Multifunctional role of thymidine phosphorylase in cancer.

Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine, maintaining nucleoside homeostasis for DNA repair and replication. In many cancers TP is expressed at high levels and promotes thymidine catabolism, ultimately generating 2-deoxyribose (2dDR) that can support multiple procancer processes, including glycation of proteins, alternative metabolism, extracellular matrix remodeling, and angiogenesis. Therefore, inhibition of TP is an attractive anticancer strategy; however, an alternative approach that exploits the catalytic activity of TP to activate 5-fluorouracil (5-FU) prodrugs has been clinically successful. Here, we review the structure, function, and regulation of TP, its multiple supporting roles in cancer growth and survival. We summarize TP inhibitor and prodrug development and propose TP-targeting strategies that could potentiate the action of current therapies.

Substituted pteridinones, pyrimidines, pyrrolopyrimidines, and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors: Pharmacophore modeling data.

The RSK2 kinase is a downstream effector of the Ras/Raf/MEK/ERK pathway that is aberrantly active in a range of cancer types and has been recognized an anticancer target. The inhibition of RSK2 kinase activity would disrupt multiple pro-cancer processes; however, there are few RSK2 inhibitors. The data have been obtained for a series of pteridinone-, pyrimidine-, purine-, and pyrrolopyrimidine-based compounds, developed to establish a structure-activity relationship for RSK inhibition. The compounds were docked into the ATP-binding site of the N-terminal domain of the RSK2 kinase using Glide. The binding conformations of these molecules was then used to generate a set of pharmacophore models to determine the structural requirements for RSK2 inhibition. Through the combination of these models, common features (pharmacophores) can be identified that can inform the development of further small molecule RSK inhibitors. The synthesis and evaluation of the pteridinone- and pyrimidine-based compounds was reported in the related articles: Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study (Casalvieri et al., 2020) and Molecular docking of substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of RSK2 and associated MM/GBSA and molecular field datasets (Casalvieri et al., 2020). [1], [2]. The synthesis and evaluation of the purine- and pyrrolopyrimidine-based compounds was reported in the related research article: N-substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors (Casalvieri et al., 2021) [3].

Evaluation of Thymidine Phosphorylase Inhibitors in Glioblastoma and Their Capacity for Temozolomide Potentiation.

A number of studies have shown high levels of thymidine phosphorylase (TP) expression in glioblastoma (GBM), with trace or undetectable TP levels in normal developed brain tissue. TP catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, maintaining nucleoside homeostasis for efficient DNA replication and cell division. The TP-mediated catabolism of thymidine is responsible for multiple protumor processes and can support angiogenesis, glycation of proteins, and alternative metabolism. In this study, we examined the effect of TP inhibition in GBM using the known nanomolar TP inhibitors 5-chloro-6-[1-(2'-iminopyrrolidin-1'-yl)methyl]uracil (TPI) and the analogous 6-[(2'-aminoimidazol-1'-yl)methyl]uracils. Although these TP inhibitors did not demonstrate any appreciable cytotoxicity in GBM cell lines as single agents, they did enhance the cytotoxicity of temozolomide (TMZ). This pontetiated action of TMZ by TP inhibition may be due to limiting the availability of thymine for DNA repair and replication. These studies support that TP inhibitors could be used as chemosensitizing agents in GBM to improve the efficacy of TMZ.

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors.

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK inhibitor, and identified pteridinones that inhibited cellular RSK2 activity that did not result in concomitant cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe complementary interactions, while retaining the 2,6-difluorophenol-4-amino group to maintain interactions with the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identified compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line. These N-substituted probes have revealed an opportunity to further examine substituents that extend from the ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

Dissociation between the pharmacokinetics and pharmacodynamics of once-daily rivaroxaban and twice-daily apixaban: a randomized crossover study.

Essentials In this crossover study the anticoagulant effects of rivaroxaban and apixaban were compared. Healthy volunteers received rivaroxaban 20 mg once daily or apixaban 5 mg twice daily. Rivaroxaban was associated with more prolonged inhibition of thrombin generation than apixaban. Rivaroxaban induced a clear prolongation of prothrombin time and activated partial thromboplastin time. SUMMARY: Background The anticoagulant actions of the oral direct activated factor Xa inhibitors, rivaroxaban and apixaban, have not previously been directly compared. Objectives To compare directly the steady-state pharmacokinetics and anticoagulant effects of rivaroxaban and apixaban at doses approved for stroke prevention in patients with non-valvular atrial fibrillation. Methods Twenty-four healthy Caucasian male volunteers were included in this open-label, two-period crossover, phase 1 study (EudraCT number: 2015-002612-32). Volunteers were randomized to receive rivaroxaban 20 mg once daily or apixaban 5 mg twice daily for 7 days, followed by a washout period of at least 7 days before they received the other treatment. Plasma concentrations and anticoagulant effects were measured at steady state and after drug discontinuation. Results Overall exposure was similar for both drugs: the geometric mean area under the plasma concentration-time curve for the 0-24-h interval was 1830 µg h L-1 for rivaroxaban and 1860 µg h L-1 for apixaban. Rivaroxaban was associated with greater inhibition of endogenous thrombin potential (geometric mean area under the curve relative to baseline during the 0-24-h interval: 15.5 h versus 17.5 h) and a more pronounced maximal prolongation relative to baseline of prothrombin time (PT) (1.66-fold versus 1.14-fold) and activated partial thromboplastin time (APTT) (1.43-fold versus 1.16-fold) at steady state than apixaban. Conclusions Despite similar exposure to both drugs, rivaroxaban 20 mg once daily was associated with greater and more sustained inhibition of thrombin generation than apixaban 5 mg twice daily. Sensitive PT and APTT assays can be used to estimate the anticoagulant effects of rivaroxaban.

Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state.

RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics.

Absence of clinically relevant interactions between rivaroxaban--an oral, direct Factor Xa inhibitor--and digoxin or atorvastatin in healthy subjects.

OBJECTIVE: To investigate potential interactions between rivaroxaban, an oral direct Factor Xa inhibitor approved for the management of thromboembolic disorders, and digoxin or atorvastatin. METHODS: Two randomized, phase 1 clinical trials were undertaken in healthy men to assess pharmacokinetic and pharmacodynamic interactions between rivaroxaban and digoxin or atorvastatin, and the safety of these drug combinations. RESULTS: Steady-state rivaroxaban did not affect the pharmacokinetic profile of steady-state digoxin (n = 17). Digoxin did not significantly influence the pharmacokinetic profile of single-dose rivaroxaban and had minimal effects on rivaroxaban-induced inhibition of Factor Xa activity and prolongation of clotting time. Similarly, steady-state atorvastatin did not affect the pharmacokinetic profile or the pharmacodynamics of rivaroxaban and vice versa (n = 19). All drugs (alone or in combination) were well tolerated. CONCLUSIONS: There were no clinically relevant pharmacokinetic or pharmacodynamic interactions between rivaroxaban and digoxin, or between rivaroxaban and atorvastatin, suggesting that rivaroxaban can be coadministered with either drug. This study also confirmed that rivaroxaban does not interact with substrates for permeability (P)-glycoprotein alone (digoxin) or P-glycoprotein and cytochrome P(450) (CYP)3A4 (atorvastatin).

Population model of the pharmacokinetics and pharmacodynamics of rivaroxaban--an oral, direct factor xa inhibitor--in healthy subjects.

OBJECTIVE: Rivaroxaban (BAY 59-7939) is an oral, direct Factor Xa (FXa) inhibitor being developed for the prevention and treatment of thromboembolic disorders. This analysis aimed to define population models for the pharmacokinetics (PK) and pharmacodynamics (PD) ofrivaroxaban in healthy males. METHODS: Non-linear, mixed-effect modeling was used to analyze rivaroxaban plasma concentration and PD data (FXa activity and clotting tests) from subjects in a phase I, multiple-ascending-dose study. Subjects received 5 mg rivaroxaban once, twice or three times daily, or 10, 20 or 30 mg rivaroxaban twice daily. RESULTS: The population PK of rivaroxaban were well described by an oral, two-compartment model with first-order absorption and elimination from the central compartment. Population mean estimates for apparent oral clearance and volume of distribution for the central compartment were 9.2 1/h and 55 1, respectively, with moderate inter-individual variability (17.4% and 30.7%, respectively). Total volume of distribution for rivaroxaban at steady state was approximately 70 1. Residual (unexplained) variability was 25%. FXa activity correlated with rivaroxaban plasma concentrations following an inhibitory Emax model; prothrombin time (PT) and rivaroxaban plasma concentrations correlated with a linear model, with a slope of 4.6 s/(100 microg/1). Inter-individual variability was low for the correlation with PT. The models derived were used to define sampling windows for population PK/PD modeling in Phase II studies. CONCLUSIONS: This analysis confirms that rivaroxaban has predictable, dose-proportional PK and PD. The linear correlation between rivaroxaban plasma concentrations and PT suggests that this test might be useful to assess rivaroxaban exposure in patients, if required.

Isospora suis: an experimental model for mammalian intestinal coccidiosis.

Piglets experimentally infected with 10,000 oocysts of Isospora suis in three identical trials (n = 50) were examined clinically and coproscopically from 5 to 11 days post-infection (d.p.i.), weighed in weekly intervals until the fourth week of life and compared to age-matched asymptomatic controls (n = 17). Furthermore, 17 infected piglets were histologically examined on days 5-14 p.i. Infected animals had a significantly lower weight gain than the controls and showed diarrhoea throughout, with maximum prevalence and intensity on 6 d.p.i. Half of the animals had diarrhoea for only 2 days or less. The number of diarrhoea days was negatively correlated with weight gain. Oocyst excretion started on 5 d.p.i. with peak prevalences and declined afterwards; a smaller peak was seen on 10 d.p.i. All animals excreted parasites at least once, and most of them excreted for 5-7 days. Oocyst excretion intensity paralleled the prevalence and ranged from 220 to 251,501 oocysts per gram of faeces (opg). Most samples contained 4 x 10(3) to 4 x 10(4) opg. The opg values were negatively correlated with faecal scores (samples with diarrhoea contained less oocysts) of the same day and the previous day. Histologically, necrosis followed by atrophy of the villi was most pronounced in the early stage of infection throughout the jejunum and ileum but declined thereafter. On 14 d.p.i., villous atrophy was still noticeable in the jejunum. Histology is difficult to quantify and requires large animal numbers, although the effects are visible for some time. Weight gain and faecal score can be affected by other factors than parasite infection. From the compiled data, we conclude that the established model is suitable to study piglet isosporosis with oocyst excretion being the most reliable parameter, although individual variations are considerable. A negative correlation between excretion and diarrhoea may be responsible for the difficulties in the detection of the parasite in field samples.

Feasibility and potential gains of enhancing the subacute rat study protocol (OECD test guideline no. 407) by additional parameters selected to determine endocrine modulation. A pre-validation study to determine endocrine-mediated effects of the antiandrogenic drug flutamide.

Groups of five male and five female Wistar rats were treated by gavage with 0, 1, 10, and 100 mg flutamide/kg body weight for at least 28 days to investigate whether proposed enhancements to the current subacute rodent OECD test guideline no. 407 could be included into the testing routine, which of the current and/or additional parameters would detect endocrine-mediated effects of flutamide reliably and sensitively, and to provide information on intra-laboratory variability. Two identical studies were performed concurrently. The enhanced protocol requests the additional determination of the specific hormones triiodothyronine, thyroxine, thyroid stimulating hormone, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, prolactin, testosterone, corticosterone; of oestrus cyclicity and necropsy of all females in the dioestrus stage; of the number of homogenization-resistant testicular spermatids and the number, motility, viability, and morphology of cauda epididymal spermatozoa; of additional organ weights (pituitary, ovaries, uterus, thyroid, male accessory reproductive organs); and of the histopathology of additional organs (pituitary, epididymides, coagulation glands, pancreas, vagina). From a technical standpoint, it was possible to conduct a study according to the enhanced protocol, however, with substantial additional effort, an increase in costs by some 67%, and logistic problems. In line with the specific pharmacological effect of flutamide, treatment-related changes were mainly found in male rats, while females were hardly affected by 100 mg/kg. In male rats, 100 mg/kg was the maximal tolerated dose resulting in reduced body weight gain, but no or little other effects on clinical, haematological, clinico-chemical, or behavioral parameters, and 1 mg/kg was the no-observed-adverse-effect level. Antagonism of peripheral androgen receptors by flutamide resulted in decreased relative organ weights of male accessory reproductive organs, changes that were reliably detected in both studies at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological changes were also detected reliably at 100 mg/kg. Antagonism of central androgen receptors by flutamide increased LH and FSH levels. LH stimulation of testicular Leydig cells in turn increased testosterone and estradiol levels. Again, all these changes were detected reliably at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological alterations (increase of LH- and FSH-secreting cells, Leydig cell hypertrophy) were detected reliably and sensitively at 10 mg/kg. Studies on liver enzymes performed outside the scope of the enhanced protocol showed that flutamide at 100 mg/kg generally induced hepatic enzyme activities, but decreased the activity of the sex-specific testosterone-dependent liver enzyme CYP2C11 in male rats. The laboratory methods employed yielded reliable results, i.e., 93.6% of the quantitative measurements obtained in both studies were in agreement. Doubling the animal number from five to ten per sex and dose does not increase the sensitivity of detection of endocrine-mediated effects above the level already provided by histopathological examination of groups of five animals. Some of the proposed enhancements evaluated (additional organgravimetry and histopathology) were helpful in detecting the endocrine-mediated effects of flutamide reliably, while others did not contribute towards this aim (spermatology resulted in doubtful effects, female cyclicity was not affected, hormone determinations provided mechanistic information). Ongoing testing according to the revised version of the enhanced OECD test guideline no. 407 protocol and using ten compounds interfering with the endocrine system by different mechanisms will result in the identification of the most appropriate enhancements.

Quantitative distribution studies in animals: cross-validation of radioluminography versus liquid-scintillation measurement.

The results of a cross-validation of the radioluminography (RLG) and liquid scintillation counting (LSC) methods are presented. The methods for the determination of radioactivity concentrations were compared in 16 organs, after administration of (14)C-labeled substances to rats. LSC measurements of two kinds were used as reference methods for RLG: (1) quantitative determination of radioactivity after conventional dissection (interindividual comparison) and (2) quantitative determination of radioactivity in tissue punches taken from the whole-body sections after they had undergone RLG measurement (intraindividual comparison). Blood standards containing known concentrations were used for calibration. For statistical evaluation log-linear regression analysis of paired concentration values and organ-specific 95% confidence intervals of the log-transformed RLG/LSC concentration quotients were compared. For most organs, the slopes of the regression lines and the means of the concentration quotients were within the defined equivalence range of 0.80-1.25. Deviations were distinctly smaller in the intraindividual comparison. For some organs, however, it became clear that found concentrations were affected by self-absorption (RLG) and by differences in sample preparation (LSC). In conclusion, quantification with RLG is a reliable and reproducible method with comparable measurement precision and greater accuracy in respect of tissue localization, compared to LSC (dissection).

Enzyme immunoassay for the measurement of human tenascin-C on the Bayer Immuno 1 analyzer.

OBJECTIVES: To evaluate a new tenascin-C assay performed on the Bayer Immuno 1 system. DESIGN AND METHODS: The precision was measured using three levels of serum pools. Linearity was tested by diluting patient serum samples containing high tenascin-C concentrations, and the minimal detectable concentration determined by repetitive analysis of the zero calibrator. Preliminary reference intervals were determined by testing serum samples from 220 healthy individuals. Biovariability was estimated in a cohort of 20 apparently healthy subjects over 18 days. The levels of tenascin-C in patients with different liver diseases was tested. RESULTS: The detection limit was 2 ng/mL. At concentrations ranging from 325 to 1957 ng/mL the assay demonstrated within-run and between-run CVs ranging from 4% to 3.6% and 8.4% to 6.7%, respectively. Dilutions of sera were linear and parallel to the standard curve with recoveries ranging from 97% to 100%. The reference interval (central 95% interval) for tenascin-C in serum of healthy adults was 199-906 ng/mL. The variability study yielded an analytical variability, CV(A), of 1.8%; a within-subject variability, CV(I), of 11.7%; and a between-subject variability, CV(G), of 39.3%. Tenascin-C concentrations in sera of liver disease patients were significantly increased. CONCLUSIONS: The novel assay provides a rapid and reliable procedure for the determination of tenascin-C levels in human sera.

A note on distribution of human plasma levels of ascorbic and dehydroascorbic acid.

Ascorbic acid concentrations in 102 human plasma samples ranged from 1 to 15 micrograms ml-1 with a mean concentration at about 8 micrograms ml-1, corresponding to the results of other authors. Dehydroascorbic acid was found only in traces, independent of ascorbic acid concentrations. The ascorbic acid concentrations in plasma of four persons, examined twice with a four-years interim period revealed no obvious differences over time. It is suggested that the variability of plasma ascorbic acid concentrations is mainly determined by long-term dietary habits.