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.     

Diabetes-induced changes of nitric oxide influence on the cardiovascular action of secretin

The modulation by condition of the lack or the excess of nitric oxide (NO) on cardiovascular action of secretin in diabetic rats was investigated. In vitro the isolated heart function and in vivo, the systolic (SBP), diastolic (DSP) blood pressure and heart rate (HR) were measured. Secretin evoked inotropic positive effect and increased coronary outflow (CO), in vivo did not increase systemic pressure and the highest dose of the peptide increased the heart rate. NO synthase inhibitor, N(G) nitro-L-arginine methyl ester (L-NAME) deeply increased the systemic pressure and in vitro decreased coronary outflow. L-arginine and sodium nitroprusside (SNP) did not influence the isolated heart function and in vivo decreased the systemic pressure. L-NAME preserved the inotropic positive effect of secretin and the increase of the coronary outflow. In vivo co-administration of L-NAME+secretin evoked hypotensive effect and abolished the increase of the heart rate after the highest dose of the peptide. L-arginine abolished inotropic positive effect of the peptide and the increase of coronary outflow. In vivo co-administration of these substances caused hypotension and attenuated the increase of the heart rate after the highest dose of secretin. Co-injection of SNP and secretin preserved the inotropic effect of secretin and abolished the increase of the coronary outflow. In vivo infusion of SNP+secretin evoked hypotension and similarly to L-arginine, SNP abolished tachycardia induced by the highest dose of secretin. Both the lack and the excess of nitric oxide changed the cardiovascular action of secretin in diabetic rats.     

Analysis of O6-methylguanine-DNA methyltransferase methylation status in sporadic colon polyps

Background/aimThe aim of our study was to check how MGMT methylation status together with known factors influenced the risk of colon cancer development.Materials and methodsWe examined patients with colon polyps. Information concerning gender, age, lifestyle, diet, anthropometry and medical information, including cancer and family history of cancer, was analyzed. Polymorphism variety of MGMT gene was investigated in another study. Genetic analysis for MGMT methylation assessment was performed for polyp tissue samples from 143 patients.ResultsPositive methylation MGMT status was found in 55 patients. There was no correlation between gender and MGMT methylation status (p = 0.43). We did not find correlation between patients younger and older than 60 (p = 0.87). There was no correlation between smoking and MGMT methylation status (p = 0.36). We did not find correlation between BMI and MGMT methylation status (p = 0.86). We did not find correlation between MGMT methylation status and colon cancer in familial history (p = 0.45).ConclusionOur study showed no correlations between methylation status of MGMT polymorphisms and clinical features like age, gender, polyp localization, smoking status, or obesity. It has been shown previously that MGMT methylation status may show nonspecific methylation in colon polyps. Gene methylation status in adenoma tissues has also been associated by other authors with the adenoma's size, histology, and degree of atypia. In our study, we evaluated the gene methylation status in colon polyps and found no association with adenoma characteristics. The present study showed no correlation for MGMT methylation in polyps in different regions of colon.     

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 1: discovery of novel orally active inhibitors of human thymidine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been prepared as inhibitors of human thymidine phosphorylase (TP). To enhance the in vivo antitumor activity of fluorinated pyrimidine 2'-deoxyribonucleosides such as 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd), a potent TP inhibitor preventing their degradation to an inactive compound, has become a target of medicinal chemistry. We present here the synthesis and evaluation of novel human TP inhibitors. Introduction of an N-substituted aminomethyl side chain at the 6-position of 5-chlorouracil has improved water solubility and enhanced inhibitory activity compared with the known TP inhibitor, 6-amino-5-chlorouracil. Compound 42 was reasonably well absorbed in mice after oral administration. When combined with F(3)dThd, compound 42 exerted its TP inhibitory potency by increasing the maximum plasma concentrations of the former as evidenced in experiments with monkeys. Positive changes in pharmacokinetic profile were accompanied by the enhanced in vivo antitumor activity of this combination when compared to F(3)dThd alone, in mice bearing human tumor xenografts. Both biochemical and pharmacological effects appeared to fit the concept as anticipated.     

Proteomics accelerating the identification of the target molecule of bioactive small molecules

Failures in many drug development programs in the past decades were related to unspecified mechanism of action and poor pharmacokinetic (PK) properties. Recent developments are focused on well defined targets, improved PK profiles, however, not much is known about off-target effects, especially those responsible for diminishing drug activity. Steadily increasing application of proteomics in drug development should expose clinically relevant proteins for the analysis of drug effects, to show what group of patients will respond and who should not be treated with an agent.     

The clinical importance of micrometastases within the lymphatic system in patients after total gastrectomy

Background

In spite of radical gastrectomy with resection of the lymphatic system, where no metastases are found during histopathological examination, about 30% of patients have relapse of the neoplastic process. This situation may be caused by micrometastases or isolated neoplastic cells in the lymphatic system which were not identified during a standard histopathological examination.

Aim

The aim of the study was to evaluate the clinical importance of micrometastases within the lymphatic system in patients with gastric cancer.

Materials and methods

A group of 20 patients treated for gastric cancer were subjected to retrospective analysis. Of all the patients who underwent surgery, a group with tumours classified as T1 or T2 was selected. No metastases within the lymphatic system were found in the standard evaluation – N0 mark. Paraffin-embedded blocks of lymph nodes were cut and new specimens were made, which were then stained again by means of immunohistochemistry. Antibodies against cytokeratin AE1/AE3 were used.

Results

A total of 319 lymph nodes were assessed in 20 patients in an H + E examination. After the immunohistochemical examination, micrometastases within the lymphatic system were found in 4 (20%) patients and isolated neoplastic cells in other 4 (20%) patients.

Conclusion

On the basis of numerous publications and our own material, we think that the presence of micrometastases may be related to a worse prognosis. The clinical importance of micrometastases within the lymphatic system in patients after total gastrectomy.     

Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

BackgroundThis project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool.Materials and methodsA TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH).ResultsThe 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment.ConclusionsThis study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases).Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics.