Therapeutic effect of uridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

CPBMF65, a synthetic human uridine phosphorylase-1 inhibitor, reduces HepG2 cell proliferation through cell cycle arrest and senescence.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

Mechanism of berberine in treating Helicobacter pylori induced chronic atrophic gastritis through IRF8-IFN-γ signaling axis suppressing.

AIMS: In this study, we will investigate the therapeutic effects of berberine (BBR) in Helicobacter pylori (H. pylori) induced chronic atrophic gastritis (CAG). Furthermore, potential mechanisms of BBR in regulating IRF8-IFN-γ signaling axis will also be investigated. MATERIALS AND METHODS: H. pylori were utilized to establish CAG model of rats. Therapeutic effects of BBR on serum supernatant indices, and histopathology of stomach were analyzed in vivo. Moreover, GES-1 cells were infected by H. pylori, and intervened with BBR in vitro. Cell viability, morphology, proliferation, and quantitative analysis were detected by high-content screening (HCS) imaging assay. To further investigate the potential mechanisms of BBR, relative mRNA, immunohistochemistry and protein expression in IRF8-IFN-γ signaling axis were measured. KEY FINDINGS: Results showed serum supernatant indices including IL-17, CXCL1, and CXCL9 were downregulated by BBR intervention, while, G-17 increased significantly. Histological injuries of gastric mucosa induced by H. pylori also were alleviated. Moreover, cell viability and morphology changes of GES-1 cells were improved by BBR intervention. In addition, proinflammatory genes and IRF8-IFN-γ signaling axis related genes, including Ifit3, Upp1, USP18, Nlrc5, were suppressed by BBR administration in vitro and in vivo. The proteins expression related to IRF8-IFN-γ signaling axis, including Ifit3, IRF1 and Ifit1 were downregulated by BBR intervention.

Human uridine phosphorylase-1 inhibitors: a new approach to ameliorate 5-fluorouracil-induced intestinal mucositis.

PURPOSE: 5-fluorouracil (5-FU) has been broadly used to treat solid tumors for more than 50 years. One of the major side effects of fluoropyrimidines therapy is oral and intestinal mucositis. Human uridine phosphorylase (hUP) inhibitors have been suggested as modulators of 5-FU toxicity. Therefore, the present study aimed to test the ability of hUP blockers in preventing mucositis induced by 5-FU. METHODS: We induced intestinal mucositis in Wistar rats with 5-FU, and the intestinal damage was evaluated in presence or absence of two hUP1 inhibitors previously characterized. We examined the loss of weight and diarrhea following the treatment, the villus integrity, uridine levels in plasma, and the neutrophil migration by MPO activity. RESULTS: We found that one of the compounds, 6-hydroxy-4-methyl-1H-pyridin-2-one-3-carbonitrile was efficient to promote intestinal mucosa protection and to inhibit the hUP1 enzyme, increasing the uridine levels in the plasma of animals. However, the loss of body weight, diarrhea intensity or neutrophil migration remained unaffected. CONCLUSION: Our results bring support to the hUP1 inhibitor strategy as a novel possibility of prevention and treatment of mucositis during the 5-FU chemotherapy, based on the approach of uridine accumulation in plasma and tissues.

Design of novel potent inhibitors of human uridine phosphorylase-1: synthesis, inhibition studies, thermodynamics, and in vitro influence on 5-fluorouracil cytotoxicity.

Uridine (Urd) is a promising biochemical modulator to reduce host toxicity caused by 5-fluorouracil (5-FU) without impairing its antitumor activity. Elevated doses of Urd are required to achieve a protective effect against 5-FU toxicity, but exogenous administration of Urd is not well-tolerated. Selective inhibitors of human uridine phosphorylase (hUP) have been proposed as a strategy to increase Urd levels. We describe synthesis and characterization of a new class of ligands that inhibit hUP type 1 (hUP1). The design of ligands was based on a possible SN1 catalytic mechanism and as mimics of the carbocation in the transition state of hUP1. The kinetic and thermodynamic profiles showed that the ligands here presented are the most potent in vitro hUP1 inhibitors developed to date. In addition, a lead compound improved the antiproliferative effects of 5-FU on colon cancer cells, accompanied by a reduction of in vitro 5-FU cytotoxicity in aggressive SW-620 cancer cells.

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity.

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer's and Parkinson's, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.

In silico/in vitro study of hybrid D-modified steroidal alkylator anticancer activity using uridine phosphorylase as target protein.

BACKGROUND: In order to reduce toxicity and to enhance anticancer activity of nitrogen mustards, three hybrid steroidal esters were synthesized and tested in vitro against human pancreatic cancer cells expressing uridine phosphorylase (UPase). The inhibition potency against a target protein implicated in the chemotherapy of solid tumors, such as UPase, is of fundamental importance in the design and synthesis of new anticancer drugs. MATERIALS AND METHODS: MTT colorimetric assay and molecular docking were employed for the in vitro and in silico drug evaluation, respectively. RESULTS: A difference in cell sensitivity was found, which followed the known different UPase expression in the cell lines. Molecular docking studies on UPase protein, revealed the tested compounds to be bound to the binding cavity of the protein, with different affinity. Between the two D-modified compounds, the D-homo-aza (lactam)-hybrid compound (C2) was found to interact with the protein in a more efficient way. CONCLUSION: The molecular docking data were in accordance with the in vitro results, where the lactam steroid alkylator showed significantly higher cytostatic and cytotoxic activity than the non-D-modified compounds, which also correlated with the level of UPase expression in the pancreatic cancer cells.

Synthesis and biological evaluation of unsaturated keto and exomethylene D-arabinopyranonucleoside analogs: novel 5-fluorouracil analogs that target thymidylate synthase.

The synthesis of pyrimidine unsaturated keto and exomethylene arabinopyranonucleoside analogs as potential antitumor and antiviral agents is described. Commercially available 1,2,3,4-tetra-O-acetyl-D-arabinopyranose (1) was condensed with silylated thymine, uracil, 5-fluorouracil, N(4)-benzoyl cytosine and 5-(trifluoromethyl)uracil, respectively, deacetylated and acetylated to afford 1-(3,4-O-isopropylidene-α-D-arabinopyranosyl)pyrimidine analogs 4. Two different synthetic routes were investigated for the conversion of compounds 4 into the new 1-(2,3,4-trideoxy-2-methylene-α-pent-3-enopyranosyl)nucleoside derivatives of thymine (10a), uracil (10b), 5-fluorouracil (10c) and N(4)-benzoyl cytosine (10d). Only the first approach could afford derivative 10d. Debenzoylation of 10d afforded 1-(2,3,4-trideoxy-2-methylene-α-pent-3-enopyranosyl)cytosine (10f). The first approach resulted also to the 2-keto-3,4-unsaturated analogs 9. The new analogs did not show inhibition of DNA and RNA virus replication in cell culture. The 2'-ketonucleoside derivatives 9 were found to be more cytostatic than the corresponding 2'-exomethylene nucleosides 10. The 5-fluorouracil unsaturated keto derivative 9c and the exomethylene derivatives 10c and 13c showed antiproliferative activity in the lower micromolar range. Experimental evidence revealed that 9c, 10c and 13c may act as novel types of 5-fluorouracil releasing prodrugs, and points to thymidylate synthase as target for their cytostatic action.

Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate.

OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.

The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy.

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either P(i) or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase.

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.

Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy.

BACKGROUND: Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents. RESULTS: In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization. CONCLUSION: The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

[Polymethylene nucleic acid derivatives with omega-functional groups: vi. [8-(2-oxocyclohexyl)-9-oxooctyl]pyrimidines as potential inhibitors of pyrimidine phosphorylases].

New polymethylene derivatives of nucleic bases with beta-diketo function in omega-position were prepared by alkylation of uracil, thymine, and cytosine. Their physicochemical properties and effect on the E. coli uridine phosphorylase were studied.

[Vasculitis with renal involvement and antineutrophil cytoplasmic antibodies (ANCA) in a child receiving benzylthiouracil]. / Vascularite avec atteinte rénale et anticorps anticytoplasme des polynucléaires neutrophiles (ANCA) après prise de benzylthio-uracile chez l'enfant.

Vasculitis associated to antineutrophil cytoplasmic antibodies (ANCA) is a rare complication of therapy with antithyroid medication. They were mainly described in patients treated with propylthiouracil (PTU), carbimazole, methimazole and rarely by benzylthiouracil (Basden). We report a case of 12-years-old girl treated by benzylthiouracil for Grave's disease who developed after 2 years vasculitis associated with cutaneous involvement (generalized ulcer necrotic purpura) and glomerulonephritis with proteinuria of 24 hours at 26 mg/kg/day, microscopic hematuria and renal failure with creatinemia level at 135 micromol/l. The ANCA type antiMPO (myeloperoxidase) was positive. The histology study of the renal needle biopsy was in favour with focal necrotizing glomerulonephritisand crescents with different evolutive stages. The discontinuation of benzylthiouracil and the treatment by the corticoids involved a disappearance of cutaneous lesions, a negative result of proteinuria, a normalization of the renal function (creatinemia=84 micromol/l) and a disappearance of hematuria and ANCA. These results permitted to announce hypothesis that benzylthiouracil was implicated in development of vasculitis associated to ANCA.

Activity and substrate specificity of pyrimidine phosphorylases and their role in fluoropyrimidine sensitivity in colon cancer cell lines.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5'-deoxy-5-fluorouridine (5'DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect. The high TP expressing Colo320TP1 cells were most sensitive to 5'DFUR and 5FU, with IC50 values of 1.4 and 0.2 microM, respectively, while SW948 and SW1398 were insensitive to 5'DFUR (IC50>150 microM for 5'DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC(50) for 5'DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5'DFUR; p<0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5'DFUR and 5FU separately. HaCaT might be a model for 5'DFUR toxicity. In the colon cancer cells 5'DFUR degradation varied from 0.4 to 50 nmol 5FU/h/10(6)cells, that of TdR from 0.3 to 103 nmol thymine/h/10(6)cells, that of Urd from 0.8 to 79 nmol uracil/h/10(6)cells, while conversion of 5FU to FUrd was from 0.3 to 46 nmol/h/10(6)cells. SW948 and SW1398 were about equally sensitive to 5'DFUR and 5FU, but SW1398 had higher phosphorylase activity (>65-fold) compared to SW948. In SW948 and HaCaT TPI and BAU inhibited TdR and Urd phosphorolysis (>80%), respectively. Both TP and UP contributed to the phosphorolysis of 5'DFUR and 5FU. In the presence of both inhibitors, still phosphorolysis of 5FU (>40%) was detected in the tumor and HaCaT cell lines, and remarkably, that of all four substrates in SW1398 cells. 5'DFUR phosphorolysis was also measured in situ, where Colo320TP1, SW1398, and HaCaT cells produced significant amounts 5FU from 5'DFUR (>10 nmol/24h/10(6)cells). In Colo320TP1 and in HaCaT cells TPI completely prevented 5FU production, but not in SW1398 cells, where BAU decreased this by 67% (p<0.01). High uracil and dUrd levels were detected in the medium. Uracil accumulation was heavily reduced in the presence of TPI for Colo320TP1 and HaCaT cells, whereas 5FU-induced dUrd production by these cell lines increased (p<0.01). In contrast, for SW1398 cells only BAU was able to reduce uracil levels, and dUrd production remained unchanged. In conclusion, overlapping substrate specificity was found for TP and UP in the cell lines, in which both enzymes were responsible for converting TdR and Urd, and 5'DFUR. 5'DFUR and 5FU were converted to their products in both the colon cancer cells and keratinocytes.

Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(Phenylthio)acyclouridine, a uridine phosphorylase inhibitor.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host-toxicity and enhancing its chemotherapeutic efficacy against human colon tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. METHODS: SCID mice bearing human colon DLD-1 or HCT-15 tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24 and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg) or their combination was administered orally 2 or 4 h after FUra injection. Another four administrations of PTAU+uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. RESULTS: Administration of the maximum tolerated dose (50 mg/kg) of FUra reduced DLD-1 and HCT-15 tumor weights by 48 and 59%, respectively, at day 38 post implantation. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not alleviate FUra host-toxicity as all the mice died. Administration of 120 mg/kg PTAUresulted in partial rescue from this lethal dose of FUra as 63% of mice survived and tumor weights were reduced by approximately 60%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra as 100% of the mice survived and tumor weights were reduced by 81-82%. Delaying the administration of the combination of PTAU plus uridine to 4 h post FUra treatment was less effective in rescuing from FUra toxicity as only 88% of the mice survived and tumor weights were reduced by only 62%. Administration of PTAU alone, under the same conditions, resulted in a 38% survival rate while the tumor weights were reduced by 47%. Treatment with uridine alone did not protect from FUra toxicity at the dose of 200 mg/kg as all mice died. At the higher dose of 300 mg/kg FUra, neither uridine nor PTAU alone, administered 2 h following the treatment with FUra, had any rescuing effect. On the other hand, the use of the PTAU plus uridine combination reduced the tumor weight by 79%, although this reduction in the tumor weight was accompanied by 37% mortality. There was no significant difference between DLD-1 and HCT-15 in their response to the different regimens employed in this study despite the fact that the tumors have different levels of UrdPase. CONCLUSIONS: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host-toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combinationin raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al., Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the large doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side-effects associated with such doses of uridine. This combination may also allow for the escalation of FUra doses for better chemotherapeutic efficacy against human colon carcinoma while avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

Structural basis for inhibition of Escherichia coli uridine phosphorylase by 5-substituted acyclouridines.

Uridine phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate and is a key enzyme in the pyrimidine-salvage pathway. Escherichia coli UP is structurally homologous to E. coli purine nucleoside phosphorylase and other members of the type I family of nucleoside phosphorylases. The structures of 5-benzylacyclouridine, 5-phenylthioacyclouridine, 5-phenylselenenylacyclouridine, 5-m-benzyloxybenzyl acyclouridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of E. coli UP have been determined, with resolutions ranging from 1.95 to 2.3 A. For all five complexes the acyclo sugar moiety binds to the active site in a conformation that mimics the ribose ring of the natural substrates. Surprisingly, the terminal hydroxyl group occupies the position of the nonessential 5'-hydroxyl substituent of the substrate rather than the 3'-hydroxyl group, which is normally required for catalytic activity. Until recently, inhibitors of UP were designed with limited structural knowledge of the active-site residues. These structures explain the basis of inhibition for this series of acyclouridine analogs and suggest possible additional avenues for future drug-design efforts. Furthermore, the studies can be extended to design inhibitors of human UP, for which no X-ray structure is available.

5-(Phenylthio)acyclouridine: a powerful enhancer of oral uridine bioavailability: relevance to chemotherapy with 5-fluorouracil and other uridine rescue regimens.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the pharmacokinetics and bioavailability of oral uridine. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU is fully absorbed after oral administration with 100% oral bioavailability. METHODS: Uridine (330, 660 or 1320 mg/kg) and/or PTAU (30, 45, 60, 120, 240 or 480 mg/kg) were orally administered to mice. The plasma levels of uridine, its catabolite uracil, and PTAU were measured using HPLC, and pharmacokinetic analysis was performed. RESULTS: Oral PTAU up to 480 mg/kg per day is not toxic to mice. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg has a prolonged plasma half-life of 2-3 h, and peak plasma PTAU concentrations (C(max)) of 41, 51, 74, 126 and 161 microM with AUCs of 70, 99, 122, 173 and 225 micromol h/l, respectively. Coadministration of uridine with PTAU did not have a significant effect on the pharmacokinetic parameters of plasma PTAU at any of the doses tested. Coadministration of PTAU (30, 45, 60 and 120 or 240 mg/kg) with uridine (330, 660 or 1320 mg/kg) elevated the concentration of plasma uridine over that following the same dose of uridine alone, a result of reduced metabolic clearance of uridine as evidenced by decreased plasma exposure (C(max) and AUC) to uracil. Plasma uridine was elevated with the increase of uridine dose at each PTAU dose tested and no plateau was reached. Coadministration of PTAU at 30, 45, 60, 120 and 240 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9-, 9.9-, 11.7- and 12.5-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol h/l) by 5.7-, 6.8-, 8.2-, 6.3-, and 6.9-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.7-, 2.4-, 2.6-, 5.2- and 4.3- fold, and that of 660 mg/kg uridine by 2.3-, 2.7-, 3.3-, 4.6- and 6.7-fold, respectively. CONCLUSION: The excellent pharmacokinetic properties of PTAU, and its extraordinary effectiveness in improving the oral bioavailability of uridine, could be useful to rescue or protect from host toxicities of 5-fluorouracil and various chemotherapeutic pyrimidine analogues used in the treatment of cancer and AIDS, as well as in the management of medical disorders that are remedied by the administration of uridine including CNS disorders (e.g. Huntington's disease, bipolar disorder), liver diseases, diabetic neuropathy, cardiac damage, various autoimmune diseases, and transplant rejection.

[Benzylthiouracil induced ANCA-positive vasculitis]. / Vascularite à anticorps anticytoplasme de polynucléaires neutrophiles induite par le benzylthio-uracile.

INTRODUCTION: Several cases of vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA) have been reported in patients treated with synthetic anti-thyroid drugs but only 2 cases have incriminated benzylthiouracil. OBSERVATION: A 36 year-old woman, 3 years after treatment with benzylthiouracil, rapidly developed progressive kidney failure, related to a pauci-immune extra-capillary glomerular nephropathy and necrotic vasculitis lesions. The search for p-ANCA was positive with anti-myeloperoxidase specificity. She was treated with corticosteroids and 6 monthly intravenous pulses of cyclophosphamide substituted by azathioprine. Renal failure and proteinuria significantly improved. However the high level of p-ANCA. CONCLUSION: ANCA vascularities are a rare but serious complication of treatment with synthetic thiouracile-type anti-thyroid drugs. The ANCA must be measured when confronted with a systemic manifestation during treatment.

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.