| BackgroundAbdominal adhesion is a common complication after surgery, especially after laparotomy. When adhesion occurs, bonds between surfaces within body cavities are formed. These bonds may cause discomfort or pain in the patient, and may lead to female infertility. In some cases severe abdominal adhesion may even tie up large area of organs to form a lumpy-like intestinal adhesion which is attached to the surface of peritoneum, hindering bowel movements and resulting in intestinal obstruction in the patient. In recent years, the development of laparoscopic techniques has reduced adhesional morbidity to some degree, but still many patients suffer from adhesion after laparoscopic surgery. Although abdominal adhesion can be alleviated by adhesiolysis surgery, the incidence of its recurrence is still high. Moreover, new adhesion may result from the damage caused by adhesiolysis surgery itself. Therefore, it is necessary to look for non-surgical treatment of adhesion.WIE (Weitong Intravenous Emulsion) is an intravenous emulsion made by the Department of Pharmacy, Nanfang Hospital. It is a new dosage form derived from the previous studies of Weitong Injection (WTI). Compared to WTI, the component of WIE is re-selected and the dosage form of the injection is changed. As the emulsion in WIE may effectively increase the solubility of indissolvable ingredient in WTI, intravenous administration of WIE is helpful to the patient who is forbidden to take food or oral drugs after the surgery, because intravenous administrated drug may rapidly activate the fibrinolytic system in vivo and suppress the inflammatory response, which facilitates recovery of the patient after surgery, thus more efficient than oral drugs.ObjectiveThe component of WIE will be determined first through preformulation researching, formulation designing and production process optimizing. Then, three issues will be addressed:(1)the stability of WIE under violent environmental conditions; (2) the toxicity of WIE in the mice; (3) the prevention effect on postoperative adhesion in animal adhesion model and the mechanism of anti-adhesion effect of WIE in molecular level.Methods1 Preformulation research of WIE1.1 Establishing the method for content analysis of the main composition of DA and DB by HPLCEstablishing the chromatographic conditions of DA:Column:C18, mobile phase:methanol-water (85:15), flow rate:1.0 mL·min-1, column temperature:30℃, the injection volume:20μl, detection wavelength:270nm; regression equation:Y= 0.00001273X+0.012698, R2=0.9999448, precision is 0.13%, the content of the sample was 984.8mg·g-1.Establishing the chromatographic conditions of DB:Column:C18, mobile phase:acetonitrile-0.6% phosphoric acid solution (40:60), flow rate:1 mL·min-1, column temperature:30℃, the injection volume:20μl, detection wavelength:220nm, the regression equation:Y= 0.0002361X+8.887164, R2=0.9998243, precision study results was 0.49%, the content of the sample was 981mg·g-1.1.2 The stability of DA and DB under violent conditionsDA and DB were placed respectively in several violent environments to study the influence of temperature stability, light stability, oxidation stability, acid stability and alkali stability.2 Research on design of formulation and production process2.1 Formulation designAccording to the results of preliminary experiment, a mixture of soybean oil and medium chain triglycerides (MCT) was used as injection oil, lecithin was selected as an emulsifier, and oleic acid was employed as stabilizer. In addition, vitamin E was used as antioxidant, and glycerol as isotonicity adjusting agent. Then, orthogonal experiment was done to find the optimum ratio of each component. As a result,20% oil phase,1.0% lecithin and 0.06% oleic acid was the best selection.2.2 Production process designThe production process was researched on the basis of the fixed prescription. In autoclave experiments, we used 105℃/30min,115℃/25min and 121℃/20min conditions in experiments and found that only under 105℃/30min condition, the demulsification did not occur, so it was selected as sterilization conditions. Under that condition, the impact of autoclaving on pH was further studied. It was found the pH was reduced when there was autoclaving. Therefore the pH of WIE was adjusted to 9.0 when we prepared WIE. Homogenization pressure and frequency experiments showed that an increase in the number of homogenization gradually minimized the change of diameter of WIE and made it a constant value and that after homogenizing 3 to 4 times the particle size was close to that constant value. The orthogonal experiment was processed to achieve optimal condition for mixed temperature, mixing time and the homogenizing pressure.3 Research on the quality of WIE3.1 The physicochemical property of WIEThe laser light scattering technique was used to determine particle size and particle size distribution. The WIE surface potential was measured by the Coulter particle size analyzer. The viscosity of WIE was measured by an Ubbelohde viscometer.3.2 The stability of WIE under violent conditionsDilution stability experiments:Saline and glucose were used respectively to dilute WIE 20 times and the average particle size was measured at Omin,30min, 1h, 2h,4h,8h,12h,24h; Shaking experiment:The WIE was placed in shaking table with 100rpm in 25℃ water bath, the average particle size was measured and the emulsion appearance was observed at 2h,6h,12h,24h,48h,72h; Centrifugal acceleration test: The WIE was centrifuged at 5000r/min for 30 minutes. After that, the emulsion appearance was observed and the particle size was measured; Low temperature experiments:The WIE was stored at 4℃ in a refrigerator for 10 days, at 0,5 and 10 days the change of appearance was observed and the variation of particle size of WIE was measured; Acceleration experiments:The WIE was put in the environment conditions of 30℃ for 6 months, sampled at 0,1,2,3,4,5,6th months respectively, the emulsion appearance was observed and the particle size was measured.4 Evaluation of the safety of WIE4.1 Hemolytic test of WIEThere are totally 7 tubes in the test. An appropriate amount of 2% erythrocyte suspension and saline was added into each tube respectively (Distilled water was added into No.7 tube as the positive control tube). All tubes were put into 37℃ water bath for 30 minutes. After that different amounts of WIE were added into all tubes (Nothing was added into No.6 tube as the blank negative control tube). All tubes were joggled gently and put back into the 37℃ water bath, the vitro erythrocyte hemolysis was observed at 0.5h,1h,2h and 3 h.4.2 Undue toxicity of WIE in miceWIE (DA content as 0.3mg·mL-1) at dose of 0.2mL/10g was injected into the tail vein of mice, whose activities and diet were closely observed after interjection for 48 hours.4.3 Research on maximum administration doseThirty mice were randomly divided into three groups (n=10, half male and half female) after being weighed. The three groups were:WIE group, blank carrier group and saline group. The mice in WIE group were intravenous administrated 0.2mL/10g of WIE, the same dose of WIE was administrated again after 10 hours. The blank carrier group and the saline group were respectively administrated with 0.2mL/10g of emulsion without drugs and saline twice at a 10h interval. The mice were observed immediately after intravenous administration and in the following 14 days. They were weighed once a day and the symptoms of poisoning, the appearance of signs and death situation were to be recorded. At the end of the experiment, the animals in each group were sacrificed, the blood of each animal was taken respectively and sent to Nanfang hospital laboratory for routine blood determination (RBC, WBC, HGB, PLT, WBC, etc.).5 Pharmacodynamic evaluation of WIE5.1 The anti-adhesion effect of WIE in ratsForty male Wistar rats were randomly divided into four groups (n=10):model control group, dexamethasone (Dex) group, high-dose (HD) group and, low-dose (LD) group of WIE. The control group was daily injected with saline 1mL·kg-1, the Dex group with 3mg·kg-1 of Dex, the HD group with 0.3mg·kg-1 DA in WIE and the LD group with 0.1mg·kg-1 DA in WIE. The following steps were taken on the seventh day after the administration:(1)Evaluate the degree of adhesion; (2) Sample blood from the abdominal aorta with scalp needle, collect 3mL into sodium heparin anticoagulant tube, centrifuge it at 3000rpm for 10min, separate plasma and store it at -70℃ ultra-low temperature refrigerator; (3) Use ophthalmic scissors to clip tissue in the abdominal wall (0.5* 0.5cm) associated with adhesions part, place it in cryovials, label and freeze it in liquid nitrogen until it is completely frozen, and store it in-70℃ refrigerator.5.2 The protein level of tPA and PAI in plasmaRats plasma were measured using enzyme-linked immunosorbent assay (ELISA), tPA and PAI protein level were assayed.5.3 Evaluation of tPA, PAI and COX-2 gene expression level in abdominal tissue by real-time fluorescence quantificationTissue RNA was extracted from the frozen tissue and reversely transcribed to cDNA. Real-time PCR method was used for detecting tPA, PAI-1, and cyclooxygenase-2 (COX-2) expression, with β-actin as an internal reference. Then the results were analyzed with 2-ΔΔAACT method to acquire relative gene expression levels.Results1 Preformulation research of WIEThe method of determining DA and DB was established. Stability studies showed that 1) DA was stable at 40℃.The chromatograms of standard substance and reserved liquid were almost the same. DB was unstable in high temperature for long time. The chromatogram showed degradation of DB at 40℃ on the 5th day; 2) Both DA and DB were sensitive to the light and need to be stored in the dark area. In chromatogram a high incidence of miscellaneous peak was found on day 5 and day 10; 3) Chromatogram showed DA in the strong oxidizing environment was not stable and therefore needed to be put in an environment without oxidation material. A strong oxidizing environment also had a great impact on DB; 4) Chromatogram showed DA was unstable in an acidic environment but stable in an alkaline environment. DB was unstable in both acidic and alkaline environment, so it must be stored in a neutral environment.2 Research on design of formulation and production processTo prepare an oil phase:2mg of DA was weighed and dispersed in the mixed oil phase (MCT 10g, soybean oil 10g),0.06g of oleic acid and 0.5g of Vitamin E were added, the oil phase was magnetically stirred to dissolve at 60℃.To prepare a water phase:20mg of DB,1.2g of egg yolk lecithin and 2.5g of glycerin were dispersed in 60mL of water phase. The mixture was dissolved with stirring at 60℃. Under conditions of high shear agitation (9500rpm/min) at 60℃, the oil phase was gradually added to the water phase dropwise. The above step took about lOmin. Then emulsion was diluted with water until it reached 100mL and transferred into a high-pressure homogenizer at 25℃. After that it was homogenized 4 times at 20000psi pressure. The pH value was adjusted to 9.0. The emulsion was bottled, nitrogenized, sealed and put into 105℃/30min autoclaving. Finally it was rapidly cooled.3 Research on the quality of WIE3.1 The physicochemical property of WIEThe experiments on WIE showed that the WIE has an average particle diameter of 191.03±2.81nm, polydispersity index of 0.11±0.01, the average surface potential of-37.5mV and the average viscosity of 1.85 mpa-s.3.2 The stability of WIE under violent conditionsThe findings of shaking experiments, centrifugal acceleration experiments, low temperature experiments and accelerating experiments prove that WIE remained stable under the influence of different factors. No stratification emerged; particle size, polydispersity index, intensity mean, volume mean and diameter all remained stable.4 Evaluation of the safety of WIE4.1 Hemolytic test of WIEThere was no hemolysis in No.1-5 tubes at each time, as well as in No.6 tube (the negative control group tube). Hemolysis occurred in the No.7 tube at each time point. The osmotic pressure of WIE in this experiment was safe and did not contain hemolysis component, thus could be used safely as an injection.4.2 Undue toxicity of WIE in miceAfter tail vein injection, the activity and eating behavior of mice were found to be normal, and no death occurred within the following 48 hours after administration. The results showed that the WIE did not have undue toxicity.4.3 Research on maximum dose administrationThe mice did not show abnormal reactions after administration of maximum dose, except that the WIE group excreted faeces with dark red color after the first day. The weight of mice did not show significant difference between each group. One-way ANOVA analysis of the results of blood routine examination showed that there were significant differences between groups concerning four indicators (P<0.05), namely Hemoglobin (HGB), Hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH). The rest of indicators were normal.5 Pharmacodynamic evaluation of WIE5.1 The anti-adhesion effect of WIE in ratsSeven days later, peritoneal adhesion levels in each group of rats were evaluated after laparotomy. Compared with the model group, all drug administrated groups witnessed a significant decrease in adhesion level. There was no significant difference between the WIE HD group and the Dex group. The anti-adhesion effect was weak in the LD WIE group. The LD group differed significantly from the HD group and the Dex groups.5.2 The protein level of tPA and PAI in plasmaAs the results showed, WIE can effectively improve the level of tPA protein in plasma. Compared with the model control group, tPA protein level of the Dex group, the HD WIE group and the LD WIE group all significantly increased (P<0.05). TPA protein levels of the HD group was significantly higher than that of the other three groups (P<0.05); PAI protein levels found in model control group was significantly lower than that of the other three groups (P<0.05). PAI protein levels in the HD WIE group, the LD WIE group and the Dex group had no significant difference (P>0.05).5.3 Evaluation of tPA, PAI and COX-2 gene expression level in abdominal tissue by real-time fluorescence quantificationCompared with the control group, tPA mRNA relative expression level of all the three administrated groups significantly increased (P<0.05). That of the HD WIE group was significantly higher (P=0.000) than that of the Dex group and the LD group. Significant difference (P=0.000) existed between the Dex group and the LD WIE group concerning the level of mRNA expression; PAI mRNA relative expression in the model control group showed significant difference when compared with the other groups (P<0.05). But mRNA expression levels of the HD and LD WIE groups and of the Dex group had no significant difference (P>0.05). The study found that compared with the control group, COX-2 mRNA relative expression levels of the administrated groups were significantly lower (P<0.05). The mRNA expression level of COX-2 in the HD WIE group was similar to that in the Dex group, but was significantly deviant from that in the LD WIE group.Conclusions1. The study of DA and DB stability in different environments showed that DA was unstable in light, oxidation and acidic environment and that DB was unstable in several violent environments, therefore DA and DB must be preserved in a dark and mild environment at low temperature. We should also pay attention to the interference of light, oxidation and other factors in preparing the production process.2. The components of WIE include:DA 0.002%, DB 0.02%, mixing the oil phase (MCT 10%, soybean oil 10%), oleic acid 0.06%, Vitamin E 0.5%, egg yolk lecithin 1.2% and glycerol 2.5%. The production process was through mixed, high-speed shear, high pressure homogenization and sterilization.3. The physicochemical property of WIE complied with intravenous emulsion requirements, the indicators showed that WIE was stable in various stability testing. 4. The hemolysis test, the abnormal toxicity test and the maximum dose experiments all showed that WIE was secure.5. The pharmacodynamic experiments proved that WIE could be effective against the formation of postoperative adhesion in abdominal cavity and that it also effectively regulated tPA and PAI protein levels, as well as tPA, PAI, COX-2 gene expression levels in abdominal tissues. |
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