| The novel drug delivery system in situ gel system which is free flowing before being used can respond to environmental stimulus and forms gel which can facilitate local drug delivery and prolong drug release compared to solutions.Thermosensitive in situ gel can transform into semi-solid gels only in response to temperature change, from ambient to body temperature. Our group had prepared thermosensitive in situ gel using the selected Thermosensitive materials and model drug.The influence of thermosensitive materials’ rations and concentrations on the properties of the gel were investigated.The erosion behavior of the gel and the drug release behavior from the gel were studied.Gelation temperature, gelation time, stability and the muscle stimulation of the gel were studied in vitro and in vivo respectively. The CPFL in situ gel was prepared for the first time in the domestic veterinary clinic.1. Selects of an optimum thermosensitive material and a suitable model drug for preparation of antiseptics in situ gel An optimum temperature-responsive material was selected based on the characteristics of drugs.With delivery times, short half-life, antimicrobial spectrum and the extent of application as indicators, suitable model drugs with good water-solubility were selected as antiseptics.Then drug loading of the thermosensitive material was investigated. It was found that P407was selected as an optimum thermosensitive material because of its unique thermo-reversible gelation properties, biocompatibility, non-toxic, good rheological characteristics and controlled release.P407at a concentration equal to or greater than17.6%formed non-chemically crosslinked hydrogel upon warming to ambient temperature.The solution-gelation conversion temperature strongly depended on P407concentration.Due to short half-life, a very broad antimicrobial spectrum, high bactericidal activity, wide range of applications and good water-solubility, Ciprofloxacin lactate (CPFL) was selected as the model drug. CPFL with up to20%concentration was dissolved in poloxamer solution,so P407has a good performance of loading drug.2. Selection of menstruum of CPFL thermosensitive in situ gel With various buffer or hydrochloric acid solution as menstruum respectively, the stability of CPFL Poloxamer solutions was investigated to select the optimized menstruum or stabilizer of in situ gel.The factors affecting the stability of CPFL poloxamer solutions were studied at the same time.Results showed that poloxamer solutions of various CPFL precipitated precipitation within1week in the experiment,when the menstruum was lactic acid buffer, lacid acid-sodium lactate buffer or Hydrochloric acid solution. Poloxamer solutions of CPFL(10%)were also unstable with various Acetate buffer as menstruum, poloxamer solution of8%CPFL kept clatify when its media was Acetate buffer(pH3.6),while5%CPFL solution of poloxamer can keep stable with Acetate buffer(pH3.6and3.8) as menstruum in the observation period. The factors affecting the quality of CPFL poloxamer solution are mainly pH, acidity regulator, temperature and concentration of CPFL.The acetate buffer (0.2mol/L)whose pH is3.6was seleted as a solvent of CPFL poloxamer in situ gel and plays an important part in keeping CPFL poloxamer solutions stable.3. Preparation and characteristics of CPFL thermosensitive in situ gel The in situ thermosensitive gel was prepared by using cold method. Temperature sensitivity of poloxamer solutions and Influence of pharmaceutical necessities or main component in the prescription on the gellation temperature of various concentration poloxamer solutions were all investigated with the method of inversion of test tube. Gelation temperature as the standards, the formulation of CPFL in situ gel was selected. Results were follows:①P407at a concentration equal to or greater than18%formed non-chemically crosslinked hydrogel upon warming to ambient temperature. The solution-gelation conversion temperature strongly depended on P407concentr-ation.②0.03%EDTA-2Na,0.15%NaHSO4and0.01%(g/mL)benzalkonium bromide were seleced as metal ion chelators, antioxidants and antiseptics of CPFL thermosensitive in situ gel, respectively.③There was no significant influence to the bases when the concentration of pharmaceutical necessities such as EDTA-2Na and NaHSO4in the prescription were added, while gelataion temperature rose a little as result of benzalkonium bromide and increased markedly as8%CPFL (g/mL)was added.④Three suitable formulations selected all included0.03%EDTA-2Na,0.15%NaHSO4,0.01%benzalkonium bromide and8%CPFL(g/mL). Furthermore,there were24%P407and2%P188in the first formulation,24%P407and3%P188in the second formulation, and23%P407and1%P188in the third formulation. Three suitable formulations selected all have suitable gelation temperature, which was32.5℃,33.5℃and31.4℃respectively.So an in situ gel with suitable gelation temperature could be achieved by adjusting the ratio of P407to PI88.4. Erosion and drug released behavior of CPFL poloxamer thermosensitive in situ gel①The method for determining CPFL from the thermosensitive in situ gel was established with UV. Recovery, precision, stability all met the requirement of technology, and determination of CPFL was not interfered by pharmaceutical necessities, such as polxamerN EDTA-2Na and so on.CPFL standard curve is linear at the range of1.0～12.0μg/mL.②Gel erosion of the in situ gels and release of CPFL from the in situ gels were investigated by membrane free method and UV method, and the following factors were included for comparison including the release area and shaking frequency.It was found that gel erosion and CPFL release all followed the Higuchi kinetic.The best prescription was selected with release time as the standard and was composed of by24%P407,1%P188,8%CPFL,0.03%EDTA-2Na,0.15%NaHSO4, and0.01%(g/mL)benzalkonium bromide. The erosion and the drug release rate were increased with the increase of release area. However, the rise of shaking frequency did not alter both the erosion and the drug release rate, and the erosion and drug released behavior of CPFL in situ gel were very similar (f2were all close to100)between the three shaking frequency.③Gel erosion and CPFL release were linear correlative(R2between0.9975and0.9998)and the slope of the linear equations between cumulative percent gel dissolved and cumulative percent drug released were close to1under various shaking frequency or release surfaces. The drug was released from the gel by a combination of diffusion and erosion.Drug release was mainly controlled by gel erosion.5. Determination of main component in thermosensitive CPFL in situ gel by RP-HPLC RP-HPLC method was established to determinate CPFL in Poloxamer in situ gel.According to scan results of CPFL solution between200and400nm with ultraviolet spectropHotometer,the detection wavelength was set at279nm.The separation was performed on Extreme C18HPLC column with mobile phase consisted of0.025mol·L-1orthophosphoric acid(adjusted to pH3.0with triethylamine)-acetonitrile(87113)at flow rate of1.0mL·min-1.The linear range of ciprofloxacin lactate were1.25～40μg·mL-1(r=1)with an average recovery of99.55%(RSD=1.68%, n=9).The method is simple, rapid, accurate, reliable and reproducible for the content determination of CPFL in poloxamer in situ gel.6. The investigation of gelation time in vitro and muscle stimulation of CPFL thermosensitive in situ gel Gelation time of CPFL in situ gels were measured with the method of inversion of test tube.Gelation in vivo was studied after administration by dorsal subcutaneous injections in a adult New Zealand rabbit.With physiological saline for injection as a control, the muscle stimulation of the CPFL in situ gel was studied with self-control method using the same body. The gelation time of CPFL poloxamer in situ gel was93-114s.Gel was observed within3-5min at application sites in the rabbit after injection.The total score of four quadriceps femoris muscles treated with0.9%Nacl was zero.The total score of four quadriceps femoris muscles treated with CPFL in situ gel was4(<10)and the difference between the highest score and the lowest was not more than2.It was found that gel phase transition of CPFL in situ gel quickly occurred at body temperature and the CPFL in situ gel can be used for intramuscular injection and should be injected deeply.7. Study on the stability of CPFL thermosensitive in situ gel The stablitity of CPFL in situ gel was studied based on the physical and chemical properties of CPFL.Results showed that the characters of this preparation including quality characters, pH, gelation temperature and contet(RSD<2%) were all stable in a dark and low or room temperature environment.The same results occured in the centrifuge test, autoclave test, preliminary accelerated stability test and preliminary long-term stability.However, some characters of its concentration (RSD=2.32%), appearance and pH(changes from0.03to0.06)has been changed in a dark and high temperature environment(40℃).It was found that this preparation can be stored in a dark and room temperature environment. |
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