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Objective: To investigate the therapeutic effect and metabolism of 5-fluorocytosine (5-FC) in human tongue squamous carcinoma cells after treatment with adenovirus-mediated cytosine deaminase (AdCMVCD)/5-FC system., Methods: Human tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5-FC system. The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay. Tumor inhibition effect and histopathological changes were observed in vivo. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of 5-FC in vitro and in vivo., Results: AdCMVCD/5-FC system had strong killing effect and bystander effect on Tca8113 cells. Both condition media and cell extracts showed two peaks identified as 5-FC and 5-fluorouracil (5-FU) by HPLC and a time-dependent generation of 5-FU and concomitant time-dependent decreases of 5-FC. Compared to the control groups, mice treated with AdCMVCD/5-FC system demonstrated significant tumor regression (P < 0.001); the tumor doubling time prolonged and inhibition rate was 92.62%. There were substantial tumor necrotic areas and infiltrative lymphocytes around necrotic areas in the AdCMVCD/5-FC treated group under light microscope. There was a significantly low concentration of 5-FC and high concentration of 5-FU in tumor tissue, but only 5-FC was found in blood., Conclusion: AdCMVCD/5-FC suicide gene system had significant in vitro and in vivo anti-tumor effect on human tongue squamous cell carcinoma due to convert 5-FC into 5-FU.

Cryptococcus neoformans is the cause of the most common life-threatening fungal infection in patients with AIDS. Thirty strains of C. neoformans were collected from inpatients and typied evaluating activity, morphotyping, serotyping, chemosensitivity and adhesivity. Cryptococcus neoformans strains showed different aspectotype profile, the sole presence of serotypes A and D, good susceptibility to azoles and Amphotericin B. Phenotypic epidemiologic markers can be used: characterization of clinical strains excludes a common source.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium expressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were detected in tumors in mice treated with both TAPET-CD and 5-FC. No 5-FU could be detected in normal tissues. Inhibition of tumor growth was observed in mice treated with either TAPET-CD alone or TAPET-CD in combination with 5-FC (TAPET-CD/5-FC), but not with 5-FC alone. TAPET-CD/5-FC inhibited tumor growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given.

Enzymatic DNA methylation of carbon 5 of cytosines is an epigenetic modification that plays a role in regulating gene expression, differentiation, and tumorigenesis. DNA (cytosine-C5)-methyltransferase-1 is the enzyme responsible for maintaining established methylation patterns during replication in mammalian cells. It is composed of a large ( approximately 1100 amino acids (a.a.)) amino-terminal region containing many putative regulatory domains and a smaller ( approximately 500 a.a.) carboxy-terminal region containing conserved, catalytic domains. In this study, murine DNA (cytosine C5)-methyltransferase-1, fused to an amino-terminal hexahistidine tag, was expressed by infecting Spodoptera frugiperda cells for 46 h with a recombinant baculovirus carrying the DNA (cytosine-C5)-methyltransferase-1 cDNA. A total of 3 x 10(8) infected S. frugiperda cells yielded approximately 1 mg of full-length, hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1, which was purified approximately 450-fold from RNase-treated S. frugiperda cell extracts by nickel affinity chromatography. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 through DNA methylation and inhibitor-binding assays indicated that the purified enzyme had at least a 30-fold higher catalytic efficiency with hemimethylated double-stranded oligodeoxyribonucleotide substrates than unmethylated substrates and was most active with small oligodeoxyribonucleotide substrates with a capacity for forming stem-loop structures. The expression and purification procedures reported here differ significantly from the original reports of baculovirus-mediated hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 expression and purification by nickel affinity chromatography and provide a consistent yield of active enzyme., (Copyright 2002 Elsevier Science (USA).)

Objective: To elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo., Method: K562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination., Results: At the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group., Conclusion: YCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population., (©2002 Elsevier Science (USA).)

The aim of this study is to investigate whether fluorouracil (5-FU) could be responsible for bone-marrow depression occurring in fluorocytosine (5-FC) treated patients. Six 5-FC treated patients were included in this pilot study. Toxicity was monitored by means of thrombocyte and leucocyte counts. 5-FC and 5-FU serum levels were measured using a high-performance liquid chromatography (HPLC) assay that allows simultaneous determination of both compounds. The amounts of 5-FU in the 34 available serum samples remained below the limit of quantitation (< 0.05 mg/L), whereas 5-FC levels could be detected in all samples. Instead, low levels of the 5-FU catabolite alpha-fluoro-beta-alanine (FBAL) were detected in several of the investigated serum samples. In case of three patients thrombocyte counts remained within the normal range during 5-FC treatment, whereas one patient developed thrombocytopenia (50 x 10(9) thrombocytes/L) during therapy. Furthermore, one patient developed leucocytopenia (2.6 x 10(9) leucocytes/L) during 5-FC therapy, whereas the remaining five patients were suffering from leucocytosis prior to 5-FC therapy. In conclusion, we found nondetectable 5-FU serum concentrations (< 0.05 mg/L) in ICU patients treated with intravenous 5-FC, making it unlikely that 5-FC-associated toxicity results from 5-FU exposure in patients receiving intravenous 5-FC therapy. These findings may be explained by the fact that our patients received 5-FC intravenously instead of orally, therefore not allowing active conversion of 5-FC to 5-FU by the human intestinal microflora.

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent., (Copyright 2002 Academic Press.)

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a approximately 100-fold and approximately 10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.

Infection of tumor cells by herpes simplex virus 1 (HSV-1) results in cell destruction and production of progeny virion in a process referred to as viral oncolysis. In this study, an HSV-1 mutant (HSV1yCD) was engineered such that the viral ribonucleotide reductase gene is disrupted by sequences encoding yeast cytosine deaminase, which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). HSV1yCD-infected cells convert 5-FC to 5-FU, which enhances cytotoxicity without significantly reducing viral replication and oncolysis. Oncolysis by a replicating HSV-1 mutant combined with therapeutic transgene delivery represents a new paradigm; HSV1yCD-infected cells are destroyed by viral replication, and uninfected cells are subjected to bystander killing from both progeny virion and extracellular diffusion of 5-FU. In contrast, HSV1yCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication. HSV1yCD administered into the portal venous system replicates preferentially in liver metastases rather than normal liver. The anti-neoplastic activity of HSV1yCD combined with systemic 5-FC administration is greater than that achieved with HSV-1 replication alone. Combination oncolysis and prodrug bioactivation leads to significant prolongation of survival in mice with diffuse liver metastases.

Colorectal cancer is a leading cause of cancer mortality in Western countries. Gene therapy has been proposed as a potential novel treatment modality for colorectal cancer, but it is still in an early stage of development. The preclinical data have been promising and numerous clinical trials are underway. This brief review aims to summarise the current status of clinical trials of different gene therapy strategies, including immune stimulation, mutant gene correction, prodrug activation and oncolytic virus therapy, for patients with colorectal cancer. Data from phase I trials have proven the safety of the reagents but have not yet demonstrated significant therapeutic benefit. In order to achieve this and extend the scope of the treatment, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery and accuracy of gene targeting.

Replicating viruses for cancer gene therapy have beneficial antitumor effects, however, in the setting of an enzyme/prodrug system, the interactions between these viruses and the activated agents are complex. A replicating vaccinia virus expressing the cytosine deaminase gene (VVCD), which converts the prodrug 5-FC into 5-FU, was characterized in vitro and in vivo for its antitumor effects and pathogenicity. Replicating VVCD (+/-5-FC) at various MOIs was used to infect MC38 murine colon adenocarcinoma cells. At high MOIs (>0.1) virus alone was able to kill the majority (65-90%) of cells by day 5 with no additional benefit from prodrug. At low MOIs only the effect of prodrug is seen. Cell lysates demonstrated 300-fold reduced viral recovery from cells treated with both VVCD and 5-FC compared with controls treated with virus alone. Nude mice bearing subcutaneous MC38 tumors were injected with VVCD (or control) and treated with 5FC or control. Mice injected with VVCD (with or without 5FC treatment) had smaller tumors than the controls, suggesting that replicating vaccinia alone is cytotoxic to tumors in vivo. The addition of 5-FC improved the antitumor response when a low dose of virus was injected into tumors. Also, compared with mice that received virus alone, those that received VVCD and 5FC had significantly prolonged survival from virus-mediated death. In conclusion, the addition of an enzyme/prodrug system to a replicating virus can improve the antitumor response and decrease viral pathogenicity. Gene Therapy (2000) 7, 1217-1223.

We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Purpose: To estimate the concentration of 5-fluorocytosine (5-FC), necessary for conversion to 5-fluorouracil (5-FU) in tumours transduced with the gene cytosine deaminase (CD), to achieve clinically significant radiosensitization to radiotherapy., Materials and Methods: Starting with a tumour control probability (TCP) of 37% from radiotherapy of 66 Gy in 2 Gy fractions, estimates were made of increase in TCP expected from sensitizer enhancement ratios (SER) of 1.1, 1.2, etc. SER values for 5-FU were obtained from a literature review. Clinical toxicity of 5-FC is also reviewed., Results: 5-FU has been reported to be an effective radiosensitizer if maintained for several days after each irradiation at concentrations of 0.6-0.9 microg/ml in surrounding medium. 5-FC is well tolerated by patients at concentrations of 25-100 microg/ml (average 60 microg/ml) for 6 weeks in standard antifungal treatment. Sufficient 5-FU should be available if conversion efficiency from 5-FC is 1-3%. SER values of 1.1 to 1.2 should be achievable with daily 2 Gy fractions. In vitro and xenograft experiments are reviewed and they do not contradict the conclusions., Conclusions: Increases in tumour control of 20 to 40% can be expected, which should be detectable in a 2-arm randomized trial of 260 (for 20%) or 60 (for 40%) patients.

Gene therapy is a novel therapeutic approach that might soon improve the prognosis of some cancers. We investigated the feasibility of cytosine deaminase (CD) suicide gene therapy in a model of peritoneal carcinomatosis. DHD/K12 colorectal adenocarcinoma cells transfected in vitro with the CD gene were highly sensitive to 5-fluorocytosine (5-FC), and a bystander effect could also be observed. Treating CD+ cells with 5-FC resulted in apoptosis as detected by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling. In vitro, several human cell lines derived from ovarian or colorectal carcinomas, as well as the rat glioblastoma 9 L cell line, responded to CD/5-FC and showed a very strong bystander effect. 5-FC treatment of peritoneal carcinomatosis generated in syngeneic BDIX rats by CD-expressing DHD/K12 cells led to a complete and prolonged response and to prolonged survival. Our study thus demonstrated the efficacy of CD suicide gene therapy for the treatment of peritoneal carcinomatosis.

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.

Background: An approach to improve current chemotherapy is the selective transduction of tumor cells with suicide genes to sensitize these cells to prodrugs of cytostatic agents., Methods: In this study, gene transfer was accomplished with the cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), able to condense plasmid-DNA by electrostatic interaction. OVCAR-3 cells were transfected with plasmids encoding E. coli-derived or human beta-glucuronidase and the transfection efficiency and inhibition by serum was determined. Next, we measured the sensitivity of OVCAR-3 cells transiently expressing beta-glucuronidase to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin., Results: OVCAR-3 cells were efficiently transfected with a plasmid encoding E. coli-derived beta-glucuronidase. The degree of transfection (30% of cells) was higher than that achieved with commercially available cationic lipids (DOTAP, Lipofectamine) without inhibition by serum. OVCAR-3 cells transiently expressing beta-glucuronidase were equally sensitive to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin itself, indicating complete conversion of prodrug to drug. Similar studies were performed with the plasmid encoding for human beta-glucuronidase, which is likely to be less immunogenic. Also in this case, OVCAR-3 cells showed an increased sensitivity to the prodrug DOX-GA3, although less pronounced than when the bacterial enzyme was used. A strong bystander effect was observed when OVCAR-3 cells transfected with beta-glucuronidase were mixed with non-transfected cells at different ratios. Complete tumor cell growth inhibition was already observed when only 15% of the cells expressed the activating enzyme., Conclusion: These studies suggest that beta-glucuronidase gene therapy using PDMAEMA as a carrier system and DOX-GA3 as the prodrug has a potential application in cancer gene therapy.

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2)(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3)(app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K(m). These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.

An adenovirus, AdCDTK, expressing both bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSVTK) was constructed and introduced into glioma cells. AdCDTK selectively rendered glioma cells sensitive to both 5-fluorocytosine (5-FCyt) and ganciclovir (GCV) (termed AdCDTK/5-FCyt-GCV). AdCDTK/5-FCyt-GCV not only potently mediated apoptosis and the arrest of glioma cell growth in vitro, but also significantly increased the survival time of glioma-bearing rats as compared with controls. The 90-day survival time was observed in 50% of rats. Interferon-alpha (IFN-alpha) further enhanced the tumor cell killing of AdCDTK/5-FCyt-GCV. In the group of AdCDTK/5-FCyt-GCV/IFN-alpha, the average survival time was significantly increased, and the average tumor size was smaller than that in the group of AdCDTK/5-FCyt-GCV. Ninety-day survival increased from 50% in the group of AdCDTK/5-FCyt-GCV to 75% in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. Complete tumor regression was observed in 50% of rats in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. The data indicate that AdCDTK/5-FCyt-GCV induces glioma cell killing greater than that induced by either CD/5-FCyt or HSVTK/GCV alone. IFN-alpha synergistically enhances this effect by increasing apoptosis.

Peripheral blood progenitor harvests of breast cancer patients are contaminated with tumor cells, suggesting a potential role for these cells in the relapse after high-dose chemotherapy. Whereas physical purging methods do not eliminate contaminating tumor cells completely, pharmacological purging, although highly efficient, is hampered by a strong nonspecific toxicity toward hematopoietic progenitor cells. Taking advantage of the high efficiency of adenovirus-mediated gene transfer to epithelial cells, we selectively loaded breast cancer cells in vitro with a cytotoxic drug by gene transfer of the prodrug-converting enzyme cytosine deaminase (AdCMV.CD) and 5-fluorocytosine (5-FC). Despite the low dose of vector administered, limited exposure to 5-FC, and transplantation only of viable tumor cells into SCID mice, all animals that received cells treated in vitro with AdCMV.CD plus 5-FC were completely free of tumor development. These data show that the selective loading of tumor cells with AdCMV.CD/5-FC might be useful for purging of autografts.

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.

The monitoring of antibody-directed enzyme-prodrug therapies requires evaluation of drug activation within the tissues of interest. We have demonstrated the feasibility of noninvasive magnetic resonance spectroscopy and spectroscopic imaging (chemical shift imaging) to detect activation of the prodrug 5-fluorocytosine (5-FCyt) to the cytotoxic species 5-fluorouracil (5-FU) by monoclonal antibody-cytosine deaminase (CD) conjugates. In vitro, L6-CD but not 1F5-CD selectively metabolized 5-FCyt to 5-FU on H2981 human lung adenocarcinoma cells because of the presence and absence of cell surface L6 and CD20 antigens, respectively. After pretreatment of H2981 tumor-bearing mice with L6-CD, in vivo metabolism of 5-FCyt to 5-FU within the tumors was detected by 19F magnetic resonance spectroscopy; the chemical shift separation between 5-FCyt and 5-FU resonances was approximately 1.2 ppm. 5-FU levels were 50-100% of 5-FCyt levels in tumors 10-60 min after 5-FCyt administration. Whole body 19F chemical shift imaging (6 x 6 mm in-plane resolution) of tumor-bearing mice demonstrated the highest signal intensity of 5-FU within the tumor region. This study supports further development of noninvasive magnetic resonance methods for preclinical and clinical monitoring of CD enzyme-prodrug therapies.

Because it appears impossible to transfer toxic genes to all the cells of a cancer, the bystander effect is critical to induce effective antitumor effects. In the present study, possible in vitro mechanisms of the bystander effect by the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) were investigated. CD-transduced cancer cells exhibited much higher sensitivity to 5-FC compared to parental cells. CD-transduced cells caused killing of neighboring parental cells in the presence of 5-FC, irrespective of direct cell-to-cell contact. Media conditioned by CD-transduced cells and 5-FC contained considerable amounts of 5-fluorouracil (5-FU) and exhibited profound cytotoxicity on parental cells. Furthermore, this killing ability of conditioned media correlated well with 5-FU levels converted from 5-FC by CD-transduced cells. CD was shown not to be secreted into media from cells. These results indicate that diffusible 5-FU plays the substantially causative role in the in vitro bystander effect caused by the CD/5-FC system.

Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were shown to be sensitive to infection by adenoviral vectors when exposed to a recombinant adenoviral vector containing the reporter gene betagalactosidase (Ad.CMV-betagal). In contrast, less than 1% of the CD34-selected cells and their more immature subsets, such as the CD34+CD38- or CD34(+)CD33- subpopulations, were positive for infection by the Ad.CMV-betagal vector, as judged by fluorescence-activated cell sorting (FACS) analysis, when exposed to the adenoviral vector under conditions that did not commit the early hematopoietic precursor cells to maturation. When artificial mixtures of hematopoietic cells and BCCs were exposed for 90 minutes to the Ad.CMV-CD vector and to 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by colony-limiting dilution assays, was observed. To test if the conditions were damaging for the hematopoietic reconstituting cells, marrow cells collected from 5-FU-treated male donor mice were incubated with the cytosine deaminase adenoviral vector and then exposed to 5-FC either for 4 days in vitro before transplantation or for 14 days immediately after transplantation in vivo. There was no significant decrease in the reconstituting capability of the male marrow cells, as measured by their persistence in female irradiated recipients for up to 6 months after transplantation. These observations suggest that adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene followed by exposure to the nontoxic pro-drug 5-FC may be a potential strategy to selectively reduce the level of contaminating BCCs in collections of hematopoietic cells used for autografts in breast cancer patients.

In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC. However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells. The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy. However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.

Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic producing 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes thc gene for Beta-galactosidase. To evaluate cytosine deaminase-mediated conversion of 5-fluorocytosine to 5-fluorouracil and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes cytosine deaminase, and exposed to 5-fluorocytosine for 6 to 8 days. Additive cytotoxicity of radiation therapy was evaluated by cobalt-60 exposure following AdCMVCD infection and 5-fluorocytosine treatment. SK-ChA-1 cells were transduced (98.4%) by AdCMVLacZ at 100 plaque-forming units per cell. Following infection with AdCMVCD and exposure to 5 to 100 microgram/ml of 5-fluorocytosine, 20% to 64% of SK-ChA-1 cells were killed. A combination of radiation and cytosine deaminase/5-fluorocytosine therapy resulted in enhanced cell killing (83.5% to 91.5%). Cholangiocarcinoma cells were transduced by recombinant adenoviral vectors and were killed by cytosine deaminase-mediated production of 5-fluorouracil. Enhanced cytotoxicity was seen with the addition of external beam radiation. These results provide a foundation for multimodality therapy for human cholangiocarcinoma that combines gene therapy technology with radiation therapy.

Background & Aims: An increase of carcinoembryonic antigen (CEA) expression is noted in about 40% of patients with gastric cancer. Adenovirus-mediated gene therapy using the CEA promoter was investigated as a way to specifically target human CEA-producing gastric tumors., Methods: Recombinant adenovirus vectors carrying a CEA promoter linked to the lacZ gene (AdCEA lacZ) or the cytosine deaminase gene (AdCEA-CD) were constructed. After infection with these vectors, CEA-producing (MKN45 and MKN28) and non-CEA-producing (MKN1) gastric cancer cells were analyzed for transgene expression and sensitivity to 5-fluorocytosine., Results: The lacZ gene was expressed selectively in CEA-producing AdCEA-lacZ-infected cells in vitro and in vivo. Transduction of the vector containing the CEA-regulated cytosine deaminase gene (AdCEA-CD) resulted in extraordinary sensitivity of MKN45 and MKN28 cells to 5-fluorocytosine. This effect was not observed in MKN1 cells. Moreover, AdCEA-CD-infected MKN45 cells showed a profound in vitro neighbor cell killing effect in the presence of 5-fluorocytosine. This effect was attributed to the diffusion of 5-fluorouracil, resulting from conversion of 5-fluorocytosine to 5-fluorouracil by the cytosine deaminase-expressing cells., Conclusions: The results of this study suggest that use of a CEA promoter in an adenovirus vector could confer selective expression of the cytosine deaminase gene in CEA-producing gastric cancer cells, rendering the transduced cells susceptible to 5 fluorocytosine. This system may be useful in gene therapy that targets CEA-producing gastric carcinomas.

To target expression of toxic genes to Epstein-Barr virus (EBV)-associated tumor cells, we have developed an EBV-driven enzyme prodrug system (EDEPS) that takes advantage of the trans-activating properties of EBNA1, a latent protein expressed in all EBV-containing cells, to direct expression of cytosine deaminase (CD) at high levels in those cells only. Plasmids were constructed in which the CD gene or a luciferase reporter gene were cloned downstream of the herpes simplex virus thymidine kinase (tk) promoter and the family of repeats (FR) sequence from the oriP region of EBV. Analysis of luciferase activity after transient transfection into a panel of EBV-negative or -positive human cell lines showed that the presence of the FR element enhanced transcription from the tk promoter in all EBV-positive cell lines, whereas transcription from tk was repressed in all EBV-negative cell lines, including B, T, and fibroblast cell lines. In clonogenicity assays following transfection with the CD vector, the presence of 5-fluorocytosine (5-FC) in the culture medium completely abolished cell growth in EBV-positive cell lines, but did not affect the growth of EBV-negative cell lines. This vector system should have wide applicability in that it allows targeted expression of any gene of interest to tumors that carry EBV, irrespective of the role EBV plays in their pathogenesis.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

DNA(cytosine-5)-methylases form tight complexes at their methylation sites when the target cytosine residue is substituted by analogs such as 5-azacytosine or 5-fluorocytosine. To test whether such complexes can block RNA transcription in vitro, template DNA-containing methylation sites were prepared, in which cytosine residues in either the (+)- or (-)-strand were substituted by the analogs. Such templates, irrespective of the strand in which substitution was made, could effectively block the elongation of RNA at specific sites when complexed with EcoRII methylase. The protein-DNA complex probably prevents the unwinding of the template strands or might directly present itself as a steric block to the advancing RNA polymerase. RNA synthesis was also inhibited at specific sites due to complex formation between azacytosine-containing DNA and two other methylases, HhaI and HpaII. The 3' ends of the interrupted transcripts were mapped and were found to lie within 13-14, 13, and 23 nucleotides of the binding sites on the (-)-strand for HhaI, HpaII, and EcoRII methylases, respectively. Exonuclease III footprinting revealed that the boundaries of the complexed methylases, HhaI, HpaII, and EcoRII, on the (-)-strand were within 2-3, 1-2, and 9-10 nucleotides, respectively, of the last nucleotide copied by the RNA polymerase.

Combination drug therapy is widely used for the management of many common medical conditions. The concurrent use of more than one drug can significantly alter the therapeutic and side-effect profile of each individual agent. Such drug-drug interactions are generally classified either as pharmacokinetic or pharmacodynamic. Oral agents used for the management of superficial fungal infections can have both types of interactions. This article describes common drug-drug interactions observed with amphotericin B, flucytosine, griseofulvin, ketoconazole, fluconazole, and itraconazole.

The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.

Cytosine deaminase (CD) is a microbial enzyme that can convert the antifungal agent 5-fluorocytosine (5-FC) into the antitumor agent, 5-fluorouracil (5-FU). The enzyme was chemically conjugated to the L6 monoclonal antibody, forming a conjugate that bound to antigens on the H2981 lung adenocarcinoma. Detailed studies were undertaken to determine the extent to which L6-CD generated 5-FU in tumor-bearing mice. Very high tumor:blood ratios of L6-CD (42:1) in vivo were obtained by injecting the conjugate followed 24 h later by an antiidiotypic antibody that could bind to circulating L6-CD but not to L6-CD that was bound to H2981 cells. As a result, significantly more 5-FC could be administered (> 800 mg/kg) than 5-FU (90 mg/kg). L6-CD converted 5-FC into 5-FU such that the L6-CD/antiidiotypic monoclonal antibody/5-FC combination resulted in 17 times more intratumoral 5-FU compared to systemic 5-FU administration. The conversion was antigen dependent since much lower intratumoral 5-FU levels were obtained in H3719 tumors that failed to localize L6-CD. The conversion of 5-FC into 5-FU was low in blood, kidneys, and liver. This demonstrates that a major increase in intratumoral drug concentrations can be attained with an monoclonal antibody-enzyme conjugate in combination with an anticancer prodrug compared to systemic drug therapy.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.

At pH 5-6 ATP-depleted washed cell preparations of strain NC233-10b[pII4-9], in which the cytosine permease was overexpressed, absorbed cytosine, hypoxanthine or fluorocytosine stoichiometrically with, respectively, about 1, 1.4 and 5 proton equivalents. The cellular pH fell proportionately. The membrane depolarization caused by each compound was assayed in the presence of glucose with a voltage-sensitive dye and increased in the same order. Fluorocytosine significantly lowered the growth yield that a 'petite' strain of the yeast formed at limiting glucose concentrations. At pH 5.6 with extracellular [K+] below 1 mM, each of the three substrates was accumulated about 200-fold from a dilute solution at the expense of the proton gradient. This concentration ratio corresponds to a solute gradient (delta mu(s)) of 13 kJ mol-1. Raising [K+]o systematically lowered the substrate accumulation ratio and delta muH. The mean ratio delta mu(s)/delta muH was 0.82 for all three substrates. It was concluded that whereas the behaviour of cytosine approximated to that expected for a symport of unit proton stoichiometry, the absorption of protons with fluorocytosine and, to a lesser extent, hypoxanthine, was only partly conserved as useful work. A possible mechanism of this novel phenomenon is outlined.

The properties of the interaction of 5-fluorocytosine-containing DNA with the EcoRII methyltransferase were studied. The DNA used was either a polymer synthesized in vitro, or a 20-mer containing one CCA/TGG sequence. The DNA could be methylated by the enzyme. In the process the enzyme formed a tight binding adduct with the DNA that could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by this interaction. The 20-mer could be used to titrate the active site of the enzyme. The DNA polymer formed a tight binding complex that could be identified following digestion of the DNA with pancreatic deoxyribonuclease or micrococcal nuclease. A peptide-DNA adduct could be isolated after digestion of the EcoRII-DNA adduct with staphylococcal protease V8 by high pressure liquid chromatography and polyacrylamide gel electrophoresis. Sequencing of the peptide indicated the DNA bound to a region of the protein that is conserved in all procaryotic DNA(cytosine-5)-methyltransferases. We have previously shown that this region contains a cysteine that can be photomethylated with adenosylmethionine. This region, in addition to forming part of, or being adjacent to, the AdoMet binding site, also forms part of the DNA binding site.

Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.

A relationship between the gut flora level, particularly gram-negative enterobacilli, and the in vivo flucytosine conversion to fluorouracil has been observed in humans from the fluorine-19 magnetic resonance spectroscopy analysis of urine from two patients treated with the flucytosine- (6 to 9 g/day) amphotericin B (1 mg/kg/day) combination. Indeed the percentage of fluorouracil metabolites was extremely low (less than 0.6% of total fluorinated compounds excreted) when the number of enterobacillary colonies was low (less than 10(3] and higher (3.5 to 8.8%) when enterobacillary colonies were under reconstitution or in the normal range (10(5) to 10(8]. The intestinal microflora assessment may therefore be of high interest to predict the risk of an additive flucytosine-induced myelotoxicity suspected to be due to fluorouracil during flucytosine chronic therapy.

Flucytosine (5-FC)-resistant strains were isolated from the haploid opportunistic pathogen Candida glabrata by UV-induced mutation and fluoropyrimidine selection. These strains were characterized biochemically, and the metabolism of fluorinated pyrimidines was studied by 19F nuclear magnetic resonance spectroscopy. No evidence was obtained from these studies for degradative metabolism of the fluorinated derivatives. In the parental susceptible strain of C. glabrata, 5-fluorouracil but not 5-FC was detected within the cells. 5-Fluorouracil was also present in the culture supernatant after incubation of the cells with 5-FC. The distribution of fluorinated derivatives within the 5-FC-resistant strains was consistent with their genotype. Two strains of C. glabrata which had only a partial loss of cytosine deaminase and UMP pyrophosphorylase activity had high levels of resistance to 5-FC. Both C. glabrata and Candida albicans were susceptible to 5-fluorouridine. This compound but not the anticancer drug 5-fluoro-2-deoxyuridine was shown to be transported into susceptible cells by a specific uridine permease.

A patient with false elevation of serum creatinine level due to 5-fluorocytosine (5-FC) is reported. 5-FC interferes with the enzymatic method used for creatinine determination in the Kodak Ektachem analyzer. Clinicians should be aware of the potential for spurious azotemia in patients receiving 5-FC therapy for fungal urinary tract infections.

Experiments were designed to determine the nature of amphotericin B (AMB)-5-fluorocytosine (5-FC) synergism against strains of Candida albicans susceptible to either drug alone. AMB strongly inhibited cell uptake of 14C-labeled 5-FC during incubation in a synthetic liquid medium. After several hours, however, this inhibition was dramatically released. Onset of 14C-5-FC uptake correlated with the known kinetics of AMB decay. These findings support the concept of sequential drug action proposed earlier. AMB apparently acts first and essentially alone until it approaches depletion. Experiments with a 5-FC-resistant mutant indicated that AMB interferes with transport of 5-FC to the cell interior.

UMP pyrophosphorylase activity was assayed in crude lysates prepared from Candida albicans. Specific activity of UMP pyrophosphorylase was high in clinical isolates which were susceptible to 5-fluorocytosine. Resistant clinical isolates displayed low activity, and partially resistant (heterozygous) isolates displayed intermediate activity. Segregation from the heterozygous state resulted in a homozygous susceptible segregant with high UMP pyrophosphorylase activity and a homozygous resistant segregant with low activity. The observed specific activities were consistent with the hypothesis that specific activity was determined by the sum of the activities due to the dominant (FCY) and recessive (fcy) alleles of the resistance gene. Strains which possessed little UMP pyrophosphorylase activity released uracil into the medium when grown in the absence of 5-fluorocytosine; this result suggested that recycling of intracellular uracil is a normal function of this enzyme.

In spite of the development in cases of deep mycoses, cases of osteoarthritis with Candida remain rare (only thirty seven cases reported). Among these, ten observations of spondylodiscitis have been collected and the authors report a new case where Candida albicans was isolated in the discovertebral focus through bone puncture. This spondylodiscitis occurring in a leukemic patient receiving antibiotics, corticosteroids, cytostatics drugs and radiotherapy, was treated successfully by an association of Amphotericin B-5-Fluorocytosine. The authors take this opportunity to report the main characteristics of the ten previously described cases specifying the rules for the uses of fungicidal drugs in the treatment of deep mycoses and for the first time, they study simultaneously seric concentration, penetration into healthy bone and into discovertebral focus of the 5-Fluorocytosine. They also justify their preference for the association of Amphotericin B-5-Fluorocytosine as a "first line" treatment after confirming the sensitivity of the strain to 5-Fluorcytosine.

Interpretation of in vitro susceptibility data for antifungal drugs is hindered by the absence of standardized test criteria. Thus, it is extremely difficult to identify a clear relation between minimal inhibitory concentrations and clinical outcome. The situation appears more readily resolvable for yeast-like than for filamentous fungi since the former are more easily quantified by standardized microbiologic techniques. Accordingly the National Committee for Clinical Laboratory Standards has initiated the process of developing standards for yeast susceptibility testing. A related issue concerns the measurement of antifungal agents in body fluids. Whereas there may be little value in measuring concentrations of amphotericin B (because of its predictable pharmacokinetics), there is value to measuring levels of flucytosine (serum concentrations may relate to bone marrow suppression and/or the development of drug resistance) and ketoconazole (which may be absorbed unpredictably from the gut). Laboratory standards for these measurements have not been established.

A patient developed a severe urinary infection with Candida albicans following a cadaveric renal transplant operation. It was necessary to remove the graft and return the patient to regular hemodialysis treatment. The fungi infection was successfully eradicated by a single oral dose of 5-Fluorocytosine (20 mg/kg) after each dialysis treatment. Pharmacokinetic studies revealed that the drug had a dialyzate clearance ratio of 0.66 when compared with creatinine; the drug was not metabolized and there was no other route of elimination.

Ketoconazole and flucytosine were administered alone and in combination for ten days to rabbits with four candida isolates growing in subcutaneously implanted semipermeable chambers. The peak concentrations of ketoconazole in serum and in the chamber were 20.3 and 3.8 mg/l, respectively, and the concentrations of flucytosine 47.7 and 37.3 mg/l, respectively. The two drugs combined resulted in better fungistatic activity than either drug alone against all four isolates. Correlation of efficacy in the rabbit model with in-vitro MICs was good for flucytosine, but poor for ketoconazole.

[18F]-5-fluorocytosine was prepared by reaction of [18F]-acetylhypofluorite with cytosine in acetic acid and was isolated in an overall radiochemical yield of 20%. Tissue distribution studies in sarcoma-bearing rats indicated in vivo stability, limited tissue uptake and rapid urinary excretion.

When the decision to treat a fungal infection is made, there are several antifungal agents available for use. AmB remains the first-line drug in the treatment of most systemic fungal infections. Miconazole should be used to treat patients who cannot tolerate AmB or who are infected with AmB-resistant organisms. Ketoconazole has a distinct advantage in that it is a relatively nontoxic oral agent and may prove very effective in treating non-life-threatening chronic fungal infections. Clinical experience with miconazole and ketoconazole is too limited at present to recommend them as first-line therapeutic agents, except in a limited number of clinical situations. 5-FC should only be used in combination with AmB to treat yeast infections.