Studies On Liposomal Formulations For Breviscapine

Breviscapine, a well-known bioactive flavonoid ingredient extracted from Erigeron breviscapus (Vant.) Hand.-Mazz., is mainly composed of scutellarin. Recent studies have shown that scutellarin possesses potent pharmacological effects in reducing blood viscosity, dilating blood vessel, increasing cerebral blood flow, inhibiting platelet aggregation, improving microcirculation, etc. The preparations of breviscapine, such as injections, tablets etc., are extensively used in clinic to treat ischemic cerebrovascular and cardiovascular diseases in China, such as cerebral infarction, apoplexy, coronary heart disease and angina pectoris, etc. Due to the prominent efficacy of breviscapine in the clinical treatment of these diseases, the research of breviscapine has become a hot topic in china in recent years. More than forty Chinese patents on breviscapine have been published since 2001. Over half of the patents focus on the renovation of the dosage form of breviscapine.It has been reported that the oral bioavailability of breviscapine is low and the residence time of breviscapine in the circulation is short. Therefore, the purposes of the present study were to increase its bioavailability after oral administration and to prolong its duration in the circulation after im administration. Several kinds of liposomes, including conventional liposomes, N-trimethyl chitosan chloride (TMC) coated liposomes, multivisicular liposomes, were used as the means of fulfilling the two purposes.Before the design of the liposomal formulations for breviscapine, the ionization constant (pKa), the true n-octanol/water partition coefficient, the solubilities of scutellarin in various organic solvent and at various pH aqueous media were determined. Meanwhile, the stability of scutellarin in aqueous media was investigated. The results indicated those as follows: Scutellarin is a weak acid, its pKa was 3.29, and its solubility was markedly affected by the pH of the environment. Its true n-octanol/water partition coefficient was 0.27. The solubilities of scutellarin in various organic solvents were low. Scutellarin was more stable at pH 2-5 at 25℃and at pH 3-5 at 37℃than at the other pH aqueous media at the same temperature. The mechanism of degradation of scutellarin can be explained by specific acid/base catalysis. The addition of EDTA-2Na or/and NaHSO₃ can improve the stability of scutellarin at aqueous media, especially the addition of both EDTA-2Na and NaHSO₃.The conventional liposomes containing breviscapine (CLB) were prepared by ethanol injection method. Based on this, the liposomes for oral and intramuscular injection administration were prepared by spray-drying-reconstitution, and lyophilization-reconstitution, respectively. Single factor experiments and orthogonal experimental design were used to optimize the formulation and investigate the effects of various factors on the encapsulation efficiency (EE) of CLB. The results indicated that the concentration of phosphatidylcholine (PC), different medium and its ionic strength had larger effect on EE than the other factors. The high EE above 80% could be obtained in the optimized formulation. Then its characteristics were investigated. The results indicated that the drug release rate from the liposomes in vitro was rapidly, and that the store stability of the liposomes at room temperature was not quite good. The effects of lyophilization and spray-drying on the characteristics of liposomes were studied. As for lyophilization-reconstitution liposomes, the higher EE could be obtained by the addition of sugars to the prepared liposomes by ethanol injection method. The particle size after reconstitution became a little larger, and the drug release in vitro from the reconstitution liposomes became faster. As for spray-drying-reconstitution liposomes, the higher EE could be obtained when the sugars were added to the prepared liposomes by ethanol injection method and the ethanol was removed before spray-drying. At appropriate amount of sugar, spray-drying had no significant effect on the particle size. No significant effects on the stability of both PC and drug were found during spray-drying.To study the pharmacokinetics behaviour of liposomal breviseapine, a stable, rapid, and accurate method was developed for the determination of scutellarin in rat plasma. By the addition of both NaHSO₃ and EDTA, the sample stability in both store and analysis process was improved to be suitable for the determination of scutellarin in the pharmacokinetic studies. The analytic time was shortened using scoparone (6, 7-dimethoxyeoumarin) as the internal standard. The results obtained from oral pharmacokinetics study of CLB indicated that the oral absolute bioavailabilities of scutellarin for CLB and breviscapine solution (BS) were 14.96 and 4.77%, respectively. The mean C_max and AUC_(0→t) of CLB were 3.3 and 3.1-fold higher than those of BS, respectively. The two parameters between CLB and BS showed a highly significant difference (P＜0.001). The results obtained from pharmacokinetics study of CLB via im administration indicated that the mean MRT_0→t of CLB had a 3.29-fold increase compared with that of BS, so the MRT_(0→t) of CLB were significantly prolonged (P＜0.001).In the study process, we felt that the molecular interaction between breviseapine and PC might exist. To confirm and then illuminate that, and to give some explains for some results obtained in earlier studies, the molecular interaction between breviscapine and PC was investigated by liposome/water partition coefficient, electron paramagnetic resonance (EPR), DSC, and IR. The results indicated those as follows: The other interaction besides hydrophobic interaction, such as ionic interaction, hydrogen bond etc, play a main role in the molecular interaction between breviscapine and PC membrane. Breviscapine was mainly located at the polar phospho-diester groups, closed to the surface of the lipid bilayers, and did not enter the hydrocarbon chain region, especially the end of the hydrophobic chain. The anion of carboxy group in scutellarin molecule and cation of quaternary amine in PC molecule might exist electrostatic interaction. Hydrogen-bond might be formed between phenolic hydroxyl group in scutellarin molecule and P-O^- group in PC molecule. All of these could be used to explain some of the earlier results partly.The oral bioavailability of breviscapine was improved using conventional liposomal formulation. However, it was still only 14.96%. To increase it further, TMC, a polymeric absorption enhancer with mucoadhesive property, was synthesized and used to prepare TMC-coated liposomes. Then the characteristics of TMC-coated liposomes containing breviscapine (TLB) were evaluated in vitro and in vivo. The results indicated those as follows: The degree of quatemization of TMC was 36.8%. The solubilities of TMC at pH 7.4 aqueous media were significantly increased compared with those of ehitosan. The particle size of TLB was increased with the increasing concentration of TMC solution used in the coating, suggesting the formation of coating layer on the surface of the liposomes. All of TLBs coated with TMCs of different concentration were stable during the six-month storage at 4℃, especially with 0.5% (w/v) TMC. The TMCs coating did not change EE significantly. The liposomes kept sphericai and no significant aggregation was observed by transmission electron microscopy after coated with 0.5% (v/w) TMC. TMCs coating made the drug release from liposome became a little slower, especially 0.5% TMC coating, however, the maximum of accumulative release became larger. TLB showed mucoadhesive property. The absolute oral bioavailability was 21.48%, which was 4.50-fold and 1.44-fold increase as compared with BS and CLB, respectively; so oral absorption of breviscapine was further improved by the TMC-coated liposome formulation.Although the mean MRT_0→t of CLB had a 3.29-fold increase compared with that of BS, it was only 6～7h. The frequency of injection administration needs to be at least once a day if CLB is used to clinic. This motivated us to design a better formulation for breviscapine in order to provide a longer sustained-delivery duration, reduce the frequency of injection administration and therefore afford patient compliance. Multivesicular liposome, a unique lipid-based depot-delivery system, was utilized as drug delivery vehicles for the purpose. Single factor experiments and orthogonal experimental design were used to optimize the formulation based on the selection of feasible preparation technology. Then the characteristics of multivesicular liposome containing breviscapine (MLB) were evaluated in vitro and in vivo. The results indicated those as follows: Within a certain scope, the amounts of PC, cholesterol, and triglycerides used in the experiment have larger effect on EE than the other factors, such as the ratio of drug/PC. The drug durations both in vitro and in vivo were significantly prolonged for MLB, and that the drug release in vitro and the absorption in vivo showed a good linear correlation (R=0.9834), which provided an evidence for the suitability to select human plasma as the medium of drug release from MLB in vitro. Drug release from MLB (triolein/tricaprylin 10/0) in vitro extended a long period of 5～6 days. The MRT_0→t obtained from the pharmacokinetics study of MLB after im administration was about 16.6-fold and 5.04-fold longer than those of BS and CLB, respectively. A duration in vivo for a period of 4～5 day was fulfilled for MLB. The results from light micrographs of pathological section of rat thigh muscle indicated that both MLB and CLB were well biocompatible.