.1. Bicyclol Protective Effect And Mechanism Of Immune Hepatic Fibrosis In Rats 2.beagle Dog Oral Fumarate Tenofovir Topiramate Furosemide Esters And Of Bp0018 After The Plasma Pharmacokinetics And Biological Utilization Of

Liver fibrosis is the pathological process of fibrous connective tissue development secondary to inflammatory necrosis of liver cells. It is not only the pathological characteristic of chronic liver disease, but also the reason which leads to the development of chronic hepatitis and cirrhosis.Studies on the mechanism of liver fibrosis and prevention against its formation have become the hot spot on all of world. CCl4, dimethyl nitrosamine, bile duct ligation and immunological liver fibrosis are the common models to study the mechanism of liver fibrosis.Since immunological liver fibrosis is more closed to the pathogenesy of liver fibrosis which is caused by chronic hepatitis in clinic, so it's used to study the immunological mechanism of action of chronic hepatitis.Liver fibrosis is a scarring process associated with an increased and altered deposition of liver extracellular matrix. It is mainly characterized by the cellular activation of HSC, aberrant activity of TGF-β1 and its downstream cellular mediators. TGF-P was known to regulate the expression of both components of ECM network, such as fibrillar collagens, fibronectin and protease inhibitors including TIMPs. Based on the previous studies, TGF-β1 signal transduction pathway has become an effective target for the prevention or treatment of liver fibrosis. As shown in the present study, bicyclol was found to reduce the expression of liver TGF-β1 with significant difference both on mRNA and protein levels. It suggested that bicyclol may protect liver from fibrogenesis through the down regulation of liver TGF-β1.Previous reports showed that TGF-P can activate and promote the transdifferentiation of HSCs to myofibroblasts via intracellular TGF-β/Smad signaling pathway during the process of liver injury. It is admitted that TGF-β/Smad pathway interacts with other signaling pathways to increase or inhibit Smad phosphorylation. Of most important is the mitogen activated protein kinase (MAPK) pathways, such as p38 MAPK. It has been proved that the phosphrylation of Smad3 is p38 MAPK-dependent. p38 MAPK is activated by various MAPK kinase kinases (MAPKKKs) in response to many stimuli. Then it promotes the progression of myoblasts differentiation at multiple levels. Our results showed that bicyclol inhibited the phosphorylation of Smad2/3, correspondently, the phosphorylation of p38 was also reduced as well. Thus, it suggested that the inhibitory effect of bicyclol on TGF-β1 expression was at least in part via the Smad2/3 and p38 signaling pathways.IL-1, as an inflammatory cytokine, was involved in the liver fibrosis mainly through the activation of p38 and up-regulation of TIMP-1 mRNA expression in HSCs. In the present study, we found that the mRNA level of IL-1βwas enhanced and bicyclol can inhibit the elevation of IL-1βin BSA-induced rats. The above results suggested that the effect of bicyclol on preventing the inflammation in liver via inhibiting p38.Interleukin-10 (IL-10) is considered as an anti-inflammatory cytokine which inhibit the production of several inflammatory cytokines, including IL-1β. Several reports demonstrated that the exogenous IL-10 can inhibited the liver fibrosis in CCl4 induced rats, the level of TGF-β1 expressed in activated HSCs cultured in vitro and mRNA level of basic fibroblast growth factor, enhanced the apoptosis of HSCs. The high mRNA expression of IL-10 in Kupffer cell, HSCs and liver-infiltrating T cells was found in the course of liver inflammation previously. It was found that the hepatic IL-10 was increased in BSA treated rats, and the overexpression of IL-10 can be reduced by bicyclol treatment. It was presumed that bicyclol significantly down regulated the gene expression of liver pro-inflammatory cytokines, and thereby attenuated feedback increase of IL-10.During the process of liver fibrosis, TNF-a may active HSCs, and play an important role in the synthesis of ECM, the release of MMPs and TIMPs. The mRNA level of TNF-a is induced in the BSA-induced liver fibrosis rats, and bicyclol can inhibite the expression of it.Previous studies indicated that the deposition and degradation of collagen in liver fibrosis were related to MMPs and TIMPs closely. The homeostasis of ECM in healthy liver is sustained by a precisely regulated permanent turn-over directed by MMPs. Upon chronic liver damage, HSCs was activated and differentiate into fibroblast-like phenotype. At the same time, the expression of TIMP-1 especially in activated HSCs is upregulated leading to the inhibition of MMP activity and subsequent accumulation of matrix proteins in extracellular space. In the present study, the treatment of bicyclol resulted in a significant reduction of MMP-2 and TIMP-1 mRNA expression. Although it is still uncertain whether the increase of collagenase activity was a result of bicyclol's inhibitory effect on TIMP-1 expression, the decrease of TIMP-1 and the increase of collagenase activity often coincide when liver fibrosis begin to reverse. In addition, it has been reported that IL-1 activated P38 MAPK and resulted in the up regulation of TIMP-1. Considered the results above all, bicyclol may protect liver from fibrogenesis through regulating TGF-β1, preventing the inflammation (as evidenced by the inhibition of IL-1βand IL-10) by suppressing the phosphorylation of p38 MAPK, thus interfering with TIMP-1 and inhibiting the collagen expression.In conclusion, bicyclol showed significant protective effect on BSA-induced liver fibrosis. The hepatoprotection of bicyclol is mostly due to the following aspects: bicyclol can modulate TGF-β1 through Smad2/3 and p38 MAPK signaling pathways; it inhibit IL-1βvia regulating TGF-β1 to protect liver from fibrogenesis, then attenuated feedback increase of IL-10; at the same time, the treatment of bicyclol lower the levels of MMP-2 and TIMP-1, it's hypothesized that bicyclol can regulate TIMP-1 and inhibit the expression of collagen by reducing the phosphrylation of p38 MAPK, the balance of fibrogenesis and fibrolysis was maintained. BP0018 is a novel anti-hepatitis drug which is developed by Jiang Su Chia-Tai Tianqing Pharmacy Co. Ltd. As a new type of nucleosidereverse transcriptase inhibitor (NRTI), BP0018 and Viread both are the prodrugs of tenofovir (PMPA) Viread was known to be efficient in inhibition of HBV and HIV, and was approved by FDA in America and Europe to cure HIV. At the same time, viread is an efficient inhibitior of anti-HBV, it can interfere with the HBV DNA polymerase to stop the virus from reproducing. As a result, the copies of hepatitis virus and serologic markers are reduced.However, for treatment of HIV or HBV, the paients need take medicine for long term, so the economic of the treatment is one of the most important elements. Viread as an import durg increases the cost of therapy and the economic burden of the patients. In China, most anti-hepatitis drugs and anti-HIV durgs are imported drugs or Generic drugs, theres no efficient new drugs with independent intellectual property in clinic. BP0018 as a new drug researched by Jiang Su Chia-Tai Tianqing Pharmacy Co. Ltd, has clinical significance. The aim of this paper is to study the plasma pharmacokinetics of viread and BP0018 after being given orally in beagle dogs. The bioavailabilities of this two drugs were analyzed to provide evidence for safety and effectivity of BP0018 in clinic.According to the instruction of chemical pharmaceutical preparations bioavailability and bioequiavailability, the plasma pharmacokinetic and bioavailabilities of BP0018 and its reference preparation viread were studied after single dose in beagle dogs.The results showed:(1) The HPLC/MS/MS analysis method for the simultaneous quantification of tenofovir, viread and BP0018 in biological samples showed good sensitivity, linearity of response, high precision and appropriate for pharmacokinetic study.(2) After single dose of BP0018 and reference preparation in beagle dogs, the concentration of drug prototype at every time point is lower than the detectable limit, but PMPA can be detected in plasma, the concentrations of PMPA vary with time.(3) Tmax of BP0018 and reference preparation PMPA are 0.25-1h and 0.25-2h, Cmax are 328.06±65.74ng/mL and 331.21±22.74ng/mL, and mean residence time (MRT) are 6.87±1.52h and 4.35±1.61h, respectively.(4) Compared with the reference preparation, the MRT and elimination half life (tl/2z) of are extended, but there's no siginificant difference of AUC(0-t),AUC(0-∞),Cmax and Tmax.(5) After single intravenous injection of PMPA, the concentration of it reached to the peak value immediately, PMPA can still be detected at 24h, the concentration of PMPA is lower the detectable limit at 36h.(6) After single dose of BP0018, the availability of it is 20.7±4.3%, and the availability of viread is 16.8±6.2%.In conclusion, study of relative system pharmacokinetic research was performed on viread and BP0018, bioavailabilities of this two drugs were analyzed to provide basis for safety and effectivity of BP0018 in clinic.