Optimization of Epigenetic Modifier Drug Combination for Synergistic Effect against Glioblastoma Multiform Cancer Cell Lines.

Glioblastoma multiforme (GBM), is a frequent class of malignant brain tumors. Epigenetic therapy, especially with synergistic combinations is highly paid attention for aggressive solid tumors like GBM. Here, RSM optimization has been used to increase the efficient arrest of U87 and U251 cell lines due to synergistic effects. Cell lines were treated with SAHA, 5-Azacytidine, GSK-126, and PTC-209 individually and then RSM was used to find most effective combinations. Results showed that optimized combinations significantly reduce cell survival and induce cell cycle arrest and apoptosis in both cell lines. Expression of cyclin B1 and cyclin D1 were decreased while caspase3 increased expression.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h-1 , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).

QbD-guided pharmaceutical development of Pembrolizumab biosimilar candidate PSG-024 propelled to industry meeting primary requirements of comparability to Keytruda®.

Pharmaceutical development of biosimilars is primarily focused on meeting the regulatory requirements for analytical comparability of the product's critical quality attributes (CQAs), concerning safety and efficacy, to those of the originator drug of interest. To this end, the early adoption of a systematic science-based approach, as guided by quality-by-design (QbD) principles, is crucial due to the blind starting point where the same insights of an originator developer into the challenges of a given biopharmaceutical and its manufacturing process are lacking. In this study, we devised a pharmaceutical QbD-guided approach to undertake the biosimilar development of Pembrolizumab (Keytruda®), the ace of therapeutic monoclonal antibodies (mAbs) in terms of approved indications and market sales, and its manufacturing process development. Quality target product profile (QTPP) for Pembrolizumab biosimilar product was assembled using publicly available information on Keytruda®. Upon preliminary analyses of four different lots of Keytruda®, the product CQAs and their acceptable ranges of specification were determined via risk assessment based on the relevant pharmaceutical development quality guidelines, particularly those of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The development and clone selection of Chinese Hamster Ovary (CHO) DG44 cell line was performed using DHFR expression vectors and Methotrexate (MTX) selective pressure. The CHO clone stably expressing relatively higher mAb titer (∼1200 mg/l) in small-scale shake-flask cultures, with the highest similarity of the CQAs charge variants contents (CVCs), N-glycan profile, and biological potency to those of Keytruda® reference standard was selected as the lead clone and the produced Pembrolizumab candidate was named PSG-024. The upstream process (USP) and downstream process (DSP) developments for production were started with the process evaluation screening experiments for the identification of critical process parameters (CPPs) founded upon the prior knowledge on different process stages, input process parameters (iPPs), output process parameters (oPPs), and their impacts on product CQAs. Thereby, screening experiments of USP fed-batch cell culture in 5-liter bioreactor resulted in improvement of PSG-024 expression titer to 2060 ± 70 mg/l and selection of the iPPs feed amount (A), glucose setpoint (B), culture temperature (C), and agitation rate (D) for the optimization design of experiments (DoEs) mainly focused on the CQA acidic CVC and the oPPs mAb expression yield. The USP optimization DoEs using response surface methodology (RSM) yielded valid prediction models and optimal conditions of A = 35%, B = 4.5 g/l, C = 37 °C, and D = 160-220 rpm, which resulted in the final PSG-024 expression titer of 3170 ± 40 mg/l without an excessive rise in acidic CVC. The DSP screening experiments led to achieving the mAb recovery rates of 94% ± 3% and 71.5% ± 3.5% for affinity (capture) and cation-exchange (polishing) chromatography stages, respectively. The capture eluate buffer and viral inactivation conditions were optimized to prevent mAb eluate turbidity and protein aggregation. Moreover, the polishing stage optimization DoEs via one-factor-at-a-time method focused on wash and elution steps for control of the acidic CVC CQA and achieving >80% mAb recovery rate. By shifting to Step elution from the primary salt gradient method and considering an additional intermediate wash step, the maximum mAb recovery of 87% ± 1.5% was achievable while maintaining the CQA acidic CVC within the acceptable range. The consistency of final analytical comparability of PSG-024 demonstrated the effectiveness of the adopted pharmaceutical QbD approach for Pembrolizumab biosimilar development, paving the way for the technology transfer to the client to proceed further development.

Acceleration of antibacterial activity of curcumin loaded biopolymers against methicillin-resistant Staphylococcus aureus: Synthesis, optimization, and evaluation.

Curcumin is a polyphenolic molecule with antibacterial, antioxidant, anti-inflammatory, and antimicrobial properties. This study aimed to prepare nanocurcumin by encapsulating in biopolymers to improve its stability, bioavailability, water-solubility, antibacterial efficiency against methicillin-resistant Staphylococcus aureus. Three effective variables of curcumin concentration, polymer concentration, and water volume on curcumin-loaded polymer nanoparticles, were optimized. The average size of polyacrylic acid (PAA), polyvinyl alcohol (PVA), and polyethyleneimine (PEI) nanoparticles were obtained 75.2, 77.1, 86.4 nm, respectively. The nanoparticles had a spherical shape, a smooth and uniform surface morphology. The MIC of PAA, PVA, and PEI nanoparticles was 0.480, 0.390, and 0.340 mg/mL, respectively and the MIC of PAA, PVA, and PEI combined with methicillin was 0.330, 0.260, and 0.200 mg/mL, respectively. According to the results, curcumin-loaded PEI nanoparticles had the highest inhibitory effect against methicillin-resistant S. aureus among the synthesized nanoparticles. The results showed that solvent volume, polymer concentration and curcumin concentration had a significant effect on particle size. The inhibitory properties of curcumin nanoparticles significantly increased due to the smaller particle size and increased penetration into the bacterium. Curcumin-loaded nanoparticles can be promising drug carriers for the treatment of infections, cancer, and other diseases.

Neuroprotective Effect of New Nanochelating-Based Nano Complex, ALZc3, Against Aß (1-42)-Induced Toxicity in Rat: a Comparison with Memantine.

PURPOSE: The current drugs for Alzheimer's disease (AD) are only used to slow or delay the progression of the pathology. So using a novel technology is a necessity to synthesize more effective medications to control this most common cause of dementia. In this study, using nanochelating technology, ALZc3 was synthesized and its therapeutic effects were evaluated in comparison with memantine on a well-known rat model of AD, which is based on Amyloid-ßeta (Aß) injection into the brain. MATERIALS AND METHODS: Aß (1-42) was injected bilaterally into the CA1 area of the hippocampus of male rats and then animals were treated daily by oral administration of Alz-C3, memantine or their vehicles. Activities of antioxidant enzymes catalase and superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) levels, as well as Bax/Bcl-2 ratio, caspase-3 activation, and TNF-α expression were evaluated 7 days after Aß injection. Finally, learning and memory of the rats were assessed by Morris water maze test. RESULTS: ALZc3 and memantine improved memory impairment and antioxidant activity and reduced TNF-α expression, caspase-3 activity and Bax/Bcl-2 ratio in the rat's hippocampus. The results showed a superiority of ALZC3 compared to memantine in reducing caspase-3, increasing CAT activity in Aß (1-42)-injected groups and improving apoptosis factor in healthy mice. CONCLUSION: These results indicated that ALZc3 could significantly prevent the memory impairment and Aß (1-42) toxicity. Thus, ALZc3 could be a promising novel anti-AD agent.

One-step separation of the recombinant protein by using the amine-functionalized magnetic mesoporous silica nanoparticles; an efficient and facile approach.

The separation process is the main stage of recombinant production. With the advancement of nanotechnology and the development of magnetic nanoparticles, these structures are increasingly used in different applications. In the present study, we produced the recombinant human growth hormone from Pichia pastoris and for protein separation provided the surfaces similar to chromatographic columns on the surface of magnetic nanoparticles. For this purpose, using a co-precipitation method, the core of Fe3O4 was prepared and coated by silica. To increase the protein availability, silica mesoporous formation and its amine functionalization were performed. The specific surface area and the pore size were determined 78.3189 m2/g and 7.44 nm. After the magnetic separation, the sample loading in SDS gel shows a reduction in protein band and the protein absorption at a wavelength of 280 nm. Finally, we evaluate the ability of amine functionalized nanoparticles for protein separation that demonstrate the adsorption capacity significantly increased compare with silica-coated nanoparticles. The amine functionalized nanoparticles provide the maximum adsorption capacity of 235.21 µg/mg and after the elution, protein concentration determined 476 mg/L. This work indicates the functionalized magnetic mesoporous silica nanoparticles can be used as the best candidate for the separation of different biological macromolecules.

Emergency Ross Procedure for Pediatric Aortic Valve Myxofibrosarcoma.

Primary cardiac tumors in children are uncommon and rarely demand surgical intervention. We report a malignant tumor arising from the aortic root in a 5-year-old boy presenting with left ventricular outflow tract obstruction and tumor embolism, its surgical management using the Ross procedure, and the unique histopathological aspects of the tumor.

Curcumin-loaded polysaccharide nanoparticles: Optimization and anticariogenic activity against Streptococcus mutans.

Curcumin was loaded into different polysaccharide nanoparticles chitosan, alginate and starch, using the desolvation method. Curcumin-loaded nanoparticles exhibited enhanced solubility in aqueous solutions comparing with free curcumin. Effects of formulation parameters such as curcumin concentration and different volumes of ethanolic solution were affected the particle size and loading efficiency. Under optimum conditions, curcumin-loaded chitosan, starch and alginate nanoparticles with mean particles sizes of 66.3, 61.1 and 78.8nm, and maximum loading efficiencies of 11.9%, 14.3% and 13.35% were achieved, respectively. Additionally, the minimum inhibitory concentration for chitosan, starch and alginate nanoparticles against the microorganism, Streptococcus mutans, were 0.114, 0.204 and 0.204mg/mL, respectively. Curcumin was observed to release from nanoparticles under physiological pH over a period of 96h. The effect of curcumin-loaded nanoparticles on S. mutans biofilms was assessed on dental models. According to the results, curcumin-loaded chitosan nanoparticles hold promises for being used in dental decay fighting products.

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of Pichia pastoris through optimization of a mixed feeding strategy by response surface methodology.

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ set glycerol and that of methanol to be 0.05 and 0.03 h(-1), respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.

Digitoflavone provokes mitochondrial biogenesis in PC12 cells: A protective approach to oxidative stress.

CONTEXT: Reactive oxygen species (ROS) are known to be one of the main causes of neurodegenerative disorders, and flavonoids play characteristic roles in a variety of biological activities, and specially are known to be antioxidant reagents. OBJECTIVE: In this study, we investigated neuroprotective effects of digitoflavone to suppress H2O2 -induced cell death in neuron-like PC12 cells. MATERIAL AND METHODS: PC12 cells were pre-treated with digitoflavone for 2 h and then cells were exposed to H2O2 for 18 h. The cells' viability was evaluated by MTT assay. Rhodamine 123 staining was used for the determination of mitochondrial membrane potential (ΔΨm). The intracellular ROS aggregation was determined by using 2',7'-dichlorofluorescein diacetate. Also, the level of mitochondrial biogenesis factors was measured by western blot. The antioxidant capacity of digitoflavone was also determined by measuring reduced glutathione (GSH) level and catalase (CAT) activity quantification. RESULTS: Digitoflavone significantly elevated cells' viability at concentrations of 10 and 20 µM. Also, digitoflavone attenuated intracellular level of ROS, and stabilized ΔΨm. Moreover, digitoflavone increased phosphorylation of AMP-activated protein kinase (AMPK) and, consequently, elevated mitochondrial biogenesis factors which were reduced after H2O2 exposure. We emphasized on the protective effect of digitoflavone through increasing mitochondrial biogenesis by specifically inhibiting AMPK. Antioxidant ability of digitoflavone was indicated by the elevation of GSH level and CAT activity. CONCLUSION: As a result, digitoflavone stabilize ΔΨm, enhanced cell viability through inducing mitochondrial biogenesis pathway, and increased antioxidant capacity of the cells which lead to better combating the oxidative stress.

Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis.

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A(pro) based on sequence comparison and homology modeling with human rhinovirus 2A(pro). The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A(pro), as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A(pro) structure.

A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures.

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 10³ to 8.08 × 10³ mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.

Optimization of PEGylation conditions for BSA nanoparticles using response surface methodology.

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and -14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of -31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.

Agmatine prevents LPS-induced spatial memory impairment and hippocampal apoptosis.

Neuroinflammation is associated with a number of neurodegenerative diseases. It is known that lipopolysaccharide (LPS) treatment induces neuroinflammation and memory deterioration. Agmatine, the metabolite of arginine by arginine decarboxylase, is suggested to be a neuroprotective agent. The aim of this study was to explore if agmatine can prevent LPS-induced spatial memory impairment and hippocampal apoptosis. Adult male Wistar rats (200-250 g) were trained in water maze for 4 days (3 days in hidden platform and the last day in visible platform task). Saline, LPS (250 microg/kg/ip) or (and) agmatine (5 or 10 mg/kg) were administered 4h before every training session. LPS treatment impaired water maze place learning while agmatine co-administration prevented it. Also western blot studies revealed that LPS induces hippocampal caspase-3 activation while agmatine treatment prevented it.

Targeting enteroviral 2A protease by a 16-mer synthetic peptide: inhibition of 2Apro-induced apoptosis in a stable Tet-on HeLa cell line.

Enteroviridae such as coxsackievirus are important infectious agents causing viral heart diseases. Viral protease 2A (2Apro) initiates the virus life cycle, and is an excellent target for developing antiviral drugs. Here, to evaluate the validity of the 2Apro as a proper therapeutic target, and based on the existing information and molecular dynamics, a 16-mer peptide was designed to specifically target the active site of protease 2Apro in order to block the activity of CVB3 2Apro. We showed that the peptide could compete with endogenous substrate in a concentration-dependent manner. Further, we established a HeLa cell line that expressed 2Apro. Expression of 2Apro resulted in significant morphological alteration and eventual cell death. Western blot and viability assay showed that the 16-mer peptide (200 microg/ml) could significantly block 2Apro activity and its cytotoxic effect. Future modification of the 16-mer peptide can improve its affinity for 2Apro and therefore develop effective antiviral drug.

5-Fluorouracil-loaded BSA nanoparticles: formulation optimization and in vitro release study.

Over the past few decades, there has been considerable interest in developing protein nanoparticles as drug delivery devices. The underlying rationale is their exceptional characteristics, namely biodegradability and nonantigenicity. Herein, phase separation method was used to prepare 5-fluorouracil-loaded bovine serum albumin (BSA) nanoparticles. Drug release was tracked by continuous flow dialysis technique. Effect of process variables on loading efficiency of 5-fluorouracil was investigated and optimized through Taguchi's M16 design with the amount of entrapped drug as response. Optimum condition was found to be 2 mg/mL of 5-fluorouracil, 3.7 mL of added ethanol, 176 microL of glutaraldehyde, drug-protein incubation time of 30 min, and pH of 8.4 for 200 mg of BSA in 2 mL drug solution. pH had the most noticeable effect on the amount of entrapped drug, but glutaraldehyde had the least. Mean diameter and zeta potential of fabricated nanoparticles under these conditions were 210 nm and -31.7 mV, respectively. Drug-loaded BSA nanoparticles suspension maintained constant release of drug for 20 h under experimental conditions, so this colloidal drug carrier is capable of releasing drug in a sustained manner.

Effect of dextromethorphan on antinociception and tolerance induced by swim-stress in the formalin test.

BACKGROUND: In the present study, the effect of dextromethorphan on antinociception and tolerance induced by water swim-stress in the formalin test was investigated. METHODS: Swim-stress at 8 masculineC induces antinociception in both phases of the formalin test. Intraperitoneal administration of dextromethorphan (60 mg/kg) also induces antinociception in the second phase of the formalin test. The lower doses of dextromethorphan (1.25, 2.5, and 5 mg/kg) which did not induce antinociception alone, but did so in combination with swim- stress (40 second), showed antinociceptive effect in both phases of the test. Exposure to water swimming stress with a period of 20 sec, once daily for three days, altered swim- stress- induced antinociception in the formalin test, when tested on the fourth day. RESULTS: In these animals, exposure to either water swimming stress alone or water swimming stress in combination with dextromethorphan showed potentiation of antinociception induced by swim- stress up to 20 second and decreased the response induced by 40 and 60 second swim- stress, indicating a tolerance induction. Dextromethorphan (20 mg/kg) did not alter the changes induced by three days exposure to swim- stress. CONCLUSION: The results may indicate a possible involvement of n-methyl-d-aspartate receptor mechanism in the antinociception but not tolerance induced by swim- stress at 8 masculineC.

Effect of lithium on swim stress-induced antinociception in naive mice and mice with subchronic administration of morphine or swim stress in formalin test.

BACKGROUND: Lithium has been shown to relieve mania and induce antinociception. In the present study, swim stress at 8C induced antinociception in both phases of the formalin test. Intraperitoneal administration of lithium chloride (LiCl) (0.05, 0.25, and 0.5 mg/kg) also induced antinociception in both phases of the formalin test. METHODS: Antinociception was assessed by the formalin test method. Swim stress was achieved in the 8C water in a container 5 cm in diameter and 20 cm tall filled with water do a depth of 11 cm. RESULTS: The drug (0.5 and 5 mg/kg) potentiated swim stress-induced antinociception in the second phase of the test. Repeated exposure to water swimming stress with a period of 40 sec, once daily for three days, in combination with lithium chloride did not alter stress-induced antinociception in either phases of the formalin test, when swim stress-induced antinociception was tested on the fourth day. Subchronic treatment with morphine (25 mg/kg), once daily for three days, in the presence or absence of lithium chloride (5 mg/kg) did not alter swim stress-induced antinociception, either, when swim stress-induced antinociception was tested on the fourth day. CONCLUSION: It may be concluded that lithium chloride potentiates swim stress-induced antinociception, but the drug has no influence on the response of subchronic administration swim stress or morphine.