Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels with evidence for circadian variation of enzyme activity and plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion.

The activity of dihydropyrimidine dehydrogenase (DPD) in peripheral blood mononuclear cells and plasma concentration of 5-fluorouracil (FUra) were simultaneously determined in cancer patients receiving FUra by protracted continuous infusion (300 mg/m2/day). Blood samples were drawn every 3 h over 24-h period and the resulting DPD and FUra values analyzed for circadian periodicity. In the seven patients studied, a circadian rhythm of DPD activity was observed (P less than 0.00001, Cosinor analysis) with the peak of activity at 1 a.m. (0.197 +/- 0.007 nmol/min/mg) and the trough at a 1 p.m. (0.113 +/- 0.007 nmol/min/mg). In addition, a circadian rhythm was observed for the plasma concentrations of FUra obtained over a 24-h period (P less than 0.00001, Cosinor analysis) with peak values (27.4 +/- 1.3 ng/ml) occurring at 11 a.m. and trough values (5.6 +/- 1.3 ng/ml) occurring at 11 p.m. The ratio of the maximum concentration of FUra to the minimum concentration observed was almost 5-fold. This study demonstrates a circadian variation of DPD activity in human peripheral blood mononuclear cells and a circadian variation of FUra plasma levels in patients receiving FUra by protracted continuous infusion. An inverse relationship between the circadian patterns of DPD activity and FUra plasma levels was also noted, suggesting that an association may exist between DPD activity and FUra plasma concentration. Further evidence of an association between DPD activity in peripheral blood mononuclear cells and plasma FUra concentration was demonstrated by a linear relationship between the two parameters in all patients (r = -0.627) and within individual patients (-0.978 less than r less than -0.742). With the recent advent of programmable pumps, information on the circadian pattern of FUra and/or DPD may be useful in planning continuous infusion schedules in order that optimal plasma drug concentration may be maintained over a 24-h cycle, thereby enhancing the therapeutic efficacy of FUra administered by continuous infusion.

Circadian variation of 5-fluorouracil catabolism in isolated perfused rat liver.

The catabolism of 5-fluorouracil (FUra) was measured in isolated perfused rat liver (IPRL) at various times of the day. IPRLs were prepared from rats sacrificed at 3-h intervals and the elimination rate of FUra and FUra catabolites (i.e., rate leaving the IPRL in the effluent perfusate) following infusion of [3H]FUra was analyzed for circadian periodicity. Animals were housed under standardized conditions of light and dark and divided into two groups of 24 animals each. The first group was housed under "normal" light conditions (lights on from 6:00 a.m. to 6:00 p.m.; off from 6:00 p.m. to 6:00 a.m.), while the second group was housed under "reverse" light conditions (lights on from 10:00 p.m. to 10:00 a.m.; off from 10:00 a.m. to 10:00 p.m.). A circadian rhythm was observed in the elimination rate of FUra and FUra catabolites by both groups (P less than 0.0001, Cosinor analysis). Under "normal" light conditions, peak and trough elimination rate of FUra was at 19 h after light onset (HALO; 183.8 +/- 3.4 nmol/min/g liver) and 7 HALO (123.8 +/- 3.4 nmol/min/g liver), respectively. There was a reciprocal relationship between the elimination rates of FUra and FUra catabolites with peak and trough values for FUra catabolites at 7 HALO (70.5 +/- 3.6 nmol/min/g liver) and 19 HALO (17.5 +/- 3.6 nmol/min/g liver), respectively. Animals housed under the "reverse" conditions of light and dark also exhibited a circadian pattern. Under the "reverse" conditions, the peak and trough elimination rate of FUra was at 18.5 HALO (170.0 +/- 1.7 nmol/min/g liver) and 6.5 HALO (130.0 +/- 1.7 nmol/min/g liver), respectively. The peak and trough elimination rate of FUra catabolites under these conditions occurred at 6.5 HALO (64.3 +/- 2.2 nmol/min/g liver) and 18.5 HALO (29.7 +/- 2.2 nmol/min/g liver), respectively. These results demonstrate that the elimination rate of FUra and FUra catabolites by IPRL varies over a 24-h period with a circadian rhythm in association with the light/dark cycle. Such a variation in the hepatic elimination rate of FUra in humans could result in a variation in the systemic level of drug during chemotherapy thus affecting the therapeutic efficacy of FUra. This study suggests that a circadian pattern in the hepatic catabolism of FUra needs to be considered when planning chemotherapeutic regimens with FUra.

Arginase activity in prostatic tissue of patients with benign prostatic hyperplasia and prostatic carcinoma.

We studied arginase activity in human prostatic tissue in 15 patients with benign hyperplasia and 27 patients with prostatic carcinoma. Arginase specific activity is greater (p less than 0.0001) in prostatic carcinomas than in hyperplastic prostates. Arginase specific activity is correlated inversely (p less than 0.0001) with the histological grade of the tumor.

Enzyme activities in prostatic carcinoma related to Gleason grades.

In an effort to identify enzymatic activities in primary prostatic carcinomas that might be complementary to histological and other clinical techniques for the prediction of prognosis, we have assayed several enzymatic activities extracted from prostatic carcinomas. We reported previously that, for each of the studied enzymes, tissues with benign prostatic hyperplasia and prostatic carcinoma showed significantly different activities. With additional patients now included and a longer interval since resection of these tumors, we have found that the histological grade (Gleason's system for grading) of the sample (prostate chip) analyzed is related to several activities extracted from the cancers including arginase (r = -0.81, p less than 0.0001), glucose-6-phosphate dehydrogenase (r = 0.72, p less than 0.0001), and the B isoenzyme of N-acetyl-beta-D-glucosaminidase (r = -0.58, p = 0.0369). Acid phosphatase (r = 0.15, p = 0.5530) and the BB isoenzyme of creatine kinase (r = -0.13, p = 0.5221) are not significantly related to histological grade. In a large series, Gleason grade is related to survival. In our series of 27 patients followed for 20 to 46 months, the Gleason grade (p = 0.22) is not related to survival, but arginase activity is related (p = 0.0392) to survival. In this small series, arginase is more valuable than the best currently proven predictor of survival, Gleason grade. Hexosaminidase B activity approaches being significantly (p = 0.0575) related to survival. Of the 11 patients whose tumors contained the lowest arginase activities, eight are dead. Of the 11 with the highest activities, only one is dead. Several of the enzyme activities in this series of 27 patients complemented each other for the prediction of Gleason grade; for example, glucose-6-phosphate dehydrogenase, arginase, and the BB isoenzyme of creatine kinase were more closely correlated (multiple correlation coefficient, r = 0.77) with the Gleason grade for all chips than was any single enzyme: arginase, r = -0.67; glucose-6-phosphate dehydrogenase, r = 0.67; creatine kinase, r = -0.16. It seems likely that enzymatic analysis may provide an approach that is qualitatively different from and complementary to histological evaluation for the prediction of prognosis in prostatic carcinoma. Verification of this hypothesis will require more patients followed over a longer period of time and will probably be facilitated by analysis of several samples from different locations in each tumor.

Metabolism of pyrimidine analogues and their nucleosides.

The pyrimidine antimetabolite drugs consist of base and nucleoside analogues of the naturally occurring pyrimidines uracil, thymine and cytosine. As is typical of antimetabolites, these drugs have a strong structural similarity to endogenous nucleic acid precursors. The structural differences are usually substitutions at one of the carbons in the pyrimidine ring itself or substitutions at on of the hydrogens attached to the ring of the pyrimidine or sugar (ribose or deoxyribose). Despite the differences noted above, these analogues, can still be taken up into cells and then metabolized via anabolic or catabolic pathways used by endogenous pyrimidines. Cytotoxicity results when the antimetabolite either is incorporated in place of the naturally occurring pyrimidine metabolite into a key molecule (such as RNA or DNA) or competes with the naturally occurring pyrimidine metabolite for a critical enzyme. There are four pyrimidine antimetabolites that are currently used extensively in clinical oncology. These include the fluoropyrimidines fluorouracil and fluorodeoxyuridine, and the cytosine analogues, cytosine arabinoside and azacytidine.

Clinical pharmacology of 5-fluorouracil.

5-Fluorouracil, first introduced as a rationally synthesized anticancer agent 30 years ago, continues to be widely used in the management of several common malignancies including cancer of the colon, breast and skin. This drug, an analogue of the naturally occurring pyrimidine uracil, is metabolised via the same metabolic pathways as uracil. Although several potential sites of antitumour activity have been identified, the precise mechanism of action and the extent to which each of these sites contributes to tumour or host cell toxicity remains unclear. Several assay methods are available to quantify 5-fluorouracil in serum, plasma and other biological fluids. Unfortunately, there is no evidence that plasma drug concentrations can predict antitumour effect or host cell toxicity. The recent development of clinically useful pharmacodynamic assays provides an attractive alternative to plasma drug concentrations, since these assays allow the detection of active metabolites of 5-fluorouracil in biopsied tumour or normal tissue. 5-Fluorouracil is poorly absorbed after oral administration, with erratic bioavailability. The parenteral preparation is the major dosage form, used intravenously (bolus or continuous infusion). Recently, studies have demonstrated the pharmacokinetic rationale and clinical feasibility of hepatic arterial infusion and intraperitoneal administration of 5-fluorouracil. In addition, 5-fluorouracil continues to be used in topical preparations for the treatment of malignant skin cancers. Following parenteral administration of 5-fluorouracil, there is rapid distribution of the drug and rapid elimination with an apparent terminal half-life of approximately 8 to 20 minutes. The rapid elimination is primarily due to swift catabolism of the liver. As with all drugs, caution should be used in administering 5-fluorouracil in various pathophysiological states. In general, however, there are no set recommendations for dose adjustment in the presence of renal or hepatic dysfunction. Drug interactions continue to be described with other antineoplastic drugs, as well as with other classes of agents.

Circadian rhythm of rat liver dihydropyrimidine dehydrogenase. Possible relevance to fluoropyrimidine chemotherapy.

The activity of dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting enzyme in pyrimidine catabolism, was measured at various times over a 24-hr period in the livers of rats housed under standardized conditions of light and dark. Under "normal" conditions, i.e. lights on from 6:00 a.m. to 6:00 p.m. and off from 6:00 p.m. to 6:00 a.m., a circadian rhythm of DPD activity was observed (P less than 0.0001, Cosinor analysis) with the peak of activity at 4:00 p.m. (2.96 nmol catabolites/min/mg) and the trough at 4:00 a.m. (0.40 nmol catabolites/min/mg). Maximum enzyme activity exceeded minimum activity by more than 7-fold. Reversing the light-dark cycle (i.e. lights on from 6:00 p.m. to 6:00 a.m. and off from 6:00 a.m. to 6:00 p.m.) resulted in a corresponding shift in enzyme activity. Under these "reverse" conditions, a circadian rhythm was observed (P less than 0.0001, Cosinor analysis) with the peak of activity at 6:00 a.m. (2.87 nmol catabolites/min/mg) and the trough at 6:00 p.m. (0.92 nmol catabolites/min/mg). These studies demonstrated that DPD activity in rat liver varies over a 24-hr period in association with the light-dark cycle.

Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency. A potentially more common pharmacogenetic syndrome.

This study describes the inheritance of a defect in pyrimidine catabolism and its association with drug-induced toxicity in a patient receiving 5-fluorouracil (FUra) as adjuvant chemotherapy for breast carcinoma. The study population included the affected patient (proband), nine of her blood relatives, and seven healthy volunteers. The activity of dihydropyrimidine dehydrogenase (DPD), the initial enzyme of pyrimidine (and FUra) catabolism, in peripheral blood mononuclear cells was measured in each subject by a specific radiometric assay using FUra as the substrate. The proband had no detectable DPD activity. When enzyme levels in the proband and relatives were compared with that in controls, an autosomal recessive pattern of inheritance was demonstrated. This is the third patient with severe FUra toxicity secondary to an alteration in pyrimidine catabolism and the second from our clinic population suggesting that the frequency of this genetic defect may be greater than previously thought. Monitoring DPD activity may be important in the management of patients experiencing severe toxicity secondary to FUra chemotherapy.

Pyruvic acid as an etching agent in clinical dentistry.

The objective of this study was to evaluate the use of pyruvic acid as an alternative etching agent to phosphoric acid (H3PO4). Solutions containing 5, 10, 15, 20, 25, and 30 m/m % pyruvic acid and 50% m/m H3PO4 were prepared. The tensile bond strengths of a composite resin to enamel surfaces etched with the respective etching agents were determined. The rates of etching of enamel surfaces by each of the etching solutions were evaluated. Unground and polished enamel surfaces were etched with the respective etching solutions and the surfaces examined by scanning electron microscopy. The tensile bond strengths of the resin to enamel surfaces etched with 10-30% pyruvic acid exceeded those obtained on enamel surfaces etched with 50% H3PO4. The rates of etching of all the pyruvic acid solutions were significantly less than of H3PO4. Well-defined etching patterns were observed on the enamel surfaces etched with all the etching solutions. The results of this laboratory study suggest that pyruvic acid may be a suitable alternative to phosphoric acid as an etching agent in clinical dentistry.

Conversion of 5-fluorocytosine to 5-fluorouracil by human intestinal microflora.

The mechanism of toxicity from 5-fluorocytosine chemotherapy is unclear. However, recent evidence suggests that the generation of 5-fluorouracil by a host may play an important role in the development of this toxicity. Using an in vitro semicontinuous culture system to mimic the intestinal microflora, we examined the capacity of this complex microbial community to convert 5-fluorocytosine to 5-fluorouracil. The system was dosed initially and after 2 weeks of chronic exposure to 5-fluorocytosine with radiolabeled 5-fluorocytosine. No detectable production of 5-fluorouracil was observed up to 8 h after the acute dose; however, at 24 h and at all time points thereafter, increasing levels of 5-fluorouracil were detected for 4 days. The chronic dose resulted in an increased rate of 5-fluorouracil production without the 8-h lag time. These findings suggest that the enzyme or enzymes responsible for the deamination of 5-fluorocytosine to 5-fluorouracil by the intestinal microflora can be induced by chronic exposure to 5-fluorocytosine and that this conversion may provide a mechanism through which 5-fluorocytosine toxicity is manifested.