Stereoselective Release From A Novel Biodegradable Injectable Implants Loaded Ketoprofen In Vitro And In Vivo

In connection with aim of prolonging the duration of action of conventional drugs in vivo,new drug delivery systems(DDS)continue to attract much attention.The controlled or sustained release of drugs represents one such approach.And in this regard report,DDS using biodegradable polymers which includes copolymer of lactic and glycolic acid(DL-PLGA)was studied.The biodegradable injectable implant using PLGA as matrix is one kind of DDS.The injectable implants is a solution with low viscosity and has a liquid consistency which facilitates injection through a needle.It can turn to a gel matrix immediately once contact with aqueous fluids and the release of the drug takes place slowly through this formed matrix.The matrix will be biodegraded by hydrolyzing into lactic and glycolic acid ultimately.In implant preparation, PLGA is used in diffusion controlled,swelling controlled and chemically controlled delivery system.This kind of DDS is able to provide pre-programmed durations of action and offer several advantages over the conventional dosage forms.A biodegradable injectable implant of Ketoprofen(KET)we developed was one kind of such DDS described above.It is expected to release slowly and maintain drug concentration within the therapeutic range for a long time.For KET is one of the most common pain relievers for Rheumatoid arthritis and t_1/2of KET is very short,to develop controlled and sustained DDS of KET is necessary.Meanwhile,although there are differences between two KET enantiomers' actions in the clinical effect,a limited number of literatures about enantioselective release of KET oral DDS have been reported.Moreover, evaluations of the enantiomer release are mainly limited to studies in vitro and little attention has been paid to the in vivo release difference between the two KET enantiomers.Onishi et al.have prepared biodegradable KET implant tablets which could keep the effective release in vivo for 4 days(administrated by inserting subcutaneously by surgical procedures in rats)using PLGA as matrix,however no attention was paid to the possible enantioselectity of the enantiomers in vivo.In order to investigate the enantioselective release,the biodegradable injectable implants containing racemaic KET(rac-KET)were elaborated with PLGA,and the enantioselective release in vitro and in vivo were observed by the special reversed-phase HPLC method developed1.Incorporation of ketprofen injectalbe implantsIn this study Poly(DL-lactide-co-glycolide)(PLGA)were used in a formulation(injectable implants)which forms a gel matrix immediatedly on contact with aqueous fluids.The biodegradable injectable implants containing a non-sterodial anti-inflammatory durg,racemic ketoprofen(rac-KET)were prepared by dissolving KET in DL-PLGA solution.To evaluate the initial fast release(initial burst)and the release profile of the formulation,the implants were prepared using the PLGA with the different lactide/glycolide ratio,PEG 400 used as additive in implants and the different drug loadings of KET,respectively.The results show that PLGA(7:3) was an ideal excipent for implants to sustain release of KETThe UV spectrophotometer method was applied to determine the drug loading of rac-KET injectable implants at 255nm,drug was extracted with the solvent,alcohol,by refluxing at 70℃. The analytical method afforded recovery ranged from 99.0～100.8%,the assay was linear from 2.0μg/ml～12.0μg/ml.The similar UV spectrophotometer method with the detected wavelength of 260nm was employed to determine the KET released from the injectable implants in vitro release,theassay was also special,accurate and simple.The different drug loadings of implants and the different amount of PEG 400 added to the implants,the formulation could provide sustained release of the drug lasting about 2 months in vitro release.The 10%drug loading of rac-KET implants,it's T80%was longer than 60 days. The initial fast release(burst effect)declined with the addition of 5%PEG400 to 5%KET implants.Compared with other KET preparations,10%rac-KET implants was a promising drug deliver system with weaker initial burst effect and the longer release period.In order to understand the drug release mode from 10%drug loading of rac-KET biodegradable injectable implants,the in vitro release data were fitted to5 power law equations,the cubic mathematical equation appeared to describe rac-KET release from the implants much better than other equations,The cubic equation(Mt/M_∞=18.5259+2.0079t-0.0238t²+0.001t³,R²=0.994; F=473.23,p＜0.O01),can only simulate the release pattern but does not describe the underlying physical drug releasing mechanism.2.The in vitro stereoselective release from rac-KET injectable implantsA stereoselective reversed-phase HPLC assay was developed that could simultaneously quantify S-(+)and R-(-)enantiomers of ketoprofen in release samples.Racemic ketoprofen(rac-KET) and its S-(+)enantiomer(S-(+)-KET)were dissolved in an injectable viscous polymer solution consisting of the biodegradable poly(D,L-lactide-co-glycolide,70:30)(D,L-PLG)and a solvent, N-methyl-2-pyrrolidone(NMP).Once injected into an aqueous environment,the polymeric mixture solidified into a solid implant due to the leaching of NMP.In vitro release studies show that such implants with ketoprofen can provide sustained release of the drug lasting about three months in a pH 7.4 release medium.Moreover,a preferential faster S-(+)-KET release over R-(-)-KET was observed for the implants containing 4%,7%,and 10%of racemic ketoprofen in the neutral pH 7.4 release medium.Stereoselective release was minimal in the first 42 days in vitro but became very pronounced at later time points.When S-(+)-KET was incorporated into the polymeric mixture,its release was also faster than that of the racemic ketoprofen,confirming the stereoselective release of ketoprofen from the D,L-PLG implants.The observed stereoselective release of KET at pH 7.4 was most likely produced by chiral interactions between KET enantiomers and transiently produced D-lactic acid or L-lactic acid rich domains within the implants during D,L-PLG degradation.However,such stereoselective release was not observed in pH 10.0 release medium,probably due to a much faster and homogeneous polymer degradation.The study suggests possible stereoselective release of racemic drugs from D,L-PLG microspheres and implants in vivo.In addition,to understand the enantiomer release mode from 7%rac-KET injectable implants, the in vitro release data were fitted to some power law equations.Higuchi equation appeared to describe S-KET release from the implants(M_t/M_∞=0.134+0.07736t^1/2)much better than other equations;and first order equation could only simulate the release pattern of R-KET(ln(1-Mt/M∞)=-0.0145t-0.23).The difference of enantiomer release between R-KET and S-KET probably causes the different release pattern. 3.The Stereoselective release from KET injectable implants in ratsA stereoselective reversed-phase HPLC method has been developed and validated to separate and quantify the S-(+)and R-(-)enantiomers of ketoprofen(KET)as their diastereoisomeric amides with(S)-(-)-alpha-(1-Naphthyl)ethylamine(S-NEA)from a novel biodegradable injectable implant for the in vivo release in rats.The method involved liquid-liquid extraction of S-(+)and R-(-)enantiomers from rat plasma,using R-(-)-flurbiprofen(FBF)as the internal standard,and employed S-NEA as a pre-column chiral derivatization reagent.The derivatized products were separated on a 5μm reversed-phase C₁₈column with a mixture of methanol and 0.01 mol·L^-1KH₂PO₄(pH4.5)with a volume ratio of 71:29 as mobile phase.The detection of ketoprofen derivatives was made atλ=244 nm with UV detector.The assay was linear from 0.03 to 10.0μg/ml for each enantiomer.The absolute recoveries for each enantiomer were greater than 77%.The intra-day and inter-day variations' were less than 13%.For each enantiomer,the limit of quantification(LOQ)was 0.03μg/ml with RSD of 15.0%(n=5)for R-(-)-KET and 13.5%(n=5)for S-(+)-KET.The reproducibility of the assay was satisfactory.The enantioselective release of the biodegradable injectable implant containing racemic KET(rac-KET)elaborated with PLGA in SD rats was investigated using pre-column chiral derivatization RP-HPLC.The rac-KET injectable implant,once injected subcutaneously in rats, produced long lasting the plasma S-enantiomer level and the plasma S-(+)-KET level was always higher than that of R-(-)-KET in rats.The difference of enantiomer concentration was related with the chiral inversion of R-(-)-KET to S-(+)-KET in rats and the biodegardable properties of the achiral excipient of Poly(D,L-lactide-co-glycolide(DL-PLGA)degraded in biological system.The rac-KET injectable implant also provided the sustained release of S-KET, the duration above the effective plasma level was about 8 weeks after a single injection.