5-Fluoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fluorouracil.

5-Fluorouracil (5-FU) is an effective antitumor agent used in treating various cancers. Because of its metabolism by intestinal and other cells, 5-FU has an inconsistent bioavailability that limits its oral use. 5-Fluoro-2-pyrimidione (5-FP), a 5-FU prodrug, was synthesized and found to be converted to 5-FU by aldehyde oxidase, an enzyme present in high concentrations in the livers of mice and humans but not in the gastrointestinal tract. Using BDF1 mice, the pharmacokinetics of 5-FP were studied and compared with those of 5-FU. The bioavailability of 5-FP given orally was 100% at a dosage of 25 mg/kg and 78% at a dosage of 50 mg/kg. The half-lives of both doses of 5-FP were at least 2-fold longer than the half-lives of the same doses of 5-FU, and the clearance rates of 5-FP were 3-fold slower. 5-FP was converted rapidly to 5-FU, in vivo. The resulting 5-FU was measured at a steady-state level of 40-70 microM in plasma, at a dosage of 25 mg/kg, that was sustained for at least 4 hr. Also, when given orally, 5-FP was shown to have potent activity against Colon 38 tumor cells and P388 leukemia cells in mice. The therapeutic index of 5-FP was similar to that of 5-FU in these mouse tumor models. The potential clinical use of 5-FP as a prodrug of 5-FU should be considered.

Pharmacokinetics of intrapericardial administration of 5-fluorouracil.

UNLABELLED: A 30-year-old patient with metastatic breast adenocarcinoma was diagnosed as having a malignant pericardial effusion. METHODS: The patient was treated with two courses of 200 mg 5-fluorouracil (5-FU) followed by 20 mg cisplatin 5 h later directly infused into the pericardial space through a catheter. The drug levels of the 5-FU were monitored during the second treatment. The half-life of 5-FU in the pericardial space was 168.6 min with a concentration of 0.113 mg/ml still detected at 5 h. The area under the curve (AUC) was estimated to be 4.739 mg h/ml. The plasma concentrations of 5-FU ranged from 0.022 to 0.04 mg/ml throughout the infusion. RESULTS: There was no significant change in the patient's blood counts or chemistry profile. She did not experience any side effects during the treatment. A pericardial window was performed 2 days later when balloon pericardiectomy was unsuccessful. The patient eventually succumbed to her disease 4 months later, but without evidence of pericardial effusion. CONCLUSIONS: We conclude that pericardial infusion of 5-FU allowed a high concentration of 5-FU to be achieved within the pericardial sac with a greatly increased half-life over that of systemic 5-FU treatment (168 min vs 6-20 min), and with little systemic toxicity.

The role of allogeneic epidermis in murine graft rejection.

Skin equivalents containing allogeneic fibroblasts in a collagen matrix and overlaid with isologous epidermal cells have been successfully grafted to rodents. By contrast, skin equivalents containing isologous fibroblasts and allogeneic epidermal cells provoke a strong rejection response, characterized by the infiltration of mononuclear cells into the epidermis at 1 week and occlusion of the microvasculature and destruction of the epidermis by lymphocytes 2 weeks after grafting. Based on these findings, skin equivalents containing allogeneic fibroblasts could be used in the treatment of burn injuries, but the epidermis should be obtained from the patient.

Characterization of conditions for the activation of endogenous guinea pig retrovirus in cultured cells by 5-bromo-2'-deoxyuridine.

Human endogenous retroviral sequences recently have been shown to be associated with breast cancer and some leukemias. These retroviral sequences have similarities to an endogenous retrovirus expressed in guinea pigs. The conditions for activation of this guinea pig retrovirus (GPRV) in cultured guinea pig embryo (GPE) cells using 5-bromo-2'-deoxyuridine (BrdU) was investigated. These studies employed the reverse transcriptase activity (RT) assay and electron microscopy (EM), in conjunction with Northern blot analysis that utilized a 2.6 kb GPRV-specific cDNA probe. Contrary to published studies, dexamethasone at concentrations ranging from 10(-8) to 10(-5) M appeared to play a minimal role in enhancing the production of GPRV. Following a 6 hr incubation with BrdU, GPRV mRNA was present in cultured GPE cells. Extracellular virion release was also observed by EM 12 hr later, although RT activity was not detected. All three methods detected viral expression at 48 hr after the addition of the drug. Additionally, after 6 hr exposure to BrdU, detectable RT and mRNA levels were maintained through 44 days after the removal of BrdU in a stationary culture condition and through 31 days in cultures that were subcultured weekly in media not containing BrdU. Low levels of extracellular viruses were detected in these cultures by electron microscopy through 49 days. Therefore, after only a 6 hr exposure to BrdU was extracellular GPRV detected 12 hr after drug removal and virus production continued for up to 49 days. This study provides information about an animal endogenous retroviral system that may be used as a model for the study of human endogenous retroviruses.

Demonstration of cytomegalovirus and retrovirus pseudotypes in cultured guinea pig cells.

A mixed viral infection with a cytomegalovirus and a retrovirus in cultured guinea pig embryo (GPE) cells was investigated. The expression of an endogenous guinea pig retrovirus (GPRV) in cultured guinea pig cells was induced by a medium containing 5-bromo-2'-deoxyuridine and dexamethasone. When the induced GPE cells were superinfected with a guinea pig cytomegalovirus (GPCMV), pseudotype virions were observed. Morphological characterization of both viruses and their locations within infected cells was achieved by examination of thin sections of infected cells with transmission electron microscopy. Immunolabeling with colloidal gold particles, 5 or 15 nm in size, permitted the identification of each virus type using GPCMV- or GPRV-specific polyclonal antibodies and the detection of a population of GPCMV and GPRV particles which expressed antigens of both viruses on their envelopes. Enhanced reverse transcriptase activity of GPRV and reduced infectivity titers of GPCMV was noted in dually infected cultures. These data suggest that interaction between GPCMV and GPRV had occurred in dually infected GPE cells and that expression of GPRV was enhanced.

Re-examination of the McCoy cell line for confirmation of its mouse origin: karyotyping, electron microscopy and reverse transcriptase assay for endogenous retrovirus.

BACKGROUND: The McCoy cell line originally derived from human synovial fluid in 1955, has been later found useful for cultivation of Chlamydia trachomatis. This cell line has been subcultured and exchanged between laboratories for many years. In recent years, the McCoy cell line has been widely used in many clinical diagnostic laboratories and has been supplied through commercial companies for the isolation and identification of Chlamydia trachomatis from clinical specimens. OBJECTIVES: Since retrovirus-like particles have been observed in McCoy cells and the species of origin of the currently used cell line has not been adequately documented, further characterization of McCoy cell line obtained from commercial sources was carried out. STUDY DESIGN: This study includes karyotypes analysis using G-banding for the confirmation of species origin of McCoy cells, electron microscopy for examination of virus particles associated with the cells and biochemical assay for reverse transcriptase activity for detection of retrovirus. RESULTS: Our results showed by karyotype analysis that McCoy cells are of mouse origin. Electron microscopic examination revealed the presence of endogenous retrovirus type-A and type-C virions. Biochemical assays of culture supernatant fluids from McCoy cells detected reverse transcriptase activity which required Mg(2+) ions. CONCLUSIONS: The present study has confirmed that McCoy cells currently used by many laboratories are mouse cells, not the original McCoy cells derived from human cells. Laboratory workers should be aware of the presence of endogenous murine retrovirus in this cell line and appropriate precautions should be taken.

Sanctuary growth of human immunodeficiency virus in the presence of 3'-azido-3'-deoxythymidine.

Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.