Characterization of plasma hydrolases and their contribution to hydrolase activities in the plasma of different species
Başlık çevirisi mevcut değil.
- Tez No: 400855
- Danışmanlar: DR. TERUKO IMAI
- Tez Türü: Doktora
- Konular: Eczacılık ve Farmakoloji, Pharmacy and Pharmacology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2013
- Dil: İngilizce
- Üniversite: Kumamoto Daigaku
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 76
Özet
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Özet (Çeviri)
Esterified compounds can be found in the human environment as medicines and nutrients. In the drug discovery stages, the esterification is frequently used to develop prodrugs and/or soft drugs. Inside the body, these drugs can be enzymatically converted to their preferred forms by esterases mainly in the liver, intestine and plasma. However, in some cases the hydrolysis process is more rapidly occurs in the plasma rather than liver and intestine. But activity of plasma esterases is markedly different in between humans and other animals resulting in differences in the pharmacokinetics of these prodrugs and creates an added difficulty during development. To understand interspecies variation in response to drugs it is important to know the identities and properties of the plasma esterases responsible for drug hydrolysis. Therefore, in the current study we have focused on plasma esterase activities and aimed to provide useful information regarding the unknown features of species differences and to propose a suitable animal model for predicting the pharmacokinetics of xenobiotics in human. For this purpose, different types of studies were undertaken and the overall findings are summarized below. In Chapter 1, expression patterns of plasma esterases were visualized on native-PAGE gels and species differences were revealed in between human plasma and the plasma of several other preclinical species such as monkey, dog, minipig, rabbit, mouse and rat. Two of the main plasma esterases, PON and BChE together with albumin, most abundant plasma protein, were observed in all tested animals. However, different contents of expressed esterases were indicated the species differences in these tested animals. Especially, these species differences were confirmed in BChE contents and it was shown that plasma of human, monkey, minipig and dog has intense BChE bands whereas rabbit, mouse and rat plasma appeared to show only weak expression. Furthermore, neither human and monkey nor dog and minipig plasma contained the other main plasma esterase, CES, which is the major component of rabbit, mouse and rat plasma. Since CES is abundantly expressed in rabbit, mouse and rat plasma, it was considered to be a possible reason for their lower BChE contents. Additionally, beside these main plasma esterases and albumin, various unknown esterases were observed in minipig and mouse plasmas. In Chapter 2, interspecies variation was evaluated by the plasma hydrolase activities. Five types of compounds (PNPA, irinotecan, oseltamivir, temocapril and propranolol derivatives) were selected for activity analysis based on their structure differences. PNPA used for the estimation of total esterase activity and minipig plasma have shown the highest activity for PNPA in spite of the fact that other substrates cannot be hydrolyzed by minipig plasma. Irinotecan is a substrate with a carbamate group and both CES and BChE showed resistance to carbamate group of this substrate. Oseltamivir, temocapril and the propranolol derivatives, are known as good substrates, especially for CES with different size of acyl moieties. High levels of hydrolase activity for oseltamivir, temocapril and propranolol derivatives were obtained in rat and mouse plasma depending on their highly expressed CES enzyme. However, only propranolol derivatives were rapidly hydrolyzed by rabbit plasma CES. In contrast, the lower hydrolysis activities that observed in human, monkey, dog and minipig plasmas were mainly due to their abundant expression of BChE enzymes. Herein, one exception was observed. It is revealed that only monkey plasma BChE was rapidly hydrolyzed O-valeryl-(R)-propranolol in spite of the fact that human and monkey plasma BChEs shows 96% homology. Sequence analysis were revealed that the acyl-binding pocket only differs in one amino acid, the Pro285 residue in human BChE is replaced by the more flexible Leu in monkey BChE. Since acyl pocket is also affect the substrate specificity of BChE, the increased flexibility of the acyl-binding pocket in monkey BChE is suggested as a possible reason for the different substrate specificities of human and monkey plasma BChEs. In Chapter 3, substrate specificities of plasma esterases were studied in more detail using aspirin as the model compound. All tested species were rapidly hydrolyzed aspirin to salicylic acid. Rabbit plasma showed faster aspirin hydrolysis than human and monkey, while mouse and rat plasma hydrolyzes aspirin slowly enough to be comparable to human. Although CES of rabbit plasma was rapidly hydrolyzed aspirin, CES activity of mouse and rat plasma was found to be low for hydrolyzing this compound. On the other hand, rapid hydrolysis in human plasma was found to be depending on its main hydrolases, PON, BChE and albumin which share the activity by 50%, 30% and 20%, respectively. Aspirin hydrolysis in monkey and dog plasma has found to be similar to that of human plasma and due to their major plasma hydrolases. The studies in order to confirm the responsible esterases in the aspirin hydrolysis activity were revealed the enhancing effect of divalent cations such as calcium and magnesium on BChE activities especially in human plasma. The increasing concentrations of divalent cations resulted in two phase of stimulating effects. It is predicted that calcium and magnesium binds to BChE and causes a change in the conformation of enzyme to more appropriate position for aspirin hydrolysis and therefore makes more active form of BChE. In Chapter 4, detailed studies were performed to understand the divalent cation effect on human plasma BChE. An allosteric effect was observed during aspirin hydrolysis by binding of calcium and the presence of two calcium binding sites with high and low binding affinities were found as dependent to its concentration. We have thought the first binding site with the high binding affinity as the EF-hand binding site which already reported in the literature. It is considered that, in the presence of low calcium concentrations (under 8 mM), calcium binds to EF-hand binding site of BChE and may cause a change in the conformation of BChE to a more appropriate position for aspirin hydrolysis. On the other hand, in the presence of higher calcium concentrations (above 8 mM), the second binding site of BChE for calcium with lower binding affinity may be creates an extra hydrolyzing environment for aspirin and therefore makes more active form of BChE. In contrast to this enhancing effect on aspirin hydrolysis, even addition of low calcium concentrations is inhibited the hydrolase activity of BChE for BTCh, the well-known BChE substrate. This inhibition effect by calcium was larger at low concentration of BTCh than that of high BTCh concentrations due to the fact that BTCh was hydrolyzed by two hydrolyzing patterns with low and high affinities. At low BTCh concentrations hydrolysis is occurred in the active site of BChE. However, if higher concentrations of BTCh are present, this substrate non-specifically bounded to BChE and creates some conformational changes and thus altering the hydrolyzing properties of BChE. In fact, at low concentrations of BTCh calcium causes an allosteric effect and change the conformation of BChE to more inappropriate position but at high BTCh concentrations calcium addition did not cause that much conformational changes in BChE. As a conclusion to overall study; the expression pattern and substrate specificities of plasma esterases of humans and several other animals were confirmed. The esterase activity observed in human plasma could not be dependably represented by rabbit, mouse, rat, monkey, dog or minipig plasma. In rabbit, mouse and rat plasma, substrates are mainly hydrolyzed by their highly active CES enzyme, but CES is not expressed in human plasma. Among the animals tested, monkey plasma appeared to be the closest to human plasma, due to its high sequence homology with the abundantly expressed esterase enzyme. The hydrolyzing pattern of human plasma was also similar to that of dog and minipig plasma. However, if both esterase expression and hydrolyzing pattern are considered, human plasma was found to be closer to dog plasma. Furthermore, if the effect of divalent cations were also considered both monkey and dog plasma was found to be closer. Nevertheless, efficient effect of divalent cations on human plasma BChE could not be dependably represented by monkey and dog plasma. It is revealed that different divalent cations have different effects on human plasma BChE activity in concentration-dependent manner. Since BChE has allosteric properties, it is very flexible to change its conformation. Therefore, divalent cations like calcium cause an allosteric effect on human plasma BChE enzyme by affecting its conformation and change it to more active or passive forms. However more detailed structural analysis will be needed. In general, these findings indicate that differences in substrate-specific hydrolase activities are found in many animals; these differences must be considered when selecting animal models in the discovery stage of the process of generating new drugs, especially ester-type prodrugs intended for human use.
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