Rapid Method to Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells
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Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells.
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In HEK cells overexpressing OCT1 exposed for 60 minutes to 5 μM 14C-metformin, higher concentrations of metformin were found versus control HEK cells (268+/-11 vs. 26.4+/-11 μM).
“Chien et al. found that higher concentrations of metformin were found in the HEK cells with overexpressed OCT1 versus control HEK cells (268+/-11 vs. 26.4+/-11 μM) (137).”
From A Critical Review of the Evidence That Metformin Is a Putative Anti-Aging Drug That Enhances Healthspan and Extends Lifespan (2021) · cites this paper as [137]
Chien et al. argue that metformin can be trapped in intracellular organelles such as microsomes and mitochondria.
“Chien et al. argue that metformin can be trapped in intracellular organelles such as microsomes and mitochondria (137).”
From A Critical Review of the Evidence That Metformin Is a Putative Anti-Aging Drug That Enhances Healthspan and Extends Lifespan (2021) · cites this paper as [137]
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Cites
How many drug targets are there?
Overington, J. P., Al-Lazikani, B., & Hopkins, A. L. (2006) · Nature Reviews Drug Discovery · doi:10.1038/nrd2199 · Cited by 3,505 · see citing works
Membrane transporters in drug development
The International Transporter Consortium, Giacomini, K. M., Huang, S.-M., Tweedie, D. J., Benet, L. Z., Brouwer, K. L. R., Chu, X., Dahlin, A., Evers, R., Fischer, V., et al. (2010) · Nature Reviews Drug Discovery · doi:10.1038/nrd3028 · Cited by 3,340 · see citing works
Self-citationCellular and molecular mechanisms of metformin: an overview
Viollet, B., Guigas, B., Garcia, N. S., Leclerc, J., Foretz, M., & Andreelli, F. (2012) · Clinical Science · doi:10.1042/cs20110386 · Cited by 1,720 · see citing works
Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action
Shu, Y., Sheardown, S. A., Brown, C., Owen, R., Zhang, S., Castro, R. A., Ianculescu, A. G., Lin, Y., Lo, J. C.-M., Burchard, E. G., et al. (2007) · Journal of Clinical Investigation · doi:10.1172/jci30558 · Cited by 971 · see citing works
Self-citationInvolvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin
Wang, D.-S., Jonker, J. W., Kato, Y., Kusuhara, H., Schinkel, A. H., & Sugiyama, Y. (2002) · The Journal of Pharmacology and Experimental Therapeutics · doi:10.1124/jpet.102.034140 · Cited by 441 · see citing works
Somlyo, A. V., Gonzalez-Serratos, H., Shuman, H., McClellan, G., & Somlyo, A. P. (1981) · The Journal of Cell Biology · doi:10.1083/jcb.90.3.577 · Cited by 436 · see citing works
Metformin is a Superior Substrate for Renal Organic Cation Transporter OCT2 rather than Hepatic OCT1
Kimura, N., Masuda, S., Tanihara, Y., Ueo, H., Okuda, M., Katsura, T., & Inui, K. (2005) · Drug Metabolism and Pharmacokinetics · doi:10.2133/dmpk.20.379 · Cited by 360 · see citing works
Tzvetkov, M. V., Vormfelde, S. V., Balen, D., Meineke, I., Schmidt, T., Sehrt, D. B., Sabolić, I., Koepsell, H., & Brockmöller, J. (2009) · Clinical Pharmacology & Therapeutics · doi:10.1038/clpt.2009.92 · Cited by 349 · see citing works
Qin, Y., Dittmer, P. J., Park, J. G., Jansen, K. B., & Palmer, A. E. (2011) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1015686108 · Cited by 307 · see citing works
Chu, X., Korzekwa, K., Elsby, R., Fenner, K., Galetin, A., Lai, Y., Matsson, P., Moss, A., Nagar, S., Rosania, G., et al. (2013) · Clinical pharmacology and therapy · doi:10.1038/clpt.2013.78 · Cited by 242 · see citing works
Chen, L., Shu, Y., Liang, X., Chen, E. C., Yee, S. W., Zur, A. A., Li, S., Xu, L., Keshari, K. R., Lin, M. J., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1314939111 · Cited by 234 · see citing works
Self-citationIntraluminal calcium as a primary regulator of endoplasmic reticulum function
Burdakov, D., Petersen, O. H., & Verkhratsky, A. (2005) · Cell Calcium · doi:10.1016/j.ceca.2005.06.010 · Cited by 228 · see citing works
Pharmacokinetics of metformin during pregnancy
Eyal, S., Easterling, T. R., Carr, D., Umans, J. G., Miodovnik, M., Hankins, G. D. V., Clark, S. M., Risler, L., Wang, J., Kelly, E. J., et al. (2010) · Drug Metabolism and Disposition · doi:10.1124/dmd.109.031245 · Cited by 209 · see citing works
Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis
Tzur, A., Moore, J. K., Jorgensen, P., Shapiro, H. M., & Kirschner, M. W. (2011) · PLoS ONE · doi:10.1371/journal.pone.0016053 · Cited by 184 · see citing works
A predictive model for the selective accumulation of chemicals in tumor cells
Trapp, S., & Horobin, R. W. (2005) · European Biophysics Journal · doi:10.1007/s00249-005-0472-1 · Cited by 180 · see citing works
Rapid Measurement of Intracellular Unbound Drug Concentrations
Mateus, A., Matsson, P., & Artursson, P. (2013) · Molecular Pharmaceutics · doi:10.1021/mp4000822 · Cited by 170 · see citing works
Quantitative modeling of selective lysosomal targeting for drug design
Trapp, S., Rosania, G. R., Horobin, R. W., & Kornhuber, J. (2008) · European Biophysics Journal · doi:10.1007/s00249-008-0338-4 · Cited by 158 · see citing works
Yamada, A., Gaja, N., Ohya, S., Muraki, K., Narita, H., Ohwada, T., & Imaizumi, Y. (2001) · The Japanese Journal of Pharmacology · doi:10.1254/jjp.86.342 · Cited by 108 · see citing works
KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology
Søgaard, R., Ljungstrøm, T., Angelo, K., Olesen, S., & Jensen, B. (2001) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.2001.280.4.c859 · Cited by 106 · see citing works
Progress Towards Prediction of Human Pharmacokinetic Parameters from In Vitro Technologies
Houston, J. B., & Galetin, A. (2003) · Drug Metabolism Reviews · doi:10.1081/dmr-120026870 · Cited by 83 · see citing works
Intracellular Drug Concentrations
Dollery, C. T. (2012) · Clinical Pharmacology & Therapeutics · doi:10.1038/clpt.2012.240 · Cited by 81 · see citing works
Klapperstück, T., Glanz, D., Klapperstück, M., et al. (2009) · Cytometry Part A · doi:10.1002/cyto.a.20735 · Cited by 54 · see citing works
Intracellular concentrations determine the cytotoxicity of adefovir, cidofovir and tenofovir
Zhang, X., Wang, R., Piotrowski, M. A., Zhang, H., & Leach, K. L. (2014) · Toxicology in Vitro · doi:10.1016/j.tiv.2014.10.019 · Cited by 34 · see citing works
Cimetidine transport in isolated luminal membrane vesicles from rabbit kidney
Gisclon, L. G., Wong, F. M., & Giacomini, K. M. (1987) · American Journal of Physiology-Renal Physiology · doi:10.1152/ajprenal.1987.253.1.f141 · Cited by 34 · see citing works
Self-citationMeasurement of intracellular volume in monolayers of cultured cells
Vasquez, B., Ishibashi, F., & Howard, B. V. (1982) · In Vitro Cellular & Developmental Biology - Plant · doi:10.1007/bf02796397 · Cited by 33 · see citing works
Brain Mitochondrial Drug Delivery: Influence of Drug Physicochemical Properties
Durazo, S. A., Kadam, R. S., Drechsel, D. A., Patel, M., & Kompella, U. B. (2011) · Pharmaceutical Research · doi:10.1007/s11095-011-0532-4 · Cited by 29 · see citing works
Ghosh, A., Maurer, T. S., Litchfield, J., Varma, M. V., Rotter, C. J., Scialis, R. J., Feng, B., Tu, M., Guimarães, C. R. W., & Scott, D. O. (2014) · Drug Metabolism and Disposition · doi:10.1124/dmd.114.058032 · Cited by 24 · see citing works
Self-citationDetection of Statin Cytotoxicity Is Increased in Cells Expressing the OATP1B1 Transporter
Zhang, X., Scialis, R. J., Feng, B., & Leach, K. L. (2013) · Toxicological Sciences · doi:10.1093/toxsci/kft085 · Cited by 24 · see citing works
K+-selective channel from sarcoplasmic reticulum of split lobster muscle fibers.
Tang, J. M., Wang, J., & Eisenberg, B. (1989) · The Journal of General Physiology · doi:10.1085/jgp.94.2.261 · Cited by 23 · see citing works
Li, Q., Yang, H., Peng, X., Guo, D., Dong, Z., Polli, J. E., & Shu, Y. (2013) · Molecular Pharmaceutics · doi:10.1021/mp400013t · Cited by 16 · see citing works
The discovery of synaptic vesicles
Wells, W. A. (2005) · The Journal of Cell Biology · doi:10.1083/jcb1681fta2 · Cited by 7 · see citing works
Ribosomes, or the particles of Palade
Wells, W. A. (2005) · The Journal of Cell Biology · doi:10.1083/jcb1681fta3 · Cited by 7 · see citing works
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Mohammed, I., Hollenberg, M. D., Ding, H., & Triggle, C. R. (2021) · Frontiers in Endocrinology · doi:10.3389/fendo.2021.718942 · Cited by 278 · see citing works
- Authors
- Chien, H.-C., Zur, A. A., Maurer, T. S., Yee, S. W., Tolsma, J., Jasper, P., Scott, D. O., & Giacomini, K. M.
- Affiliations
- University of California, San Francisco (US)
- Corresponding author
- Giacomini, Kathleen M
- Venue
- Drug Metabolism and Disposition
- Publisher
- Elsevier BV
- Published
- 2016-03
- Volume
- 44
- Issue
- 3
- Pages
- 356-364
- ISSN
- 0090-9556 (ISSN-L)
- PMC
- PMC6047207
- OpenAlex
- W2265806198
- Cited by
- 63
- References
- 32
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 4 · 2023: 2 · 2022: 5 · 2021: 8 · 2020: 8 · 2019: 8 · 2018: 11 · 2017: 6 · 2016: 4 · 2014: 1
- FWCI
- 3.06
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Drug Metabolism and Disposition (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Pfizer
- University of California, San Francisco
- National Institute of General Medical Sciences (R01GM117163)
- Topics
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Drug Solubulity and Delivery Systems
- Keywords
- intracellular drug concentration, unbound drug concentration, metformin, OCT1, HEK293 cells, intracellular space, extracellular space, potassium concentration, endoplasmic reticulum, mitochondria, drug uptake
- MeSH terms
- Biological Transport, Cell Line, Endoplasmic Reticulum, Humans, Metformin, Mitochondria, Transfection, Organic Cation Transporter 1, HEK293 Cells
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