Lyophilized preparation of prostaglandin E1 methyl ester for injection and production and use thereof

Abstract

The disclosure provides a lyophilized preparation of prostaglandin E1 methyl ester for injection and production and use thereof. The lyophilized preparation comprises the following components by weight: 0.1-10 parts of prostaglandin E1 methyl ester, 500-4000 parts of an oil for injection, 500-2000 parts of an emulsifier, 0-10 parts of a co-emulsifier, 5000-50000 parts of a lyoprotectant, and 200-1500 parts of glycerin. The prostaglandin E1 methyl ester lyophilized agent of the disclosure has less blood vessel irritation, good drug stability, and superior drug activity and therapeutic effect than similar prostaglandin E1 products.

Claims

1-10. (canceled)

11. A lyophilized preparation of prostaglandin E1 methyl ester for injection, wherein the lyophilized preparation comprises the following components by weight: 0.1-10 parts of prostaglandin E1 methyl ester, 500-4000 parts of an oil for injection, 500-2000 parts of an emulsifier, 0-10 parts of a co-emulsifier, 5000-50000 parts of a lyoprotectant, and 200-1500 parts of glycerin.

12. The lyophilized preparation according to claim 11, wherein the lyophilized preparation comprises the following components by weight: 0.1-10 parts of prostaglandin E1 methyl ester, 500-4000 parts of the oil for injection, 500-1500 parts of the emulsifier, 0-10 parts of the co-emulsifier, 5000-20000 parts of the lyoprotectant, and 200-1500 parts of glycerin.

13. The lyophilized preparation according to claim 11, wherein the lyophilized preparation comprises the following components per 100 ml before lyophilization: 0.1-10 mg of prostaglandin E1 methyl ester, 0.5-4 g of soybean oil, 0.5-1.5 g of lecithin, 0-0.01 g of sodium oleate, 5-20 g of lactose, and 0.2-1.5 g of glycerin.

14. The lyophilized preparation according to claim 11, wherein the weight ratio of the lyoprotectant to prostaglandin E1 methyl ester is 20-500:0.01.

15. The lyophilized preparation according to claim 11, wherein the oil for injection is selected from one or more of soybean oil, medium chain oil, olive oil, tea oil, corn oil or castor oil; the emulsifier is selected from lecithin and/or soybean phospholipid; the co-emulsifier is selected from one or more of oleic acid, palmitic acid, stearic acid, linolenic acid, linoleic acid and sodium oleate; the lyoprotectant is selected from one or more of lactose, sucrose, trehalose, mannitol, glucose and maltose.

16. A method for producing a lyophilized preparation of prostaglandin E1 methyl ester for injection, wherein the method includes producing the lyophilized preparation with the following components as raw materials in weight percentage: 0.0001-0.01% of prostaglandin E1 methyl ester, 0.5-4% of an oil for injection, 0.5-2% of an emulsifier, 0-0.01% of a co-emulsifier, 5-50% of a lyoprotectant, 0.2-1.5% of glycerin, an appropriate amount of pH adjuster, and a balance of water for injection.

17. The method according to claim 16, wherein the method includes producing the lyophilized preparation with the following components as raw materials in weight percentage: 0.0001-0.01% of prostaglandin E1 methyl ester, 0.5-4% of the oil for injection, 0.5-1.5% of the emulsifier, 0-0.01% of the co-emulsifier, 5-20% of the lyoprotectant, 0.2-1.5% of the glycerin, an appropriate amount of pH adjuster, and a balance of water for injection.

18. The method according to claim 16, wherein the method includes producing the lyophilized preparation with the following components as raw materials by weight: 0.1-10 parts of prostaglandin E1 methyl ester, 500-4000 parts of soybean oil, 500-1500 parts of lecithin, 0-10 parts of sodium oleate, 5000-20000 parts of lactose, 20-1500 parts of glycerin, an appropriate amount of sodium citrate or hydrochloric acid, and a balance of water for injection based on 100×10.sup.3 parts of the total weight of raw materials.

19. The method according to claim 16, wherein the method includes the following steps: a. dispersing prostaglandin E1 methyl ester and ⅓ of the emulsifier uniformly in an oily solvent as an oil phase; b. dissolving glycerin and the lyoprotectant and the remaining emulsifier in an appropriate amount of water for injection as a water phase, and adjusting the pH of the water phase to 4.5-6.5 with a pH regulator; c. dissolving the co-emulsifier in the oil or water phase; d. adding the oil phase to the water phase under stirring, or adding the water phase to the oil phase under stirring, and obtaining an initial emulsion by shearing, then diluting to full volume with water for injection; e. homogenizing the initial emulsion to obtain a uniform emulsion; f. sterilizing the obtained emulsion by filtration, packing, lyophilizing, and sealing to obtain the lyophilized preparation.

20. The method according to claim 19, wherein step d comprises shearing at a constant temperature of 20° C.-50° C.

21. The method according to claim 19, wherein in step f the lyophilizing includes pre-freezing at −50° C. to −35° C. for 100-200 minutes, and then vacuuming at 70-90 mTorr for 420-540 minutes at −25° C. to −15° C., then vacuuming at 50-70 mTorr for 240-360 minutes at 5° C. to 15° C., and then vacuuming at 35-45 mTorr for 300-420 minutes at 35° C. to 45° C.

22. The method according to claim 16, wherein the oil for injection is selected from one or more of soybean oil, medium chain oil, olive oil, tea oil, corn oil or castor oil; the emulsifier is selected from lecithin and/or soybean phospholipid; the co-emulsifier is selected from one or more of oleic acid, palmitic acid, stearic acid, linolenic acid, linoleic acid and sodium oleate; the lyoprotectant is selected from one or more of lactose, sucrose, trehalose, mannitol, glucose and maltose.

23. A method for dilating blood vessels, comprising administering to a subject the lyophilized preparation of prostaglandin E1 methyl ester for injection according to claim 11.

24. The method according to claim 23, wherein, the method is a method for treating microcirculation disorders, coronary heart disease, angina pectoris, heart failure, pulmonary heart disease, cerebral infarction, amniotic fluid embolism, or scleroderma.

25. The method according to claim 24, wherein, the microcirculation disorders are caused by thromboangiitis obliterans, arteriosclerosis obliterans, diabetes, frostbite, burns or bedsores.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is a curve showing the relationship between the inhibitory rate of the compound on ADP-induced platelet aggregation and the incubation time in Experimental Example 1;

[0048] FIG. 2 is a curve showing the relationship between the diastolic efficacy of the vascular ring and the concentration of prostaglandin E1 in Experimental Example 2;

[0049] FIG. 3 is a curve showing the relationship between the diameter of the mesenteric arterioles and the administration time in Experimental Example 3;

[0050] FIG. 4 is the plasma concentration-time curve of alprostadil after intravenous injection of 40 μg/kg alprostadil in rats.

DETAILED DESCRIPTION

[0051] The technical solutions of the disclosure will be described in detail below in conjunction with the drawings and examples, but the protection scope of the disclosure includes but is not limited to these.

Example 1

Synthesis of Compound 1 of the Disclosure (methyl [(1R,2R,3R)-3-hydroxy-2-(S,E)-3-hydroxyoct-1-enyl)-5-oxocyclopentyl]heptanoate) (prostaglandin E1 methyl ester

[0052] ##STR00001##

[0053] The starting material PGE1 (63 mg, 0.18 mmol) was added to a three-necked flask, and then a prepared 1M dry THF/Et.sub.2O solution was added and stirred to dissolve. Under ice bath conditions, MeI (26 mg, 1M) solution was slowly added dropwise to the reaction solution, and after the completion of dropwise addition, KOH (10 mg, 0.18 mmol) and Bu.sub.4NBr (6 mg, 0.018 mmol) were added. After the reaction solution was stirred for 1 h, it was heated to room temperature and monitored by TLC until the end of the reaction. The reaction was quenched by adding 20 ml of water. It was extracted with EtOAc (10 mL×3), and the organic phases were combined, dried over anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (eluent n-hexane/EA=1/1) to obtain a white solid product (24.8 mg, 38% yield).

[0054] LCMS (MS Found: 391.3 [M+Na].sup.+).

[0055] .sup.1HNMR (400 MHZ, DMSO) (ppm): 5.46 (s, 2H), 5.01 (s, 1H), 4.57 (s, 1H), 3.88 (s, 2H), 3.57 (s, 3H), 1.9-2.3 (m, 5H) 1.2-1.48 (m, 19H), 0.85 (s, 3H).

Example 2

Synthesis of Compound 2: (ethyl [(1R,2R,3R)-3-hydroxy-2-(S,E)-3-hydroxyoct-1-enyl)-5-oxocyclopentyl]heptanoate) (Prostaglandin E1 ethyl ester

[0056] ##STR00002##

[0057] The starting material PGE1 (63 mg, 0.18 mmol) was added to a three-necked flask, and then a prepared 1M dry THF/Et.sub.2O solution was added and stirred to dissolve. Under ice bath conditions, EtBr (20 mg, 1M) solution was slowly added dropwise to the reaction solution, and after the completion of dropwise addition, KOH (10 mg, 0.18 mmol) and Bu.sub.4NBr (6 mg, 0.018 mmol) were added. After the reaction solution was stirred for 1 h, it was heated to room temperature and monitored by TLC until the end of the reaction. The reaction was quenched by adding 20 ml of water. It was extracted with EtOAc (10 mL×3), and the organic phases were combined, dried over anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (eluent n-hexane/EA=1/1) to obtain a white solid product (20.5 mg, 29.8% yield).

[0058] LCMS (MS Found: 405 [M+Na].sup.+.

[0059] .sup.1HNMR (400 MHZ, DMSO) (ppm): 5.46 (s, 2H), 5.01 (s, 1H), 4.57 (s, 1H), 3.88 (s, 2H), 3.57 (s, 3H), 1.9-2.3 (m, 7H) 1.2-1.48 (m, 19H), 0.85 (s, 3H).

[0060] Lyophilized Preparations of Prostaglandin E1 Methyl Ester with Different Main Drug Content

Example 3

[0061]

TABLE-US-00001 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 1.5 g Sodium oleate 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0062] The preparation process is as follows:

[0063] Oil phase: 2 g of soybean oil was weighted, 0.5 g of lecithin, and 0.5 mg of prostaglandin E1 methyl ester were added, and dissolved under shearing at 50° C.;

[0064] Water phase: 90 g of water for injection was weighted, 0.75 g of glycerin, 12.5 g of lactose, 0.01 g of sodium oleate were added, and mixed uniformly under shearing. The pH was adjusted to 6.5 with 0.1 M sodium citrate, then 2 g of phospholipid was added, and continued shearing for 10 min.

[0065] The oil phase was slowly added to the water phase, continued shearing at 50° C. for 10 minutes to obtain an initial emulsion, and water was balanced to 100 mL;

[0066] The initial emulsion was passed through a homogenizer and homogenized 8 times at a pressure of 850 bar;

[0067] The homogenized emulsion was sterilized by filtration, packaged, pre-frozen at −40° C. for 150 minutes, vacuumed at 80 mTorr for 480 minutes at −20° C., vacuumed at 60 mTorr for 300 minutes at 10° C., vacuumed at 40 mTorr for 360 minutes at 40° C., and capped under vacuum to obtain the lyophilized powder for injection.

Example 4

[0068]

TABLE-US-00002 Component content Prostaglandin E1 methyl ester 0.1 mg Soybean oil 2 g Lecithin 15 g Sodium oleate 0.01 g Lactose 5 g Glycerin 15 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0069] The preparation process was the same as Example 3

Example 5

[0070]

TABLE-US-00003 Component content Prostaglandin E1 methyl ester 10 mg Soybean oil 2 g Lecithin 15 g Sodium oleate 0.01 g Lactose 20 g Glycerin 0.5 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0071] The preparation process was the same as Example 3

[0072] Lyophilized Preparations of Prostaglandin E1 Methyl Ester with Different Oils for Injection, Phospholipids and Contents Thereof

Example 6

[0073]

TABLE-US-00004 Component content Prostaglandin E1 methyl ester 0.5 mg Olive oil 0.5 g Lecithin 0.5 g Sodium oleate 0.005 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0074] The preparation process was the same as Example 3

Example 7

[0075]

TABLE-US-00005 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 1 g Lecithin 0.8 g Sodium Oleate 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0076] The preparation process was the same as Example 3

Example 8

[0077]

TABLE-US-00006 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 4 g Soybean phospholipid 2 g Sodium Oleate 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0078] The preparation process was the same as Example 3

Example 9

[0079]

TABLE-US-00007 Component content Prostaglandin E1 methyl ester 0.5 mg Tea oil 2 g Lecithin 15 g Sodium oleate 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0080] The preparation process was the same as Example 3

Example 10

[0081]

TABLE-US-00008 Component content Prostaglandin E1 methyl ester 0.5 mg Corn oil 2 g Soybean phospholipid 1 g Sodium oleate 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0082] The preparation process was the same as Example 3

[0083] Lyophilized Preparations of Prostaglandin E1 Methyl Ester with Different Co-Emulsifiers and Contents Thereof

Example 11

[0084]

TABLE-US-00009 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 1.5 g Oleic acid 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0085] The preparation process was the same as Example 3

Example 12

[0086]

TABLE-US-00010 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 1.5 g Stearic acid 0.005 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0087] The preparation process was the same as Example 3

Example 13

[0088]

TABLE-US-00011 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 1.5 g Linoleic acid 0.01 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0089] The preparation process was the same as Example 3

Example 14

[0090]

TABLE-US-00012 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 15 g Lactose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0091] The preparation process was the same as Example 3

[0092] Lyophilized Preparations of Prostaglandin E1 Methyl Ester with Different Lyoprotectants and Contents Thereof

Example 15

[0093]

TABLE-US-00013 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 15 g Sodium oleate 0.01 g Sucrose 12.5 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0094] The preparation process was the same as Example 3

Example 16

[0095]

TABLE-US-00014 Component content Prostaglandin E1 methyl ester 1 mg Soybean oil 2 g Lecithin 1.5 g Sodium oleate 0.01 g Trehalose 20 g Glycerin 0.5 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0096] The preparation process was the same as Example 3

Example 17

[0097]

TABLE-US-00015 Component content Prostaglandin E1 methyl ester 0.5 mg Soybean oil 2 g Lecithin 1.5 g Sodium oleate 0.01 g Mannitol 10 g Glycerin 0.75 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0098] The preparation process was the same as Example 3

Example 18

[0099]

TABLE-US-00016 Component content Prostaglandin E1 methyl ester 5 mg Soybean oil 2 g lecithin 1.5 g Sodium oleate 0.01 g Lactose 50 g Glycerin 0.2 g Sodium citrate/Hydrochloric acid Appropriate amount Water for Injection Balanced to 100 mL

[0100] The preparation process was the same as Example 3

Comparative Example 1

[0101] It is prepared according to Example 2 in U.S. Pat. No. 4,849,451.

Comparative Example 2

[0102] The lyoprotectant used in the formulation of the disclosure was added to the preparation obtained in Comparative Example 1 for lyophilization.

Experimental Example 1

The Effect in the Anti-Platelet Aggregation Test In Vitro

[0103] When healthy adult SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, fresh whole blood was collected from the abdominal aorta and added to a centrifuge tube anticoagulated with 3.8% sodium citrate solution, and centrifuged at 900 rpm for 10 minutes to remove the upper platelet-rich plasma (PRP) for use. The tube having PRP removed was further centrifuged at 4000 rpm for 10 minutes, and the upper clarified plasma (PPP) was removed for use. In the experiment, Techlink model LBY-NJ4 4-channel platelet aggregator was used to determine the anticoagulant efficacy of each compound.

[0104] Into a sample cup containing 300 μL of PRP, 2 μL of 100 μM PGE1, Compound 1 of Example, Compound 2 of Example and methanol (solvent) were first added. After incubated for different periods (0, 1, 2, 4, 7, 10, 15 min), 204, of aggregation inducer 180 μM ADP solution was added. The aggregation rate of each sample was measured, and the inhibitory rate of the compound on ADP-induced platelet aggregation was calculated.

Inhibition rate %=(solvent aggregation rate−compound aggregation rate)/solvent aggregation rate×100%

[0105] From the results (shown in FIG. 1), it can be seen that the additions of PGE1 and Compound 1 of Example to PRP take effect immediately, and the inhibition rates were equivalent. With prolonged incubation time, the anticoagulant efficacy of the compound of the Example slowly decreased, and the inhibition rate is still 43.66% after 10 minutes, but the inhibition rate of PGE1 is only 4.93% after 4 minutes of incubation. Compound 2 of Example does not take effect immediately after the addition, and the efficacy gradually increased over time, and reaches the maximum inhibition rate of 51.86% after 10 minutes of incubation. Therefore, Compound 1 of Example is an active non-prodrug compound, and its anticoagulant effect is 2 times longer than that of PGE1, and Compound 2 of Example is a typical prodrug compound.

Experimental Example 2

Vasodilation Test In Vitro

[0106] In the experiment, rabbits were selected to prepare isolated aortic ring specimens: New Zealand white rabbits, male, weighing (2.5±0.3) kg. The rabbit was stunned with a blunt instrument, fixed on the rabbit dissecting table, and the thoracic aorta was quickly separated, and placed in a petri dish filled with saturated Kerbs solution (containing NaCl 6.9 g, KCl 0.35 g, MgSO.sub.4.7H.sub.2O 0.29 g, KH.sub.2PO.sub.4 0.16 g, NaHCO.sub.3 2.1 g, CaCl.sub.2 0.28 g, glucose 2 g per 1000 mL) at 37° C. and continuously introduced with mixed gas (95% O.sub.2, 5% CO.sub.2). The remaining blood in the blood vessel was squeezed out, and the peripheral fat and connective tissue were carefully peeled off, and it was cut into 0.5 cm long arterial rings for use. Two stainless steel L-shaped hooks were used to pierce through the vascular lumen of the vascular ring, and the vascular ring was hung horizontally in a 20 mL bath tube, fixed at the bottom, and connected to a tension transducer with a thin steel wire at the top. The resting tension was first adjusted to 0.00 g, and after stabilization for 20 minutes, 3.00 g tension was applied, and the tension level was continuously adjusted to maintain it at about 3.00 g and stabilized for 2 h (replacing the Kerbs solution along the wall of the bath every 15 minutes).

[0107] BL-420S biological function experiment system (Chengdu Techman Technology) was used to record the variation of vascular ring tension. After the vascular ring contraction was stable, prostaglandin E1 and the compounds of the Examples were accumulatively added to successively increase the final mass concentration of prostaglandin E1 in the bath tube to 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 nM, and the diastolic efficacies of the vascular ring were recorded.

[0108] The results (shown in FIG. 2) show that under the experimental conditions, the diastolic efficacy of the compound of Example 1 on isolated rabbit blood vessels (EC.sub.50=1.090 nM) is significantly stronger than that of PGE1 (EC.sub.50=9.767 nM).

Experimental Example 3

Mesenteric Microcirculation Disorder Model

[0109] Rats were anesthetized with 2.5% sodium pentobarbital 30 mg/kg ip, fixed in the dorsal position, a 3 to 4 cm long incision was made in the midline of the abdomen, a section of small mesentery was gently pulled back to the cecum, and placed in an organic glass constant temperature water bath filled with physiological saline at 37° C. The mesentery was kept moist and laid flat on the organic convex observation table in the center of the bath, and pressed with a fixing plate. The video image under the microscope was collected by the camera of the biological microscope (magnification 40×), and the BI-2000 microcirculation observation system was used to real-time analyze the collected video images under the microscope.

[0110] A visual field was fixed and balanced for 10 minutes, and then the diameter and flow velocity of arterioles and venules in the selected area were observed. Then, 100 μL of a 1:100 diluted epinephrine hydrochloride physiological saline solution was added dropwise into the selected area, and an equal volume of physiological saline was added in the normal control group. At the same time, 10 μg/kg of the preparation of Example and Udil were immediately administered via the tail vein. The diameters of mesenteric arterioles were measured at 0.5, 1.2, 4, 6, 8, 10, 12, 15 min after administration.

[0111] As shown in FIG. 3, by measuring the diameter, the preparation group of Example 3 can significantly improve the microcirculation disorder caused by epinephrine, and there were significant differences between the preparation group of Example 3 and the model group from 0.5 to 10 minutes after administration (P<0.05). Compared with the model control group, the Udil group had no significant difference (P>0.05) at 4 min. The efficiency of improving microcirculation disorder caused by epinephrine in the Example group was significantly higher than that of Udil, and there was a significant difference between the two at 0.5-10 min (P<0.05).

Experimental Example 4

Determination of Free Drug in the Preparation

[0112] Sample processing method: the lyophilized emulsion of each Example was added with 10 ml for reconstitution, and one part was simultaneously diluted 10 times with water, and an appropriate amount was accurately metered and placed in a 20 ml brown test tube with a stopper. 2.5 ml of tetrahydrofuran was added, mixed well, and 15 ml of phosphoric acid solution (1.fwdarw.1000) was added, mixed well and passed through the pretreatment column [the filler was ODS with a particle size of 70 μm, (ϕ) 10 mm×9 mm polypropylene tube (SEP-PAK C18 column, Waters), rinsed with 10 ml of methanol and 10 ml of water before use]. The test tube was rinsed with 10 ml of water and passed through the pretreatment column. Then 7 ml methanol was used for elution, and all the eluate was transferred into a 10 ml brown distillation flask and distilled under reduced pressure at 50° C. for 10 minutes. The solvent was evaporated, and the residue was dissolved with 1 ml internal standard solution and shaken well to obtain the sample.

[0113] The ODS was used as a filler, 0.0067 mol/L phosphate buffer (pH=6.3) (9.07 g of potassium dihydrogen phosphate was taken, and water was added to dissolve it to make 1000 ml solution; 9.46 g of anhydrous disodium hydrogen phosphate was separately taken, and water was added to dissolve it to make 1000 ml solution; the latter was added to the former until the pH was 6.3, 100 ml of this solution was taken and water was added to reach 1000 ml, and shaken well to get the buffer)-acetonitrile (70:30) was used as a mobile phase. The flow rate was 1 ml per minute; the post-column reaction solution was 1 mol.Math.L−1 KOH solution, and the post-column reaction tube was a polytetrafluoroethylene tube (φ0.5 mm×10 m); column temperature was 60° C.; detection wavelength was 278 nm. 20 μl of each test substance and reference substance solution were injected, and calculated according to the internal standard peak area method.

[0114] Examples 3 to 18 and Udil were reconstituted with physiological saline to reach a concentration of 1 μg/mL, and all samples were diluted 10 times to 0.1 μg/mL, and appropriate amounts of the above-mentioned reconstituted preparations were taken and placed in an ultrafiltration centrifuge tube. After centrifugal ultrafiltration at 2800 rpm for 30 minutes, 1 ml of the filtrate was taken to prepare a sample solution according to the above sample processing method, and 20 μl of sample was injected. The measured content of each drug is the free drug content.

[0115] Meanwhile, the free rates of 1 mL of reconstituted emulsion (lx) and 10-fold dilution (lox) of the samples of each Example were measured.

Free rate %=total amount of free drug/total drug amount×100%

[0116] The measured free rate of each example is shown in Table 1 below:

TABLE-US-00017 TABLE 1 Free rate (%) Sample 1 × (1 μg/ml) 10 × (0.1 μg/ml) Udil 2.98 11.25 Example 3 0.21 0.86 Example 4 0 0 Example 5 0.47 0.55 Example 6 0.19 0.22 Example 7 0.12 0.18 Example 8 0.09 0.11 Example 9 0.31 0.29 Example 10 0.36 0.27 Example 11 0.28 0.35 Example 12 0.29 0.27 Example 13 0.15 0.19 Example 14 0.25 0.29 Example 15 0.28 0.41 Example 16 0.26 0.33 Example 17 0.39 0.34 Example 18 0.37 0.47

[0117] The experimental results show that the free drug of Examples 3-18 was significantly lower than that of Udil after reconstitution. When Udil was diluted by 10 times according to the instructions, the free rate increased significantly. However, there was no significant increase in the free drug of the preparations prepared in the Examples of the disclosure.

Experimental Example 5

Vascular Irritation Test

[0118] Twenty New Zealand white rabbits were selected and divided into 2 groups with a weight of about 2 kg. Both the preparations of the Examples and Udil were reconstituted with physiological saline to reach 1 μg/ml, and further diluted with physiological saline to 0.1 μg/ml. According to the clinical dosage of prostaglandin E1, 0.5 μg/kg was instilled slowly in the ear vein, and the same volume of physiological saline was injected in the ear vein at the opposite side, once a day for 7 days. Two hours after the last administration, the degree of vascular irritation was visually evaluated. All animals were sacrificed, and then the injection site was taken for histopathological examination. The results are shown in Table 2 and Table 3 below.

TABLE-US-00018 TABLE 2 General checklist of vascular irritation test Lesion area Score No obvious reaction 0 Mild hyperemia 1 Mild to moderate hyperemia, swelling 2 Mild to moderate hyperemia, swelling, 3 drooping ears Mild to moderate hyperemia, swelling, 4 drooping ears with mild to moderate necrosis Mild to moderate hyperemia, swelling, 5 drooping ears with severe extensive necrosis

[0119] The results showed that the Example group was substantially non-irritative (P>0.05), while the Udil group had obvious vascular irritation (P<0.05).

TABLE-US-00019 TABLE 3 Group Vascular irritation score (Mean ± SD) Preparation of Administered ear 0.40 ± 0.52 .sup.  Example Control ear 0 Udil Administered ear 2.6 ± 0.84*.sup.# Control ear 0 *P < 0.05 compared with the control ears in this group; .sup.#P < 0.05 compared with the administered ears in the Example group

Experimental Example 6

Preparation Stability Test

[0120] The appearance and particle size of the samples prepared in Examples 3 to 18 and Comparative Examples 1 and 2 are shown in Table 4 below:

TABLE-US-00020 TABLE 4 Average particle >5 μm size of the final droplets Appearance product (nm) (%) Example 3 Loose lumps, good 94 0.0021 reconstitution Example 4 Loose lumps, good 93 0.0019 reconstitution Example 5 Loose lumps, good 96 0.0022 reconstitution Example 6 Loose lumps, good 77 0.0018 reconstitution Example 7 Loose lumps, good 89 0.0019 reconstitution Example 8 Loose lumps, good 148 0.0053 reconstitution Example 9 Loose lumps, good 93 0.0037 reconstitution Example 10 Loose lumps, good 104 0.0018 reconstitution Example 11 Loose lumps, good 86 0.0025 reconstitution Example 12 Loose lumps, good 86 0.0022 reconstitution Example 13 Loose lumps, good 101 0.0019 reconstitution Example 14 Loose lumps, good 113 0.0033 reconstitution Example 15 Loose lumps, good 92 0.0024 reconstitution Example 16 Loose lumps, good 99 0.0018 reconstitution Example 17 Loose lumps, good 91 0.0038 reconstitution Example 18 Loose lumps, good 89 0.0023 reconstitution Comparative White homogeneous 206 0.2502 Example 1 milky liquid Comparative Unable to shaped / / Example 2

[0121] 380ZLS particle size analyzer (dynamic light scattering method) from PSS corporation was used to determine the average particle size, and the Accusizer 780 instrument (light blockage method) from PSS corporation was used to determine the large-size emulsion droplets, and the percentage of emulsion droplets larger than 5 μm was calculated. The results showed that the samples prepared in Examples 3-18 had a good appearance after lyophilization, an average particle size after reconstitution significantly smaller than that of Comparative Example 1, and a percentage of large emulsion droplets (>5 μm) much less than 0.05%, while the large particle size (>5 μm) of Comparative Example 1 exceeds the standard (>0.05%, USP standard), and Comparative Example 2 cannot be lyophilized.

[0122] The samples prepared in Examples 3, 5, 8, and 15 were taken for a 24-month long-term stability test (temperature 25° C.±2° C., humidity 60%±10%). The results are shown in Table 5 below:

TABLE-US-00021 TABLE 5 Average particle drug Storage size content time Characters (nm) % Example 0 month Good appearance, 92 103.2% 3 normal reconstitution 3 months Good appearance, 94 103.3% normal reconstitution 6 months Good appearance, 95 103.0% normal reconstitution 9 months Good appearance, 94 102.8% normal reconstitution 12 months Good appearance, 96 102.6% normal reconstitution 18 months Good appearance, 95 102.7% normal reconstitution 24 months Good appearance, 97 102.4% normal reconstitution Example 0 month Good appearance, 88 101.5% 5 normal reconstitution 3 months Good appearance, 87 101.3% normal reconstitution 6 months Good appearance, 93 101.4% normal reconstitution 9 months Good appearance, 91 101.1% normal reconstitution 12 months Good appearance, 93 100.8% normal reconstitution 18 months Good appearance, 95 100.4% normal reconstitution 24 months Good appearance, 94 100.4% normal reconstitution Example 0 month Good appearance, 136 104.3% 8 normal reconstitution 3 months Good appearance, 135 104.0% normal reconstitution 6 months Good appearance, 141 104.0% normal reconstitution 9 months Good appearance, 139 103.8% normal reconstitution 12 months Good appearance, 145 103.9% normal reconstitution 18 months Good appearance, 152 103.5% normal reconstitution 24 months Good appearance, 147 103.4% normal reconstitution Example 0 month Good appearance, 93 102.5% 15 normal reconstitution 3 months Good appearance, 95 102.5% normal reconstitution 6 months Good appearance, 94 102.1% normal reconstitution 9 months Good appearance, 99 102.2% normal reconstitution 12 months Good appearance, 97 101.5% normal reconstitution 18 months Good appearance, 103 101.7% normal reconstitution 24 months Good appearance, 103 101.4% normal reconstitution

[0123] The results showed that the lyophilized preparations of prostaglandin E1 methyl ester prepared in the Examples of the disclosure had stable product quality in the 24-month long-term stability test.

Experimental Example 7

Pharmacokinetic Study of Examples 3, 6, 7, 8 and Comparative Example 1 in Rats

[0124] In the pharmacokinetic experiment, 12 male SD rats were used in each group, fasted for 12 hours before administration, and given free drinking water. The two preparations were administered intravenously at a dose of 40 μg/kg respectively, and blood was collected from the orbit at 0.5 min, 1.25 min, 3 min, 5 min, 8 min, 12 min, 16 min, 20 min, 30 min, and 45 min after administration. The blood concentration (the sum of prostaglandin E1 methyl ester and prostaglandin) measured at each time point is shown in FIG. 4.

[0125] The pharmacokinetic parameters of the two preparations were calculated according to the blood concentration curve, the peak blood concentration after administration of Example 3 was significantly higher than that of Comparative Example 1, the half-life was significantly prolonged, and the bioavailability was also significantly improved (see Table 6).

TABLE-US-00022 TABLE 6 The pharmacokinetic parameters of 40 μg/kg intravenous injection of the preparations of Examples 3, 6-8 and Comparative Example 1 in rats Cmax (ng/mL) AUC(0-t) (ng/mL*min) t½(min) Example 3 178.1 ± 24.5  606.1 ± 100.4 6.6 ± 1.3 Example 6 165.3 ± 31.8  671.1 ± 110.9 6.2 ± 0.9 Example 7 168.5 ± 26.9 625.3 ± 82.4 7.1 ± 1.6 Example 8 151.6 ± 24.6 591.6 ± 68.2 6.7 ± 1.1 Comparative 125.3 ± 41.9 233.3 ± 78.8 2.3 ± 0.3 Example 1

[0126] Compared with the preparation of Comparative Example 1, the preparations of Examples 3 and 6-8 have an obvious improvement in the pharmacokinetic behavior in vivo. This may be due to the inhibition of the metabolic inactivation of the drug in the pulmonary circulation, and this phenomenon may be related to the particle size of the drug.