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CN112370529A - A kind of compound preparation for treating pulmonary arterial hypertension and preparation method - Google Patents

A kind of compound preparation for treating pulmonary arterial hypertension and preparation method Download PDF

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CN112370529A
CN112370529A CN202011372665.7A CN202011372665A CN112370529A CN 112370529 A CN112370529 A CN 112370529A CN 202011372665 A CN202011372665 A CN 202011372665A CN 112370529 A CN112370529 A CN 112370529A
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cholesterol
tgf
inhibitor
compound preparation
liposome
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CN112370529B (en
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何伟
王孝春
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China Pharmaceutical University
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Abstract

本发明公开了一种治疗肺动脉高压的复方制剂及其制备方法和应用，属药物制剂，药物递送领域。所述的复方制剂粒径为130‑200 nm，主要包括以下两类药物：胆固醇前药、TGF‑β1抑制剂。本发明涉及的复方制剂，通过胆固醇修饰水溶性药物(如：二氯乙酸)实现脂质体同时递送两种水溶性药物的目标，不仅可以高效递送水溶性药物，还可降低水溶性药物大量递送带来的全身毒性，提高患者用药顺从性；此外，两种具有协同作用的药物共同递送可有效增强疗效，实例证明该复方制剂具有优良的抗肺动脉高压(PAH)效果。

Figure 202011372665

The invention discloses a compound preparation for treating pulmonary arterial hypertension, a preparation method and application thereof, and belongs to the field of pharmaceutical preparations and drug delivery. The particle size of the compound preparation is 130-200 nm, and mainly includes the following two types of drugs: cholesterol prodrugs and TGF-β1 inhibitors. The compound preparation involved in the present invention achieves the goal of simultaneously delivering two water-soluble drugs by liposomes by modifying the water-soluble drugs (eg, dichloroacetic acid) with cholesterol, which can not only efficiently deliver the water-soluble drugs, but also reduce the mass delivery of the water-soluble drugs. The systemic toxicity brought by the drug improves the compliance of patients with medication; in addition, the co-delivery of two synergistic drugs can effectively enhance the curative effect, and the example proves that the compound preparation has excellent anti-pulmonary hypertension (PAH) effect.

Figure 202011372665

Description

Compound preparation for treating pulmonary hypertension and preparation method thereof
Technical Field
The invention relates to a prescription composition of a compound preparation for treating pulmonary hypertension by injection and a preparation method thereof, belonging to the fields of medicinal preparations and medicament delivery.
Background
Liposomes are widely used for delivery of hydrophobic drugs (such as paclitaxel) and macromolecular drugs (such as proteins and genes) due to a plurality of excellent properties, however, the liposome has limited inner core space which is difficult to deliver two water-soluble drugs simultaneously, and the liposome has low entrapment rate for delivering small-molecule water-soluble drugs and is easy to leak, so that the research on co-delivery of two water-soluble drugs by the liposome is very little. At present, the administration mode of water-soluble chemotherapeutic drugs is mainly oral administration, the bioavailability is difficult to improve, and the subsequent toxic and side effects can damage the normal tissues of a human body and reduce the physiological function of a patient, so the high-efficiency co-delivery of the water-soluble chemotherapeutic drugs is an effective strategy for solving the defects.
Cholesterol is widely present in various organs of the animal body, with the highest levels in brain and nervous tissue, followed by kidney, spleen, skin, liver and bile. Cholesterol has been reported in the literature to target insulin-like growth factor 2 receptor (IGF2R), Toll-like receptor (TLR), and the like. Because the micromolecule drug has low cell-entering efficiency, low encapsulation rate, easy leakage, large oral dosage and low safety, the cholesterol is used for carrying out hydrophobic modification and preparing the prodrug, the micromolecule drug not only can target to a focus part, solve the cell-entering problem and obviously improve the activity of the drug, but also can reduce the toxic and side effect caused by singly using a large amount of water-soluble drug.
Transforming growth factor-beta (TGF-beta) belongs to a group of TGF-beta superfamilies that regulate cell growth and differentiation. The signal channel participates in a plurality of biological processes, such as cell differentiation, proliferation, migration, extracellular matrix remodeling, apoptosis and the like, and plays a promoting role in the development process of diseases such as vascular cancer, gastric cancer, pancreatic cancer, bladder cancer, lung cancer, liver cancer, tumor, hepatic fibrosis, renal fibrosis, pulmonary hypertension, leukemia and the like. Therefore, inhibition of the action and activity of TGF-. beta.s may be effective in treating the above-mentioned diseases.
In conclusion, the invention intends to develop a compound preparation system, and the cholesterol-water-soluble drug prodrug is assembled on the outer layer of the liposome carrying the TGF-beta 1 inhibitor, so that the co-delivery of two water-soluble chemotherapeutic drugs is realized, and the inflammatory diseases such as pulmonary hypertension and the like are treated. The compound preparation acts on two targets simultaneously, can realize a spatial synergistic effect, can improve the uptake rate of a water-soluble medicament by an organism, reduce the administration dosage, reduce the toxic and side effects, and can obviously improve the treatment effect.
Disclosure of Invention
The invention provides a prescription composition of a compound preparation for treating pulmonary hypertension and a preparation method thereof. Aims to realize the co-delivery of two water-soluble medicines, breaks through the defects caused by the mass delivery of the water-soluble medicines and is expected to improve the combined treatment index of a plurality of medicines.
In order to achieve the above object, the present invention provides the following technical solutions:
1) cholesterol and dichloroacetic acid are used as raw materials to synthesize the cholesterol-dichloroacetic acid prodrug.
Cholesterol and dichloroacetic acid are used as raw materials, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are used as catalysts, and dichloromethane is used as an organic solvent (DCA: Chol: DCC: DMAP ═ 4:1:2:0.095, n/n/n/n). Weighing DCC with the prescription amount, filling the DCC with a dry round-bottom flask, adding DCM under the ice bath condition to completely dissolve the DCC, then adding DCA with the prescription amount, and sealing the flask and stirring in the ice bath for 2 hours. After the end of the time, Chol and DMAP were added, DCM was added until complete dissolution, and the flask was sealed and allowed to react overnight at room temperature. And (3) separating and purifying the cholesterol-dichloroacetic acid prodrug by using column chromatography, wherein the yield of the cholesterol-dichloroacetic acid is 40-75%.
The other water-soluble drugs can be synthesized by the above reaction, i.e. cholesterol and water-soluble drugs are used as reactants, and dicyclohexylcarbodiimide and 4-dimethylamino are used as catalysts.
The local hydrophilic inner core space of the liposome is difficult to deliver two water-soluble drugs, so that cholesterol is utilized to synthesize a cholesterol-water-soluble drug prodrug to be loaded into a hydrophobic membrane of the liposome; furthermore, it has been reported in the literature that pulmonary hypertension smooth muscle cells highly express insulin-like growth factor 2 receptor (IGF2R), and cholesterol can target IGF2R receptor. In conclusion, the water-soluble drugs are subjected to hydrophobic modification by cholesterol to prepare the prodrug, so that the liposome can co-deliver two water-soluble drugs, the problem that the water-soluble drugs are difficult to enter cells is solved, the toxic and side effects caused by singly using a large amount of water-soluble drugs are reduced, and the prodrug can be used for targeted treatment of related diseases to a certain extent.
2) The liposome carrying the TGF-beta 1 inhibitor is prepared by adopting a cell friability apparatus and an ultrasonic method.
Acid-sensitive liposome
Prescription amounts of DOPE, mPEG2000-DSPE, CHEMS were weighed separately into a dry round bottom flask, and 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); TGF-beta 1 inhibitor bulk drug is dissolved in sodium hydroxide solution (150 mu g/mL) and is dissolved by ultrasonic through a common ultrasonic instrument. After the phospholipid bilayer membrane is hydrated by TGF-beta 1 inhibitor solution, the phospholipid bilayer membrane is transferred to a cell friability apparatus and is subjected to ultrasonic treatment for 10-15min by using a No. 3 amplitude transformer, and the power is 100-120W. Filtering with 0.22 μm filter membrane to obtain liposome carrying TGF-beta 1 inhibitor, wherein the particle size is 75-100nm, the potential is-35 to-15 mV, the entrapment rate of TGF-beta 1 inhibitor is 56-65.91%, and the drug-loading rate is 1.8-5.5%.
Temperature responsive liposome
The prescribed amounts of DPPC, 1-StePC and DSPE-PEG2000 were weighed separately into a dry round bottom flask, and 5mL of chloroform-methanol solution was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); the membrane was hydrated at 45 ℃ by adding 5ml of a pH 6.5 PBS solution containing a TGF-. beta.1 inhibitor. Performing ultrasonic treatment on the probe for 10min at 120W for 2s to form a bluish opalescent solution, and filtering the solution with a 0.22 μm filter membrane at 4 ℃ for later use.
Common type liposome
Egg yolk lecithin was weighed separately in the prescribed amount into a dry round bottom flask, 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); 5mL of PBS solution containing TGF-. beta.1 inhibitor was added to hydrate the membrane at 45 ℃. Performing ultrasonic treatment on the probe for 10min at 120W, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and filtering the ultrasonic mode through a 0.22 mu m filter membrane to store the ultrasonic mode in a dark place at 4 ℃ for later use.
3) Preparation of compound preparation of cholesterol prodrug and TGF-beta 1 inhibitor
The preferable cholesterol-dichloroacetic acid prodrug is dissolved in 0.25mL of absolute ethyl alcohol, added into the liposome encapsulating the TGF-beta 1 inhibitor under the stirring condition and mixed evenly by a mechanical method. And (3) stirring the suspension at room temperature for 30min (800rpm) to obtain the compound preparation of the cholesterol prodrug and the TGF-beta 1 inhibitor, wherein the particle size is 130-200nm, the potential is-8-2 mV, the encapsulation rate of the cholesterol prodrug is 59.93-77.5%, and the drug loading is 6.85-8.86%. Wherein the mechanical method is vortex (2min), cell disruption apparatus sonication (100-.
4) The invention finally provides the application of the cholesterol prodrug and TGF-beta 1 inhibitor compound preparation in an anti-pulmonary hypertension combined administration preparation.
The invention has the beneficial effects that: the compound preparation of the cholesterol prodrug and the TGF-beta 1 inhibitor is expected to deliver water-soluble drugs efficiently, overcomes the difficulties of delivery of high water-soluble drugs (easy leakage, low encapsulation rate, difficult cell entry and the like), realizes targeted therapy by modifying cholesterol, enhances the targeting property of a drug delivery system, and reduces the systemic toxicity of the drugs. In addition, the compound preparation has the advantages of classical model medicine selection and simple preparation process, is expected to provide a new medicine carrying technology, and expands the application of the compound preparation in the field of biological medicine.
Abbreviations
1-StePC stearoyl lysolecithin
6-TAMRA 6-carboxytetramethyl rhodamine
Chol cholesterol
Chol-DCA cholesterol-dichloroacetic acid prodrugs
CHEMS Cholesterol succinate monoester
CDPs cholesterol prodrug and TGF-beta 1 inhibitor compound preparation
CI combination therapeutic index
DCA Dichloroacetic acid
DCC dicyclohexylcarbodiimide
DMAP 4-dimethylaminopyridine
DCM dichloromethane
DOPE dioleoyl L-alpha-phosphatidylethanolamine
DPPC dipalmitoyl phosphatidylcholine
DMSO dimethyl sulfoxide
DLS dynamic light scattering method
Inhibition rate of Fa cells
IGF2R insulin-like growth factor 2 receptor
MTT tetramethyl azodicarbonyl blue
mPEG2000-DSPE distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000
mPA mean pulmonary arterial pressure
PASMCs pulmonary arterial hypertension smooth muscle cells
PAH pulmonary hypertension
PFD pirfenidone
PFD-pH-Lips pirfenidone acid-sensitive response type liposome
PDI polydispersity index
RVHI right ventricular hypertrophy index
TGF-beta transforming growth factor-beta
TLR Toll-like receptors
TEM transmission scanning electron microscope
WT model group
Saline physiological Saline
Drawings
FIG. 1 is a graph showing the particle size distribution of the TGF- β 1 inhibitor-loaded liposome and the TGF- β 1 inhibitor combined preparation of example 1, wherein A is PFD-pH-Lips, and B is CDPs;
FIG. 2 is a potential diagram of the TGF- β 1 inhibitor-loaded liposome of example 1 and a TGF- β 1 inhibitor combined preparation, wherein A is PFD-pH-Lips, and B is CDPs;
FIG. 3 is a transmission electron micrograph of the TGF- β 1 inhibitor liposome-loaded and cholesterol prodrug-loaded TGF- β 1 inhibitor complex formulation of example 1, wherein A is PFD-pH-Lips, and B is CDPs;
FIG. 4 is a chart of the serum stability study of the combined formulation of cholesterol prodrug and TGF- β 1 inhibitor of example 3;
FIG. 5 is an acid sensitivity profile of the co-formulation of cholesterol prodrug and TGF- β 1 inhibitor of example 4;
FIG. 6 is a study of the synergistic therapeutic effect of two drugs in the combination formulation of cholesterol prodrug and TGF-beta 1 inhibitor of example 1, wherein A is the result of cytotoxicity and B is the indication graph of synergistic effect;
FIG. 7 is a targeting study of cholesterol;
FIG. 8 is a pharmacodynamic study in animals of the compound formulation of cholesterol prodrug and TGF- β 1 inhibitor of example 1, wherein A is the mPAP study and B is the RVHI study;
fig. 9 is a schematic diagram of the compound preparation of the invention.
Detailed Description
Example 1
Prescription:
cholesterol-dichloroacetic acid prodrug 4mg
Acid-sensitive response type liposome 30mg
Pirfenidone 1mg
The preparation method comprises the following steps:
prescription amounts of DOPE, mPEG2000-DSPE, CHEMS were weighed separately into a dry round bottom flask, and 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); the film was hydrated by the addition of 5mL of 15% NaOH (m/v) containing pirfenidone at 45 ℃. Performing ultrasonic treatment on the probe for 10min at 120W for 2s to form a bluish opalescent solution, and filtering the solution with a 0.22 μm filter membrane at 4 ℃ for later use. Weighing the cholesterol-dichloroacetic acid prodrug with the prescription amount, dissolving the cholesterol-dichloroacetic acid prodrug in 250 mu L of absolute ethyl alcohol, dropwise adding the cholesterol-dichloroacetic acid prodrug into the liposome suspension under the stirring state, carrying out ultrasonic treatment on a probe for 10W for 3min, switching on an ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and stirring at room temperature for 30min to obtain the cholesterol-dichloroacetic acid prodrug.
Wherein, the cholesterol-dichloroacetic acid prodrug is prepared by the following steps: takes cholesterol and dichloroacetic acid as reactants, DCC and DMAP as catalysts, DCM as a solvent, and takes esterification reaction at room temperature, and finally the product is obtained by purifying through a chromatographic column.
The particle size, Polydispersity index (PDI) and Zeta potential of the TGF-beta 1 inhibitor-loaded liposome, the cholesterol prodrug and the TGF-beta 1 inhibitor compound preparation are measured by a Dynamic Light Scattering (DLS) method at room temperature. Each sample was repeated 3 times. TEM is adopted to observe the appearance of the TGF-beta 1 inhibitor-loaded liposome, the cholesterol prodrug and the TGF-beta 1 inhibitor compound preparation, and the method comprises the following steps: and (3) sucking 20 mu L of sample liquid to be measured on the surface of a special copper mesh by a liquid transfer gun, standing for 10min, sucking redundant solution by using filter paper, completely drying the liquid on the surface of the copper mesh, taking 20 mu L of 2% phosphotungstic acid solution to the surface of the copper mesh, dyeing for 1min, drying at room temperature, and then carrying out TEM shooting.
The particle sizes of the TGF-beta 1 inhibitor-carrying liposome and the cholesterol prodrug prepared in the embodiment 1 and the TGF-beta 1 inhibitor compound preparation are 91.42 +/-0.62 and 178.13 +/-2.49 nm respectively, the PDI is 0.266 +/-0.005 and 0.214 +/-0.038 respectively, and the Zeta potential is-31.03 +/-4.78 and-1.36 +/-2.45 mv respectively. The particle size distribution diagram of the TGF-beta 1 inhibitor-loaded liposome prepared in example 1, the cholesterol prodrug and the TGF-beta 1 inhibitor compound preparation is shown in figure 1; the potential diagram is shown in FIG. 2; the transmission electron micrograph is shown in FIG. 3.
Example 2
Prescription:
cholesterol-beraprost prodrug 4mg
Temperature responsive liposome 30mg
Horse
The prescribed amounts of DPPC, 1-StePC and DSPE-PEG2000 were weighed separately into a dry round bottom flask, and 5mL of chloroform-methanol solution was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); the film was hydrated at 45 ℃ by adding 5ml of a PBS solution of pH 6.5 containing horse. Performing ultrasonic treatment on the probe for 10min at 120W for 2s to form a bluish opalescent solution, and filtering the solution with a 0.22 μm filter membrane at 4 ℃ for later use. Dropwise adding the cholesterol-beraprost prodrug solution into the liposome suspension under stirring, carrying out ultrasonic treatment by a probe for 120W for 3min, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and stirring at room temperature for 30min to obtain the cholesterol-beraprost prodrug solution.
Wherein, the cholesterol-beraprost prodrug is prepared by the following steps: cholesterol and beraprost are taken as reactants, DCC and DMAP are taken as catalysts, DCM is taken as a solvent, esterification reaction is carried out at room temperature, and finally the product is obtained by purifying through a chromatographic column.
Example 3
Prescription:
cholesterol-epoprostenol prodrug 4mg
Normal liposome 30mg
Horse
Egg yolk lecithin was weighed separately in the prescribed amount into a dry round bottom flask, 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); the film was hydrated at 45 ℃ by adding a 5ml PBS solution containing horse. Performing ultrasonic treatment on the probe for 10min at 120W, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and filtering the ultrasonic mode through a 0.22 mu m filter membrane to store the ultrasonic mode in a dark place at 4 ℃ for later use. And (3) dropwise adding the cholesterol-epoprostenol prodrug solution with the prescription amount into the liposome suspension under the stirring state, carrying out ultrasonic treatment by a probe for 120W for 3min, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and stirring at room temperature for 30min to obtain the cholesterol-epoprostenol prodrug.
The serum stability of the combination preparation was examined by incubating the combination preparation prepared in example 3 with a PBS solution containing 10% bovine serum at 37 ℃ and measuring the particle size and PDI at a specific time. The results show that the particle size and PDI of the compound preparation are stable within 12h, the particle size does not exceed 200nm, and the PDI does not exceed 0.3, and the experimental results are shown in figure 4.
Wherein the cholesterol-epoprostenol prodrug is prepared by the following steps: takes cholesterol and epoprostenol as reactants, DCC and DMAP as catalysts, DCM as a solvent, and takes esterification reaction at room temperature, and finally the product is obtained by purifying through a chromatographic column.
Example 4
Prescription:
cholesterol-treprostinil prodrug 4mg
Acid-sensitive response type liposome 30mg
Pirfenidone 1mg
Prescription amounts of DOPE, mPEG2000-DSPE, CHEMS were weighed separately into a dry round bottom flask, and 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); the film was hydrated by the addition of 5mL of 15% NaOH (m/v) containing pirfenidone at 45 ℃. Ultrasonic treating with probe at 120W for 10min, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and filtering with 0.22 μm filter membrane at 4 deg.C for storage. And (3) dropwise adding the cholesterol-treprostinil prodrug solution with the prescription amount into the liposome suspension under the stirring state, carrying out ultrasonic treatment on the liposome suspension for 120W for 3min by using a probe, switching on the ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and stirring the mixture at room temperature for 30min to obtain the cholesterol-treprostinil prodrug solution.
The acid sensitivity of the compound preparation prepared in example 4 was examined by HPLC. The mixture was packed in dialysis bags and finally placed in media of different pH (pH 5.0, 6.0 and 7.4) and shaken in a water bath shaker at 37 ℃ and 100 rpm. The release medium was removed and replaced with an equal volume of medium at the specified time and finally the concentration of Pirfenidone (PFD) in the release medium was measured by HPLC. The results show that the compound preparation releases faster at pH 5.0 than at pH 6.0 and 7.4, which shows that the compound preparation has acid-sensitive characteristics, and the results are shown in FIG. 5.
Wherein the cholesterol-treprostinil prodrug is prepared by the following steps: the preparation method comprises the steps of carrying out esterification reaction at room temperature by using cholesterol and treprostinil as reactants, DCC and DMAP as catalysts and DCM as a solvent, and finally purifying the product by using a chromatographic column.
Example 5
Prescription:
cholesterol-dichloroacetic acid prodrug 4mg
Normal liposome 30mg
Salvianolic acid B1 mg
Egg yolk lecithin was weighed separately in the prescribed amount into a dry round bottom flask, 5mL of chloroform was removed and dissolved by vortexing. Evaporating under reduced pressure at 45 deg.C and 100rpm in thermostatic water bath to remove organic solvent (1 h); 5mL of PBS containing salvianolic acid B was added to hydrate the film at 45 ℃. Performing ultrasonic treatment on the probe for 10min at 120W for 2s to form a bluish opalescent solution, and filtering the solution with a 0.22 μm filter membrane at 4 ℃ for later use. Weighing the cholesterol-dichloroacetic acid prodrug with the prescription amount, dissolving the cholesterol-dichloroacetic acid prodrug in 250 mu L of absolute ethyl alcohol, dropwise adding the cholesterol-dichloroacetic acid prodrug into the liposome suspension under the stirring state, carrying out ultrasonic treatment by a probe for 120W for 3min, switching on an ultrasonic mode for 2s, switching off the ultrasonic mode for 2s, and stirring at room temperature for 30min to obtain the cholesterol-dichloroacetic acid prodrug.
Example 6
Application example 1 Compound formulation of Cholesterol prodrug and TGF-. beta.1 inhibitor the in vitro synergistic therapeutic effects of TGF-. beta.1 inhibitor and cholesterol prodrug in this system were investigated. Rat pulmonary artery high pressure smooth muscle cells (PASMCs) in good growth state are digested, centrifuged, counted and resuspended, inoculated into a 96-well plate at the cell density of 5000 cells/well, and placed in a cell culture box for 24 h. Adding 20 μ L of each preparation with different concentrations and 180 μ L of RPMI 1640 culture medium, setting 5 multiple wells for each test group, setting blank well and control well, culturing for 48 hr, adding 20 μ L of MTT (5mg/mL) solution into each well, incubating for 4 hr at 37 deg.C, discarding culture medium in the wells, adding 200 μ L of dimethyl sulfoxide (DMSO) to dissolve methyl chloride in the wells
Figure BDA0002806584540000121
Crystallize, and put 96-well plate on the shaker to shake gently for 10 min. The absorbance at 570nm was measured by a microplate reader, and the Cell viability (Cell viability) was calculated by the following formula.
According to cell viability, the inhibition rate (Fa) of cells was plotted on the abscissa and the combination therapy index (CI) on the ordinate using the CompuSyn software. Wherein abs (treated), abs (untreated) and abs (blank) represent absorbance values for the test, control and blank wells, respectively. Various formulations include Chol-DCA, PFD, Chol-DCA/PFD-pH-lip (CDPs). Chol-DCA was administered at concentrations of 2.5, 4, 5, 8, 10 and 12.5. mu.g/mL for each formulation, with a ratio of PFD concentration added to Chol-DCA concentration of 1.25: 1.
Combination therapy index (CI) is a simple method to quantify the additive, synergistic or antagonistic effect of a combination of multiple drugs, and is commonly used to measure the effect of a combination. When the CI value is equal to 1, the combination of the two medicines shows additive effect; when the CI value is more than 1, the combination of the two medicines shows antagonism; when the CI value is less than 1, the combination of the two drugs shows synergistic effect. The above results show that: for the cytotoxicity inhibition capability of the PASMCs, the CDPs inhibition capability is strongest and is obviously higher than that of free medicines. The compound preparation is proved to be capable of successfully delivering two water-soluble medicines of DCA and PFD, more importantly, the CI values are less than 1 under the condition of higher administration concentration, namely high inhibition rate, and the synergistic effect is shown when Chol-DCA and PFD are combined in a Chol-DCA/PFD-pH-Lips system. The results of the experiment are shown in FIG. 6.
Example 7
The cholesterol prodrug and the TGF-beta 1 inhibitor compound preparation in the example 1 are applied to carry out cholesterol targeting investigation. Rat pulmonary hypertension smooth muscle cells (PASMCs) in good growth state are digested, centrifuged, counted and resuspended, inoculated in a 12-well plate at a cell density of 1 x 10^ 5/well, and placed in a cell incubator for 24 h. The medium in the well plate was discarded, washed three times with pre-cooled PBS, 1mL of Chol solution at 5mg/mL was added and incubated in the incubator for 1h, and the control group was pre-incubated with PBS without Chol for 2 h. After the incubation time was over, the Chol solution was discarded, 900. mu.L of RPMI 1640 medium and 100. mu.L of 6-TAMRA-Chol were added, and the mixture was incubated for 4 hours in a cell incubator. Wherein the final concentrations of 6-TAMRA are 0.5. mu.g/mL, 5. mu.g/mL and 10. mu.g/mL, respectively.
After the incubation time is over, the fluorescent medium in the pore plate is discarded, the cell is washed three times by precooled PBS, 400 mu L of pancreatin digestive cells containing EDTA is added, the digestion is stopped by PBS containing serum, the cell suspension is collected at 1500rpm and 4 ℃, the cell suspension is centrifuged for 3min, the cell is resuspended by 250 mu L of PBS after the PBS is washed three times, and the cell is sieved and detected by a flow cytometer B2 channel.
The results show that after IGF2R receptor is blocked by Chol solution with high concentration, the uptake of 6-TAMRA-Chol by the PASMCs is obviously reduced at the same time, and the influence of the blocking of IGF2R receptor by the Chol incubation in advance is obvious no matter at low concentration or high concentration. The results are shown in FIG. 7.
Example 8
Pharmacodynamic studies were performed using the cholesterol prodrug and TGF- β 1 inhibitor combination formulation of example 1. Each rat was given a subcutaneous injection of monocrotaline (MCT,60mg/kg) three weeks later. Rats meeting the requirements were randomly divided into 5 groups of 6 rats each, and each rat was given intravenous injections of 0.5mL of each type of preparation or physiological saline. The groups include a model group (WT), a Saline group (Saline), a DCA group, a Chol-DCA group and a CDPs group; wherein the administration dose of DCA in each preparation is 1mg/kg, the administration dose of PFD is 5mg/kg, and the administration frequency is 3 d/time and 5 times. After the dosing period, pulmonary artery pressure was measured for each rat and mean pulmonary artery pressure (mPAP) was calculated; the rats were then decapped, and the rat hearts removed and the Right Ventricle (RV), left ventricle plus ventricular septum (LV + S) and weighed to calculate the Right Ventricle Hypertrophy Index (RVHI).
The results show that after the drug treatment, the average pulmonary artery pressure and the right ventricular hypertrophy index of the rats suffering from the pulmonary hypertension are reduced, and on the contrary, the mPAP and the RVHI of the model group are higher because the drug is not effectively treated. Among these, the cholesterol-dichloroacetic acid prodrug works best in combination with pirfenidone therapy, i.e., the group of CDPs inhibits mPAP and RVHI. The results show that the active targeting of the cholesterol prodrug is combined with the excellent passive targeting of the liposome, and the effective treatment of pulmonary hypertension can be realized by the combined co-delivery treatment with pirfenidone, and the experimental results are shown in fig. 8.

Claims (8)

1.一种复方制剂，其特征在于，所述的复方制剂包括包封于脂质体内核的TGF-β1抑制剂和嵌于脂质体双层分子膜的胆固醇前药。1. a compound preparation, is characterized in that, described compound preparation comprises the TGF-β1 inhibitor that is encapsulated in liposome core and the cholesterol prodrug embedded in liposome bilayer molecular membrane. 2.根据权利要求1所述的复方制剂，其特征在于所述的胆固醇前药为胆固醇-水溶性药物，其中水溶性药物包括依前列醇、曲前列环素、伊洛前列素、贝前列素、法舒地尔、盐酸伊马替尼、波生坦、马西替坦、安倍生坦、利奥西瓜、塞乐西帕、环磷酰胺、雷默司汀、西地那非、柔红糖胺和二氯乙酸。2. compound preparation according to claim 1 is characterized in that described cholesterol prodrug is cholesterol-water-soluble drug, wherein water-soluble drug comprises epoprostenol, treprostinil, iloprost, beraprost , fasudil, imatinib hydrochloride, bosentan, macitentan, ambesentan, rio watermelon, celecipa, cyclophosphamide, ramustine, sildenafil, soft brown sugar amine and dichloroacetic acid. 3.根据权利要求1所述的复方制剂，其特征在于所述的复方制剂载体为脂质体，包括普通脂质体、温度响应型脂质体、磁场响应脂质体、酶敏感脂质体和酸敏感响应型脂质体。3. compound preparation according to claim 1 is characterized in that described compound preparation carrier is liposome, comprises common liposome, temperature-responsive liposome, magnetic field-responsive liposome, enzyme-sensitive liposome and acid-sensitive responsive liposomes. 4.根据权利要求1所述的复方制剂，其特征在于所述的TGF-β1抑制剂包括丹酚酸B、氧化苦参碱、三七皂苷R1、陈皮素、马力马司它、吡非尼酮，2,3-二取代吡啶、地司特泰、二氢吡咯吡唑取代苯并咪唑、加努塞蒂布、盐酸瓦托沙和LY3200882。4. compound preparation according to claim 1 is characterized in that described TGF-β1 inhibitor comprises salvianolic acid B, oxymatrine, Panax notoginseng saponin R1, tangerine peel, marimastat, pirfenib ketones, 2,3-disubstituted pyridines, destastat, dihydropyrrole pyrazole substituted benzimidazoles, ganucetib, vatoxa hydrochloride and LY3200882. 5.根据权利要求2所述的复方制剂，其特征在于所述的胆固醇-二氯乙酸前药由如下步骤制备获得：5. compound preparation according to claim 2 is characterized in that described cholesterol-dichloroacetic acid prodrug is prepared by following steps: 以Chol、DCA为原料，以DCC)、DMAP为催化剂，以DCM为有机溶剂，其中DCA: Chol: DCC:DMAP=4: 1: 2: 0.095, n/n/n/n；称取处方量的DCC装于干燥的圆底烧瓶，冰浴条件下加入DCM使其溶解完全，随后加入处方量DCA，密封烧瓶冰浴搅拌2 h；时间结束后加入Chol及DMAP，补加DCM直至完全溶解，密封烧瓶室温反应过夜；Take Chol, DCA as raw material, take DCC), DMAP as catalyzer, take DCM as organic solvent, wherein DCA: Chol: DCC: DMAP=4: 1: 2: 0.095, n/n/n/n; Weigh recipe quantity The DCC was placed in a dry round-bottomed flask, and DCM was added under ice bath conditions to make it completely dissolved, then the recipe amount of DCA was added, and the flask was sealed in an ice bath and stirred for 2 h; after the time was over, Chol and DMAP were added, and DCM was added until it was completely dissolved, The sealed flask was reacted at room temperature overnight; 利用柱层析分离纯化胆固醇-二氯乙酸前药。Cholesterol-dichloroacetic acid prodrug was isolated and purified by column chromatography. 6.根据权利要求3所述的复方制剂，其特征在于所述的载TGF-β1抑制剂脂质体的制备方法，其特征在于，包括以下步骤：6. compound preparation according to claim 3 is characterized in that the preparation method of described liposome loaded with TGF-β1 inhibitor, is characterized in that, comprises the following steps: （1） 将TGF-β1抑制剂原料药溶于氢氧化钠溶液150 μg/mL中普通超声溶解；(1) Dissolve the TGF-β1 inhibitor API in a sodium hydroxide solution of 150 μg/mL by ordinary ultrasonic dissolution; （2） 采用旋转蒸发仪制备磷脂双分子层膜，仪器参数为45 ℃，100 rpm，1 h；(2) The phospholipid bilayer film was prepared by a rotary evaporator, and the instrument parameters were 45 °C, 100 rpm, 1 h; （3） 经上述TGF-β1抑制剂溶液水化薄膜后，移至细胞脆碎仪中采用3号变幅杆超声10-15 min，功率为100-120 W；过0.22 μm滤膜得载TGF-β1抑制剂脂质体；(3) After the film was hydrated by the above TGF-β1 inhibitor solution, it was transferred to a cell crusher and ultrasonicated with a No. 3 horn for 10-15 min at a power of 100-120 W; TGF-b was obtained by passing through a 0.22 μm filter membrane. - β1 inhibitor liposomes; 其中包载于脂质体中的TGF-β1抑制剂浓度为0.2-2 mg/mL。The concentration of TGF-β1 inhibitor encapsulated in liposome is 0.2-2 mg/mL. 7.根据权利要求1所述的复方制剂制备方法，其特征在于，包括以下步骤：7. compound preparation preparation method according to claim 1 is characterized in that, comprises the following steps: （1）将胆固醇-水溶性药物前药溶于0.25 mL无水乙醇，在搅拌条件下逐滴加入到包载TGF-β1抑制剂的脂质体中，经机械方法使其混合均匀；(1) Dissolve cholesterol-water-soluble drug prodrugs in 0.25 mL of absolute ethanol, add dropwise to the liposomes encapsulating the TGF-β1 inhibitor under stirring conditions, and mix them uniformly by mechanical methods; 其中机械方法包括涡旋2 min，细胞脆碎仪超声100-120 W，3-5 min；Among them, mechanical methods include vortexing for 2 min, and ultrasonication of cell crusher at 100-120 W for 3-5 min; （2）将步骤（1）中的胆固醇-水溶性药物前药与包载TGF-β1抑制剂脂质体混悬液室温搅拌30 min，800 rpm，得胆固醇-水溶性药物前药与TGF-β1抑制剂复方制剂；(2) The cholesterol-water-soluble drug prodrug in step (1) and the liposome suspension containing TGF-β1 inhibitor were stirred at room temperature for 30 min and 800 rpm to obtain cholesterol-water-soluble drug prodrug and TGF-β1 inhibitor. β1 inhibitor compound preparation; 其中胆固醇-水溶性药物前药于包载TGF-β1抑制剂脂质体混悬液中的浓度为125-1000μg/mL。The concentration of cholesterol-water-soluble drug prodrug in liposome suspension containing TGF-β1 inhibitor is 125-1000 μg/mL. 8.根据权利要求1-6任意一项所述的复方制剂在制备抗肺动脉高压药物中的应用。8. The application of the compound preparation according to any one of claims 1-6 in the preparation of an anti-pulmonary hypertension drug.
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CN114732789A (en) * 2022-05-05 2022-07-12 中国药科大学 Compound long-acting drug delivery system for treating pulmonary hypertension and preparation thereof
WO2024253511A1 (en) * 2023-06-06 2024-12-12 Excelsior Pharmatech Labs. Method for preventing or treating pulmonary arterial hypertension by using 7-dehydrocholesterols or salts, oxides, metabolites, or derivatives thereof

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TW201922724A (en) * 2017-09-29 2019-06-16 德商拜耳廠股份有限公司 Substituted 3-phenylquinazolin-4(3H)-ones and uses thereof
CN109248145A (en) * 2018-09-28 2019-01-22 中国药科大学 A kind of combined system carrying small-molecule drug and macromolecular drug altogether

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CN114732789B (en) * 2022-05-05 2023-08-22 中国药科大学 A compound long-acting drug delivery system for treating pulmonary hypertension and its preparation
WO2024253511A1 (en) * 2023-06-06 2024-12-12 Excelsior Pharmatech Labs. Method for preventing or treating pulmonary arterial hypertension by using 7-dehydrocholesterols or salts, oxides, metabolites, or derivatives thereof

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