WO2016141503A1 - 一种稳定的不含溶剂化物的阿普司特晶型ii及其制备方法 - Google Patents

一种稳定的不含溶剂化物的阿普司特晶型ii及其制备方法 Download PDF

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WO2016141503A1
WO2016141503A1 PCT/CN2015/000566 CN2015000566W WO2016141503A1 WO 2016141503 A1 WO2016141503 A1 WO 2016141503A1 CN 2015000566 W CN2015000566 W CN 2015000566W WO 2016141503 A1 WO2016141503 A1 WO 2016141503A1
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disease
crystal form
crystal
sarcoidosis
acetone
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PCT/CN2015/000566
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English (en)
French (fr)
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罗军芝
年静
高思原
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上海优拓医药科技有限公司
罗军芝
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Priority to RU2017126491A priority Critical patent/RU2673889C1/ru
Priority to EP15884186.6A priority patent/EP3269711B1/en
Priority to JP2017553290A priority patent/JP6457658B2/ja
Priority to AU2015385707A priority patent/AU2015385707B2/en
Publication of WO2016141503A1 publication Critical patent/WO2016141503A1/zh
Priority to US15/638,384 priority patent/US9850205B2/en

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Definitions

  • the present invention relates to apricot crystal form II, in particular to a crystal form II of a small molecule inhibitor of phosphodiesterase 4 (PDE4), a pharmaceutical composition, a preparation method thereof, and a method for preparing the same
  • PDE4 phosphodiesterase 4
  • the invention belongs to the field of medicinal chemistry.
  • the drug was approved for marketing in the United States in March 2014 for the treatment of psoriatic arthritis; it was approved for the treatment of psoriasis in September.
  • the literature on the crystal form of the drug mainly includes the Chinese patent CN102702070A, which has reported a total of seven forms of solid forms of the solid forms of Aptast in the form A, B, C, D, E, F and G.
  • the crystal form, according to its reported XRPD chart, DSC chart and TGA chart and data, is summarized as follows, see Table 1.
  • the crystal forms C, D, E and G are solvates, which are not suitable for medicinal use, and the crystal forms A, B and F are unsolvated or substantially free of solvents.
  • the patent also reports interconversion phenomena and results between various crystal forms, and there is no specific embodiment for the preparation method of each crystal form, and thus it is difficult to reproduce.
  • Patent CN102702070A and Celgene's registration information submitted to EMEA clearly indicate that among the above seven solid forms or crystal forms, Form B is the most thermodynamically stable crystalline form and is suitable as a pharmaceutically acceptable crystalline form, easy to store, suitable for formulation. machining.
  • Celgene's commercial Otezla is valid for about one year, which is extremely good for commercial products. It is unfavorable. Therefore, it is necessary to find a thermodynamically more stable crystal form, which is beneficial to the long-term storage of raw materials and preparations without affecting the activity in the body, and even the activity in vivo is superior to the existing pharmaceutical form B.
  • the present invention provides a novel stable solvate-free apricot crystal form II, composition and use, and the present invention also provides a simple and industrially suitable method for preparing apricot crystal form II.
  • the present invention also provides a method for preparing an apulite mixed crystal, that is, a method for preparing a mixed crystal containing aphetite crystal form II and form B.
  • the Aphrodite Form II of the present invention is superior in thermodynamic stability to the various crystalline forms of the existing Props, such as Forms A, B, C, D, E, F or G.
  • Form II of the present invention exhibits greater in vivo activity than existing pharmaceutically acceptable Form B.
  • Patent CN201410335852.6 relates to an industrialization method for preparing apriste and an intermediate thereof, and the patent CN201410420960.3 relates to a stable solvate-free apricot crystal form I and a preparation method thereof, all of which are technically applicable to the present application.
  • the related patent application the entire disclosure of which is incorporated herein by reference.
  • DSC differential thermal analysis
  • TG thermogravimetric
  • the crystalline form II of the present invention has a melting point of 146 to 151 °C.
  • the present invention provides a process for the preparation of the above-mentioned solvate-free Form II of pristone, characterized in that the solvent used is a mixed solvent composed of acetone and water or THF and water or three. A mixed solvent of acetone and water is preferred.
  • the preparation method described above is characterized by comprising:
  • the apulstat or its solvate is heated and dissolved in 2 to 10 times acetone (ml/g), preferably 3-5 times acetone, and then cooled to 40 ° C or lower, and slowly added dropwise to the amount of acetone corresponding to 0.5 to 0.5 ° 2 times the amount of water, with or without the seed crystal of crystal form II, then stir for 0.5-3 hours, then add water equivalent to 2-6 times the amount of acetone, add, and stir at 20 ° C to reflux temperature 1 - 24 hours, filtered, washed with water and dried to give a white solid, ie, aplast crystal form II.
  • the powder X-ray diffraction pattern of Form II obtained by the difference in the stirring time after adding water is slightly different, but the main characteristic peak of 2 ⁇ is between 0 and 20 degrees, no difference, see Fig. 2 and Fig. 5, the differential thermal analysis and heat
  • the weight loss map is basically indistinguishable.
  • the present inventors have surprisingly found that the temperature at which crystallization is crystallized with acetone/water or THF/water, the amount of initial water added, and agitation have an important influence on the type of crystal form formed, and a temperature of less than 40 ° C is advantageous for obtaining Form II. More advantageously, the addition of seed crystals of Form II during the crystallization process is more advantageous for the rapid formation of Form II.
  • After the initial addition of water stir for 1-3 hours to allow the crystal form II to be analyzed, and then add 2-6 times the amount of water corresponding to the amount of acetone or THF, and stir at 20 ° C to reflux temperature for 1-24 hours to make The Form II powder is finer, then cooled, filtered, and dried to give Form II.
  • the present invention also provides a second method for preparing Form II, that is, other crystal forms of Appast, such as Form A, B, C, D, E, F, G or crystal, of suitable particle size.
  • One or more of the formula I is suspended in acetone and water or THF and water or a mixed solvent of three, preferably suspended in a mixed solvent of acetone and water, followed by stirring at 30 ° C - reflux temperature 72 hours or longer, then cooled, filtered, and dried to give Form II.
  • the ratio of acetone to water is preferably from 1:1 to 1:4.
  • crystallizing with acetone/water can also obtain an apricot mixed crystal, that is, a mixture of Aphrodite crystal form B and crystal form II, that is, by controlling the ratio of acetone to water, the drop rate of water or adding Time can make the ratio of crystal form B to form II in the mixed crystal It may be arbitrary, that is, the proportion of the crystal form II in the mixed crystal is between 0 and 100%.
  • the X powder diffraction pattern (XRPD) of the mixed crystal will vary greatly due to the different ratios of the two, and the DSC spectrum will also show two absorption peaks at 150 ⁇ 3°C and 157 ⁇ 3°C. The intensity of the absorption peak will vary depending on the ratio of the two.
  • the preparation of the apulite mixed crystal can also be carried out by suspending other crystalline forms of apricot, such as crystal form A, B, C, D, E, F or G, in acetone and water, THF. It is obtained by mixing with water or a mixed solvent of three, and then stirring at 20 ° C - reflux temperature for different times. Similarly, various ratios of mixed crystals can be obtained by controlling the stirring time.
  • the Appast blended crystal referred to in the present invention means two or more crystal forms, at least one of which is apricot crystal form II, and the other one is selected from other crystal forms reported in the literature, such as Prostat crystal form A, B, C, D, E, F or G, preferably Form B or Form F.
  • the apulite mixed crystal according to the present invention is preferably a mixed crystal composed of the crystalline form II and the crystalline form B, and the ratio of the two may be arbitrary.
  • a mixed crystal composed of Form II and Form B can also be used as a pharmaceutically active ingredient.
  • the white or off-white product of the aplitt crystal form II can be obtained by crystallization with acetone/water, and a high purity, more than 99.8%, a single impurity of less than 0.1% of the apster product, and the product are obtained.
  • the optical isomers do not substantially change.
  • the resulting Appastene Form B which was crystallized from acetone or acetone/ethanol, was slightly yellow in color, but the most unfavorable was that the R-isomer per crystallization of the product increased accordingly.
  • Chinese patent CN102702070 reports that the optical purity of the product prepared by the optical purity of 99.2% intermediate is only 98%, and the obtained product is easy to agglomerate during drying, and it is easy to form larger crystals when the amount of ethanol is large.
  • the granules are also difficult to pulverize and are difficult to directly use in the processing of the preparation.
  • the aplast crystal form II obtained in the present invention is usually in the form of a powder, and in some cases the particle size D 90 is less than 15 um.
  • the resulting product can be used for formulation processing without pulverization.
  • thermogravimetric map shows that the crystal form II with no or substantially no solvate is obtained, and the X-ray diffraction pattern data is completely different from the crystal forms A, B, C, D, E, F and G reported by Chinese patent CN102702070.
  • XRPD X-ray powder diffraction pattern
  • IR infrared spectrum
  • DSC differential thermal analysis
  • TGA thermogravimetric map
  • the X-ray powder diffraction pattern shows that Form II has the following characteristic absorption peak at a reflection angle of 2 ⁇ ⁇ 0.2, Figure 8:
  • the X-ray powder diffraction pattern of the aplitt crystal form II has a main characteristic absorption peak at a reflection angle of 2 ⁇ ⁇ 0.2 or less of 11.2, 13.2, 13.5, 13.8, 14.7, 16.2, 17.9, 18.7, 20.2, 20.7, 27.0.
  • the X-ray powder diffraction pattern of the Aphrodite Form II has the following five main characteristic absorption peaks at the reflection angle of 2 ⁇ ⁇ 0.2: 11.2, 13.2, 13.5, 13.8, 14.7.
  • the X-ray powder diffraction pattern of Appastene Form II has the following two main characteristic absorption peaks at a reflection angle of 2 ⁇ ⁇ 0.2: 11.2, 14.7.
  • the characteristic absorption peak of the crystal form II obtained by using acetone and water at a reflection angle of 2 ⁇ 0.2 in the range of 20-30° may cause a certain difference due to the measurement error, but whether the above-mentioned main one is caused by the reflection angle of the obtained product 2 ⁇ 0.2
  • the characteristic absorption peak can be used to judge whether the product contains part or all of Form II. It is particularly important that the characteristic absorption peaks of Form II in the range of 2 ⁇ ⁇ 0.2 in the range of 10-20° are substantially identical, see Figure 2, Figure 5, Figure 6, Figure 8, Figure 12, Figure 14, attached Figure 16 and Figure 17.
  • the DSC spectra were not much different, showing a single absorption peak at 100-180 ° C, located at 150 ⁇ 3 ° C.
  • the preparation containing the aplast crystal form II as the active component also exhibits the above-mentioned main characteristic absorption peaks at the reflection angle 2 ⁇ 0.2, such as 11.2, 13.2, 13.5, 13.8, 14.7, etc., or 11.2, 14.7.
  • the differential thermal analysis (DSC) pattern of the Aphrodite Form II shows a single peak at 100-180 ° C, 150 ⁇ 3 ° C; the thermogravimetric (TG) pattern of Form II shows no or substantially no crystalline solvent or Water, weight loss begins to decompose at around 250 °C, see Figure 3; Form II has a melting point of 146-151 °C.
  • the crystal form II and the crystal form B of the pulverized apulast are suspended in water, respectively, stirred at 60 ° C and 100 ° C for 24-48 hours, then cooled and filtered, and dried to test the X powder diffraction pattern, DSC chart, Melting point and related substances, etc., the results are shown in Table 4.
  • thermodynamic stability of Form B of Apostatin is inferior to that of Form II of Apostat, and the X-ray diffraction pattern and DSC chart of Form B after heating show the characteristic absorption peak of Form II.
  • Form II has almost no change.
  • the electrostatic effect of Form II is weak or almost absent, while Form B clearly produces a greater electrostatic effect.
  • the crystal form B is suspended in water at 100 ° C for 24 hours, filtered, and the electrostatic effect of the crystal form B is extremely serious after drying, while the crystal form II has almost no electrostatic effect, and the electrostatic effect is greater on the preparation of the preparation. The more unfavorable.
  • the pure crystalline form II can be obtained by suspending the pulverized crystal form A, B, C, D, E, F and/or G in acetone/water and stirring at 50-reflow temperature for 1-48 hours. This again shows that the thermodynamic stability of Form II is better than Form B.
  • Form B is considered to be the most thermodynamically stable crystal form and is suitable for medicinal use.
  • Our existing experiments show that Form II is more stable in thermodynamic stability than Form B, which is more conducive to long-term storage, and is more suitable as a medicinal crystal form for the processing of preparations.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned solvate-free form of Form II of ampastide as a pharmaceutically active ingredient and a pharmaceutically acceptable carrier, wherein Pharmaceutical active ingredient
  • the content of the special crystal form II is 1-100%.
  • the solvate-free Appastene Form II prepared according to the present invention can also be used for the preparation of a medicament for treating a disease or condition which can be ameliorated by inhibiting TNF- ⁇ production.
  • a disease or condition selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, rheumatoid arthritis, atopic dermatitis, Behcet's mouth ulcer, chronic cutaneous sarcoidosis , giant cell arteritis, Parkinson's disease, nodular pruritus, lichen planus, complex oral disease, lupus, hepatitis, uveitis, Sjogren's syndrome, depression, interstitial cystitis, vulvar pain, prostatitis, bone Arthritis, diffuse large B-cell tumor, polymyositis, dermatomyositis, inclusion body myositis, erosive osteoarthritis,
  • the solvate-free form of Form I of solvate prepared in accordance with the present invention is equally useful in the manufacture of a medicament for treating a disease or condition ameliorated by inhibition of PDE4, wherein the disease or The condition is selected from: HIV, hepatitis, adult respiratory distress syndrome, bone resorption disease, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, dermatitis, inflammatory skin disease, atopic dermatitis, cystic fibrosis, septic shock , sepsis, endotoxic shock, hemodynamic shock, sepsis syndrome, post-ischemic reperfusion injury, meningitis, psoriasis, fibrotic disease, psoriatic arthritis, cachexia, transplant rejection, graft versus host Disease, autoimmune disease, rheumatoid spondylitis, arthritic condition, ankylosing spondylitis, rheumatoid arthritis, osteoarthritis, osteoporosis, segmental enter
  • the solvate-free form of Form I of solvate prepared according to the present invention can also be used for the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of multiple myeloma, malignant melanoma, malignant Glioma, leukemia and solid tumors.
  • the use of the solvate-free Appastene Form II prepared according to the present invention for the preparation of a medicament for treating sarcoidosis wherein the sarcoidosis is selected from the group consisting of cardiac sarcoidosis and skin sarcoidosis , hepatic sarcoidosis, oral sarcoidosis, nervous system sarcoidosis, nasal sinus sarcoidosis, Lovegren's syndrome, frostbite-like lupus, uveitis or chronic skin sarcoidosis.
  • solvate-free APS crystal form II can be used as an active ingredient for the treatment of the above diseases or symptoms.
  • suitable pharmaceutical dosage forms include tablets, capsules, dispersible tablets, orally disintegrating tablets, etc. Not limited to lactose, mannitol, crospovidone, micronized silica gel, magnesium stearate, microcrystalline cellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, croscarmellose sodium, dioxide Silicon and so on. Preferred are lactose, microcrystalline cellulose, croscarmellose sodium, micronized silica gel, magnesium stearate and the like.
  • the apulast mentioned in the present invention can be referred to existing literature such as US6020358, US6962940 or CN201410335852.6.
  • the reported method is easy to prepare.
  • Figure 1 X-ray powder diffraction pattern (XRPD) of Aphrodite Form B
  • Figure 2 X-ray powder diffraction pattern (XRPD) of Aptast Form II, stirred for 4 h after adding water
  • FIG. 3 Thermal weight loss (TG) pattern and differential thermal analysis (DSC) pattern of Aptast Form II, stirred for 23 hours after adding water
  • Figure 5 X-ray powder diffraction pattern (XRPD) of Appastene Form II, stirred for 23 h after adding water
  • Figure 6 X-ray powder diffraction pattern (XRPD) of Aptast Form II, stirred at 60 ° C for 48 h
  • Figure 7 X-ray powder diffraction pattern (XRPD) of Apostatin Form B, stirred at 60 ° C for 48 h
  • Figure 8 X-ray powder diffraction pattern (XRPD) of Appastene Form II, stirred at 100 ° C for 24 h
  • Figure 9 X-ray powder diffraction pattern (XRPD) of Apostatin Form B, stirred at 100 ° C for 24 h
  • Figure 10 DSC chart of Aphrodite Form II, stirred at 100 ° C for 24 h
  • Figure 11 DSC chart of Aptast Form B, stirred at 100 ° C for 24 h
  • Figure 12 Aptast Form B suspended in acetone/water, stirred at 70 ° C for 36 h, X-ray powder diffraction pattern (XRPD) of the product obtained
  • Figure 13 The differential thermal analysis (DSC) pattern of the product obtained by suspending the propionate Form B in acetone/water and stirring at 70 ° C for 36 h.
  • Figure 14 X-ray powder diffraction pattern (XRPD) of the product obtained by suspending the propionate Form D in acetone/water and stirring at 70 ° C for 10 h.
  • XRPD X-ray powder diffraction pattern
  • FIG. 15 Differential thermal analysis (DSC) of the product obtained by suspending the propionate Form D in acetone/water and stirring at 70 ° C for 10 h.
  • Figure 16 X-ray powder diffraction pattern (XRPD) of Aptast Form II
  • Figure 17 X-ray powder diffraction pattern (XRPD) of Aptast Form II
  • Figure 18 X-ray powder diffraction pattern (XRPD) of apulite mixed crystals (i.e., Form II and Form B), the ratio of which is approximately 1:1
  • Figure 19 Plasma concentration-time curve after intragastric administration of 10 mg/kg APST-B suspension in rats
  • Figure 20 Plasma concentration-time curve after intragastric administration of 10 mg/kg APST-II suspension in rats
  • Figure 21 Plasma concentration-time curve after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions in male rats
  • Figure 22 Plasma concentration-time curve after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions in female rats
  • Figure 23 Plasma concentration-time curve after oral administration of 30 mg APST-B and APST-II API powder capsules in a single cycle of beagle dogs (the upper graph is constant coordinates, the lower graph is semi-logarithmic coordinates)
  • ⁇ type aluminum ⁇ (needle piercing)
  • Purge gas high purity nitrogen, 20mL/min
  • Purge gas high purity nitrogen, 20mL/min
  • the main drug passes through a 200 mesh sieve, the filler and the disintegrant pass through the 80 mesh sieve, and the prescribed amount of the filler and the disintegrant are uniformly mixed, and then the prescribed amount of the main drug is uniformly mixed with the equal amount of the addition method, and the prescription is added.
  • the amount of glidant and lubricant, after mixing evenly, the tablet is obtained.
  • the main drug passes through a 200 mesh sieve, the filler and the disintegrant pass through the 80 mesh sieve, and the prescribed amount of the filler and the disintegrant are uniformly mixed, and then the prescribed amount of the main drug is uniformly mixed with the equal amount of the addition method, and the prescription is added.
  • the amount of glidant and lubricant, after mixing evenly, the tablet is obtained.
  • Crystallize Form II and Form B of Apostatin were suspended in water, stirred at 60-100 ° C for 48 h, sampled and filtered at intervals, dried to test the change of melting point, and sampled and dried at 24 h, 48 h.
  • Apulcit crystal form B (5.0 g, particle size below 300 mesh) was suspended in 60 ml of acetone/water (1:3), heated to reflux, stirred for 36 h, then cooled under stirring, filtered, dried at 60 ° C. Obtained about 4.88 g of Appastene Form II, mp: 148.3-150.3 °C.
  • the X-ray diffraction pattern and the DSC pattern showed complete conversion to Form II.
  • the apster crystal form D (5.0 g, particle size below 200 mesh) was suspended in 60 ml of acetone/water (1:3), heated to reflux, stirred for 8 to 10 h, then cooled under stirring, filtered, 60 ° C Drying, about 4.60 g of Appastene Form II, mp: 148.3-150.5 °C.
  • the X-ray diffraction pattern and the DSC pattern showed complete conversion to Form II.
  • Example 9 Comparative study of pharmacokinetics of aplast crystal form B and form II in SD rats
  • the particle size of the two crystal forms is controlled by micronization as follows:
  • the pharmacokinetic behavior of the two crystal forms in rats was selected by intragastric administration.
  • the doses of both crystal forms were 10 mg/kg (solvent was 1% carboxymethyl cellulose). ). Fasting was given for 12 h before the test, and food was provided 4 h after the administration. The whole experiment was not water-free.
  • the concentration of APST in plasma was determined by LC-MS/MS method.
  • the linear range of APST in plasma was determined to be 2 ⁇ 1000 ng/ml, and the linearity was good.
  • the plasma concentration-time curves of each group of animals were shown in Figures 19 to 22 after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions, respectively.
  • the main pharmacokinetic parameters of APST in rats after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions were as follows: peak time Tmax was 1-6 h (median 2.5 h) and 1-6h (median 3h), the peak concentration Cmax was 523.05 ⁇ 417.46 and 506.90 ⁇ 451.89ng/ml, respectively.
  • the area under the plasma concentration-time curve AUC0-t was 3766.48 ⁇ 3617.82 and 5533.11 ⁇ 5613.02ng ⁇ h. /ml, the exposure of 10mg/kg APST-B in rats was about 68.07% of APST-II.
  • the difference between the two groups was large. Because the exposure was very different, the standard deviation between the two groups was larger. There was no statistically significant difference in exposure. Therefore, the in vivo pharmacokinetics of male and male and female rats were further analyzed.
  • the main pharmacokinetic parameters of APST in male rats after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions were as follows: peak time Tmax was 1 h (median 1 h) and 1-2 h ( The median was 1 h), the peak concentrations of Cmax were 142.4 ⁇ 13.96 and 107.63 ⁇ 16.05 ng/ml, respectively.
  • the area under the plasma concentration-time curve AUC0-t was 530.44 ⁇ 70.05 and 445.59 ⁇ 81.25 ng ⁇ h/ml, respectively.
  • the Cmax and AUC0-t of male/kgAPST-B in male rats were 132.3% and 119.0% of APST-II, respectively.
  • the results showed that the absorption of APST-B in male rats was higher than that of APST-II, but the difference was not statistical. Learning meaning.
  • the main pharmacokinetic parameters of APST in female rats after intragastric administration of 10 mg/kg APST-B and APST-II suspension solutions the peak time Tmax was 2-6 h (median 4 h) and 4 -6h (median 6h), the peak concentration Cmax was 903.7 ⁇ 28.47 and 906.17 ⁇ 178.89ng/ml, respectively.
  • the area under the plasma concentration-time curve AUC0-t was 7002.52 ⁇ 1140.54 and 10620.62 ⁇ 1053.56ng ⁇ h/
  • the AUC0-t of APST-B in female rats was 65.9% of APST-II.
  • the results showed that the absorption of APST-B in female rats was lower than that of APST-II, and the difference was statistically significant ( P ⁇ 0.05).
  • Example 10 Comparative Study on Pharmacokinetics of Apostatin Form B and Form II in Beagle Dogs
  • the oral administration is selected, and the dosage is 30 mg/time/time.
  • the powders of the two crystal forms are weighed about 30 mg respectively, and then filled with a capsule shell, and 10 ml water is used for administration. clothes. Beagle dogs were fasted for 12 hours before the test, and food was provided 4 hours after the administration. The whole experiment was not water-free.
  • the specific drug administration group design is shown in the following table.
  • venous blood was taken from the small saphenous vein for about 0.5 ml, and centrifuged at 8000 rpm for 5 minutes. The plasma was separated into anti-freezing plastic test tubes and placed in a -20 ° C refrigerator for free storage.
  • the concentration of APST in plasma was determined by LC-MS/MS method, and the linear range of APST in plasma was determined to be 5 to 5000 ng/ml, and the linearity was good.

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Abstract

一种稳定的不含溶剂化物的阿普司特(式I)晶型II及其制备方法、药物组合物和医药用途，以及包含晶型II和晶型B的混晶及其制备方法。所述的阿普司特晶型II的X-射线粉末衍射图(XRPD)的2θ±0.2具以下五个特征吸收峰：11.2，13.2，13.5，13.8，14.7，其DSC图谱显示单一吸热峰，位于150±3℃。晶型II的活性不低于晶型B，热力学稳定性方面优于晶型B，其制备方法操作简单，所用结晶溶剂安全并易于除去。

Description

一种稳定的不含溶剂化物的阿普司特晶型II及其制备方法 技术领域
本发明涉及阿普司特晶型II，具体地说，涉及一种磷酸二酯酶4(PDE4)的小分子抑制剂阿普司特的晶型II、药物组合物及其制备方法，以及使用该晶型治疗各种疾病或病症的用途。本发明属于药物化学领域。
背景技术
众所周知，结晶溶剂不同或结晶方法差异，如结晶温度，冷却速度的不同，搅拌或静置等，甚至同样的溶剂，操作的微妙差异都会产生不同的晶型，而不同的晶型具有不同的稳定性和溶解性，有时甚至在体内的生物利用度不一样。因而在药物开发中需要寻找一种获得高纯度的且热力学稳定的晶型非常必要，并且该方法易于重现且适合工业大规模的制备。而在晶型研究中，X-射线粉末衍射图(XRPD)、红外图谱(IR)、差热分析图谱(DSC)以及热失重图谱(TG)是确定晶型的一种有效手段。
阿普司特(Apremilast，化合物I，化学名：(+)-2-[1-(3-乙氧基-4-甲氧基苯基)-2-甲磺酰基乙基]-4-乙酰基氨基异吲哚啉-1，3-二酮)是一种磷酸二酯酶4(PDE4)的小分子抑制剂，特定作用于单磷酸环腺苷酸(cAMP)，PDE4抑制可导致细胞内cAMP水平升高，能够减轻关节肿胀并改善关节部位的生理机能。结构式如下：
Figure PCTCN2015000566-appb-000001
该药物已于2014年3月在美国获准上市，用于治疗银屑病性关节炎；9月有获准用于治疗银屑病。然而，关于该药物的晶型文献报道主要有中国专利CN102702070A，该专利总共报道了阿普司特的固体形式有形式A，B，C，D，E，F和G，共7种固体形式或晶型，根据其报道的有关XRPD图，DSC图以及TGA图及数据，现总结如下，见表一。
其中晶型C、D、E及G为溶剂化物，不适合药用，晶型A、B及F为非溶剂化物或基本不含溶剂。该专利同时报道了各种晶型之间的互变现象和结果，而对于每一种晶型的制备方法并无具体实施例，因而难以重现。
表一、CN102702070A报道的阿普司特晶型的数据
Figure PCTCN2015000566-appb-000002
专利CN102702070A及Celgene公司提交给EMEA注册资料均明确指出上述7种固体形式或晶型中，晶型B是热力学最稳定的晶型并适于作为药用晶型，易于储存，适合用于制剂的加工。然而，Celgene的市售商品Otezla的有效期大约为一年，这对商业化的产品来说是极 为不利的。因而需要找到一种热力学更稳定的晶型，有利于原料和制剂的长期储存，同时又不影响体内的活性，甚至体内活性优于现有药用晶型B。
发明内容
本发明提供了一种新颖的稳定的不含溶剂化物的阿普司特晶型II、组合物及用途，本发明还提供简单的适合工业化的制备阿普司特晶型II的方法。另外，本发明也提供了一种阿普司特混晶的制备方法，即制备含阿普司特晶型II和晶型B的混晶的方法。
尤为重要的是，本发明的阿普司特晶型II在热力学稳定性方面均要优于现有报道的阿普司特各种晶型，如晶型A，B，C，D，E，F或者G。
并且，在某些方面，与现有的药用晶型B相比，本发明的晶型II显示更强的体内活性。
专利CN201410335852.6涉及制备阿普司特及其中间体的产业化方法，专利CN201410420960.3涉及一种稳定的不含溶剂化物阿普司特晶型I及其制备方法，均为本申请技术上相关的专利申请，其专利申请全文将引入本申请。
本发明不含溶剂化物的阿普司特晶型II特征如下：
一种式(I)的不含溶剂化物的阿普司特晶型II，
Figure PCTCN2015000566-appb-000003
其X-射线粉末衍射图(XRPD)在2θ±0.2具如下典型特征吸收峰，图8：
# 2-Theta d(A) Height I％ Area I％ FWHM
1 10.666 8.2876 568 10.9 1090 2.4 0.077
2 11.290 7.8312 3576 68.5 45434 100.0 0.234
3 12.573 7.0346 833 15.9 2789 6.1 0.111
4 13.203 6.7001 3140 60.1 31411 69.1 0.192
5 13.541 6.5338 2509 48.0 22813 50.2 0.174
6 13.874 6.3775 1946 37.3 17299 38.1 0.193
7 14.724 6.0114 5224 100.0 42800 94.2 0.149
8 16.223 5.4592 2582 49.4 21876 48.1 0.163
9 17.924 4.9448 2862 54.8 24450 53.8 0.164
10 18.751 4.7285 2068 39.6 15517 34.2 0.153
11 20.290 4.3731 1268 24.3 7487 16.5 0.139
12 20.725 4.2822 832 15.9 3836 8.4 0.143
13 21.531 4.1238 1573 30.1 8335 18.3 0.132
14 21.989 4.0388 1666 31.9 18358 40.4 0.277
15 22.778 3.9007 1887 36.1 19298 42.5 0.244
16 23.194 3.8317 1404 26.9 19738 43.4 0.375
17 25.265 3.5221 1412 27.0 22193 48.8 0.382
18 25.641 3.4714 1702 32.6 19926 43.9 0.268
19 26.587 3.3499 1904 36.4 20529 45.2 0.244
20 27.022 3.2970 3644 69.8 44458 97.9 0.234
21 27.596 3.2297 962 18.4 5166 11.4 0.194
22 28.226 3.1590 1147 22.0 7133 15.7 0.170
23 29.112 3.0648 581 11.1 973 2.1 0.103
24 29.667 3.0087 463 8.9 696 1.5 0.133
25 30.934 2.8884 482 9.2 1767 3.9 0.207
26 32.035 2.7916 486 9.3 1075 2.4 0.150
27 33.023 2.7103 450 8.6 1343 3.0 0.215
28 33.597 2.6653 473 9.1 2192 4.8 0.261
29 34.207 2.6191 509 9.7 2957 6.5 0.263
30 34.915 2.5676 404 7.7 1621 3.6 0.275
31 36.222 2.4779 336 6.4 662 1.5 0.174
32 39.625 2.2726 318 6.1 343 0.8 0.087
33 40.351 2.2334 311 6.0 690 1.5 0.156
34 41.080 2.1954 288 5.5 1946 4.3 0.583
35 43.804 2.0650 372 7.1 1599 3.5 0.190
其差热分析(DSC)图谱显示在100-180℃之间有一个吸热峰，为150±3℃。其热失重(TG)图谱显示不含或基本不含结晶水或结晶溶剂。
本发明所述的晶型II的熔点为146～151℃。
另一方面，本发明还提供了制备上述的不含溶剂化物的阿普司特的晶型II的方法，其特征在于所用溶剂是由丙酮和水或者THF和水或三者组成的混合溶剂，优选丙酮和水组成的混合溶剂。
具体地说，上述所述的制备方法，其特征在于包括：
i)将阿普司特或其溶剂化物加热用丙酮或THF溶解，然后冷却至40℃以下，接着
ii)搅拌下缓慢滴加相当于0.5～2倍丙酮或THF量的水，加入或者不加入阿普司特晶型II的晶种，搅拌30～180分钟，然后
iii)继续加入相当于2～6倍丙酮或THF量的水，于20℃至回流温度下搅拌1～24小时，然后iv)过滤，烘干得到阿普司特晶型II。
其中，作为优选，上述所述的制备方法具体为：
将阿普司特或其溶剂化物加热溶解于2～10倍丙酮(ml/g)中，优选3-5倍丙酮，接着冷却至40℃以下，搅拌下缓慢滴加相当于丙酮量的0.5～2倍量的水，加或者不加晶型II的晶种，然后保温搅拌0.5-3小时后再加入相当于2～6倍丙酮量的水，加毕，于20℃至回流温度下搅拌1-24小时，过滤，用水洗涤，烘干得到白色固体，即阿普司特晶型II。加水之后搅拌的时间的不同得到的晶型II的粉末X衍射图谱略有不同，但其2θ位于0-20度之间主要特征峰无区别，见图2和图5，其差热分析和热失重图谱基本无区别。
本发明人惊奇的发现，用丙酮/水或THF/水结晶析晶时的温度、初始加入水的量及搅拌对生成晶型类型有重要影响，温度小于40℃有利于得到晶型II。更为有利的是，在析晶过程中加入晶型II的晶种更有利于快速生成晶型II。初始加水后，搅拌1-3小时以使晶型II充分析出，然后再加入相当于丙酮或THF量的2-6倍量的水，于20℃至回流温度下保温搅拌1-24小时以使晶型II粉末更细腻，然后冷却，过滤，烘干得到晶型II。
另一方面，本发明也提供了第二种制备晶型II的方法，即将粒径适当的阿普司特其他晶型，如晶型A，B，C，D，E，F，G或晶型I中的一种或几种混悬于丙酮和水或THF和水或者三者的混合溶剂中，优选混悬于丙酮和水的混合溶剂中，接着于30℃-回流温度下搅拌1-72小时或更长，然后冷却，过滤，烘干得到晶型II。研究表明粒径越小转化为晶型II所需时间越短，越不稳定的晶型越容易转化为晶型II。丙酮和水的比例优选1∶1～1∶4。
另外，用丙酮/水结晶也可以得到一种阿普司特混晶，即阿普司特晶型B与晶型II的混晶，即通过控制丙酮和水的比例、水的滴加速度或加入时间可以使混晶中晶型B与晶型II的比例 可以是任意的，即混晶中晶型II所占比例在0-100％之间。所述混晶的X粉末衍射图谱(XRPD)会因两者的比例不一样而差别较大，且其DSC图谱也会在150±3℃及157±3℃显示两个吸收峰，这两个吸收峰的强度会因为两者比例不同而强度各不相同。总之，通过X粉末衍射图谱是否含有晶型II的上述特征吸收峰即可判断混晶中是否含有晶型II，并通过DSC图谱在150±3℃是否有吸收峰来进一步确认是否含有晶型II。
显然，阿普司特混晶的制备也可通过将粒径适当的阿普司特其他晶型，如晶型A，B，C，D，E，F或G混悬于丙酮和水、THF和水或三者的混合溶剂中，然后于20℃-回流温度下搅拌不同时间得到。同样地，通过控制搅拌时间，温度也可以得到各种比例的混晶。
本发明所指的阿普司特混晶指含有两种或两种以上的晶型，其中至少一种为阿普司特晶型II，另外一种选自文献报道的其它晶型，如阿普司特晶型A，B，C，D，E，F或G，优选晶型B或晶型F。
进一步地，本发明所述的阿普司特混晶优选是由晶型II与晶型B组成的混晶，且两者的比例可以是任意的。显然，由晶型II与晶型B组成的混晶也可作为药用活性成分。但从热力学稳定性角度来讲，优选用单一的晶型II作为药用活性成分。
有利的是，通过用丙酮/水结晶可以得到白色或类白色的阿普司特晶型II产物，并获得高纯度，达99.8％以上，单杂小于0.1％的阿普司特成品，且产物的光学异构体基本不会发生变化。而用丙酮或丙酮/乙醇结晶的得到的阿普司特晶型B颜色略呈黄色，但最为不利的是每结晶一次产物的R-异构体均会相应增加。如中国专利CN102702070报道由光学纯度为99.2％中间体制备得到的产物的的光学纯度仅为98％，且所得产物烘干过程中易结块，在乙醇用量大的时候易生成颗粒较大的晶体，颗粒大也不易粉碎，难以直接用于制剂的加工。
更为有利的是本发明所得阿普司特晶型II通常呈粉末状，某些情况下粒径D90低于15um。所得产物无需粉碎即可用于制剂加工。
具体地，由本发明制备的阿普司特晶型II经X-射线粉末衍射图谱(XRPD)、红外光谱(IR)(KBr压片)、差热分析图谱(DSC)以及热失重图谱(TGA)确证。热失重图谱(TGA)表明得到的是不含或基本不含溶剂化物的晶型II，其X衍射图谱数据与中国专利CN102702070报道的晶型A，B，C，D，E，F和G完全不同，为一种新型的晶型，测试结果如下：
X-射线粉末衍射图显示晶型II在反射角2θ±0.2有如下显著特征吸收峰，图8：。
# 2-Theta d(A) Height I％ Area I％ FWHM
1 10.666 8.2876 568 10.9 1090 2.4 0.077
2 11.290 7.8312 3576 68.5 45434 100.0 0.234
3 12.573 7.0346 833 15.9 2789 6.1 0.111
4 13.203 6.7001 3140 60.1 31411 69.1 0.192
5 13.541 6.5338 2509 48.0 22813 50.2 0.174
6 13.874 6.3775 1946 37.3 17299 38.1 0.193
7 14.724 6.0114 5224 100.0 42800 94.2 0.149
8 16.223 5.4592 2582 49.4 21876 48.1 0.163
9 17.924 4.9448 2862 54.8 24450 53.8 0.164
10 18.751 4.7285 2068 39.6 15517 34.2 0.153
11 20.290 4.3731 1268 24.3 7487 16.5 0.139
12 20.725 4.2822 832 15.9 3836 8.4 0.143
13 21.531 4.1238 1573 30.1 8335 18.3 0.132
14 21.989 4.0388 1666 31.9 18358 40.4 0.277
15 22.778 3.9007 1887 36.1 19298 42.5 0.244
16 23.194 3.8317 1404 26.9 19738 43.4 0.375
17 25.265 3.5221 1412 27.0 22193 48.8 0.382
18 25.641 3.4714 1702 32.6 19926 43.9 0.268
19 26.587 3.3499 1904 36.4 20529 45.2 0.244
20 27.022 3.2970 3644 69.8 44458 97.9 0.234
21 27.596 3.2297 962 18.4 5166 11.4 0.194
22 28.226 3.1590 1147 22.0 7133 15.7 0.170
23 29.112 3.0648 581 11.1 973 2.1 0.103
24 29.667 3.0087 463 8.9 696 1.5 0.133
25 30.934 2.8884 482 9.2 1767 3.9 0.207
26 32.035 2.7916 486 9.3 1075 2.4 0.150
27 33.023 2.7103 450 8.6 1343 3.0 0.215
28 33.597 2.6653 473 9.1 2192 4.8 0.261
29 34.207 2.6191 509 9.7 2957 6.5 0.263
30 34.915 2.5676 404 7.7 1621 3.6 0.275
31 36.222 2.4779 336 6.4 662 1.5 0.174
32 39.625 2.2726 318 6.1 343 0.8 0.087
33 40.351 2.2334 311 6.0 690 1.5 0.156
34 41.080 2.1954 288 5.5 1946 4.3 0.583
35 43.804 2.0650 372 7.1 1599 3.5 0.190
阿普司特晶型II的X-射线粉末衍射图在反射角2θ±0.2具以下主要特征吸收峰为11.2，13.2，13.5，13.8，14.7，16.2，17.9，18.7，20.2，20.7，27.0。
尤为重要的是，阿普司特晶型II的X-射线粉末衍射图在反射角2θ±0.2处同时具以下五个主要特征吸收峰：11.2，13.2，13.5，13.8，14.7。
最后，阿普司特晶型II的X-射线粉末衍射图在反射角2θ±0.2处同时具以下两个主要特征吸收峰：11.2，14.7。
另一方面，利用丙酮和水所得晶型II在反射角2θ±0.2位于20-30°范围的特征吸收峰会因测量误差会产生一定差异，但通过所得产物反射角2θ±0.2处是否有上述主要特征吸收峰即可判断产物是否含有部分或全部晶型II。尤为重要的是晶型II在2θ±0.2位于10-20°范围的特征吸收峰基本一致，参见附图2，附图5，附图6，附图8，附图12，附图14，附图16和附图17。其DSC图谱均无太大的差异，在100-180℃显示单一吸收峰，位于150±3℃。
与空白辅料相比，以阿普司特晶型II作活性组分的制剂也会同时在反射角2θ±0.2处出现上述主要特征吸收峰，如11.2，13.2，13.5，13.8，14.7等，或者11.2，14.7。
阿普司特晶型II的差热分析(DSC)图谱在100-180℃显示单峰，为150±3℃；晶型II的热失重(TG)图谱显示不含或基本不含结晶溶剂或水，250℃左右开始失重并分解，见附图3；晶型II的熔点为146-151℃。
红外(IR)光谱显示晶型II具如下显著特征吸收峰：
3002，2932，1763，1697，1621，1519，1480，1428，1394，1367，1340，1297，1269，1234，1163，1139，1095，1044，1028，971，908，859，826，774，750
下面对晶型I、晶型II和晶型B进行了一系列的比较研究，具体如下：
1、晶型I、晶型II和晶型B特征比较：
阿普司特的晶型I、晶型II和晶型B具体比较见表2。
表2、阿普司特的晶型I、晶型II和晶型B差别比较
Figure PCTCN2015000566-appb-000004
2、晶型I、晶型II和晶型B的影响因素试验：
另外，同一批原料分别用丙酮/乙醇结晶得晶型B、丙酮/水结晶得晶型I以及晶型II，比较产物外观颜色，并对三种晶型进行影响因素研究。结果表明，阿普司特晶型I、晶型II、晶型B在高湿、高温条件下均稳定，光照实验表明两者稳定性差不多，具体比较见下表3。
表3、晶型I、晶型II和晶型B的稳定性实验研究结果
Figure PCTCN2015000566-appb-000005
3、晶型II和晶型B热力学稳定性研究
通过将粉碎的阿普司特的晶型II和晶型B悬浮于水中，分别于60℃和100℃下搅拌24-48小时，然后冷却过滤，烘干测试其X粉末衍射图谱、DSC图、熔点及有关物质等，结果见表4。
表4、阿普司特的晶型II和晶型B热力学稳定性研究
Figure PCTCN2015000566-appb-000006
上述实验结果说明阿普司特的晶型B的热力学稳定性不如阿普司特的晶型II，加热后的晶型B的X衍射图谱和DSC图会显示晶型II的独有特征吸收峰，而晶型II基本无变化。更为有利的是，同样微粉化，晶型II的静电效应弱或几乎没有，而晶型B明显产生更大的静电效应。另外，将晶型B于100℃悬浮于水中搅拌24小时，过滤，烘干后晶型B的静电效应更是极为严重，而晶型II几乎没有静电效应，而静电效应越大对制剂的加工越为不利。100℃搅拌48小时，两者光学异构体基本无明显变化，有关物质N-去乙酰物均有所增加，晶型B的增加幅度略大于晶型II，分别增加0.059％和0.046％，两者差异不太明显。
4、晶型A，B，C，D，E，F，G和晶型II转化研究
通过将粉碎的粒径适当的晶型A，B，C，D，E，F和/或G悬浮于丙酮/水中，于50-回流温度下搅拌1-48小时，可以得到纯的晶型II。这再次说明晶型II的热力学稳定性优于晶型B。
先前Celgene报道的晶型A，B，C，D，E，F和G中，晶型B被认为是热力学最为稳定的晶型，适合药用。而我们现有实验表明晶型II在热力学稳定性上比晶型B更为稳定，这更有利于长期储存，更适合作为药用晶型用于制剂的加工。
5、晶型B与晶型II溶解性试验
比较了粒径接近的晶型II与晶型B在丙酮，丁酮，乙醇，甲醇，乙酸乙酯，乙腈，二氯甲烷，四氢呋喃，石油醚，正己烷，水等常见溶剂中的溶解性，实验表明晶型II与晶型B基本无区别。
6、晶型B与晶型II动物体内的药代动力学研究
1)大鼠体内吸收试验
在SD大鼠体内比较研究粒径基本接近的阿普司特晶型B与晶型II的药代动力学，结果表明本发明的阿普司特晶型II与晶型B均显示出较大的雌雄差异，两者的Tmax，Cmax，T1/2基本一致。在雌性大鼠体内，晶型II的暴露量约为晶型B的1.5倍，因此晶型II显示更强的体内活性。
2)比格犬体内吸收实验
在比格犬体内比较研究粒径基本接近的阿普司特晶型B与晶型II的药代动力学，结果表明本发明的阿普司特晶型II与晶型B的Tmax，Cmax，T1/2及AUC0-t基本一致。
7、药物组合物
作为本发明内容，本发明还提供一种药物组合物，其包含上述所述的不含溶剂化物的阿普司特的晶型II作为药物活性成分和药学上可接受的载体，其中所述的药物活性成分中阿普司 特晶型II的含量为1-100％。
显然，与晶型B的适应症等用途一样，按照本发明制备的不含溶剂化物的阿普司特晶型II同样可用于制备治疗可通过抑制TNF-α产生来改善的疾病或病症的药物中的用途，其中所述的疾病或病症选自银屑病，银屑病关节炎，强直性脊柱炎、类风湿性关节炎、特应性皮炎、白塞病口腔溃疡、慢性皮肤肉样瘤，巨细胞动脉炎、帕金森病、结节性痒疹，扁平苔藓、复杂性口腔病、狼疮、肝炎、葡萄膜炎、干燥综合征、抑郁、间质性膀胱炎、外阴痛、前列腺炎、骨关节炎、弥漫性大B细胞瘤、多肌炎、皮肌炎、包涵体肌炎、侵蚀性骨关节炎、子宫内膜异位、神经病根、和坏疽性脓皮病或慢性阻塞性肺病。
另一方面，按照本发明制备的不含溶剂化物的阿普司特的晶型II也同样可用于制备治疗可通过抑制PDE4来改善的疾病或病症的药物中的用途，其中所述的疾病或病症选自：HIV、肝炎、成人呼吸窘迫综合征、骨吸收疾病、慢性阻塞性肺病、慢性肺部炎性疾病、皮炎、炎症性皮肤疾病、特应性皮炎、囊性纤维化、感染性休克、败血症、内毒性休克、血流动力学休克、败血症综合征、缺血后再灌注损伤、脑膜炎、银屑病，纤维化疾病、银屑病关节炎，恶病质、移植排斥、移植物抗宿主病、自体免疫性疾病、类风湿性脊椎炎、关节炎病症、强直性脊柱炎、类风湿性关节炎、骨关节炎、骨质疏松症、节段性肠炎、溃疡性结肠炎、炎性肠病、多发性硬化、全身性红斑狼疮、麻风病中的麻风结节性红斑、辐射损伤和高氧肺泡损伤。
再者，按照本发明制备的不含溶剂化物的阿普司特的晶型II还可用于制备治疗癌症的药物中的用途，其中所述的癌症选自多发性骨髓瘤、恶性黑色素瘤、恶性胶质瘤、白血病和实体瘤。
最后，按照本发明制备的不含溶剂化物的阿普司特晶型II可用于制备治疗结节病的药物中的用途，其中所述的结节病选自心脏结节病、皮肤结节病、肝结节病、口腔结节病、神经系统结节病、鼻腔鼻窦结节病、洛夫格伦综合症、冻疮样狼疮、葡萄膜炎或慢性皮肤结节病。
显然，不含溶剂化物的阿普司特晶型II可作为用于上述疾病或症状的治疗的活性成分，合适的药物剂型包括片剂，胶囊，分散片，口崩片等，辅料选自但不限于乳糖，甘露醇，交联聚维酮，微粉硅胶，硬脂酸镁，微晶纤维素，羧甲基纤维素钠，羟丙基纤维素，交联羧甲基纤维素钠，二氧化硅等。优选乳糖，微晶纤维素，交联羧甲基纤维素钠，微粉硅胶，硬脂酸镁等辅料。
本发明所提到的阿普司特可参考现有文献如US6020358、US6962940或CN201410335852.6 报道的方法轻易制备。
附图说明
本申请中包括的附图是构成说明书的一部分，附图与说明书和权利要求项一起用于说明本发明的实质内容，用于更好地理解本发明。
图1：阿普司特晶型B的X-射线粉末衍射图(XRPD)
图2：阿普司特晶型II的X-射线粉末衍射图(XRPD)，加水后搅拌4h
图3：阿普司特晶型II的热失重(TG)图谱和差热分析(DSC)图谱，加水后搅拌23h
图4：阿普司特晶型II的红外图谱(IR)，加水后搅拌23h
图5：阿普司特晶型II的X-射线粉末衍射图(XRPD)，加水后搅拌23h
图6：阿普司特晶型II的X-射线粉末衍射图(XRPD)，60℃搅拌48h
图7：阿普司特晶型B的X-射线粉末衍射图(XRPD)，60℃搅拌48h
图8：阿普司特晶型II的X-射线粉末衍射图(XRPD)，100℃搅拌24h
图9：阿普司特晶型B的X-射线粉末衍射图(XRPD)，100℃搅拌24h
图10：阿普司特晶型II的DSC图，100℃搅拌24h
图11：阿普司特晶型B的DSC图，100℃搅拌24h
图12：阿普司特晶型B悬浮于丙酮/水，70℃搅拌36h，所得产物的X-射线粉末衍射图(XRPD)
图13：阿普司特晶型B悬浮于丙酮/水，70℃搅拌36h，所得产物的差热分析(DSC)图谱
图14：阿普司特晶型D悬浮于丙酮/水，70℃搅拌10h，所得产物的X-射线粉末衍射图(XRPD)
图15：阿普司特晶型D悬浮于丙酮/水，70℃搅拌10h，所得产物的差热分析(DSC)图谱
图16：阿普司特晶型II的X-射线粉末衍射图(XRPD)
图17：阿普司特晶型II的X-射线粉末衍射图(XRPD)
图18：阿普司特混晶(即晶型II和晶型B)的X-射线粉末衍射图(XRPD)，两者比例大致1∶1
图19：大鼠灌胃10mg/kg APST-B混悬溶液后血浆浓度-时间曲线
图20：大鼠灌胃10mg/kg APST-II混悬溶液后血浆浓度-时间曲线
图21：雄性大鼠分别灌胃10mg/kg APST-B和APST-II混悬溶液后血浆浓度-时间曲线
图22：雌性大鼠分别灌胃10mg/kg APST-B和APST-II混悬溶液后血浆浓度-时间曲线
[根据细则91更正 08.09.2015]　
图23：比格犬单周期分别经口给予30mg APST-B和APST-II原料药粉末胶囊后血浆浓度-时间曲线(上图为常量坐标，下图为半对数坐标)
测试仪器及方法：
1、X射线粉末衍射(XRPD)：
仪器型号：Bruker D8 ADVANCE粉末X射线衍射仪
实验条件：光源：CuKα 40kV 40mA；发散狭缝：1mn；索拉狭缝：0.4mm；扫描方式：连续扫描；扫描范围：3°～45°；采样间隔：0.02°；扫描速度：8°/min。
2、红外(IR)：
仪器型号：NICOLET 670-FTIR，实验条件：KBr压片
3、DSC parmeters：
仪器型号：NETZSCH DSC 204 F1
坩埚类型：铝坩埚(针刺穿孔)
吹扫气：高纯氮，20mL/min
保护气：高纯氮，60mL/min
升温速度：10℃/min
4、TG parmeters：
仪器型号：NETZSCH TG 209 F1
坩埚类型：氧化铝坩埚
吹扫气：高纯氮，20mL/min
保护气：高纯氮，10mL/min
升温速度：10℃/min
5、熔点：
RD-1熔点测试仪，天津旭阳仪器设备有限公司。
6、粒径测试：
Mastersizer 2000粒径测试仪，马尔文仪器有限公司
具体实施方式
下面结合实施例对本发明作进一步阐述，但这些实施例不对本发明构成任何限制，任何温度，溶剂比例的调整都在本发明保护范围内。
实施例1：阿普司特晶型II
将阿普司特(10.0g)，丙酮35ml加入三口烧瓶，加热溶解，然后冷却至35℃以下，缓慢滴加0.5-3.0倍量纯化水，加入少量晶型II作为晶种，搅拌1小时至产物析出后，继续加2倍 量纯化水(70ml)，于15-20℃继续搅拌过夜，共约24小时，过滤，水洗，60℃烘干，得到阿普司特晶型II约9.32克，mp：147.2-149.8℃。
实施例2：阿普司特晶型II
将阿普司特(400.0g)，丙酮1200ml加入三口烧瓶，加热溶解，缓慢滴加0.5-2.0倍量纯化水，加入少量晶型II作为晶种，搅拌1小时至产物析出后，继续加2倍量纯化水(2.4L)，于10-60℃继续搅拌过夜，共约18小时，过滤，水洗，60℃烘干，得到阿普司特晶型II约392.3克，mp：147.2-150.2℃。
实施例3：阿普司特晶型B
将阿普司特(10.0g)，加入丙酮(30ml)加入三口烧瓶中，加热溶解，然后冷却至30℃下缓慢加入10ml纯化水，冷却下搅拌至产物析出，搅拌2小时，继续缓慢滴加水(100ml)，继续保温搅拌过夜，约24小时，过滤，水洗，60℃烘干，得到阿普司特晶型B约9.45克，mp：156.2-157.8℃。
实施例4：
Figure PCTCN2015000566-appb-000007
主药过200目筛，填充剂、崩解剂过80目筛，称取处方量的填充剂、崩解剂混合均匀，然后将处方量的主药与其按照等量递加法混合均匀，加入处方量的助流剂和润滑剂，混合均匀后，压片即得。
实施例5：
Figure PCTCN2015000566-appb-000008
主药过200目筛，填充剂、崩解剂过80目筛，称取处方量的填充剂、崩解剂混合均匀，然后将处方量的主药与其按照等量递加法混合均匀，加入处方量的助流剂和润滑剂，混合均匀后，压片即得。
实施例6：晶型II与晶型B的热稳定性研究
分别将阿普司特的晶型II和晶型B悬浮于水，于60-100℃下搅拌48h，每隔一段时间取样过滤，烘干测试熔点变化情况，并在24h，48h取样过滤干燥测试X粉末衍射图谱，DSC图，熔点，静电效应及有关物质变化情况。
实施例7：阿普司特晶型B的转化研究
将阿普司特晶型B(5.0g，粒径300目以下)悬浮于60ml丙酮/水(1∶3)中，加热至回流，保温搅拌36h，然后搅拌下冷却，过滤，60℃烘干，得到阿普司特晶型II约4.88克，mp：148.3-150.3℃。
X衍射图谱和DSC图谱显示完全转化为晶型II。
实施例8：阿普司特晶型D的转化研究
将阿普司特晶型D(5.0g，粒径200目以下)悬浮于60ml丙酮/水(1∶3)中，加热至回流，保温搅拌8～10h，然后搅拌下冷却，过滤，60℃烘干，得到阿普司特晶型II约4.60克，mp：148.3-150.5℃。
X衍射图谱和DSC图谱显示完全转化为晶型II。
实施例9：阿普司特晶型B与晶型II在SD大鼠体内的药代动力学比较研究
1、晶型的粒径控制
通过微粉化控制两种晶型的粒径如下：
  D0.1 D0.5 D0.9
阿普司特晶型B(APST-B) 1.052um 6.792um 55.073um
阿普司特晶型II(APST-II) 1.395um 8.722um 58.942um
2、方法：
SD大鼠12只，雌雄各半，体重200-220g，随机分为2组，每组6只且雌雄各半，编为A和B组。
Figure PCTCN2015000566-appb-000009
参照FDA文献中给药途径和剂量，选择灌胃给药考察两种晶型在大鼠体内药代行为，两种晶型给药剂量均为10mg/kg(溶剂为1％羧甲基纤维素)。试验前禁食12 h，给药后4 h后提供食物，整个实验过程不禁水。
给药后10，30min，1，2，4，6，8，10，12和24h经眼底静脉丛取血约0.3ml，采集好的血液放置于冰上，经8000rpm离心5min后，分离血浆，放入-20℃冰箱中冷冻暂存。
采用LC-MS/MS法测定血浆中APST的浓度，测定血浆中APST的线性范围为2～1000ng/ml，线性良好。
3、实验结果
大鼠分别灌胃给予10mg/kg APST-B和APST-II混悬溶液后，每组动物的血浆浓度-时间曲线如图19至22所示。
4、结果分析：
大鼠分别灌胃给予10mg/kg APST-B和APST-II混悬溶液后，大鼠体内APST的主要药动学参数：达峰时间Tmax分别为1-6h(中位数为2.5h)和1-6h(中位数为3h)，达峰浓度Cmax分别为523.05±417.46和506.90±451.89ng/ml，血浆浓度-时间曲线下面积AUC0-t分别为3766.48±3617.82和5533.11±5613.02ng·h/ml，10mg/kgAPST-B的大鼠体内暴露量约为APST-II的68.07％，两组差距较大，由于暴露量雌雄差异很大，两组的标准偏差较大导致两组之间的暴露量差异无统计学意义。因此进一步对雄性与雌雄大鼠的体内药动学进行比较分析。
大鼠灌胃给予10mg/kgAPST-B和APST-II混悬溶液后，雄性大鼠体内APST的主要药动学参数：达峰时间Tmax分别为1h(中位数为1h)和1-2h(中位数为1h)，达峰浓度Cmax分别为142.4±13.96和107.63±16.05ng/ml，血浆浓度-时间曲线下面积AUC0-t分别为530.44±70.05和445.59±81.25ng·h/ml，10mg/kgAPST-B在雄性大鼠体内Cmax和AUC0-t分别为APST-II的132.3％和119.0％，结果显示在雄性大鼠体内APST-B的吸收程度高于APST-II，但该差异无统计学意义。
大鼠灌胃给予10mg/kg APST-B和APST-II混悬溶液后，雌性大鼠体内APST的主要药动学参数：达峰时间Tmax分别为2-6h(中位数为4h)和4-6h(中位数为6h)，达峰浓度Cmax分别为903.7±28.47和906.17±178.89ng/ml，血浆浓度-时间曲线下面积AUC0-t分别为7002.52±1140.54和10620.62±1053.56ng·h/ml，10mg/kgAPST-B在雌性大鼠体内AUC0-t为APST-II的65.9％，结果显示在雌性大鼠体内APST-B的吸收程度低于APST-II，且该差异有统计学意义(P＜0.05)。
实施例10：阿普司特晶型B与晶型II在比格犬体内的药代动力学比较研究
1、晶型的粒径控制
同实施例9。
2、方法
健康Beagle犬6只，雌雄各半，体重6-8kg。6只犬随机分为2组，每组3只。
参照临床给药途径和剂量，选择口服给药，给药剂量30mg/只/次，两种晶型的原料药粉末分别称重约30mg后，采用胶囊壳灌装，给药时以10ml水送服。比格犬试验前禁食12h，给药4h后提供食物，整个实验过程不禁水。具体给药组别设计如下表所示。
Figure PCTCN2015000566-appb-000010
给药前(0小时)及给药后10，30min，1，2，4，6，8，10，12，24，48，72h经小隐静脉取静脉血约0.5ml，经8000rpm离心5min后，分离血浆至抗冷冻塑料试管中，放入-20℃冰箱中冷冻暂存。
采用LC-MS/MS法测定血浆中APST的浓度，测定血浆中APST的线性范围为5～5000ng/ml，线性良好。
3、实验结果
[根据细则91更正 08.09.2015]　
比格犬分别经口给予30mg APST-B和APST-II原料药粉末胶囊后，每组动物的血浆浓度-时间曲线如图23所示。
4、结果分析
比格犬单周期分别经口给予30mg APST-B和APST-II料药粉末胶囊后主要药动学参数汇总如下：
Figure PCTCN2015000566-appb-000011
对药动学参数Cmax和AUC0-t经对数转化后进行t检验，APST-B与APST-II组的差异均无显著性意义(p＞0.05)。

Claims (13)

  1. 一种式(I)所示阿普司特的晶型II，
    Figure PCTCN2015000566-appb-100001
    其特征在于：
    i)差热分析(DSC)显示在100-180℃之间只有一个吸热峰，为150±3℃；
    ii)热失重(TG)显示不含结晶溶剂；
    iii)熔点为146-151℃。
  2. 根据权利要求1所述的阿普司特晶型II，其特征在于其X-射线粉末衍射图(XRPD)在2θ±0.2同时具以下两个特征吸收峰：11.2，14.7；优选其X-射线粉末衍射图(XRPD)在2θ±0.2同时具以下五个特征吸收峰：11.2，13.2，13.5，13.8，14.7；更优选其X-射线粉末衍射图(XRPD)在2θ±0.2同时具以下特征吸收峰：11.2，13.2，13.5，13.8，14.7，16.2，17.9，18.7，20.2，20.7，27.0。
  3. 根据权利要求1-2所述的阿普司特晶型II，其特征在于其X-射线粉末衍射图(XRPD)在2θ±0.2具如下特征吸收峰：
    # 2-Theta d(A) Height I％ Area I％ FWHM 1 10.666 8.2876 568 10.9 1090 2.4 0.077 2 11.290 7.8312 3576 68.5 45434 100.0 0.234 3 12.573 7.0346 833 15.9 2789 6.1 0.111 4 13.203 6.7001 3140 60.1 31411 69.1 0.192 5 13.541 6.5338 2509 48.0 22813 50.2 0.174 6 13.874 6.3775 1946 37.3 17299 38.1 0.193 7 14.724 6.0114 5224 100.0 42800 94.2 0.149 8 16.223 5.4592 2582 49.4 21876 48.1 0.163 9 17.924 4.9448 2862 54.8 24450 53.8 0.164 10 18.751 4.7285 2068 39.6 15517 34.2 0.153 11 20.290 4.3731 1268 24.3 7487 16.5 0.139 12 20.725 4.2822 832 15.9 3836 8.4 0.143 13 21.531 4.1238 1573 30.1 8335 18.3 0.132 14 21.989 4.0388 1666 31.9 18358 40.4 0.277 15 22.778 3.9007 1887 36.1 19298 42.5 0.244 16 23.194 3.8317 1404 26.9 19738 43.4 0.375
    17 25.265 3.5221 1412 27.0 22193 48.8 0.382 18 25.641 3.4714 1702 32.6 19926 43.9 0.268 19 26.587 3.3499 1904 36.4 20529 45.2 0.244 20 27.022 3.2970 3644 69.8 44458 97.9 0.234 21 27.596 3.2297 962 18.4 5166 11.4 0.194 22 28.226 3.1590 1147 22.0 7133 15.7 0.170 23 29.112 3.0648 581 11.1 973 2.1 0.103 24 29.667 3.0087 463 8.9 696 1.5 0.133 25 30.934 2.8884 482 9.2 1767 3.9 0.207 26 32.035 2.7916 486 9.3 1075 2.4 0.150 27 33.023 2.7103 450 8.6 1343 3.0 0.215 28 33.597 2.6653 473 9.1 2192 4.8 0.261 29 34.207 2.6191 509 9.7 2957 6.5 0.263 30 34.915 2.5676 404 7.7 1621 3.6 0.275 31 36.222 2.4779 336 6.4 662 1.5 0.174 32 39.625 2.2726 318 6.1 343 0.8 0.087 33 40.351 2.2334 311 6.0 690 1.5 0.156 34 41.080 2.1954 288 5.5 1946 4.3 0.583 35 43.804 2.0650 372 7.1 1599 3.5 0.190
  4. 根据权利要求1-2所述的阿普司特晶型II，其特征在于其具有如附图2，附图5，附图6，附图8，附图12，附图14，附图16或附图17所示的类似的X-射线粉末衍射图(XRPD)。
  5. 一种制备权利要求1-4所述的阿普司特晶型II的方法，其特征在于所用溶剂是由丙酮和水或者THF和水或者三者组成的混合溶剂，优选丙酮和水组成的混合溶剂。
  6. 根据权利要求5所述的方法，其特征在于：
    i)将阿普司特或其溶剂化物加热用丙酮或THF溶解，然后冷却至40℃以下，接着
    ii)搅拌下缓慢滴加相当于0.5-3倍丙酮或THF量的水，加入或者不加入晶型II的晶种，继续搅拌30～180分钟，然后
    iii)继续加入相当于2-6倍丙酮或THF量的水，于20℃至回流温度下搅拌1-24小时，然后
    iv)过滤，烘干得到阿普司特晶型II。
  7. 根据权利要求5所述的方法，其特征在于将阿普司特其他晶型混悬于丙酮/水或者THF/水的混合溶剂中，加热搅拌1-72小时，然后冷却过滤，烘干得到阿普司特晶型II。
  8. 一种药物组合物，其含有权利要求1-4所述的阿普司特晶型II作为药物活性成分和药学上可接受的载体。
  9. 根据权利要求8所述的药物组合物，其特征在于所述的药物活性成分中阿普司特晶型II的含量为1-100％。
  10. 权利要求1-4所述的阿普司特晶型II在制备用于治疗可通过抑制TNF-α产生来改善的疾病或病症的药物中的用途，其中所述的疾病或病症选自银屑病，银屑病关节炎，强直性脊柱炎、类风湿性关节炎、特应性皮炎、白塞病患者口腔溃疡、慢性皮肤肉样瘤，巨细胞动脉炎、帕金森病、结节性痒疹，扁平苔藓、复杂性口腔病、狼疮、肝炎、葡萄膜炎、干燥综合征、抑郁、间质性膀胱炎、外阴痛、前列腺炎、骨关节炎、弥漫性大B细胞瘤、多肌炎、皮肌炎、包涵体肌炎、侵蚀性骨关节炎、子宫内膜异位、神经病根、和坏疽性脓皮病或慢性阻塞性肺病。
  11. 权利要求1-4所述的阿普司特晶型II在制备用于治疗可通过抑制PDE4来改善的疾病或病症的药物中的用途，其中所述的疾病或病症选自：HIV、肝炎、成人呼吸窘迫综合征、骨吸收疾病、慢性阻塞性肺病、慢性肺部炎性疾病、皮炎、炎症性皮肤疾病、特应性皮炎、囊性纤维化、感染性休克、败血症、内毒性休克、血流动力学休克、败血症综合征、缺血后再灌注损伤、脑膜炎、银屑病，纤维化疾病、银屑病关节炎，恶病质、移植排斥、移植物抗宿主病、自体免疫性疾病、类风湿性脊椎炎、关节炎病症、强直性脊柱炎、类风湿性关节炎、骨关节炎、骨质疏松症、节段性肠炎、溃疡性结肠炎、炎性肠病、多发性硬化、全身性红斑狼疮、麻风病中的麻风结节性红斑、辐射损伤和高氧肺泡损伤。
  12. 权利要求1-4所述的阿普司特晶型II在制备用于治疗癌症的药物中的用途，其中所述的癌症选自多发性骨髓瘤、恶性黑色素瘤、恶性胶质瘤、白血病和实体瘤。
  13. 权利要求1-4所述的阿普司特晶型II在制备用于治疗结节病的药物中的用途，结节病选自心脏结节病、皮肤结节病、肝结节病、口腔结节病、神经系统结节病、鼻腔鼻窦结节病、洛夫格伦综合症、冻疮样狼疮、葡萄膜炎或慢性皮肤结节病。
PCT/CN2015/000566 2014-11-24 2015-08-05 一种稳定的不含溶剂化物的阿普司特晶型ii及其制备方法 WO2016141503A1 (zh)

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