Structure prediction of linear and cyclic peptides using CABS-flex.

The structural modeling of peptides can be a useful aid in the discovery of new drugs and a deeper understanding of the molecular mechanisms of life. Here we present a novel multiscale protocol for the structure prediction of linear and cyclic peptides. The protocol combines two main stages: coarse-grained simulations using the CABS-flex standalone package and an all-atom reconstruction-optimization process using the Modeller program. We evaluated the protocol on a set of linear peptides and two sets of cyclic peptides, with cyclization through the backbone and disulfide bonds. A comparison with other state-of-the-art tools (APPTEST, PEP-FOLD, ESMFold and AlphaFold implementation in ColabFold) shows that for most cases, AlphaFold offers the highest resolution. However, CABS-flex is competitive, particularly when it comes to short linear peptides. As demonstrated, the protocol performance can be further improved by combination with the residue-residue contact prediction method or more efficient scoring. The protocol is included in the CABS-flex standalone package along with online documentation to aid users in predicting the structure of peptides and mini-proteins.

Measurements of cadmium levels in relation to tobacco dependence and as a function of cytisine administration.

Cigarette smoking delivers a number of heavy metals, including cadmium (Cd), into the body. Bioaccumulation may result in an increase in pathological consequences over time. The assessment of changes in serum Cd concentrations during the treatment of cigarette dependence with cytisine was performed for the first time. Parameters assessing smoking habits, strength of addiction, and effectiveness of therapy were analyzed. Cd was determined before, during, and after the end of treatment. The serum Cd levels were significantly higher in the smokers than in the nonsmokers. Significant differences in Cd concentrations between sampling times in smokers were observed. Individuals who stopped smoking had significantly lower Cd concentrations compared to baseline. A significant positive correlation between the serum Cd before treatment and smoking urges was also obtained. Additionally, salivary Cd determinations were performed before treatment to evaluate the use of this method to assess cigarette addiction. Our findings indicate that Cd can be used as a biomarker of smoking addiction, and provide an alternative assessment of tobacco smoke exposure to other methods. The results provide new knowledge related to Cd concentrations in human body fluids and may play a role in monitoring and assessing the efficacy of cytisine for smoking cessation.

Development and Validation of LC-MS/MS Method for Determination of Cytisine in Human Serum and Saliva.

Cytisine (CYT) is a quinolizidine alkaloid used for nicotine addiction treatment. Recent clinical trial data regarding cytisine confirm its high effectiveness and safety as a smoking cessation treatment. CYT's popularity is growing due to its increased availability and licensing in more countries worldwide. This increased use by smokers has also resulted in an urgent need for continued drug research, including developing appropriate analytical methods for analyzing the drug in biological samples. In this study, a simple, fast, and reliable method combining hydrophilic interaction liquid chromatography and electrospray ionization quadrupole time of flight mass spectrometry (HILIC/ESI-QTOF-MS) for the determination of CYT in human serum and saliva was developed and validated. This was undertaken after the previous pre-treatment of the sample using solid-phase extraction (SPE). A hydrophilic interaction liquid chromatography (HILIC) column with a silica stationary phase was used for chromatographic analysis. In a linear gradient, the mobile phase consisted of acetonitrile (ACN) and formate buffer at pH 4.0. The proposed method was fully validated and demonstrated its sensitivity, selectivity, precision, and accuracy. The method was successfully applied to determine CYT in serum and, for the first time, in saliva. The findings indicate that saliva could be a promising non-invasive alternative to measure the free concentration of CYT.

MAPIYA contact map server for identification and visualization of molecular interactions in proteins and biological complexes.

Correct identification and effective visualization of interactions in biomolecular structures facilitate understanding of their functions and molecular design. In response to the practical needs of structure-based analysis, we have created a Mapiya web server. The Mapiya integrates four main functionalities: (i) generation of contact maps - intramolecular and intermolecular-for proteins, nucleic acids, and their complexes; (ii) characterization of the interactions physicochemical nature, (iii) interactive visualization of biomolecular conformations with automatic zoom on selected contacts using Molstar and (iv) additional sequence- and structure-based analyses performed with third-party software and in-house algorithms combined into an easy-to-use interface. Thus, Mapiya offers a highly customized analysis of the molecular interactions' in various biological systems. The web server is available at: http://mapiya.lcbio.pl/.

Development of the Validated Stability-Indicating Method for the Determination of Vortioxetine in Bulk and Pharmaceutical Formulation by HPLC-DAD, Stress Degradation Kinetics Studies and Detection of Degradation Products by LC-ESI-QTOF-MS.

Vortioxetine (VOR) is a new antidepressant drug used to treat major depressive disorder. In this work, a novel, simple, rapid, accurate, precise, selective, stability-indicating, and fully validated high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed to determine VOR in bulk and pharmaceutical formulations. A Polar-RP column was used, with a mobile phase consisting of acetonitrile (ACN), methanol (MeOH), acetate buffer pH 3.5, and addition of diethylamine (DEA) in the isocratic elution mode. Assessing the stability of the VOR is fundamental to guarantee the efficacy, safety, and quality of drug products. In this study, the VOR active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, using several degrading agents (acid, alkaline, water, heat, light, and oxidation agents). The developed HPLC-DAD method allows the collection of all the essential data to determine degradation kinetics. It was found that the decomposition of vortioxetine is fragile towards oxidative conditions and photolysis, yielding the first-order and second-order kinetic reaction in the above stress conditions, respectively. The degradation products (DPs) were identified by the high-resolution liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) method. The HPLC-DAD method was successfully applied for the quantification of VOR in tablets. Additionally, in silico toxicity prediction of the DPs was performed.

Determination of Cytisine and N-Methylcytisine from Selected Plant Extracts by High-Performance Liquid Chromatography and Comparison of Their Cytotoxic Activity.

Quinolizidine alkaloids exhibit various forms of biological activity. A lot of them were found in the Leguminosae family, including Laburnum and Genista. The aim of the study was the optimization of a chromatographic system for the analysis of cytisine and N-methylcytisine in various plant extracts as well as an investigation of the cytotoxic activities of selected alkaloids and plant extracts obtained from Laburnum anagyroides, Laburnum anagyroides L. quercifolium, Laburnum alpinum, Laburnum watereri, Genista germanica, and Genista tinctoria against various cancer cell lines. The determination of investigated compounds was performed by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD), while High Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight-Mass Spectrometry (HPLC-QTOF-MS) was applied for the qualitative analysis of plant extracts. The retention, separation selectivity, peaks shape, and systems efficiency obtained for cytisine and N-methylcytisine in different chromatographic systems were compared. The application of columns with alkylbonded and phenyl stationary phases led to a very weak retention of cytisine and N-methylcytisine, even when the mobile phases containing only 5% of organic modifiers were used. The strongest retention was observed when hydrophilic interaction chromatography (HILIC) or especially when ion exchange chromatography (IEC) were applied. The most optimal system in terms of alkaloid retention, peak shape, and system efficiency containing an strong cation exchange (SCX) stationary phase and a mobile phase consisted of 25% acetonitrile and formic buffer at pH 4.0 was applied for investigating alkaloids analysis in plant extracts. Cytotoxic properties of the investigated plant extracts as well as cytisine and N-methylcytisine were examined using human tongue squamous carcinoma cells (SCC-25), human pharyngeal squamous carcinoma cells (FaDu), human triple-negative breast adenocarcinoma cell line (MDA-MB-231), and human breast adenocarcinoma cell line (MCF-7). The highest cytotoxic activity against FaDu, MCF-7, and MDA-MB cancer cell lines was observed after applying the Genista germanica leaves extract. In contrast, the highest cytotoxic activity against SCC-25 cell line was obtained after treating with the seed extract of Laburnum watereri. The investigated plant extracts exhibit significant cytotoxicity against the tested human cancer cell lines and seem to be promising for further research on its anticancer activity.

Comparison of Various Chromatographic Systems for Identification of Vortioxetine in Bulk Drug Substance, Human Serum, Saliva, and Urine Samples by HPLC-DAD and LC-QTOF-MS.

Background: Determination of psychotropic drugs in clinical study is significant, and the establishment of methodologies for these drugs in biological matrices is essential for patients' safety. The search for new methods for their detection is one of the most important challenges of modern scientific research. The methods for analyzing of psychotropic drugs and their metabolites in different biological samples should be based on combining a very efficient separation technique including high-performance liquid chromatography (HPLC), with a sensitive detection method and effectively sample preparation methods. Objective: Retention, peaks symmetry and system efficiency of vortioxetine on Hydro RP, Polar RP, HILIC A (with silica stationary phase), HILIC-B (with aminopropyl stationary phase), and ACE HILIC-N (with polyhydroxy stationary phase and SCX columns were investigated. Various mobile phases containing methanol or acetonitrile as organic modifiers and different additives were also applied to obtained optimal retention, peaks shape, and systems efficiency. The best chromatographic procedure was used for simultaneous analysis of vortioxetine and its metabolites in human serum, urine and saliva samples. Methods: Analysis of vortioxetine was performed in various chromatographic systems: Reversed phase (RP) systems on alkylbonded or phenyl stationary phases, hydrophilic interaction liquid chromatography (HILIC), and ion-exchange chromatography (IEC). Based on the dependence of log k vs the concentration of the organic modifier, log kw values for vortioxetine in various chromatographic systems were determined and compared with calculated log P values. Solid phase extraction (SPE) method was applied for sample pre-treatment before HPLC analysis. HPLC-QTOF-MS method was applied for confirmation of presence of vortioxetine and some its metabolites in biological samples collected from psychiatric patient. Conclusions: Differences were observed in retention parameters with a change of the applied chromatographic system. The various properties of stationary phases resulted in differences in vortioxetine retention, systems' efficiency, and peaks' shape. Lipophilicity parameters were also determined using different HPLC conditions. The most optimal systems were chosen for the analysis of vortioxetine in biological samples. Both serum and urine or saliva samples collected from patients treated with vortioxetine can be used for the drug determination. For the first time, vortioxetine was detected in patient's saliva. Obtained results indicate on possibility of application of saliva samples, which collection are non-invasive and painless, for determination and therapeutic drug monitoring in patients.

Comparison of Various Chromatographic Systems for Analysis of Cytisine in Human Serum, Saliva and Pharmaceutical Formulation by HPLC with Diode Array, Fluorescence or Mass Spectrometry Detection.

Background: Identification and quantitative determination of cytisine, especially in biological samples and pharmaceutical formulations, is still a difficult analytical task. Cytisine is an alkaloid with a small and very polar molecule. For this reason, it is very weakly retained on reversed phase (RP) stationary phases, such as commonly used alkyl-bonded phases. The very weak retention of cytisine causes it to be eluted together with the components of biological matrices. Objective: Comparison and evaluation of various chromatographic systems for analysis of cytisine in different matrices-serum, saliva and pharmaceutical formulation-by high performance liquid chromatography (HPLC) with diode array (DAD), fluorescence (FLD) and mass spectrometry (MS) detection. Methods: The analyses were performed using HPLC in reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) modes. Different sample pre-treatment methods were tested: Protein precipitation (with acetone, methanol (MeOH) or acetonitrile (ACN), and solid phase extraction (SPE) using cartridges with octadecyl (C18), hydrophilic-lipophilic balanced copolymer (HLB) or strong cation exchange sorbents (Strata X-C). Conclusion: Significant differences were observed in retention parameters with a change of the used chromatographic system. The various properties of stationary phases resulted in differences in analyte retention, peaks' shape and systems' efficiency. The weakest retention was observed using RP systems; however, the use of the Polar RP phase can be an alternative for application in green chromatography. In the strongest retention was observed using a strong cation exchange (SCX) phase. The most optimal systems were chosen for the analysis of cytisine in the pharmaceutical preparation, serum and saliva after sample pre-treatment with the new SPE procedure. Due to the sensitivity, the use of HPLC-DAD or HPLC-FLD is the most optimal for drug analysis in pharmaceutical preparations, whereas HPLC-MS is suitable for analysis of cytisine in biological samples.

DETERMINATION OF VENLAFAXINE, VILAZODONE AND THEIR MAIN ACTIVE METABOLITES IN HUMAN SERUM BY HPLC-DAD AND HPLC-MS.

A high performance liquid chromatography (HPLC) method for simultaneous analysis of venlafaxine and its major metabolite 0-desmethylvenlafaxine and vilazodone and its methabolite M10 have been devel- oped and validated. Chromatography was performed on the Phenyl-Hexyl column with mobile phase containing methanol, acetate buffer at pH 3.5 and diethylamine. The application of stationary phase with 7r-7c moieties and mobile phase containing diethylamine as silanol blocker lets to obtain double protection against silanols and thus very high theoretical plate numbers were obtained. The good separation selectivity, good peaks' symmetry and very high systems efficiency for all investigated compounds were obtained in applied chromatographic system. The method is very efficient and suitable for the analysis of investigated drugs and their metabolites in human serum for patients' pharmacotherapy control.

RP-HPLC ANALYSIS OF ACIDIC AND BASIC DRUGS IN SYSTEMS WITH DIETHYLAMINE AS ELUENTS ADDITIVE.

The chromatographic behavior of some basic and acidic drugs was studied on Cl 8, Phenyl-Hexyl and Polar RP columns with methanol or acetonitrile as organic modifiers of aqueous mobile phases containing addition of diethylamine. Diethylamine plays a double function of silanol blocker reagent in analysis of basic drugs and ion-pair reagent in analysis of acidic drugs. Most symmetrical peaks and highest system efficiency were obtained on Phenyl-Hexyl and Polar RP columns in tested mobile phase systems compared to results obtained on C18 column. A new rapid, simple, specific and accurate reverse phase liquid chromatographic method was developed for the simultaneous determination of atorvastatin - antihyperlipidemic drug and amlodipine - calcium channel blocker in one pharmaceutical formulation. Atorvastatin is an acidic compounds while amlodipine is a basic substance. The chromatographic separation was carried out on Phenyl-Hexyl column by gradient elution mode with acetonitrile as organic modifier, acetate buffer at pH 3.5 and Q.025 M/L diethylamine. The proposed method was validated for specificity, precision, accuracy, linearity, and robustness. The linearity range of atorvastatin and amlodipine for 5 - 100 µg/mL was obtained with limits of-detection (LOD) 3.2750 gg/mL and 3.2102 µg/mL, respectively. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

Ion-exchange vs reversed-phase chromatography for separation and determination of basic psychotropic drugs.

Ion exchange chromatography, an alternative to reversed-phase (RP) chromatography, is described in this paper. We aimed to obtain optimal conditions for the separation of basic drugs because silica-based RP stationary phases show silanol effect and make the analysis of basic analytes hardly possible. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types of buffers at acidic pH and with the addition of organic modifiers: methanol and acetonitrile. The obtained results reveal a large influence of the salt cation used for buffer preparation and the type of organic modifier on the retention behavior of the analytes. These results were also compared with those obtained on an XBridge C18 column. The obtained results demonstrated that SCX stationary phases can be successfully used as alternatives to C18 stationary phases in the separation of basic compounds. The most selective and efficient chromatographic systems were applied for the quantification of some psychotropic drugs in fortified human serum samples.

Effect of chromatographic conditions on retention behavior and system efficiency for HPTLC of selected psychotropic drugs on chemically bonded stationary phases.

Selected psychotropic drug standards have been chromatographed on RP18, CN and diol layers with a variety of aqueous and nonaqueous mobile phases. The effect of buffers at acidic or basic pH, acetic acid, ammonia and diethylamine (DEA) in aqueous mobile phases on retention, efficiency and peak symmetry was examined. Improved peak symmetry and separation selectivity for investigated compounds were observed when ammonia or DEA were used as mobile phase additives. The effect of diethylamine concentration in aqueous eluents on retention, peak symmetry and theoretical plate number obtained on CN plates was also investigated. Because of the strong retention of these basic drugs on stationary phases bonded on silica matrix, nonaqueous eluents containing medium polar diluents, strongly polar modifiers and silanol blockers (ammonia or diethylamine) were applied. Aqueous and nonaqueous eluent systems with the best selectivity and efficiency were used for separate psychotropic drug standards' mixture on CN layer by 2D TLC.

Comparison of chromatographic conditions for analysis of selected psychotropic drugs in human serum.

Retention parameters of psychotropic drug standards were determined on different stationary phases: octadecyl silica, polar octadecyl silica, cyanopropyl silica and phenyl-hexyl silica using aqueous eluent systems containing acetonitrile, methanol or mixture of acetonitrile and methanol as organic modifiers; acetic buffer at pH 3.5 and diethylamine. The influence of stationary phases, kind of organic modifier and concentration of methanol and acetonitrile in mobile phases on retention, separation selectivity, peak symmetry and system efficiency was examined. These chromatographic parameters were significantly changed when analyses were performed on different stationary phases, when acetonitrile or methanol was used as organic modifier and when proportions of acetonitrile and methanol in eluent were different. The most efficient and selective systems were applied for quantification of the selected psychotropic drugs in fortified samples of human serum.

Review of awakening agents.

Brain injuries are a serious burden of illness to Canada and the US. Advances in managing head trauma have allowed more patients to emerge from decreased levels of consciousness and helped them cope with neurocognitive, neurobehavioural, and neuropsychiatric deficits. In this article, we review the current (1986-2002) evidence surrounding the pharmacological management of arousal states and the aforementioned neurological sequelae of head injury in either acute or chronic conditions. This article will review the evidence for the use of psychostimulants (methylphenidate), antidepressants (amitriptyline, selective serotonin reuptake inhibitors, and buproprion), Parkinson's medications (amantadine, bromocriptine, carbidopa/levodopa), anticonvulsants (valproic acid), modafinil (Provigil), lactate, hyperbaric oxygen chamber, electroconvulsive therapy, and transmagnetic stimulation, in patients following a head injury. The review did not include all anticonvulsants, neuroleptics, beta-blockers, benzodiazepines, azospirones or cognitive enhancers. Unfortunately, the quality of the evidence is generally poor, and sometimes conflicting, which in turn results in indecisive guidelines for treating patients. Accepting the inherent flaws in the evidence we feel that this paper may serve as a stepping-stone for future researchers to improve data gathering that targets neurocognitive, neurobehavioural and neuropsychiatric symptoms following a head injury.