Dipyridamole nanosuspension, preparation method of dipyridamole nanosuspension and dipyridamole redissolved suspension

Abstract

The invention relates to the technical field of pharmaceutical combination preparations, in particular to a dipyridamole nanosuspension, a preparation method of the dipyridamole nanosuspension and a dipyridamole redissolved suspension. The dipyridamole dry nanosuspension is prepared from the following raw materials: a crystallization inhibitor and dipyridamole, and the mass ratio of the crystallization inhibitor to the dipyridamole is (1 to 10): 1. The dipyridamole nanosuspension disclosed by the invention has relatively good solubility in water, and has relatively excellent bioavailability and patient adaptability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical combination formulation technology, and more specifically, to dipyridamole nanosuspension, its preparation method, and dipyridamole reconstituted suspension. Background Technology

[0002] Dipyridamole (DIP) is a platelet phosphodiesterase inhibitor, and its structural formula is shown below:

[0003]

[0004] It has the potential to treat a variety of diseases. For example, it can alleviate or treat Covid-19-mediated symptoms. Specifically, it possesses anti-SARS-CoV-2, anti-inflammatory, and antiplatelet properties, which can directly alleviate or treat Covid-19-mediated symptoms; it can also indirectly alleviate or treat Covid-19-mediated symptoms by increasing extracellular adenosine levels, thereby exerting anti-inflammatory and immunomodulatory effects. In particular, it can be used as an adjunct to coumarin anticoagulants in preventing thromboembolic complications after heart valve replacement surgery, and was approved by the US Food and Drug Administration (FDA) in 1961. Recent research results show that platelets are activated in the colonic mucosa of children with colitis or inflammatory bowel disease (IBD). In a preliminary study, DIP promoted mucosal healing in nine children with colitis. Therefore, DIP has great potential for clinical application.

[0005] However, DIP is soluble in dilute acids, methanol, and chloroform, but almost insoluble in water and intestinal fluid, resulting in low bioavailability. Furthermore, commercially available products are not suitable for pediatric use, which significantly limits its practical application.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide dipyridamole nanosuspension, its preparation method, and a dipyridamole reconstituted suspension. The dipyridamole nanosuspension provided in this invention exhibits good solubility in water, as well as excellent bioavailability and patient suitability.

[0008] This invention is implemented as follows:

[0009] In a first aspect, the present invention provides a dipyridamole dry nano-suspension, the raw materials of which include a crystallization inhibitor and dipyridamole, wherein the mass ratio of the crystallization inhibitor to the dipyridamole is (1-10):1.

[0010] In an optional embodiment, the mass ratio of the crystallization inhibitor to the dipyridamole is (1-8):1; preferably (1.2-5):1.

[0011] Preferably, the crystallization inhibitor is selected from polymers.

[0012] Preferably, the crystallization inhibitor is selected from cellulose polymers, ethylene polymers, and ethylene glycol polymers;

[0013] More preferably, it is at least one of polyvinylpyrrolidone, vinylpyrrolidone / vinyl acetate copolymer, polyvinyl alcohol, polyethylene glycol, cellulose ethers, polyacrylic acid polymers, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, Gelucire 44 / 14, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer;

[0014] The preferred option is a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

[0015] In an optional embodiment, the raw material further includes a surfactant; the mass ratio of the surfactant to the dipyridamole is (0.1-1):1;

[0016] The preferred ratio is (0.2-0.8):1, and the more preferred ratio is (0.3-0.5):1.

[0017] In an optional embodiment, the surfactant comprises at least one of sodium dodecyl sulfate, calcium stearate, triethanolamine stearate, magnesium lauryl sulfate, sodium octadecyl fumarate, sodium taurocholate, sodium ursodeoxycholate, lecithin, sodium soybean lecithin deoxycholate, alkylbenzene sulfonate, benzalkonium chloride, benzalkonium bromide, poloxamer, Tween, Span, benzyl sulfadiazine, methyl sulfadiazine, polyoxyethylene castor oil, and polyethylene glycol ester.

[0018] Preferably, the surfactant comprises at least one of sodium dodecyl sulfate and Tween;

[0019] Further preferred is Tween, and most preferred is Tween 80.

[0020] In an optional embodiment, the raw materials include a freeze-drying protectant, wherein the mass ratio of the freeze-drying protectant to the dipyridamole is (1-5):1.

[0021] In an optional embodiment, the freeze-drying protectant is selected from polyols, preferably C3-C10 polyols, and more preferably mannitol.

[0022] In an optional embodiment, the raw materials include Tween, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and dipyridamole, wherein the mass ratio of Tween, the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and dipyridamole is (0.3-0.5):(1.2-3):1;

[0023] Preferably, the Tween includes Tween 80.

[0024] Secondly, the present invention provides a method for preparing dipyridamole dry nano-suspension, comprising: mixing and wet-grinding a crystallization inhibitor and dipyridamole and then freeze-drying.

[0025] In an optional implementation, the conditions for wet grinding include: a rotation speed of 400-600 rpm; a duration of 8-12 minutes, an interval of 3-7 minutes, and a total running time of 1-1.5 hours.

[0026] In an optional embodiment, the freeze-drying conditions include: a freeze-drying temperature of -60 to -80°C, a pressure of 0.2 to 0.4 mbar, and a time of 70 to 80 hours;

[0027] Preferably, pre-freezing is performed before freeze-drying;

[0028] Preferably, the pre-freezing conditions include a temperature of -60 to -80°C and a time of 10 to 15 hours.

[0029] Thirdly, the present invention provides a dipyridamole reconstituted suspension, which includes a reconstituted solvent and the dipyridamole dry nano-suspension described in the foregoing embodiments.

[0030] The present invention has the following beneficial effects: The embodiments of the present invention specifically use Tween, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer to form a dry nano-suspension with dipyridamole. This new improved dosage form gives dipyridamole excellent solubility, bioavailability and patient adaptability, thus expanding the scope of use of dipyridamole. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The detection result diagram provided in Test Example 1 of the present invention;

[0033] Figure 2-3The detection result diagram provided in Test Example 2 of the present invention;

[0034] Figure 4 The detection result diagram provided in Test Example 3 of the present invention;

[0035] Figure 5 This is a test result diagram provided for Test Example 4 of the present invention;

[0036] Figure 6 The detection result diagram provided in Test Example 5 of the present invention;

[0037] Figure 7 The image shows the detection results of the dissolution experiment provided in Test Example 6 of this invention;

[0038] Figure 8 The image shows the detection results of dynamic light scattering provided in Test Example 6 of the present invention;

[0039] Figure 9 The image shows the detection results of powder X-ray diffraction provided in Test Example 6 of the present invention;

[0040] Figure 10 The image shows the detection results of a field emission scanning electron microscope provided in Test Example 6 of this invention;

[0041] Figure 11 A graph showing the experimental results of an in vivo study provided in Test Example 6 of the present invention;

[0042] Figure 12 The simulation results of PBPK provided in Test Example 6 of this invention are shown in the figure.

[0043] Figure 13 The graph shows the results of simulated plasma drug concentrations in a pediatric population under dose-escalation conditions, as provided in Test Example 6 of this invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0045] In a first aspect, the present invention provides a dipyridamole dry nano-suspension, the raw materials of which include a crystallization inhibitor and dipyridamole, wherein the mass ratio of the crystallization inhibitor to the dipyridamole is (1-10):1. For example, it is any value or a range between any two values ​​of (1-10):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1, etc., preferably (1-8):1; more preferably (1.2-5):1.

[0046] Specifically, the crystallization inhibitor is selected from polymers, preferably from cellulose polymers, ethylene polymers, and ethylene glycol polymers; for example, including but not limited to at least one of polyvinylpyrrolidone, vinylpyrrolidone / vinyl acetate copolymer, polyvinyl alcohol, polyethylene glycol, cellulose ethers, polyacrylic acid polymers, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, Gelucire 44 / 14, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer; most preferably, it is a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

[0047] The raw materials also include a surfactant; the mass ratio of the surfactant to the dipyridamole is (0.1-1):1. For example, it can be any value or range between any two values ​​between (0.1-1):1, such as 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, and 1:1. For example, it is preferably (0.2-0.8):1, and more preferably (0.3-0.5):1.

[0048] The surfactant includes, but is not limited to, at least one of sodium dodecyl sulfate, calcium stearate, triethanolamine stearate, magnesium lauryl sulfate, sodium octadecyl fumarate, sodium taurocholate, sodium ursodeoxycholate, lecithin, sodium soybean lecithin deoxycholate, alkylbenzene sulfonate, benzalkonium chloride, benzalkonium bromide, poloxamer, Tween, Span, benzyl ether, meropenem, polyoxyethylene castor oil, and polyethylene glycol ester; preferably, the surfactant includes at least one of sodium dodecyl sulfate and Tween; more preferably, Tween, for example, including but not limited to Tween 80.

[0049] Furthermore, the raw materials include a freeze-drying protectant, wherein the mass ratio of the freeze-drying protectant to the dipyridamole is (1-5):1. For example, it can be any value between (1-5):1 or any range between any two values, such as 1:1, 2:1, 3:1, 4:1, and 5:1.

[0050] Furthermore, the freeze-drying protectant is selected from polyols, preferably C3-C10 polyols, and more preferably mannitol.

[0051] Using mannitol will not decrease solubility, but it will not increase it either. Therefore, the preferred cost-effective solution does not include lyophilization protectants. If cost is not a concern, or if it is to increase sweetness, mannitol can be added. However, using other lyophilization protectants, such as lactic acid, may lead to decreased solubility; therefore, lyophilization protectants should not be added arbitrarily.

[0052] Specifically, the raw materials for forming dipyridamole dry nano-suspension include Tween, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and dipyridamole, wherein the mass ratio of Tween, the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and dipyridamole is (0.3-0.5):(1.2-3):1; preferably, the Tween includes Tween 80.

[0053] In a second aspect, the present invention provides a method for preparing dipyridamole dry nano-suspension, comprising: mixing raw materials (e.g., crystallization inhibitor, surfactant, water and dipyridamole) by wet grinding and then freeze-drying.

[0054] Specifically, the crystallization inhibitor, dipyridamole, and water are mixed, wherein the concentration of the crystallization inhibitor in the water is 50-80 mg / ml. For example, any value between 50-80 mg / ml, such as 50 mg / ml, 60 mg / ml, 70 mg / ml, and 80 mg / ml.

[0055] It should be noted that although the unit for the concentration of polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer in water is specified as "mg / ml" in the embodiments of the present invention, it is understood that different units can be converted according to the proportion, such as mg / l, g / l and kg / l.

[0056] Next, after wet grinding, the material is pre-frozen, and then freeze-dried.

[0057] The conditions for wet grinding include: a rotation speed of 400-600 rpm; a duration of 8-12 minutes, with an interval of 3-7 minutes, and a total running time of 1-1.5 hours. The conditions for pre-freezing include: a temperature of -60 to -80℃ and a time of 10-15 hours. The conditions for freeze-drying include: a freeze-drying temperature of -60 to -80℃, a pressure of 0.2-0.4 mbar, and a time of 70-80 hours.

[0058] Thirdly, the present invention provides a dipyridamole reconstituted suspension, comprising a reconstituted solvent and the dipyridamole dry nano-suspension described in the foregoing embodiments. The reconstituted solvent may, for example, be deionized water.

[0059] Fourthly, the present invention provides a method for preparing a dipyridamole reconstituted suspension, comprising: mixing and reconstituted a reconstituted solvent and the dipyridamole dry nano-suspension described in the foregoing embodiments.

[0060] This invention also provides an optimal method for screening crystallization inhibitors, including screening using molecular dynamics simulation technology.

[0061] Molecular dynamics (MD) simulations are widely used in drug discovery and development. This technique is typically used to simulate molecular interactions, enabling tasks such as screening potential drug candidates and explaining formulation mechanisms at the molecular level. Commonly used molecular dynamics simulation software tools include Gromacs, Amber, and Materials Studio. All-atom molecular dynamics models involve interacting particles, represented by atoms, which simultaneously represent solute and solvent. These atoms are placed in a sufficiently large simulation chamber, and their motion follows Newton's laws of motion. Commonly used algorithms, such as velocity-Verlet or leap-frog, are used to describe the dynamic behavior of the system. Therefore, molecular dynamics is a classic tool for gaining in-depth understanding of entire systems.

[0062] Specifically, the active ingredient (e.g., dipyridamole), the crystallization inhibitors to be screened (e.g., HPMC, P188, Soluplus, and Gelucire44 / 14), and the surfactant (e.g., Tween) and their proportions are determined.

[0063] Molecular topology parameters were determined: MD simulations employed a universal Amber force field (GAFF). Both the active ingredient and the crystallization inhibitors to be screened were optimized using Gaussian 16 at B3YLP / 6-311G** level. The active ingredient model was then generated using sobtop software. The topology files for the crystallization inhibitors to be screened were developed based on monomer units using the ztop software package. The RESP charge of all molecules was calculated using Multiwfn software.

[0064] All-atom molecular dynamics simulations were performed on several different systems using GROMACS 2021. Specifically, NPT simulations were performed on each pure polymer chain in a vacuum chamber to reduce the simulation size by causing structural collapse. Then, compounds were randomly inserted into the initial chamber according to their mass ratio, and the chamber was filled with SPC water model solvent. The maximum energy was reduced to 100 kJ / mol using the steepest descent method and the conjugate gradient energy minimization method. -1 nm -1 The following steps were performed. Subsequently, NPT pre-equilibration was conducted using a Nosé-Hoover thermostat and Berendsen isotropic pressure coupling. During production, 100 ns NPT simulations were performed at 310 K using a v-rescale thermostat and a Parrinello-Rahman barostat, and the results were analyzed for this trajectory segment. MD simulation results were displayed using VMD visualization software. The optimal crystal inhibitor was determined.

[0065] Next, dissolution experiments were conducted on the suspension formulation containing the crystallization inhibitor to evaluate the effect of the crystallization inhibitor and determine that the optimal crystallization inhibitor screened by molecular dynamics simulation technology has the best dissolution effect.

[0066] The dry nano-suspension was prepared using wet grinding and freeze-drying techniques.

[0067] The optimal formulation of dry nanosuspensions for crystal inhibitor formation was evaluated in different dissolution media. The characterization techniques used included, but were not limited to, dynamic light scattering (DLS), powder X-ray diffraction (PXRD), and field emission scanning electron microscopy (FESEM).

[0068] Next, cell and animal experiments were conducted to evaluate the in vivo performance of the dry nanosuspension formulation with the optimal crystal inhibitor formation. A physiologically based pharmacokinetic (PBPK) model was also employed to predict in vivo behavior in adults and children.

[0069] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0070] Example 1

[0071] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: Tween 80 50mg, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 250mg, dipyridamole 200mg and water 5ml.

[0072] This embodiment provides a method for preparing dipyridamole nanosuspension, including:

[0073] After mixing the above raw materials, wet grinding was performed. The parameters for the wet grinding process were set as follows: 500 rpm for 10 minutes, followed by a 5-minute interval, and a total running time of 1 hour.

[0074] Next, the sample was frozen in a freezer at -80°C for 12 hours; then, it was placed in a freeze dryer, with the temperature maintained at -80°C and the pressure stabilized at 0.3 mBar, and freeze-dried for 72 hours.

[0075] Example 2

[0076] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: Tween 80 50mg, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 400mg, dipyridamole 200mg and water 5ml.

[0077] The preparation method is the same as in Example 1.

[0078] Example 3

[0079] This invention provides a dipyridamole nano-suspension with the following raw material composition: Tween 80 50mg, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 250mg, dipyridamole 200mg, water 5ml and mannitol 500mg.

[0080] The preparation method is the same as in Example 1.

[0081] Example 4

[0082] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: 200 mg Tween 80, 200 mg polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, 200 mg dipyridamole, and 5 ml water.

[0083] Example 5

[0084] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: Tween 80 20mg, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 200mg, dipyridamole 200mg and water 5ml.

[0085] Example 6

[0086] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: Tween 80 200mg, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 2000mg, dipyridamole 200mg and water 25ml.

[0087] Example 7

[0088] This invention provides a dipyridamole nano-suspension, the raw material composition of which is as follows: Tween 80 20mg, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer 2000mg, dipyridamole 200mg and water 25ml.

[0089] Test 1

[0090] Methods: The equilibrium solubility test was conducted in a water bath shaker at 37°C. Excess dipyridamole was added to three different media: water, acetate buffer (pH 4.5), and phosphate buffer (pH 6.8). Three samples were prepared in parallel for each medium. After shaking for 72 hours, the absorbance was measured at 283 nm using a UV-Vis spectrophotometer to calculate the concentration of dipyridamole (DIP).

[0091] See results Figure 1 ,according to Figure 1 It was found that DIP had significantly higher solubility in acetate buffer at pH 4.5 compared to water and phosphate buffer at pH 6.8. These findings indicate that DIP solubility varies with pH, ​​suggesting that it may dissolve in gastric fluid but crystallize and precipitate in intestinal fluid, indicating that its absorption in intestinal fluid may be limited.

[0092] Test 2

[0093] Methods: Polymers from HPMC, P188, Soluplus, and Gelucire44 / 144 were selected for simulation. The mass ratio of dipyridamole, polymer, and Tween 80 was 4:5:1.

[0094] MD simulations employed a universal Amber force field (GAFF). The dipyridamole molecule and all polymer monomer units were optimized using Gaussian 16 at the B3YLP / 6-311G** level. The dipyridamole model was then generated using sobtop software. Topology files for HPMC, P188, Soluplus, Gelucire44 / 14, and Tween 80 were developed based on the monomer units using the ztop software package. The RESP charge of all molecules was calculated using Multiwfn software.

[0095] As shown in Table 1, all-atom molecular dynamics simulations were performed on five different systems using GROMACS 2021. First, NPT simulations were performed on each pure polymer chain in a vacuum chamber to cause structural collapse and reduce the simulation size. Then, the compounds were randomly inserted into the initial chamber according to their mass ratio, and the chamber was filled with SPC water model solvent. The maximum energy was reduced to 100 kJ / mol using the steepest descent method and the conjugate gradient energy minimization method. -1 nm -1 The following steps were performed. Subsequently, NPT pre-equilibration was conducted using a Nosé-Hoover thermostat and Berendsen isotropic pressure coupling. During production, 100 ns NPT simulations were performed at 310 K using a v-rescale thermostat and a Parrinello-Rahman barostat, and the results were analyzed for this trajectory segment. The MD simulation results were displayed using VMD visualization software.

[0096] Table 1 Initial System Composition

[0097]

[0098] See results Figure 2 and Figure 3 . Figure 2 In Figure A, we have the self-assembly state of pure DIP in water. Figure B shows the root mean square deviation (RMSD) of the four formulations and pure DIP over a period of 100 ns. Figure C shows the aggregation state of the four formulations, where gray represents Tween 80, yellow represents Gelucire 44 / 14, red represents P188, purple represents HPMC, and blue represents Soluplus. Figure D shows the distribution of DIP after polymer removal. Figure E shows the vector of possible aggregation sites. Figure F shows the possible clusters of aggregates in the four formulations, with each bead representing one DIP molecule. Blue circles indicate clusters of three molecules.

[0099] The optimal crystallization inhibitor effectively prevents the recrystallization of the active pharmaceutical ingredient (API). Therefore, the degree of influence of the polymer on the self-assembly of the pure drug is a comparative indicator. Generally, the greater the influence, the stronger the ability of the crystallization inhibitor. To quantify this influence, the distance between the geometric centers of adjacent drug molecules and the angle between the perpendicular rigid ring vectors are introduced. Figure 2 (E). Subsequently, the distance and angle differences between the pure drug and the formulation self-assembly were compared to assess the polymer's ability to disrupt crystals.

[0100] according to Figure 2 As shown in section A, due to the interaction (π-π stacking) between parallel aromatic rings, dipyridamole molecules were observed to form clusters. Two characteristics were measured as references: the distance between the geometric centers of adjacent molecules and the angle between the perpendicular rigid ring vectors. During the self-assembly of pure dipyridamole, the average values ​​of the distance and angle were... 12° and 12° are set as critical values. If the test value in the polymer-formed formulation is below the threshold, these drug molecules are considered self-assembled drugs.

[0101] All systems ( Figure 2 The root mean square deviation (RMSD) of B) did not show significant fluctuations after 40 ns, indicating that its aggregate conformation is relatively stable. Figure 2 Figure C shows the aggregation states of the four test formulations. After removing the polymer molecules, the state of the dipyridamole molecules is as follows: Figure 2As shown in Figure D, this figure clearly illustrates the self-assembly trends of the P188 and HPMC systems. After comparison with reference values, molecules meeting the threshold in the self-assembly system are considered potential clusters. This indicates that P188 and HPMC have poorer crystallization inhibition effects than Soluplus, and their DIP formulations have poorer dissolution effects than the Soluplus formulation.

[0102] Figure 2 The graph in Figure F shows the possible clusters in all systems, with each drug molecule represented by a bead. Regarding the number of molecules in each cluster, only the Gelucire 44 / 14 and HPMC systems contain a cluster of three molecules, marked with a blue dashed circle. Meanwhile, Soluplus and Gelucire44 / 14 have the fewest clusters, with only three pairs. Generally, the number of clusters and the number of molecules within each cluster are inversely proportional to the ability to inhibit crystallization. Therefore, based on the above analysis, it can be concluded that Soluplus, provided by the embodiments of this invention, exhibits optimal performance in inhibiting drug crystallization.

[0103] Figure 3 In this diagram, A represents the intermolecular energy between the DIP molecule and the polymer. B represents the number of hydrogen bonds between the DIP molecule and the polymer.

[0104] Figure 3 Figure A shows the intermolecular energy between dipyridamole molecules and the polymer. The strength decreases in the following order: Soluplus > P188 > HPMC > Gelucire44 / 14. Furthermore, the number of hydrogen bonds decreases in the following order: Gelucire44 / 14 > Soluplus > P188 > HPMC. Based on the above analysis, the interaction between Soluplus and dipyridamole is the strongest, further illustrating that Soluplus provided in this embodiment of the invention is the optimal crystallization inhibitor, while the other three crystallization inhibitors are less effective.

[0105] Test Example 3

[0106] A suspension was prepared according to the formulation shown in Table 2, and then a dissolution test was performed. 1 mL of the suspension was dissolved in 1 mL of HCl solution to form a clear solution. This solution was then added to 200 mL of phosphate buffer solution with a pH of 6.8. The mixture was stirred at 200 rpm on a magnetic stirrer, and the temperature was maintained at 37°C. The absorbance was measured at 283 nm using UV spectrophotometry to calculate the DIP concentration.

[0107] Table 2 Formula

[0108]

[0109] A suspension was prepared according to the formulation shown in Table 3, and then a dissolution experiment was conducted.

[0110] Table 3. Soluplus formulations with different dosages

[0111]

[0112] See results Figure 4 ,according to Figure 4 As can be seen from A, Soluplus has significant advantages over other polymers (including various models of HPMC, P188, and Gelucire). According to... Figure 4 As can be seen from B, the 50mg / ml Soluplus and 80mg / ml Soluplus formulations have similar advantages compared to the 20mg / ml Soluplus formulation.

[0113] Test Example 4

[0114] A suspension was formed according to the formulation shown in Table 4, and a dry nano-suspension was formed according to the preparation method of Example 1. Dissolution tests were then performed, and the results are shown below. Figure 5 .

[0115] Table 4 Formulations of different lyophilization protectants

[0116]

[0117] according to Figure 5 It was observed that after 10 minutes, the lactose-containing formulation (F11) began to precipitate, and its solubility decreased accordingly. In the 60-minute dissolution test, the solubility of the formulation containing only Soluplus (F10) and the formulation with added mannitol (F12) was comparable. Considering the principle of minimizing the required amount of excipients, F10, containing only Soluplus, was selected as the optimal formulation.

[0118] Test Example 5

[0119] The stability of the nanosuspension formed by wet milling in Example 1 was tested. The physical stability of the nanosuspension was evaluated by sedimentation volume ratio (F), resuspending properties, and particle size distribution.

[0120] Because nanosuspensions are thermodynamically unstable, their stability must be assessed before any curing process (e.g., lyophilization). Particle size distribution, including average particle size and polydispersity index, was determined by dynamic light scattering. The nanosuspensions were then stored at room temperature, and samples were analyzed after one week. Sedimentation volume ratio and resuspendability were also measured.

[0121] The ratio F represents the relationship between the final settling volume and the initial volume, and can be used as an indicator of the overall aggregation-flocculation state of the system.

[0122] F = Vi / V f

[0123] Vi represents the initial volume, and Vf represents the final settling volume.

[0124] The calculated ratio F is 1, indicating that the nano-suspension has high suspension properties.

[0125] Resusceptibility refers to the ability of particles to be resuspended or redispersed after settling or precipitation. An optimal suspension should be able to resuspend within a short time. Measurements of a nano-suspension stored for one week showed that it could be resuspended within 15 seconds, and no significant precipitation was observed. This indicates that the suspension can be easily redispersed.

[0126] See results Figure 6 The particle size distribution measurements showed that after one week of storage at room temperature, the average particle size increased slightly, while the polydispersity index decreased slightly. These changes may be attributed to Oswald ripening.

[0127] Based on the above results, the DIP nano-suspension after wet milling exhibited satisfactory physical stability within one week, ensuring quality assurance before freeze-drying.

[0128] Test Example 6

[0129] The dipyridamole nanosuspension of Example 1 was characterized or tested as follows:

[0130] (1) Dissolution test:

[0131] The optimal dipyridamole nanosuspension (hereinafter also referred to as OF) prepared in Example 1 and the raw material DIP were dissolved and tested in different media: water, acetate buffer at pH 4.5, and phosphate buffer at pH 6.8. Dissolution tests were conducted using a paddle method on 100 mg DIP and an equivalent formulation. The test medium volume was 900 mL, the temperature was 37 °C, and the rotation speed was 50 rpm.

[0132] See results Figure 7 Where A represents the dissolution curves of OF and DIP in acetate buffer at pH 4.5; B represents the dissolution curves of OF and DIP in water; and C represents the dissolution curves of OF and DIP in phosphate buffer at pH 6.8.

[0133] exist Figure 7 In the pH 4.5 acetate buffer (represented by A), OF dissolves to approximately 100% within 10 minutes, while DIP dissolves to only about 80% after 60 minutes. In water (… Figure 7After 1 hour, the dissolution rate of OF was approximately 60%, about three times that of DIP. Both showed low dissolution rates in phosphate buffer at pH 6.8, especially DIP, which dissolved only about 4.5% within 60 minutes. In contrast, OF's dissolution rate was approximately 36%, indicating that OF increased the dissolution rate of DIP by about 8 times (e.g., ☐ ... Figure 7 (As shown in C).

[0134] (2) Dynamic light scattering:

[0135] The dry nano-suspension equivalent to 4 mg DIP from Example 1 was diluted with water to 4 mL, and then 0.25 mL of the suspension was further diluted with water to 4 mL. Sufficient sample was added to a cuvette. Three experiments were conducted, 14 times each, over a 60-second equilibration interval. The polydispersity index and average particle size were determined.

[0136] In addition, a dissolution test was conducted to investigate the particle size distribution of the formulation in intestinal fluid. Similar to the screening process for crystallization inhibitors, the dry nano-suspension of Example 1 was dissolved in 0.1M HCl solution and then added to PBS solution at pH 6.8 to simulate the process of the drug in the gastrointestinal tract. Appropriate samples were taken at 0.5 hours, 1 hour, 2 hours and 4 hours, respectively, and the particle size was determined using a Malvern Zetasizer Nano ZSP system (Malvern, UK).

[0137] See results Figure 8 ,in Figure 8 In this context, A represents the average particle size, and B represents the particle size distribution index. According to... Figure 8 It can be seen that the particle size of the reconstituted suspension (463.6 nm) is similar to that of the suspension before freeze-drying (434.2 nm). Furthermore, the polydispersity index of the reconstituted suspension (0.466) is lower than that of the suspension before freeze-drying (0.581). This indicates that the particle size distribution of the reconstituted nano-suspension after drying is comparable to that of the original nano-suspension.

[0138] The dissolved sample solution was analyzed using dynamic light scattering (DLS) over a 4-hour period. The results showed an average particle size of less than 80 nm and a narrow polydispersity index. These results suggest that DIP may exist in nanoscale form in intestinal fluid, potentially promoting absorption.

[0139] (3) Powder X-ray diffraction:

[0140] See results Figure 9 Where A represents the X-ray diffraction pattern of DIP; B represents the X-ray diffraction pattern of Soluplus; and C represents the X-ray diffraction pattern of the dipyridamole nanosuspension of Example 1.

[0141] according to Figure 9 As shown in Figure A, the unique crystal diffraction peaks of pure DIP in the range of 5° to 40° indicate its clear crystal structure. Conversely, Figure 9 The B-value shows that Soluplus did not exhibit any peaks across the entire range from 5° to 90°. Figure 9 In Example C, the dipyridamole nano-suspension powder of Example 1 showed a peak similar to that of pure DIP. Although its intensity was not as strong as that of the pure DIP pattern, this similarity indicates that the DIP in the formulation exists in a crystalline state.

[0142] (4) Field emission scanning electron microscopy

[0143] See results Figure 10 , Figure 10 In the image, A represents a field emission scanning electron microscope (FESEM) image of DIP; B represents a field emission scanning electron microscope (FESEM) image of dipyridamole nanosuspension from Example 1.

[0144] from Figure 10 As can be seen in image A, the DIP particles exhibit irregular shapes and are approximately 3-4 μm in size. Figure 10 The DIP size of the dipyridamole nanosuspension particles in Example 1 of Chinese B is approximately 500 nm, which is close to the result of DLS (400-500 nm).

[0145] (5) In vivo studies

[0146] (5.1) Cell Culture and Cell Research

[0147] 5.1.1 Caco-2 cell culture

[0148] Caco-2 cells (Procell, Wuhan, China) (passage number 20-30) were cultured in Dulbecco modified Eagle medium (DMEM) (Gibco, Beijing, China) supplemented with 20% fetal bovine serum (FBS) (TransGen Biotech, China), 1% penicillin / streptomycin (Gibco, USA), and 1% non-essential amino acids (Gibco, USA). The incubator (Thermo, USA) was set to 5% CO2. Cells were harvested when confluence reached 90%, and the medium was changed every 48 hours.

[0149] 5.1.2 Cellular uptake

[0150] Caco-2 cells were seeded into 6-well plates (2 × 10⁻⁶ cells per well). 5Cells were incubated in wells (cells / well) and allowed to adhere overnight. When cells reached 90% confluence, the culture medium was replaced with those containing DIP (50 μg / mL), a physical mixture of DIP (PM, 50 μg / mL DIP, 12.5 μg / mL Tween 80, 62.5 μg / mL Soluplus), and the dipyridamole nanosuspension from Example 1 (DIP-OF, containing 50 μg / mL DIP). After 3 hours of incubation, the treated cells were washed with ice-cold PBS, lysed three times in PBS, and then collected by centrifugation at 12,000 rpm for 20 minutes. The DIP content in the cell lysate was quantified by HPLC.

[0151] 5.1.3 Transcellular transport research

[0152] Collect Caco-2 cells and divide them into 5×10 5 Inoculated onto Corning polycarbonate inserts (0.4 μm pore size, 4.67 cm² growth area) at a ratio of [number] cells / well. 2 After culturing for 21 days, transcellular transport studies were conducted using the previously recommended methods. Caco-2 monolayer cells with a transepithelial electrical resistance (TEER) exceeding 600 Ω were selected as experimental cells. DIP (25 μg / mL), a physical mixture of DIP (25 μg / mL DIP, 6.25 μg / mL Tween80, 31.25 μg / mL Soluplus), and the dipyridamole nanosuspension from Example 1 (containing 25 μg / mL DIP) were loaded into the top chamber containing 1.5 mL of Hank balanced salt solution (HBSS). At specified time points, 1 mL of sample was taken from the basal chamber (containing 2.6 mL HBSS) and replenished with an equal volume of fresh HBSS. The DIP concentration was determined by HPLC.

[0153] 5.2 Pharmacokinetic Studies

[0154] Male SD rats (6 weeks old, 225±25g) were provided by the Guangdong Provincial Center for Laboratory Animal Science (Foshan, China). The rats were housed under standard conditions: temperature 22℃, humidity 60%±10%, and a 12-hour light-dark cycle. They had free access to water and rodent feed. The rats were used in experiments one week after acclimatization to their new environment. All experimental protocols complied with the National Research Council's Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Research Ethics Committee of Shenzhen University.

[0155] Overnight-fasted SD rats (n=5) were administered DIP (5 mg / kg, IV), DIP (40 mg / kg, PO), commercially available DIP tablets (DIP-CT, Yabao Pharmaceutical Co., Ltd., equivalent to 40 mg / kg, PO), and the dipyridamole nanosuspension from Example 1 (DIP-OF, equivalent to 40 mg / kg DIP, PO). Blood samples were collected in heparinized tubes at specified time points, and analyzed by LC-MS / MS (Waters, USA) with glipizide as an internal standard. The relationship between plasma concentration and time was analyzed using DAS software (Shanghai, China).

[0156] See results Figure 11 And Table 5, in which, Figure 11 In the diagram, A represents cellular uptake in Caco-2 cells after 3 hours of culture (n=3). B represents a transmembrane transport study conducted on a Caco-2 cell monolayer, lasting 2 hours (n=3). C represents the pharmacokinetic curves of DIP, DIP-, and DIP-OF (n=5).

[0157] Table 5. Pharmacokinetic parameters of DIP in rats after administration.

[0158]

[0159]

[0160] *p<0.5,**p<0.01,***p<0.001,****p<0.0001

[0161] like Figure 11 As shown in Figure A, the optimal DIP formulation (DIP-OF) showed uptake in Caco-2 cells that was 2.3 times and 3.1 times that of pure DIP and the physical mixture (PM), respectively, indicating that DIP-OF promotes intestinal absorption. The cumulative permeability of pure DIP in Caco-2 monolayer cells was less than 0.1%. Figure 11 (B) In contrast, after 2 hours of culture, the cumulative permeability of DIP-OF was approximately 13.6%, while only 0.1% of PM penetrated the Caco-2 cell monolayer. Consistent with the results of transcellular transport studies, DIP-OF significantly improved the oral bioavailability of DIP compared to pure DIP and commercially available DIP tablets (DIP-CT). Figure 11 In the study of DIP, DIP-OF significantly increased Cmax (3.0-fold and 1.5-fold) and AUC0-t (4.5-fold and 2.3-fold) (Table 5). The absolute bioavailability of pure DIP, DIP-OF, and DIP-CT were 9.51%, 18.17%, and 42.15%, respectively. All these results indicate that DIP-OF can enhance the intestinal absorption of DIP.

[0162] (6) PBPK Simulation

[0163] The overall strategy for DIP PBPK modeling and pediatric simulation followed a stepwise approach. An adult intravenous PBPK model was established to determine the distribution and elimination of DIP in vivo. Subsequently, absorption characteristics in rats were extrapolated to humans, and the intravenous PBPK model was integrated to establish an adult oral PBPK model. Finally, a pediatric PBPK model was derived based on the maturation of metabolic enzymes and organs.

[0164] 6.1 PBPK data source for model development

[0165] A systematic search of intravenous and oral tablet pharmacokinetic studies for dipyridamole (DIP) was conducted on PubMed, using the filter term "Humanadults" (Dipyridamole AND Pharmacokinetic). Based on appropriate routes of administration, dosage forms, data availability, and study participants, two intravenous studies and five oral tablet studies were selected. Table 6 summarizes the study details and participant statistical characteristics.

[0166] Table 6. Demographic characteristics and modeling results of participants in the intravenous and oral administration studies.

[0167]

[0168] a Absolute average folding error

[0169] b Average folding error

[0170] 6.2 PBPK Model

[0171] The PBPK model was developed using PK-Sim (V11.2; Bayer AG, Germany). Drug release curves were deconvolved using the RIVIVC R software package (V0.9.1). PK observation data from scientific literature were digitized using GetData Graph Digitizer (V2.26; Kogarah, Australia), with an error tolerance of less than 2% between values. Table 7 lists the physicochemical parameters of DIP in the PBPK model.

[0172] Table 7. Drug parameters used in the dipyridamole PBPK model.

[0173]

[0174]

[0175] a Adjusted by PK-SIM software

[0176] b The default Kp values ​​for other organs in PK-Sim software

[0177] 6.2.1 Intravenous PBPK Model

[0178] Dipyridamole is metabolized in the liver to form a glucuronide conjugate, which is excreted in bile. In the absence of information on specific metabolic enzymes, the systemic clearance rate was used in the intravenous PBPK model. Tissue distribution values ​​in rats were used to calculate Kp in the intravenous PBPK model. Distribution and elimination parameters were derived from literature, and their final values ​​are detailed in Table 7. All other software settings were default values.

[0179] 6.2.2 Oral PBPK Model

[0180] Based on the intravenous PBPK model, and considering drug release and absorption, an adult oral PBPK model of DIP-OF was established. Then, a pediatric PBPK model of DIP-OF was deduced from the adult PBPK model. First, the release amount of DIP in different dosage forms in humans was inferred using the in vivo release amount in rats. Based on the rat pharmacokinetic studies of DIP, the absorption of DIP in rats was determined using the deconvolution method. The results were fitted using a Weibull function (as shown in Equation 1) and then applied to human absorption.

[0181]

[0182] Where %DoseReleased represents the cumulative percentage of drug release at time t, A is the time scale factor, and b is the shape factor. In the final PBPK model, the Weibull parameters are b = 0.43, A = 8.80 (DIP-CT), b = 0.36, and A = 4.89 (DIP-OF).

[0183] The oral PBPK model of DIP-CT was used to verify the scaling ratio of drug absorption from rats to humans. Secondly, drug solubility and permeability were set in the software, while other relevant parameters were left at default. Thirdly, pediatric extrapolation considered the maturity and clinical application range of uridine 5'-diphospho-glucuronosyl transferase (UGT). Newborns have low UGT levels and insufficient glucuronidation capacity at birth, reaching adult levels only at least 3-4 years of age. The traditional allometric scaling method was used to describe the pediatric elimination of DIP, as shown in Equation 2.

[0184] The DIP-OF PK simulation is for children aged 4-18 years.

[0185]

[0186] Where CL represents drug clearance rate and WT represents body weight.

[0187] 6.3 Verification

[0188] Model performance was evaluated using goodness-of-fit plots, absolute average folding error (AAFE), and average folding error (AFE). As shown in Equation 3, the closer the AAFE is to 1, the better the model fit. Furthermore, as shown in Equation 4, an AFE value greater than 1 indicates that the predicted values ​​are overestimated on average; conversely, a value less than 1 indicates that the predicted values ​​are underestimated. In our study, both AAFE and AFE were calculated for the complete dataset (including all observations and predictions) and for each study (including observations and predictions from individual studies).

[0189]

[0190] Among them O i For observation data, P i For the predicted data, n is the number of time points during sampling.

[0191] See results Figure 12 .in, Figure 12 In Figure A, the comparison between the predicted and actual plasma concentrations of the DIP PBPK model is shown. The black dashed line represents a 2-fold bias, and the black dotted line represents a 3-fold bias. Figure B shows the deconvolution of the DIP-CT and DIP-OF absorption curves in rats and their fitting using the Weibull function. Figure C shows the comparison of the pharmacokinetic (PK) curves of DIP-CT and DIP-OF.

[0192] The intravenous PBPK model reasonably describes the concentration-time curves for the 20-45 mg dose range. The goodness-of-fit plot between predicted and observed plasma concentrations is shown in [reference needed]. Figure 12 In the middle A, the calculated total AAFE and AFE values ​​were 1.47 and 1.36, respectively. Table 5 lists the calculated AAFE and AFE values ​​for each intravenous PBPK model. Furthermore, 84.44% of all predicted concentrations were within the 2-fold range, and 97.78% were within the 3-fold range.

[0193] The in vivo absorption curves of DIP-CT and DIP-OF were determined using the deconvolution method, such as... Figure 12 As shown in Figure B, the results fitted using the Weibull function are also presented. The oral PBPK model effectively captures the concentration-time curves within the 50-200 mg dose range. Figure 12 Figure A shows the goodness-of-fit plot between the predicted and actual measured plasma concentrations. Figure 12Table C shows a visual comparison of the predicted and actual measured plasma concentration curves for each oral study. The overall AAFE and AFE values ​​were calculated to be 1.75 and 1.26, respectively. The calculated AAFE and AFE values ​​for each oral PBPK model are shown in Table 5. Furthermore, 78.95% of all predicted concentrations were within the 2-fold range, and 89.47% of the predicted concentrations were within the 3-fold range. Figure 12 The results showed that, at the same dose, the predicted pharmacokinetic (PK) curve of DIP-OF was superior to that of DIP-CT.

[0194] PBPK simulations were performed on DIP-OF after oral administration at doses of 3 mg / kg, 4 mg / kg, and 5 mg / kg once daily for 12 weeks. The simulated age groups included 4 years (infants), 8 years (children), and 12 years (adolescents). Mature UGT expression was assumed in all age groups, and systemic clearance was estimated using allometric scaling. The default sex was male. Steady-state simulated pharmacokinetic (PK) parameters are listed in Table 8, and the simulated PK curves are shown in [Table data missing]. Figure 13 middle.

[0195] Table 8 Steady-state pediatric pharmacokinetic parameters of DIP-OF after 12 weeks of once-daily oral administration at doses of 3, 4, and 5 mg / kg.

[0196]

[0197] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dipyridamole nano-suspension characterized in that, The raw material comprises a crystallization inhibitor and dipyridamole, wherein the mass ratio of the crystallization inhibitor to the dipyridamole is (1-10):

1.

2. The dipyridamole nano-suspension of claim 1, wherein, The mass ratio of the crystallization inhibitor to the dipyridamole is (1-8):1; preferably (1.2-5):

1.

3. The dipyridamole dry nano-suspension according to claim 1 or 2, characterized in that, The crystallization inhibitor is selected from polymers, Preferably, the crystallization inhibitor is selected from cellulose-based polymers, ethylene-based polymers and ethylene glycol-based polymers; More preferably, at least one of polyvinylpyrrolidone, vinylpyrrolidone / vinyl acetate copolymer, polyvinyl alcohol, polyethylene glycol, cellulose ethers, polyacrylic acid-based polymers, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, Gelucire 44 / 14, hydroxypropyl cellulose, hypromellose and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. Most preferably, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

4. The dipyridamole nano-suspension of claim 1, wherein The raw material further comprises a surfactant; the mass ratio of the surfactant to the dipyridamole is (0.1-1):1; Preferably (0.2-0.8):1, more preferably (0.3-0.5):1; Preferably, the surfactant comprises at least one of sodium dodecyl sulfate, calcium stearate, triethanolamine stearate, magnesium lauryl sulfate, sodium octadecyl fumarate, sodium taurocholate, sodium ursodeoxycholate, lecithin, soybean phospholipid deoxycholate sodium, alkyl benzene sulfonate, benzalkonium chloride, benzalkonium bromide, poloxamer, Tween, Span, Brij, Cremophor, polyoxyethylene castor oil and polyethylene glycol fat. Preferably, the surfactant comprises at least one of sodium dodecyl sulfate and Tween; Further preferably, Tween, most preferably Tween 80.

5. The dipyridamole nano-suspension of claim 1, wherein The raw material comprises a lyophilization protective agent, and the mass ratio of the lyophilization protective agent to the dipyridamole is (1-5):

1.

6. The dipyridamole nano-suspension of claim 5, wherein The lyophilization protective agent is selected from polyols, preferably C3-C10 polyols, more preferably mannitol.

7. The dipyridamole nano-suspension of claim 1, wherein The raw material comprises Tween, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and dipyridamole, wherein the mass ratio of the Tween, the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and the dipyridamole is (0.3-0.5):(1.2-3):1; Preferably, the Tween comprises Tween 80.

8. A process for the preparation of the dipyridamole nano suspension of claim 1, characterized by, Comprise: Mixing the crystallization inhibitor and dipyridamole by wet grinding and then lyophilizing.

9. The preparation method according to claim 8, characterized in that, The conditions of wet grinding comprise: rotation speed 400-600 rpm; duration 8-12 minutes, intermittent 3-7 minutes, total running time 1-1.5 h; The conditions of lyophilization comprise: lyophilization temperature -60 to -80℃, pressure 0.2-0.4 mbar, time 70-80 hours; Preferably, pre-freezing is performed before lyophilization; Preferably, the conditions of pre-freezing comprise: temperature -60 to -80℃, time 10-15 hours; Preferably, it further comprises mixing the crystallization inhibitor, dipyridamole and water, wherein the concentration of the crystallization inhibitor in water is 50-80 mg / ml.

10. A dipyridamole reconstituted suspension characterized in that, It comprises a reconstituting solvent and the dipyridamole dry nanosuspension according to claim 1.

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