Bmp-2 As A Target For Anti-osteoporosis Drug Screening And Transfers 04-5195a, No.39. No.3 Vitro And In Vivo Research

It is estimated that about one hundred million people are currently suffering fromosteoporosis in the world, so there is an increasingly need to perform reseach to findnew drugs to treat this disease.Up to the present, many studies performed in this area have revealed that BoneMorphogenetic Protein-Ⅱ(BMP-2), a member of the BMP family, can increase theexpression of asteocalcin and alkali phosphatase, a marker enzyme for osteoblastdifferentiation, subsequently improves the formation of bone nodule and bone layer,therefore it plays an important role in the osteoblast differentiation and the boneformation. But some recent studies also reveal that the bone morphogenetic protein(BMP-2) can inhibit activity of osteoclast. Lorenz C et al found BMP-2 couldincrease OPG mRNA levels in a fetal osteoblastic cell line (hFOB). In addition, thefact that BMP-2 bind to BMP1A receptor by Smadl could stimulate OPG promoteractivity had been proved. OPG has recently been identified as a novel member ofthe TNF-receptor (TNF-R) superfamily, and it is a soluble decoy receptor for RANKLand could inhibit osteoclast by blocking RANKL binding to RANK.In order to search new agents that enhance the expression of the BMP-2, ascreening model targeted on bmp-2 promoter has been established in our laboratory.In this model, the effects on the promoter are assessed by the luciferase activity whichis driven by the bmp-2 promoter of the mouse. With the model, thousands samples ofnatrural products library were screened and some positive results came out. On thebasis of previous research works, a active strain having the potency of increasingexpression of the BMP-2 was further investigated in this paper.In the first part of this thesis, the fermentation broth of the strain 04-5195 ofNocardia.sp.nov (other publication) was isolated and purified. The structure of mainactive component 04-5195A was identified. 04-5195A was proved as same asAmicoumacin B which was one of oligopeptide antibiotics and producted by Bacillus brevis. Amicoumacin B was reported to exhibit protection to ulcer caused bynervous and to have action of anti-inflammatory. However, It is the first time tofind that Amicoumacin B could improve expression of BMP-2 and can be productedby Actinomycetes. Then, the activity of 04-5195A in vitro to increasingconcentrations of BMP-2 mRNA in MG63 cells was confirmed by means ofSemiquantitative RT-PCR analysis, its action on increasing concentrations of BMP-2concentrations in MG63 cells was proved using flowcytometry.The second part of the thesis deals with the two compounds NO.39 and NO.3highly up-regulating BMP-2, which were screened out from the chemical compoundslibrary with the model targeting bmp-2 promoter. The effect of NO.39 in vitro wasconfirmed by increasing concentrations of BMP-2 mRNA and expression of BMP-2.The activity in vivo of NO.39 and NO.3 were investigated with an osteoporosis modelof SD Rats variectomized (OVX). The static parameter and kinetic parameter ofcancellous bone of tibial metaphyseal sections were determined after oraladministration of the active compounds at the dose of 10mg.d.^-1kg^-1 for 90 days. Theresults showed that both NO.39 and NO.3 could prevent bone loss, inhibit osteoclastactivity and decrease bone turnover rate.In the third part, a HPLC method was developed and validated for determination ofthe concentration of NO.39 in rat plasma and tissues. As to determining concentrationof NO.39 in plasma, the HPLC method exhibits good linearity in the range of0.1-75.0μg.mL^-1(r=0.9968) with the average extraction recoveries 99.8-103.7%.Furthermore, this method was used to measure the main pharmacokinetics parameterssuch as CL, t_1/2, T_max and Vd_ss of NO.39 in rat plasma. The result showed that NO.39could be absorbed and cleaned quickly after oral dose of 4.0-30.0mg.kg^-1. And theconcentration of NO.39 in plasma increased with dose levels after different oral dose,exhibiting a linear pharmacokinetic profile. As to determining concentration of NO.39in rat tissues, the HPLC method also showed good linearity in the range of0.05-25.0μg.mL^-1 (r＞0.992) with the average extraction recoveries 99.8-108.5%for NO.39. Then, this method was used to measure the concentration of NO.39 in rattissue after a simple oral dose rang of 10.0 mg.kg^-1. The highest level was observed in liver, then droping down successively in lung, in kidney, in spleen and in heart, thelowest level was found in brain. It was not found that the NO.39 accumulated in rattissues.The results we got here indicated the model established for screeninganti-osteoporosis drug is feasible and efficient. NO.39 exhibits higher concentrantionin rat tissue, therefore it is worthy to be further studied to develop a new anti-osteoporosis drug.