Article Figures & Data
Figures
- Fig. 1.
In vitro transport study of m1A. The uptake of m1A (100 μM) by organic cation transporters was determined in the absence (solid lines) or presence (dotted lines) of inhibitor (5 mM TEA). The incubation buffer also contained 10 μM dipyridamole to inhibit the ENT-mediated m1A uptake. Each symbol and bar represents the mean ± S.E. (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, the absence vs. presence of 5 mM TEA in transporter-overexpressing cells, Student’s two-tailed unpaired t test.
- Fig. 2.
Effect of various inhibitors on hOCT2- and hMATE2-K-mediated m1A uptake. The uptake of m1A (100 μM) for 60 minutes in HEK293 cells stably expressing hOCT2 or hMATE2-K was determined in the absence and presence of inhibitors (trimethoprim, pyrimethamine, and cimetidine) at the designated concentrations. The transporter-specific uptake of m1A was calculated by subtracting the uptake by empty vector–transfected cells from that by transporter-expressing cells and shown as a proportion to the uptake value in the absence of inhibitors. The solid line represents the fitted line obtained by nonlinear regression analysis as described in Materials and Methods. Each symbol and bar represents the mean ± S.E. (n = 3).
- Fig. 3.
m1A uptake by renal cortical slices of WT and Oct1/2 dKO mice. The uptake of m1A (100 μM) for 10 minutes in renal cortical slices of WT and Oct1/2 dKO mice was determined in the absence or presence of 250 μM probenecid or 5 mM TEA. Each bar represents the mean ± S.E. (n = 3). **P < 0.01, Dunnett’s post hoc test.
- Fig. 4.
m1A infusion assay using WT and Oct1/2 dKO mice. Effect of Oct1/2 dKO on the plasma and concentrations and urinary excretion of exogenously given m1A (100 nmol/min per kilogram) in mice. The kinetic parameters were calculated as described in Materials and Methods. Each symbol and bar represents the mean and S.E. (n = 3). *P < 0.05; **P < 0.01, Student’s two-tailed unpaired t test.
- Fig. 5.
Effect of PYR on the kinetics of m1A in mice. Plasma concentrations and urinary excretion of m1A were determined in control and PYR-treated mice. PYR (20 μmol/kg) was given to mice by bolus injection 30 minutes before starting intravenous infusion of (A) m1A (100 nmol/min per kilogram) and (B) rhodamine 123 (1 nmol/min per kilogram). The kinetic parameters were calculated as described in Materials and Methods. Each symbol and bar represents the mean and S.E. (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s two-tailed unpaired t test.
- Fig. 6.
In vivo inhibition study of OCT2 and MATEs using DX-619. Plasma concentration profiles of m1A, metformin, creatinine, and DX-619 were determined after oral administration of metformin (5 mg/kg) with (□) and without (■) DX-619 pretreatment (30 mg/kg) in cynomolgus monkeys. Each symbol and bar represents the mean and S.E. (n = 3). P value was calculated by Student’s two-tailed paired t test.
- Fig. 7.
Plasma concentrations and renal clearance of m1A in younger and older volunteers. Plasma concentration profiles and urinary excretion of m1A and creatinine (A) in younger and older volunteers (B) were determined. Each symbol and bar represents the mean ± S.D. of eight and seven subjects. *P < 0.05; **P < 0.01; ***P < 0.001, younger vs. older, Student’s two-tailed unpaired t test.
Tables
- TABLE 1
List of endogenous compounds of interest in the metabolomics analysis performed on WT and Oct1/2 dKO mice
Identity Platform Protonated Molecule Fold Change (Oct1/2 dKO/WT) Plasma Urine Pipecolate LC-MS Pos 130.1 1.43*** 1.1 Cadaverine GC-MS 174 N/D 0.43* 3-methylglutarylcarnitine LC-MS Pos 290.1 2.65** 1.9 S-methylcysteine GC-MS 162.1 1.04 0.54* Putrescine GC-MS 174 0.93 0.62** Docosahexaenoate LC-MS Neg 327.3 1.35** N/D Dihomolinolenate LC-MS Neg 305.4 1.26* N/D Arachidonate LC-MS Neg 303.4 1.28* N/D Docosapentaenoate LC-MS Neg 329.4 2.23** N/D Mead acid LC-MS Neg 305.4 1.51** N/D 12,13-DiHOME LC-MS Neg 313.4 1.69** N/D Choline LC-MS Pos 104.2 1.21* N/D 1-Docosahexaenoylglycerophosphocholine LC-MS Pos 568.4 1.14* N/D 1-Docosahexaenoyl-glycerophosphoethanolamine LC-MS Neg 524.3 1.31* N/D N1-methyladenosine LC-MS Pos 282.1 2.78** 0.84 3-Ureidopropionate LC-MS Pos 133.1 0.55* 0.66* Glycolate (hydroxyacetate) GC-MS 177 1.23* 1.19 X-11478a LC-MS Neg 165.2 2*** N/D X-11909a LC-MS Neg 297.3 1.86** N/D X-12257a LC-MS Neg 269.1 3.13* 0.77 X-16570a LC-MS Neg 198.2 2.95* 1.29 X-16575a LC-MS Neg 293.1 2.04*** N/D X-16581a LC-MS Pos 304.1 2.36*** 2.77** X-17307a LC-MS Pos 162.1 N/D 0.48** X-18628a LC-MS Pos 931.4 2.16* N/D X-20568a LC-MS Pos 188.1 N/D 0.56* GC-MS, gas chromatography/mass spectrometry; LC-MS, liquid chromatography/mass spectrometry; Neg, negative; Pos, positive; 12, 13-DiHOME, (±)12,13-dihydroxy-9Z-octadecenoic acid. N/D, Not detected. *P < 0.05; **P < 0.01; ***P < 0.001, Welch's two-sample t test; value in Oct1/2 dKO mice vs. WT mice.
- TABLE 2
Comparison of in vitro inhibition constant of various drugs for OCT2- and MATE2-K–mediated uptake
Each value was determined from the data shown in Fig. 2. The equations for the calculations are described in Materials and Methods. Each value represents the mean ± computer-calculated S.D.
Inhibitor hOCT2 hMATE2-K m1A 100 μM Creatinine 100 μM Metformin 10 μM m1A 100 μM Creatinine 100 μM Metformin 10 μM Trimethoprim 20 ± 5 26 ± 2 20 ± 2 2.4 ± 0.4 0.58 ± 0.11 0.92 ± 0.04 Pyrimethamine 0.64 ± 0.18 0.93 ± 0.04 0.61 ± 0.04 0.22 ± 0.06 0.35 ± 0.05 0.22 ± 0.02 Cimetidine 1.8 ± 0.4 36 ± 3 59 ± 1.4 5.3 ± 1.6 24 ± 5 5.2 ± 0.8 - TABLE 3
Pharmacokinetic parameters of m1A in WT and Oct1/2 dKO mice
The equations to calculate the kinetic parameters are described in Materials and Methods. Each value represents the mean ± S.E. (n = 3).
Parameter WT Oct1/2 dKO Cp,120 min (μM) 1.63 ± 0.23* 2.45 ± 0.11* AUCp,0–120 min (µmol per min/l) 138 ± 15* 203 ± 9* Xurine (nmol) 220 ± 27* 103 ± 13* Cliver (μM) 3.31 ± 0.49* 5.63 ± 0.28* Kp,liver (ml/g of liver) 2.08 ± 0.36 2.30 ± 0.09 Ckidney (μM) 15.6 ± 6.7 9.27 ± 0.91 Kp,kidney (ml/g of kidney) 8.81 ± 2.70 3.76 ± 0.23 CLtot,p (ml/min) 2.10 ± 0.13** 1.11 ± 0.04** CLtot,p (ml/min per kilogram) 61.1 ± 2.7** 36.4 ± 1.3** CLr,p (ml/min) 1.61 ± 0.16** 0.511 ± 0.077** CLr,p (ml/min per kilogram) 46.9 ± 4.9** 16.6 ± 2.6** GFR (Jonker et al., 2003) 0.462 ± 0.045 0.462 ± 0.065 Ckidney, kidney concentration; Cp,120 min, plasma concentration of compound at 120 minutes after administration; GFR, glomerular filtration rate; Kp,liver, liver-to-plasma ratio; Xurine, amount of compound excreted in urine.
↵* P < 0.05; **P < 0.01; ***P < 0.001, Student’s two-tailed unpaired t test; value in Oct1/2 dKO mice vs. WT mice.
- TABLE 4
Pharmacokinetic parameters of m1A and rhodamine 123 in control and PYR-treated mice
The equations to calculate the kinetic parameters are described in Materials and Methods. Each value represents the mean ± S.E. (n = 4); Student’s two-tailed unpaired t test; value in control mice vs. PYR-treated mice.
Parameter m1A Rhodamine 123 Control PYR Unit Control PYR Unit Cp,120 min 1.64 ± 0.10 2.31 ± 0.13** μM 5.61 ± 0.46 5.01 ± 0.39 nM AUCp,0–120 min 140 ± 9 182 ± 11* μmol per min/l 564 ± 27 512 ± 12 nmol per min/l Xurine 170 ± 10 126 ± 5** nmol 477 ± 32 21.2 ± 3.4*** pmol Ckidney 7.22 ± 1.82 34.0 ± 5.4** μM 0.800 ± 0.074 1.86 ± 0.21 μM Kp,kidney 4.4 ± 1.1 14.7 ± 2.0** ml/g of kidney 143 ± 6 381 ± 70 ml/g of kidney CLtot,p 2.32 ± 0.12 1.78 ± 0.13* ml/min 3.93 ± 0.18 4.07 ± 0.05 ml/min CLtot,p 80.4 ± 4.9 61.9 ± 3.6* ml/min per kilogram 136 ± 8 142 ± 2 ml/min per kilogram CLr,p 1.23 ± 0.11 0.698 ± 0.035** ml/min 0.851 ± 0.073 0.0416 ± 0.0073 ml/min CLr,p 42.6 ± 4.1 24.3 ± 0.6** ml/min per kilogram 29.6 ± 3.1 1.44 ± 0.21 ml/min per kilogram GFR (Jonker et al., 2003) 0.462 ± 0.045 0.462 ± 0.065 ml/min Ckidney, kidney concentration; Cp,120 min, plasma concentration of compound at 120 minutes after administration; GFR, glomerular filtration rate; Xurine, amount of compound excreted in urine.
↵* P < 0.05; **P < 0.01; ***P < 0.001.
Treatment 1 2 3 Mean ± S.E. m1A (nM·h) Control 364 216 219 266 ± 49 +DX-619 468 361 464 431 ± 35 Fold change 1.29 1.67 2.12 1.72 ± 0.24 Metformin (μM·h) Control 43.5 11.1 38.3 31.0 ± 10.0 +DX-619 91.9 26.5 77.6 65.3 ± 19.9 Fold change 2.11 2.39 2.03 2.18 ± 0.11 - TABLE 6
Pharmacokinetic parameters of m1A in younger and older Japanese volunteers
Each value was determined from the data shown in Fig. 7. The equations to calculate the kinetic parameters are described in Materials and Methods. Each value represents the mean ± S.D. of eight and seven subjects; Student’s two-tailed unpaired t test; younger vs. older. Subject information is shown in Supplemental Table 4.
Parameter m1A Creatinine Younger Older Unit Younger Older Unit AUC0–24 h 1.22 ± 0.22 1.29 ± 0.14 μmol per h/l 1.05 ± 0.12 1.04 ± 0.16 mmol per h/l Xurine, 0–24 h 17.2 ± 1.4 13.0 ± 0.9*** μmol 8.06 ± 0.79 6.61 ± 1.17 mmol CLr,p 244 ± 58 169 ± 22** ml/min 129 ± 18 108 ± 21 ml/min AUC0–24 h, area under the plasma concentration-time curve from 0 to 24 hours; Xurine, 0–24 h, amount excreted into the urine from 0 to 24 hours.
↵** P < 0.01; ***P < 0.001.
Data Supplement
- Supplemental Data -
Supplementary Materials and Methods
Supplementary References
Supplementary Figure 1 - The effect of dipyridamole on the m1A uptake in HEK293 cells and the function of renal cation transporters.
Supplementary Figure 2 - The effect of G808T mutation to the uptake of m1A by hOCT2.
Supplementary Figure 3 - The affinity of m1A to renal cation transporters.
Supplementary Figure 4 - The uptake of m1A by cynomolgus monkey OCT2.
Supplementary Figure 5 - The effect of gut flora to the systemic exposure of m1A.
Supplementary Figure 6 - m1A infusion assay using WT and Mdr1a/1b/Abcg2 TKO mice.
Supplementary Table 1 - Participants' information.
Supplementary Table 2 - Analytical conditions for test compounds in cell, mice and human biological specimens.
Supplementary Table 3 - Analytical conditions for metformin and DX-619 in monkey plasma specimens.
Supplementary Table 4 - Pharmacokinetic parameters of m1A between wild-type and Mdrla/1b/Abcg2 TKO mice.
Supplementary Table 5 - OCT2 variants associated with serum m1A in GWAS study (Shin et al., 2014)
- Supplemental Data -
In this issue
Endogenous Probe of Organic Cation Transporters
