Quaternized Chitosan-Coated Montmorillonite Interior Antimicrobial Metal-Antibiotic in Situ Coordination Complexation for Mixed Infections of Wounds.

Conventional drug delivery systems for natural clay materials still face critical challenges in their practical application, including multiple bacterial infections, combined infection of bacteria and fungi, and low sterilization efficiency. In this work, we address these challenges using the multifunctional montmorillonite nanosheet-based (MMT-based) drug nanoplatform, which involves the antibiotic 5-fluorocytosine (5-FC), antibacterial metal copper ions, and quaternized chitosan (QCS). Composite material QCS/MMT/5-FCCu can can strongly inhibit Staphylococcus aureus (a typical Gram-positive bacterium), Escherichia coli (a typical Gram-negative bacterium), and Candida albicans (a fungus) because 5-FC coordinates with copper ions in situ and due to the deposition of QCS. The subsequent drug release behavior of 5-FCCu was studied, and the results show an initial high concentration kills microorganisms and long-acting sustained release inhibition. Moreover, in vivo wound experiments and toxicity experiments show the promotion of wound healing and excellent biocompatibility. As a demonstration of the utility of the latter, we have shown that the MMT-based smart platform can be used for the treatment of mixed infections of wounds.

Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.

We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a ß-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

Discovery of a bacterial 5-methylcytosine deaminase.

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 10(5), 2.9 × 10(4), and 1.1 × 10(3) M(-1) s(-1), respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 10(5), 6.8 × 10(4), and 2.0 × 10(2) M(-1) s(-1), respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85 ), 5-fluorocytosine (PDB id: 4R88 ), and phosphonocytosine (PDB id: 4R7W ) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

Induction of a thoracolumbar supernumerary rib in rat developmental toxicity studies: A short discussion on the critical window.

Thoracolumbar supernumerary ribs (TSRs) are classified as less severe skeletal anomalies in rat developmental toxicity studies, although their incidence is relatively high in rodent studies. To investigate the characteristics of the critical window for chemically-induced TSR, in this study, rats were administered 5-fluorocytocine (5-FC) or sodium salicylate (SAL) at one of three time periods on gestational day (GD) 9, early morning (7:00 am), midday (12:00 pm to 1:00 pm), or late afternoon (4:00 pm or 7:00 pm). The incidence of TSR and other anomalies were assessed in GD20 fetuses. A single treatment with both chemicals on GD9-induced TSR, with the incidence highest when administered at 7:00 Am, decreasing gradually when administered later. This trajectory was clearer in rats treated with 5-FC than with SAL. The critical period of TSR induction is shorter in rats administered 5-FC than SAL. The characteristics of the critical window may cause variability in the incidence of TSR observed in developmental toxicity studies.

Developmental toxicity of flucytosine following administration to pregnant rats at a specific time point of organogenesis.

To investigate the abnormalities that are specific to administration of flucytosine at one time point during embryonic organogenesis, flucytosine was administered orally to pregnant Sprague Dawley (SD) rats in a single dose on day 11 of pregnancy at 25 or 35 mg/kg. Fetuses on day 20 of pregnancy were externally, viscerally, and skeletally examined. Maternal body weight gain and food consumption were suppressed the day after administration of a 35 mg/kg. Fetal examinations revealed various alterations in both dose groups: externally preaxial polydactyly in the hind limb; skeletally fused lumbar centrum, absent sacral centrum, supernumerary sacral vertebra, and absent ribs. Our findings indicated that specific types of external and skeletal anomalies were induced following flucytosine administration on day 11 of pregnancy.

Liposomal formulation of 5-fluorocytosine in suicide gene therapy with cytosine deaminase--for colorectal cancer.

It is generally accepted that successful gene therapy depends on two major factors: tumor-specific expression of a therapeutic gene and the efficient transfer of a therapeutic gene to tumor cells. For gene-directed enzyme prodrug therapy (GDEPT) involving Escherichia coli cytosine deaminase (CD) and 5-fluorocytosine (5-FC), several tumor-specific promoters and virus-based vectors were used. No attention whatsoever was paid to the way of 5-FC delivery to solid tumors, despite the fact that the delivery of drugs to such tumors is generally low because of their insufficient transfer from the blood. To compare the effectiveness of GDEPT with free and liposomal 5-FC, the prodrug was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (1:1). When the liposomal form of 5-FC was administered i.v., mice treated with a dose of 5mg of liposomal 5-FC/kg body weight for 10 days, showed complete regression of transplanted tumors and complete cure was observed, whereas in animals treated with the same amounts of the free prodrug, 50% tumor regression and only insignificantly prolonged median survival were found. In summary, these results showed a remarkable enhancement of the antitumor effects of the liposomal form of 5-FC in comparison with the free prodrug. Therapy with liposomal 5-FC thus represents a new approach to achieving a high local concentration of the prodrug for suicide gene therapy using E. coli CD.

Coexpression of the uracil phosphoribosyltransferase gene with a chimeric human nerve growth factor receptor/cytosine deaminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine.

Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.

Osteoclasts direct bystander killing of cancer cells in vitro.

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells (P < 0.006) and GFP-4T1 cells (P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.

In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase.

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.

Bioactive Core-Shell Nanofiber Hybrid Scaffold for Efficient Suicide Gene Transfection and Subsequent Time Resolved Delivery of Prodrug for Anticancer Therapy.

Nanofiber scaffold's ability to foster seemingly nonexistent interface with the cells enables them to effectively deliver various bioactive molecules to cells in the vicinity. Among such bioactive molecules, therapeutically active nucleic acid has been the most common candidate. In spite of such magnanimous efforts in this field, it remains a paradox that suicide gene delivery by nanofibers has never been sought for anticancer application. To investigate such a possibility, in the present work, a composite core-shell nanofiberous scaffold has been realized which could efficiently transfect suicide gene into cancer cells and simultaneously deliver prodrug, 5-Fluorocytosine (5-FC) in a controlled and sustained manner. The scaffold's ability to instigate apoptosis by suicide gene therapy in nonsmall lung cancer cells (A549) was ascertained at both phenotypic and genotypic levels. A cascade of events starting from suicide gene polyplex release from nanofibers, transfection, and expression of cytosine deaminase-uracil phosphoribosyltransferase (CD::UPRT) suicide gene by A549; subsequent prodrug release; and its metabolic conversion into toxic intermediates which finally culminates in host cells apoptosis has been monitored in a time-dependent manner. This work opens up new application avenues for nanofiber-based scaffolds which can effectively manage cancer prognosis.

Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity.

To ascertain whether concomitant expression of Escherichia coli deaminase (CD) and herpes simplex virus type-1 thymidine kinase (HSV-1 TK) could mediate greater levels of cytotoxicity beyond that observed with either suicide gene alone, 9L gliosarcoma cells were transduced with a retrovirus encoding a CD/HSV-1 TK fusion gene. The resultant CD/HSV-1 TK fusion protein (CDglyTK) was found to be bifunctional via CD and HSV-1 TK enzymatic assays, and conferred upon cells prodrug sensitivities equivalent to or better than that observed for each enzyme independently (ganciclovir [GCV] and bromovinyldeoxyuridine [BVdU] for HSV-1 TK and 5-fluorocytosine [5-FC] for CD). Simultaneous treatment of CDglyTK-expressing cells with prodrugs specific for HSV-1 TK and CD (GCV/5-FC or BVdU/5-FC) resulted in slight synergistic toxicity, two- to three-fold greater than that expected if the cytotoxic effects of each prodrug were purely additive. More importantly, co-treatment with HSV-1 TK- and CD-specific prodrugs was found to increase greatly the radiosensitivity of CDglyTK-expressing cells. Sensitivity enhancement ratios of 2.44 (GCV/5-FC) and 3.90 (BVdU/5-FC) were achieved. The results suggest that double suicide gene therapy, using a bifunctional CD/HSV-1 TK fusion gene, coupled with radiotherapy may provide a highly efficient means of selectively treating cancer.

Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy.

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.

The use of suicide gene systems in vascular cells in vitro.

To investigate the efficiency of suicide gene systems on vascular cells, HSV-tk/GCV and EC-CD/5-FC systems were established on vascular endothelial cells in vitro by retroviral transduction. Both modified cell lines were highly sensitive to prodrugs, the IC50 for GCV was less than 0.4 microM, and IC50 for 5-FC was less than 75 microM, while the parental endothelial cells were insensitive even at the highest concentrations of prodrugs in this experiment. Mixed cellular assay showed that significant bystander effect was exhibited in modified endothelial cells. When only 10% or 30% of the mixed cells were tk positive and exposed to 20 microM GCV for 6 days, more than 60% or 90% of the whole population was killed. Similar result was also found in CD positive cells. These results indicated that both HSV-tk/GCV and EC-CD/5-FC systems could efficiently suppress endothelial cell growth in vitro.

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.

Selection of therapeutic agents for intraocular proliferative disease. II. Differing antiproliferative activity of the fluoropyrimidines.

We confirm the potent antiproliferative effects of the fluoropyrimidines on cellular proliferation in vitro in three different nonmalignant cell types. All fluoropyrimidines tested, except for fluorocytosine, decrease proliferation of human dermal fibroblasts, bovine aortic vascular endothelial cells, and human retinal pigment epithelial cells in vitro. Fluorouridine, an intracellular metabolite of fluorouracil, is nearly 100-fold more potent than fluorouracil and its deoxymetabolite. Human dermal fibroblasts are more sensitive to the inhibitory effects of deoxymetabolites than the cells of either human retinal pigment epithelium or bovine aortic vascular endothelium. Fluorouridine and other fluoropyrimidines may prove to be valuable second-generation drugs in the treatment of intraocular proliferative disorders.

5-fluorocytosine-related bone-marrow depression and conversion to fluorouracil: a pilot study.

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.

Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro.

To develop a suitable suicide gene/prodrug therapy for the treatment of thyroid carcinomas, the relative therapeutic efficacy of four different suicide gene/prodrug combinations was compared in thyroid carcinomas in vitro. Herpes simplex virus thymidine kinase and ganciclovir (HSV-TK/GCV), Escherichia coli cytosine deaminase and 5-fluorocytosine (CD/5FC), E coli nitroreductase and CB1954 (NTR/CB1954), and human deoxycytidine kinase and cytosine arabinoside (dCK/AraC) were employed. The suicide genes were transduced into two thyroid carcinoma cell lines with retroviral vectors in which all the suicide genes were under the control of the same promoter. When the relative efficacy of four suicide gene/prodrugs was compared with therapeutic index and degree of bystander effect, we found a clear dissociation between these two parameters. Thus, HSV-TKIGCV demonstrated the widest therapeutic index, while CD/5FC and NTR/CB1954 showed the stronger bystander effect than HSV-TK/GCV. dCK/AraC had little efficacy. Advantages and limitations of each suicide gene/prodrug combinations are discussed.

Effects of a hybrid recombinant human alpha interferon (A/D) on in vitro cytotoxicity and in vivo localization of monoclonal antibody L6-cytosine deaminase conjugate in a colon cancer model.

L6 is an IgG2a murine monoclonal antibody which we have demonstrated binds well to HT29 human colon carcinoma cells by flow cytometry, whole cell ELISA, and mixed hemadsorption. In vitro cytotoxicity studies revealed that the monoclonal antibody L6-cytosine deaminase (L6-CD) immunoconjugate plus the nontoxic prodrug, 5-fluorocytosine (5-FC), is equivalent to 5-fluorouracil (5-FU) in its ability to kill HT29 cells. Human alpha-interferon (A/D) was able to enhance this cytotoxic effect. The I.C.50's revealed that very small amounts of L6-CD are needed for this cytotoxic effect (approximately, 5 pg/ml resulted in 50% viability). The limiting factor was the amount of 5-FC employed with L6-CD (3 microM yielded 50% cell viability). alpha-Interferon (A/D) lowered the requirement of 5-FC to 1 microM to achieve 50% cell lethality. In vivo biodistribution experiments indicated that 1 microgram of L6-CD is nonspecifically taken up by the liver and spleen and cleared rapidly from the blood. Significant localization of L6-CD to HT29 tumors occurred only when 99 micrograms of unlabeled L6-CD was added to 1 microgram of 125I-labeled immunoconjugate injected intravenously. Further augmentation of tumor/blood ratios without reduction in percent injected dose per gram of tumor was possible with the intravenous injection of 100 micrograms of anti-idiotypic monoclonal antibody 13B, 24 hours after L6-CD, which bound unreacted L6-CD and cleared it from the blood. The addition of 100,000 U of alpha-interferon (A/D) given intraperitoneally every day increased the clearance of L6-CD by the liver and spleen, but impaired tumor localization (percent injected dose per gram). These studies demonstrated that in vivo localization of the L6-CD conjugate to HT29 tumors could be optimized by injecting excess L6-CD followed by an equal amount of L6 anti-idiotype mAb 13B, 24 hours after L6-CD.

Pulsed versus continuous application of the prodrug 5-fluorocytosine to enhance the oncolytic effectiveness of a measles vaccine virus armed with a suicide gene.

Abstract Oncolytic virotherapy with measles vaccine virus (MeV) already has been demonstrated to be safe. However, early clinical results pointed out the necessity for an enhancement of oncolytic effectiveness of MeV-based virotherapeutics. In our work, we are developing an armed measles vaccine virus (MeV-SCD) encoding a suicide fusion gene of yeast cytosine deaminase/uracil phosphoribosyltransferase, converting the nontoxic prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic drug 5-fluorouracil (5-FU). To preclinically investigate what an optimal prodrug-assisted therapeutic regimen might look like, we added 5-FC at various time points after infection with MeV-SCD and either let the prodrug remain in the tumor cell culture medium continuously for various time periods ("continuous" 5-FC application) or applied it only temporarily for defined shorter periods of time ("pulsed" 5-FC application); we also varied the time point at which 5-FC was added after infection with MeV-SCD. As a result, addition of the prodrug at early times postinfection (e.g., at 3 hr postinfection) was found to be inferior concerning the overall oncolytic effectiveness when compared with addition of 5-FC at later time points (e.g., at 24 hr postinfection). Next, oncolytic effectiveness was found to correlate positively with the overall duration of incubation of MeV-infected tumor cells with 5-FC. Of note, this was true despite our finding that addition of the prodrug could also exert an inhibitory effect on the generation of infectious progeny viral particles, that is, on virus replication. These findings should be helpful for the rational design of further trials (preclinical, clinical) using suicide gene armed virotherapeutics, such as MeV-SCD.