Current Pharmaceutical Biotechnology - Volume 18, Issue 13, 2017

Background: One of the most important causes of death in the modern lifestyle is acute ischemic stroke, which is related to thrombosis in the blood vessels. Staphylokinase (SAK), a fibrinolytic agent, which is produced mainly by Staphylococcus aureus, is an indirect activator of plasminogen and belongs to the third generation of fibrinolytic enzymes. Methods: Considering the very low level of production and immunogenicity concerns of natural SAK produced by Staphylococcus aureus, attempts have been made to produce recombinant SAKs with high production levels, more fibrinolytic activities and low immunogenicity. Results and Conclusion: In this review, we summarized a number of expression systems based on recombinant DNA technology and protein-engineering approaches, which have been developed for the production of engineered recombinant SAK molecules with higher fibrinolytic activities and lower antigenicity.

Background: RNA-guided endonuclease as a versatile genome editing technology opened new windows in various fields of biology. The simplicity of this revolutionary technique provides a promising future for its application in a broad range of approaches from functional annotation of genes to diseases, to genetic manipulation and gene therapy. Besides the site-specific activity of Cas9 endonuclease, the unintended cleavage known as off-target effect is still a major challenge for this genome editing technique. Methods: Various strategies have been developed to resolve this bottleneck including development of new softwares for designing optimized guide RNA (gRNA), engineering Cas9 enzyme, improvement in off-target detection assays, etc. Results: This review dedicated to discuss on methods that have been used for optimizing Cas9, specificity with the aim of improving this technology for therapeutic applications. Conclusion: In addition, the applications and novel breakthroughs in the field of CRISPR technology will be described.

Background: Microfungi are causal agents of numerous diseases and disorders of agricultural plants, farm mushrooms and animals as well as human, which results are serious global reduction of the food amount, decrease of life quality, the severe life-threatening diseases and enormous economic losses. Methods: In spite of organism innate ability to combat against pathogens, in invasions of some pathogens, support of additional antimycotic agents to defence system is required. Nowadays, common “fighters” against the microfungi are numerous synthetic fungicides that, besides benefits, have also side effects on host and environment and can cause the development of fungicide resistance in the pathogens. Therefore, the creation of new natural fungicides with different modes of action, strengthening the defense system and increase of organism resistance to pathogens are the main requirements of modern society. Results: Numerous mushrooms produce chemically various intra- and extracellular metabolites with antifungal potential, among which the most potent ones are polysaccharides, proteins, and phenolic compounds. They act as immunostimulators, inhibitors of pathogen development and virulence and/or activators of pathogens` autolytic system. Conclusion: Therefore, mushroom-based antimycotic agents could be successfully applied in the diseases treatments as accessories or alternatives to commercial therapies and in such a way contribute to environmentally friendly combat against pathogens, i.e. decrease or complete substitution of commercial synthetic fungicides with natural ones.

Background: Recent studies have shown that rutin presents a heightened interest due to the plethora of biological activities. The major drawback for this phytocompound is the poor water solubility, a parameter that limits its bioavailability. The study aimed to prepare and assess the inclusion complexes of rutin with β-cyclodextrin (BCD) and hydroxypropyl-β-cyclodextrin (HPBCD), followed by the evaluation of the antioxidant, antiproliferative and pro-apoptotic activity against B164A5 murine melanoma cell line. Methods: Inclusion complexes were prepared by kneading method. Scanning electron microscopy, differential scanning calorimetry and X-ray powder diffraction were used in order to assess their formation. Antioxidant activity was determined by DPPH assay. Antiproliferative and pro-apoptotic effects where tested by MTT, respectively Annexin V-PI assays. Physico-chemical methods proved that incorporation took place. Results: Results show that rutin presents antioxidant activity, and complexation increases this property. After 72 h of incubation at the concentration of 100 μM rutin proved to be an active antiproliferative and pro-apoptotic compound against B164A5 cells. Conclusion: Incorporation in BCD and HBCD maintained the activity, thus representing an important finding and open path for in vivo experiments.

Background: Thrombospondin (TSP) 1 and 4 are extracellular matrix glycoproteins that mediate cell proliferation, platelet aggregation and inflammatory response. Conflicting data addressed the possible contribution of TSP-1 and TSP-4 gene polymorphisms to acute myocardial infarction (AMI). Objective: Our study aimed to examine the association of TSP-1 (N700S) and TSP-4 (A387P) genetic variants with the incidence of AMI in Egyptians. It also correlated TSP-1 variants to TSP-1 and TNF-α serum concentrations while TSP-4 variants to IL-8 concentration identifying TSPs' contribution to vascular inflammation. Methods: Genotyping was done in 214 subjects; 114 AMI patients and 100 controls using PCR-RFLP analysis. Serum Tsp-1, TNF-α and IL-8 levels were measured by ELISA assay. Results: For TSP-4, (GC and CC) genotype distribution and the (C) allele frequency were significantly higher in AMI patients than controls (p = 0.0186), (p = 0.0117) respectively. In contrast, TSP-1 genotypes and allele frequencies showed no significant difference between AMI and controls (p = 0.7124 and p = 0.7201, respectively). Serum TSP-1, TNF-α and IL-8 concentrations were significantly elevated in AMI compared to controls (p = 0.0146, p < 0.0001 and p = 0.0057) respectively. Serum IL-8 levels had a significant difference among TSP-4 genotypes (p= 0.0368), being highest in the mutant C allele. Serum TSP-1 and TNF-α concentrations showed no significant difference among TSP-1 genotypes, but there was a positive correlation between both concentrations in AMI patients (p = 0.0014), (r = 0.4125). Conclusion: TSP-4 A387P polymorphism, but not TSP-1 polymorphism, is an independent risk factor for AMI in the Egyptians.

Background: In mammals, the cholinergic nervous system plays a crucial role in neuronal regulation of physiological processes. It acts on cells by two types of receptors – nicotinic and muscarinic receptors. Both signal transmission pathways also operate in the central and peripheral cholinergic nervous system of insects. Method: In our pharmacological experiments, we studied the effects of two muscarinic agonists (carbachol, pilocarpine) and two muscarinic antagonists (atropine, scopolamine) on the muscle contractile activity of visceral organs in the beetle, Tenebrio molitor. Results: Both antagonists, when injected to haemolymph at concentration 10-5 M, caused delayed and prolonged cardioinhibitory effects on heart contractility in ortho- and antidromic phases of heart activity in T. molitor pupa what was observed as negative chrono- and inotropic effects. Agonist of muscarinic receptors – carbachol evoked opposite effect and increased contraction rate but only in antidromic phase. Pilocarpine, the second agonist induced weak negative chronotropic effects in the antiand orthodromic phases of heart activity. However, neither agonists had an effect on semi-isolated beetle heart in vitro. Only atropine at the highest tested concentrations slightly decreased the frequency of myocardial contractions. These suggest the regulation of heart activity by muscarinic system indirectly. The tested compounds also affected the contractility of the oviduct and hindgut, but the responses of these organs were varied and depended on the concentration of the applied compounds. Conclusion: These pharmacological experiments suggest the possible modulation of insect visceral muscle contractility by the cholinergic nervous system and indirectly indicate the presence of muscarinic receptor(s) in the visceral organs of the beetle T. molitor.

Background: Phenytoin has very challenging pharmacokinetic properties. To prevent its toxicity and ensure efficacy, continuous therapeutic monitoring is required. It is hard to get a simple, accurate, rapid, easily available, economical and highly sensitive assay in one method for therapeutic monitoring of phenytoin. Objective: The present study is directed towards establishing and validating a simpler, rapid, an accurate, highly sensitive, novel and environment friendly liquid chromatography/mass spectrometry (LC/MS) method for offering rapid and reliable TDM results of phenytoin in epileptic patients to physicians and clinicians for making immediate and rational decision. Methods: 27 epileptics patients with uncontrolled seizures or suspected of non-compliance or toxicity of phenytoin were selected and advised for TDM of phenytoin by neurologists of Qilu Hospital Jinan, China. The LC/MS assay was used for performing of therapeutic monitoring of phenytoin. The Agilent 1100 LC/MS system was used for TDM. The mixture of Ammonium acetate 5mM: Methanol at (35: 65 v/v) was used for the composition of mobile phase. The Diamonsil C18 (150mm.6mm, 5μm) column was used for the extraction of analytes in plasma. The samples were prepared with one step simple protein precipitation method. The technique was validated with the guidelines of International Conference on Harmonisation (ICH). Results: The calibration curve demonstrated decent linearity within (0.2-20 μg/mL) concentration range with linearity equation, y= 0.0667855 x +0.00241785 and correlation coefficient (R2) of 0.99928. The specificity, recovery, linearity, accuracy, precision and stability results were within the accepted limits. The concentration of 0.2 μg/mL was observed as lower limit of quantitation (LLOQ), which is 12.5 times lower than the currently available enzyme-multiplied immunoassay technique (EMIT) for measurement of phenytoin in epilepsy patients. Conclusion: A rapid, simple, economical, precise, highly sensitive and novel LC/MS assay has been established, validated and applied successfully in TDM of 27 epileptics patients. It was alarmingly found that TDM results of all these patients were out of safe range except two patients. However, it needs further evaluation. Besides TDM, the stated method can also be applied in bioequivalence, pharmacokinetics, toxicokinetics and pharmacovigilance studies.

Background: Lonicera macranthoides is a Chinese herb that contains a large number of bioactive spanions possessing important pharmacological activities, such as anti-tumour activity. However, detailed information about their anti-tumor activity and bioactive compounds is limited. Methods: In order to evaluate the scientific basis, the method of high-speed counter-current chromatography (HSCCC) combined with high performance liquid chromatography mass spectrometry (HPLC-ESI-QTOF/MS) has been developed to separate, purify and analyze saponins from Lonicera macranthoides. Four main saponins, Macranthoidin B (I), Macranthoidin A (II), Macranthoides B (III) and Akebia saponin D (IV) were separated by HSCCC with the solvent systems of ethyl acetate-nbutanol- water (3:2:5) and n-butanol-water-methanol-ethyl acetate (1:6:0.5:4). The purities of these four bioactive ingredients (I-IV) identified and detected by HPLC-ESI-QTOF/MS were 95.1%, 92.7%, 91.8% and 96.3%, respectively. The separated saponins were evaluated for their cytotoxic activities against six tumor cell lines (MCF-7, Hela, A549, HepG2, HT29 and Eca109). Results: Results show that compounds I-IV exhibited particular significant anti-tumor activities against human mammary adenocarcinoma MCF-7 cell with IC50 values ranging from 12.7 to 30.8 μM. Conclusion: It was demonstrated that the combinative method using HPLC-ESI-QTOF/MS and HSCCC was suitable for rapid screening and isolating saponins of Lonicera macranthoides and the isolated compounds have great potential for the development of new antitumor drugs.