Regional differences in physicians' behavior and factors influencing the intensity of PCSK9 inhibitor therapy with alirocumab: a subanalysis of the ODYSSEY APPRISE study.

Background: Despite better accessibility of the effective lipid-lowering therapies, only about 20% of patients at very high cardiovascular risk achieve the low-density lipoprotein cholesterol (LDL-C) goals. There is a large disparity between European countries with worse results observed for the Central and Eastern Europe (CEE) patients. One of the main reasons for this ineffectiveness is therapeutic inertia related to the limited access to appropriate therapy and suitable dosage intensity. Thus, we aimed to compare the differences in physicians' therapeutic decisions on alirocumab dose selection, and factors affecting these in CEE countries vs. other countries included in the ODYSSEY APPRISE study. Methods: ODYSSEY APPRISE was a prospective, single-arm, phase 3b open-label (≥12 weeks to ≤30 months) study with alirocumab. Patients received 75 or 150 mg of alirocumab every 2 weeks, with dose adjustment during the study based on physician's judgment. The CEE group in the study included Czechia, Greece, Hungary, Poland, Romania, Slovakia, and Slovenia, which we compared with the other nine European countries (Austria, Belgium, Denmark, Finland, France, Germany, Italy, Spain, and Switzerland) plus Canada. Results: A total of 921 patients on alirocumab were involved [modified intention-to-treat (mITT) analysis], including 114 (12.4%) subjects from CEE countries. Therapy in CEE vs. other countries was numerically more frequently started with lower alirocumab dose (75 mg) at the first visit (74.6 vs. 68%, p = 0.16). Since week 36, the higher dose was predominantly used in CEE patients (150 mg dose in 51.6% patients), which was maintained by the end of the study. Altogether, alirocumab dose was significantly more often increased by CEE physicians (54.1 vs. 39.9%, p = 0.013). Therefore, more patients achieved LDL-C goal at the end of the study (<55 mg/dl/1.4 mmol/L and 50% reduction of LDL-C: 32.5% vs. 28.8%). The only factor significantly influencing the decision on dose of alirocumab was LDL-C level for both countries' groups (CEE: 199.2 vs. 175.3 mg/dl; p = 0.019; other: 205.9 vs. 171.6 mg/dl; p < 0.001, for 150 and 75 mg of alirocumab, respectively) which was also confirmed in multivariable analysis (OR = 1.10; 95% CI: 1.07-1.13). Conclusions: Despite larger unmet needs and regional disparities in LDL-C targets achievement in CEE countries, more physicians in this region tend to use the higher dose of alirocumab, they are more prone to increase the dose, which is associated with a higher proportion of patients reaching LDL-C goals. The only factor that significantly influences decision whether to increase or decrease the dose of alirocumab is LDL-C level.

Identification and structural characterization of three psychoactive substances, phenylpiperazines (pBPP and 3,4-CFPP) and a cocaine analogue (troparil), in collected samples.

PURPOSE: New psychoactive substances (NPSs) still appear on the market, mainly due to their legal status. This situation indicates and alarms that permanent recognition of the designer drug scene should be conducted. In this paper, we describe the detection of three psychoactive substances in samples collected from drug users. METHODS: Qualitative characterization was performed using liquid chromatography-high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyzer, gas chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: In this study, we reported the detection and structural elucidation of three psychoactive substances: 1-(4-bromophenyl)piperazine (pBPP), 1-(3-chloro-4-fluorophenyl)piperazine (3,4-CFPP) and methyl 8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-4-carboxylate (troparil). CONCLUSIONS: To the best of our knowledge, this is the first report that presents an identification methodology for these substances found in illegal products. Comprehensive characterization of the NPSs presented in this paper facilitates their detection and identification by forensic and clinical laboratories.

The occurrence of non-anatomical therapeutic chemical-international nonproprietary name molecules in suspected illegal or illegally traded health products in Europe: A retrospective and prospective study.

The General European Official Medicines Control Laboratory (OMCL) Network (GEON), co-ordinated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), regularly organises market surveillance studies on specific categories of suspected illegal or illegally traded products. These studies are generally based on a combination of retrospective and prospective data collection over a defined period of time. This paper reports the results of the most recent study in this context with the focus on health products containing non-Anatomical Therapeutic Chemical-International Nonproprietary Name (ATC-INN) molecules. In total 1104 cases were reported by 16 countries for the period between January 2017 and the end of September 2019. The vast majority of these samples (83%) were collected from the illegal market, while only 3% originated from a legal source. For the rest of the samples, categorisation was not possible. Moreover, 69% of all the reported samples were presented as medicines, including sexual performance enhancers, sports performance enhancers, physical performance enhancers and cognitive enhancers or nootropic molecules that act on the central nervous system (CNS). Although the popularity of anabolics, PDE-5 inhibitors and CNS drugs in illegal products has already been reported, the study showed some new trends and challenges. Indeed, 11% of the samples contained molecules of biological origin, that is, research peptides, representing the second most reported category in this study. Furthermore, the study also clearly shows the increasing popularity of Selective Androgen Receptor Modulators and nootropics, two categories that need attention and should be further monitored.

Controlled Drug Release and Cytotoxicity Studies of Beta-Lapachone and Doxorubicin Loaded into Cyclodextrins Attached to a Polyethyleneimine Matrix.

This work presents a new look at the application of cyclodextrins (CD) as a drug nanocarrier. Two different cyclodextrins (αCD, ßCD) were covalently conjugated to branched polyethylenimine (PEI), which was additionally functionalized with folic acid (PEI-ßCD-αCD-FA). Here, we demonstrated that the combination of αCD and ßCD enabled to load and control release of two anticancer drugs: doxorubicin (DOX) and beta-lapachone (beta-LP) (DOX in ß-CD and beta-LP into α-CD) via host-guest inclusion. The PEI-ßCD(DOX)-αCD-FA nanoconjugate was used to transport anticancer drugs into A549 lung cancer cells for estimation the cytotoxic and antitumor effect of this nanoconjugate. The presence of FA molecules should facilitate the penetration of studied nanoconjugate into the cell. Whereas, the non-cellular experiments proved that the drugs are released from the carrier mainly in the pH 4.0. The release mechanism is found to be anomalous in all studied cases.

Supramolecular Interactions between ß-Cyclodextrin and the Nucleobase Derivatives of Ferrocene.

Novel conjugates of ferrocene with uracil, 5-fluorouracil, tegafur, or acyclovir are reported. Their synthesis involved (i) the azide-alkyne 1,3-dipolar cycloaddition or (ii) the formation of the ester linkage. For the first time, we present an in-depth insight into the supramolecular interactions between ß-cyclodextrin and ferrocene-nucleobase derivatives. Spectroscopic and voltammetric analyses performed within this work suggested that the ferrocene or adamantane unit of the conjugates interacted with the ß-cyclodextrin's inner cavity. The methods applied for the supramolecular studies included 1H-1H ROESY NMR, 1H NMR titration, Fourier-transform infrared spectroscopy, cyclic voltammetry, fluorescence spectra titration, and 1H DOSY NMR. 1H DOSY NMR was also employed to evaluate the apparent binding constants for all the complexes. The ferrocene-acyclovir conjugate Fc-5 featured the highest apparent binding constant value among all the complexes tested.

Nanocomposites as biomolecules delivery agents in nanomedicine.

Nanoparticles (NPs) are atomic clusters of crystalline or amorphous structure that possess unique physical and chemical properties associated with a size range of between 1 and 100 nm. Their nano-sized dimensions, which are in the same range as those of vital biomolecules, such as antibodies, membrane receptors, nucleic acids, and proteins, allow them to interact with different structures within living organisms. Because of these features, numerous nanoparticles are used in medicine as delivery agents for biomolecules. However, off-target drug delivery can cause serious side effects to normal tissues and organs. Considering this issue, it is essential to develop bioengineering strategies to significantly reduce systemic toxicity and improve therapeutic effect. In contrast to passive delivery, nanosystems enable to obtain enhanced therapeutic efficacy, decrease the possibility of drug resistance, and reduce side effects of "conventional" therapy in cancers. The present review provides an overview of the most recent (mostly last 3 years) achievements related to different biomolecules used to enable targeting capabilities of highly diverse nanoparticles. These include monoclonal antibodies, receptor-specific peptides or proteins, deoxyribonucleic acids, ribonucleic acids, [DNA/RNA] aptamers, and small molecules such as folates, and even vitamins or carbohydrates.

Reduced graphene oxide doping with nanometer-sized ferrocene moieties - New active material for glucose redox sensors.

Herein, we present that the reduced graphene oxide (rGO) doped with nanometer-sized ferrocene moieties is a new, excellent active material for redox sensors. Two distinct approaches were utilized for the modification of rGO. The first method was based on the covalent decoration of rGO via the addition of azomethine ylide generated from the ferrocenecarboxaldehyde oxime. The second approach utilized the adsorption of 1,1'-ferrocenedicarboxylic acid on the graphene sheet via the π-π stacking. The morphology of the synthesized graphene materials was studied by application of microscopic techniques, whereas the Raman data allowed the characteristics of the tested materials in terms of their structural properties. The tested graphene materials doped with ferrocene moieties were used as a bioactive platform for glucose oxidase (GOx) immobilization. The enzyme was immobilized onto the rGO materials in two ways: (i) using a crosslinking agent - glutaraldehyde (GA) and (ii) by formation of the amide bonds between carboxylic groups of rGO-Fc(COOH)2 and amine groups from enzyme. Ferrocene moieties present at the graphene surface play the role of mediator in the electron transfer between the redox center of GOx and the electrode surface. The functionality of the constructed biosensors has been tested on real samples. The results of the recovery rates showed a satisfying degree of accuracy toward determination of glucose concentration. Examination of the potential interfering species has demonstrated favorable sensitivity and selectivity of the designed biosensor for the detection of glucose.

Identification of a novel growth hormone releasing peptide (a glycine analogue of GHRP-2) in a seized injection vial.

Growth hormone releasing peptides (GHRPs) are synthetic peptides with the ability to stimulate human growth hormone (hGH) secretion. Several GHRPs have been developed as drug candidates; however, only one of them, GHRP-2 (Pralmorelin), has received a clinical approval. Nevertheless, they are distributed on the black market and misused by cheating athletes, due to their performance-enhancing effects. Hence, GHRPs have been included in the World-Anti-Doping-Agency's Prohibited List as forbidden substances in sport. Predominantly, analytical methods for detection and unequivocal identification of doping substances are based on mass spectrometry. Therefore, in the present work, a qualitative analysis by liquid chromatography coupled to high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyzer was performed to identify a new heptapeptide (MW = 874.02 Da) - a glycine analogue of GHRP-2. Structure determination using de novo sequencing is described here in detail. The results of this study may indicate a new approach to circumvent a detection of doping practices.

Functionalization of graphene: does the organic chemistry matter?

Reactions applying amidation- or esterification-type processes and diazonium salts chemistry constitute the most commonly applied synthetic approaches for the modification of graphene-family materials. This work presents a critical assessment of the amidation and esterification methodologies reported in the recent literature, as well as a discussion of the reactions that apply diazonium salts. Common misunderstandings from the reported covalent functionalization methods are discussed, and a direct link between the reaction mechanisms and the basic principles of organic chemistry is taken into special consideration.

Distribution of polyethylenimine in zebrafish embryos

Background: Polyethylenimine (PEI) plays important roles in the pharmaceutical design of non-viral gene delivery systems. Due to a set of unique physicochemical properties this cationic polymer has a great potential in modern gene therapies. Objective: The aim of the present study was to determine the distribution of branched PEI (0.8 kDa) in zebrafish embryos (Danio rerio). Material and methods: Zebrafish embryos at 3 hours post-fertilization (hpf) were incubated with PEI (10 µg/ml) for 24 and 48 hours and studied using the confocal laser microscopy. Results: The obtained results show that PEI effectively distributed into the layers of the chorion and yolk sac in developing embryos due to first 24 hours of exposure. In contrast, PEI was found in the yolk, head, trunk and tail of the embryos due to prolonged treatments (48 hours). Conclusion: The study evidences a high distribution of the branched PEI (0.8 kDa) polymer in the zebrafish embryo tissues.

Recent developments in the synthesis and applications of graphene-family materials functionalized with cyclodextrins.

The introduction of cyclodextrin species to graphene-family materials (GFMs) constitutes an important area of research, especially in terms of the development of applied nanoscience. The chemistry of cyclodextrins is the so-called host-guest chemistry, which has impacted on many fields of research, including catalysis, electrochemistry and nanomedicine. Cyclodextrins are water-soluble and biocompatible supramolecules, and therefore they may introduce new interesting properties to GFMs and may enhance the physicochemical/biological features of native GFMs. The reported methods for the conjugation of cyclodextrins to GFMs utilize either covalent or non-covalent approaches. The recent progress in the applications of GFMs functionalized with cyclodextrins, with the respect to the chemistry and features of these conjugates, is discussed. Special consideration is also given to the recent developments in (i) nanomedicine, (ii) electrochemistry, (iii) adsorption and (iv) catalysis. Examples of these materials are discussed in this work, together with the future outlook on the impact of GFM-cyclodextrin conjugates in the development of applied nanoscience.

Covalent mechanochemical functionalization of carbon-encapsulated iron nanoparticles towards the improvement of their colloidal stability.

The mechanochemical covalent functionalization of carbon-encapsulated iron nanoparticles (CEINs) is reported. Unprotected sugars (mannose, galactose, ß-cyclodextrin) and amino sugars (glucosamine and chitosan) were successfully conjugated to the surface of CEINs. The developed grinding-induced methods employ (i) the 1,3-dipolar cycloadditions of nitrile oxides or azomethine ylides and (ii) amidation-type reactions with the inclusion of carboxyl-functionalized CEINs and amino sugars. All the developed mechanochemical processes are fast (reaction time 10 min) and result in high degrees of coverage (7.3-31.5 wt%). The presented functionalization routes also constitute easy to perform and environmentally improved protocols. Moreover, the use of toxic organic solvents is not required. A comprehensive study on the colloidal stability of the sugar-functionalized CEINs is also included in this work. The results of the turbidimetric analyses reveal that both grinding-induced formation of amide bonds and the cycloadditions of sugar moieties to the surface of CEINs result in the significant improvement of their colloidal stability. The highest stability of the aqueous dispersion was found for CEINs functionalized with ß-cyclodextrin. The comparative studies between the classical wet approach and the grinding-induced functionalization of CEINs show that the herein developed environmentally improved method increases the colloidal stability three times. The crucial role of the mechanochemical approach in the covalent functionalization of CEINs and the improvement of their colloidal stability is discussed in this work.

Occlusion phenomenon of redox probe by protein as a way of voltammetric detection of non-electroactive C-reactive protein.

Simple, selective and sensitive analytical devices are of a great importance for medical application. Herein, we developed highly selective immunosensor for electrochemical detection of C-reactive protein (CRP) in blood sample. Branched polyethylenimine functionalized with ferrocene residues (PEI-Fc) was the main element of the recognition layer, which allowed: (i) covalent binding of an antibody in its most favorable orientation and (ii) voltammetric detection of the C-reactive protein. Anchoring of PEI-Fc to the electrode surface through the electrodeposition process leads to the formation of thin, stable and reproducible layers, which is extremely important in the case of electrochemical immunosensing. The proposed analytical device is characterized by high selectivity and sensitivity and can be successfully used in the concentration range of CRP from 1 to 5·104 ng mL-1. The determined limit of detection was circa 0.5 and 2.5 ng mL-1 for voltammetric and impedance analysis, respectively. The developed analytical device has also been successfully applied for the analysis of CRP level in rat blood samples.

Sulfonated carbon-encapsulated iron nanoparticles as an efficient magnetic nanocatalyst for highly selective synthesis of benzimidazoles.

Surface functionalized carbon-encapsulated iron nanoparticles (CEINs) were found to be a magnetic nanocatalyst for the efficient and highly selective synthesis of benzimidazoles. CEINs were covalently decorated with carboxyl or sulfonyl groups and their catalytic activity was examined. Carboxyl-modified CEINs were obtained via the radical or oxidative treatment, whilst the sulfonated CEINs were obtained using the one-step diazotization approach with sulfanilic acid and isoamyl nitrite. The content of surface acidic groups varied between the obtained materials and was found to be the highest for sulfonyl-modified CEINs. CEINs functionalized with sulfonyl groups were the most efficient and the most selective nanocatalyst for the synthesis of benzimidazoles. Various benzimidazoles were obtained in very high yields (92.5-97.0%). Both metallocene, aliphatic, heterocyclic and aromatic aldehydes substituted with different functional groups were subjected to the synthesis process. The reaction proceeded in a short time, which varied from 25 min to 65 min depending on the aldehyde used. Additionally, the mechanism of the studied catalytic condensation by applying sulfonated CEINs as the catalyst was discussed. Importantly, the developed magnetic nanocatalysts could be easily separated from the reaction mixture using a permanent magnet. The nanocatalysts can be used up to six reaction cycles without any significant loss of their catalytic activity. This work opens up new ways for very efficient and simple synthesis of benzimidazoles - an important class of organic compounds for various biomedical applications.

Nanoconjugates of ferrocene and carbon-encapsulated iron nanoparticles as sensing platforms for voltammetric determination of ceruloplasmin in blood.

The nanoparticles comprising of iron core and carbon shell were decorated with ferrocene derivatives: ferrocenecarboxaldehyde (Fc-1) and ferrocenecarboxaldehyde oxime (Fc-2). A microdrop of suspension of the nanoconjugate was placed on a glassy-carbon electrode to prepare the recognition/sensing layer. Drying and purification of the sensing layer resulted in a well-defined and stable square-wave voltammogram of the ferrocene moiety. The height of the voltammetric peak increased in the presence of ceruloplasmin. That increase was linearly dependent on the logarithmic concentration of ceruloplasmin in blood. The applied external magnetic field was a factor which yielded better sensitivity and repeatability of the sensor response. The linearity of sensor response was found to be between 0.001 and 10µgdL-1 and 0.05-10µgdL-1 for both nanoconjugates: Fe@C-Fc-1 and Fe@C-Fc-2, in the presence and absence of the magnet, respectively. The obtained detection limit (LOD) for Fe@C-Fc-1 was found to be 0.60 and 0.10µgdL-1 in the absence and presence of magnetic field, respectively, whilst for Fe@C-Fc-2 was 0.4 and 0.07µgdL-1 in the absence and presence of a magnet, respectively. The proposed method is selective because the presence of common antioxidants in blood did not interfere significantly with the determination of the concentration of ceruloplasmin.

New Insight into the Synthesis and Biological Activity of the Polymeric Materials Consisting of Folic Acid and ß-Cyclodextrin.

This work presents a very new look at folate targeting and is focused on synthesizing and assessing the biological activity of folic acid-targeted drug delivery materials based on ß-cyclodextrin. Both folic acid and ß-cyclodextrin have been covalently conjugated to branched polyethylenimine as the polymeric vector. Host-guest inclusion of folic acid into a ß-cyclodextrin cavity, demonstrated by means of the spectroscopic methods (2-D NMR, IR, UV-Vis), is found to be of crucial importance for biological activity of nanotherapeutics. This paper describes the very first example of the versatile synthetic approach to create the polymeric biosystems, where folic acid activity is not limited by the inclusion phenomenon. Cytotoxicity of the obtained polymeric materials against Lewis lung carcinoma cells is determined by neutral red uptake assay. Folate receptor-binding studies reveal that the developed synthetic approach enables full exploitation of the potential of folic acid as a targeting ligand.

Addition of azomethine ylides to carbon-encapsulated iron nanoparticles.

The Prato reaction was applied for the covalent introduction of a variety of organic moieties onto carbon-encapsulated iron nanoparticles. The developed method is versatile and employs a broad range of commercially available reactants, including both aromatic and aliphatic aldehydes. The reported functionalization route provides high functionalization yields (ca. 12-21 wt%).

Novel non-covalent stable supramolecular ternary system comprising of cyclodextrin and branched polyethylenimine.

ABSTRACT: The synthesis of a novel supramolecular system comprising of branched polyethylenimine and cyclodextrin, is presented. The synthesis route is based on the self-assembly phenomena with the inclusion of solvent molecules. The systems are formed by a hydrogen-bonding network and host-guest type interactions between the building blocks. It was found that the native cyclodextrin and polyethylenimine are able to form stable systems when the reaction medium constitutes a polar solvent forming host-guest type complexes with cyclodextrin. A special consideration was paid on the detailed spectroscopic analyses of the obtained water-soluble constructs, including ROESY and diffusion-ordered (DOSY) NMR spectroscopy studies. The versatility and significance of DOSY technique for the analysis of the cyclodextrin complexes and its non-covalent systems with branched polymers, were presented. It was also found that the guest molecules that were incorporated in the complexes exhibited enhanced thermal stability. The morphological details in the solid state were obtained by scanning electron microscope.

Internalization and cytotoxicity effects of carbon-encapsulated iron nanoparticles in murine endothelial cells: Studies on internal dosages due to loaded mass agglomerates.

Carbon-encapsulated iron nanoparticles (CEINs) qualified as metal-inorganic hybrid nanomaterials offer a potential scope for an increasing number of biomedical applications. In this study, we have focused on the investigation of cellular fate and resulting cytotoxic effects of CEINs synthesized using a carbon arc route and studied in murine endothelial (HECa-10) cells. The CEIN samples were characterized as pristine (the mean diameter between 47 and 56nm) and hydrodynamic (the mean diameter between 270 and 460nm) forms and tested using a battery of methods to determine the cell internalization extent and cytotoxicity effects upon to the exposures (0.0001-100µg/ml) in HECa-10 cells. Our studies evidenced that the incubation with CEINs for 24h is accompanied with substantial changes of Zeta potential in cells which can be considered as a key factor for affecting the membrane transport, cellular distribution and cytotoxicity of these nanoparticles. The results demonstrate that CEINs have entered the endothelial cell through the endocytic pathway rather than by passive diffusion and they were mainly loaded as agglomerates on the cell membrane and throughout the cytoplasm, mitochondria and nucleus. The studies show that CEINs induce the mitochondrial and cell membrane cytotoxicities in a dose-dependent manner resulting from the internal dosages due to CEIN agglomerates. Our results highlight the importance of the physicochemical characterization of CEINs in studying the magnetic nanoparticle-endothelial cell interactions because the CEIN mass agglomerates can sediment more or less rapidly in culture models.

Sulfhydrylated graphene-encapsulated iron nanoparticles directly aminated with polyethylenimine: a novel magnetic nanoplatform for bioconjugation of gamma globulins and polyclonal antibodies.

This study presents for the first time the direct amination of graphene-encapsulated iron nanoparticles (GEINs) with polyethylenimine (PEI) via radical-type reaction. This work describes the first example of a direct addition of N-centered radical species onto the graphene layer. The pristine PEI and the PEI attached to GEINs have also been derivatized to introduce sulfhydryl functionalities. The proposed two-step protocol constitutes a novel, versatile and low cost method for the synthesis of polymer derivatives decorated with SH moieties. The derivatives of pristine polyethylenimine were analyzed by means of spectroscopic methods (NMR and IR), while the obtained carbon materials were studied by thermogravimetry, infrared spectroscopy, dynamic light scattering, and transmission electron microscopy. Finally, the concomitant part of this work focused on the bioconjugation type reactions of various biocompounds, including bovine gamma-globulins and human polyclonal antibodies of class IgG, with the as-obtained sulfhydrylated GEINs-PEI nanoplatform. The presence of immobilized molecules was confirmed by thermogravimetry, protein and fluorescence assays as well as confocal microscopy images.