| Uricase (URI) is a key enzyme in purine degradation pathways oforganisms. When purine metabolism is disorder or excretion of uric acidprocess is blocked, the concentration of serum uric acid will significantlyincreased and cause hyperuricemia. Then it may degenerate into gout andseries of disease syndrome. URI could decrease the concentration ofserum uric acid and it can be used for the treatment of hyperuricemia.However, URI have some weak points just like poor stability, short activehalf-life, easily degradation by trypsin and lower active in vivo etc.Liposomes（LPs）have lipid bilayer which could encapsulate the drug inbilayer to form ultra micro spherical carrier preparation. LPs show theadvantages of the similarity between biological membranes, improve thedrug stability, reduce drug toxicity and immunogenicity. And it also couldimprove the drug bioavailability, prolonged drug half-life, target effectsand delayed release. We selected four different buffers to prepare LPswhich are containing URI. And then we study on the activity, stability, invivo pharmacodynamic and pharmacokinetic of free URI and ULPs.Method:1. ULPs were prepared by reverse-phase evaporation method. ULP1,ULP2, ULP3and ULP4respectively stand for ULPs prepared by Tris-HCl,Bicine-NaOH, Tricine-NaOH and Boric acid-Borax buffer. 2. Entrapment efficiency of ULPs were determined by gel exclusionchromatography performed with Sephadex column. Particle size and Zetapotential of ULPs were measured by Malvern particle size analyzer.3. Study on the in vitro activity of ULPs. Determining the optimumtemperature and optimum pH of free URI and ULPs and then comparetheir activity.4. Study on the in vitro stability of ULPs. Through analyzed thestorage stability, thermal stability, pH stability and proteolytic stability tocompare the stability of free URI and ULPs.5. Study on the in vivo pharmacodynamics of ULPs. Establishedhyperuricemia mice model, and measuring the serum uric acid level in it.6. Study on the in vivo pharmacokinetic properties of ULP2bymeasuring the activity in serum of URI in SD rats.Result:1. ULP1, ULP2, ULP3and ULP4were successfully prepared in thisstudy, and the products have uniform appearance and good stability.2. The average entrapment efficiency of ULP1, ULP2, ULP3andULP4were51.88%,56.45%,51.82%and54.19%, which ULP2is thehighest. The average particle size of ULP1, ULP2, ULP3and ULP4were330.8nm,280.5nm,263.1nm and265.7nm. The average Zeta potentialwere-18.9mV,-19.0mV,-14.3mV and-29.0mV.3. The optimum temperature of free URI were40℃. Meanwhile,ULP1, ULP2, ULP3and ULP4were40℃,50℃,40℃and40℃. Theoptimum pH of free URI were8.5, which URI2is the highest. Moreover,ULPs were8.0, which ULP2has the highest activity. The results of invitro activity show that ULPs were higher than free URI. 4. The results of in vitro stability show that the stability of ULPs weresignificantly higher than free URI. In the storage stability and proteolyticstability, URI2and ULP2were the most stable in free URI and ULPs.5. Preliminary pharmacodynamic study in vivo show that ULPs havea better effect of lowering the serum uric acid level in mice. URI2andULP2are the most obvious in free URI and ULPs.6. The result of pharmacokinetic shows that the LPs which preparedby Bicine-NaOH buffer can improve the pharmacokinetic properties of thefree URI, and it also can improve the bioavailability of free URI.Conclusion:In this study, we successfully prepared liposomes containing URIwith four different buffers. Experiment results show that the buffer typeshave influence on activity, stability and pharmacodynamic of URI.Bicine-NaOH buffer is more suitable for the study of URI in vitro and invivo than the other three buffers. Pharmacokinetic results showed thatULP2have higher activity than the free URI in vivo. The result ofpharmacokinetic showed that the ULP2can improve the bioavailability offree URI. The results show that ULPs can improve the activity, stabilityand the ability to lower serum uric acid levels of URI. |
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