A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Platelet inhibition with standard vs. lower maintenance dose of ticagrelor early after myocardial infarction (ELECTRA): a randomized, open-label, active-controlled pharmacodynamic and pharmacokinetic study.

AIMS: Currently available data indicate that reduction of ticagrelor maintenance dose (MD) 1-3 years after acute myocardial infarction (AMI) not only provides sufficient platelet inhibition but also can improve ticagrelor's safety profile. The aim of this study was to compare the antiplatelet effect of reduced and standard ticagrelor MD in stable patients beginning 1 month after AMI. METHODS AND RESULTS: In a single-centre, randomized, open-label, active-controlled trial, on Day 30 following AMI, 52 patients (26 in each study arm) were assigned in a 1:1 ratio to receive either reduced (60 mg b.i.d) or standard (90 mg b.i.d) ticagrelor MD for the following 2 weeks. On Day 45 after AMI the antiplatelet effect of ticagrelor was evaluated with the VASP assay and Multiplate, and there were no significant differences in platelet inhibition between patients on reduced vs. standard MD [VASP: 10.4 (5.6-22.2) vs. 14.1 (9.4-22.1) platelet reactivity index; P = 0.30; Multiplate: 30.0 (24.0-39.0) vs. 26.5 (22.0-35.0) U; P = 0.26]. Likewise, no differences were found regarding the prevalence of on-ticagrelor high platelet reactivity between patients on ticagrelor 60 mg b.i.d vs. 90 mg b.i.d (VASP: 4% vs. 8%; P = 0.67; Multiplate: 15% vs. 8%; P = 0.54). Administration of reduced MD resulted in proportionally lower plasma concentrations of ticagrelor and its active metabolite on Day 45 after AMI. CONCLUSION: These results suggest that lowering ticagrelor MD 1 month after AMI confers an adequate antiplatelet effect that is comparable to the standard dose. The tested strategy warrants further research to assess its clinical efficacy and safety. CLINICALTRIALS.GOV IDENTIFIER: NCT03251859.

METoclopramide Administration as a Strategy to Overcome MORPHine-ticagrelOr Interaction in PatientS with Unstable Angina PectorIS-The METAMORPHOSIS Trial.

Extensive search for methods of overcoming morphine-related delay of the absorption and onset of action of oral P2Y12 inhibitors in patients presenting with acute coronary syndrome is on-going. The aim of the trial was to investigate whether metoclopramide co-administration could reduce this delay and improve the pharmacokinetics (PKs) and pharmacodynamics (PDs) of ticagrelor and its active metabolite AR-C124900XX. Plasma concentration of both compounds and platelet reactivity were evaluated in nine pre-defined time points within 6 hours after administration of ticagrelor loading dose. The results of our study show that mean platelet activity within the first hour was noticeably higher in metoclopramide-naive patients. Moreover, ticagrelor mean plasma concentration was significantly higher within the initial four time points (15, 30, 45, 60 minutes) in patients receiving metoclopramide (p = 0.039; p = 0.009; p = 0.005; p = 0.008, respectively). To conclude, the co-administration of metoclopramide in patients presenting with unstable angina and treated with morphine, has a beneficial effect on the PK/PD profile of ticagrelor and its metabolite; however, its impact on ST-elevation myocardial infarction patients requires further investigation.

Impact of levosimendan on platelet function.

Levosimendan has been developed for treatment of severe heart failure. The favorable hemodynamic effect of levosimendan is related to its unique dual mechanism of action - increase of the contractile force of the myocardium caused by enhanced sensitivity of myofilaments to calcium combined with vasodilatation caused by the opening of adenosine triphosphate - dependent potassium channels. Due to the structural similarities to phosphodiesterase inhibitors it may partly exert its action via inhibition of phosphodiesterase inhibitors III. Inhibition of the phosphodiesterase inhibitors III leads to an increase of intracellular concentration of cyclic adenosine monophosphate causing an anti-aggregatory effect. There are some contradictory or indirect and inconclusive reports related to the impact of levosimendan on platelet function. The aim of this systematic review was to critically discuss the impact of levosimendan on platelet function according to currently available knowledge based on the findings of experimental as well as observational and randomized clinical studies.

Comparison of Ticagrelor Pharmacokinetics and Pharmacodynamics in STEMI and NSTEMI Patients (PINPOINT): protocol for a prospective, observational, single-centre study.

INTRODUCTION: The most common classification of acute myocardial infarction (AMI) is based on electrocardiographic findings and distinguishes ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI). Both types of AMI differ concerning their epidemiology, clinical approach and early outcomes. Ticagrelor is a P2Y12 receptor inhibitor, constituting the first-line treatment for STEMI and NSTEMI. According to available data, STEMI may be associated with lower plasma concentration of ticagrelor in the first hours of AMI, but currently there are no studies directly comparing ticagrelor pharmacokinetics or antiplatelet effect in patients with STEMI versus NSTEMI. METHODS AND ANALYSIS: The PINPOINT study is a phase IV, single-centre, investigator-initiated, prospective, observational study designed to compare the pharmacokinetics and pharmacodynamics of ticagrelor in patients with STEMI and NSTEMI assigned to the invasive strategy of treatment. Based on an internal pilot study, the trial is expected to include at least 23 patients with each AMI type. All subjects will receive a 180 mg loading dose of ticagrelor. The primary end point of the study is the area under the plasma concentration-time curve (AUC(0-6)) for ticagrelor during the first 6 hours after the loading dose. Secondary end points include various pharmacokinetic features of ticagrelor and its active metabolite (AR-C124910XX), and evaluation of platelet reactivity by the vasodilator-stimulated phosphoprotein assay and multiple electrode aggregometry. Blood samples for the pharmacokinetic and pharmacodynamic assessment will be obtained at pretreatment, 30 min, 1, 2, 3, 4, 6 and 12 hours post-ticagrelor loading dose. ETHICS AND DISSEMINATION: The study received approval from the Local Ethics Committee (Komisja Bioetyczna Uniwersytetu Mikolaja Kopernika w Toruniu przy Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy; approval reference number KB 617/2015). The study results will be disseminated through conference presentations and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT02602444; Pre-results.

Crushed sublingual versus oral ticagrelor administration strategies in patients with unstable angina. A pharmacokinetic/pharmacodynamic study.

Oral administration of crushed ticagrelor tablets turned out to be an efficacious method that improves its pharmacokinetics and pharmacodynamics. This strategy, however, is unlikely to eliminate the drug-drug interaction in patients receiving intravenous morphine, as the impairment of the P2Y12 inhibitor absorption related to decreased propulsive motility of the gastro-intestinal tract is the most likely mechanism of interaction. Thus, we designed a pharmacokinetic and pharmacodynamic study setting the feasibility of platelet inhibition with a loading dose of ticagrelor given as crushed tablets sublingually compared with two other ticagrelor loading dose administration strategies: integral tablet given orally and crushed tablet given orally in patients with unstable angina. Ticagrelor and its metabolite AR-C124900XX plasma concentration was evaluated in nine time points (time frame of 6 hours) using liquid chromatography coupled with mass spectrometry; platelet reactivity was evaluated using multiple electrode aggregometry. The area under the plasma concentration-time curve for ticagrelor and AR-C124900XX was significantly higher in patients treated with crushed tablets given orally compared with crushed tablets given sublingually only within the first hour after loading dose (936.9 ± 898.0 vs 368.0 ± 422.4, p=0.042 and 103.4 ± 120.8 vs 31.3 ± 43.9, p=0.031, respectively). Moreover, we showed significantly stronger platelet inhibition in patients receiving crushed ticagrelor orally vs. sublingually at 30 and 45 min after the loading dose (p=0.024 and p=0.016, respectively). Therefore, the administration strategy of ticagrelor determines the pharmacokinetic and pharmacodynamic profile of both ticagrelor and its active metabolite AR-C124900XX.

Which platelet function test best reflects the in vivo plasma concentrations of ticagrelor and its active metabolite? The HARMONIC study.

Aim of this study was assessment of the relationship between concentrations of ticagrelor and its active metabolite (AR-C124910XX) and results of selected platelet function tests. In a single-centre, cohort study, patients with myocardial infarction underwent blood sampling following a 180 mg ticagrelor loading dose intake (predose, 1, 2, 3, 4, 6, 12, 24 hours postdose) to perform pharmacokinetic and pharmacodynamic assessments. Platelet reactivity was evaluated using the VASP-assay, the VerifyNow device and the Multiplate analyzer. Analysis of 36 patients revealed high negative correlations between ticagrelor concentrations and platelet reactivity evaluated with all three platelet function tests (the VASP-assay: RS=-0.722; p<0.0001; the VerifyNow device: RS=-0.715; p<0.0001; the Multiplate analyzer: RS=-0.722; p<0.0001), with no significant differences between correlation coefficients. Similar results were found for AR-C124910XX. Platelet reactivity values assessed with all three methods generally correlated well with each other; however, a significantly higher correlation (p<0.02) was demonstrated between the VerifyNow and Multiplate tests (RS=0.707; p<0.0001) than in other assay combinations (the VASP-assay and the VerifyNow device: RS=0.595; p<0.0001; the VASP-assay and the Multiplate analyzer: RS=0.588; p<0.0001). With respect to the recognition of high platelet reactivity, we found higher measurement concordance between the VerifyNow and Multiplate tests compared with other assay combinations, while for low platelet reactivity, only results of the VerifyNow and Multiplate assay were related to each other. Platelet reactivity measurements performed with the VASP, VerifyNow and Multiplate tests show comparably strong negative correlations with ticagrelor and AR-C124910XX concentrations.

Morphine delays and attenuates ticagrelor exposure and action in patients with myocardial infarction: the randomized, double-blind, placebo-controlled IMPRESSION trial.

AIMS: The currently available data indicate a drug-drug interaction between morphine and oral P2Y12 receptor inhibitors, when administered together. The aim of this trial was to assess the influence of infused morphine on pharmacokinetics and pharmacodynamics of ticagrelor and its active metabolite (AR-C124910XX) in patients with acute myocardial infarction. METHODS AND RESULTS: In a single-centre, randomized, double-blind trial, patients were assigned in a 1:1 ratio to receive intravenously either morphine (5 mg) or placebo, followed by a 180 mg loading dose of ticagrelor. Pharmacokinetics was determined with liquid chromatography tandem mass spectrometry and ticagrelor antiplatelet effects were measured with up to three different platelet function tests: vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode aggregometry and VerifyNow. The pharmacokinetic and pharmacodynamic assessment was performed in 70 patients (35 in each study group). Morphine lowered the total exposure to ticagrelor and its active metabolite by 36% (AUC(0-12): 6307 vs. 9791 ng h/mL; P = 0.003), and 37% (AUC(0-12): 1503 vs. 2388 ng h/mL; P = 0.008), respectively, with a concomitant delay in maximal plasma concentration of ticagrelor (4 vs. 2 h; P = 0.004). Multiple regression analysis showed that lower AUC(0-12) values for ticagrelor were independently associated with the administration of morphine (P = 0.004) and the presence of ST-segment elevation myocardial infarction (P = 0.014). All three methods of platelet reactivity assessment showed a stronger antiplatelet effect in the placebo group and a greater prevalence of high platelet reactivity in patients receiving morphine. CONCLUSIONS: Morphine delays and attenuates ticagrelor exposure and action in patients with myocardial infarction. ClinicalTrials.gov Identifier: NCT02217878.

Alpha-methylacyl-CoA racemase and hepsin as urinary prostate cancer markers.

BACKGROUND: Because of the numerous limitations of prostate-specific antigen (PSA), α-methylacyl-CoA racemase (AMACR) and hepsin have recently been suggested as potential biomarkers in prostate cancer (PC). This report presents a comparison study of the presence of AMACR and hepsin in urine collected before and after digital rectal examination (DRE) as a previously suggested diagnostic marker for PC. METHODS: Seventy-six urine samples (38 before and 38 after prostate massage) from patients with benign prostate hyperplasia (BPH) and 66 urine samples (33 before and 33 after prostate massage) from patients with PC were analyzed. PC was confirmed by prostate biopsy. Urinary levels of AMACR and hepsin were determined by ELISA and related to the tumor stage, Gleason score and PSA level. RESULTS: AMACR and hepsin levels in urine collected after prostate massage were higher only in the PC group. There were no correlations between AMACR levels, hepsin levels, tumor stage and Gleason score. AMACR and hepsin did not differentiate between BPH and PC with better true positive and false negative rates than serum PSA. CONCLUSIONS: AMACR and hepsin were unable to diagnose PC with better true positive and false negative rates than PSA. An additional procedure combined with other markers should be applied for the reliable diagnosis of PC.

Engrailed-2 protein as a potential urinary prostate cancer biomarker: a comparison study before and after digital rectal examination.

This study was designed to compare and evaluate the presence of engrailed-2 (EN2) protein in urine collected before and after prostate massage as a diagnostic marker for prostate cancer (PCa). We analysed and compared 76 urine samples (38 before and 38 after prostate massage) from the benign group (BPH) and 66 urine samples (33 before and 33 after prostate massage) from patients with PCa confirmed by prostate biopsy. EN2 levels from the PCa and men with BPH (age range 50-82) were related to the tumour stage, Gleason score and prostate-specific antigen. EN2 levels were determined by enzyme-linked immunosorbent assay in urine. The median EN2 levels in urine after prostate massage were significantly different from those determined in urine before prostate massage (1.25 ng/ml in the PCa group and 0.34 ng/ml in the BPH). The mean EN2 levels in PCa patients were 3.76-fold higher than those in non-PCa patients after prostate massage. The distinct influence of prostate massage on EN2 levels was found to be related to the Gleason score and tumour stage. EN2 may be considered a marker of PCa with certain limitations, such as those related to tumour staging. The specificity and sensitivity of the protocol are highly dependent on prostate massage.

Siderophore-drug complexes: potential medicinal applications of the 'Trojan horse' strategy.

The ability of bacteria to develop resistance to antimicrobial agents poses problems in the treatment of numerous bacterial infections. One method to circumvent permeability-mediated drug resistance involves the employment of the 'Trojan horse' strategy. The Trojan horse concept involves the use of bacterial iron uptake systems to enter and kill bacteria. The siderophore-drug complex is recognized by specific siderophore receptors and is then actively transported across the outer membrane. The recently identified benefits of this strategy have led to the synthesis of a series of siderophore-based antibiotics. Several studies have shown that siderophore-drug conjugates make it possible to design antibiotics with improved cell transport and reduce the frequency of resistance mutants. Growing interest in siderophore-drug conjugates for the treatment of human diseases including iron overload, cancer, and malaria has driven the search for new siderophore-drug complexes. This strategy may have special importance for the development of iron oxide nanoparticle-based therapeutics.

Tropomyosin-1 protects endothelial cell-cell junctions against cigarette smoke extract through F-actin stabilization in EA.hy926 cell line.

The aim of the study was to estimate the effect of cigarette smoke extract (CSE) on EA.hy926 endothelial cells in culture in the context of maintenance of cell-cell junctions through the structural stabilization of the actin cytoskeleton. In the present study, F-actin was stabilized by the overexpression of tropomyosin-1, which is known to stabilize actin filaments in muscle and non-muscle cells. Our study showed that the stabilization of F-actin significantly increased the survival of cells treated with 25% CSE. In addition, after stabilization of F-actin the migratory potential of EA.hy926 cells subjected to CSE treatment was increased. Our results also showed increased fluorescence intensity of alpha- and beta-catenin after CSE treatment in cells which had stabilized F-actin. Analysis of fluorescence intensity of Zonula occludens-1 did not reveal any significant differences when EA.hy926 cells overexpressing tropomyosin-1 were compared with those lacking overexpression. It would appear that overexpression of tropomyosin-1 preserved the structure of actin filaments in the cells treated with CSE. In conclusion, the present study demonstrates that stabilization of F-actin protects EA.hy926 cells against CSE-induced loss of both adherens and tight junctions. The data presented in this study suggest that overexpression of tropomyosin-1 stabilizes the organizational structure of actin filaments and helps preserve the endothelial barrier function under conditions of strong oxidative stress.

Importance of retention data from affinity and reverse-phase high-performance liquid chromatography on antitumor activity prediction of imidazoacridinones using QSAR strategy.

Quantitative structure-activity relationships (QSAR) studies for prediction of cytotoxic and antitumor activity of imidazoacridinones (IA) based on experimentally obtained high-performance liquid chromatography (HPLC) retention data and calculated parameters using computational (molecular modeling) medicinal chemistry methods were proposed. The RP-HPLC and affinity-HPLC chromatographic techniques with four diversified HPLC systems applying columns with octadecylsilanes (C18), phosphatidylcholine (IAM), as well as α(1)-glycoprotein (AGP) and albumin (HSA) were used for the determination of the retention constants logk and logk(w) which characterize lipophilicity and protein affinity of IA. Moreover, molecular modeling studies were performed using HyperChem and Dragon software's, and structural descriptors were calculated and subsequently used. The QSAR equations using multiple linear regression (MLR) analysis method were derived which indicated that in vivo antileukemia activity of IA depends on cytotoxic activity against leukemia cells, whereas this cytotoxic activity depends on logk and logk(w) parameters obtained on all HPLC systems. Moreover, the QSRR equations were derived and indicated that logk and logk(w) parameters depend on calculated non-empirical structural parameters. The predictive power of obtained QSAR and QSRR equations allowed the prediction of cytotoxic and antitumor activity of IA and also their HPLC retention parameters. Finally, the equations can be used for prediction of antileukemia activity of IA without the necessity of carrying out experimental measurements.

A new approach to determine camptothecin and its analogues affinity to human serum albumin.

A novel and fast method for the determination of the binding kinetic data of ligand to protein has been developed. A new tool including human serum albumin-coated magnetic beads (HSA-MB) was used to determine the affinity of camptothecin (CPT) and its analogues to HSA. From the biological activity point of view, these compounds have potential anticancer activity. However, the numerous studies indicate that some of these analogues have a strong affinity to plasma proteins stopping their effective therapy. Thus, the problem of plasma protein binding behavior of CPT's analogues was the subject of this study.

Contribution of artificial intelligence to the knowledge of prognostic factors in Hodgkin's lymphoma.

Hodgkin's lymphoma is one of the most curable malignancies and most patients achieve a lasting complete remission. In this study, artificial neural network (ANN) analysis was shown to provide significant factors with regard to 5-year recurrence after lymphoma treatment. Data from 114 patients treated for Hodgkin's disease were available for evaluation and comparison. A total of 31 variables were subjected to ANN analysis. The ANN approach as an advanced multivariate data processing method was shown to provide objective prognostic data. Some of these prognostic factors are consistent or even identical to the factors evaluated earlier by other statistical methods.