Periodic Reporting for period 1 - EVPERS (Extracellular vesicles as a tool to predict drug clearance and improve precision pharmacotherapy)
Berichtszeitraum: 2022-01-01 bis 2023-12-31
What is the problem/issue being addressed?
Drug transporters of the ABC (ATP-binding cassette) family are membrane proteins expressed in epithelia of pharmaco-toxicological relevance (intestine, kidney, bile canaliculi, blood-brain-barrier). Due to their critical role in the efflux of a wide range of drugs, they constitute a major factor determining therapeutic efficacy and toxicity. The most studied member of the ABC family is the P-glycoprotein (P-gp). In addition, the relevance of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) is also well-acknowledged.
The expression and activity of ABC transporters are highly variable between individuals. Several factors (e.g. drugs, hormones, diet) lead to major changes in the transporter expression and, therefore, in drug pharmacokinetics. So far, extraction of biopsies would be the only option to measure ABC transporter expression in relevant tissues. However, due to its invasive character, this is neither performed routinely, nor in those cases where serious side effects could be expected due to inappropriate dosing.
Extracellular vesicles (EVs) are nanoparticles released by all cell types of the organism. They carry proteins, RNA and lipids, the array of these components frequently reflecting the conditions of the cell of origin. The presence of EVs from a wide range of tissues in the blood allows a minimally invasive access to these nanoparticles. However, the limited amount of EV material requires advanced analytical methods (e.g. liquid chromatography coupled to tandem mass spectrometry, UPLC-MS/MS). So far, it is not known whether EVs constitute a good surrogate for changes in the protein levels of ABC transporters.
Why is it important for society?
Drug pharmacokinetics is one of the pillars determining the efficacy and safety of a therapy. An incorrect dosing might have, in some cases, fatal consequences. So far, there is no analytical strategy available to estimate the expression of ABC transporters or their changes before or during a treatment. Analyzing the potential of EVs as dynamic surrogates for ABC transporter expression can deliver useful knowledge towards developing biomarkers of drug clearance.
What are the overall objectives?
The general objective of this project was to investigate the potential of the ABC transporter-cargo in EVs as a biomarker of drug efflux. For this purpose, a UPLC-MS/MS method shall be developed and validated. This method shall be used to quantify the expression of ABC transporters in different types of models (i.e. primary and immortalized cells, cell lines) under treatment with different modulators. This way, the dynamic association between ABC transporter expression and activity in the cells of origin and in the EVs shall be investigated. Finally, the potential of EVs to assess cell-specific ABC transporter expression in a multicellular system (i.e. organoids) shall be investigated.
Drug transporters of the ABC (ATP-binding cassette) family are membrane proteins expressed in epithelia of pharmaco-toxicological relevance (intestine, kidney, bile canaliculi, blood-brain-barrier). Due to their critical role in the efflux of a wide range of drugs, they constitute a major factor determining therapeutic efficacy and toxicity. The most studied member of the ABC family is the P-glycoprotein (P-gp). In addition, the relevance of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) is also well-acknowledged.
The expression and activity of ABC transporters are highly variable between individuals. Several factors (e.g. drugs, hormones, diet) lead to major changes in the transporter expression and, therefore, in drug pharmacokinetics. So far, extraction of biopsies would be the only option to measure ABC transporter expression in relevant tissues. However, due to its invasive character, this is neither performed routinely, nor in those cases where serious side effects could be expected due to inappropriate dosing.
Extracellular vesicles (EVs) are nanoparticles released by all cell types of the organism. They carry proteins, RNA and lipids, the array of these components frequently reflecting the conditions of the cell of origin. The presence of EVs from a wide range of tissues in the blood allows a minimally invasive access to these nanoparticles. However, the limited amount of EV material requires advanced analytical methods (e.g. liquid chromatography coupled to tandem mass spectrometry, UPLC-MS/MS). So far, it is not known whether EVs constitute a good surrogate for changes in the protein levels of ABC transporters.
Why is it important for society?
Drug pharmacokinetics is one of the pillars determining the efficacy and safety of a therapy. An incorrect dosing might have, in some cases, fatal consequences. So far, there is no analytical strategy available to estimate the expression of ABC transporters or their changes before or during a treatment. Analyzing the potential of EVs as dynamic surrogates for ABC transporter expression can deliver useful knowledge towards developing biomarkers of drug clearance.
What are the overall objectives?
The general objective of this project was to investigate the potential of the ABC transporter-cargo in EVs as a biomarker of drug efflux. For this purpose, a UPLC-MS/MS method shall be developed and validated. This method shall be used to quantify the expression of ABC transporters in different types of models (i.e. primary and immortalized cells, cell lines) under treatment with different modulators. This way, the dynamic association between ABC transporter expression and activity in the cells of origin and in the EVs shall be investigated. Finally, the potential of EVs to assess cell-specific ABC transporter expression in a multicellular system (i.e. organoids) shall be investigated.
1. Development and validation of an analytical strategy to quantify ABC transporters in EVs.
We developed a UPLC-MS/MS method aimed at the simultaneous quantification of P-gp, MRP2 and BCRP. The method was validated according to international standards (EMA, FDA). Surrogate peptides for each transporter were identified and the best chromatographic conditions for these analytes and our UPLC-MS/MS hardware were identified. Linearity, specificity and selectivity were confirmed.
2. Determination of ABC transporter expression in MDCK cells and EVs
Our analytical method was tested on MDCK cells (i.e. renal epithelial cells) with and without overexpression of each individual transporter. EVs and fully reproduced the transporter overexpression of the cells of origin. These results, confirm the suitability of our analytical method for the simultaneous detection of ABC transporters in cells and their derived EVs.
3. Determination of ABC transporter expression and activity in cell lines, immortalized cells and their derived-EVs under situations of up- and down-regulation
We applied our UPLC-MS/MS method to determine whether EVs reflected changes in the transporter expression in the cells of origin. First of all, suitable situations of regulation had to be identified. We used rifampicin, as modulator. Eight different cells were screened. For a cell type to be suitable for further analysis, at least one transporter needed to be expressed and regulated in the cells and detected in EVs. LS180 (up-regulation of P-gp), HepG2 (down-regulation of MRP2) and MCF7 cells (up-regulation of BCRP) fulfilled the conditions.
In HepG2 cells, the decrease in MRP2 expression by rifampicin (and hypericin, a second compound used for confirmation purposes), was fully reflected by the EVs. In MCF7 cells, instead, the BCRP up-regulation by rifampicin was not reproduced by EVs. Although, our results support the potential of EVs as surrogate for ABC transporter regulation, important cell- and transporter factors need to be considered.
4. EVs as surrogates for ABC transporter expression in a multicellular system
We used blood-brain-barrier organoids, consisting of endothelial cells, pericytes and astrocytes, as a model to investigate whether cell-specific EVs can be isolated and, analyzed.
First of all, the expression of P-gp, MRP2 and BCRP was determined in lysates from the constituent cells and their EVs. While endothelial cells exhibited detectable levels of all transporters, none of them was detected in pericytes, astrocytes or EVs. The lack of transporter expression in EVs, specially in the case of astrocytes and pericytes, is likely attributed to the intrinsically low expression in the cells of origin. Thus, blood-brain-barrier organoids do not constitute a good model to study ABC transporters in EVs.
We developed a UPLC-MS/MS method aimed at the simultaneous quantification of P-gp, MRP2 and BCRP. The method was validated according to international standards (EMA, FDA). Surrogate peptides for each transporter were identified and the best chromatographic conditions for these analytes and our UPLC-MS/MS hardware were identified. Linearity, specificity and selectivity were confirmed.
2. Determination of ABC transporter expression in MDCK cells and EVs
Our analytical method was tested on MDCK cells (i.e. renal epithelial cells) with and without overexpression of each individual transporter. EVs and fully reproduced the transporter overexpression of the cells of origin. These results, confirm the suitability of our analytical method for the simultaneous detection of ABC transporters in cells and their derived EVs.
3. Determination of ABC transporter expression and activity in cell lines, immortalized cells and their derived-EVs under situations of up- and down-regulation
We applied our UPLC-MS/MS method to determine whether EVs reflected changes in the transporter expression in the cells of origin. First of all, suitable situations of regulation had to be identified. We used rifampicin, as modulator. Eight different cells were screened. For a cell type to be suitable for further analysis, at least one transporter needed to be expressed and regulated in the cells and detected in EVs. LS180 (up-regulation of P-gp), HepG2 (down-regulation of MRP2) and MCF7 cells (up-regulation of BCRP) fulfilled the conditions.
In HepG2 cells, the decrease in MRP2 expression by rifampicin (and hypericin, a second compound used for confirmation purposes), was fully reflected by the EVs. In MCF7 cells, instead, the BCRP up-regulation by rifampicin was not reproduced by EVs. Although, our results support the potential of EVs as surrogate for ABC transporter regulation, important cell- and transporter factors need to be considered.
4. EVs as surrogates for ABC transporter expression in a multicellular system
We used blood-brain-barrier organoids, consisting of endothelial cells, pericytes and astrocytes, as a model to investigate whether cell-specific EVs can be isolated and, analyzed.
First of all, the expression of P-gp, MRP2 and BCRP was determined in lysates from the constituent cells and their EVs. While endothelial cells exhibited detectable levels of all transporters, none of them was detected in pericytes, astrocytes or EVs. The lack of transporter expression in EVs, specially in the case of astrocytes and pericytes, is likely attributed to the intrinsically low expression in the cells of origin. Thus, blood-brain-barrier organoids do not constitute a good model to study ABC transporters in EVs.
In the frame of this work, a method for the simultaneous quantification of low concentrations of ABC transporters at the protein level was validated. This way data for different transporters could be obtained without requiring independent samples for each analyte. Thus, costs and processing time can be saved.
Our study detected for the first-time the expression of two different drug transporters in EVs at the protein level. Protein levels are more closely related to the function of the transporter and, therefore, to their activity. In fact, our data clearly showed an association between transporter levels in EVs, transporter levels in the cells and transporter activity in HepG2 cells
Our observations support, at least for MRP2, the potential of EVs to predict transporter changes in the cells of origin. The next step would be to investigate the expression levels of MRP2 in plasma EVs from individuals treated with rifampicin or other drugs known to regulate transporter levels. The application of our analytical protocol to this type of sample could allow to confirm at clinical level whether EVs represent indeed a good surrogate for the transporter expression. If this is the case, EVs may be used to estimate the drug clearance capacity of an individual before the therapy and adjust the dosing to prevent situations of under or overdosing and guarantee the therapy efficacy and safety.
Our study detected for the first-time the expression of two different drug transporters in EVs at the protein level. Protein levels are more closely related to the function of the transporter and, therefore, to their activity. In fact, our data clearly showed an association between transporter levels in EVs, transporter levels in the cells and transporter activity in HepG2 cells
Our observations support, at least for MRP2, the potential of EVs to predict transporter changes in the cells of origin. The next step would be to investigate the expression levels of MRP2 in plasma EVs from individuals treated with rifampicin or other drugs known to regulate transporter levels. The application of our analytical protocol to this type of sample could allow to confirm at clinical level whether EVs represent indeed a good surrogate for the transporter expression. If this is the case, EVs may be used to estimate the drug clearance capacity of an individual before the therapy and adjust the dosing to prevent situations of under or overdosing and guarantee the therapy efficacy and safety.