Icariin and urea co-crystal

Abstract

The application provides an icariin-urea co-crystal, and relates to the technical field of crystal form drug molecules. The co-crystal uses Cu-K alpha radiation, and the X-ray diffraction spectrum expressed in 2theta has characteristic peaks at 6.08+ / -0.2 degrees, 11.44+ / -0.2 degrees, 13.13+ / -0.2 degrees, 22.23+ / -0.2 degrees, 22.31+ / -0.2 degrees, 24.57+ / -0.2 degrees, 29.28+ / -0.2 degrees, 29.34+ / -0.2 degrees, 31.59+ / -0.2 degrees, 35.41+ / -0.2 degrees and 35.57+ / -0.2 degrees; the crystallographic measurement parameters are as follows: monoclinic crystal system, space group P21 / c; the cell parameters are as follows: alpha=90 degrees, beta=110.872(6) degrees, gamma=90 degrees, and the cell volume is provided with related preparation methods and applications. The icariin-urea co-crystal of the application has achieved great improvement in stability, bioavailability and the like.

Description

Technical Field

[0001] This invention relates to the field of crystalline drug molecular technology, and particularly to the field of icariin organic drug cocrystal technology, specifically to a drug cocrystal of icariin and urea, its preparation method and application. Background Technology

[0002] Epimedium, also known as icariin, has the molecular formula C 21 H 20 O6, with a relative molecular mass of 368.126, has the following structure:

[0003]

[0004] Epimedium extract is the main pharmacologically active monomeric component of Epimedium, belonging to the flavonoid class of compounds. Flavonoids are a class of polyphenolic compounds with a basic parent nucleus of benzopyranone (C6-C3-C6), widely found in various plants in free form or bound to sugars as glycosides. They possess a variety of pharmacological activities, including antioxidant, antitumor, antiviral, neuroprotective, free radical scavenging, anti-inflammatory, cardiovascular protective, and α-glucosidase inhibition. For example, the journal *Chinese Journal of Experimental Traditional Medical Formulae*, Vol. 18, No. 14, 2012, published an article on "The Effect of Epimedium Extract on Estrogen-Dependent Breast Cancer MCF-7 Cells," which revealed that the combined action of epimedium extract and estradiol inhibited the proliferation of E2-induced human breast cancer MCF-7 cells. The journal *Chinese Journal of Comparative Medicine*, No. 6, 2011, published an article on "The In Vitro Anti-Lymphoma Cell Proliferative Effect of Epimedium Extract," which revealed the effect of epimedium extract on tumor cell proliferation. Although icariin shows promising clinical application prospects, its poor water solubility significantly limits its clinical application. Icariin is only slightly soluble in water, and this poor water solubility results in poor oral absorption and low bioavailability. Statistics show that over 40% of potential drug candidates fail to enter clinical trials due to poor water solubility, poor permeability, and low bioavailability.

[0005] Recent studies have found that different drug crystal forms can alter their physicochemical properties (density, hardness, solubility, stability, optical properties, and electrical properties), dissolution rate, and biological effects. Therefore, the study of drug crystal forms is of great value in medicine.

[0006] Currently reported icariin flavonoids include icariin, epimedium glycoside, and dehydrated epimedium glycoside, all of which suffer from poor water solubility and low bioavailability. For class IV icariin with low solubility and low permeability, the literature "Study on the Polymorphism of Icariin and the Crystallization-Induced Asymmetric Transformation of Sanguisorbin Methanol" (Nanchang University, Jia Lina) studied and prepared a series of icariin hydrates and amorphous forms. Patent CN104844668A discloses the preparation of an anhydrous α-crystalline form of icariin, and patents CN104829667A and CN104804053A further prepared icariin hydrate crystal forms H1 and H2 with slightly improved dissolution rates through crystal form studies. The literature "Transformation History of the Material Basis in the Processing of Epimedium" reports on three crystal forms (A, B, and C) of dehydrated epimedium, as well as crystal form D of epimedium. Patent CN103936705A discloses four epimedium solvates and one amorphous form B, and reports that the solvate crystal forms are unstable and prone to transformation into amorphous form B. Patents CN104230870A and CN104945364A disclose two hydrated crystal forms of epimedium and study the light stability of the hydrated crystal form and amorphous form B, reporting that amorphous form B is unstable under light, while the hydrated crystal form shows some improvement in light stability. Patent CN112294765A discloses an amorphous form of icariin. Compared with the crystal form of icariin, the amorphous icariin has improved solubility and bioavailability. However, the amorphous form generally has poor stability, which is not conducive to its practical application in drugs.

[0007] Analysis of currently reported icariin crystal forms reveals that improvements in medicinal efficacy are not ideal. For example, amorphous form B shows some improvement in physical stability, but its chemical stability, particularly light stability, and bioavailability are poor. Amorphous icariin has achieved some improvement in solubility and bioavailability, but its stability is poor. Given these issues, further research and development of optimal icariin crystal forms suitable for pharmaceutical use remains a problem that needs to be solved. Summary of the Invention

[0008] To address the problems of existing technologies, this invention provides an icariin-urea eutectic with precise crystallographic parameters and atomic spatial positions; another aspect of this invention provides a method for preparing the eutectic.

[0009] The specific technical solution of this invention is as follows:

[0010] In a first aspect, the present invention provides an icariin-urea eutectic, wherein the molar ratio of icariin to urea in the eutectic is 1:1, and one molecule of icariin and one molecule of urea constitute the basic unit of the crystal form, as shown in Formula I:

[0011]

[0012] Preferably, the epimedium-urea eutectic, when subjected to Cu-Kα radiation, exhibits characteristic peaks in its X-ray diffraction pattern (denoted as 2θ) at 6.08±0.2°, 22.23±0.2°, 22.31±0.2°, 29.28±0.2°, 29.34±0.2°, and 35.41±0.2°.

[0013] Preferably, the epimedium-urea eutectic, when subjected to Cu-Kα radiation, exhibits characteristic peaks in its 2θ X-ray diffraction pattern at 6.08±0.2°, 11.44±0.2°, 13.13±0.2°, 22.23±0.2°, 22.31±0.2°, 24.57±0.2°, 29.28±0.2°, 29.34±0.2°, 31.59±0.2°, 35.41±0.2°, and 35.57±0.2°.

[0014] Preferably, the icariin-urea eutectic, when subjected to Cu-Kα radiation, exhibits characteristic peaks that conform to the following... Figure 1 The X-ray powder diffraction pattern shown is shown.

[0015] Preferably, the icariin-urea eutectic has the following crystallographic parameters: monoclinic crystal system, space group P21 / c; and the following unit cell parameters: α = 90°, β = 110.872(6)°, γ = 90°, cell volume

[0016] Secondly, the present invention provides a method for preparing icariin-urea eutectic, comprising the following steps:

[0017] Icariin and urea were placed in an organic solvent, heated and stirred, cooled to crystallize, filtered, washed and dried to obtain icariin-urea eutectic.

[0018] Preferably, the molar ratio of epimedium to urea is 1:1 to 1.5, more preferably 1:1.2.

[0019] Preferably, the organic solvent is one or a combination of acetonitrile, acetone, tetrahydrofuran, methanol, ethanol, and isopropanol; more preferably, it is one or a combination of acetonitrile and acetone; if the organic solvent is a mixture of two solvents, the volume ratio of the less polar solvent to the more polar solvent is preferably 1:1 to 1.5.

[0020] Preferably, the mass-to-volume ratio of epimedium to organic solvent is 5-7:1, wherein the mass is expressed in mg and the volume in mL.

[0021] Preferably, the heating temperature is 45–75°C; and the heating and stirring time is 2–6 hours.

[0022] Preferably, the cooling and crystallization temperature is 0–15°C; more preferably, it is 5–10°C.

[0023] Preferably, the washing solvent is one or two of ethanol, acetone, and acetonitrile.

[0024] Thirdly, the present invention provides a pharmaceutical composition comprising the epimedium-urea cocrystal described herein and other pharmaceutically acceptable components.

[0025] Preferably, the other pharmaceutically acceptable components include other active ingredients, excipients, fillers, etc., that can be used in combination.

[0026] Preferably, the pharmaceutical compositions of the present invention can be prepared by combining the compounds of the present invention with a pharmaceutically acceptable solid or liquid carrier using standard and conventional techniques, and by combining them with pharmaceutically acceptable excipients and formulations to prepare a usable dosage form.

[0027] Preferably, the pharmaceutical composition is a spray, tablet, capsule, powder for injection, liquid for injection, etc.

[0028] Fourthly, the present invention provides the application of icariin-urea cocrystal as an active ingredient in the preparation of therapeutic antitumor drugs and its application as an active ingredient in the preparation of α-glucosidase inhibitor drugs.

[0029] The beneficial effects of this invention are:

[0030] The icariin-urea eutectic prepared by this invention exhibits significant improvements in stability and bioavailability. The preparation method of this icariin-urea eutectic is simple and suitable for industrial production; the resulting eutectic has a regular crystal structure, uniform particle size, and well-defined crystallographic parameters and precise atomic spatial positions, making it suitable for large-scale application. Attached Figure Description

[0031] Figure 1 X-ray powder diffraction pattern of icariin-urea eutectic.

[0032] Figure 2 : ORTEP diagram of epimedium-urea eutectic.

[0033] Figure 3 : Epimedium-urea eutectic packing diagram.

[0034] Figure 4 DSC-TGA image of icariin-urea eutectic. Detailed Implementation

[0035] The present invention will be further illustrated below through embodiments. It should be understood that the embodiments of the present invention are merely for illustrating the invention and not for limiting the invention. Therefore, simple improvements to the present invention under the premise of the method of the present invention are all within the scope of protection of the present invention.

[0036] Materials used in the experiment: Icariin can be purchased or prepared according to existing methods. The icariin crystal form required for the comparative experiment can be prepared according to existing techniques; other materials used in this experiment without specified source and specifications are all commercially available analytical grade or chemically pure.

[0037] Example 1

[0038] Approximately 3.7 g of icariin and 0.72 g of urea were placed in 700 ml of acetonitrile, heated to 75 °C and stirred for 4 hours. The mixture was then slowly cooled to 5–10 °C and crystallized under controlled temperature. After crystallization, the mixture was filtered, and the filter cake was washed with ethanol and dried to obtain icariin-urea eutectic with a yield of 95.6% and a purity of 99.95%.

[0039] Example 2

[0040] Approximately 3.7 g of icariin and 0.6 g of urea were placed in 550 ml of acetone, heated to 50 °C and stirred for 3 hours. The mixture was then slowly cooled to 0–5 °C and crystallized under controlled temperature. After crystallization, the mixture was filtered, and the filter cake was washed with acetone and dried to obtain icariin-urea eutectic crystals with a yield of 93.4% and a purity of 99.92%.

[0041] Example 3

[0042] Approximately 3.7g of icariin and 0.9g of urea were placed in 750ml of acetone / acetonitrile (V 丙酮 V 乙腈 In a mixed solvent of 1:1, the mixture was heated to 55°C and stirred for 6 hours. Then, the temperature was slowly lowered to 5-10°C to control crystallization. After crystallization, the mixture was filtered, the filter cake was washed with acetonitrile, and dried to obtain icariin-urea eutectic with a yield of 94.3% and a purity of 99.90%.

[0043] Example 4

[0044] Approximately 3.7 g of icariin and 0.75 g of urea were placed in 600 ml of ethanol, heated to 70 °C and stirred for 5 hours. The mixture was then slowly cooled to 10–15 °C and crystallized under controlled temperature. After crystallization, the mixture was filtered, the filter cake was washed with ethanol, and dried to obtain icariin-urea eutectic with a yield of 94.8% and a purity of 99.93%.

[0045] Example 5

[0046] Approximately 3.7g of icariin and 0.82g of urea were placed in 650ml of tetrahydrofuran / ethanol (V 四氢呋喃 V 乙醇 In a mixed solvent of 1:1.5, the mixture was heated to 60°C and stirred for 4 hours. Then, the temperature was slowly lowered to 5-10°C to control crystallization. After crystallization, the mixture was filtered, the filter cake was washed with ethanol, and dried to obtain icariin-urea eutectic with a yield of 93.8% and a purity of 99.87%.

[0047] Example 6

[0048] Approximately 3.7 g of icariin and 0.65 g of urea were placed in 650 ml of methanol, heated to 55 °C and stirred for 6 hours. The mixture was then slowly cooled to 0–5 °C and crystallized under controlled temperature. After crystallization, the mixture was filtered, the filter cake was washed with ethanol and dried to obtain icariin-urea eutectic crystals with a yield of 94.2% and a purity of 99.91%.

[0049] Characterization of icariin-urea cocrystal

[0050] The X-ray powder diffraction testing instrument and conditions involved in this invention are as follows: X-ray powder diffractometer: PANalytical EMPYREA; Cu-Kα; sample stage: flat plate; incident light path: BBHD; diffraction light path: PLXCEL; voltage 45kV, current 40mA; divergence slit: 1 / 4; anti-scattering slit: 1; Solar slit: 0.04rad; step size: 0.5s; scanning range: 3~50°. The characteristic peaks in the corresponding X-ray secretion diffraction pattern (Cu-Kα) are detailed in the appendix. Figure 1 And Table 1.

[0051] Table 1. PXRD peaks of the icariin-urea eutectic

[0052]

[0053]

[0054] The present invention provides X-ray single-crystal diffraction analysis of the epimedium-urea eutectic. The X-ray single-crystal diffraction instrument and testing conditions involved in the present invention are: Rigaku XtaLAB Synergy X-ray single-crystal diffractometer, testing temperature 293(2)K, CuKa radiation, data collection in ω-scan mode and Lp correction. The structure is resolved by direct method, all non-hydrogen atoms are identified by difference Fourier method, all hydrogen atoms on carbon and nitrogen are obtained by theoretical hydrogenation, and the structure is refined by least squares method.

[0055] The crystallographic parameters of the icariin-urea eutectic prepared by this invention, as tested and analyzed, are: monoclinic crystal system, space group P21 / c; cell parameters are: α = 90°, β = 110.872(6)°, γ = 90°, cell volume The ORTEP of the icariin-urea eutectic of the present invention Figure 2 This indicates that one molecule of icariin combines with one molecule of urea to form a eutectic. A packing diagram of the icariin-urea eutectic of this invention is attached. Figure 3 As shown.

[0056] Table 2. Main crystallographic data of the icariin-urea eutectic.

[0057]

[0058]

[0059] The TGA / DSC thermal analysis instrument and testing conditions in this invention are as follows: TGA / DSC thermal analyzer: METTLER TOLEDOTGA / DSC3+; dynamic temperature range: 30–300℃; heating rate: 10℃ / min; programmed gas N2; gas flow rate: 50 mL / min; crucible: 40 μl aluminum crucible. Its differential scanning calorimetry (DSC / TGA) curve and thermogravimetric analysis (DSC / TGA) plot are shown below. Figure 4 As shown.

[0060] The samples in Examples 1 to 6 all conform to X-ray powder diffraction patterns, crystallographic parameters, differential scanning calorimetry curves, and thermogravimetric analysis (DSC / TGA) diagrams.

[0061] Comparative Example 1

[0062] Dissolve 6.5g of icariin in 200ml of acetone, filter, add 100ml of distilled water to the filtrate, reflux at 75℃ to dissolve, let crystallize at 20℃ for 24 hours, filter, and dry continuously at 80℃ until the weight no longer changes, to obtain icariin crystal form B, with a yield of 90.3% and a purity of 99.86%.

[0063] Comparative Example 2

[0064] Take about 1g of icariin and add chloroform / methanol (V) to it. 氯仿 V 甲醇 After the mixture of solvents (1:1) was completely dissolved, it was placed in an environment with a temperature of 30°C and a relative humidity of 90% to evaporate and crystallize. After crystallization, it was filtered to obtain crystal form D with a yield of 82.4% and a purity of 99.83%.

[0065] Comparative Example 3

[0066] 10g of icariin and 700mL of acetone were placed in a 5.0L flask and dissolved by stirring in a warm water bath. Then, 4L of purified water was quickly added while stirring, and stirring was continued for 10min to induce crystallization. After the solution cooled to room temperature, it was filtered. The filter cake was placed in a 25℃ forced-air drying oven and dried for 48 hours to obtain icariin monohydrate with a yield of 92.8% and a purity of 99.88%.

[0067] Comparative Example 4

[0068] Take 10g of icariin and 700mL of acetone and place them in a 1L beaker. Dissolve them by stirring in a warm water bath. Then, quickly add the icariin acetone solution to a flask containing 4L of purified water at room temperature. Stir vigorously for 10 minutes to induce crystallization. After the solution cools to room temperature, filter it. Place the filter cake in a 25℃ forced-air drying oven and dry it for 48 hours to obtain icariin hemihydrate with a yield of 90% and a purity of 99.85%.

[0069] Comparative Example 5

[0070] 2.4 g of icariin was placed in 120 ml of sodium hydroxide aqueous solution (0.2 mol / L) and stirred to dissolve, yielding an icariin alkaline solution. 3.84 g of polymer Soluplus was placed in 480 ml of hydrochloric acid aqueous solution (0.05 mol / L) and stirred to dissolve, yielding a polymer Soluplus hydrochloric acid aqueous solution. The icariin alkaline solution was added to the hydrochloric acid aqueous solution using a peristaltic pump, and the mixture was stirred at 1000 r / min for 20 min. After filtration, the precipitate was collected, pre-frozen at -40℃, and freeze-dried for 24 hours to obtain amorphous icariin particles with a yield of 73.2% and a purity of 99.80%.

[0071] Verification Example:

[0072] The present invention investigated the stability and bioavailability of the prepared icariin crystal form, and the specific implementation details are as follows:

[0073] Stability test

[0074] 1. Light test: Referring to the "Guidelines for Stability Testing of Raw Materials and Preparations of Chinese Pharmacopoeia 2020 Edition, Part IV - 9001", appropriate amounts of the icariin crystal form of Example 1 and Comparative Examples 1-5 were taken and dispersed in open containers (thickness not exceeding 3 mm). The containers were placed in a light box and tested under an illuminance of 4500 lx ± 500 lx. Samples were taken on days 0, 5, 10 and 30 to test the purity.

[0075] 2. High temperature test: Referring to the "Guidelines for Stability Testing of Raw Materials and Preparations of Chinese Pharmacopoeia 2020 Edition, Part IV - 9001", appropriate amounts of the icariin crystal form of Example 1 and Comparative Examples 1-5 were taken and placed in a 60℃ closed constant temperature incubator. Samples were taken on days 0, 5, 10 and 30 to test the purity.

[0076] Table 3. Results of photostability test on icariin crystal form

[0077]

[0078]

[0079] The results of light and high-temperature stability tests showed that icariin in its amorphous form had poor stability and significantly degraded after light and high-temperature testing. Icariin crystal forms B and D also exhibited poor light stability and significantly degraded after light-induced stability testing. The icariin-urea eutectic prepared in this invention showed good stability under both light and high-temperature conditions. Similar stability test results were found in Examples 1-6.

[0080] Comparison of relative humidity (RH) and high temperature stability

[0081] The icariin crystal forms prepared in Example 1 and Comparative Examples 1-5 were stored at 40℃, 75%RH and 80℃ for 1 week, respectively, and then subjected to PXRD tests to test the relative humidity and high temperature stability of each crystal form. The results are shown in Table 4.

[0082] Table 4. Results of relative humidity stability of icariin crystal forms

[0083]

[0084] Note: √ indicates that the storage conditions are stable and the PXRD pattern remains unchanged; ╳ indicates that the storage conditions are unstable.

[0085] The results of the relative humidity (RH) stability comparison show that the epimedium-urea eutectic prepared in this invention has good stability under high humidity and high temperature environments, and the crystal form does not change.

[0086] Beagle pharmacokinetics study

[0087] Healthy male beagle dogs (weighing 6-8 kg, aged 1-2 years) were placed in the same environment and allowed free access to food and water. They were fasted for 24 hours before administration and then orally administered the icariin-urea cocrystal of Example 1, the icariin crystal forms of Comparative Examples 1-5, and the control (icariin nanocrystals from Shandong New Era Pharmaceutical Co., Ltd.). The dosage was 10 mg / kg (calculated as icariin). Blood samples were collected from peripheral veins before administration and at 10 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 8 h, 10 h, 12 h, and 24 h after administration. After collection, the blood was centrifuged for 10 min (3000 r / min) to separate the plasma, which was then frozen at -20℃ for later analysis.

[0088] Take 50 μL of plasma sample, add 150 μL of 0.05 mol / L ammonium dihydrogen phosphate aqueous solution, vortex for 1 min, take 30 μL and add 125 μL of 200 U β-glucuronidase, mix well, incubate at 37 °C for 1 hour, add 50 μL of baicalein methanol solution as internal standard, extract with 1.2 mL of methyl tert-butyl ether-n-hexane (2:1), vortex for 2 min, centrifuge at 4 °C and 12000 r / min for 5 min, take 960 μL of the upper organic phase into a glass test tube, blow dry with nitrogen in a 40 °C water bath, add 100 μL of 70% methanol aqueous solution, vortex to reconstitute, after reconstitution, centrifuge at 4 °C and 12000 r / min for 10 min, take 50 μL of the supernatant for LC-MS / MS quantitative analysis.

[0089] Table 5. Pharmacokinetic parameters of beagle dogs after oral administration (n=3)

[0090]

[0091] Mouse glucose tolerance test

[0092] Eighty mice were randomly divided into eight groups of ten each. Epimedium crystals from Examples 1 and Comparative Examples 1-5 were administered via gavage at a dose of 50 mg / kg. A negative control group and a positive control group were established. The negative control group received physiological saline, while the positive control group received acarbose at a dose of 50 mg / kg via gavage. The administration was repeated for seven consecutive days. Mice were fasted for eight hours before the last administration. One hour after administration, starch (5 g / kg) was administered via gavage. Blood samples were collected from the orbital venous sinus at 0, 0.5, 1, 2, and 3 hours after starch administration. Blood glucose levels were measured using glucose oxidase test strips (unit: mmol / L). The results are shown in Table 6.

[0093] Table 6 Results of glucose tolerance test in mice using icariin crystal forms

[0094]

[0095] In addition, in vitro cell tests revealed that the epimedium-urea cocrystal of the present invention has an inhibitory effect on a series of tumor cells, such as the inhibition of liver cancer Huh-7 cells (IC50 = 3.2 μM), breast cancer MCF-7 cells (IC50 = 2.3 μM), and acute myeloid leukemia MV-4-11 cells (IC50 = 4.8 μM).

[0096] The icariin-urea eutectic prepared by this invention has achieved beneficial effects in terms of stability and bioavailability, and its overall performance has made significant progress compared with the icariin crystal form reported in the prior art.

Claims

1. An icariin-urea eutectic, characterized in that, The co-crystal is composed of the active pharmaceutical ingredient icariin and the co-crystal ligand urea; the molar ratio of icariin to urea in the co-crystal is 1:1, and the co-crystal has the X-ray powder diffraction pattern shown in Figure 1, with the structure as follows: 。 2. The icariin-urea eutectic as described in claim 1, characterized in that, The eutectic basic unit is composed of one molecule of epimedium and one molecule of urea. Its crystallographic parameters are: monoclinic crystal system, space group P21 / c; cell parameters a=10.0320(5) Å, b=32.8535(11) Å, c=7.1847(4) Å, α=90 °, β=110.872(6) °, γ=90 °, cell volume V=2212.6(2) Å. 3 .

3. A method for preparing the icariin-urea eutectic as described in any one of claims 1-2, characterized in that, The method includes the following steps: placing icariin and urea in an organic solvent, heating and stirring, cooling to crystallize, filtering, washing, and drying to obtain icariin-urea eutectic; wherein the organic solvent is one or a combination of acetonitrile, acetone, tetrahydrofuran, methanol, ethanol, and isopropanol.

4. The method for preparing the icariin-urea eutectic according to claim 3, characterized in that, The molar ratio of epimedium to urea is 1:1 to 1.5; the mass-to-volume ratio of epimedium to organic solvent is 5 to 7:1, where mass is expressed in mg and volume in mL.

5. Use of the icariin-urea eutectic according to any one of claims 1-2 in the preparation of medicaments for treating liver cancer, breast cancer, and acute myeloid leukemia.

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