| 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/cm2of 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/cm2. 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/cm2every 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-1for 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 with103CFU·mL-1compared to105CFU·mL-1and107CFU·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/cm2, 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; tmax(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; tmax(1.17±0.55),(1.28±0.39), (1.28±0.39) h；AUC(0-12h)(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/cm2, and0.2mg/cm2for the damaged skin group. The LOAEL was among the range of0.2to0.4mg/cm2. 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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