Methionine tumor starvation by erythrocyte-encapsulated methionine gamma-lyase activity controlled with per os vitamin B6.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma-lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)-dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U-87 MG-luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event-free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half-life was increased from <24 h to 9-12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation-sensitive tumors.

Negligible effect of oral garlic oil on the oral absorption of pyridoxine in metadoxine in rats.

Metadoxine [an ion-pair between pyridoxine and pyrrolidone carboxylate (PCA)] plus garlic oil treatment synergistically reduces alcoholic steatosis compared to each agent alone. We evaluated the effect of garlic oil on the pharmacokinetics of pyridoxine. After the oral administration of metadoxine, the total area under the plasma concentration-time curve from time zero to time infinity (AUC) and the peak plasma concentration (C(max)) of pyridoxine were significantly greater (by 40.6%) and higher (by 63.9%), respectively, than after oral administration of pyridoxine plus PCA. Oral metadoxine plus garlic oil also gave larger AUC (31.8%) and higher C(max) (64.9%) than pyridoxine plus PCA. However, garlic oil did not change the AUC or C(max) of pyridoxine in metadoxine. Thus, garlic oil does not enhance the metadoxine activity by affecting the absorption of pyridoxine.

Uptake, hydrolysis, and metabolism of pyridoxine-5'-beta-D-glucoside in Caco-2 cells.

An important dietary source of vitamin B-6, pyridoxine-5'-beta-D-glucoside (PNG), exhibits only partial bioavailability, which is limited by the extent of enzymatic cleavage of the beta-glucosidic bond to release metabolically available pyridoxine (PN). This laboratory showed that the intestinal hydrolysis of PNG is catalyzed by cytosolic PNG hydrolase (PNGH) and brush border lactase-phlorizin hydrolase (LPH). LPH-catalyzed PNG hydrolysis in vitro is competitively inhibited by lactose. In the present study, the uptake and hydrolysis of PNG were examined in Caco-2 human colon carcinoma cells, which express a functional LPH but exhibit no PNGH activity. PNG uptake at 37 degrees C was linear over 5-500 micromol/L PNG. Uptake was not significantly reduced when Na(+) was substituted with K(+), Li(+), or Tris in the medium. Increasing PNG concentration in the medium did not change intracellular concentrations of PN, pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), or pyridoxal 5'-phosphate (PLP); however, intracellular pyridoxal (PL) concentration increased. Intracellular PNG concentration was not significantly reduced in the presence of lactose, but the concentration of PL declined in proportion to extracellular lactose (P = 0.01). These results indicate that PNG can be absorbed intact in a Na(+)-independent process and is taken up by passive diffusion. The presence of lactose in this in vitro model of intestinal uptake reduced the enzymatic hydrolysis of PNG by lactase.

Mechanism and regulation of vitamin B(6) uptake by renal tubular epithelia: studies with cultured OK cells.

The kidneys play an important role in regulating vitamin B(6) body homeostasis, but limited information exists regarding the mechanism of pyridoxine uptake by renal epithelial cells, and no study exists on its regulation. To address these issues, we used the renal opossum-derived tubular epithelial (opossum kidney; OK) cells and found pyridoxine uptake to 1) be temperature and energy dependent, 2) be pH dependent, with a higher uptake at alkaline or neutral buffer pH compared with acidic pH, 3) be Na(+) independent, 4) involve a saturable component (apparent Michaelis- Menten constant of 2.40 +/- 0.23 microM), 5) be inhibited by structural analogs, and 6) be amiloride sensitive. Maintaining OK cells in a vitamin B(6)-deficient growth medium (for 48 h) led to a significant upregulation of pyridoxine uptake. This upregulation was found to be specific for pyridoxine, inhibited by cyclohexamide and actinomycin D, reversible, and mediated via an increase in maximal velocity. Pretreating OK cells with modulates of a Ca(2+)/calmodulin-mediated pathway led to a significant downregulation in pyridoxine uptake via inhibition of maximal velocity. These results demonstrate that pyridoxine uptake by renal tubular epithelial OK cells is via a specialized pH-sensitive carrier-mediated mechanism. This mechanism appears to be regulated by extracellular vitamin B(6) levels and an intracellular Ca(2+)/calmodulin-mediated pathway.

Vitamin B6 and cancer: adenosine-N6-diethylthioether N1-pyridoximine 5'-PO4, a circulating human tumor marker.

Studies in my laboratory on the metabolism and utilization of labeled vitamin B6 by tumor-bearing animals and tumor cells in culture have shown the production and presence in the circulation of a novel pyridoxal 5'-phosphate conjugate metabolite (1-5). Using specific radiolabeling and analytical tests, its structure was tentatively identified (now confirmed) as adenosine-N6-diethylthioether-N1-pyridoximine 5'-phosphate (4-6). The novel vitamin conjugate was isolated from the plasma of patients and control subjects following extraction with perchloric acid and separation by pair-ion HPLC. This communication reports the chromatographic separation of the novel compound and its plasma levels in control subjects, cancer patients and patients with other ailments. The results show that the novel metabolite is a circulating human tumor marker with levels of up to 4x or greater seen in cancer patients compared to controls and to subjects with other ailments. The results indicate strongly that the novel circulating vitamin B6 conjugate compound can be successfully used for the detection of different malignancies in humans by evaluating its level in circulation.

Crisis convulsivas de difícil manejo secundarias a dependencia de piridoxina: presentación de un caso

Se presenta el caso de un lactante, con crísis clónicas inicialmente y luego tónicoclónicas que lo llevan finalmente a status epiléptico, secundarias a déficit de piridoxina. El caso debe alertar sobre la posibilidad de procesos infrecuentes, como el de este ejemplo, como causa de cuadros convulsivos de difícil manejo.

Vitamin B6 metabolism by human liver.

The B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) are primarily metabolized in liver to pyridoxal 5'-phosphate (PLP) and the deadend catabolite 4-pyridoxic acid. We have built on the elegant early work of Snell and others to describe the activities of the human liver enzymes responsible for vitamin B6 metabolism and to develop a model of the relative rates of these interconversions in vivo. This model is consistent with changes in plasma B6 after a load, the clearance of different vitamers (e.g., pyridoxine versus pyridoxal), and with the low plasma PLP in patients with cirrhosis. Because cirrhotics were found to be capable of PLP synthesis, we have used oral supplementation with pyridoxine to restore plasma PLP to the normal range, and have evaluated the effects of this intervention on amino acid metabolism. No significant differences were observed in plasma or urinary clearance of methionine (or cystathionine) after an oral load, nor in amino acid clearance from circulation after a protein load for cirrhotic patients before and after restoration of normal plasma PLP. Hence, the abnormal metabolism of vitamin B6 does not appear to be an important factor in the deranged amino acid metabolism in this disease. Nonetheless, this approach may be generally useful in assessing the importance of PLP in other abnormalities.

[Anticarcinogenic action of vitamins PP and B6 in the natulan initiation of malignant growth in mice]. / Antikantserogennoe deistvie vitaminov PP i B6 pri initsiatsii natulanom zlokachestvennogo rosta u myshei.

Parenteral administration of vitamins PP and B6 at the initiation stage of natulan-induced carcinogenesis was shown to significantly inhibit formation of lung adenomas. The preventive effect was found to depend on treatment schedule. Biochemical aspects of anticarcinogenic action of the vitamins require special investigation.