创伤涂膜剂的药理学研究及安全性评价
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摘要
目的：创伤涂膜剂是由白求恩国际和平医院药剂科自主研发的一种具有镇痛、抑菌和抗感染作用的新型复方涂膜剂，主药成分包括替硝唑、盐酸达克罗宁和醋酸氯己定等。本研究的主要目的是对该制剂的药效学、药代动力学和安全性进行系统的评价，包括镇痛试验、体外抑菌试验、在体抗感染试验、药代动力学研究、皮肤刺激试验、急性毒性和长期毒性试验，为报批临床研究提供数据支持。同时结合该制剂的剂型特点，对其在血液和皮肤组织的药代动力学特征进行深入细致的研究，为临床应用选择合适的用法用量提供科学依据。
方法：
镇痛试验采用豚鼠背部皮肤烫伤和小鼠尾部福尔马林刺激两种致痛模型，并与盐酸达克罗宁胶浆剂进行比较。将80℃水浴中加热至平衡的砝码在豚鼠裸露背部皮肤烫5s制备烫伤模型。豚鼠随机分为5组，每组6只，分别是空白对照组（基质组），创伤涂膜剂低、中、高3个剂量组，阳性对照组（盐酸达克罗宁胶浆），以20μL/cm~2的剂量涂抹药物或基质，采用针刺法进行痛觉测定，评价指标为平均起效时间、平均持续时间、有效率。鼠尾福尔马林试验分组同豚鼠试验，每组12只。将小鼠尾部浸入药液3min后，皮内注射20%福尔马林溶液20μL，然后观察并记录小鼠舔舐注射部位及尾根部的时间（sec）。
体外抑菌试验采用试管二倍稀释法，对金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌、奇异变形杆菌四种需氧菌标准菌株，脆弱拟杆菌、产气荚膜梭菌两种厌氧菌标准菌株和200种临床分离菌株进行了体外抑菌效果的评价，分别测定了创伤涂膜剂的最低抑菌浓度（MIC）、最低杀菌浓度（MBC），同时考察了pH值、细菌接种量、血清蛋白、附加药物对MIC的影响。
在药代动力学研究中，首先建立同时测定替硝唑、达克罗宁、氯己定浓度的HPLC/MS/MS分析方法，条件如下：色谱柱为Phenomenex GeminiC_(18)（50mm×2.0mm，5μm），流动相为甲醇-10mmol·L~(-1)甲酸铵（含0.2%甲酸）（56:44，v/v），流速0.2mL·min~(~(-1))，柱温25℃，通过电喷雾离子化（ESI）三级四极杆串联质谱进行正离子多反应选择监测（MRM）。用该法分别测定了大鼠血浆和皮肤微透析样品的药物浓度变化情况。实验前一天，将大鼠背部皮肤用电动剃毛器脱毛，给药前用砂纸摩擦至渗血制备破损皮肤模型，按20μL/cm2的给药剂量涂于3cm×3cm的创面，测定给药后0.25，0.5，1，1.5，2，3，4，6，8，10，12，14，24h血浆中的药物浓度经时变化情况，用DAS2.0.1程序进行数据的模拟分析。利用在体皮肤微透析技术，以5%的葡萄糖溶液（含0.1%甲酸）作为灌流液，使用线性微透析探针，在1.5μL·min~(~(-1))灌流速度下收集样品，每20min收集一次，持续12h，透析液样品直接进行HPLC/MS/MS分析，研究了创伤涂膜剂中各药物成分在皮肤组织的实时浓度变化情况。
皮肤刺激试验以家兔为模型，分为完整皮肤和破损皮肤组，进行了单次和多次给药试验。给药前将兔的背部脱毛，给药面积左右各3cm×3cm。实验采用同体左右侧自身比较，将0.5mL受试药物涂布于左侧，右侧涂布同体积溶剂作为对照。破损皮肤组给药前使用高压灭菌砂纸磨擦用药部位以渗血为度。单次给药试验持续24h，多次给药试验每天给药1次，连续给药7天。给药结束后用灭菌水清洁给药部位除去药物，分别于除去药物后1h、24h、48h和72h，肉眼观察并记录涂敷部位有无红斑和水肿等情况,并进行刺激反应评分。如存在持久性损伤，则延长观察至14d，每天观察一次。对出现中度及以上皮肤刺激性的动物，在观察期结束时对给药局部取材，进行组织病理学检查。
毒性试验包括急性毒性和长期毒性试验。采用大鼠背部皮肤给药方法，给药面积两侧各5cm×4cm，给药量20μL/cm~2/次。急性毒性试验设8个试验组，每组10只，分别是完整皮肤高剂量涂膜剂组，破损皮肤高、中、低3个剂量的复方涂膜剂组，3个高浓度的单方涂膜剂组和溶剂对照组，共给药2次，间隔6h；长毒试验设9个试验组，比急毒试验增加一个完整皮肤空白对照组，每天给药1次，连续给药4周。每次给药前用温热至37℃无菌蒸馏水除去残留受试物。试验过程中每天记录大鼠反应情况，试验结束后，所有动物被处死，做相应的血液、解剖学和组织病理学检查。
结果：
镇痛试验：豚鼠背部皮肤烫伤试验中，各试验组的有效镇痛时间范围为：高剂量组>中剂量组>低剂量组>阳性对照组。成组t检验分析显示，创伤涂膜剂各剂量组在起效时间、持续时间和有效率方面均明显优于阳性对照组（P＜0.01），各组镇痛作用强弱顺序依次为：高剂量组>中剂量组>低剂量组>阳性对照组。由镇痛有效率的卡方分析可知，四个药物组镇痛作用的有效率差异显著；创伤涂膜剂各剂量组均与阳性对照组的总体有效率差异显著，且均高于阳性对照组；创伤涂膜剂各剂量组间的有效率差异显著。创伤涂膜剂三个剂量组呈现良好的线性关系，r=0.9679。鼠尾福尔马林试验中，创伤涂膜剂各剂量组的小鼠舔尾时间与空白对照组比较差异显著（P＜0.05或P＜0.01），与阳性对照组相比也明显减少。
体外抑菌试验：创伤涂膜剂对所考察的6种标准菌株的MIC和MBC如下：金黄色葡萄球菌0.0625，0.125μg·mL~(-1)；大肠埃希氏菌2，2μg·mL~(-1)；铜绿假单胞菌4，4μg·mL~(-1)；奇异变形杆菌8，8μg·mL~(-1)；脆弱拟杆菌1，1μg·mL~(-1)；产气荚膜梭菌1，2μg·mL~(-1)。培养基pH5.0和7.0时对金黄色葡萄球菌、铜绿假单胞菌、脆弱拟杆菌MIC无影响；pH9.0时金黄色葡萄球菌、铜绿假单胞菌MIC降低，脆弱拟杆菌不生长；对产气荚膜梭菌MIC影响不同。细菌接种量105CFU·mL~(-1)和10~7CFU·mL~(-1)时对金黄色葡萄球菌、铜绿假单胞菌、脆弱拟杆菌、产气荚膜梭菌MIC均无影响；接种量10~3CFU·mL~(-1)时四种标准菌株MIC均有所降低。含25%的胎牛血清营养肉汤，对脆弱拟杆菌MIC无影响，含量为50%、75%时，脆弱拟杆菌MIC均升高；以上三种含量的胎牛血清营养肉汤，对金黄色葡萄球菌、产气荚膜梭菌MIC均升高，铜绿假单胞菌均＞32μg·mL~(-1)。
药代动力学研究：使用本实验建立的液质分析方法，空白血浆及空白透析液中内源性物质不干扰待测物的测定，血浆中替硝唑、达克罗宁、氯己定的线性范围均为2～1000ng·mL~(-1)，日内RSD均＜11.0%，日间RSD均＜14.1%，准确度在7.2～2.9%范围内，提取回收率均＞80%，在室温放置24h、-20℃冻存3d、冻融3次所测得的RE%均在±15%以内。微透析液中三种药物的线性范围均为1～200ng·mL~(-1)，日内RSD均＜6.9%，日间RSD均＜12.8%，准确度在5.0～2.7%范围内，在室温放置24h、0～4℃冷藏3d所测得的RE%均在±15%以内。两种样品的方法学验证结果均符合目前生物样品的测定要求。
创伤涂膜剂在大鼠背部破损皮肤，按20μL/cm~2剂量给药后，替硝唑、达克罗宁、氯己定在血浆中的达峰浓度分别为（97.59±47.74）、（20.34±14.20）、（10.00±5.54）ng·mL~(-1)；达峰时间分别为（1.33±0.41）、（1.67±0.26）、（1.42±0.38） h； AUC_(0-24h)分别为（376.25±53.27）、（63.41±27.15）、（69.52±29.02）ng·h·mL~(-1)，血药浓度数据经DAS软件模拟，符合二室模型。同时采用微透析技术测得皮肤组织中的药物达峰浓度分别为（363.24±276.03）、（419.33±184.85）、（15.26±6.51）ng·mL~(-1)；达峰时间分别为（1.17±0.55）、（1.28±0.39）、（1.28±0.39）h；AUC_((0-24h))分别为（606.12±225.44）、（871.36±369.31）、（78.45±36.82）ng·h·mL~(-1)。
创伤涂膜剂单次和多次给药皮肤刺激试验：全部动物（包括完整皮肤组和破损皮肤组），给药侧与对照侧相比，无明显差异，均未见红斑、水肿，两侧皮肤刺激评分均为0。
急性毒性试验：完整皮肤给药后全部动物未见明显异常表现，创伤涂膜剂的最大无毒性反应剂量（以达克罗宁的含量计算）为0.4mg/cm~2。在破损皮肤给药的动物中，创伤涂膜剂的最大无毒性反应剂量（NOAEL）为0.2mg/cm~2，最小毒性反应剂量（LOAEL）在0.2～0.4mg/cm~2范围内。创伤涂膜剂高剂量组中有8只动物于给药后8min～40min依次出现自发活动减少、俯卧不动，站立行走不稳，侧卧不动，上述表现均在给药后50min～1h内恢复。盐酸达克罗宁组动物给药后6min～19min出现相似的表现，于给药后50min～1.5h恢复。其余各组动物均未见明显异常表现。给药后第14d大体解剖，所有动物肉眼观察各脏器均未见明显异常改变。
长期毒性试验：在连续4周的给药和2周的恢复期间，未发生与药物毒性相关的动物死亡。在第一次给药后，创伤涂膜剂高、中、低剂量组、各单方组与溶剂对照组比较，动物均出现自发活动减少，盐酸达克罗宁组部分动物甚至出现运动失调和强直性抽搐，症状持续时间约1h。但是出现这些症状的时间逐日缩短，至第4周时，各组给药后均未见异常表现。给药期间，各给药组雄性动物体重明显减轻，但试验结束后可以恢复，雌性动物盐酸达克罗宁单方组肾上腺重量及肾上腺/体重系数明显增加，试验结束后亦可恢复，这两种现象考虑与操作应激有关。其余各组一般体征、体重、摄食量、血液学、血液生化及组织病理学检查均未见明显异常。
结论：
豚鼠背部皮肤烫伤和小鼠尾部福尔马林刺激试验结果表明，创伤涂膜剂对物理刺激和化学刺激引起的动物皮肤损伤有显著的镇痛作用，并呈现良好的线性关系，与达克罗宁胶浆剂相比，涂膜剂的镇痛效果更好。
体外抑菌试验表明该制剂对革兰氏阳性菌和革兰氏阴性菌均有良好的抑菌效果。
建立的同时测定替硝唑、达克罗宁、氯己定浓度的HPLC/MS/MS分析方法，灵敏、准确、稳定，能够满足大鼠在体皮肤微透析样品和血浆样品中三种药物的测定要求；大鼠破损皮肤给药后，血液和皮肤组织中药物的吸收速度相似，但皮肤组织中替硝唑和达克罗宁的浓度均明显高于血液中的药物浓度，有利于发挥局部抗菌和镇痛作用，氯己定在血液和皮肤组织中的吸收程度均较小。
皮肤刺激试验表明创伤涂膜剂对家兔完整皮肤及破损皮肤均无明显刺激性。
急性毒性试验结果表明，创伤涂膜剂高剂量组出现的神经系统毒性主要来自于组方中的盐酸达克罗宁，但涂膜剂复方的毒性小于单方盐酸达克罗宁的毒性，且这种反应是一过性的，可于给药1h内恢复正常。
长期毒性试验对SD大鼠进行创伤涂膜剂连续4周给药及停药恢复2周观察后，暂未见与药物相关的明显毒理学改变。
Objective: New type of film-forming solution which have analgesic,antibacterial and anti-infection effect were independently developed byBethune International Peace Hospital. The three main components of thefilm-forming solution are tinidazole, dyclonine and chlorhexidine. The aim ofthis study is to make a systematic evaluation on the pharmacodynamics,pharmacokinetics and safety of the preparation, which including analgesia test,in vitro antibacterial test, in vivo anti-infection test, skin stimulation test, acuteand long-term toxicity test. The results can provide preclinical data to applyfor clinical research. At the same time, a deep and detailed pharmacokineticresearch of the film-solution was made to provide references for selectingappropriate dosage in clinical application.
Methods:
Two animal models，back skin burn in guinea pig and tail formalin test inmice, were applied to evaluate the analgesic effects of the film-formingsolution for trauma. The dyclonine hydrochloride plasmagel was used as thepositive control drug. Back skin burn model of guinea was made by exposion5s to a hot counterweight which removed immediately from a thermostaticwaterbath at80℃.Then the burned guinea pigs were divided into5groups,which are blank control group (solvent), the film-forming solution groups athigh, middle and low concentration, positive control group (dyclonineplasmagel),6guinea pigs per group. A dosage of20μL/cm~2of drug solutionsor solvent was spreaded on the burned skin. Pain sensation was measured byacupuncture. Evaluation index is mean onset time, mean duration time andefficient rate. Tail formalin test in mice include the same groups to guinea pigstest,12mice per group. The mouse tail was immersed in drug solution for3min, administered intradermally20μL of20%formalin solution. The pain behavior was quantified by determining the time (s) the mouse lick theinjected site, over40min using5min stage.
Double-dilution method was used during in vitro antibacterial test of thefilm-forming solution. Minimal inhibitory concentration (MIC) and minimalbactericidal concentration (MBC) were determined for six standard strainsincluding four aerobe and two anaerobe, which are staphylococcus aureusATCC25923, escherichia coli ATCC25922, pseudomonas aeruginosaATCC27853, proteus mirabilis ATCC12453, bacteroides fragilis ATCC25285and clostridium perfringens CICC22949. In addition,200clinical separationstrains were tested with the studied drug solution. The effects of pH value,bacterial load, serum protein and additional drugs on antibacterial activitieswere investigated.
A rapid and sensitive HPLC/MS/MS method for simultaneousdetermination of tinidazole, dyclonine and chlorhexidine was developed andapplied in the pharmacokinetics research of the film-forming solution. It wasused for the concentration determination of plsama and microdialysis samples.Separation was achieved on a Phenomenex Gemini C18column (50mm×2.0mm,5μm) using an isocratic mobile phase system composed ofmethanol-ammonium formate (10mM)-formic acid (56:44:0.2, v/v/v) at aflow rate of0.2mL·min~(-1). Analytes were determined by tandem massspectrometry with electrospray positive ionization and multiple-reactionmonitoring (MRM) mode. Rats were depilated on the back skin to an area of3cm×3cm by animal shaver the day before experiment. Before administration,the depilated skin was fricted by sandpaper to the degree of bleeding. The dosewas20μL/cm~2. The plasma drug congcentration at0.25，0.5，1，1.5，2，3，4，6，8，10，12，14，24h after adminstration were determined. Thepharmacokinetic parameters were calculated by DAS2.0.1program.Microdialysis technique was used for the determination of drug concetrationin subcutaneous tissue with5%of the glucose solution (including0.1%formic acid) as perfusion fluid. The perfusion speed was1.5μL·min~(-1).Microdialysis samples were collected by linear probe every20min until12h and injected directly for HPLC/MS/MS analysis.
Skin irritation test was made with rabbit. All animals were depilated twoareas of3cm×3cm on the left and right side of back skin by animal shaverbefore administration. And they are divided into complete and damaged skingroups, the latter were fricted by autoclaving sandpaper to the degree ofbleeding before administration.0.5mL of test solution was coated on the leftside and the same volume of solvent was coated on the right side as a control.Single test just administrate once and the film was kept for24h. Multiple testadministrate once every day and last for7days. Before next administration,the formed film was washed out with sterile water. After the lastadministration, a record of erythema and edema was made at1,24,48and72h. If persistent injury was found by eyes, the observation period will beextended to14days and made records everyday. At the end of observation,histopathological examination should be made for those animals which havemoderate and serious skin irritation.
Toxicity test include acute and long-term toxicity test. The drug solutionwas spread on two areas of5cm×4cm of the bilateral back skin of rats whichwere depilated before experiment. The dose was20μL/cm~2every time. Acutetoxicity test set8groups, which include one complete skin group and sevendamaged skin groups. The former was just given the compound film-formingsolution at high concentration. The damaged groups were given the compoundfilm-forming solution at three concentrations, three single drug film-formingsolution at high congcentration and a solvent control group. Acute toxicity testwas given test solutions twice during an interval of6h. Long-term toxicitytest have9groups, adding a blank control group with complete skin comparedto acute test. The rats were given test solutions once a day and lasting for4weeks. Before application of the test solution, the last residual drug need to beremoved with sterile distilled water heated to37℃. During the experiment,the reactions of rats were recorded every day. When the experiment is finished,all animals were sacrificed and made blood, gross anatomy andhistopathological examination.
Results:
Results of analgesic test: In guinea pig back skin burn test, the effectiveanalgesic time range for different test groups are the following: highconcentration (H)＞median concentration (M)＞low concentration (L)＞positive control (C) group. t-test analysis of the efficacy data showed that thefilm-forming solution of each concentration group were significantly betterthan the positive control group according to the onset time, duration time andefficiency (P＜0.01). The order of analgesic effect from strong to weak was H＞M＞L＞C group. Through the chi-square analysis of analgesic efficiency,there were significant differences between the four drug groups. The overallefficiency of each concentration group of the film-forming solution was betterthan that of C group. The curve for negative reaction rate was linear in theconcentration range of0.25%～1%with correlation coefficient values was0.9679. For mice tail formalin test, the licking time for both the thefilm-forming solution group and the positive control group were significantlyreduced compared to that for the blank control group (P＜0.05or P＜0.01)，and the film-forming solution group was better than the positive control group.
In vitro antibacterial test: MIC and MBC of the film-forming solution fortrauma was0.0625and0.125μg·mL~(-1),2and2μg·mL~(-1),4and4μg·mL~(-1),8and8μg·mL~(-1),1and1μg·mL~(-1),1and2μg·mL~(-1)for staphylococcus aureus,escherichia coli, pseudomonas aeruginosa, proteus mirabilis, bacteroidesfragilis and clostridium perfringens respectively. Culture media pH5.0and7.0had no effects on the activity against staphylococcus aureus, pseudomonasaeruginosa and bacteroides fragilis, but MIC against staphylococcus aureusand pseudomonas aeruginosa decreased and bacteroides fragilis no growthwhen pH9.0. pH values of culture media had different effects on the MICagainst clostridium perfringens. The inoculum size of bacteria had no effect onthe MIC against staphylococcus aureus, pseudomonas aeruginosa, bacteroidesfragilis and clostridium perfringens, but had reduced on MIC against with10~3CFU·mL~(-1)compared to10~5CFU·mL~(-1)and10~7CFU·mL~(-1). MIC againstbacteroides fragilis in nutrient broth which contained25%fetal calf had no effect and contained50%,75%fetal calf had increased. MIC againststaphylococcus aureus and clostridium perfringens in nutrient broth whichcontained25%,50%and75%fetal calf had increased. MIC againstpseudomonas aeruginosa in nutrient broth which contained25%,50%and75%fetal calf serum exceeded32μg·mL~(-1).
Pharmacokinetics study of the film-forming solution applied on damagedskin: For the developed HPLC/MS/MS method, no endogenous impurity fromblank plasma and dialysate was found to interfere with analytes. Thecalibration curves of tinidazole, dyclonine, chlorhexidine in rat plasma werelinear in the concentration range of2～1000ng·mL~(-1). The intra-day RSDs areless than11.0%, the inter-day RSDs are less than14.1%and the RE valueswere-7.2～2.9%. The mean extraction recoveries were all above80%. Nosignificant matrix effect was found during the determination. Stability testshowed the RE%for plasma samples kept at room temperature for24h, afterthree freeze–thaw cycles and below20°C for3days were all within±15%.The calibration curves of three components in dialysate were linear in theconcentration range of1～200ng·mL~(-1). The intra-day RSDs are less than6.9%, the inter-day RSDs are less than12.8%and the RE values were-5.0～2.7%. The RE%for dialysate samples kept at room temperature for24h and0-4°C for3days were all within±15%. Both validation results for plasma anddialysate samples meet the criteria for biological samples analysis.
After the film-forming solution was applied once on the damaged skin ofrats with the dose of20μL/cm~2, the main pharmarcokinetic parameters oftinidazole, dyclonine and chlorhexidine in plasma were as follows: Cmax(97.59±47.74),(20.34±14.20),(10.00±5.54) ng·mL~(-1); t_(max)(1.33±0.41),(1.67±0.26),(1.42±0.38) h; AUC_((0-24h))(376.25±53.27),(63.41±27.15),(69.52±29.02) ng·h·mL~(-1). The concentration-time curves were fitted totwo-compartment models. The main pharmarcokinetic parameters oftinidazole, dyclonine and chlorhexidine in skin tissue determined bymicodialysis technique were as follows: Cmax(363.24±276.03),(419.33±184.85),(15.26±6.51) ng·mL~(-1); t_(max)(1.17±0.55),(1.28±0.39), (1.28±0.39) h；AUC(0~(-1)2h)(606.12±225.44),(871.36±369.31),(78.45±36.82)ng·h·mL~(-1).
Single and multiple administrate on skin irritation test: No significantdifference was found between the administration side and control side for theanimals whether in complete or damaged skin group, both sides had noerythema, edema and score of skin irritation were0.
Acute toxicity test: For complete skin groups, all animals had no obviousabnormal performance after administration and the NOAEL (calculated bydyclonine) of the compound film-forming solution was0.4mg/cm~2, and0.2mg/cm~2for the damaged skin group. The LOAEL was among the range of0.2to0.4mg/cm~2. Eight animals in the film-forming solution group at highconcentration showed reduced spontaneous movement, pronation andimmobility, unsteadystanding and walking, sidelying and immobility8to40min after administration. And the above performances recovered during50min to1h. All animals in the middle and low concentration group showed noobvious abnormalities after administration. High concentration of dycloninehydrochloride group showed the same performance with those rats in the highconcentration group of film-forming solution6to19min after administrationand recovered during50min to1.5h. Chlorhexidine acetate and tinidazolegroup had no obvious abnormalities. All organs were not found abnormalchange by eyes when gross anatomy was made14days after administration.
Long-term toxicity test: No death related with test drugs happened during4weeks administration and2weeks restoration period for all the animals.After the first administration, spontaneous movement of rats in compoundfilm-forming solution group and single recipe film-forming solution groupwere all reduced compared to those in solvent control group. Some rats indyclonine group showed dyskinesias and even tetanic convulsion, thesymptom could last1h. But the time of abnormal symptoms released day byday. After4weeks, no abnormal symptoms happened after administration，somaybe the animals produced tolerance to the drug. During the administration,body weight of male animals which were treated by test solutions released significantly compared to those in blank control group, but recoveried afterexperiment. Adrenal weight and adrenal gland/body weight index of femaleanimals significantly increased for dyclonine hydrochloride group and alsorecoveried after experiment. These two symptoms were considered to correlatewith operating stress. General signs, body weight, food consumption,hematology, blood biochemistry and histopathology examination of the othergroups did not show significant abnormality.
Conclusion:
The test results of back skin burn in guinea pig and tail formalin test inmice showed that the studied film-forming solution for trauma had asignificant analgesic effect on the skin damage caused by physical andchemical stimulation compared with dyclonine plasmagel and its analgesiceffect had a dose-response relationship.
The studied film-forming solution for trauma had strong antibacterialactivity against gram-positive bacteria and gram-negative bacteria in the invitro antibacterial experiment.
A sensitive, accurate and stable HPLC/MS/MS method was developedand validated for simultaneous determination of tinidazole, dyclonine andchlorhexidine, in rat plasma and skin microdialysis sample. When applied thefilm-forming solution on rat’s damaged back skin, the drug absorption rateinto blood was similar with that into skin tissue, but drug concentration of skintissue higher than that in blood, which would have profit to produce localanti-infection and analgesic effect. Chlorhexidine absorbed little whether inplasma or skin.
Skin irritation test showed that the studied film-forming has nosignificantly irritation with complete and damaged skin of the rabbit.
Acute toxicity test showed that the nervous system toxicity of the studiedfilm-forming solution at high concentration might correlate with the dycloninehydrochloride in formula. However, the toxicity of the compoundfilm-forming solution less than that of dyclonine solution, and this reactionwas temple, could recover within1h.
Long-term toxicity test made with SD rat showed that during the periodof4weeks when it is continuously applying the studied film-forming solutionand2weeks when the drug was withdrawed, no obvious toxicology alterationcorrelated with drug.
方法：
镇痛试验采用豚鼠背部皮肤烫伤和小鼠尾部福尔马林刺激两种致痛模型，并与盐酸达克罗宁胶浆剂进行比较。将80℃水浴中加热至平衡的砝码在豚鼠裸露背部皮肤烫5s制备烫伤模型。豚鼠随机分为5组，每组6只，分别是空白对照组（基质组），创伤涂膜剂低、中、高3个剂量组，阳性对照组（盐酸达克罗宁胶浆），以20μL/cm~2的剂量涂抹药物或基质，采用针刺法进行痛觉测定，评价指标为平均起效时间、平均持续时间、有效率。鼠尾福尔马林试验分组同豚鼠试验，每组12只。将小鼠尾部浸入药液3min后，皮内注射20%福尔马林溶液20μL，然后观察并记录小鼠舔舐注射部位及尾根部的时间（sec）。
体外抑菌试验采用试管二倍稀释法，对金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌、奇异变形杆菌四种需氧菌标准菌株，脆弱拟杆菌、产气荚膜梭菌两种厌氧菌标准菌株和200种临床分离菌株进行了体外抑菌效果的评价，分别测定了创伤涂膜剂的最低抑菌浓度（MIC）、最低杀菌浓度（MBC），同时考察了pH值、细菌接种量、血清蛋白、附加药物对MIC的影响。
在药代动力学研究中，首先建立同时测定替硝唑、达克罗宁、氯己定浓度的HPLC/MS/MS分析方法，条件如下：色谱柱为Phenomenex GeminiC_(18)（50mm×2.0mm，5μm），流动相为甲醇-10mmol·L~(-1)甲酸铵（含0.2%甲酸）（56:44，v/v），流速0.2mL·min~(~(-1))，柱温25℃，通过电喷雾离子化（ESI）三级四极杆串联质谱进行正离子多反应选择监测（MRM）。用该法分别测定了大鼠血浆和皮肤微透析样品的药物浓度变化情况。实验前一天，将大鼠背部皮肤用电动剃毛器脱毛，给药前用砂纸摩擦至渗血制备破损皮肤模型，按20μL/cm2的给药剂量涂于3cm×3cm的创面，测定给药后0.25，0.5，1，1.5，2，3，4，6，8，10，12，14，24h血浆中的药物浓度经时变化情况，用DAS2.0.1程序进行数据的模拟分析。利用在体皮肤微透析技术，以5%的葡萄糖溶液（含0.1%甲酸）作为灌流液，使用线性微透析探针，在1.5μL·min~(~(-1))灌流速度下收集样品，每20min收集一次，持续12h，透析液样品直接进行HPLC/MS/MS分析，研究了创伤涂膜剂中各药物成分在皮肤组织的实时浓度变化情况。
皮肤刺激试验以家兔为模型，分为完整皮肤和破损皮肤组，进行了单次和多次给药试验。给药前将兔的背部脱毛，给药面积左右各3cm×3cm。实验采用同体左右侧自身比较，将0.5mL受试药物涂布于左侧，右侧涂布同体积溶剂作为对照。破损皮肤组给药前使用高压灭菌砂纸磨擦用药部位以渗血为度。单次给药试验持续24h，多次给药试验每天给药1次，连续给药7天。给药结束后用灭菌水清洁给药部位除去药物，分别于除去药物后1h、24h、48h和72h，肉眼观察并记录涂敷部位有无红斑和水肿等情况,并进行刺激反应评分。如存在持久性损伤，则延长观察至14d，每天观察一次。对出现中度及以上皮肤刺激性的动物，在观察期结束时对给药局部取材，进行组织病理学检查。
毒性试验包括急性毒性和长期毒性试验。采用大鼠背部皮肤给药方法，给药面积两侧各5cm×4cm，给药量20μL/cm~2/次。急性毒性试验设8个试验组，每组10只，分别是完整皮肤高剂量涂膜剂组，破损皮肤高、中、低3个剂量的复方涂膜剂组，3个高浓度的单方涂膜剂组和溶剂对照组，共给药2次，间隔6h；长毒试验设9个试验组，比急毒试验增加一个完整皮肤空白对照组，每天给药1次，连续给药4周。每次给药前用温热至37℃无菌蒸馏水除去残留受试物。试验过程中每天记录大鼠反应情况，试验结束后，所有动物被处死，做相应的血液、解剖学和组织病理学检查。
结果：
镇痛试验：豚鼠背部皮肤烫伤试验中，各试验组的有效镇痛时间范围为：高剂量组>中剂量组>低剂量组>阳性对照组。成组t检验分析显示，创伤涂膜剂各剂量组在起效时间、持续时间和有效率方面均明显优于阳性对照组（P＜0.01），各组镇痛作用强弱顺序依次为：高剂量组>中剂量组>低剂量组>阳性对照组。由镇痛有效率的卡方分析可知，四个药物组镇痛作用的有效率差异显著；创伤涂膜剂各剂量组均与阳性对照组的总体有效率差异显著，且均高于阳性对照组；创伤涂膜剂各剂量组间的有效率差异显著。创伤涂膜剂三个剂量组呈现良好的线性关系，r=0.9679。鼠尾福尔马林试验中，创伤涂膜剂各剂量组的小鼠舔尾时间与空白对照组比较差异显著（P＜0.05或P＜0.01），与阳性对照组相比也明显减少。
体外抑菌试验：创伤涂膜剂对所考察的6种标准菌株的MIC和MBC如下：金黄色葡萄球菌0.0625，0.125μg·mL~(-1)；大肠埃希氏菌2，2μg·mL~(-1)；铜绿假单胞菌4，4μg·mL~(-1)；奇异变形杆菌8，8μg·mL~(-1)；脆弱拟杆菌1，1μg·mL~(-1)；产气荚膜梭菌1，2μg·mL~(-1)。培养基pH5.0和7.0时对金黄色葡萄球菌、铜绿假单胞菌、脆弱拟杆菌MIC无影响；pH9.0时金黄色葡萄球菌、铜绿假单胞菌MIC降低，脆弱拟杆菌不生长；对产气荚膜梭菌MIC影响不同。细菌接种量105CFU·mL~(-1)和10~7CFU·mL~(-1)时对金黄色葡萄球菌、铜绿假单胞菌、脆弱拟杆菌、产气荚膜梭菌MIC均无影响；接种量10~3CFU·mL~(-1)时四种标准菌株MIC均有所降低。含25%的胎牛血清营养肉汤，对脆弱拟杆菌MIC无影响，含量为50%、75%时，脆弱拟杆菌MIC均升高；以上三种含量的胎牛血清营养肉汤，对金黄色葡萄球菌、产气荚膜梭菌MIC均升高，铜绿假单胞菌均＞32μg·mL~(-1)。
药代动力学研究：使用本实验建立的液质分析方法，空白血浆及空白透析液中内源性物质不干扰待测物的测定，血浆中替硝唑、达克罗宁、氯己定的线性范围均为2～1000ng·mL~(-1)，日内RSD均＜11.0%，日间RSD均＜14.1%，准确度在7.2～2.9%范围内，提取回收率均＞80%，在室温放置24h、-20℃冻存3d、冻融3次所测得的RE%均在±15%以内。微透析液中三种药物的线性范围均为1～200ng·mL~(-1)，日内RSD均＜6.9%，日间RSD均＜12.8%，准确度在5.0～2.7%范围内，在室温放置24h、0～4℃冷藏3d所测得的RE%均在±15%以内。两种样品的方法学验证结果均符合目前生物样品的测定要求。
创伤涂膜剂在大鼠背部破损皮肤，按20μL/cm~2剂量给药后，替硝唑、达克罗宁、氯己定在血浆中的达峰浓度分别为（97.59±47.74）、（20.34±14.20）、（10.00±5.54）ng·mL~(-1)；达峰时间分别为（1.33±0.41）、（1.67±0.26）、（1.42±0.38） h； AUC_(0-24h)分别为（376.25±53.27）、（63.41±27.15）、（69.52±29.02）ng·h·mL~(-1)，血药浓度数据经DAS软件模拟，符合二室模型。同时采用微透析技术测得皮肤组织中的药物达峰浓度分别为（363.24±276.03）、（419.33±184.85）、（15.26±6.51）ng·mL~(-1)；达峰时间分别为（1.17±0.55）、（1.28±0.39）、（1.28±0.39）h；AUC_((0-24h))分别为（606.12±225.44）、（871.36±369.31）、（78.45±36.82）ng·h·mL~(-1)。
创伤涂膜剂单次和多次给药皮肤刺激试验：全部动物（包括完整皮肤组和破损皮肤组），给药侧与对照侧相比，无明显差异，均未见红斑、水肿，两侧皮肤刺激评分均为0。
急性毒性试验：完整皮肤给药后全部动物未见明显异常表现，创伤涂膜剂的最大无毒性反应剂量（以达克罗宁的含量计算）为0.4mg/cm~2。在破损皮肤给药的动物中，创伤涂膜剂的最大无毒性反应剂量（NOAEL）为0.2mg/cm~2，最小毒性反应剂量（LOAEL）在0.2～0.4mg/cm~2范围内。创伤涂膜剂高剂量组中有8只动物于给药后8min～40min依次出现自发活动减少、俯卧不动，站立行走不稳，侧卧不动，上述表现均在给药后50min～1h内恢复。盐酸达克罗宁组动物给药后6min～19min出现相似的表现，于给药后50min～1.5h恢复。其余各组动物均未见明显异常表现。给药后第14d大体解剖，所有动物肉眼观察各脏器均未见明显异常改变。
长期毒性试验：在连续4周的给药和2周的恢复期间，未发生与药物毒性相关的动物死亡。在第一次给药后，创伤涂膜剂高、中、低剂量组、各单方组与溶剂对照组比较，动物均出现自发活动减少，盐酸达克罗宁组部分动物甚至出现运动失调和强直性抽搐，症状持续时间约1h。但是出现这些症状的时间逐日缩短，至第4周时，各组给药后均未见异常表现。给药期间，各给药组雄性动物体重明显减轻，但试验结束后可以恢复，雌性动物盐酸达克罗宁单方组肾上腺重量及肾上腺/体重系数明显增加，试验结束后亦可恢复，这两种现象考虑与操作应激有关。其余各组一般体征、体重、摄食量、血液学、血液生化及组织病理学检查均未见明显异常。
结论：
豚鼠背部皮肤烫伤和小鼠尾部福尔马林刺激试验结果表明，创伤涂膜剂对物理刺激和化学刺激引起的动物皮肤损伤有显著的镇痛作用，并呈现良好的线性关系，与达克罗宁胶浆剂相比，涂膜剂的镇痛效果更好。
体外抑菌试验表明该制剂对革兰氏阳性菌和革兰氏阴性菌均有良好的抑菌效果。
建立的同时测定替硝唑、达克罗宁、氯己定浓度的HPLC/MS/MS分析方法，灵敏、准确、稳定，能够满足大鼠在体皮肤微透析样品和血浆样品中三种药物的测定要求；大鼠破损皮肤给药后，血液和皮肤组织中药物的吸收速度相似，但皮肤组织中替硝唑和达克罗宁的浓度均明显高于血液中的药物浓度，有利于发挥局部抗菌和镇痛作用，氯己定在血液和皮肤组织中的吸收程度均较小。
皮肤刺激试验表明创伤涂膜剂对家兔完整皮肤及破损皮肤均无明显刺激性。
急性毒性试验结果表明，创伤涂膜剂高剂量组出现的神经系统毒性主要来自于组方中的盐酸达克罗宁，但涂膜剂复方的毒性小于单方盐酸达克罗宁的毒性，且这种反应是一过性的，可于给药1h内恢复正常。
长期毒性试验对SD大鼠进行创伤涂膜剂连续4周给药及停药恢复2周观察后，暂未见与药物相关的明显毒理学改变。
Objective: New type of film-forming solution which have analgesic,antibacterial and anti-infection effect were independently developed byBethune International Peace Hospital. The three main components of thefilm-forming solution are tinidazole, dyclonine and chlorhexidine. The aim ofthis study is to make a systematic evaluation on the pharmacodynamics,pharmacokinetics and safety of the preparation, which including analgesia test,in vitro antibacterial test, in vivo anti-infection test, skin stimulation test, acuteand long-term toxicity test. The results can provide preclinical data to applyfor clinical research. At the same time, a deep and detailed pharmacokineticresearch of the film-solution was made to provide references for selectingappropriate dosage in clinical application.
Methods:
Two animal models，back skin burn in guinea pig and tail formalin test inmice, were applied to evaluate the analgesic effects of the film-formingsolution for trauma. The dyclonine hydrochloride plasmagel was used as thepositive control drug. Back skin burn model of guinea was made by exposion5s to a hot counterweight which removed immediately from a thermostaticwaterbath at80℃.Then the burned guinea pigs were divided into5groups,which are blank control group (solvent), the film-forming solution groups athigh, middle and low concentration, positive control group (dyclonineplasmagel),6guinea pigs per group. A dosage of20μL/cm~2of drug solutionsor solvent was spreaded on the burned skin. Pain sensation was measured byacupuncture. Evaluation index is mean onset time, mean duration time andefficient rate. Tail formalin test in mice include the same groups to guinea pigstest,12mice per group. The mouse tail was immersed in drug solution for3min, administered intradermally20μL of20%formalin solution. The pain behavior was quantified by determining the time (s) the mouse lick theinjected site, over40min using5min stage.
Double-dilution method was used during in vitro antibacterial test of thefilm-forming solution. Minimal inhibitory concentration (MIC) and minimalbactericidal concentration (MBC) were determined for six standard strainsincluding four aerobe and two anaerobe, which are staphylococcus aureusATCC25923, escherichia coli ATCC25922, pseudomonas aeruginosaATCC27853, proteus mirabilis ATCC12453, bacteroides fragilis ATCC25285and clostridium perfringens CICC22949. In addition,200clinical separationstrains were tested with the studied drug solution. The effects of pH value,bacterial load, serum protein and additional drugs on antibacterial activitieswere investigated.
A rapid and sensitive HPLC/MS/MS method for simultaneousdetermination of tinidazole, dyclonine and chlorhexidine was developed andapplied in the pharmacokinetics research of the film-forming solution. It wasused for the concentration determination of plsama and microdialysis samples.Separation was achieved on a Phenomenex Gemini C18column (50mm×2.0mm,5μm) using an isocratic mobile phase system composed ofmethanol-ammonium formate (10mM)-formic acid (56:44:0.2, v/v/v) at aflow rate of0.2mL·min~(-1). Analytes were determined by tandem massspectrometry with electrospray positive ionization and multiple-reactionmonitoring (MRM) mode. Rats were depilated on the back skin to an area of3cm×3cm by animal shaver the day before experiment. Before administration,the depilated skin was fricted by sandpaper to the degree of bleeding. The dosewas20μL/cm~2. The plasma drug congcentration at0.25，0.5，1，1.5，2，3，4，6，8，10，12，14，24h after adminstration were determined. Thepharmacokinetic parameters were calculated by DAS2.0.1program.Microdialysis technique was used for the determination of drug concetrationin subcutaneous tissue with5%of the glucose solution (including0.1%formic acid) as perfusion fluid. The perfusion speed was1.5μL·min~(-1).Microdialysis samples were collected by linear probe every20min until12h and injected directly for HPLC/MS/MS analysis.
Skin irritation test was made with rabbit. All animals were depilated twoareas of3cm×3cm on the left and right side of back skin by animal shaverbefore administration. And they are divided into complete and damaged skingroups, the latter were fricted by autoclaving sandpaper to the degree ofbleeding before administration.0.5mL of test solution was coated on the leftside and the same volume of solvent was coated on the right side as a control.Single test just administrate once and the film was kept for24h. Multiple testadministrate once every day and last for7days. Before next administration,the formed film was washed out with sterile water. After the lastadministration, a record of erythema and edema was made at1,24,48and72h. If persistent injury was found by eyes, the observation period will beextended to14days and made records everyday. At the end of observation,histopathological examination should be made for those animals which havemoderate and serious skin irritation.
Toxicity test include acute and long-term toxicity test. The drug solutionwas spread on two areas of5cm×4cm of the bilateral back skin of rats whichwere depilated before experiment. The dose was20μL/cm~2every time. Acutetoxicity test set8groups, which include one complete skin group and sevendamaged skin groups. The former was just given the compound film-formingsolution at high concentration. The damaged groups were given the compoundfilm-forming solution at three concentrations, three single drug film-formingsolution at high congcentration and a solvent control group. Acute toxicity testwas given test solutions twice during an interval of6h. Long-term toxicitytest have9groups, adding a blank control group with complete skin comparedto acute test. The rats were given test solutions once a day and lasting for4weeks. Before application of the test solution, the last residual drug need to beremoved with sterile distilled water heated to37℃. During the experiment,the reactions of rats were recorded every day. When the experiment is finished,all animals were sacrificed and made blood, gross anatomy andhistopathological examination.
Results:
Results of analgesic test: In guinea pig back skin burn test, the effectiveanalgesic time range for different test groups are the following: highconcentration (H)＞median concentration (M)＞low concentration (L)＞positive control (C) group. t-test analysis of the efficacy data showed that thefilm-forming solution of each concentration group were significantly betterthan the positive control group according to the onset time, duration time andefficiency (P＜0.01). The order of analgesic effect from strong to weak was H＞M＞L＞C group. Through the chi-square analysis of analgesic efficiency,there were significant differences between the four drug groups. The overallefficiency of each concentration group of the film-forming solution was betterthan that of C group. The curve for negative reaction rate was linear in theconcentration range of0.25%～1%with correlation coefficient values was0.9679. For mice tail formalin test, the licking time for both the thefilm-forming solution group and the positive control group were significantlyreduced compared to that for the blank control group (P＜0.05or P＜0.01)，and the film-forming solution group was better than the positive control group.
In vitro antibacterial test: MIC and MBC of the film-forming solution fortrauma was0.0625and0.125μg·mL~(-1),2and2μg·mL~(-1),4and4μg·mL~(-1),8and8μg·mL~(-1),1and1μg·mL~(-1),1and2μg·mL~(-1)for staphylococcus aureus,escherichia coli, pseudomonas aeruginosa, proteus mirabilis, bacteroidesfragilis and clostridium perfringens respectively. Culture media pH5.0and7.0had no effects on the activity against staphylococcus aureus, pseudomonasaeruginosa and bacteroides fragilis, but MIC against staphylococcus aureusand pseudomonas aeruginosa decreased and bacteroides fragilis no growthwhen pH9.0. pH values of culture media had different effects on the MICagainst clostridium perfringens. The inoculum size of bacteria had no effect onthe MIC against staphylococcus aureus, pseudomonas aeruginosa, bacteroidesfragilis and clostridium perfringens, but had reduced on MIC against with10~3CFU·mL~(-1)compared to10~5CFU·mL~(-1)and10~7CFU·mL~(-1). MIC againstbacteroides fragilis in nutrient broth which contained25%fetal calf had no effect and contained50%,75%fetal calf had increased. MIC againststaphylococcus aureus and clostridium perfringens in nutrient broth whichcontained25%,50%and75%fetal calf had increased. MIC againstpseudomonas aeruginosa in nutrient broth which contained25%,50%and75%fetal calf serum exceeded32μg·mL~(-1).
Pharmacokinetics study of the film-forming solution applied on damagedskin: For the developed HPLC/MS/MS method, no endogenous impurity fromblank plasma and dialysate was found to interfere with analytes. Thecalibration curves of tinidazole, dyclonine, chlorhexidine in rat plasma werelinear in the concentration range of2～1000ng·mL~(-1). The intra-day RSDs areless than11.0%, the inter-day RSDs are less than14.1%and the RE valueswere-7.2～2.9%. The mean extraction recoveries were all above80%. Nosignificant matrix effect was found during the determination. Stability testshowed the RE%for plasma samples kept at room temperature for24h, afterthree freeze–thaw cycles and below20°C for3days were all within±15%.The calibration curves of three components in dialysate were linear in theconcentration range of1～200ng·mL~(-1). The intra-day RSDs are less than6.9%, the inter-day RSDs are less than12.8%and the RE values were-5.0～2.7%. The RE%for dialysate samples kept at room temperature for24h and0-4°C for3days were all within±15%. Both validation results for plasma anddialysate samples meet the criteria for biological samples analysis.
After the film-forming solution was applied once on the damaged skin ofrats with the dose of20μL/cm~2, the main pharmarcokinetic parameters oftinidazole, dyclonine and chlorhexidine in plasma were as follows: Cmax(97.59±47.74),(20.34±14.20),(10.00±5.54) ng·mL~(-1); t_(max)(1.33±0.41),(1.67±0.26),(1.42±0.38) h; AUC_((0-24h))(376.25±53.27),(63.41±27.15),(69.52±29.02) ng·h·mL~(-1). The concentration-time curves were fitted totwo-compartment models. The main pharmarcokinetic parameters oftinidazole, dyclonine and chlorhexidine in skin tissue determined bymicodialysis technique were as follows: Cmax(363.24±276.03),(419.33±184.85),(15.26±6.51) ng·mL~(-1); t_(max)(1.17±0.55),(1.28±0.39), (1.28±0.39) h；AUC(0~(-1)2h)(606.12±225.44),(871.36±369.31),(78.45±36.82)ng·h·mL~(-1).
Single and multiple administrate on skin irritation test: No significantdifference was found between the administration side and control side for theanimals whether in complete or damaged skin group, both sides had noerythema, edema and score of skin irritation were0.
Acute toxicity test: For complete skin groups, all animals had no obviousabnormal performance after administration and the NOAEL (calculated bydyclonine) of the compound film-forming solution was0.4mg/cm~2, and0.2mg/cm~2for the damaged skin group. The LOAEL was among the range of0.2to0.4mg/cm~2. Eight animals in the film-forming solution group at highconcentration showed reduced spontaneous movement, pronation andimmobility, unsteadystanding and walking, sidelying and immobility8to40min after administration. And the above performances recovered during50min to1h. All animals in the middle and low concentration group showed noobvious abnormalities after administration. High concentration of dycloninehydrochloride group showed the same performance with those rats in the highconcentration group of film-forming solution6to19min after administrationand recovered during50min to1.5h. Chlorhexidine acetate and tinidazolegroup had no obvious abnormalities. All organs were not found abnormalchange by eyes when gross anatomy was made14days after administration.
Long-term toxicity test: No death related with test drugs happened during4weeks administration and2weeks restoration period for all the animals.After the first administration, spontaneous movement of rats in compoundfilm-forming solution group and single recipe film-forming solution groupwere all reduced compared to those in solvent control group. Some rats indyclonine group showed dyskinesias and even tetanic convulsion, thesymptom could last1h. But the time of abnormal symptoms released day byday. After4weeks, no abnormal symptoms happened after administration，somaybe the animals produced tolerance to the drug. During the administration,body weight of male animals which were treated by test solutions released significantly compared to those in blank control group, but recoveried afterexperiment. Adrenal weight and adrenal gland/body weight index of femaleanimals significantly increased for dyclonine hydrochloride group and alsorecoveried after experiment. These two symptoms were considered to correlatewith operating stress. General signs, body weight, food consumption,hematology, blood biochemistry and histopathology examination of the othergroups did not show significant abnormality.
Conclusion:
The test results of back skin burn in guinea pig and tail formalin test inmice showed that the studied film-forming solution for trauma had asignificant analgesic effect on the skin damage caused by physical andchemical stimulation compared with dyclonine plasmagel and its analgesiceffect had a dose-response relationship.
The studied film-forming solution for trauma had strong antibacterialactivity against gram-positive bacteria and gram-negative bacteria in the invitro antibacterial experiment.
A sensitive, accurate and stable HPLC/MS/MS method was developedand validated for simultaneous determination of tinidazole, dyclonine andchlorhexidine, in rat plasma and skin microdialysis sample. When applied thefilm-forming solution on rat’s damaged back skin, the drug absorption rateinto blood was similar with that into skin tissue, but drug concentration of skintissue higher than that in blood, which would have profit to produce localanti-infection and analgesic effect. Chlorhexidine absorbed little whether inplasma or skin.
Skin irritation test showed that the studied film-forming has nosignificantly irritation with complete and damaged skin of the rabbit.
Acute toxicity test showed that the nervous system toxicity of the studiedfilm-forming solution at high concentration might correlate with the dycloninehydrochloride in formula. However, the toxicity of the compoundfilm-forming solution less than that of dyclonine solution, and this reactionwas temple, could recover within1h.
Long-term toxicity test made with SD rat showed that during the periodof4weeks when it is continuously applying the studied film-forming solutionand2weeks when the drug was withdrawed, no obvious toxicology alterationcorrelated with drug.
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