Repurposing of Antifungal Drug Flucytosine/Flucytosine Cocrystals for Anticancer Activity against Prostate Cancer Targeting Apoptosis and Inflammatory Signaling Pathways.

This study aimed to repurpose the antifungal drug flucytosine (FCN) for anticancer activity together with cocrystals of nutraceutical coformers sinapic acid (SNP) and syringic acid (SYA). The cocrystal screening experiments with SNP resulted in three cocrystal hydrate forms in which two are polymorphs, namely, FCN-SNP F-I and FCN-SNP F-II, and the third one with different stoichiometry in the asymmetric unit (1:2:1 ratio of FCN:SNP:H2O, FCN-SNP F-III). Cocrystallization with SYA resulted in two hydrated cocrystal polymorphs, namely, FCN-SYA F-I and FCN-SYA F-II. All the cocrystal polymorphs were obtained concomitantly during the slow evaporation method, and one of the polymorphs of each system was produced in bulk by the slurry method. The interaction energy and lattice energies of all cocrystal polymorphs were established using solid-state DFT calculations, and the outcomes correlated with the experimental results. Further, the in vitro cytotoxic activity of the cocrystals was determined against DU145 prostate cancer and the results showed that the FCN-based cocrystals (FCN-SNP F-III and FCN-SYA F-I) have excellent growth inhibitory activity at lower concentrations compared with parent FCN molecules. The prepared cocrystals induce apoptosis by generating oxidative stress and causing nuclear damage in prostate cancer cells. The Western blot analysis also depicted that the cocrystals downregulate the inflammatory markers such as NLRP3 and caspase-1 and upregulate the intrinsic apoptosis signaling pathway marker proteins, such as Bax, p53, and caspase-3. These findings suggest that the antifungal drug FCN can be repurposed for anticancer activity.

Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers.

Cancer in dogs has increased in recent years and is a leading cause of death. We have developed a retroviral replicating vector (RRV) that specifically targets cancer cells for infection and replication. RRV carrying a suicide gene induced synchronized killing of cancer cells when administered with a prodrug after infection. In this study, we evaluated two distinct RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) in canine tumor models both in vitro and in vivo. Despite low infection rates in normal canine cells, both RRVs efficiently infected and replicated within all the canine tumor cells tested. The efficient intratumoral spread of the RRVs after their intratumoral injection was also demonstrated in nude mouse models of subcutaneous canine tumor xenografts. When both RRVs encoded a yeast cytosine deaminase suicide gene, which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil, they caused tumor-cell-specific 5-FC-induced killing of the canine tumor cells in vitro. Furthermore, in the AZACF- and AZACH-cell subcutaneous tumor xenograft models, both RRVs exerted significant antitumor effects. These results suggest that RRV-mediated suicide gene therapy is a novel therapeutic approach to canine cancers.

Comparison of the antifungal activity of the pyrimidine analogs flucytosine and carmofur against human-pathogenic dematiaceous fungi.

Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi.

Temporal trends in antifungal susceptibility of Cryptococcus neoformans isolates from a reference laboratory in the United States, 2011-2021.

BACKGROUND: There are no established clinical breakpoints for antifungal agents against Cryptococcus species; however, epidemiological cut-off values can help distinguish wild-type (WT) isolates without any acquired resistance from non-WT strains, which may harbour resistance mechanisms. PATIENTS/METHODS: We describe the trends of antifungal MICs and percentages of WT C. neoformans species complex (CNSC) isolates processed in our reference laboratory from November 2011 to June 2021. There were only nine isolates in 2011, thus, we included them in the year 2012 for data analysis. Clinical data is also described when available. RESULTS: We identified 632 CNSC, the majority collected from blood (n = 301), cerebrospinal fluid (n = 230), and respiratory (n = 71) sources. The overall percentage of WT isolates for amphotericin B (AMB), 5-flucytosine, and fluconazole was 77%, 98%, and 91%, respectively. We noticed a statistically significant change in the percentage of AMB WT isolates over the years, with 98% of isolates being WT in 2012 compared to 79% in 2021 (p < .01). A similar change was not observed for other antifungal agents. Clinical data was available for 36 patients, primarily non-HIV immunocompromised patients with disseminated cryptococcosis. There were no statistically significant differences in the clinical characteristics and outcomes between patients with WT (58.3%) versus non-WT (41.7%) isolates, but we noticed higher mortality in patients infected with an AMB non-WT CNSC isolate. CONCLUSIONS: We observed an increase in the percentage of AMB non-WT CNSC isolates in the past decade. The clinical implications of this finding warrant further evaluation in larger studies.

Escherichia coli cytosine deaminase: Structural and biotechnological aspects.

Suicide gene therapy involves introducing viral or bacterial genes into tumor cells, which enables the conversion of a nontoxic prodrug into a toxic-lethal drug. The application of the bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) approach has been beneficial and progressive within the current field of cancer therapy because of the enhanced bystander effect. The basis of this method is the preferential deamination of 5-FC to 5-fluorouracil by cancer cells expressing cytosine deaminase (CD), which strongly inhibits DNA synthesis and RNA function, effectively targeting tumor cells. However, the poor binding affinity of toward 5-FC compared to the natural substrate cytosine and/or inappropriate thermostability limits the clinical applications of this gene therapy approach. Nowadays, many genetic engineering studies have been carried out to solve and improve the activity of this enzyme. In the current review, we intend to discuss the biotechnological aspects of Escherichia coli CD, including its structure, functions, molecular cloning, and protein engineering. We will also explore its relevance in cancer clinical trials. By examining these aspects, we hope to provide a thorough understanding of E. coli CD and its potential applications in cancer therapy.

Analysis of the relationship between drug susceptibility of Cryptococcus neoformans isolates and mortality in HIV-negative cryptococcal meningitis.

OBJECTIVES: The relationship between antifungal susceptibility and mortality of cryptococcal meningitis (CM) in HIV-negative patients is poorly understood. METHODS: We conducted a retrospective analysis of 1-year follow-up of 200 HIV-negative CM patients with an initial cerebrospinal fluid (CSF) culture for Cryptococcus neoformans. According to the cut-off values of minimum inhibitory concentration (MIC), two groups of five antifungal agents were classified: amphotericin B (AmB), ≤0.5 µg/mL, >0.5 µg/mL; 5-flucytosine (5-FC), ≤4 µg/mL, >4 µg/mL; fluconazole (FLU), ≤4 µg/mL, >4 µg/mL; itraconazole (ITR), ≤0.125 µg/mL, >0.125 µg/mL; and voriconazole (VOR), <0.25 µg/mL, ≥0.25 µg/mL. Comparisons were performed to analyse clinical features, laboratory, modified Rankin Scale (mRS) scores, and CSF findings under different prognosis outcomes in 1-year. RESULTS: All of Cryptococcus neoformans isolates were sensitive to AmB and VOR, most of them were sensitive to 5-FC and FLU (95.5% and 90.5%, respectively) while only 55.0% of them were susceptible to ITR. Minimum inhibitory concentrations of ITR and VOR were significantly related to baseline mRS scores. All-cause mortality was not significantly related to MICs in Cryptococcus neoformans strains. The combination of actual antifungal agents and two groups of the MICs values for antifungal agents had no significant effects on all-cause mortality. CONCLUSION: Most Cryptococcus neoformans isolates were sensitive to AmB, VOR, 5-FC, and FLU. Because of the small number of deaths, we are not able to comment on whether MIC is associated with mortality of CM in HIV-negative patients.

Synergistic effect of the verapamil and amphotericin B against Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis and cryptococcal meningitis, which conventional treatment involves antifungal drugs such as polyenes, flucytosine, azoles, and their combinations. However, the high cost, toxicity, and increase in fungi resistance to antifungal agents stimulate the search for therapeutic strategies such as drug repurposing and combination therapy. This study evaluated the activity of the antihypertensive verapamil (VEH) alone and combined with amphotericin B (AmB) against C. neoformans. VEH exhibited antifungal activity against C. neoformans with minimum inhibitory concentration and minimum fungicidal concentration of 118 µg per mL. The combination of VEH and AmB exhibited synergism, reducing at least eightfold both drugs' concentrations. Moreover, the combination decreased the size and glucuronoxylomannnan content of C. neoformans capsule. However, no difference was observed in ergosterol levels of C. neoformans after treatment with VEH and AmB in combination. Altogether, VEH in combination with AmB exhibits potential as a candidate as for the development of anti-cryptococcal drug.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

BACKGROUND/AIM: Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. MATERIALS AND METHODS: Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. RESULTS: Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. CONCLUSION: RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Cross-feeding affects the target of resistance evolution to an antifungal drug.

Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.

Synergistic antifungal activity and potential mechanism of action of a glycolipid-like compound produced by Streptomyces blastmyceticus S108 against Candida clinical isolates.

AIM: The present study aimed to investigate a novel antifungal compound produced by Streptomyces blastmyceticus S108 strain. Its effectiveness against clinical isolates of Candida species and its synergistic effect with conventional antifungal drugs were assessed, and its molecular mechanism of action was further studied against Candida albicans. METHODS AND RESULTS: A newly isolated strain from Tunisian soil, S. blastmyceticus S108, showed significant antifungal activity against Candida species by well diffusion method. The butanolic extract of S108 strain supernatant exhibited the best anti-Candida activity with a minimal inhibitory concentration (MIC) value of 250 µg ml-1, determined by the microdilution method. The bio-guided purification steps of the butanolic extract were performed by chromatographic techniques. Among the fractions obtained, F13 demonstrated the highest level of activity, displaying a MIC of 31.25 µg ml-1. Gas chromatography-mass spectrometry and electrospray ionization mass spectrometry analyses of this fraction (F13) revealed the glycolipidic nature of the active molecule with a molecular weight of 685.6 m/z. This antifungal metabolite remained stable to physicochemical changes and did not show hemolytic activity even at 4MIC corresponding to 125 µg ml-1 toward human erythrocytes. Besides, the glycolipid compound was combined with 5-flucytosine and showed a high synergistic effect with a fractional inhibitory concentration index value 0.14 against C. albicans ATCC 10231. This combination resulted in a decrease of MIC values of 5-flucytosine and the glycolipid-like compound by 8- and 64-fold, respectively. The examination of gene expression in treated C. albicans cells by quantitative polymerase chain reaction (qPCR) revealed that the active compound tested alone or in combination with 5-flucytosine blocks the ergosterol biosynthesis pathway by downregulating the expression of ERG1, ERG3, ERG5, ERG11, and ERG25 genes. CONCLUSION AND IMPACT OF THE STUDY: The new glycolipid-like compound, produced by Streptomyces S108 isolate, could be a promising drug for medical use against pathogenic Candida isolates.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Correlation of antifungal susceptibility and sequence types within Cryptococcus neoformans VNI from HIV patients, and ERG11 gene polymorphism.

INTRODUCTION: Here we tested the correlation between minimum inhibitory concentrations (MICs) of major antifungal agents and sequence types (STs) within Cryptococcus neoformans VNI isolates, and explored the ERG11 gene of included strains. MATERIALS AND METHODS: We analysed 23 C. neoformans strains categorised into two groups according to the distribution of the ST profile in Kinshasa clinics (Democratic Republic of Congo): major ST [ST93 (n = 15)], and less common STs [ST659 (n = 2), ST5 (n = 2), ST4 (n = 1), ST 53 (n = 1), ST31 (n = 1), and ST69 (n = 1)]. The MICs of the major antifungal agents [amphotericin B (AMB), 5-fluorocytosine (5FC) and fluconazole (FCZ)] were determined following EUCAST guidelines. ERG11 gene sequences were extracted from whole genome sequence of the isolates and compared with the wild-type gene sequence of the C. neoformans VNI. RESULTS: Although major ST isolates appeared to have lower median MICs for AMB and 5FU than less common ST isolates (0.50 vs. 0.75 mg/L for AMB, 2 vs. 4 mg/L for 5FU, respectively), FCZ susceptibility was similar in both groups (4 mg/L) (p-value >0.05). The susceptibility profile of C. neoformans strains separately considered did not significantly affect the patients' clinical outcomes (p-value >0.05). Furthermore, two structural modalities of the ERG11 gene were observed: (1) that of the reference gene, and (2) that containing two exonic silent point substitutions, and one intronic point substitution located in a sequence potentially involved in pre-mRNA splicing (c.337-22C > T); with no association with the MICs of the isolates (p-value >0.05). CONCLUSIONS: The lack of association/correlation found in this study calls for further investigations to better understand the mechanisms of C. neoformans resistance to antifungal agents.

Searching for new antifungals for the treatment of cryptococcosis.

There is a consensus that the antifungal repertoire for the treatment of cryptococcal infections is limited. Standard treatment involves the administration of an antifungal drug derived from natural sources (i.e., amphotericin B) and two other drugs developed synthetically (i.e., flucytosine and fluconazole). Despite treatment, the mortality rates associated with fungal cryptococcosis are high. Amphotericin B and flucytosine are toxic, require intravenous administration, and are usually unavailable in low-income countries because of their high cost. However, fluconazole is cost-effective, widely available, and harmless with regard to its side effects. However, fluconazole is a fungistatic agent that has contributed considerably to the increase in fungal resistance and frequent relapses in patients with cryptococcal meningitis. Therefore, there is an unquestionable need to identify new alternatives or adjuvants to conventional drugs for the treatment of cryptococcosis. A potential antifungal agent should be able to kill cryptococci and "bypass" the virulence mechanism of the yeast. Furthermore, it should have fungicidal action, low toxicity, high selectivity, easily penetrate the central nervous system, and widely available. In this review, we describe cryptococcosis, its conventional therapy, and failures arising from the use of drugs traditionally considered to be the reference standard. Additionally, we present the approaches used for the discovery of new drugs to counteract cryptococcosis, ranging from the conventional screening of natural products to the inclusion of structural modifications to optimize anticryptococcal activity, as well as drug repositioning and combined therapies.

Heightened Efficacy of Anidulafungin When Used in Combination with Manogepix or 5-Flucytosine against Candida auris In Vitro.

Candida auris is an emerging, multidrug-resistant fungal pathogen that causes refractory colonization and life-threatening, invasive nosocomial infections. The high proportion of C. auris isolates that display antifungal resistance severely limits treatment options. Combination therapies provide a possible strategy by which to enhance antifungal efficacy and prevent the emergence of further resistance. Therefore, we examined drug combinations using antifungals that are already in clinical use or are undergoing clinical trials. Using checkerboard assays, we screened combinations of 5-flucytosine and manogepix (the active form of the novel antifungal drug fosmanogepix) with anidulafungin, amphotericin B, or voriconazole against drug resistant and susceptible C. auris isolates from clades I and III. Fractional inhibitory concentration indices (FICI values) of 0.28 to 0.75 and 0.36 to 1.02 were observed for combinations of anidulafungin with manogepix or 5-flucytosine, respectively, indicating synergistic activity. The high potency of these anidulafungin combinations was confirmed using live-cell microfluidics-assisted imaging of the fungal growth. In summary, combinations of anidulafungin with manogepix or 5-flucytosine show great potential against both resistant and susceptible C. auris isolates.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment.

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR.

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment.

BACKGROUND: Mesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene. METHODS: Modified MSCs were preserved in a commercially available, GMP-grade cryopreservative-CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC). RESULTS: We found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers. CONCLUSION: This study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use.

Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically measure the effect of all amino acid substitutions in the yeast cytosine deaminase Fcy1, the target of the antifungal 5-fluorocytosine (5-FC, flucytosine). We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilization of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose-response functions of 5-FC and cytosine. Finally, we observe the same trade-off shape for the orthologue of FCY1 in Cryptoccocus neoformans, a human pathogen. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance-function trade-offs.

Enzyme Prodrug Therapy with Photo-Cross-Linkable Anti-EGFR Affibodies Conjugated to Upconverting Nanoparticles.

In this work, we demonstrate that a photo-cross-linkable conjugate of upconverting nanoparticles and cytosine deaminase can catalyze prodrug conversion specifically at tumor sites in vivo. Non-covalent association of proteins and peptides with cellular surfaces leads to receptor-mediated endocytosis and catabolic degradation. Recently, we showed that covalent attachment of proteins such as affibodies to cell receptors yields extended expression on cell surfaces with preservation of protein function. To adapt this technology for in vivo applications, conjugates were prepared from upconverting nanoparticles and fusion proteins of affibody and cytosine deaminase enzyme (UC-ACD). The affibody allows covalent photo-cross-linking to epidermal growth factor receptors (EGFRs) overexpressed on Caco-2 human colorectal cancer cells under near-infrared (NIR) light. Once bound, the cytosine deaminase portion of the fusion protein converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). NIR covalent photoconjugation of UC-ACD to Caco-2 cells showed 4-fold higher retention than observed with cells that were not irradiated in vitro. Next, athymic mice expressing Caco-2 tumors showed 5-fold greater UC-ACD accumulation in the tumors than either conjugates without the CD enzyme or UC-ACDs in the absence of NIR excitation. With oral administration of 5-FC prodrug, tumors with photoconjugated UC-ACD yielded 2-fold slower growth than control groups, and median mouse survival increased from 28 days to 35 days. These experiments demonstrate that enzyme-decorated nanoparticles can remain viable after a single covalent photoconjugation in vivo, which can in turn localize prodrug conversion to tumor sites for multiple weeks.

[Cryptococcal Meningitis with Inadequate Response to Antifungal Therapy or Drug-Induced Side Effects That Make It Difficult to Continue Treatment: Eleven Clinical Issues in Medical Practice].

Cryptococcal meningoencephalitis is mainly caused by Cryptococcus neoformans and accounts for 90% of fungal meningitis cases in Japan. Cryptococcal meningoencephalitis is a rare disease, and similar to tuberculosis meningitis. It often exhibits subacute or chronic progression symptoms such as headache, fever, coma, personality changes, and memory disturbance. Cryptococcal meningoencephalitis often develops in immunosuppressed hosts, but can sometimes occur in healthy individuals, and the mortality rate is 10-25%, indicating a poor prognosis. For the treatment of cryptococcal meningoencephalitis, introduction therapy using a combination of liposomal amphotericin B and flucytosine is recommended. However, in practice, cryptococcal meningoencephalitis is refractory and often requires prolonged treatment; therefore, it is the most difficult to treat among the central nervous system infections. We discuss the following 11 issues: I. Sustainability of first-line treatments, II. Treatment options in case of decreased renal function, III. Association with increased intracranial pressure IV. Causes of visual impairment, V. Necessary steps when symptoms/laboratory findings worsen during antifungal treatment, VI. Cerebral infarction, VII. Difficulty in controlling underlying and comorbid diseases, VIII. Indications for lumbar and ventricular drainage (Ommaya reservoir placement), IX. Pros and cons of concomitant use of corticosteroids, X. Treatment evaluation index: usefulness of head MRI, and XI. Determining the end of treatment and the need for preventive medication.