CN117510447A - Dapagliflozin-pioglitazone co-amorphous substance and preparation method and application thereof - Google Patents

Dapagliflozin-pioglitazone co-amorphous substance and preparation method and application thereof Download PDF

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CN117510447A
CN117510447A CN202311479792.0A CN202311479792A CN117510447A CN 117510447 A CN117510447 A CN 117510447A CN 202311479792 A CN202311479792 A CN 202311479792A CN 117510447 A CN117510447 A CN 117510447A
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pioglitazone
dapagliflozin
amorphous
solvent
dap
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王静
任赛赛
郝汉
郭伟
任甜甜
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Hebei Medical University
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Hebei Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/10—Oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Abstract

The invention relates to a dapagliflozin-pioglitazone co-amorphous substance, a preparation method and application thereof, wherein the molar ratio of dapagliflozin to pioglitazone in the dapagliflozin-pioglitazone co-amorphous substance is 1:2-2:1, and an X-ray powder diffraction spectrum of the dapagliflozin-pioglitazone co-amorphous substance does not have sharp diffraction peaks, so that the formation of a co-amorphous form is shown. The co-amorphous is obtained by utilizing a solvent-assisted grinding method and a rotary evaporation method, and the experiment proves that the co-amorphous has good solubility, dissolution and bioavailability, and the in-vivo and in-vitro hypoglycemic activity experiment proves that the co-amorphous is obviously superior to the activity of single application of dapagliflozin or pioglitazone, so that the dapagliflozin-pioglitazone in the co-amorphous supermolecular system has a synergistic hypoglycemic effect.

Description

Dapagliflozin-pioglitazone co-amorphous substance and preparation method and application thereof
Technical Field
The invention relates to the technical field of pharmacy, in particular to a dapagliflozin-pioglitazone co-amorphous substance, and a preparation method and application thereof.
Background
The clinical insulin sensitizer thiazolidinedione hypoglycemic agent pioglitazone is PPAR-gamma receptor agonist, belongs to insulin dependent medicine, and has curative effect reduced with the decrease of islet beta cell function in the progress of type II diabetes (T2 DM). At the same time, its increase in body weight further exacerbates insulin resistance. The selective SGLT-2 inhibitor dapagliflozin is a new antidiabetic drug, and mainly reduces the blood sugar of a T2DM patient by specifically inhibiting the reabsorption of proximal tubular glucose, and the blood sugar reducing mechanism is independent of insulin secretion state and continuously and stably controls the blood sugar, so that the pancreatic beta cells can be prevented from causing drug resistance and islet hypofunction due to excessive stimulation secretion, and the antidiabetic drug has the function of potentially protecting the beta cells. However, the long-term administration of dapagliflozin presents the risk of inducing recurrent urinary infections. In conclusion, clinical dapagliflozin and pioglitazone double-target combined administration has important significance for stably controlling blood sugar and reducing side effect risks in the later stage of disease.
The construction of the medicine-medicine supermolecule combines the supermolecule technology with the combined medicine, the single medicine administration can achieve the aim of the combined medicine, improves the bad physicochemical properties of each component, and importantly, the synergistic effect of the two-component supermolecule systems with different action targets on the molecular level has the advantages of further synergism and toxicity reduction, and promotes the penetration of the combined medicine technology and theory. The invention aims to explore a novel form (technical innovation) of combined medication by constructing a dapagliflozin-pioglitazone double-target supermolecule system and taking the improvement of the bioavailability of the dapagliflozin-pioglitazone double-target supermolecule system as a starting point; meanwhile, the synergistic action mechanism (theoretical innovation) of two components in a supermolecular system is deeply researched from the molecular level, and an innovative clinical intervention strategy is provided for treating type II diabetes.
Disclosure of Invention
The invention aims to provide DAP-pioglitazone co-amorphous substance (DAP-PIO) with good dissolution property and bioavailability, and simultaneously provides a preparation method and application thereof.
The invention provides a dapagliflozin-pioglitazone co-amorphous substance, which is characterized by comprising dapagliflozin and pioglitazone, wherein the molar ratio of dapagliflozin to pioglitazone is 1:2-2:1.
As a further improvement of the present invention, the co-amorphous material was irradiated with Cu-kα, and the X-ray powder diffraction pattern expressed in 2-theta showed a camelid halo, and the crystallization diffraction peak disappeared. Preliminary evidence of the formation of co-amorphous forms. TMDSC and TG analyses, as shown in FIG. 2, an endothermic step appears in the reversible heat flow signal curve of each proportion of samples prepared by DAP, which proves the formation of amorphous forms of each system of DAP-PIO.
As a further improvement of the present invention, the X-ray powder diffraction pattern is shown in FIG. 1-A.
As a further improvement of the present invention, the glass transition temperature of the co-amorphous material is 19.75 to 24.77 ℃.
As a further improvement of the present invention, the co-amorphous is prepared by a solvent-assisted milling method or a rotary evaporation method.
The invention also provides a preparation method of the dapagliflozin-pioglitazone co-amorphous substance.
Scheme one: adopts a solvent auxiliary grinding method: grinding dapagliflozin and pioglitazone, mixing, adding solvent, grinding, and drying.
As a further improvement of the invention, the solvent is selected from 100% ethanol, and the ratio of the solvent to the material (dapagliflozin and pioglitazone) is 0.3-1 mL:200mg; the grinding time of adding solvent is 30-40 min, and the grinding rotating speed is 1200-1500 rpm.
As a further improvement of the present invention, the molar ratio of dapagliflozin to pioglitazone is 2:1, respectively.
Scheme II: the rotary evaporation method is adopted: adding dapagliflozin and pioglitazone into a solvent, ultrasonically extracting a sample for 30-40 min at a solid-to-liquid ratio of 5-8 mg/mL, and performing rotary evaporation at 38+/-2 ℃ to obtain co-amorphous.
As a further improvement of the present invention, the solvent is selected from methanol, ethanol, a mixture of methanol and water, and the concentration of the mixture of ethanol and water is 50% or more.
As a further improvement of the invention, the molar ratio of the dapagliflozin to the pioglitazone is 1:1, 1:2 and 2:1 respectively.
In a further aspect, the invention provides application of the dapagliflozin-pioglitazone co-amorphous substance in preparation of medicines for treating diabetes and obesity.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the method takes dapagliflozin and pioglitazone as raw materials, and obtains the co-amorphous substance through a solvent-assisted grinding method and an ultrasonic rotary steaming method, and the experiment proves that the co-amorphous substance has good dissolution, solubility and bioavailability.
The co-amorphous form provided by the invention is proved by in vitro and in vivo activity tests for treating diabetes and obesity to be obviously superior to the activity of the two medicaments which are independently applied, so that the co-amorphous form can enhance the hypoglycemic activity of dapagliflozin and pioglitazone and reduce the weight.
Drawings
FIG. 1 is a graph depicting the characterization of the co-amorphous form of dapagliflozin and pioglitazone obtained in example 2 of the present invention: (A) XRD, (B) IR, (C) Raman characterization map;
FIG. 2 is a TMDSC-TG representation of the co-amorphous obtained in example 2 of the present invention;
FIG. 3 is a dissolution chart of test example 2 of the present invention: (a) DAP solubility, (B) PIO solubility, (C) powder PXRD after 72h at ph=1;
Note:*P<0.05(vs.DAP or PIO)；**P<0.01(vs.DAP or PIO), ★ P<0.05(vs.PM)and ★★ P<0.01(vs.PM)；
FIG. 4 is a dissolution profile of test example 3 of the present invention in 0.1mol/L hydrochloric acid medium (38deg.C, n=3);
fig. 5 is a drug-time curve (n=6) of experimental example 4 of the present invention in SD rats;
FIG. 6 is a graph showing the statistics of glucose elimination and glucose consumption rate of different concentration drugs in the cell insulin resistance model of test example 5 according to the present invention: H9C2 (FIGS. A-C) and HepG2 (FIGS. D-F);
FIG. 7 glucose uptake by H9C2 cell insulin resistance model supramolecular system (DAP-PIO) in test example 6 of the present invention (note: blue is DAPI; green is 2-NBDG);
FIG. 8 is a graph showing the effect of DAP-PIO co-amorphous on HFD-induced diabetic mice metabolic parameters and glucose homeostasis in test example 7 of the present invention;
FIG. 9 is a graph showing the effect of DAP-PIO co-amorphous on liver and heart lipid levels in HFD and HFD treated mice in accordance with test example 7 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be clearly and completely described in connection with the following specific embodiments.
EXAMPLE 1 preparation of dapagliflozin-pioglitazone co-amorphous
Precision weighing Dapagliflozin (DAP) 60mg and Pioglitazone (PIO) 28.83mg (molar ratio DAP/PIO 2: 1), placing in a 2mL grinding tube, adding two 5mm zirconia grinding balls, uniformly mixing, adding 100% ethanol (v/v) 0.2mL, keeping the sample in slurry form, grinding at 1200rpm for 30min, and suspending every 5min to cool the material. Grinding, and drying in a vacuum drying oven at 45 ℃ overnight to obtain dapagliflozin-pioglitazone co-amorphous.
EXAMPLE 2 Co-amorphous preparation of dapagliflozin-pioglitazone
Rotary evaporation method: 200.00mg of each physical mixture containing dapagliflozin and pioglitazone (the molar ratio is 1:2,1:1 and 2:1) is placed in a 100mL round-bottom flask, 40mL of ethanol is respectively added, the solution is completely dissolved by ultrasonic treatment at room temperature for 30min, the solution is subjected to rotary reaction at 38 ℃ for 20min (the rotating speed is 50 rpm), the solvent is evaporated under the same condition, and dapagliflozin-pioglitazone co-amorphous matters with different molar ratios are obtained by vacuum drying for standby.
Experimental example 1 structural characterization of Co-amorphous forms
In this test example, dapagliflozin-pioglitazone co-amorphous obtained in example 2 was used in different molar ratios and named COA-i (DAP: pio=1:1), COA-ii (DAP: pio=2:1), COA-iii (DAP: pio=1:2), and their corresponding physical mixtures were PM-i (DAP: pio=1:1), PM-ii (DAP: pio=2:1), PM-iii (DAP: pio=1:2).
(1) X-ray powder diffraction detection
The dapagliflozin, pioglitazone and the co-amorphous prepared in example 2 in different molar ratios were weighed on a table, and the X-ray diffraction patterns of the respective samples were determined. The scanning range is 5 to 40 degrees (2 theta), the step length is 0.02 degrees, the scanning speed is 10 degrees/min, and the PXRD map is shown in figure 1A.
From the graph, crystalline PIO has obvious characteristic diffraction peaks, and PXRD patterns of different proportions of the DAP-PIO supermolecular system show a camel-shaped halo without sharp characteristic peaks, and the crystalline diffraction peaks disappear, so that the formation of a co-amorphous form is initially shown, and the amorphous substance prepared in the example 1 is consistent with the dapagliflozin-pioglitazone 2:1 pattern prepared in the example 2.
(2) FT-IR and Raman detection
DAP, PIO and DAP-PIO co-set FT-IR spectra were measured on a Spectrum Two FT-IR (PerkinElmer Company, USA) using KBr pellet. About 1mg of DAP, PIO and co-amorphous powder were weighed separately, about 160mg of KBr was weighed separately, mixed in equal increments and ground thoroughly, and tableted. With blank KBr sheet as reference, the resolution is 4cm -1 Scanning range 4000 to 400cm -1 The total number of scans is 40 and the signals are cumulatively averaged.
DAP, PIO and co-amorphous were measured between 3500 and 400cm using an XploRA Plus Raman imaging spectrometer (Horiba France Sas, france) -1 Raman spectrum in the range. The excitation wavelength is 638nm; RTD time was 1s, scanned 3 times and averaged together.
The FT-IR spectrum is shown in FIG. 1B: the telescopic vibration of the DAP-PIO supermolecular system, which is attributed to C=O in the pioglitazone structure, is represented by 1744cm -1 Move to 1750cm -1 From 1694cm -1 Respectively move to 1696cm -1 ，1698cm -1 Moving to the high field. And the N-H telescopic vibration is from 3084cm -1 Respectively move to 3036cm -1 ，3034cm -1 Move to the low field. It is inferred that c=o and N-H in pioglitazone form hydrogen bonds with certain groups in the dapagliflozin structure. The structure of dapagliflozin is characterized in that the O-H telescopic vibration is 3378cm -1 Respectively move to 3386cm -1 ，3388cm -1 ，3422cm -1 Moving to the high field. Description of O-H participation in dapagliflozin in Co-amorphous supramoleculesThe formation of hydrogen bonds between the net and pioglitazone.
As shown in Raman spectrum of fig. 1C, PIO drug substance c=o is 1610cm -1 DAP has-OH of 873cm -1 . Overlapping peaks of DAP and PIO can be observed in the raman spectrum of the physical mixture. In contrast, the co-amorphous peaks all vary in intensity, width and position. Spectrum 2934cm in co-amorphous -1 、3067cm -1 The peak at this point is clearly broadened, indicating the formation of disordered structures. At the same time, the-OH oscillations of the DAP-PIO supermolecule were combined and transferred to 827cm -1 . These obvious variations and combinations also indicate that the co-amorphous is likely to be formed by intermolecular hydrogen bonding between-OH of DAP and c=o of PIO.
(2) DSC-TG detection
TMDSC analysis: about 4mg of DAP, PIO and DAP-PIO co-amorphous powder with different molar ratios are respectively precisely weighed in an aluminum crucible, and TMDSC spectrograms of all samples are measured by taking an empty crucible as a reference. Temperature: heating rate at 0-250 ℃): 2.000K/min, cycle: 60 seconds, amplitude 0.5K.
Thermogravimetric (TG) analysis: about 4mg of DAP, PIO and DAP-PIO co-amorphous powder were precisely weighed into an aluminum crucible, and the N2 flow rate was set: 40mL/min, temperature range: 40-900 ℃, and the temperature rising rate is as follows: 10K/min.
As shown in fig. 2A and 2B, an endothermic step appears in the reversible heat flow signal curves of the DAP preparation samples in each proportion, which proves the formation of amorphous forms of each system of DAP-PIO. Meanwhile, the Tg of the co-amorphous COA-II is higher than that of the single DAP amorphous, and the formation of the co-amorphous COA-II can be preliminarily judged to improve the stability of the DAP by comparing the values of the Tg.
As can be seen from fig. 2C-2F, in the co-amorphous TG profile, the DTG profile clearly shows only one thermal decomposition peak for pioglitazone, indicating that the decomposition process is one-step decomposition, and two thermal decomposition peaks for dapagliflozin, indicating that the decomposition process is two-step decomposition, and that there is a difference between the physical mixture and the co-amorphous, indicating that the physical mixture is only a single mixture of samples, whereas the co-amorphous is essentially altered.
Test example 2 solubility determination of dapagliflozin-pioglitazone
The equilibrium solubility of dapagliflozin-pioglitazone co-amorphous in ph=1, ph=1.2, ph=4, ph=6.8 was determined using the shake flask method. Adding excessive sample to be tested into shake flask containing 10mL medium, placing on air bath shake table, setting air bath shake table at constant temperature of 37+ -0.5deg.C, and measuring 150 r.min -1 Shaking until the concentrations of dapagliflozin and pioglitazone in the solution are not changed any more, namely the saturated solution. After 72h reach equilibrium, the suspension is sucked by a disposable syringe and filtered by a microporous filter membrane with the size of 0.22 mu m, and the equilibrium solubility of pioglitazone hydrochloride, dapagliflozin and co-amorphous substances (1:1; 2:1; 1:2) with different molar ratios is measured by a high performance liquid phase. The solid phase was collected, dried and measured for PXRD. All experiments were performed in triplicate.
The equilibrium solubility of DAP, PIO and DAP-PIO co-amorphous in PH (1, 1.2, 4, 6.8) medium is shown in fig. 3A and 3B, with overall decreasing drug solubility with increasing PH, but with significant differences in co-amorphous solubility over physical mixtures. After the equilibrium solubility measurement of ph=1, solid samples were collected and PXRD was performed, as shown in fig. 3-C, the co-amorphous COA-ii did not show a new diffraction peak, which indicates that the co-amorphous COA-ii was stable after repeated dissolution and precipitation in a solvent, and no pioglitazone free base was precipitated, thereby playing a role in preventing disproportionation of pioglitazone hydrochloride. At pH of 1, physical mixture of dapagliflozin and pioglitazone forms co-amorphous after 72h action, which increases solubility and facilitates absorption of drug.
Test example 3 dissolution rate measurement of dapagliflozin-pioglitazone
The dissolution rates of dapagliflozin, pioglitazone, physical mixtures, co-amorphous were determined on an RC-808D dissolution apparatus (Tianjin Tianda, inc.) using the second method of the fourth section of pharmacopoeia (Paddle method). 30mg of DAP, 28.83mg of PIO and 58.83mg of PM-I (comprising 30mg of DAP and 28.83mg of PIO) are precisely weighed, 58.83mg of COA-I (comprising 30mg of DAP and 28.83mg of PIO) are taken as dissolution media, 500mL of 0.1mol/L hydrochloric acid solution is used as dissolution media, the dissolution media are stirred at 100rpm at 37+/-0.5 ℃ and sampled at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300 and 360min, the dissolution media with the same volume and the same temperature are supplemented, and after the sample is filtered by a microporous filter membrane with 0.22 mu m, the concentration is measured by using a high performance liquid phase after proper dilution, and all the tests are performed in parallel three times.
As can be seen from FIG. 4, the overall dissolution rate of the drug after formation of co-amorphous forms is improved, and in particular, the dissolution rate of the drug after formation of co-amorphous COA-I and co-amorphous COA-III is significantly improved, which contributes to the improvement of the in vivo bioavailability of the drug. From the figure, it is clear that the dissolution of the physical mixture is improved, probably due to the interaction between the substances of DAP and PIO in the 0.1mol/L hydrochloric acid medium, and the dissolution of each other is increased.
Test example 4 pharmacokinetic assay of dapagliflozin-pioglitazone
Male SD rats 24 weighing about 250+ -25 g were selected as test animals (Experimental animal center, hebei province, SPF, license number: SCXK (Ji) 2022-001). Fasted, free drinking water was taken from 12h before the test to the end of the test. All animal facilities follow the requirements of the international laboratory animal administration review and certification association. Experimental animal feeding environment: the temperature is 22-24 ℃ and the relative humidity is 50+/-5%. And the light is irradiated for 12 hours and then is dark for 12 hours.
24 SD rats were randomly divided into 4 groups of 6 rats each, each group being orally administered DAP, PIO, PM-II and COA-II, and the rats were kept in an environment at a temperature of 22-24℃and a relative humidity of 50.+ -. 5%. The rats were weighed prior to the experiment and the dose (equivalent to 83mg/kg dapagliflozin, 80mg/kg pioglitazone; etc.) was calculated, fasted for 12 hours prior to dosing, free drinking water, single gastric administration, 0.3mL of blood was taken from the orbit at 5, 15, 30min, 1, 1.5, 2, 3, 5, 8, 12, 16, 24 hours, in an anticoagulant tube containing dry heparin, centrifuged at 3500rpm for 10min at 4 ℃, and the plasma supernatant was stored at-20 ℃. After the plasma sample is treated, the supernatant is precisely measured to 10 mu L, and the supernatant is injected into a liquid chromatograph to record a chromatogram. Taking blood taking time as an abscissa, taking the concentration of DAP and PIO in a plasma sample as an ordinate, drawing a medicine-time curve, calculating pharmacokinetic parameters such as AUC0-t, cmax and the like by using DAS2.0 software, and carrying out statistical analysis on the pharmacokinetic parameters by using SPSS. The final result was at a test level of 0.05, with statistical differences considered when p < 0.05.
The concentration-time curves of DAP and PIO in rat plasma are shown in fig. 5. AUC calculated using DAS2.0 software 0-t 、C max 、t 1/2 And T max See tables 1 and 2. For DAP, the co-amorphous material is compared to PM-II and amorphous DAP, C max About 1.52-fold and 3.09-fold increases, AUC, respectively 0-t The AUC is increased by 1.33 times and 3.06 times respectively 0-∞ Respectively improves by 1.28 times and 3.19 times, T max Advance by about 1.16h and 0.58h; the results demonstrate that DAP-PIO co-amorphization has higher bioavailability than DAP alone or both PM. For PIO, the co-amorphous form is compared to PM and PIO, C max No too great change in AUC 0-t ，AUC 0-∞ Slightly lower, but T max Advanced by about 1.17h and 0.58h, t 1/2α The half-life of the distribution is advanced by about 0.94h and 0.87h. t is t 1/2β The elimination half-life was delayed by about 5.88h and 2.33h, indicating that the drug distribution in vivo was faster and the elimination in vivo was slower following the PIO preparation as co-amorphous.
TABLE 1 pharmacokinetic mean parameters of DAP in rats after dosing
Table 4 Mean pharmacokinetic parameters of DAPin rats after administration
Note:ns＝no significance(vs.DAP or PM),*P<0.05(vs.DAP)；**P<0.01(vs.DAP), ★ P<0.05(vs.PM)and ★★ P<0.01(vs.PM)
TABLE 2 pharmacokinetic mean parameters of PIO in rats after dosing
Table 5 Mean pharmacokinetic parameters of PIOin rats after administration
Note:ns＝no significance(vs.PIO or PM),*P<0.05(vs.PIO)；**P<0.01(vs.PIO), ★ P<0.05(vs.PM)and ★★ P<0.01(vs.PM)
Test example 5 evaluation of in vitro cell level efficacy
Cell culture and treatment:
human HepG2 cells and H9c2 rat myocardial myoblasts were supplied from the university of Hebei medical college of Hebei (Hebei Shijia of China) and cultured in DMEM medium containing 25mM glucose supplemented with 10% FBS and 1% penicillin/streptomycin at 37℃and a humidity of 5% CO 2 . Cells were passaged using trypsin-EDTA. Cells were seeded into 24-well plates and after 60% confluence, the cells were maintained overnight in DMEM with normal glucose. Cells with 25mM DMEM were considered as control. Conditions of 30mM glucose plus 5. Mu.M insulin (HGHI) for 28 hours were used to mimic T2DM in vitro. Cells under HGHI conditions were treated in the absence or presence of different concentrations of DAP, PIO, PM and COA. For the mechanism study, PI3K inhibitor LY294002 and AMPK inhibitor compound C were pre-treated for 2 hours prior to HGHI incubation.
Glucose consumption rate determination:
conditions of 30mM glucose plus 5. Mu.M insulin (HGHI) for 28 hours were used to mimic T2DM in vitro. Cells under HGHI conditions were treated for 24 hours in the absence or presence of different concentrations of DAP, PIO, PM and COA. At the time point of 24 hours, the glucose concentration of each group of media was measured by a glucose measuring reagent (built in south Beijing, china). The glucose consumption rate was calculated by (glucose concentration of each group-glucose concentration of control group)/glucose concentration of control group 100%. From fig. 6, it is clear that the co-amorphous drug administration group had better glucose lowering effect than PM for H9C2 and HepG2 cells, and that the consumption of glucose in the cells was increased, indicating that the glucose utilization rate of the cells was increased, and preliminarily confirmed the co-amorphous synergistic glucose lowering effect. However, as the concentration of the drug increases, the consumption of glucose decreases to some extent, which means that the drug produces side effects as the concentration of the drug increases, so that the optimal therapeutic effect can be achieved only by selecting a drug with a proper concentration, and the effect is best when the concentration ratio is (10. Mu.M: 5. Mu.M) in the co-amorphous system COA-II and COA-III, and the subsequent blood glucose reducing effect study is correspondingly performed by selecting (10. Mu.M: 10. Mu.M) in the co-amorphous system COA-I.
Test example 6 in vitro cell level efficacy evaluation of insulin resistance glucose uptake assay of HepG2 and H9c2 cells
Glucose uptake assay in H9C2 cell insulin resistance model
Cells were cultured in DMEM medium containing 25mM glucose, supplemented with 10% fbs and 1% penicillin/streptomycin. trypsin-EDTA was used for cell passaging. Cells were seeded in 6-well plates and after 60% confluence, the medium was changed. T2DM was simulated in vitro using 30mM glucose plus 5. Mu.M insulin (HGHI) for 28 hours. Cells under HGHI conditions were incubated for 24 hours in the absence or presence of different drugs for glucose uptake assay experiments (n=3). 2-NBDG (fluorescent labeled glucose analog for detecting glucose uptake in cells). After the cells were treated and washed, 500. Mu.L of PBS containing 60. Mu.M 2-NBDG was added to each well, and the cells were further cultured for 30 minutes. After incubation, each well was washed twice with ice-cold PBS to stop glucose uptake and ensure no 2-NBDG remained. The fluorescence intensity of 2-NBDG was determined by confocal microscopy at 488nm excitation and 550nm emission wavelength. From fig. 7, it can be seen that the glucose uptake of the DAP-PIO co-amorphous supramolecular system is higher than the physical mixture and the pure drug uptake, demonstrating that the DAP-PIO co-amorphous supramolecular system improves insulin resistance of the cells.
Test example 7 in vivo improvement of HFD-induced diabetic mice hyperglycemia and insulin sensitivity by dapagliflozin-pioglitazone supermolecular system
Male C57BL/6J mice of 4 weeks old were obtained from St Bei Fu Biotechnology Inc. and placed in a room at 25℃and 50% relative humidity with 12 hours light/12 hours dark cycle, food and water were freely available. The normal control group was fed with a standard diet and the diabetes model group was fed with a high fat diet (HFD, research diet, D12492, 60% calories from fat). The establishment of T2DM and IR began with HFD feeding for two months. Mice with high body weight and hyperglycemia (fasting blood glucose. Gtoreq.11.1 mmol/L) were classified as diabetic.
DAP, PIO, PM and COA-i were orally administered daily by gavage after T2DM establishment and administration continued for 2 weeks (n=6 per group). Diabetic model groups and treated diabetic mice were fed with HFD and normal control groups were fed with normal diet.
Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT):
at the end of the experiment, the fasting blood glucose level of the mice was measured by an Accu-Chek Performa glucometer (Swiss Roche). For the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT), mice were fasted overnight for the last two weeks, respectively, and then either 1g/kg glucose was orally administered or 0.75U/kg insulin was intraperitoneally injected. Glucose baseline levels were then measured at 0, 15, 30, 60, 90, 120min and calculated by area under the curve (AUC). After mice were sacrificed, plasma samples and liver tissue were collected. Liver and heart indices of body weight, liver weight and heart weight were recorded.
Fasting glycemia (FBG) levels in HFD mice increased significantly (. Gtoreq.11.1 mmol/L) after 2 months of HFD feeding, indicating successful establishment of T2DM. Fasting glycemia (FBG) levels in HFD mice increased significantly (. Gtoreq.11.1 mmol/L) after 2 months of HFD feeding, indicating successful establishment of T2DM. HFD mice treated with dapagliflozin-pioglitazone supramolecular system improved systemic glucose tolerance and insulin sensitivity compared to control group as assessed by OGTT and ITT assays (fig. 8). The dapagliflozin-pioglitazone supramolecular system improved glucose tolerance impairment and systemic IR in HFD-induced diabetic mice, indicating its beneficial effects on glycemic control.
HFD causes swelling of hepatocytes, cell disorders, lipid accumulation, microvesicles and large bleb lipid deposition in the liver, resulting in an increase in liver volume. However, in the dapagliflozin-pioglitazone supramolecular system treatment group, an increase in liver weight and liver index was avoided (fig. 9A, fig. 9B). The heart of HFD mice is prone to diabetes-induced myocardial hypertrophy, resulting in significant increases in cardiac index, cardiomyocyte size, and left ventricular mass. Whereas treatment with dapagliflozin-pioglitazone supramolecular system significantly reduced the cardiac index of the diabetic heart (fig. 9C, fig. 9D).
Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The dapagliflozin-pioglitazone co-amorphous substance is characterized by comprising dapagliflozin and pioglitazone, and the molar ratio of dapagliflozin to pioglitazone is 1:2-2:1.
2. A dapagliflozin-pioglitazone co-amorphous according to claim 1, wherein said co-amorphous uses Cu-ka radiation and has no sharp diffraction peaks in the X-ray powder diffraction spectrum expressed in 2Θ.
3. The dapagliflozin-pioglitazone co-amorphous material according to claim 1, wherein the glass transition temperature of the co-amorphous material is 19.75-24.77 ℃.
4. The dapagliflozin-pioglitazone co-amorphous product of claim 1, wherein the co-amorphous product is prepared by a solvent-assisted milling method or a rotary evaporation method.
5. A process for the preparation of dapagliflozin-pioglitazone co-amorphous material as claimed in any one of claims 1 to 4, wherein solvent assisted milling is employed: grinding dapagliflozin and pioglitazone, mixing, adding solvent, grinding, and drying;
the solvent is selected from 100% ethanol, and the ratio of the solvent to the dapagliflozin and pioglitazone is 0.3-1 mL/200 mg; the grinding time of the added solvent is 30-40 min, and the grinding rotating speed is 1200-1500 rpm.
6. The method for preparing dapagliflozin-pioglitazone co-amorphous according to claim 5, wherein the molar ratio of dapagliflozin to pioglitazone is 2:1.
7. A process for the preparation of dapagliflozin-pioglitazone co-amorphous material as claimed in any one of claims 1 to 4, wherein rotary evaporation is used: adding dapagliflozin and pioglitazone into a solvent, ultrasonically extracting a sample for 30-40 min at a solid-to-liquid ratio of 5-8 mg/mL, and performing rotary evaporation at 38+/-2 ℃ to obtain co-amorphous.
8. The method for preparing dapagliflozin-pioglitazone co-amorphous substance as claimed in claim 7, wherein the solvent is selected from methanol, ethanol, a mixture of methanol and water, and the concentration of the mixture of ethanol and water is greater than or equal to 50%.
9. The method for preparing dapagliflozin-pioglitazone co-amorphous according to claim 7, wherein the molar ratio of dapagliflozin to pioglitazone is 1:1, 1:2 and 2:1 respectively.
10. Use of dapagliflozin-pioglitazone co-amorphous material as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of diabetes and obesity.
CN202311479792.0A 2023-11-08 2023-11-08 Dapagliflozin-pioglitazone co-amorphous substance and preparation method and application thereof Pending CN117510447A (en)

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