Ecofriendly single-step HPLC and TLC methods for concurrent analysis of ternary antifungal mixture in their pharmaceutical products.

Two accurate, sensitive, and selective methods for simultaneous determination of miconazole nitrate (MIC), nystatin (NYS), and metronidazole (MET) in pure state or drug product were established and verified. First, RP-HPLC-DAD was designed. Separation was accomplished using a ZOBRAX Eclipse Plus RP-C8 column that was running under an isocratic elution of methanol: 0.05% aqueous solution of sodium dodecyl sulphate (40: 60 v/v), with a flow rate that was regulated at 0.8 mL/min. The column temperature was adjusted at 25 °C and diode array detector was monitored at 220 nm. The linearity range of the proposed method was achieved at the concentration of 5-50, 4-50, and 4-40 µg/mL and the attained retention time for the studied drugs was 2.52, 3.52 and 4.99 min for MIC, NYS, and MET, correspondingly. Second, a TLC-densitometric approach was used to resolve the three compounds. Resolution of the three cited drugs was carried out using TLC aluminum plates pre-coated with 0.25 mm silica gel 60 F254. A developing solvent comprised ethyl acetate: toluene: methanol: triethyl amine: formic acid (3: 1: 7: 0.3: 0.1 by volume) (pH = 5.5) was utilized and scanning of the resolved bands at 215 nm. Linearity of the developed TLC method was evaluated and evident to be 0.4-2, 0.4-2.2, and 0.4-2 µg/band for MIC, NYS, and MET, in that order. The suggested chromatographic methods were verified according to ICH directives. The findings of the developed chromatographic procedures were statistically compared with the results of the reported ones using student's t-test and F-test. Furthermore, two green assessment tools evaluated the indicated methods' level of greenness (GAPI and AGREE).

Comparative Study of Augmented Classical Least Squares Models for UV Assay of Co-Formulated Antiemetics Together with Related Impurities.

The classical least squares (CLS) model and three augmented CLS models are adopted and validated for the analysis of pyridoxine HCl (PYR), cyclizine HCl (CYC), and meclizine HCl (MEC) in a quinary mixture with two related impurities: the CYC main impurity, Benzhydrol (BEH), which has carcinogenic and hepatotoxic effects, and the MEC official impurity, 4-Chlorobenzophenone (BEP). The proposed augmented CLS models are orthogonal signal correction CLS (OSC-CLS), direct orthogonal signal correction CLS (DOSC-CLS), and net analyte processing CLS (NAP-CLS). These models were applied to quantify the three active constituents in their raw materials and their corresponding dosage forms using their UV spectra. To evaluate the CLS-based models sensibly, we design a comparative study involving two sets: the training set to construct models and the validation set to assess the prediction abilities of these models. A five-level, five-factor calibration design was established to produce 25 mixtures for the calibration set. In addition, 16 experiments were performed for a test set distributed equally between the in-space and out-space samples. The primary criterion for comparing the models' performance was the validation set's root mean square error of prediction (RMSEP) value. Finally, augmented CLS models showed acceptable results for assaying the three analytes. The results were compared statistically with the reported HPLC methods; however, the DOSC-CLS model proved the best for assaying the dosage forms.

A computational investigation of thymidylate synthase inhibitors through a combined approach of 3D-QSAR and pharmacophore modelling.

Thymidylate synthase (TS) is a crucial target of cancer drug discovery and is mainly involved in the De novo synthesis of the DNA precursor thymine. In the present study, to generate reliable models and identify a few promising molecules, we combined QSAR modelling with the pharmacophore hypothesis-generating technique. Input molecules were clustered on their similarity, and a cluster of 74 molecules with a pyrimidine moiety was chosen as the set for 3D-QSAR and pharmacophore modelling. Atom-based and field-based 3D-QSAR models were generated and statistically validated with R2 > 0.90 and Q2 > 0.75. The common pharmacophore hypothesis(CPH) generation identified the best six-point model ADHRRR. Using these best models, a library of FDA-approved drugs was screened for activity and filtered via molecular docking, ADME profiling, and molecular dynamics simulations. The top ten promising TS-inhibiting candidates were identified, and their chemical features profitable for TS inhibitors were explored.Communicated by Ramaswamy H. Sarma.

Luteolin 4'-Neohesperidoside Inhibits Clinically Isolated Resistant Bacteria In Vitro and In Vivo.

Multidrug resistance (MDR) pathogens are usually associated with higher morbidity and mortality rates. Flavonoids are good candidates for the development of new potential antimicrobials. This research investigated whether luteolin 4'-neohesperidoside (L4N) has antibacterial and synergistic activities against four antibiotic-resistant pathogens: methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, fosA-positive shiga toxin producing the Escherichia coli serogroup O111 (STEC O111), and Bacillus cereus. In vitro antimicrobial susceptibility testing revealed highly potent anti-MRSA (MIC of 106.66 ± 6.95 µg/mL), anti-K. pneumoniae (MIC of 53.33 ± 8.47 µg/mL) and anti-STEC O111 (MIC of 26.66 ± 5.23 µg/mL) activities. Significant synergistic combination was clearly noted in the case of gentamycin (GEN) against Gram-negative bacteria. In the case of B. cereus, the combination of vancomycin (VAN) with L4N could efficiently inhibit bacterial growth, despite the pathogen being VAN-resistant (MIC of 213.33 ± 7.9 µg/mL). In vivo evaluation of L4N showed significant decreases in K. pneumoniae and STEC shedding and colonization. Treatment could significantly diminish the levels of pro-inflammatory markers, tumor necrosis factor-alpha (TNF-α), and immunoglobulin (IgM). Additionally, the renal and pulmonary lesions were remarkably enhanced, with a significant decrease in the bacterial loads in the tissues. Finally, this study presents L4N as a potent substitute for traditional antibiotics with anti-STEC O111 and anti-K. pneumoniae potential, a finding which is reported here for the first time.

Ameliorative Effect of Curcumin Nanoparticles against Monosodium Iodoacetate-Induced Knee Osteoarthritis in Rats.

Aim: This study is aimed at evaluating the use of curcumin-loaded polylactic-co-glycolic acid nanoparticles (CUR-loaded PLGA NPs) as a treatment against monosodium iodoacetate- (MIA-) induced knee OA. Materials and Methods: Eighteen rats were assigned to three groups (n = 6), namely, normal control group that received intra-articular injections (IAIs) of saline, an OA control group that received an IAIs of MIA (2 mg/50 µL), and a treatment group (MIA+CUR-loaded PLGA NPs) that received IAIs of CUR-loaded PLGA NPs (200 mg/kg b.wt). Results: The CUR NP treatment against knee OA alleviated radiographic alternations and histopathological changes and inhibited the upregulation in the serum levels of interleukin-1ß, tumor necrosis factor-α, interleukin-6, and transforming growth factor-beta and the downregulation in interleukin-10. CUR NP-treated joints also decreased the mRNA expression of nuclear factor-kappa B and inducible nitric oxide synthase and the protein expression of matrix metalloproteinase-13 and caspase-3. Finally, CUR-loaded PLGA NP treatment mitigated the loss of type II collagen, which resulted in a significant reduction in malondialdehyde level and increased the glutathione content and superoxide dismutase activity compared with that of the OA group. Conclusion: This study demonstrated that the administration of CUR NPs could provide effective protection against MIA-induced OA and knee joint histological deteriorated changes due to its anti-inflammatory, antioxidant, and antiapoptotic properties.

Assessment of the Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells against a Monoiodoacetate-Induced Osteoarthritis Model in Wistar Rats.

Osteoarthritis (OA) of the knee is a debilitating condition that can severely limit an individual's mobility and quality of life. This study was designed to evaluate the efficacy of bone marrow-derived mesenchymal stem cell (BM-MSC) treatment in cartilage repair using a rat model of monoiodoacetate- (MIA-) induced knee OA. OA was induced in the knee joint of rats by an intracapsular injection of MIA (2 mg/50 µL) on day zero. The rats were divided into three groups (n = 6): a normal control group, an osteoarthritic control group, and an osteoarthritic group receiving a single intra-articular injection of BM-MSCs (5 × 106 cells/rat). The knee diameter was recorded once per week. By the end of the performed experiment, X-ray imaging and enzyme-linked immunosorbent assay analysis of serum inflammatory cytokines interleukin-1beta (IL-ß), IL-6, and tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokines interleukin-10 and transforming growth factor-beta (TGF-ß) were carried out. In addition, RT-PCR was used to measure nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and type II collagen mRNA levels and Western blot analysis was used to determine caspase-3 protein levels in all treated groups. Finally, hematoxylin/and eosin stains were used for histopathological investigation. Administration of BM-MSCs significantly downregulated knee joint swelling and MIA-induced (IL-1ß, IL-6, and TNF-α) and upregulated IL-10 and TGF-ß as well. Moreover, BM-MSC-treated osteoarthritic rats exhibited decreased expression of NF-κB, iNOS, and apoptotic mediator (caspase-3) and increased expression of type II collagen when compared to rats treated with MIA alone. The hematoxylin/eosin-stained sections revealed that BM-MSC administration ameliorated the knee joint alterations in MIA-injected rats. BM-MSCs could be an effective treatment for inflamed knee joints in the MIA-treated rat model of osteoarthritis, and the effect may be mediated via its anti-inflammatory and antioxidant potential.

Rutin and Quercetin Counter Doxorubicin-Induced Liver Toxicity in Wistar Rats via Their Modulatory Effects on Inflammation, Oxidative Stress, Apoptosis, and Nrf2.

The presented study was performed to verify whether rutin and/or quercetin can inhibit liver injury induced by doxorubicin (DXR) in male Wistar rats. In this study, male Wistar rats were treated via the oral route with rutin and quercetin (50 mg/kg) either alone or in combination every other day for five weeks concomitant with receiving intraperitoneal DXR (2 mg/kg) two times a week for five successive weeks. Quercetin, rutin, and their combination significantly improved the deteriorated serum AST, ALT, and ALP activities and total bilirubin level, as well as albumin, AFP, and CA 19.9 levels in DXR-injected rats. Treatments of the DXR-injected group with quercetin and rutin prevented the elevation in liver lipid peroxidation and the reduction in superoxide dismutase, glutathione-S-transferase and glutathione peroxidase activities, and glutathione content. Treatments with quercetin and rutin significantly repressed the elevated expression of liver p53 and TNF-α and enhanced Nrf2 expression. Furthermore, the treatments significantly reduced DXR-induced liver histological changes. In conclusion, rutin and quercetin either alone or in combination may have potential preventive effects against DXR-induced hepatotoxicity through inhibiting oxidative stress, inflammation, and apoptosis as well as modulating the Nrf2 expression.

Isatin Counteracts Diethylnitrosamine/2-Acetylaminofluorene-Induced Hepatocarcinogenesis in Male Wistar Rats by Upregulating Anti-Inflammatory, Antioxidant, and Detoxification Pathways.

Hepatocellular carcinoma (HCC) represents around 85% of all known types of liver cancers and is estimated to be the fifth most common cause of cancer-related death worldwide. The current study assessed the preventive efficacy of isatin on diethylnitrosamine (DENA)/2-acetylaminofluorene (2-AAF)-induced hepatocarcinogenesis in male Wistar rats and investigated the underlying cellular and molecular mechanisms. HCC was initiated by intraperitoneal injection of DENA (150 mg/kg/week) for two weeks, followed by oral 2-AAF (20 mg/kg) every other day for three successive weeks. Oral isatin or vehicle (control) was administered at 25 mg/kg for 20 weeks during and following HCC induction. Isatin ameliorated the deleterious effects of DENA/2-AAF on liver function as evidenced by reduced serum levels of AST, ALT, total bilirubin, albumin, and liver tumor biomarkers (CA19.9 and AFP) compared to control DENA/2-AAF-treated rats. Histopathological evaluations demonstrated that isatin-mediated protection against hepatocarcinogenesis was accompanied by a decline in hepatic lipid peroxidation, a marker of oxidative stress, and enhanced antioxidant capacity, as evidenced by increased glutathione and superoxide dismutase expression. Isatin treatment also upregulated expression of the major stress-response transcription factor Nrf2 and the detoxifying enzymes NAD(P)H quinine oxidoreductase and glutathione-S-transferase alpha 2 and downregulated expression of the proliferation marker Ki67. Moreover, isatin significantly reduced the DENA/2-AAF-induced decrease in hepatic expression of anti-apoptotic Bcl2 and the DENA/2-AAF-induced increases in pro-inflammatory and pro-apoptotic factors (TNF-α, NF-κB p50, NF-κB p65, p53, and caspase 3). Thus, it can be concluded that isatin may protect against chemically induced hepatocarcinogenesis by enhancing cellular antioxidant, anti-inflammatory, and detoxification mechanisms, in part through upregulation of the Nrf2 signaling pathway.

Protective effects of perindopril against indomethacin-induced gastric mucosal damage through modulation of DDAH-1/ADMA and ACE-2/ANG-(1-7) signaling pathways.

Indomethacin is a widely used nonsteroidal anti-inflammatory drug; however, its clinical utility is accompanied by serious adverse reactions including peptic ulcers. The current study aims to investigate the protective potential of perindopril against indomethacin-induced gastric injury in rats. Perindopril (4 mg/kg) was administered orally for 7 days and indomethacin (60 mg/kg, single oral dose) was administered on the 7th day, 1 h after perindopril administration. Pantoprazole was used as a standard agent. Ulcer index (UI), preventive index ratio (PI), histopathological examination, oxidative stress, and inflammatory biomarkers were investigated. Perindopril significantly decreased UI while increased PI and counteracted histopathological aberrations induced by indomethacin. It alleviated indomethacin-induced oxidative stress by lowering NO while increasing GSH content and superoxide dismutase activity. Perindopril significantly downregulated TNF-α and asymmetric dimethylarginine (ADMA), while significantly upregulated COX-2, PGE-2, dimethylarginine dimethylaminohydrolase-1 (DDAH-1), ANG-(1-7), and ACE-2 expression. Together, these findings suggest the gastroprotective effects of perindopril through modulation of DDAH-1/ADMA and ACE-2/ANG-(1-7) signaling.HIGHLIGHTSPerindopril attenuated gastric histopathological damage.It increased GSH content and SOD activity while decreased NO content.It modulated gastric ADMA and DDAH-1 activity.It reduced TNF-α, while increased COX-2 and PGE-2 expression.It upregulated ACE-2 activity and ANG-(1-7) protein expression.

Chemical characteristics and targeted encapsulated Cordia myxa fruits extracts nanoparticles for antioxidant and cytotoxicity potentials.

A promising Cordia myxa fruit (CMF) extract targets an additional incorporation in functional foods. It retains appropriate health welfares owing to its antioxidant properties with limited incorporation in food matrices due its hydrophobicity. Therefore, CMF extract micro- and nanocapsulation was performed to protect and facilitate consistency of produced hydrophobic foods matrices. Furthermore, to determine its phytochemicals, antioxidant, and cytotoxic effects by applying analytical HPLC, FRAP and SRB assay, respectively. HPLC analysis of the tested extracts revealed the presence of, 25.59 ± 1.78 mg catechin/g, 69.68 ± 4.20 mg quercetin/g, and 112.72 ± 8.38 mg gallic acid/g extract. The CMF extract displayed a potent DPPH radicals' scavenger and FRAP high reduction capability in a dose-dependent manner. The potent pharmacological activities of CMF extract may be ascribed to high concentration of polyphenolics including flavonoids which strongly reported to possess an antitumor and antioxidant activities. To confirm the efficient CMF incorporation in micro- and nanosystems and their thermal stabilities to withstand the high temperatures applied during some food processing. DSC of the apparent melt of non-capsulated CMF and encapsulated forms (MCMF and NCMF) in sodium alginate gel and beads was studied. Results showed that melting point of CMF extract (86.17 °C) indicating its inability whereas the MCMF and NCMF melting points (226.45 and 383.87 °C, respectively) demonstrating the capability of expending alginate - packaging material to shield the vital active compounds of C. myxa fruit to be applied in different targeted delivery products especially that disclosed to high thermal treatments.

Response surface methodology for optimization of micellar-enhanced spectrofluorimetric method for assay of foretinib in bulk powder and human urine.

This work investigates a sensitive and precise enhanced spectrofluorimetric assay for assay of foretinib (FTB); a tyrosine kinase inhibitor drug used for treatment of breast cancer, in tablets and urine through response surface optimization by micelle mediated protocol. The basis of the described method is the enhancement of the fluorescence behavior of FTB in Cremophor RH 40 (Cr RH 40) micellar medium and measuring the fluorescence of FTB at 344 nm after excitation at 245 nm. Optimization was performed through evaluation of diluting solvent, types of organized media, buffer type and its relevant pH. Response surface methodology was applied to obtain the optimized values of variables that mostly affect interaction of Cr RH 40 with FTB using Box-Behnken design. ICH guidelines were adhered for the validation of merit figures. Acceptable linear relationship was obtained between relative fluorescence intensity (RFI) and FTB concentrations in the range of 50 - 1000 µg L-1, with correlation coefficient of 0.998. Accuracy was ≥ 99.82% and calculated limit of detection (LOD) was 10.60 µg L-1. Method applications included FTB assaying in pure bulk powder. Furthermore, applications on urine samples were performed with accuracy of 100.59 ± 3.40%. The method represents echo-friendly approach and effective alternating methodology to the relevant analytical ones for FTB assaying.

Green HPLC-DAD and HPTLC Methods for Quantitative Determination of Binary Mixture of Pregabalin and Amitriptyline Used for Neuropathic Pain Management.

First analytical methods were herein developed for determination of pregabalin (PGB) and amitriptyline (AMT) as an active binary mixture used for management of neuropathic pain whether in pure forms or in human biological fluids (plasma/urine). First method is green high-performance liquid chromatography-diode array detector (HPLC-DAD) after derivatization of PGB with ninhydrin (NIN) on a reversed-phase C18 column using a mobile phase consisting of ethanol:water (97:3%, v/v) pumped isocratically at 0.8 mL/min; AMT were scanned at 215 nm, whereas PGB-NIN was scanned at 580 nm. Second method is High-performance thin-layer chromatography (HPTLC), where PGB and AMT were separated on silica gel HPTLC F254 plates, using ethanol:ethyl acetate:acetone:ammonia solution (8:2:1:0.05, by volume) as a developing system. AMT peaks were scanned at 220 nm, whereas PGB peaks were visualized by spraying 3% (w/v) ethanolic NIN solution and scanning at 550 nm. Linear calibration curves were obtained for human plasma and urine spiked with PGB and AMT over the ranges of 5-100 µg/mL and 0.2-2.5 µg/band for PGB, and 1-100 µg/mL and 0.1-2.0 µg/band for AMT for HPLC-DAD and HPTLC methods, respectively. The suggested methods were validated according to Food and Drug Administration guidelines for bioanalytical methods validation and they can be applied for routine therapeutic drug monitoring for the concerned drugs.

Validated spectral manipulations for determination of an anti-neoplastic drug and its related impurities including its hazardous degradation product.

Innovative and specific double dual wavelength, dual ratio subtraction spectrophotometric methods were carried out along with a successive ratio subtraction spectrophotometric method for determination of dacarbazine and its related impurities including toxic and hazardous ones. For determination of dacarbazine by the double dual wavelength method, the absorbance differences between 323 and 350 nm of the zero order absorption spectra of dacarbazine were used. The values of absorbance difference between 267.2 and 286.2 nm of the zero order spectra of 5-amino-imidazole-4 carboxamide were used for its determination by the dual ratio subtraction method. The zero order absorption spectrum of 2-azahypoxanthine at 235 nm was used for its determination after applying the successive ratio subtraction method. ICH guidelines were followed for validation of the developed methods, where linear relationships were obtained in the range of 4-20, 1-16, and 2-20 µg mL-1 for dacarbazine, 5-amino imidazole-4-carboxamide and 2-azahypoxanthine, respectively. Accurate, precise, and specific results were obtained upon applying the proposed methods according to ICH guidelines. Furthermore, the developed methods were successfully applied for determination of dacarbazine in its pharmaceutical formulation. Comparing the results of the developed methods with those of the official USP spectrophotometric method statistically showed no significant difference. The developed methods don't need any sophisticated techniques so they are considered cost effective methods. Moreover, the introduced methods have the advantages of being green where water was used as a solvent. The methods proved to be more economic, fast and simple than other reported HPLC methods.

Development of pyridazine derivatives as potential EGFR inhibitors and apoptosis inducers: Design, synthesis, anticancer evaluation, and molecular modeling studies.

Novel hybrids of pyridazine-pyrazoline were synthesized aiming to develop new antiproliferative candidates. All compounds were submitted to the National Cancer Institute (NCI), USA, and many were proved to have significant antiproliferative activity. In addition, in vitro studies of the epidermal growth factor receptor (EGFR) inhibition showed that compounds IXn, IXg, IXb and IXl exhibited excellent inhibitory effect (IC50 = 0.65, 0.75, 0.82 and 0.84 µM, respectively) compared to Erlotinib (IC50 = 0.95 µM). The mechanistic effectiveness in cell cycle progression, apoptotic induction and gene regulation were assessed for the promising compounds IXg and IXn due to their significant EGFR inhibition. Flow cytometeric analysis indicated that compounds IXg and IXn result in increased cell numbers in phase G2/M, suggesting cell cycle arrest in phase G2/M in UO-31cells. Furthermore, real time PCR assay illustrated that compounds IXg and IXn elevated Bax/Bcl2 ratio which confirmed the mechanistic pathway of them. Moreover, the apoptotic induction of UO-31 renal cancer cells was enhanced effectively through activation of caspase-3 by compounds IXg and IXn. On the other hand, molecular docking study was performed to investigate binding mode of interaction of compounds with EGFR-PK in the active site with the aim of rationalizing its promising inhibitory activity. Finally, based on the aforementioned findings, compounds IXg and IXn could be considered as effective apoptosis modulators and promising leads for future development of new anti-renal cancer agents.

Development and Validation of HPTLC and Green HPLC Methods for Determination of a New Combination of Quinfamide and Mebendazole.

Two selective and sensitive chromatographic methods were developed for simultaneous determination of new combination of quinfamide and mebendazole in bulk powder and in pharmaceutical formulation. The first method is HPTLC by which separation was obtained using silica gel HPTLC F254 plates and a simple mobile phase consisting of methanol:toluene (2:6, v/v) and the separated bands were scanned at 254 nm. The second method RP-HPLC that comprised isocratic separation of both drugs on a Phenomenex C18 column using a green mobile phase consisting of double distilled water:methanol (30:70, v/v) at a flow rate of 0.8 mL/min and UV detection at 254 nm. The developed methods were validated and proved to meet ICH guidelines. Successful application of the developed methods was carried out for determination of quinfamide and mebendazole in Vermox Plus® tablets. Statistical comparison between the developed chromatographic methods and the reported simultaneous equation spectrophotometric method showed that there was no significant difference between them, proving the ability of applying the proposed methods in quality control testing of the studied drugs. The developed methods are considered the first chromatographic methods for simultaneous determination of quinfamide and mebendazole; moreover, they offered sensitive and selective eco-friendly methods for analysis of the studied drugs.

HPTLC and RP-HPLC methods for simultaneous determination of Paracetamol and Pamabrom in presence of their potential impurities.

Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and quantitation of the studied drugs on aluminum plates pre-coated with silica gel 60 F254 as a stationary phase using chloroform:methanol:ethyl acetate:glacial acetic acid (8:0.8:0.6:0.2, v/v/v/v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05 M): methanol:acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL/min for first 7.5 min and (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 min. The proposed methods were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found; indicating the ability of proposed methods to be used for routine quality control analysis of these drugs.