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1.
Drug Metab Dispos ; 29(4 Pt 1): 394-400, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11259322

ABSTRACT

The hepatic uptake and biliary excretion of a novel methotrexate derivative, N-[4-[(2,4-diamminopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl-L-homoglutamic acid (MX-68), were examined in rats in vitro using isolated hepatocytes and bile canalicular membrane vesicles (CMVs), respectively. The uptake of MX-68 by isolated rat hepatocytes showed a saturable component (Km = 2.15 microM and Vmax = 2.34 pmol/min/mg of protein) and was inhibited by ATP-depletors and anionic compounds such as taurocholate and probenecid. [3H]MX-68 uptake was also inhibited by folate analogs such as methotrexate and 5CH3-tetrahydrofolate, but the effect of these compounds was slightly less than that of unlabeled MX-68. On replacing Na+ with choline, MX-68 uptake remained unchanged, whereas the methotrexate uptake was reduced. Uptake of MX-68 increased as the extracellular pH fell from 7.5 to 5.5. These results suggest that MX-68 is taken up via active transport systems. The uptake of MX-68 by CMVs prepared from normal rats exhibited clear ATP dependence, whereas ATP had only a minimal effect on the uptake by CMVs from Eisai-hyperbilirubinemic rats with a hereditary deficiency in canalicular multispecific organic anion transporter (cMOAT). The initial uptake rate of ATP-dependent MX-68 transport showed saturation with kinetic parameters similar to those of methotrexate. MX-68 inhibited the ATP-dependent transport of 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, the inhibition constant (162 microM) being comparable with the Km of ATP-dependent MX-68 transport. These results suggest that the biliary excretion of MX-68 via the bile canalicular membrane is mediated mainly by cMOAT. In conclusion, active transport systems are involved in membrane penetration of MX-68 both at sinusoidal and canalicular sides in the liver, the latter being mainly involved with methotrexate (MTX) whereas the former differs partially from that for MTX.


Subject(s)
2-Aminoadipic Acid/analogs & derivatives , 2-Aminoadipic Acid/pharmacokinetics , Carrier Proteins/metabolism , Folic Acid Antagonists/pharmacokinetics , Methotrexate/analogs & derivatives , Methotrexate/pharmacokinetics , Animals , Anion Transport Proteins , Anions/pharmacology , Antimetabolites/pharmacology , Biological Transport , Cells, Cultured , Drug Carriers , Folic Acid/analogs & derivatives , Folic Acid/pharmacology , Glutathione/analogs & derivatives , Glutathione/metabolism , Hepatocytes/metabolism , Hydrogen-Ion Concentration , Male , Rats , Sodium/pharmacology , Time Factors , Transport Vesicles/metabolism , Tritium
2.
J Pharmacol Exp Ther ; 291(1): 204-12, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10490906

ABSTRACT

The excretion and tissue distribution kinetics of a novel antifolate, MX-68, were evaluated under conditions of a continuous steady-state infusion in Sprague-Dawley rats (SDRs). The biliary excretion clearance defined with respect to the hepatic concentration (CL(bile, h)) was much lower in Eisai hyperbilirubinemic rats with a hereditary deficiency in canalicular multispecific organic anion transporter than that in SDRs, suggesting the involvement of canalicular multispecific organic anion transporter in its transport across the bile canalicular membrane. The CL(bile, h) in SDRs increased as the infusion rate increased; this can be largely explained by saturation of the intracellular binding of MX-68. On the other hand, the urinary excretion clearance defined with respect to the renal concentration (CL(urine, k)) was comparable for the two strains but showed an increase and subsequent decrease as the renal concentration increased. This nonlinear profile was also found even when the CL(urine, k) was normalized by the unbound fraction in kidney. Therefore, this kinetic profile represents the saturation of both reabsorption and secretion. Reabsorption of MX-68 in kidney was supported by its saturable transport by renal brush border membrane vesicles at an inward H(+) gradient. The liver-to-plasma unbound concentration ratio decreased as the steady-state plasma concentration increased, suggesting that MX-68 is taken up by a saturable mechanism or mechanisms. Thus, the saturation of transport systems across several plasma membranes and intracellular binding in both the liver and kidney produce the nonlinear disposition of MX-68.


Subject(s)
2-Aminoadipic Acid/analogs & derivatives , Folic Acid Antagonists/pharmacokinetics , Methotrexate/analogs & derivatives , 2-Aminoadipic Acid/chemistry , 2-Aminoadipic Acid/pharmacokinetics , 2-Aminoadipic Acid/urine , Administration, Oral , Animals , Anion Transport Proteins , Binding Sites , Carrier Proteins/metabolism , Cell Membrane/metabolism , Cytosol/metabolism , Dose-Response Relationship, Drug , Folic Acid Antagonists/chemistry , Folic Acid Antagonists/urine , Ion Transport , Kidney Cortex/metabolism , Liver/metabolism , Male , Metabolic Clearance Rate , Methotrexate/chemistry , Methotrexate/pharmacokinetics , Methotrexate/urine , Microvilli/metabolism , Rats , Rats, Sprague-Dawley , Tissue Distribution
3.
Biopharm Drug Dispos ; 20(8): 361-7, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10870092

ABSTRACT

Binding and transport properties of methotrexate (MTX) and its novel derivative, MX-68, were examined in brush-border membrane vesicles (BBMVs) isolated from rat kidneys. The uptake of MTX, MX-68 and folic acid by BBMVs was stimulated by an inwardly-directed H(+) gradient. Such H(+)-dependent uptake of folic acid is compatible with a previous report (Bhandari et al., Biochim Biophys Acta 1988; 937: 211). The MTX uptake exhibits saturation with a K(m) of 0.834 microM. Although the uptake of these three compounds at optimal pH depended on the osmolarity of the medium, a substantial portion of the uptake was osmolarity-insensitive. By changing the medium osmolarity, the uptake by BBMVs could be separately discriminated as osmolarity-sensitive and insensitive portions, representing transport into the intravesicular space and binding to the surface of BBMVs, respectively. For all three compounds, the binding increased in a time-dependent manner, while the amount transported reached a maximum after a relatively short incubation period. The transport of folic acid, but not its binding, exhibited an overshoot phenomenon under conditions of an inward H(+) gradient. The present results suggest that reabsorption of MTX and MX-68 in the kidney is governed by both their binding and transport mechanisms, with a similar kinetic profile to that of folic acid. The involvement of a transport system seems to make a relatively small contribution to the reabsorption of MTX assessed in BBMVs compared with MX-68 and folic acid.


Subject(s)
2-Aminoadipic Acid/analogs & derivatives , Folic Acid Antagonists/pharmacokinetics , Kidney/metabolism , Methotrexate/analogs & derivatives , Methotrexate/pharmacokinetics , 2-Aminoadipic Acid/pharmacokinetics , Animals , Biological Transport , Dose-Response Relationship, Drug , Folic Acid/pharmacokinetics , Hydrogen-Ion Concentration , Kidney/ultrastructure , Male , Microvilli/metabolism , Osmolar Concentration , Rats , Rats, Sprague-Dawley
4.
Am J Clin Nutr ; 68(4): 827-39, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9771859

ABSTRACT

BACKGROUND: This is a continuation of investigations of the relations between amino acid kinetics and amino acid dietary requirements in healthy adults. OBJECTIVE: The aim was to investigate the 24-h pattern and rate of the metabolism of an L-[1-13C]-2-aminoadipic acid ([13C]AAA) tracer and of whole-body L-[1-13C]lysine ([13C]lysine) oxidation and balance in healthy, young adults receiving a generous intake of lysine. DESIGN: Thirteen healthy adults were given an adequate, L-amino acid-based diet supplying 77 mg lysine x kg(-1) x d(-1) for 6 d before the tracer studies. Two subjects received [13C]AAA intravenously and 2 received it orally; 3 subjects received [13C]lysine intravenously and 6 received it orally. We measured 13CO2 output, plasma [13C]AAA and [13C]lysine enrichment, and urinary [13C]AAA. RESULTS: [13C]AAA oxidation was estimated to be higher after the orally administered than after the intravenously administer tracer; plasma [13C]AAA was similar to urinary [13C]AAA. Whole-body lysine oxidation showed a rhythm that was induced by meal feeding. The intravenous [13C]lysine tracer gave mean estimates of lysine balances (lysine intake minus oxidation) that apparently were too low (-15.7 mg x kg(-1) x d(-1)) or too high (16.6 mg x kg(-1) x d(-1), P < 0.05 from zero balance) on the basis of urinary [13C]AAA or plasma [13C]lysine estimates of oxidation, respectively. For the orally administered tracer and plasma [13C]lysine enrichment, the mean balance was slightly positive (8.7 mg x kg(-1) x d(-1), P < 0.05 from zero). CONCLUSIONS: Use of urinary [13C]AAA as an index of the enrichment of the precursor pool did not appear to significantly improve the estimate of the fasting and feeding components of daily lysine balance. For estimates of daily, whole-body lysine oxidation, we propose use of plasma [13C]lysine with a 24-h, orally administered tracer protocol.


Subject(s)
2-Aminoadipic Acid/pharmacokinetics , Lysine/pharmacokinetics , Nitrogen/administration & dosage , Nutritional Requirements , 2-Aminoadipic Acid/administration & dosage , 2-Aminoadipic Acid/metabolism , Administration, Oral , Adult , Carbon Isotopes , Female , Food , Humans , Infusions, Intravenous , Kinetics , Lysine/administration & dosage , Lysine/metabolism , Male , Oxidation-Reduction
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