Protective effects of microbial biosurfactants produced by Bacillus halotolerans and Candida parapsilosis on bleomycin-induced pulmonary fibrosis in mice: Impact of antioxidant, anti-inflammatory and anti-fibrotic properties via TGF-ß1/Smad-3 pathway and miRNA-326.

Idiopathic pulmonary fibrosis (IPF) is an irreversible disease which considered the most fatal pulmonary fibrosis. Pulmonary toxicity including IPF is the most severe adverse effect of bleomycin, the chemotherapeutic agent. Based on the fact that, exogenous surfactants could induce alveolar stabilization in many lung diseases, the aim of this study was to explore the effects of low cost biosurfactants, surfactin (SUR) and sophorolipids (SLs), against bleomycin-induced pulmonary fibrosis in mice due to their antioxidant, and anti-inflammatory properties. Surfactin and sophorolipids were produced by microbial conversion of frying oil and potato peel wastes using Bacillus halotolerans and Candida parapsilosis respectively. These biosurfactants were identified by FTIR, 1H NMR, and LC-MS/MS spectra. C57BL/6 mice were administered the produced biosurfactants daily at oral dose of 200 mg kg-1 one day after the first bleomycin dose (35 U/kg). We evaluated four study groups: Control, Bleomycin, Bleomycin+SUR, Bleomycin+SLs. After 30 days, lungs from each mouse were sampled for oxidative stress, ELISA, Western blot, histopathological, immunohistochemical analyses. Our results showed that the produced SUR and SLs reduced pulmonary oxidative stress and inflammatory response in the lungs of bleomycin induced mice as they suppressed SOD, CAT, and GST activities also reduced NF-κß, TNF-α, and CD68 levels. Furthermore, biosurfactants suppressed the expression of TGF-ß1, Smad-3, and p-JNK fibrotic signaling pathway in pulmonary tissues. Histologically, SUR and SLs protected against lung ECM deposition caused by bleomycin administration. Biosurfactants produced from microbial sources can inhibit the induced inflammatory and fibrotic responses in bleomycin-induced pulmonary fibrosis.

The beneficial effects of l-carnitine and infliximab in methotrexate-induced hepatotoxicity: Emphasis on Notch1/Hes-1 signaling.

Methotrexate (MTX)-induced hepatotoxicity is a serious adverse effect that may limit its use. Therefore, eligible drugs to ameliorate MTX-induced hepatotoxicity are required. l-Carnitine (LC) is a natural molecule with beneficial metabolic effects and infliximab (INF) is an anti-inflammatory monoclonal antibody against tumor necrosis factor-alpha (TNF-α). Recently, Notch1/Hes-1 signaling was found to play a key role in the pathogenesis of liver injury. However, its role in MTX-induced hepatotoxicity is unclear. This study aimed to evaluate the modulatory effects of LC or INF on MTX-induced hepatotoxicity and to explore the underlying mechanism with emphasis on the Notch1/Hes-1 signaling pathway. Sixty rats were randomized into six groups (n = 10): (1) control (saline); (2) MTX (20 mg/kg MTX, intraperitoneal [ip], once); (3) LC group (500 mg/kg ip, 5 days); (4) INF (7 mg/kg INF ip, once); (5) MTX+LC (20 mg/kg ip, once, 500 mg/kg ip, 5 days, respectively); (6) MTX+INF (20 mg/kg ip, once, 7 mg/kg INF ip, once, respectively). Oxidative stress, inflammatory markers, and Notch1/Hes-1 were investigated. MTX induced the expression of Notch1 and Hes-1 proteins and increased the levels of TNF-α, interleukin (IL)-6, and IL-1ß in the liver. Cotreatment with LC or INF showed apparent antioxidant and anti-inflammatory effects. Interestingly, the downregulation of Notch1 and Hes-1 expression was more prominent in LC cotreatment as compared with INF. In conclusion, LC or INF attenuates MTX-induced hepatotoxicity through modulation of Notch1/Hes-1 signaling. The LC ameliorative effect against MTX-induced hepatotoxicity is significantly better than that of INF. Therefore, LC cotreatment may present a safe and therapeutically effective therapy in alleviating MTX-induced hepatotoxicity.

Selective spectrofluorimetric determination of two corticosteroids along with their co-formulated drugs and degradation products in ophthalmic solution and aqueous humour.

Prednisolone acetate (PNO) and fluorometholone (FRT) are corticosteroids, co-formulated with moxifloxacin HCl (MFX) and cromolyn sodium (CML), respectively. PNO has a negligible quantum yield and its hydrolytic degradation products have enhanced fluorescence, which is 250-fold greater. FRT is a nonfluorescent drug, but its hydrolytic degradation products show reasonable fluorescence; MFX and CML have native fluorescence. Two methods were proposed based on the determination of PNO and FRT via their hydrolytic degradation products in the presence of other degradation products. Method (A) was developed for simultaneous determination of PNO and MFX in the presence of PNO degradation products by measuring peak amplitudes of the first derivative (1 D) of its enhanced fluorescence; PNO and MFX were measured at 345 and 473 nm, respectively. Method (B) is a synchronous fluorescence spectroscopic method for simultaneous determination of FRT and its co-formulated drug CML in the presence of its degradation products. Fluorescence intensities were measured at λem 283 and 347 nm for FRT and CML, respectively, using Δλ = 99.20 nm. Validation of the proposed methods was conducted as per International Council for Harmonisation (ICH) guidelines. The proposed methods were successfully applied for the determination of the proposed drugs in bulk powder, ophthalmic solution, and rabbit's aqueous humour.

Optimization of localized surface plasmon resonance hot spots in surface-enhanced infrared absorption spectroscopy aluminum substrate as an optical sensor coupled to chemometric tools for the purity assay of quinary mixtures.

Surface-enhanced infrared absorption spectroscopy offers an alternative to conventional IR spectroscopy and utilizes the signal enhancement exerted by the plasmon resonance of nanostructured metal thin films. Citrate-capped silver nanoparticles were prepared in a single-step method, and their morphology was identified using transmission electron microscopy, scanning electron microscopy, ultraviolet/visible spectrophotometry, and Zetasizer. The nanoparticles generated were deposited on the surface of cheap aluminum slides for different durations aiming for the selection of the best time producing a thin film, suitable to act as a lab-on-a-chip SEIRA substrate. These substrates were coupled to partial least squares regression tools for simultaneous resolving of the quinary mixture in commercial dosage forms of bisoprolol, perindopril, bisoprolol acid degradation product, bisoprolol alkali degradation product, and perindoprilat in concentration ranges of 15-75, 60-300, 15-55, 12-60, and 20-80 µg/mL with limits of detection values of 0.69, 3.43, 0.97, 1.25, and 1.09 µg/mL, respectively. Overall, we could demostrate that the localized surface plasmon resonance sensor coupled to chemometrics provides cheap, simple, selective, multiplex, rapid, and molecular specific procedures for impurity detection, which would be beneficial in many applications for quality control and quality accuracy of active pharmaceutical ingredients.

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability-indicating orthogonal testing protocol.

Forced degradation studies are crucial for the evaluation of the stability and biosimilarity. Here, adalimumab was subjected to oxidation, pH, temperature, agitation and repeated freeze-thaw in order to generate all possible degradation products. An orthogonal stability-indicating testing protocol comprising SE-HPLC, RP-HPLC, TapeStation gel electrophoresis, dynamic light scattering (DLS), and functional receptor binding assay was developed and validated. The assay protocol was used for the assessment of the pattern and kinetics of aggregation/degradation of adalimumab. SE-HPLC and DLS were used to show the formation of aggregates/fragments of adalimumab under nondenaturing conditions. TapeStation electrophoresis was performed under denaturing conditions to reveal the nature of aggregates. Results of the receptor binding assay agreed to those of SE-HPLC and DLS which indicated that it can be used as an activity-indicating assay for adalimumab. RP-HPLC demonstrated excellent selectivity for adalimumab in the presence of its oxidized forms. The kinetics of degradation was studied in each case and the results showed that it followed the first-order reaction kinetics. Correlation between the results supported the quality assessment of the tested product in industrial and clinical settings. This orthogonal protocol is a useful tool in stability assessment of monoclonal antibodies and a key criterion for the biosimilarity assessment.

Stability-indicating determination of rebamipide in the presence of its acid degradation products.

Four sensitive and precise stability-indicating methods for the determination of rebamipide (REB) in the presence of its acid-degradation products and in a pharmaceutical formulation were developed and validated. Method A used the first derivative of the ratio spectra (1DD) spectrophotometric method by measuring the peak amplitude at 249.4 nm (maximum) and at 259 nm (minimum), and at the total peak amplitude (from 249.4 to 259 nm, 1DD(249.4 + 259 nm)) in the range of 2-14 microg/mL. This method yielded mean recoveries of 99.87 +/- 0.83, 100.04 +/- 0.75, and 100.28 +/- 1.11%, respectively. Method B is a dual wavelength method, which allows the determination of REB in presence of its acid-degradation products by measuring the absorbance difference between 254 and 269 nm within a linearity range of 5-65 microg/mL; it showed a mean recovery of 99.84 +/- 1.06. Method C is a TLC-densitometric procedure in which REB was separated from its degradation products using a developing solution of methanol-chloroform-ammonia (8.5 + 1.5 + 0.5, v/v/v). The quantitative evaluation of REB at 329 nm was linear over the concentration range of 0.50-4.5 microg/band, with a mean recovery of 99.49 +/- 0.99% even in the presence of up to 90% degradation products. Method D is an RP-HPLC procedure. It provided the complete separation of REB from its degradation products on an Xterra C18 column using phosphate buffer (pH 6, 0.01 M)-methanol (1 + 1, v/v) as the mobile phase (UV detection at 254 nm). Recovery was 99.28 +/- 0.78% within the range of 10-190 microg/mL. The selectivity of the proposed methods was checked using laboratory-prepared mixtures. The proposed methods have been successfully applied to the analysis of REB in pharmaceutical dosage forms without interference from other dosage form excipients.

Possible potentiation by certain antioxidants of the anti-inflammatory effects of diclofenac in rats.

In the present study, we investigated the potential beneficial impact of the addition of antioxidant supplements to diclofenac regimen in a model of carrageenan-induced paw. Rats were treated daily with antioxidants, that is, a-lipoic acid (50 mg/kg), selenium (2.5 mg/kg), vitamin C (1 g/kg), vitamin E (300 mg/kg), or zinc (25 mg/kg) on seven successive days and then received a single treatment with diclofenac or saline before carrageenan was injected to induce paw inflammation. The results indicated that these combinations did not significantly affect the percentage inhibition of paw edema caused by diclofenac alone; however, some combination treatments ameliorated signs of concomitant oxidative stress (such as alterations in plasma malondialdehyde (MDA) levels, hemolysate reduced glutathione levels, and erythrocytic superoxide dismutase enzyme activities) imparted by diclofenac alone. In some cases, few tested antioxidants in combination with diclofenac resulted in increased plasma levels of interleukin- (IL-) 6 and C-reactive protein (CRP). In conclusion, the results of these studies suggested to us that the added presence of natural antioxidants could be beneficial as standard anti-inflammatory therapeutics for a patient under diclofenac treatment, albeit that these effects do not appear to significantly build upon those that could be obtained from this common anti-inflammatory agent per se.

Niacin, an innovative protein kinase-C-dependent endoplasmic reticulum stress reticence in murine Parkinson's disease.

AIMS: Niacin (NIA) supplementation showed effectiveness against Parkinson's disease (PD) in clinical trials. The depletion of NAD and endoplasmic reticulum stress response (ERSR) are implicated in the pathogenesis of PD, but the potential role for NAD precursors on ERSR is not yet established. This study was undertaken to decipher NIA molecular mechanisms against PD-accompanied ERSR, especially in relation to PKC. METHODS: Alternate-day-low-dose-21 day-subcutaneous exposure to rotenone (ROT) in rats induced PD. Following the 5th ROT injection, rats received daily doses of either NIA alone or preceded by the PKC inhibitor tamoxifen (TAM). Extent of disease progression was assessed by behavioral, striatal biochemical and striatal/nigral histopathological/immunohistochemical analysis. KEY FINDINGS: Via activating PKC/LKB1/AMPK stream, NIA post-treatment attenuated the ERSR reflected by the decline in ATF4, ATF6 and XBP1s to downregulate the apoptotic markers, CHOP/GADD153, p-JNK and active caspase-3. Such amendments congregated in motor activity/coordination improvements in open field and rotarod tasks, enhanced grid test latency and reduced overall PD scores, while boosting nigral/striatal tyrosine hydroxylase immunoreactivity and increasing intact neurons (Nissl stain) in both SNpc and striatum that showed less neurodegeneration (H&E stain). To different extents, TAM reverted all the NIA-related actions to prove PKC as a fulcrum in conveying the drug neurotherapeutic potential. SIGNIFICANCE: PKC activation is a pioneer mechanism in the drug ERSR inhibitory anti-apoptotic modality to clarify NIA promising clinical and potent preclinical anti-PD efficacy. This kinase can be tagged as a druggable target for future add-on treatments that can assist dopaminergic neuronal aptitude against this devastating neurodegenerative disease.

Dimethyl fumarate abrogates striatal endoplasmic reticulum stress in experimentally induced late-stage Huntington's disease: Focus on the IRE1α/JNK and PERK/CHOP trajectories.

Introduction: Dimethyl fumarate (DMF) is FDA-approved for use in patients with relapsing multiple sclerosis, and it processes neuroprotection in several experimental settings; however, its impact on combating Huntington's disease (HD) remains elusive. This study aimed to explore the role of DMF post-treatment on HD mediated endoplasmic reticulum (ER) stress response in a selective striatal degeneration HD model. Methods: Rats, exposed to 3-nitropropionic acid, were either left untreated or post-treated with DMF for 14 days. Results and Discussion: DMF reduced locomotion deficits in both the open field and beam walk paradigms, boosted the striatal dopamine (DA) content, improved its architecture at the microscopic level, and hindered astrogliosis. Mechanistically, DMF limited the activation of two of the ER stress arms in the striatum by reducing p-IRE1α, p-JNK, and p-PERK protein expressions besides the CHOP/GADD153 content. Downstream from both ER stress arms' suppression, DMF inhibited the intrinsic apoptotic pathway, as shown by the decrease in Bax and active caspase-3 while raising Bcl-2. DMF also decreased oxidative stress markers indicated by a decline in both reactive oxygen species and malondialdehyde while boosting glutathione. Meanwhile, it enhanced p-AKT to activate /phosphorylate mTOR and stimulate the CREB/BDNF/TrkB trajectory, which, in a positive feedforward loop, activates AKT again. DMF also downregulated the expression of miRNA-634, which negatively regulates AKT, to foster survival kinase activation. Conclusion: This study features a focal novel point on the DMF therapeutic ability to reduce HD motor manifestations via its ability to enhance DA and suppress the IRE1α/JNK and PERK/CHOP/GADD153 hubs to inhibit the mitochondrial apoptotic pathway through activating the AKT/mTOR and BDNF/TrkB/AKT/CREB signaling pathways and abating miRNA-634 and oxidative stress.

Stability assessment of Polatuzumab vedotin and Brentuximab vedotin using different analytical techniques.

Antibody-drug conjugates (ADC) are considered to be fast-growing innovative biopharmaceuticals. The science used for conjugating potent cytotoxic payload to the targeted monoclonal antibody through a chemical linker has played a great value in the area of oncology treatment. In this study; Polatuzumab vedotin (POLA) and Brentuximab vedotin (SGN-35) were subjected to various stress conditions enclosing different pH, thermal stress, agitation, and successive cycles of freeze and thaw in order to produce potential degradation by-products and guarantee the appropriateness of the applied testing protocol. Different analytical techniques were established and validated to be used in the quantitation of the degraded products from different perspectives. The formation of ADC aggregates and fragments was monitored using SE-HPLC as well as dynamic light scattering (DLS). The drug antibody ratio (DAR) and ADC conjugation profile were determined using hydrophobic interaction chromatography (HIC-HPLC). In addition to performing a statistical interpretation of HIC-HPLC results by principal component analysis (PCA) to explicate the obtained data. Also, the quantity of the unconjugated toxic drug was quantified using RP-HPLC. Testing the binding activity of ADC to their target receptor ADC was conducted using ELISA. Results presented that used assay protocol had worked as a complementary design for characterization and stability assessment of the used ADC. Variances in the stability profile of both products were observed which could be attributed to the usage of different formulation buffers. This highlighted the importance of using multiple techniques for the assessment of the quality attributes of such sophisticated products. The analytical assay protocol should be used for the evaluation of the quality and stability of several ADC.

Spectrum subtraction as a complementary method for six resolution techniques resolving overlapping spectra; application to multicomponent veterinary formulation with greenness and whiteness assessment.

Mathematical filtration is an efficient tool to resolve the overlapping spectra of binary mixtures in zero or first order form. Herein, a comparative study was conducted between six economic, accurate and precise spectrophotometric methods for determination of Triclabendazole (TCB) and Levamisole HCl (LVM). Each component was resolved with minimum mathematical steps in its zero-order absorption spectrum by ratio subtraction, constant multiplication, and the recent factorized response method; coupled with spectrum subtraction. In addition, the mixture was resolved in its first derivative form by derivative subtraction, D1 constant multiplication, and the recent D1 factorized response method; coupled with spectrum subtraction. Results obtained were also compared to those obtained from constant value, concentration value, and derivative ratio methods. The linearity range was found to be either 1.0-10.0 µg/mL or 2.0-20.0 µg/mL for TCB, and 2.0-14.0 µg/mL for LVM with LOD of 0.08 µg/mL and 0.19 µg/mL, respectively. Validation of the proposed methods was performed according to VICH guidelines. Results obtained from the statistical data showed no significant difference regarding accuracy and precision compared to the reported methods. The developed spectrophotometric methods followed the principles of green analytical chemistry, in which the green assessment was done through four tools, called, National Environmental Methods Index (NEMI), Analytical Eco-Scale (AES), Green Analytical Procedure Index (GAPI) and Analytical greenness metric (AGREE). Also, a white assessment was performed using RGB model. The proposed methods could offer an economic alternative for the routine analysis of bulk materials and combined veterinary dosage form.

Novel eco-friendly spectrophotometric approaches for resolution of fixed dose formulation of phenylbutazone with minor component of dexamethasone: Greenness assessment by AGREE tool.

Spectrophotometric resolution of severely overlapped binary mixtures with minor component is challenging. Herein, coupling of mathematical manipulation steps with sample enrichment was conducted on the binary mixture spectrum of Phenylbutazone (PBZ) and Dexamethasone sodium phosphate (DEX) to resolve, for the first time each component separately. Simultaneous determination of both components in a mixture ratio of 1:0.002 was achieved in their zero or first order spectra by the recent factorized response method along with ratio subtraction and constant multiplication methods; all coupled with spectrum subtraction. In addition, a novel second derivative concentration value and second derivative constant value methods were developed for PBZ determination. The concentration of the minor component DEX was obtained, without preliminary separation steps by derivative ratio after sample enrichment by either spectrum addition or standard addition. Spectrum addition approach showed superior characteristics compared to standard addition technique. All proposed methods were placed through a comparative study. Linear correlation was found to be 1.5-18.0 µg/mL for PBZ, and 4.0-45.0 µg/mL for DEX. The proposed methods were validated in accordance with ICH guidelines. The greenness assessment of the proposed spectrophotometric methods was evaluated by AGREE software. Results obtained from the statistical data were evaluated by comparing to one another as well as the official USP methods. These methods offer a cost and time effective platform to analyze bulk materials and combined veterinary formulation.

Regulation of NOX/p38 MAPK/PPARα pathways and miR-155 expression by boswellic acids reduces hepatic injury in experimentally-induced alcoholic liver disease mouse model: novel mechanistic insight.

Alcoholic liver disease (ALD) refers to hepatic ailments induced by excessive alcohol intake. The pathogenesis of ALD comprises a complex interplay between various mechanistic pathways, among which inflammation and oxidative stress are key players. Boswellic acids (BAs), found in Boswellia serrata, have shown hepatoprotective effects owing to their antioxidant and anti-inflammatory activities, nevertheless, their therapeutic potential against ALD has not been previously investigated. Hence, this study was performed to depict the possible protective effect of BAs and detect their underlying mechanism of action in an experimentally-induced ALD mouse model. Male BALB/c mice were equally categorized into six groups: control, BAs-treated, ALD, and ALD that received BAs at three-dose levels (125, 250, and 500 mg/kg) by oral gavage for 14 days. Results showed that the high dose of BAs had the most protective impact against ALD according to histopathology examination, blood alcohol concentration (BAC), and liver function enzymes. Mechanistic investigations revealed that BAs (500 mg/kg) caused a significant decrease in cytochrome P450 2E1(CYP2E1), nicotine adenine dinucleotide phosphate oxidase (NOX) 1/2/4, p38 mitogen-activated protein kinase (MAPK), and sterol regulatory element-binding protein-1c (SREBP-1c) levels, and the expression of miR-155, yet increased peroxisome proliferator-activated receptor alpha (PPARα) levels. This led to an improvement in lipid profile and reduced hepatic inflammation, oxidative stress, and apoptosis indices. In summary, our study concludes that BAs can protect against ethanol-induced hepatic injury, via modulating NOX/p38 MAPK/PPARα pathways and miR-155 expression.

Sophorolipids produced by Yarrowia lipolytica grown on Moringa oleifera oil cake protect against acetic acid-induced colitis in rats: impact on TLR-4/p-JNK/NFκB-p65 pathway.

OBJECTIVES: Toll-like receptor-4 (TLR-4) activation plays a major role in triggering oxidative stress (OS) and inflammation implicated in the pathogenesis of ulcerative colitis (UC). Due to sophorolipids (SLs) antioxidant and anti-inflammatory properties, they are interestingly becoming more valued for their potential effectiveness in treating a variety of diseases. This study was designed to explore the effect of SLs produced by microbial conversion of Moringa oleifera oil cake using isolated yeast Yarrowia lipolytica against UC induced by acetic acid (AA) in rats. METHODS: The produced SLs were identified by FTIR, 1H NMR and LC-MS/MS spectra, and administered orally for 7 days (200 mg/kg/day) before AA (2 ml, 4% v/v) to induce UC intrarectally on day eight. Biochemically, the levels of TLR-4, c-Jun N-terminal kinase (JNK), nuclear factor kappa B-p65 (NFκB-p65), interleukin-1beta (IL-1ß), malondialdehyd, glutathione, Bax/Bcl2 ratio and the immunohistochemical evaluation of inducible nitric oxide synthase and caspase-3 were assayed. KEY FINDINGS: SLs significantly reduced OS, inflammatory and apoptotic markers in AA-treated rats, almost like the reference sulfasalazine. CONCLUSIONS: This study provided a novel impact for SLs produced by microbial conversion of M. oleifera oil cake against AA-induced UC in rats through hampering the TLR-4/p-JNK/NFκB-p65 signalling pathway.

The mechanistic interplay between Nrf-2, NF-κB/MAPK, caspase-dependent apoptosis, and autophagy in the hepatoprotective effects of Sophorolipids produced by microbial conversion of banana peels using Saccharomyces cerevisiae against doxorubicin-induced hepatotoxicity in rats.

BACKGROUND: Doxorubicin (DOX) is a well-known chemotherapeutic agent which causes serious adverse effects due to multiple organ damage, including cardiotoxicity, nephrotoxicity, neurotoxicity, and hepatotoxicity. The mechanism of DOX-induced organ toxicity might be attributed to oxidative stress (OS) and, consequently, activation of inflammatory signaling pathways, apoptosis, and blockage of autophagy. Sophorolipids (SLs) as a glycolipid type of biosurfactants, are natural products that have unique properties and a wide range of applications attributed to their antioxidant and anti-inflammatory properties. AIMS: Production of low-cost SLs from Saccharomyces cerevisiae grown on banana peels and investigating their possible protective effects against DOX-induced hepatotoxicity. MAIN METHODS: The yeast was locally isolated and molecularly identified, then the yielded SLs were characterized by FTIR, 1H NMR and LC-MS/MS spectra. Posteriorly, thirty-two male Wistar rats were randomly divided into four groups; control (oral saline), SLs (200 mg/kg, p.o), DOX (10 mg/kg; i.p.), and SL + DOX (200 mg/kg p.o.,10 mg/kg; i.p., respectively). Liver function tests (LFTs), oxidative stress, inflammatory, apoptosis as well as autophagy markers were investigated. KEY FINDINGS: SLs were produced with a yield of 49.04% and treatment with SLs improved LFTs, enhanced Nrf2 and suppressed NF-κB, IL-6, IL-1ß, p38, caspase 3 and Bax/Bcl2 ratio in addition to promotion of autophagy when compared to DOX group. SIGNIFICANCE: Our results revealed a novel promising protective effect of SLs against DOX-induced hepatotoxicity in rats.

Advanced chemometric methods as powerful tools for impurity profiling of drug substances and drug products: Application on bisoprolol and perindopril binary mixture.

Impurity profiling has a rising importance nowadays due to the increased health problems associated with impurities and degradation products found in several drug substances and formulations. Three advanced, accurate and precise chemometric methods were developed as impurity profiling methods for a mixture of bisoprolol fumarate (BIS) and perindopril arginine (PER) with their degradation products which represent drug impurity or a precursor to such impurity. The methods applied were Partial Least Squares (PLS-1), Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Artificial Neural Networks (ANN). Genetic Algorithm (GA) was used as a variable selection tool to select the most significant wavelengths for the three chemometric models. For proper analysis, a 5-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products (impurities). The validity of the proposed methods was assessed using an independent validation set. The designed models were able to predict the concentrations of the drugs and the degradation products/impurities in the validation set and pharmaceutical formulation. The proposed methods presented a powerful alternative to traditional and expensive chromatographic methods as impurity profiling tools.

LCZ696 (sacubitril/valsartan) protects against cyclophosphamide-induced nephrotoxicity in adult male rats: Up-regulation of Apelin-13/ACE2, miR-200, and down-regulation of TGF-ß/SMAD 2/3 and miR-192.

BACKGROUND: Cyclophosphamide (CP) is a widely used chemotherapeutic drug. However, the associated nephrotoxicity restricts its clinical use. AIM: The present research was designed to study the impact of LCZ696 (LCZ); which is a combination of Sacubitril/Valsartan compared to valsartan (VAL) on CP-induced nephrotoxicity. MAIN METHODS: Sixty adult male Wistar rats were randomly and equally assigned into 6 groups as follows: Control, LCZ (30 mg/kg, p.o.), VAL (15 mg/kg, p.o.), CP (200 mg/kg, single dose, i.p.), CP/LCZ, and CP/VAL groups. LCZ and VAL were given once daily for 6 days prior to CP (groups 5 & 6). At the end of the experiment, kidney functions, oxidants/antioxidants, inflammatory and fibrotic biomarkers in renal tissues were assessed. Further, immunohistochemical, and histomorphometric analyses were carried out. KEY FINDINGS: In comparison with CP-treated rats, LCZ resulted in a significant reduction in serum urea (26.6 %) and creatinine (63 %), moreover it decreased renal content of reactive oxygen species (ROS), zinc finger E-box-binding homeobox (ZEB)-1, SMAD2/3, plasminogen activator inhibitor (PAI)-1, fibronectin, histone deacetylase (HDAC)-4, nuclear factor-kappa B (NF-κB) and miR-192 expression by ~40-60 % as well as the immunohistological expressions of transforming growth factor-ß (TGF-ß) and anti-phospho Histone (H2AX) by ~75 % reduction. Whereas the renal total antioxidant capacity (TAC), apelin-13, miR-200 expression, and the immunoreactivity of angiotensin-converting enzyme 2 (ACE2) were enhanced by ~3-4-folds. Noteworthy, the prophylactic effect of LCZ was superior to VAL on the histomorphometric and immunohistological levels. SIGNIFICANCE: Prophylactic administration of LCZ protected against CP-induced nephrotoxicity via up-regulating apelin-13/ACE2, miR-200, and down-regulating TGF-ß/SMAD 2/3 and miR-192.

Development and validation of a reversed-phase column liquid chromatographic method for the determination of five cephalosporins in pharmaceutical preparations.

A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters microBondapak C18 column (250 x 4.6 mm id, 10 pm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)-acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30-300, 3-30, and 15-120 microg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.

Simultaneous determination of diloxanide furoate and metronidazole in presence of diloxanide furoate degradation products.

Three methods are presented for the simultaneous determination of diloxanide furoate (DLX) and metronidazole (MTR), used for their antiprotozoal and antiamoebic effect, in the presence of DLX alkaline degradates and in pharmaceutical formulations, without previous separation. The first method is chemometric-assisted spectrophotometry, in which principal component regression and partial least squares were applied. These two approaches were successfully applied to quantify each drug in the mixture using the information included in, the absorption spectra in the range of 225-320 nm. The second method is TLC-densitometry, in which the binary mixture and degradates were separated on silica gel plates using a chloroform-acetone-glacial acetic acid (9.5 + 0.5 + 0.07, v/v/v) mobile phase and the bands were scanned at 254 nm. The last method is HPLC, in which DLX, MTR, and degradates were separated using the mobile phase acetonitrile-0.05 M dibasic potassium phosphate (25 + 75, v/v), adjusted to pH 4 with orthophosphoric acid, at a flow rate of 1 mL/min, on a C18 analytical column. Detection was at 254 nm. The proposed methods were successfully applied for the analysis of DLX and MTR in pharmaceutical formulations, and the results were statistically compared with a reported spectrophotometric method.

Implementation of Two Chromatographic Methods for Simultaneous Quantitation of Thioctic Acid, Benfotiamine and Cyanocobalamin.

Two accurate and sensitive chromatographic methods have been introduced and validated for the simultaneous determination of thioctic acid, benfotiamine and cyanocobalamin in bulk powders and in their pharmaceutical formulation. Method A is reversed-phase ultra performance liquid chromatographic method with an isocratic elution, where a rapid separation was accomplished on a Zorbax C8 column using a mobile phase of acetonitrile:0.05 M phosphate buffer (pH 6 adjusted by o-phosphoric acid) (23:77, v/v). The retention times (tR) were 0.578, 0.852 and 1.376 for cyanocobalamin, benfotiamine and thioctic acid, respectively. The separated peaks were revealed at 210.0 nm. Method B is a thin-layer densitometric method where the separation of the studied drugs was carried out on silica gel plates using methanol-chloroform-heptane-1-sulphonic acid sodium salt (0.4%) (7:3:0.1, by volume) as a mobile phase, and scanning of the separated bands was done at 240.0 nm. The retardation factor (Rf) values were 0.17, 0.48 and 0.75 for cyanocobalamin, benfotiamine and thioctic acid, respectively. Validation of the methods was achieved following ICH guidelines and the applied methods succeeded to determine the cited drugs in their pure forms and capsules. Results were statistically compared to the manufacturer's method where no significant difference was observed.