcohol phase;

[0033] Polyoxyethylene-15-hydroxystearate was added to the ester-alcohol phase and stirred until uniformly dispersed.

[0034] Slowly add the isotonic water-carrier to the final volume while stirring, so that the C1-C2 alcohol concentration decreases to 1.0% v / v to 1.5% v / v;

[0035] The resulting solution was filtered through a 0.22 μm filter membrane to obtain a clear solution;

[0036] Repackaging yields a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids.

[0037] Thirdly, the present invention provides the use of the solubilizing agent in the preparation of an intramuscular dosing kit for nonclinical DMPK / TK evaluation of ionizable cationic lipids.

[0038] Optionally, the application includes: administering the solubilizing agent intramuscularly to a non-clinical species at a volume of 0.05 mL to 0.5 mL per injection point, and collecting muscle, plasma, and major organs at the administration site within 0 to 336 hours after administration for quantitative analysis by LC-MS / MS.

[0039] Furthermore, the solubilizing system can be extended to safety evaluation studies required for the registration of new excipients for ionizable cationic lipids (such as CDE excipient registration / DMF data), and used as a standardized intramuscular injection reference solvent system.

[0040] The key technical points of this invention are as follows:

[0041] 1. Precise locking of the recipe window

[0042] This invention, through systematic screening, has for the first time discovered and identified a synergistic window between C1-C2 alcohols (1.0%-1.5% v / v) and Solutol HS15 (10%-15% v / v). This window is the only feasible range that can simultaneously satisfy the three mutually restrictive conditions of "complete dissolution of ionizable cationic lipids," "phase stability after dilution," and "hemolysis rate ≤ 5%." Deviations from this window will prevent the achievement of the invention's objectives; for example, an ethanol volume concentration < 1.0% will result in incomplete lipid dissolution, while > 1.5% will lead to hemolysis; a Solutol HS15 volume concentration < 10% will cause phase separation after dilution, while > 15% offers no additional benefit and increases risk.

[0043] 2. The key role of filtration process

[0044] This invention discovers that under the boundary formulation (ethanol 1.2% v / v + Solutol HS15 12% v / v), the initial mixture is in a metastable mist state. After filtration through a 0.22 μm filter membrane, a clear and stable formulation can be obtained. This process step makes the formulation that was originally at the dissolution boundary practical, broadens the operating window, and reduces production sensitivity.

[0045] 3. For the first time, DMPK / TK modeling after intramuscular injection of ionizable cationic lipids was achieved.

[0046] The solvent of this invention shortens the Tmax of lipids after intramuscular injection from 16-18 days to within half a day, and the elimination half-life from 27-73 days to 1.5-1.8 days, thereby reducing all PK parameters (Tmax, Tmin, and Glucosamine). 1 / 2 The parameters (AUC, CL, Vd) can all be calculated completely, thus completely solving the industry pain point of "inability to calculate appropriate pharmacokinetic parameters" caused by the depot effect in existing soybean oil solvents.

[0047] 4. The formula is simple and has strong applicability.

[0048] The solubilizing formulation provided by this invention requires only three excipients (C1-C2 alcohol, Solutol HS15, and isotonic carrier) plus one-step filtration, without the need for complex LNP assembly, and is suitable for various ionizable cationic lipids such as SM-102, DLin-MC3-DMA, and ALC-0315.

[0049] The ionizable cationic lipids targeted in this invention typically include, but are not limited to, SM-102, DLin-MC3-DMA, ALC-0315, and the company's self-developed ester-degradable ionizable cationic lipids (pKa generally between 5.5 and 7.5). These are highly hydrophobic amphiphilic molecules, almost insoluble in aqueous or conventional isotonic carriers. If injected intramuscularly in their free state or with inappropriate solvents, they can lead to long-term lipid deposition at the administration site, causing local TK curve tailing for weeks to months, making it impossible to obtain modelable clearance kinetics, and consequently affecting NOAEL derivation and excipient safety conclusions. This invention addresses this by rationally selecting excipients: C1-C2 alcohol, polyoxyethylene-15-hydroxystearate, and an isotonic aqueous carrier, and precisely limiting the C1-C2 alcohol concentration (1.0%-1.5% v / v) and Solutol HS15 concentration (10%-15% v / v), and introducing a 0.22 μm filtration process. Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. For the first time, DMPK / TK modeling after intramuscular injection of ionizable cationic lipids has been achieved.

[0051] Existing soybean oil solvents suffer from prolonged lipid retention at the injection site, resulting in a Tmax of 16-18 days and an elimination half-life of 27-73 days. The original report explicitly stated that "insufficient elimination-phase blood drug concentrations were not obtained, making it impossible to calculate appropriate pharmacokinetic parameters." This invention, however, uses C1-C2 alcohols, Solutol HS15, and an isotonic carrier to formulate a solubilizing agent for ionizable cationic lipids, shortening the Tmax to 7-13 hours (an increase of approximately 30-60 times) and the elimination half-life to 37-43 hours. Tmax can be fully calculated for all dosage groups. 1 / 2 The invention of all PK parameters, including AUC, CL, and Vd, makes it possible for the first time to evaluate intramuscularly injected DMPK / TK of ionizable cationic lipids.

[0052] 2. While ensuring complete dissolution, control the risk of hemolysis within an acceptable range.

[0053] In the solubilizing formulation of this invention, lipids cannot be completely dissolved (the solution is mist-like) when the C1-C2 alcohol concentration is below 1.0% v / v, and the hemolysis test is positive when it is above 1.5% v / v. This invention strictly limits the C1-C2 alcohol concentration to the range of 1.0%-1.5% v / v, achieving both complete dissolution and a hemolysis rate ≤ 5% (micro-positive). This window is the only feasible range found after extensive screening.

[0054] 3. Ensure the phase stability of the diluted tissue fluid to avoid secondary deposition.

[0055] In the solubilizing formulation of this invention, the number of micelles is insufficient when the Solutol HS15 concentration is below 10% v / v, leading to lipid re-precipitation after dilution; above 15% v / v, the risk of irritation increases without any additional benefit. This invention limits the Solutol HS15 concentration to the range of 10%-15% v / v, ensuring that it remains clear and transparent without phase separation even after dilutions of 20 times or more.

[0056] 4. The filtration process broadens the practicality of the formulation window.

[0057] At the boundary of a formulation with 1.2% v / v ethanol and 12% v / v Solutol, the initial mixture was slightly misty, but a clear and stable formulation could be obtained after filtration through a 0.22 μm filter. This process step makes a formulation that was originally at the dissolution boundary usable, reducing the sensitivity to batch-to-batch variations of raw materials in industrial production.

[0058] 5. The formula is simple and has strong applicability.

[0059] The solubilizing formulation of this invention requires only three excipients (C1-C2 alcohol, Solutol HS15, and isotonic carrier) plus one-step filtration, without the need for LNP assembly, making it suitable for rapid batch formulation by CROs. It has been verified to be applicable to TQ-0005, and based on structural similarity, it can be expected that this system is also applicable to various ionizable cationic lipids such as DLin-MC3-DMA and ALC-0315. Attached Figure Description

[0060] Figure 1 Results of hemolysis tests for different prescriptions;

[0061] Figure 2 The results of dissolution tests for different formulations;

[0062] Figure 3 Blood concentration versus time curves of TQ-0005 administered intramuscularly to Beagle dogs using soybean oil as the solvent;

[0063] Figure 4 This is a graph showing the blood concentration versus time curve of the solvent TQ-0005 injected intramuscularly into Beagle dogs according to the present invention. Detailed Implementation

[0064] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0065] This invention provides a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids, comprising the following components:

[0066] Ionizable cationic lipids, C1-C2 alcohols, polyoxyethylene-15-hydroxystearate (Solutol HS15), and isotonic water-carriers;

[0067] Furthermore, the solubilizing agent satisfies the following conditions:

[0068] (1) The volume concentration of the C1-C2 alcohol in the final state system is 1.0%-1.5% v / v;

[0069] (2) The volume concentration of the polyoxyethylene-15-hydroxystearate in the final system is 10%-15% v / v;

[0070] (3) The concentration of the ionizable cationic lipid in the final state system is 0.1-50 mg / mL;

[0071] (4) The solubilizing agent is filtered through a 0.22 μm filter membrane during the preparation process.

[0072] In some examples, the C1-C2 alcohol is anhydrous ethanol.

[0073] In some examples, the ionizable cationic lipid is an ionizable cationic lipid with an ester-degradable structure and a pKa value of 5.5 to 7.5, such as SM-102, DLin-MC3-DMA, ALC-0315 or structural analogs thereof.

[0074] In some examples, the isotonic carrier is selected from at least one of 5% glucose injection, 0.9% sodium chloride injection, and Ringer's solution.

[0075] In some examples, the method for preparing the solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids includes the following steps:

[0076] Ionizable cationic lipids are dissolved in C1-C2 alcohols to form an ester-alcohol phase;

[0077] Polyoxyethylene-15-hydroxystearate was added to the ester-alcohol phase and stirred until uniformly dispersed.

[0078] Slowly add the isotonic water-carrier to the final volume while stirring, so that the C1-C2 alcohol concentration decreases to 1.0% v / v to 1.5% v / v;

[0079] The resulting solution was filtered through a 0.22 μm filter membrane to obtain a clear solution;

[0080] Repackaging yields a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids.

[0081] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in the art.

[0082] Example 1

[0083] This embodiment focuses on the solubilization formulation of the ionizable cationic lipid TQ-0005 for intramuscular administration, and investigates the effects of ethanol concentration, Solutol HS15 concentration, type of solubilizer, and cyclodextrin addition on solubility and hemolysis.

[0084] Using TQ-0005 as the target lipid, with a fixed final concentration of 30 mg / mL, solubilizing agents with different compositions were prepared. The appearance of the solutions (clear / misty / turbid / precipitated) was observed, and the preparations were carried out according to USP. <797> The method involved performing an in vitro hemolysis test, with results categorized into three levels: negative, slightly positive, and positive. Details of the formulation and test results are shown in Tables 1-3. Photographs of some hemolysis test results are available in [link to table]. Figure 1 See photos of partial dissolution test results. Figure 2 . Figure 1 Test tubes 1, 2, 3, 4, 5, and 6 correspond to formulas A, C, J, I, D, and H, respectively. Figure 2 Test tubes 1 and 2 in the diagram correspond to formulas B and D, respectively.

[0085] Table 1: Screening Results of Solvent Systems

[0086]

[0087] Table 2: Screening Results of the DMSO / Solutol HS15 System

[0088]

[0089] Table 3: Screening Results of Cyclodextrin-Assisted Systems

[0090]

[0091] Table 1 shows that in the ethanol / Solutol HS15 / 5% glucose injection system, lipids cannot be completely dissolved (misting) when the ethanol concentration is below 1.0% v / v, and hemolysis becomes positive when it is above 1.5% v / v. Solutol HS15 concentration is insufficiently dissolved when below 10% v / v, and provides no additional benefit and increases the risk of hemolysis when above 15% v / v. Only formulation D (ethanol 1.2% v / v + Solutol HS15 12% v / v) produces a clear solution after filtration with only a slight positive result for hemolysis, making it the only formulation that simultaneously meets the requirements of solubility and low hemolysis. Table 2 shows that although the DMSO / Solutol HS15 / 5% glucose injection system has good solubility, all formulations are positive for hemolysis and are not suitable as a solubilizer for injection. Table 3 shows that using a cyclodextrin-assisted system also fails to solve the hemolysis problem.

[0092] Based on the above, the optimal solubilizing formulation for the ionizable cationic lipid TQ-0005 (30 mg / mL) is determined to be: 1.2% v / v anhydrous ethanol, 12% v / v Solutol HS15, and 5% glucose injection to make up to 100%. The preparation process requires filtration through a 0.22 μm filter membrane.

[0093] Example 2

[0094] This embodiment provides a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipid TQ-0005, the formulation of which is shown in Table 4.

[0095] Table 4: Solubilizing formulations for non-clinical evaluation of intramuscular administration of ionizable cationic lipid TQ-0005

[0096]

[0097] The preparation steps of the solubilizing agent are as follows:

[0098] (1) Dissolve 300 mg TQ-0005 in 1.2 mL of anhydrous ethanol and vortex until completely dissolved to obtain the ester-alcohol phase;

[0099] (2) Add 12 g Solutol HS15 to the ester-alcohol phase and stir until evenly dispersed;

[0100] (3) Slowly add 5% glucose injection solution to a total volume of 100 mL while stirring, so that the final concentration of ethanol is reduced to 1.2% v / v;

[0101] (4) The resulting solution was initially slightly misty. After filtration through a 0.22 μm filter membrane, a colorless and clear solution was obtained.

[0102] (5) Aseptically dispensed to obtain a 3 mg / mL solubilizing formulation for administration.

[0103] The formulation properties of the solubilizing agent are as follows:

[0104] Appearance: Colorless, clear liquid; transmittance at 600 nm ≥ 95%;

[0105] pH: 6.5-7.0;

[0106] Osmotic pressure: 280-320 mOsm / kg;

[0107] Hemolysis test: slightly positive (hemolysis rate approximately 3%-5%).

[0108] Dilution stability: It remained clear and showed no phase separation after being diluted 20 times with 5% glucose injection.

[0109] Example 3: Comparative Pharmacokinetic Study in Beagle Dogs

[0110] This embodiment compares the pharmacokinetic behavior of the solvent of the present invention with that of existing soybean oil solvent in TQ-0005 after intramuscular injection in Beagle dogs, and verifies the improvement effect of the solvent of the present invention on depot problems.

[0111] Animal grouping (4 females and 4 males per group, weighing 8-12 kg) and dosing details are shown in Table 5.

[0112] Table 5: Animal grouping and administration details

[0113]

[0114] In the existing soybean oil solvent group, blood samples were collected from 0 to 504 h after drug administration. In the solvent group of this invention, blood samples were collected from 0 to 336 h after drug administration. The concentration of TQ-0005 in plasma was determined by LC-MS / MS. The results are as follows: Figure 3-4 As shown in the figure; and the pharmacokinetic parameters were calculated using WinNonlin software, and the results are shown in Tables 6 and 7.

[0115] Table 6: Pharmacokinetic parameters of soybean oil solvent TQ-0005 administered intramuscularly / intravenously in Beagle dogs

[0116]

[0117] Note: " / " indicates that the calculation could not be performed because sufficient elimination phase blood drug concentrations were not obtained, and "NA" indicates that the calculation was not performed.

[0118] Table 7: Pharmacokinetic parameters of the solvent TQ-0005 for Beagle dogs (intramuscular / intravenous injection) of the present invention

[0119]

[0120] In Table 7, the multiple administration refers to administration once a week for 5 consecutive weeks, for a total of 5 administrations; samples were collected before administration on D29 and at 0.083 h (± 1 min), 0.5 h (± 1 min), 2 h (± 5 min), 4 h (± 5 min), 8 h (± 10 min), 24 h (± 10 min, D30), 48 h (± 15 min, D31), 72 h (± 15 min, D32), 96 h (± 15 min, D33), 120 h (± 15 min, D34), 168 h (± 30 min, D36), 240 h (± 30 min, D39), and 336 h (± 30 min, D43).

[0121] As shown in Tables 6 and 7, regarding absorption rate, the Tmax after intramuscular injection of soybean oil solvent is as long as 395-504 hours (16-21 days), indicating that lipids form a long-term deposit at the injection site and are released slowly; while the Tmax after intramuscular injection of the solvent of this invention is only 7-13 hours (peaking within half a day), with an absorption rate increased by about 30-60 times. Regarding elimination half-life, the Tmax after intramuscular injection of soybean oil solvent is... 1 / 2 The duration of treatment is as long as 655-1784 hours (27-74 days), and due to severe tailing, insufficient elimination phase blood drug concentrations are not obtained, making it impossible to calculate appropriate pharmacokinetic parameters; while the T after intramuscular injection of the solvent of this invention... 1 / 2 The exposure time was 36-43 hours (approximately 1.5-1.8 days), and all pharmacokinetic parameters could be fully calculated for all dose groups. Regarding systemic exposure, taking the medium-dose group as an example, the Cmax of 5 mg / kg IM in soybean oil solvent was 0.35 μg / mL, and the AUC was... 0-504 h The Cmax of the solvent in this invention (1.0 mg / kg IM, a dose only 1 / 5 of the former) was 104.33 h·μg / mL; while the Cmax of the solvent in this invention (1.0 mg / kg IM, a dose only 1 / 5 of the former) was 8.33 μg / mL (an increase of approximately 122-fold after dose normalization), and the AUC was... 0-336 h The concentration was 498.25 h·μg / mL (an increase of approximately 23 times after dose normalization). These results indicate that the solvent of the present invention enables lipids to enter the systemic circulation efficiently and completely eliminates the depot effect.

[0122] Regarding bioavailability, the absolute bioavailability (F) of the soybean oil solvent was 49.67%, with nearly half of the drug remaining at the injection site; while the F of the solvent of this invention was 137.63% (over 100%), because the lipids in group IV were rapidly cleared (T... 1 / 2 (≈35 h), while the IM group achieved a higher total exposure due to rapid and complete absorption, further demonstrating that the depot effect was completely eliminated.

[0123] Furthermore, in cases of multiple administrations, after multiple administrations of solvent D29 of the present invention, T 1 / 2 The duration of action was 73.11 h, slightly longer than with a single dose, but the AUC was [not specified]. 0-336 The h value was 486.15 h·μg / mL, which was comparable to the single medium-dose group, indicating that no significant accumulation occurred after 5 doses.

[0124] In summary, the solubilizing formulation provided by this invention is completely dissolved during the preparation stage, with a hemolysis rate ≤ 5%. Furthermore, it exhibits a short residual time at the administration site after intramuscular injection, completely resolving the depot problem of ionizable cationic lipids after intramuscular injection. This allows the lipids to peak within half a day and be eliminated within 1.5-1.8 days. All PK parameters can be fully calculated, achieving modeling and evaluation of the intramuscular DMPK / TK of this type of lipid for the first time. Compared with existing soybean oil solvents, the absorption rate is increased by 30-60 times, systemic exposure is increased by 20-120 times, and bioavailability is increased from 49% to 137%, demonstrating significant progress and practical value.

[0125] Example 4: Applicability verification of different ionizable cationic lipids

[0126] Based on the similar physicochemical properties (high hydrophobicity, pKa 5.5-7.5, and degradable ester bonds) of ionizable cationic lipids such as SM-102, DLin-MC3-DMA, and ALC-0315 to TQ-0005, it is reasonable to expect that the solvent of this invention is also suitable for the evaluation of intramuscular injection DMPK / TK of the above lipids. Specific verification experiments were conducted according to the method in Example 3.

[0127] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids, characterized in that, Includes the following components: The mixture comprises an ionizable cationic lipid, a C1-C2 alcohol, a polyoxyethylene-15-hydroxystearate, and an isotonic water-resistant carrier; wherein the concentration of the ionizable cationic lipid is 0.1-50 mg / mL, the volume concentration of the C1-C2 alcohol is 1.0%-1.5% v / v, and the volume concentration of the polyoxyethylene-15-hydroxystearate is 10%-15% v / v.

2. The solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 1, characterized in that, The C1-C2 alcohol is anhydrous ethanol.

3. The solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 2, characterized in that, The volume concentration of anhydrous ethanol in the final state system is 1.2% v / v.

4. The solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 1, characterized in that, The isotonic water-bearing carrier is selected from at least one of 5% glucose injection, 0.9% sodium chloride injection, and Ringer's solution.

5. A solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 1, characterized in that, The ionizable cationic lipid is an ionizable cationic lipid with a degradable ester bond structure and a pKa value of 5.5 to 7.

5.

6. A solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 5, characterized in that, The ionizable cationic lipids include, but are not limited to, SM-102, DLin-MC3-DMA, ALC-0315, or their structural analogs.

7. A solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to claim 1, characterized in that, The volume concentration of the polyoxyethylene-15-hydroxystearate is 12% v / v.

8. The method for preparing a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids according to any one of claims 1-7, characterized in that, Includes the following steps: Ionizable cationic lipids are dissolved in C1-C2 alcohols to form an ester-alcohol phase; Polyoxyethylene-15-hydroxystearate was added to the ester-alcohol phase and mixed thoroughly. Add isotonic water-carrier to the final volume; The resulting solution was filtered through a 0.22 μm filter membrane to obtain a solubilizing formulation for non-clinical evaluation of intramuscular administration of ionizable cationic lipids.

9. The use of the solubilizing formulation according to any one of claims 1-7 or the solubilizing formulation prepared by the preparation method according to claim 8 in the preparation of an intramuscular dosing kit for non-clinical DMPK / TK evaluation of ionizable cationic lipids.

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