UGTs and glucuronidation analyses in Caco-2 cells, human microsomes and recombinant enzymes

Forskningsoutput: AvhandlingDoktorsavhandlingSamling av artiklar

Sammanfattning

Glucuronidation of drugs and several endogenous compounds is catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, membrane proteins of the endoplasmic reticulum that are variably expressed in the liver, intestine and few other tissues.

In this PhD research it was first studied UGTs expression and glucuronidation activity in Caco-2 cells, a common model system for drug absorption in the intestine. Caco-2 cells differentiation significantly stimulated UGTs expression and glucuronidation activity. However, the activity of most UGTs, with the exception of UGT1A6, was very low in comparison to the human intestinal or liver microsomes (HIM and HLM, respectively), indicating that these cells do not provide a good model system for drug glucuronidation in the intestine.

The second part of this thesis studied the different aspects of improving the use of recombinant UGTs and the in vitro glucuronidation assays. It included the possible use of DMSO, up to 10% (volume per volume), in studying the glucuronidation of highly lipophilic substrates, and the effect of alamethicin addition that is now recommended mainly for the assays using HLM or HIM and in combination of only low DMSO concentrations. In addition, it was found out that glucuronidation experiments with compounds containing free carboxylic acid groups should be done at both pH 6.0 and pH 7.4. The lower pH value is recommended for reducing acyl migration and stimulating the activity of many UGTs, whereas the higher pH would be needed to test whether or not UGT2B15, the only UGT that is inactive at pH 6.0, catalyzes the test compound.

A method to quantitatively evaluate the contributions of individual UGTs to the combined activity of a tissue sample, the relative activity factor (RAF), approach was also examined. This study, done in collaboration with a Chinese laboratory from the Dalian Institute of Chemical Physics, turned out to be very good, if selective substrates or inhibitors for the different UGTs that catalyze the test reaction are available.

Finally, I also tried to improve the quality of recombinant UGTs that were produced in baculovirus-infected Spodoptera fragiperda 9 (Sf9) insect cells, mainly by further improving the infection optimization tests that were developed in order to reduce the fraction of inactive recombinant UGT in the produced samples. Another part of that study was to examine the possible effects of the C-terminal fusion peptide that ends with 6 His residues (His-tag) on the enzyme kinetics of the recombinant UGTs. The detailed studies revealed that there were no significant adverse effects of this fusion peptide, therefore prompting us to continue using it due to the possibilities it opens for relatively easy testing of the expression level of the different recombinant UGTs.

Taken together, this thesis contains several topics, all of which are related to the use of UGT enzymes in glucuronidation studies, and largely productive efforts to improve them.

In addition to the specific findings with several different UGTs, the results are expected to be useful for future in vitro studies in the field.
Originalspråkengelska
UtgivningsortHelsinki
Förlag
Tryckta ISBN978-952-10-8265-8
StatusPublicerad - 2012
MoE-publikationstypG5 Doktorsavhandling (artikel)

Vetenskapsgrenar

  • 317 Farmaci

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title = "UGTs and glucuronidation analyses in Caco-2 cells, human microsomes and recombinant enzymes",
abstract = "Glucuronidation of drugs and several endogenous compounds is catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, membrane proteins of the endoplasmic reticulum that are variably expressed in the liver, intestine and few other tissues. In this PhD research it was first studied UGTs expression and glucuronidation activity in Caco-2 cells, a common model system for drug absorption in the intestine. Caco-2 cells differentiation significantly stimulated UGTs expression and glucuronidation activity. However, the activity of most UGTs, with the exception of UGT1A6, was very low in comparison to the human intestinal or liver microsomes (HIM and HLM, respectively), indicating that these cells do not provide a good model system for drug glucuronidation in the intestine. The second part of this thesis studied the different aspects of improving the use of recombinant UGTs and the in vitro glucuronidation assays. It included the possible use of DMSO, up to 10{\%} (volume per volume), in studying the glucuronidation of highly lipophilic substrates, and the effect of alamethicin addition that is now recommended mainly for the assays using HLM or HIM and in combination of only low DMSO concentrations. In addition, it was found out that glucuronidation experiments with compounds containing free carboxylic acid groups should be done at both pH 6.0 and pH 7.4. The lower pH value is recommended for reducing acyl migration and stimulating the activity of many UGTs, whereas the higher pH would be needed to test whether or not UGT2B15, the only UGT that is inactive at pH 6.0, catalyzes the test compound. A method to quantitatively evaluate the contributions of individual UGTs to the combined activity of a tissue sample, the relative activity factor (RAF), approach was also examined. This study, done in collaboration with a Chinese laboratory from the Dalian Institute of Chemical Physics, turned out to be very good, if selective substrates or inhibitors for the different UGTs that catalyze the test reaction are available. Finally, I also tried to improve the quality of recombinant UGTs that were produced in baculovirus-infected Spodoptera fragiperda 9 (Sf9) insect cells, mainly by further improving the infection optimization tests that were developed in order to reduce the fraction of inactive recombinant UGT in the produced samples. Another part of that study was to examine the possible effects of the C-terminal fusion peptide that ends with 6 His residues (His-tag) on the enzyme kinetics of the recombinant UGTs. The detailed studies revealed that there were no significant adverse effects of this fusion peptide, therefore prompting us to continue using it due to the possibilities it opens for relatively easy testing of the expression level of the different recombinant UGTs. Taken together, this thesis contains several topics, all of which are related to the use of UGT enzymes in glucuronidation studies, and largely productive efforts to improve them. In addition to the specific findings with several different UGTs, the results are expected to be useful for future in vitro studies in the field.",
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author = "Hongbo Zhang",
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publisher = "University of Helsinki",
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UGTs and glucuronidation analyses in Caco-2 cells, human microsomes and recombinant enzymes. / Zhang, Hongbo.

Helsinki : University of Helsinki, 2012. 74 s.

Forskningsoutput: AvhandlingDoktorsavhandlingSamling av artiklar

TY - THES

T1 - UGTs and glucuronidation analyses in Caco-2 cells, human microsomes and recombinant enzymes

AU - Zhang, Hongbo

PY - 2012

Y1 - 2012

N2 - Glucuronidation of drugs and several endogenous compounds is catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, membrane proteins of the endoplasmic reticulum that are variably expressed in the liver, intestine and few other tissues. In this PhD research it was first studied UGTs expression and glucuronidation activity in Caco-2 cells, a common model system for drug absorption in the intestine. Caco-2 cells differentiation significantly stimulated UGTs expression and glucuronidation activity. However, the activity of most UGTs, with the exception of UGT1A6, was very low in comparison to the human intestinal or liver microsomes (HIM and HLM, respectively), indicating that these cells do not provide a good model system for drug glucuronidation in the intestine. The second part of this thesis studied the different aspects of improving the use of recombinant UGTs and the in vitro glucuronidation assays. It included the possible use of DMSO, up to 10% (volume per volume), in studying the glucuronidation of highly lipophilic substrates, and the effect of alamethicin addition that is now recommended mainly for the assays using HLM or HIM and in combination of only low DMSO concentrations. In addition, it was found out that glucuronidation experiments with compounds containing free carboxylic acid groups should be done at both pH 6.0 and pH 7.4. The lower pH value is recommended for reducing acyl migration and stimulating the activity of many UGTs, whereas the higher pH would be needed to test whether or not UGT2B15, the only UGT that is inactive at pH 6.0, catalyzes the test compound. A method to quantitatively evaluate the contributions of individual UGTs to the combined activity of a tissue sample, the relative activity factor (RAF), approach was also examined. This study, done in collaboration with a Chinese laboratory from the Dalian Institute of Chemical Physics, turned out to be very good, if selective substrates or inhibitors for the different UGTs that catalyze the test reaction are available. Finally, I also tried to improve the quality of recombinant UGTs that were produced in baculovirus-infected Spodoptera fragiperda 9 (Sf9) insect cells, mainly by further improving the infection optimization tests that were developed in order to reduce the fraction of inactive recombinant UGT in the produced samples. Another part of that study was to examine the possible effects of the C-terminal fusion peptide that ends with 6 His residues (His-tag) on the enzyme kinetics of the recombinant UGTs. The detailed studies revealed that there were no significant adverse effects of this fusion peptide, therefore prompting us to continue using it due to the possibilities it opens for relatively easy testing of the expression level of the different recombinant UGTs. Taken together, this thesis contains several topics, all of which are related to the use of UGT enzymes in glucuronidation studies, and largely productive efforts to improve them. In addition to the specific findings with several different UGTs, the results are expected to be useful for future in vitro studies in the field.

AB - Glucuronidation of drugs and several endogenous compounds is catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, membrane proteins of the endoplasmic reticulum that are variably expressed in the liver, intestine and few other tissues. In this PhD research it was first studied UGTs expression and glucuronidation activity in Caco-2 cells, a common model system for drug absorption in the intestine. Caco-2 cells differentiation significantly stimulated UGTs expression and glucuronidation activity. However, the activity of most UGTs, with the exception of UGT1A6, was very low in comparison to the human intestinal or liver microsomes (HIM and HLM, respectively), indicating that these cells do not provide a good model system for drug glucuronidation in the intestine. The second part of this thesis studied the different aspects of improving the use of recombinant UGTs and the in vitro glucuronidation assays. It included the possible use of DMSO, up to 10% (volume per volume), in studying the glucuronidation of highly lipophilic substrates, and the effect of alamethicin addition that is now recommended mainly for the assays using HLM or HIM and in combination of only low DMSO concentrations. In addition, it was found out that glucuronidation experiments with compounds containing free carboxylic acid groups should be done at both pH 6.0 and pH 7.4. The lower pH value is recommended for reducing acyl migration and stimulating the activity of many UGTs, whereas the higher pH would be needed to test whether or not UGT2B15, the only UGT that is inactive at pH 6.0, catalyzes the test compound. A method to quantitatively evaluate the contributions of individual UGTs to the combined activity of a tissue sample, the relative activity factor (RAF), approach was also examined. This study, done in collaboration with a Chinese laboratory from the Dalian Institute of Chemical Physics, turned out to be very good, if selective substrates or inhibitors for the different UGTs that catalyze the test reaction are available. Finally, I also tried to improve the quality of recombinant UGTs that were produced in baculovirus-infected Spodoptera fragiperda 9 (Sf9) insect cells, mainly by further improving the infection optimization tests that were developed in order to reduce the fraction of inactive recombinant UGT in the produced samples. Another part of that study was to examine the possible effects of the C-terminal fusion peptide that ends with 6 His residues (His-tag) on the enzyme kinetics of the recombinant UGTs. The detailed studies revealed that there were no significant adverse effects of this fusion peptide, therefore prompting us to continue using it due to the possibilities it opens for relatively easy testing of the expression level of the different recombinant UGTs. Taken together, this thesis contains several topics, all of which are related to the use of UGT enzymes in glucuronidation studies, and largely productive efforts to improve them. In addition to the specific findings with several different UGTs, the results are expected to be useful for future in vitro studies in the field.

KW - 317 Pharmacy

M3 - Doctoral Thesis

SN - 978-952-10-8265-8

T3 - Dissertationes Biocentri Viikki Universitatis Helsingiensis

PB - University of Helsinki

CY - Helsinki

ER -

Zhang H. UGTs and glucuronidation analyses in Caco-2 cells, human microsomes and recombinant enzymes. Helsinki: University of Helsinki, 2012. 74 s. (Dissertationes Biocentri Viikki Universitatis Helsingiensis; 36/2012).