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Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

Myint K, Li Y, Paxton J, McKeage M - PLoS ONE (2015)

Bottom Line: However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear.In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum.Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.

ABSTRACT
The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

No MeSH data available.


Related in: MedlinePlus

Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r2 = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).
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pone.0130727.g007: Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r2 = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).

Mentions: The possible degradation of oxaliplatin in membrane vesicle incubation buffer during incubations was studied to identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport mechanism. HPLC chromatograms of authentic standards of oxaliplatin, glutathione and Pt(DACH)Cl2 showed acceptable separation of analytes with retention times of 6.5, 12.5 and 10.5 minutes, respectively, with no interference from any components of blank membrane vesicle incubation buffer, under these HPLC conditions (Fig 6). After incubation of oxaliplatin for 20 min with or without glutathione (pH 7.4, 37˚C), the HPLC-UV chromatograms appeared more-or-less unchanged from the start of the incubation. No new peaks appeared and a very small (10%) reduction in the oxaliplatin peak area was observed at this time point. With an increased incubation time, the oxaliplatin peak areas progressively were reduced and new peaks appeared, corresponding to Pt(DACH)Cl2 in the solution without glutathione, and an unknown compound eluting at 9.5 min in the solution with glutathione. Kinetic analysis of the oxaliplatin concentration time-course gave a degradation half-life of 2.24 h (95%CI, 2.08 to 2.43 h) during incubation (pH 7.4, 37˚C) in membrane vesicle incubation buffer containing glutathione (Fig 7). Interpolation of the nonlinear regression fit to the oxaliplatin degradation data indicated that 95% of the added oxaliplatin (95%CI, 94.6 to 95.4%) remained intact after a 10 min incubation, and after 20 min, 90.2% of the added oxaliplatin (95%CI, 89.5 to 90.9%) remained intact, in membrane vesicle incubation buffer containing glutathione.


Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

Myint K, Li Y, Paxton J, McKeage M - PLoS ONE (2015)

Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r2 = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488857&req=5

pone.0130727.g007: Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r2 = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).
Mentions: The possible degradation of oxaliplatin in membrane vesicle incubation buffer during incubations was studied to identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport mechanism. HPLC chromatograms of authentic standards of oxaliplatin, glutathione and Pt(DACH)Cl2 showed acceptable separation of analytes with retention times of 6.5, 12.5 and 10.5 minutes, respectively, with no interference from any components of blank membrane vesicle incubation buffer, under these HPLC conditions (Fig 6). After incubation of oxaliplatin for 20 min with or without glutathione (pH 7.4, 37˚C), the HPLC-UV chromatograms appeared more-or-less unchanged from the start of the incubation. No new peaks appeared and a very small (10%) reduction in the oxaliplatin peak area was observed at this time point. With an increased incubation time, the oxaliplatin peak areas progressively were reduced and new peaks appeared, corresponding to Pt(DACH)Cl2 in the solution without glutathione, and an unknown compound eluting at 9.5 min in the solution with glutathione. Kinetic analysis of the oxaliplatin concentration time-course gave a degradation half-life of 2.24 h (95%CI, 2.08 to 2.43 h) during incubation (pH 7.4, 37˚C) in membrane vesicle incubation buffer containing glutathione (Fig 7). Interpolation of the nonlinear regression fit to the oxaliplatin degradation data indicated that 95% of the added oxaliplatin (95%CI, 94.6 to 95.4%) remained intact after a 10 min incubation, and after 20 min, 90.2% of the added oxaliplatin (95%CI, 89.5 to 90.9%) remained intact, in membrane vesicle incubation buffer containing glutathione.

Bottom Line: However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear.In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum.Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.

ABSTRACT
The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

No MeSH data available.


Related in: MedlinePlus