Treatment with Paclitaxel Orotate and Carboxyamidotriazole Orotate in SC-Implanted OVCAR-5 Human Ovarian Tumor Xenografts

Background: Paclitaxel (PTX) is approved for the treatment of refractory ovarian cancer and breast cancer, but is problematic due to severe, dose-dependent, potentially irreversible neurotoxicity. Alternative formulations using nanoparticles and liposomes have been developed to avoid solvent-related toxicity. These formulations allow improved delivery;however, toxicity, compensatory signaling, and drug resistance still pose challenges. Conversion of cytotoxic agents to their orotate compounds offers a potentially improved approach by increasing bioavailability and reducing toxicity. Orotate salts are neutral and acquire lipophilic properties, easing diffusion through lipid membranes. The orotate salt of PTX (PTXO) may yield an improved safety profile. Combination therapy with cytotoxic drugs, antiangiogenics and/or signal transduction pathway inhibitors has shown better efficacy than cytotoxic monotherapy. The combination of carboxyamidotriazole orotate (CTO, a calcium signal transduction pathway inhibitor) and PTX may be more effective than PTX alone at non-toxic doses. Materials and Methods: PTXO alone, and combinations of CTO with PTX and PTXO were first tested in female athymic NCr-nu/nu mice to evaluate tolerance of the combinations. The tolerated combinations, PTX monotherapy, and PTXO monotherapy were then tested to evaluate their antitumor activity in female athymic NCr-nu/nu mice with subcutaneously implanted OVCAR-5 human ovarian tumor. Antitumor activity was measured by median time to doubling, median tumor growth delay, and mean percent body weight loss. Results: CTO, PTX, and PTXO showed significant inhibition of growth of the human OVCAR-5 ovarian tumor xenografts. The combination of low PTX and CTO, or high PTXO monotherapy, had significant efficacy and it was less toxic than high PTX as measured by body weight loss. Conclusions: Low-dose CTO is effective and has low toxicity, suggesting the potential for maintenance therapy for ovarian cancer. PTXO offers efficacy and a strategy for minimizing body weight loss, and may improve outcomes for patients who demonstrate toxicity to PTX.

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.

Comparative Response of SC CAKI-1 Renal Tumor to Treatment with Doxorubicin HCl and Doxorubicin Orotate

Background: Doxorubicin (DOX) is an effective treatment for many cancers across the age spectrum, but its therapeutic potential is limited because of dose-dependent relation to both progressive and irreversible cardiomyopathy leading to congestive heart failure. While decreases in cardiotoxicity have been reported with liposomal doxorubicin, the long-term cardiac effects are not known. Orotate salts of cytotoxic drugs have been shown to confer antitumor effects with a better safety profile than unconverted drug, and therefore may offer an improved approach to cancer treatment. Materials and Methods: Male, athymic NCr-nu/nu mice with subcutaneously implanted CAKI-1 human renal tumor xenografts were treated with DOX and its orotate salt (DOX-O) to evaluate antitumor activity, measured by median tumor mass doubling time and tumor weight. Nontumored male, athymic NCr-nu/nu mice were treated with DOX, DOX-O and liposomal doxorubicin formulations to determine DOX concentration in liver and heart;and to evaluate their effect on body weight. Non-tumored female, athymic NCr-nu/nu mice were treated with daunorubicin and daunorubicin orotate to evaluate tolerance. Results: DOX and DOX-O exhibited significant, similar levels of antitumor activity. Mice treated with DOX-O had a lower percentage body weight loss. In the animals treated with DOX, DOX-O, or liposomal doxorubicin, liposomal doxorubicin was associated with the lowest percentage of body weight loss, but the highest concentration of DOX in heart. In daunorubicin tolerance experiments, animals showed a better tolerance for daunorubicin orotate as measured by a smaller percentage change in body weight. Conclusions: DOX-O is effective as an antitumor therapy and may offer a less toxic alternative to DOX for maintaining therapy. The lower percentage of body weight loss in animals treated with DOX-O and daunorubicin orotate is a measure of improved tolerance and may translate into better patient outcomes.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

AIM:To determine whether expression of certain enzymes related to 5-fluorouracil(5-FU)metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma(CCA).METHODS:The histoculture drug response assay(HDRA)was performed using surgically resected CCA tissues.Tumor cell viability was determined morphologically with hematoxylin and eosin-and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues.The mRNA expression of thymidine phosphorylase(TP),orotate phosphoribosyl transferase(OPRT),thymidylate synthase(TS),and dihydropyrimidine dehydrogenase(DPD)was determined with realtime reverse transcriptase-polymerase chain reaction.The levels of gene expression and the sensitivity to 5-FU were evaluated.RESULTS:Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital,Khon Kaen University from 2007 to 2009.HDRA was used to determine the response of these CCA tissues to 5-FU.Based on the dose-response curve,200μg/mL 5-FU was selected as the test concentration.The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU.When the relationship between TP,OPRT,TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined,only OPRT mRNA expression was significantly correlated with the response to 5-FU.The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group(0.41±0.25 vs 0.22±0.12,P<0.05).CONCLUSION:OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA.Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.

Hydrothermal Synthesis and Crystal Structure of Lanthanum Orotate Supermolecule with A Honeycomb Structure Constructed from Mononuclear Molecules via Hydrogen Bonds

A new complex of lanthanum orotate [La（C5H3N2O4）2（OH）（H2O）6 ]·2（C5H4N2O4）·9H2O was synthesized using a hydrothermal method. The crystal structure was determined with help of single crystal X-ray diffraction analysis. The complex crystallized in orthorhombic system, Cmc2（1） space group. In the complex, the center lanthanum （Ⅲ） ion was nine-coordinated with coordination geometry of a distorted monocapped square antiprism, in which two of the nine coordinated oxygen atoms were from two orotate ligands in a monodentate mode, one oxygen atom from a hydroxyl, and the others from six coordinated water molecules. The mononuclear complex [La（C5H3N2O4）2（OH）（H2O）6 ]·2（C5H4N2O4）·9H2O was linked through the hydrogen bonds into an infinite supermolecular honeycomb structure.

A predictive factor for the response to S-1 plus cisplatin in gastric cancer

AIM:To prove that the protein expression level of thymidylate synthase is a predictive factor for the response to S-1/cisplatin(CDDP)chemotherapy in gastric cancer.METHODS:We measured the protein expression levels of thymidylate synthase(TS),dihydropyrimidine dehydrogenase(DPD),and orotate phosphoribosyltrans-ferase(OPRT)in advanced gastric cancer.Before S-1/CDDP chemotherapy,tumor specimens from primary sites were obtained by endoscopic biopsy and analyzed by enzyme-linked immunosorbent assay.The chemo-therapeutic effects on the primary sites were evaluated by endoscopic biopsy performed more than once after S-1/CDDP chemotherapy.The effects are a predictive factor for the response to S-1/CDDP chemotherapy inpatients with advanced gastric cancer,as evaluated by endoscopic biopsy over time.RESULTS:The protein expression level of TS was significantly higher(P<0.05)in the tumor than in the normal tissue,and significantly lower(P<0.05)in the responders than in the non-responders.We were able to evaluate the correlation between changes in the protein expression levels of TS,DPD and OPRT and chemotherapeutic responses in 7 patients by assessing tumor tissues more than twice.In the responders,the protein expression level of TS was<40 ng/mg protein.However,there were significant increases in the protein expression levels of TS(P<0.01)and DPD(P<0.05)after chemotherapy in 3 patients.In these cases,the patient assessment changed from"responder"to"non-responder".In the non-responders,the protein expression level of TS was>40 ng/mg protein.CONCLUSION:We have confirmed that the protein expression level of TS is a predictive factor for the response to S-1/CDDP chemotherapy in patients with advanced gastric cancer.

OPRT Is a Potential Predictive Factor for the Response to S-1 in Gastric Cancer

Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric cancer patients received S-1 (80 mg/m2/day;days 1-14) and oxaliplatin (130 mg/m2;day 1) every 3 weeks and subsequently received gastrectomy with D1/D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase using quantitative reverse-transcriptase polymerase chain reaction. Results: Between December 2009 and October 2010, thirty-five patients were enrolled in this study. 24 (68.5%) patients had clinical tumor response and 10 (28.6%) patients achieved histological response. Quantitative reverse-transcriptase polymerase chain reaction results showed that orotate phosphoribo-syltransferase (OPRT) mRNA expression was significantly higher in histological responders than non-responders (3.75 vs. 1.81, P = 0.005). Diffuse-type gastric cancer patients demonstrated higher orotate phosphoribosyltransferase (OPRT) expression levels than intestinal-type ones (2.79 vs. 1.60, P = 0.014). Similar results were not found when comparing thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression levels. Conclusion: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy. Mini Abstract: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy.

Measurement of Orotic Acid in Urine by Supercritical Fluid Chromatography

This work presents a simple, rapid and reliable supercritical fluid chromatography (SFC) method for a sensitive measurement of orotic acid in human urine. The samples were diluted with deionized water and analyzed directly without any pretreatment.

Synthesis and Crystal Structure of [Ni(C_5H_2N_2O_4)(2,2'bipy)(H_2O)_2]·H_2O

The crystal structure of [Ni(C5H2N2O4)(2, 2?bipy)(H2O)2]?H2O 1 has been determined by X-ray diffraction. Crystal data: triclinic system, space group P with a = 7.9424(3), b = 9.9417(3), c = 12.1867(3) ? a = 84.771(1), b = 77.375(2), g = 68.993(2)? C15H18N4O8Ni, Mr = 440.7, V = 876.16(5) 3, Z = 2, Dc = 1.672 g/cm3, F(000) = 456, m(MoKa) = 1.162 mm-1, the final R = 0.0464 and wR = 0.1055 for 3026 observed reflections with I > 2s(I). In the title compound, the nickel ion is coordinated by a nitrogen atom and an oxygen atom from the orotate ligand, two nitrogen atoms from 2, 2?bipy and two oxygen atoms from the coordinated water molecules in a distorted octahedral geometry. The presence of intermolecular hydrogen bonding and p-p stacking interaction of aromatic rings from 2, 2?bipy results in a 3D structure.

Synthesis, Molecular Spectroscopy, Computational, Thermal Analysis and Biological Activity of Some Orotic Acid Complexes

Binary orotic acid metal complexes of Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Hg(II), and two mixed metals complexes of (Co(II), Ni(II)) and (Ni(II), Cu(II)) were synthesized and characterized by elemental analysis, IR, electronic spectra, magnetic susceptibility, and ESR spectra. The Analysis proved that the ligand has different coordination modes and the complexes were of octahedral, tetrahedral, and trigonal bipyramidal geometries. Molecular modeling techniques and quantum chemical methods have been performed for orotic acid to calculate charges, bond lengths, bond angles, dihedral angles, electronegativity (χ), chemical potential (μ), global hardness (η), softness (σ) and the electrophilicity index (ω). The thermal decomposition of the complexes was monitored by TGA, DTA, and DSC techniques under the N2 atmosphere. The thermal decomposition mechanisms of the complexes were suggested. The biological activity of orotic acid and some of the complexes are tested against antibacterial and antifungal organisms.

Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis

It is timely to consider the many facets of the small molecule orotic acid （OA）, which is well-known as an essential intermediate of pyrimidine de novo synthesis. In addition, it can be taken up by erythrocytes and hepatocytes for conversion into uridine and for use in the pyrimidine recycling pathway. We discuss the link between dietary orotate and fatty liver in rats, and the potential for the alleviation of neonatal hyperbilirubinaemia. We address the development of orotate derivatives for application as anti-pyrimidine drugs, and of com- plexes with metal ions and organic cations to assist therapies of metabolic syndromes. Recent genetic data link human Miller syndrome to defects in the dihydroorotate dehydrogenase （DHODH） gene, hence to depleted orotate production. Another defect in pyrimidine biosynthesis, the orotic aciduria arising in humans and cattle with a deficiency of UMP synthase （UMPS）, has different symptoms. More recent work leads us to conclude that OA may have a role in regulating gene transcription.

Preparation and application of a novel orotic acid chelating resin for removal of Cu(Ⅱ) in aqueous solutions

A novel chelating resin OABA,capable of removing Cu（Ⅱ） from aqueous solution,was synthesized via the reaction of macroporous chloromethylated PS-DVB copolymer beads with orotic acid.The elemental analysis（EA）,Fourier transform infrared spectroscopy（FT-IR）,and scanning electron microscopy microscope-energy dispersive X-ray spectroscopy（SEM-EDS） were used in the characterization of the synthesized chelating resin.Multiple,static batch adsorption experiments were conducted at different initial concentrations and temperatures.OABA showed good adsorption capacity for Cu（Ⅱ） and the equilibrium data could be well matched with the Freundlich isotherm model.Coexisting sodium chloride and calcium chloride in solutions favored the Cu（Ⅱ） adsorption.Moreover,the desorption process of Cu（Ⅱ） was tested and over 90%regeneration efficiency for the spent OABA was achieved at ammonia concentrations ranging from 1.0%to 2.0%.The results suggested that OABA would be a potential alternative adsorbent for Cu（Ⅱ）,even with other heavy metal ion treatments of wastewater.