| Background and objective:Postoperative pain is a major problem for the healing process over surgical period.Injection of local anesthetics is the most sitedirected and effective analgesic modality for the management of postoperative pain.Local anesthetics reversibly block nerve conduction near their site of application or injection thus producing temporary loss of sensation over a specific area of the body,howerever,clinical application of water-soluble local anesthetics can not achieve a long period analgesia by a single injection.The longest duration of local anesthetics acting are not more than 12 hours after administration.Current clinical methods to prolong analgesia include mutiple injections of short acting local anesthetics solutions and implantation catheter into body but are time-consuming and demand expensive equipment and monitoring. Administration of local anesthetics frequently can cause excessive accumulation of drugs,which can lead to inhibition of respiration and circulation or toxicity of local anesthetic. Application of local anesthetics via a catheter can produce neural blockade for days to weeks,howerever,catheters may become infected,blocked or migrate. Therefore,these metholds can not be an ideal mean to prolong analgesia.This lack of efficient treatment for postoperative analgesia highlights the need for new therapeutic principles in this area.About 40 years Microcap technology has been applied to the field of pharmaceutical preparations,which characterized by using of natural or synthetic copolymers as drug carrier and extending the duration of drugs through prolonging the release of drugs.Microcapsule has been applied to anticancer drugs,antibiotics,eye diseases,gene therapy,diabetes treatment and many other studies.The PLA and PLGA microspheres have been applied to the study of local anesthetics for about thirty years. Early in 1981, Wakiyama N prepared the PLA microspheres containing several local anesthetics; In 1994, Le Corre et al studied the preparation and properties of bupivacaine PLA and PLGA microspheres,and both of them proved that it is feasible to take biodegradable material to preparare of the sustained-release microspheres containing local anesthetics,which can extend the acting duration of local anesthetics significantly. Nineties, Jean MM, Fletcher D, Passcal LC, Jean PE and other many scholars studied pharmacokinetics and pharmacodynamic characteristics of local anesthetics slow-release microspheres with different animals.The results showed that local anesthetics slow-release microspheres can significantly extend analgesic acting time of the single dose of local anesthetics and reduce the fluctuation of drug plasma concentration.1996 Joanne C,1998 years Christiane D,et al demonstrated bupivacanine microspheres containing small amounts of dexamethasone produced further longer period analgesia compared with microspheres contained plain bupivacaine.But there is no report about research of sustained-release ropivacaine microspheres in the world at present. Structure,nature and metabolic pathways of ropivacaine is similar to bupivacaine, but its neurotoxicity and cardiotoxicity is lower than bupivacaine.A more prominent characteristic of ropivacaine are the high degree separation of feeling-motor nerve block, low drug concentration produces almost exclusively sensory nerve block,increasing the possibility of exercise quickly for patients. It is more suitable for intractable pain treatment such as postoperative analgesia, advanced cancer and trigeminal neuralgia.For these reasons metioned above, our team studied the preparation of PLGA ropivacaine sustainde-release microspheres and the characteristic of its release in vitro and in vivo.The animal experiment proved that it is feasible to take completely biodegradable materials poly-lactic acid-glycolic(PLGA) as carrier to prepare the sustained-release microspheres contained ropivacaine,which produce further longer period action significantly.The purpose of our study is to prepare the sustained-release Rop-Dex-PLGA microspheres by W/O/W emulsion-solvent evaporation method and to evaluate Pharmacokinetic and Pharmacodynamic characterization of Rop-Dex-PLGA microspheres by implanted ropivacaine-dexamethasone PLGA microspheres near the sciatic nerve in mice to observe the change of hot pain threshold and the blood concentration of ropivacaine and dexamethasone.Research methods:1. Preparation of Rop-Dex-PLGA-MS and Rop-PLGA-MS:Prepare ropivacaine microspheres with or without dexamethasone by W1/O/W2 double emulsion-solvent evaporation.The steps are as follows:(1)Taking a certain amount ropivacaine and dexamethasone dissolved with double-distilled water and methanol respectively as internal aqueous phase; (2) Taking appropriate amount of PLGA and emulsiflers dissolved in dichloromethane as the organic phase,then mix (1) wth (2) by high-speed disperser homogenizer for 90s to make colostrum;(3)Move colostrum to the external water phase(PVA aqueous solution) with a syring and homogening with high-speed disperser homogenizer for 90s,the system was double emulsion;(4)The double emulsion was stirred with a magnetic stirrer continuously, after centrifuged, filtered, washed three times with double distilled water then collectt microspheres, freeze-drying stored at a temperature of 4℃of the dryer for use.Ropivacaine content and (or) dexamethasone of Rop-PLGA-MS and Rop-Dex-PLGA-MS was measured by high performance liquid chromatography.2. Animal group:165 Kunming female mice weigthing 25-30g were randomly assigned into three groups, namely the Dexamethasone-loaded ropivacaine microspheres group(group A, n=55), ropivacaine microspheres(group B, n=55)and PLGA microspheres(group C, n=55). The mice in three groups was divided into 11 groups according to the time interval set point at 0.5,1,2,4,8,12,24,36,48,72,96 h after anesthesia.Each mice was measured five times at interval of 5 min.3. Adminstration methods and sample collection:Each mouse in three groups was implanted surgically near the sciatic nerve of mice with 400 mg/kg of Rop-Dxm-MS, Rop-MS and PLGA-MS respectively after anesthesia. A hot plate was used to observe the latency to withdraw the implanted hind paw at 0.5,1,2,4,8,12,24, 36,48,72,96 h set time point. After hot-plate test each mouse in three groups was executed to abtain 0.8～1.0 ml blood by digged eyes.Blood was anticoagulated with 0.1ml 1/2000 heparin and centrifugation for 8 minuter.0.5ml blood plasma was extraxted and stored at-20℃for detected by HPLC.4.Sample detection and parameter analysis:Plasma was taken out to melt at room temperature for detection.The condition of HPLC:Column:Shimpack VP-ODS 250×4.6mm;column temperature:room temperature; mobile phase:methanol: acetonitrile:water (20mMNaH2PO4, pH= 4.6)= 18:26:56 (V:V:V), flow rate:1.0 ml.min-1; detection wavelength:Ropivacaine and Bupivacaine for 210 nm; Dexamethasone for 240 nm; internal standard:bupivacaine solution; quantitative methods:external standard method. Sample volume:20 ul, the minimum detection limit of 0.010 ug.ml-1. The parameter of pharmacokinetics such as AUC,Cmax,Tmax. t1/2 was analysized by WinNonlin3P87DAS2.1.1.5.Statistical analysis:All measuremental data were expessed as mean±SD or median.Experimental data and charts were used statistical software SPSS13.0 and image software Origin6.0; parameters of pharmacodynamics and pharamacokinetics were analyzed using a factorial analysis of variance.Comparison of plasma concentration between two groups was analyzed using unpaired T test.A p value<0.05 is considered significant.Results1. Dexamethasone-containing ropivacaine micorspheres prepared by W/O/W emulsion-solvent evaporation method was stable and reproducible. Ropivacaine Drugs loading of microspheres was (7.60±0.08)%,encapsulation efficiency of microspheres was (74.10±1.42)%;Dexamethasone Drugs loading of microspheres was (1.54±0.02)%; encapsulation efficiency of microspheres was (60.16±1.55)%.2. Results of pharmacodynamics:There was significant difference statistically about latency to withdraw the drug hind paw at different time among three groups in mice(P<0.001);there exist across effect about latency to withdraw the drug hind paw at different time among three groups (P=0.000);there was significant difference about latency to withdraw the drug hind paw between Rop-Dex-PLGA-MS and PLGA-MS at 2h,4h,8h,12h,24h,36h,48h,72h set point(P< 0.001).there was significant difference about latency to withdraw the drug hind paw between Rop-PLGA-MS and PLGA-MS at 2h,4h,8h,12h,24h,36h set point(P<0.001);Latency to withdraw hind paw of heat pain has signicant differece at 48h and 72h between Rop-Dex-PLGA-MS and Rop-PLGA-MS.3. Results of pharmacokinetics:Plasma ropivacaine concentration has significant difference statistically at different time between Rop-Dex-PLGA-MS and Rop-PLGA-MS(P<0.001). Cmax,Tmax,AUC,MRT,t1/2 for Rop-Dex-PLGA-MS and Rop-PLGA-MS was 117.8 ng.ml-1 and 119.8 ng.ml-1,24h and 12 h,8793.2 ng.h.ml-1 and 6861.15 ng.h.ml-1,43.7491h and 38.7743 h,65.799h and 46.166h,respectively. Plasma dexamathasone concentration was not been detected in three groups and plasma ropivacaine and dexamathasone concentration was not been detected in C group.Conclusion1. It is feasible to take completely biodegradable materials poly-lactic acid-glycolic(PLGA) as carrier to prepare the sustained-release microspheres contained ropivacaine by W/O/W emulsion-solvent evaporation method.2. The study of pharmacokinetic demonstrated that:(1) PLGA as carrier did not produce analgesic effect;(2) Rop-Dex-PLGA-MS and Rop-PLGA-MS has not been found obviously motor blockade;(3)The duration of analgesic effect was 72h and 36h for Rop-Dex-PLGA-MS and Rop-PLGA-MS,respectively.Dexamethasone-containing ropivacaine microspheres can significantly extend the duration of analgesia of ropivacaine in mice.3. The study of parmacodynamics investigated that the release drug curve of Rop-Dex-PLGA-MS and Rop-PLGA-MS in vivo in mice was double-peak mode.T1/2 and mean rest time(MRT)of the former are longer signifficantly than the latter,and Tmax for Rop-Dex-PLGA-MS was two times of Rop-PLGA-MS. |
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