Pharmaceutical composition of semaglutin, preparation method and application thereofTechnical Field
The invention relates to a pharmaceutical composition of semaglutin, a preparation method and application thereof.
Background
Glucagon-like peptide-1 (GLP-1) receptor agonist is used as a new generation hypoglycemic agent, has the hypoglycemic effect which is inferior to that of insulin, and has multiple clinical advantages of strong hypoglycemic effect, low risk of hypoglycemia, obvious weight-losing effect, cardiovascular benefit and the like. Diabetes is a progressive disease, and GLP-1 receptor agonist is used as a transitional therapy between oral hypoglycemic agents and insulin treatment, has biological effects of delaying disease progression, and is one of the hypoglycemic agents with the market potential worldwide at present from the current clinical application trend.
Semaglutin (structure as above), also known as cord Ma Lutai, is a glucagon-like peptide-1 (GLP-1) receptor agonist that stimulates insulin secretion in a glucose-dependent manner and reduces glucagon secretion, thereby reducing blood glucose. In the prior art, the semaglutin injection is usually adopted, is administrated in a subcutaneous injection mode, has a long-acting effect, and can realize the administration frequency once a week so as to better exert the curative effect of the medicine, but the subcutaneous injection is an invasive administration mode, and can cause the problems of pain, discomfort at an injection position and the like, thereby bringing physiological and psychological discomfort to patients. Meanwhile, the injection pen for administration cannot be reused, and the cost of the injection pen occupies a large part of the costs of developing, producing and using the injection. In addition, the subcutaneous injection has the problems of complicated filling process and the like.
The cable Ma Lutai injection (trade name: ozempic) in 2017 shows advantages in aspects of reducing blood sugar, reducing weight, cardiovascular system and the like after being marketed, the Norand Nord company overcomes the technical limit that peptide medicines are easy to degrade by digestive enzymes, and the developed oral preparation cable Ma Lutai tablets (trade name: rybelsus) are formally approved by the United states Food and Drug Administration (FDA) in 2019 for controlling blood sugar of adult T2DM patients. Sodium N- (8- (2-hydroxybenzoyl) amino) octoate (SNAC) technology can prevent the damage of semaglutin in stomach, and promote cell absorption through gastric membrane, so that semaglutin can completely reach the systemic circulation. Oral semaglutin enhances patient compliance, but the bioavailability is far lower than that of subcutaneous injection, and the administration frequency is frequent, and the administration needs to be carried out once a day.
Disclosure of Invention
The invention aims to overcome the defect of limited preparation types of semaglutin in the prior art, and provides a semaglutin pharmaceutical composition, a preparation method and application thereof. The compositions of semaglutin of the present invention have one or more of the following beneficial effects of higher bioavailability than oral administration, avoidance of the risk of symptoms such as cough that may be caused by FDKP (fumaryldiketopiperazine) and the lack of invasive means such as injection.
The invention solves the technical problems by the following technical scheme:
The invention provides a pharmaceutical composition which comprises an active substance X and a pharmaceutical excipient A, wherein the active substance X is semaglutin or pharmaceutically acceptable salt thereof, and the pharmaceutical excipient A is a diketopiperazine compound shown in a formula I or pharmaceutically acceptable salt thereof:
Wherein Ra and Ra' are independently selected from 3-8 membered cycloalkyl, said 3-8 membered cycloalkyl is preferably monocyclic cycloalkyl or bicyclo [ n, m, l ] alkyl, 2≤n+m≤7, more preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, -bicyclo [1.1.1] pentanyl-, -bicyclo [1.1.2] hexanyl-, -bicyclo [1.2.2] heptanyl-or bicyclo [2.2.2] octanyl, for example
In one scheme, the diketopiperazine compound shown as the formula I is
In a certain scheme, the pharmaceutically acceptable salt of the diketopiperazine compound shown in the formula I can be ammonium salt, preferably diammonium salt, and ammonium ion in the ammonium salt is preferably NH4+.
In a certain scheme, the mass ratio of the active substance X to the pharmaceutical auxiliary material A is preferably 1:1-1:100, more preferably 1:1-1:10, for example 1:2, 1:5 or 1:7.
In one embodiment, the pharmaceutical composition is in the form of a suspension in a dispersion medium.
In one embodiment, the pharmaceutical composition is in solid form, for example in the form of a solid dispersion and/or solid particles.
In one embodiment, when the pharmaceutical composition is in the form of a suspension in a dispersion medium, the pharmaceutical composition is in the form of microparticles in the suspension.
In one embodiment, when the pharmaceutical composition is in the form of a suspension in a dispersion medium, the dispersion medium of the suspension may be a dispersion medium conventional in the art, preferably water, more preferably distilled water, and the ratio of the total mass of the active substance X and the pharmaceutical excipients a to the mass of the dispersion medium may be 1% to 20%, preferably 1% to 10%, for example 3%.
In one embodiment, the pharmaceutical composition is in the form of a suspension in a dispersion medium, wherein the suspension further comprises a pH regulator, the pH regulator can be ammonia water, and the mass fraction of the ammonia water can be 1% -40%, for example 25%. The dosage of the pH regulator is preferably limited by regulating the pH value of the pharmaceutic adjuvant A in the suspension in a dispersion medium to 7-9, and is preferably limited by 7.5-8.
In a certain scheme, when the pharmaceutical composition exists in the form of solid particles, the pharmaceutical auxiliary material A is used as a carrier, microspheres are formed by self-assembly, and the active substance X is uniformly dispersed in the carrier microspheres in a highly dispersed state.
In a certain scheme, when the pharmaceutical composition exists in the form of solid microspheres, a scanning electron microscope of the pharmaceutical composition shows that the blank microspheres are in the shape of spheres, are complete in shape, have multiple pores on the surfaces, and are smoother in surface after drug loading, and active substances are adsorbed and filled in the pores on the surfaces of the blank microspheres.
In one embodiment, when the pharmaceutical composition is in the form of solid particles, the particle size of the pharmaceutical composition may be conventional in the art, preferably D50 is 2-20 μm, more preferably 5-10 μm.
In a certain embodiment, when the pharmaceutical composition is in the form of solid particles, the X50 of the pharmaceutical composition is 1-10 μm, preferably 1-4, e.g. 3 μm.
In one embodiment, when the pharmaceutical composition is in the form of solid particles, the pharmaceutical composition has respirable particles (FPF) of 30% to 50%, preferably 40% to 50%, for example 43.65%.
In a certain scheme, the pharmaceutical composition consists of the active substance X and the pharmaceutical auxiliary material A.
In one embodiment, the pharmaceutical composition comprises the active substance X, the pharmaceutical excipient a and the dispersion medium.
In one embodiment, the pharmaceutical composition comprises the active substance X, the pharmaceutical excipient a, the dispersion medium and the pH adjuster.
In a certain scheme, the pharmaceutical composition is prepared by a preparation method comprising the following steps of (1) mixing the pharmaceutic adjuvant A, a dispersion medium and the pH regulator to obtain a suspension 1 containing the pharmaceutic adjuvant A;
(2) Sequentially adding a dispersion medium and the active substance X into the suspension 1 to obtain a suspension containing the active substance X and the pharmaceutic adjuvant A;
Preferably, the method further comprises (3) lyophilizing the suspension to obtain a solid of the pharmaceutical composition.
In one embodiment, the dispersion medium is a dispersion medium conventional in the art, preferably water, more preferably distilled water.
In one embodiment, the mass ratio of the dispersion medium in step (1) and step (2) may be (0.1-3): 1, preferably (0.5-2.5): 1, for example 1.5:1.
In one embodiment, the mass ratio of the total mass of the active substance X and the pharmaceutical auxiliary material a to the dispersion medium (total mass of two additions) is conventional in the art, preferably 1% to 20%, more preferably 1% to 10%, for example 3%.
In one embodiment, the pH adjustor can be aqueous ammonia.
In one scheme, the mass fraction of the ammonia water can be 1% -40%, such as 25%.
In a certain scheme, the dosage of the pH regulator is not particularly limited, and the pH value of the suspension 1 containing the pharmaceutical excipients A is regulated to 7-9, preferably 7.5-8.
In one embodiment, when the pharmaceutical composition is prepared by the preparation method as described above, the raw materials of the pharmaceutical composition consist of the substance X, the pharmaceutical excipients a, the dispersion medium and the pH adjuster.
In one embodiment, the lyophilization is direct (vacuum) lyophilization of the suspension, or alternatively (e.g., -40 degrees) freezing to ice cubes and thawing, followed by vacuum lyophilization.
In one embodiment, when the pharmaceutical composition is prepared by the method as described above, the preparation method may further comprise the step of pulverizing the solid of the pharmaceutical composition obtained in the step (3) to obtain solid particles containing the active substance X and the pharmaceutical excipients A.
In one embodiment, the conditions and operations of the comminution are those conventional in the art.
In one embodiment, the comminution is carried out at a humidity of from 0 to 100%, preferably from 30% to 70%.
In one embodiment, the comminution is carried out at a pressure of from 1 to 10bar, preferably from 2 to 6 bar.
In one embodiment, the comminution is preferably a screw jet mill comminution, for example by a screw jet mill model 50AS from Mikroorganism, inc.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps of (1) mixing the pharmaceutical auxiliary material A, a dispersion medium and a pH regulator to obtain a suspension 1 containing the pharmaceutical auxiliary material A;
(2) Sequentially adding a dispersion medium and an active substance X into the suspension 1 to obtain a suspension containing the active substance X and a pharmaceutic adjuvant A;
preferably, the method further comprises (3) lyophilizing the suspension to obtain a solid of the pharmaceutical composition;
wherein the substance X, the pharmaceutic adjuvant A, the dispersion medium and the pH regulator are all as described above;
The steps and conditions in the preparation method are as described above.
The invention also provides an inhalation formulation comprising the pharmaceutical composition of any of the above aspects.
The invention also provides an application of the pharmaceutical composition or the inhalation preparation in preparing a GLP-1 receptor agonist or preparing a medicament for treating and/or preventing diseases related to a GLP-1 receptor.
In one embodiment, the GLP-1 receptor associated disease is diabetes, obesity, cardiovascular disease, or nonalcoholic fatty liver disease.
The invention also provides an application of the pharmaceutical composition or the inhalation preparation in preparing medicines for treating and/or preventing diabetes, obesity, cardiovascular diseases or nonalcoholic fatty liver.
The present invention also provides a method of treating and/or preventing a disease associated with the GLP-1 receptor comprising administering to a patient an effective amount of the above pharmaceutical composition or inhaled formulation.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The pharmaceutical composition of the invention has the positive progress effects of (1) avoiding invasion of preparation, (2) having high bioavailability, (3) having good physicochemical properties (for example, having high FPF value and/or lung deposition amount), (4) being convenient to operate, and (5) avoiding side effects such as cough and the like possibly caused by FDKP.
Drawings
FIG. 1 is a FL scanning electron micrograph (10 μm field of view) of Compound 1.
FIG. 2 is a FL scanning electron micrograph (200 μm field of view) of Compound 1.
FIG. 3 is a scanning electron micrograph of Compound 1-SMG obtained in step 3 of example 1.
FIG. 4 is a photograph of compound 1-SMG after lyophilization.
FIG. 5 is a graph of the NGI assay for compound 1-SMG dry powder of example 1.
FIG. 6 is a graph of the NGI assay for the compound S1-SMG dry powder.
FIG. 7 is a graph of the NGI assay for compound S2-SMG dry powder.
FIG. 8 is a graph of the NGI assay for the compound S3-SMG dry powder.
FIG. 9 is a graph of the NGI assay for compound S4-SMG dry powder.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1
The first step is that the semaglutin-diketopiperazine suspension is prepared:
1g of 3,3' - ((((2S, 5S) -3, 6-dioxopiperazine-2, 5-diyl) bis (butane-4, 1-diyl)) bis (azadiyl)) bis (carbonyl)) bis (bicyclo [1.1.1] pentane-1-carboxylic acid) (compound 1, scanning electron microscopy spectra as shown in FIGS. 1 and 2) were placed in a 100 ml single-necked flask, 23 ml of distilled water was added at room temperature, 25% by mass of aqueous ammonia was added, and the pH was 7.5-8. The particles were crushed by a spoon for 5 minutes and continued to be sonicated for 5 minutes, stirred for 4 hours, 15 ml distilled water was added, stirred for 20 minutes, 143 mg semaglutin (SMG, 98%) was added and stirred overnight at room temperature.
Secondly, preparing semaglutin-diketopiperazine freeze-dried powder:
The suspension is poured into a 100ml plastic test tube, and is freeze-dried by a vacuum freeze dryer in a conventional operation to obtain powdery particles. The freeze-dried product is a uniform white solid, and the freeze-dried sample has good appearance and no atrophy and collapse. (as in FIG. 4)
Third, crushing the semaglutin-diketopiperazine freeze-dried powder:
The above packaged granule is slowly added into pulverizer under 30-70% humidity and 2-6bar pressure for pulverizing to obtain pulverized product (compound 1-SMG, with scanning electron microscope spectrogram shown in figure 3). The crushed grain size is controlled to D50- (5-10 μm). Then the powder is crushed by a screw jet mill with the model of 50AS by the company of fine Sichuan Mikron powder machinery, and the crushing grain diameter is controlled to be D50- (1-10 mu m).
Example 2
First step, preparation of a semaglutin-diketopiperazine suspension
2G of 3,3' - ((((2S, 5S) -3, 6-dioxopiperazine-2, 5-diyl) bis (butane-4, 1-diyl)) bis (azadiyl)) bis (bicyclo [1.1.1] pentane-1-carboxylic acid) were placed in a 250 ml single necked flask, 46 ml of distilled water was added at room temperature, sonicated for 5 minutes, 25% aqueous ammonia was added, pH was 7.5-8, sonicated for 5 minutes, the particles were crushed with a spoon, sonicated for 5 minutes, stirring was continued for 30 minutes, 30 ml of distilled water was added, stirring was carried out for 4 hours, 286 mg of semaglutin was added, and stirring was carried out at room temperature overnight.
Secondly, preparing semaglutin-diketopiperazine freeze-dried powder:
Freezing the suspension into ice cubes in a refrigerator at the temperature of-40 ℃, thawing, pouring into two 100ml plastic test tubes, and freeze-drying by a vacuum freeze dryer in a conventional operation to obtain powdery particles. The freeze-dried product is a uniform white solid, and the freeze-dried sample has good appearance and no atrophy and collapse. (its morphology is as in FIG. 4)
Third, crushing the semaglutin-diketopiperazine freeze-dried powder:
the above sub-packaged granules are slowly added into a pulverizer under the pressure of 2-6bar at 30-70% humidity to obtain pulverized product (compound 1-SMG). The crushed grain size is controlled to D50- (5-10 μm).
Example 3
The first step is that the semaglutin-diketopiperazine suspension is prepared:
1g of 3,3' - ((((2S, 5S) -3, 6-dioxopiperazine-2, 5-diyl) bis (butane-4, 1-diyl)) bis (azadiyl)) bis (carbonyl)) bis (bicyclo [1.1.1] pentane-1-carboxylic acid) (compound 1, scanning electron microscopy spectra as shown in FIGS. 1 and 2) were placed in a 100 ml single-necked flask, 23 ml of distilled water was added at room temperature, 25% by mass of aqueous ammonia was added, and the pH was 7.5-8. The particles were crushed by a spoon for 5 minutes and continued to be sonicated for 5 minutes, stirred for 4 hours, 15 ml distilled water was added, stirred for 20 minutes, 143 mg semaglutin (SMG, 98%) was added and stirred overnight at room temperature.
Secondly, preparing semaglutin-diketopiperazine freeze-dried powder:
The suspension is poured into a 100ml plastic test tube, and is freeze-dried by a vacuum freeze dryer in a conventional operation to obtain powdery particles. The freeze-dried product is a uniform white solid, and the freeze-dried sample has good appearance and no atrophy and collapse. (as in FIG. 4)
Third, crushing the semaglutin-diketopiperazine freeze-dried powder:
The subpackaged granules are slowly added into fine Sichuan Mikron powder mechanical Co., ltd under the pressure of 2-6bar at 30-70% humidity, and crushed by a screw jet mill with the model of 50AS to obtain a crushed product (compound 1-SMG), and the particle size is controlled to D50- (1-10 μm).
Effect example 1
SD rats were supplied by Shanghai Laike laboratory animal Limited with animal production license number SCXK (Shanghai) 2022.0004 and animal use license number SCXK (Shanghai) 2019-0027.
Rats are placed in a constant-temperature room with the temperature of 20-24 ℃ and the humidity of 40-70% and the illumination of 12 hours according to the species, fasted for 4 hours and water forbidden for 2 hours during the drug administration and blood collection period of the drug generation experimental animals. No water is forbidden during the period of drug efficacy test.
The administration and blood sampling modes for different groups are respectively as follows:
1. Compound 1-SMG dry powder nebulization transpulmonary administration group
The specific experimental operation steps are as follows, SD rats are anesthetized by chloral hydrate, the chloral hydrate solution is administered by intraperitoneal injection at a dose of 3mL/Kg, and after the SD rats are completely anesthetized, the SD rats are fixed on the inclined plane of the triangular flat plate and are 60 degrees away from the horizontal plane. Fixing SD rat, pulling out tongue of SD rat with forceps, observing trachea position with endoscope, inserting 18G floating needle cannula into trachea, loading 1-SMG dry powder (obtained in step 3 of example 1) of compound with set dose (6 mg/kg) at the tail of cannula, connecting 5mL syringe, compressing and purging 3 times with syringe, sucking dry powder into lung of SD rat, fixing SD rat for 5min after administration, removing fixing SD rat, and placing SD rat back into squirrel cage.
The orbits were bled at time points 0.083h, 0.25h, 0.5h, 1h, 2h, 3h, 6h, 9h, 24h, 32h and 48h, respectively, after dosing, and blood was collected in 1.5ml EDTA K2 anticoagulants. Whole blood was rapidly placed in a high speed centrifuge and centrifuged at 8000rpm for 10min at 4 ℃ and plasma was pipetted into a blank centrifuge tube and cryopreserved at-20 ℃.
2. Lung administration group atomized by aqueous solution of Semaglutin (SMG)
Reference compound 1-SMG dry powder nebulization pulmonary administration group.
3. Serraglutide (SMG) intravenous injection group
An appropriate amount of SMG powder was weighed, dissolved in 0.9% physiological saline, diluted to a drug concentration of 0.2mg/mL, and the drug solution was administered by intravenous injection at 1 mL/Kg.
Table 1 main pharmacokinetic parameters of semaglutin in plasma after semaglutin administration in SD rats
AUC (0-t) refers to the area under the blood concentration time curve from time 0 to the last time point selected;
AUC (0-inf) _obs represents the area under Area (AUC) of the observed Cumulative Distribution Function (CDF) over the interval 0 to positive infinity (0-inf);
V_obs represents a version object library;
cl_obs represents observations of drug effects or side effects in the trial;
MRT (0-t) represents the average residence time from the start of drug administration to time point t;
MRT (0-inf) _obs represents the observed period average residence time from the start of administration to infinite time.
F represents bioavailability.
The above pharmacokinetic data indicate that the compound 1-SMG dry powder has good bioavailability (16.02%).
Effect example 2
The respirable particles (FINE PARTICLE fraction, FPF) of the dry powder were evaluated using a new generation of pharmaceutical multistage impactors (Next Generation Pharmaceutical Impactor, NGI). And (3) dripping the prepared glycerol solution on the trays of each level to reduce the occurrence of bouncing and secondary entrainment of particles in the test.
The glycerol solution is prepared by dissolving 5g Tween 80 in 20mL ethanol, removing 1mL solution, adding 5g glycerol, and shaking.
The coating mode is that two drops are dropped on a first disk, one drop is dropped on a second, third, fourth, fifth, sixth and seventh disks respectively, the coating is uniformly carried out, a glass cellulose filter membrane is placed on a detection disk (MOC) disk, and the mixture is kept stand for 30min.
The throat is connected to the adapter and the inhalation device is connected to the adapter. The vacuum pump is opened, and the flow regulating valve is regulated to make the reading of the flowmeter connected to the L-shaped connecting pipe 80+/-4L/min. The drug powder was weighed into a destaticized capsule and Breezhaler capsules were placed in a Dry Powder Inhaler (DPI) inhalation device to begin the NGI assay.
After 3.0 seconds from the end of the measurement, the vacuum pump was turned off, and the suction was repeated 3 times. The device was disassembled, and the drugs of each level and the related parts were recovered with 10mmol/L phosphate buffer in such a manner that the inhalation device and the capsule shell were diluted with 25mL of solvent, the adapter was diluted with 25mL of solvent, the throat was diluted with 50mL of solvent, the 1 st, 2 nd, 3 rd, 4 th, 5 th plates were diluted with 25mL of solvent, the 6 th, 7 th plates were diluted with 10mL of solvent, the filter membrane of the MOC layer was sonicated with 10mL of solvent for 10min. Sample injection analysis was performed according to chromatographic conditions, data processing was performed using Copley INHALER TESTING DATA ANALYSIS Software (CITDAS) version 3.10, and FPF of inhalation powder was calculated.
Compound 1-SMG dry powder (prepared from example 3) was tested for NGI with an FPF of 43.65% to achieve the desired deposition site as shown in figure 5.
The following compounds S1-S4 were prepared by reference (CN 113527216A) and compound S1-SMG dry powder, compound S2-SMG dry powder, compound S3-SMG dry powder and compound S4-SMG dry powder were prepared, respectively, according to the procedure of example 3 above. The FPF of the test by NGI is shown in the following table.
TABLE 2
Effect example 3
The particle size of compound 1-SMG dry powder (prepared in example 1), compound S1-SMG dry powder, compound S2-SMG dry powder, compound S3-SMG dry powder, compound S4-SMG dry powder (using a laser particle sizer model H4605, HELOS| MYTOS Co.) was measured. Because the physical state of the drug particles has a large impact on the performance of Dry Powder Inhalants (DPIs), the aerodynamic diameter of the dry powder inhalant particles is generally preferably 1.0-5.0 μm. Studies show that atomized particles with a particle size of 1-4 μm can be effectively inhaled and deposited in bronchioles and alveoli of the lung.
The compound 1-SMG dry powder (prepared from example 3) had an inhaled mass powder particle size X50 of about 3 μm and was effectively inhaled and deposited in the bronchioles and alveoli of the lungs.
A dry powder of the compound FDKP-SMG was prepared according to the method of example 3 above. The particle size X50 of the powder of the FDKP-SMG dry powder inhalation material is about 8.8 mu m, and the particles are larger.
The powder particle size X50 of the dry powder inhalation material of the compound S1-SMG is about 22 mu m, and the particles are larger.
The powder particle size X50 of the dry powder inhalation material of the compound S2-SMG is about 5.5 mu m, and the particles are bigger.
The powder particle size X50 of the dry powder inhalation material of the compound S3-SMG is about 7.5 mu m, and the particles are large.
The powder particle size X50 of the dry powder inhalation material of the compound S4-SMG is about 5.1 mu m, and the particles are larger.