Absorption, metabolism, and excretion of 14C-temozolomide following oral administration to patients with advanced cancer.
The purpose of this study is to characterize the absorption, metabolism, and excretion of carbon 14-labeled temozolomide (14C-TMZ) administered p.o. to adult patients with advanced solid malignancies. On day 1 of cycle 1, six patients received a single oral 200-mg dose of 14C-TMZ (70.2 microCi). Whole blood, plasma, urine, and feces were collected from days 1-8 and on day 14 of cycle 1. Total radioactivity was measured in all samples. TMZ, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC), and 4-amino-5-imidazole-carboxamide (AIC) concentrations were determined in plasma, and urine and plasma samples were profiled for metabolite/degradation products. Maximum TMZ plasma concentrations were achieved between 0.33 to 2 h (mean, 1.2 h), and half-life, apparent volume of distribution, and oral clearance values averaged 1.9 h, 17 liters/m2, and 104 ml/min/m2, respectively. A first-order absorption, one-compartment linear model, which included first-order formation of MTIC from TMZ and elimination of MTIC via degradation to AIC, and a peripheral distribution compartment for AIC, adequately described the plasma TMZ, MTIC, and AIC concentrations. MTIC systemic clearance was estimated to be 5384 ml/min/m2, and the half-life was calculated to be 2.5 min. Metabolite profiles of plasma at 1 and 4 h after treatment showed that 14C-derived radioactivity was primarily associated with TMZ, and a smaller amount was attributed to AIC. Profiles of urine samples from 0-24 h revealed that 14C-TMZ-derived urinary radioactivity was primarily associated with unchanged drug (5.6%), AIC (12%), or 3-methyl-2,3-dihydro-4-oxoimidazo[5,1-d]tetrazine-8-carboxyl ic acid (2.3%). The recovered radioactive dose (39%) was principally eliminated in the urine (38%), and a small amount (0.8%) was excreted in the feces. TMZ exhibits rapid oral absorption and high systemic availability. The primary elimination pathway for TMZ is by pH-dependent degradation to MTIC and further degradation to AIC. Incomplete recovery of radioactivity may be explained by the incorporation of AIC into nucleic acids. (+info
Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6-bisphosphate.
Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose 2,6-bisphosphate, or for fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon. (+info
Effect of AMPK activation on muscle glucose metabolism in conscious rats.
The effect of AMP-activated protein kinase (AMPK) activation on skeletal muscle glucose metabolism was examined in awake rats by infusing them with 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR; 40 mg/kg bolus and 7.5 mg. kg-1. min-1 constant infusion) along with a variable infusion of glucose (49.1 +/- 2.4 micromol. kg-1. min-1) to maintain euglycemia. Activation of AMPK by AICAR caused 2-deoxy-D-[1,2-3H]glucose (2-DG) uptake to increase more than twofold in the soleus and the lateral and medial gastrocnemius compared with saline infusion and occurred without phosphatidylinositol 3-kinase activation. Glucose uptake was also assessed in vitro by use of the epitrochlearis muscle incubated either with AICAR (0.5 mM) or insulin (20 mU/ml) or both in the presence or absence of wortmannin (1.0 microM). AICAR and insulin increased muscle 2-DG uptake rates by approximately 2- and 2.7-fold, respectively, compared with basal rates. Combining AICAR and insulin led to a fully additive effect on muscle glucose transport activity. Wortmannin inhibited insulin-stimulated glucose uptake. However, neither wortmannin nor 8-(p-sulfophenyl)-theophylline (10 microM), an adenosine receptor antagonist, inhibited the AICAR-induced activation of glucose uptake. Electrical stimulation led to an about threefold increase in glucose uptake over basal rates, whereas no additive effect was found when AICAR and contractions were combined. In conclusion, the activation of AMPK by AICAR increases skeletal muscle glucose transport activity both in vivo and in vitro. This cellular pathway may play an important role in exercise-induced increase in glucose transport activity. (+info
5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes. (+info
Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.
Insulin increases glucose uptake through the translocation of GLUT-4 via a pathway mediated by phosphatidylinositol 3-kinase (PI3K). In contrast, myocardial glucose uptake during ischemia and hypoxia is stimulated by the translocation of GLUT-4 to the surface of cardiac myocytes through a PI3K-independent pathway that has not been characterized. AMP-activated protein kinase (AMPK) activity is also increased by myocardial ischemia, and we examined whether AMPK stimulates glucose uptake and GLUT-4 translocation. In isolated rat ventricular papillary muscles, 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, as well as cyanide-induced chemical hypoxia and insulin, increased 2-[(3)H]deoxyglucose uptake two- to threefold. Wortmannin, a PI3K inhibitor, did not affect either the AICAR- or the cyanide-stimulated increase in deoxyglucose uptake but eliminated the insulin-stimulated increase in deoxyglucose uptake. Immunofluorescence studies demonstrated translocation of GLUT-4 to the myocyte sarcolemma in response to stimulation with AICAR, cyanide, or insulin. Preincubation of papillary muscles with the kinase inhibitor iodotubercidin or adenine 9-beta-D-arabinofuranoside (araA), a precursor of araATP (a competitive inhibitor of AMPK), decreased AICAR- and cyanide-stimulated glucose uptake but did not affect basal or insulin-stimulated glucose uptake. In vivo infusion of AICAR caused myocardial AMPK activation and GLUT-4 translocation in the rat. We conclude that AMPK activation increases cardiac muscle glucose uptake through translocation of GLUT-4 via a pathway that is independent of PI3K. These findings suggest that AMPK activation may be important in ischemia-induced translocation of GLUT-4 in the heart. (+info
Improvement by 5-amino-4-imidazole carboxamide riboside of the contractile dysfunction that follows brief periods of ischemia through increases in ecto-5-nucleotidase activity and adenosine release in canine hearts.
5-Amino-4-imidazole carboxamide (AICA) riboside increases adenosine release in ischemic myocardium, suggesting that AICA riboside improves contractile dysfunction. In 49 open-chest dogs, contractile function assessed by fractional shortening (FS) was observed 3 h after the onset of reperfusion following 15 min of occlusion of the left anterior descending coronary artery. During reperfusion, the treatment with AICA riboside increased adenosine concentration in the coronary venous blood (536+/-44 vs. 281+/-21 pmol/ml at 3 min of reperfusion, p<0.001) and peak coronary hyperemic flow (367+/-13 vs. 300+/-21 ml/100 g per min, p<0.001) when compared with the untreated group. FS at 3h of reperfusion increased in the AICA riboside group (21.1+/-2.3 vs. 12.8+/-0.6% in the untreated group, p<0.001). AICA riboside increased myocardial ecto-5'-nucleotidase activity. Administration of adenosine also augmented coronary hyperemic flow and increased FS to the levels of the AICA riboside group. Either 8-phenyltheophylline (an antagonist of adenosine receptors) or alpha,beta-methylene-adenosine 5'-diphosphate (an inhibitor of ecto-5'-nucleotidase) completely abolished the increased coronary hyperemic flow and improvements of myocardial contractile function due to AICA riboside. Thus it was concluded that AICA riboside improves the contractile dysfunction that follows a brief period of ischemia via adenosine-dependent mechanisms. (+info
Effect of methotrexate on blood purine and pyrimidine levels in patients with rheumatoid arthritis.
OBJECTIVE: The mechanism of anti-inflammatory effects of methotrexate (MTX) at low dose may relate to a decrease in availability of the purine precursor or it may depend on accumulation of 5-aminoimidazole-4-carboxamide (AICAR) and the anti-inflammatory nucleoside adenosine. The aim of this study was to evaluate the possible mechanism of action by analysis of changes in blood concentrations of purine and pyrimidine metabolites during MTX treatment. METHODS: Venous blood samples were collected from rheumatoid arthritis patients before and at different times for up to 7 days after the start of MTX treatment. Whole blood concentrations of adenosine, uridine, hypoxanthine, uric acid and erythrocyte nucleotides were measured by HPLC. RESULTS: The initial blood adenosine concentration was 0.073 +/- 0.013 microM and no differences were observed during MTX treatment. However, a decrease in uric acid concentration was observed from 205.5+/-13.5 to 160. 9+/-13.5 microM (P<0.05) within 24 h after MTX administration. The hypoxanthine concentration decreased in parallel with uric acid, while the uridine concentration decreased 48 h after MTX administration. No accumulation of AICAR-triphosphate (ZTP) was observed in the erythrocytes. CONCLUSIONS: MTX decreases circulating purine and pyrimidine concentrations, and their availability for DNA and RNA synthesis, which may affect immune cell proliferation and protein (cytokine) expression. The absence of adenosine concentration changes and lack of ZTP formation is evidence against an AICAR/adenosine mechanism, although localized adenosine concentration changes cannot be excluded. (+info
Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.
This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased approximately 2. 5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training. (+info