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Objective. To evaluate the influence of food on the bioavailability of an oral dose of methotrexate in patients with rheumatoid arthritis.Methods. Methotrexate (15 mg) was given intravenously and orally, with or without a meal, to 10 patients. Blood samples were drawn at specific intervals to evaluate drug levels.Results. Food reduced the peak concentration, from a mean of 0.71 pmoleshiter to 0.49 pmoles/liter (P < 0.02), and slightly increased the time to peak concentration, from a mean of 1.3 hours to 2.0 hours. Bioavailability was highly variable (range 28-9470) but was not affected by food intake. Conclusion. Bioavailability of oral methotrexate shows marked interindividual variation but is not affected by the presence of food.Methotrexate, a potent inhibitor of dihydrofolate reductase, was introduced into human pharmacology as an antineoplastic agent. It is increasingly being employed at low doses (5-25 mg weekly) as a secondline drug for rheumatoid arthritis that has been refractory to corticosteroid therapy (1) the high doses typically given to combat malignant diseases (2). Relatively little is known about the pharmacokinetics of low doses of this agent (3-5). Oral doses of less than 30 mg/m2 of body surface have been shown to be well absorbed (2), but milk is known to affect the methotrexate absorption in children with leukemia (6).Only one published study has addressed the bioavailability of methotrexate in patients with rheumatoid arthritis (4). In that study, the bioavailability of an oral dose of methotrexate was highly variable, ranging from 4% to 191%. Whether food affects the bioavailability of low-dose methotrexate is unknown. Although oral administration of methotrexate could be desirable for patients with rheumatoid arthritis, therapeutic success depends on adequate absorption. We therefore studied the bioavailability of methotrexate, and whether it was influenced by food, in patients with rheumatoid arthritis. PATIENTS AND METHODSPatient population. The bioavailability of methotrexate was studied in 10 patients (5 men and 5 women) with classic rheumatoid arthritis (7). The patients had a mean 2 SD body weight of 69 1-11 kg (range 56-90) and were 58 5 8 (mean 2 SD) years old (range 45-66). Eight patients were taking nonsteroidal antiinflammatory agents, and 5 were taking prednisone. All medications were withheld the day of the study. No other concomitant drugs, in particular, no drugs known t o induce or inhibit the microsomal oxygenase system, were taken.All patients had normal renal and hepatic function, as determined by assessing plasma levels of creatinine, liver function tests, and galactose elimination capacity. Four patients had been taking methotrexate for more than 1 year and 4 for less than 2 weeks. The study was approved by the

Chronic infection with hepatitis B virus (HBV) is associated with the development of human hepatocellular carcinoma (HCC). One of the HBV genes, HBx, may have transforming potential, but this issue is still the subject of controversy. One of the major difficulties in addressing this question is the lack of a suitable in vitro model. We used a nontransformed, differentiated murine hepatocyte cell line (AML12) to transfect the HBx gene and examine its transforming capabilities. Because mutations of the p53 gene, in particular at codon 249, have been implicated in HCC development in geographical areas with high incidence of the tumor, we also studied the putative cooperative role in transformation between HBx and mutated p53 by cotransfecting HBx with a murine p53 mutant equivalent to human ser249 (ser246p53). Transfection with HBx plasmids containing the HBx gene under the control of two different promoters resulted in fewer colonies than in control plasmids. The toxic effect of HBx on colony formation was abolished by cotransfection with 246p53, suggesting that the inhibitory effect requires functionally intact p53. Clonal cell lines that stably expressed HBx messenger RNA (mRNA) (HBX lines) were tested for their growth characteristics and their ability to grow in soft agar and form tumors in nude mice. At passages 19-27 after transfection, one of four HBx-expressing lines showed the capacity for anchorage-independent growth in soft agar and produced poorly differentiated hepatocellular carcinomas in 8 of 13 sites of injection in nude mice. HBX lines as well as clonal cell lines of cells transfected with 246p53 (246 cell lines), cotransfected with HBx and 246p53 (246x lines) or transfected with control plasmids, were analyzed by flow cytometry to determine the fraction of cells in S phase (SPF). 246 and 246X lines had similar SPFs that were approximately twofold greater than control or HBX lines. 246x lines showed morphological changes in culture such as marked cellular heterogeneity, cell crowding, and the presence of multinucleated giant cells, but their tumorigenicity was not increased compared with the HBX lines. These data show that HBx has a weak tumorigenicity in murine hepatocytes and that the addition of mutation of p53 at codon 249 to HBx expression does not increase tumorigenicity in AML12 cells.

The p53 gene is frequently mutated in human tumors; in hepatocellular carcinomas, there is a high frequency of a specific mutation at codon 249 in regions with significant aflatoxin exposure. To assess the role of this p53 mutation in the development of hepatocellular carcinoma, a mutant murine p53 gene, p53ser246, which corresponds to human codon 249, was transfected into a differentiated, nontransformed hepatocyte cell line AML12. Expression of p53ser246 in this line resulted in a growth advantage when compared with either a control vector (which contains a large p53 deletion) or with a different p53 mutant, val135, not found in hepatocellular carcinoma. Overall, there was a threefold increase in colony formation after transfection with p53ser246 as compared with the control or p53val135 vectors, and the p53ser246 plates developed consistently larger colonies. Whereas clones expressing the control or p53val135 constructs showed no significant morphological changes, clones expressing p53ser246 showed increased heterogeneity (large multinucleated cells and areas of small crowded cells) without focus formation. In addition, the ser246 mutation imparted a growth advantage in serumfree media, suggesting less dependence on specific factors present in serum. None of the mutant p53 or control lines were capable of growth in soft agar or tumor formation in nude mice. Thus in this model, in which endogenous wild-type p53 expression is retained, a high level of mutant p53 expression is not sufficient to transform hepatocytes. Our findings indicate that p53ser246 has effects on hepatocytes that may result in a clonal growth advantage and suggest that additional factors are required for the development of hepatocellular carcinoma.