A new fluorescence probe for sofosbuvir analysis in dosage form and spiked human plasma.

A simple, rapid, and low-cost technique was developed to allow reliable analysis of the anti-hepatitis C drug sofosbuvir in bulk, tablet form, and spiked human plasma. This method depends on the ability of sofosbuvir to quench the fluorescence of the newly synthesized 2-amino-3-cyano-4,6-dimethylpyridine (reagent 3). Elemental analysis and spectral data were used to validate the structure of the synthesized reagent. The newly synthesized reagent exhibited a satisfactory level of fluorescence emission at 365 nm after excitation at 247 nm. All experimental variables that might affect the quenching process were analyzed and optimized. Linearity, range, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) were all validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The concentration range was shown to be linear between 0.1 and 1.5 µg/mL. The technique was effectively utilized for sofosbuvir analysis in both its tablet dosage form and spiked human plasma, with mean percentage recoveries of 100.13 ± 0.35 and 94.26 ± 1.69, respectively.

Bioactivity Studies of Hesperidin and XAV939.

The present work aimed to evaluate the reactivity of natural bioflavonoid hesperidin (HSP) and synthetically derived XAV939 (XAV) against human hepatocellular carcinoma (HepG2), human breast cancer (MDA-MB231) cancer cell lines, and related molecular and pathological profiles. Data recorded revealed that the cytotoxic potential of the tested products was found to be cell type- and concentration-dependent. The half-maximal inhibitory concentration (IC50) value of the HSP-XAV mixture against MDA-MB231 was significantly decreased in the case of using the HSP-XAV mixture against the HepG2 cell line. Also, there was a significant upregulation of the phosphotumor suppressor protein gene (P53) and proapoptotic genes such as B-cell lymphoma-associated X-protein (Bax, CK, and Caspase-3), while antiapoptotic gene B-cell lymphoma (Bcl-2) was significantly downregulated compared with the untreated cell control. The cell cycle analysis demonstrated that DNA accumulation was detected mainly during the G2/M phase of the cell cycle accompanied with the elevated reactive oxygen species level in the treatment of HepG2 and MDA-MB231 cell lines by the HSP-XAV mixture, more significantly than that in the case of cell control. Finally, our finding suggests that both HSP and XAV939 and their mixture may offer an alternative in human liver and breast cancer therapy.

In vitro evaluation of electroporated gold nanoparticles and extremely-low frequency electromagnetic field anticancer activity against Hep-2 laryngeal cancer cells.

INTRODUCTION: The extremely-low frequency electromagnetic field (ELFEMF) has been proposed for use in cancer therapy since it was found that magnetic waves interfere with many biological processes. Gold nanoparticles (Au-NPs) have been widely used for drug delivery during cancer in vitro studies due to their low cytotoxity and high biocompatibility. The electroporation of cancer cells in a presence of Au-NPs (EP Au-NPs) can induce cell apoptosis, alterations of cell cycle profile and morphological changes. The impact of ELFEMF and EP Au-NPs on morphology, cell cycle and activation of apoptosis-associated genes on Hep-2 laryngeal cancer cell line has not been studied yet. MATERIALS AND METHODS: ELFEMF on Hep-2 cells were carried out using four different conditions: 25/50 mT at 15/30 min, while Au-NPs were used as direct contact (DC) or with electroporation (EP, 10 pulses at 200V, equal time intervals of 4 sec). MTT assay was used to check the toxicity of DC Au-NPs. Expression of CASP3, P53, BAX and BCL2 genes was quantified using qPCR. Cell cycle was analyzed by flow cytometry. Hematoxylin and eosin (HE) staining was used to observe cell morphology. RESULTS: Calculated IC50 of DC Au-NPs 24.36 µM (4.79 µg/ml) and such concentration was used for further DC and EP AuNPs experiments. The up-regulation of pro-apoptotic genes (CASP3, P53, BAX) and decreased expression of BCL2, respectively, was observed for all analyzed conditions with the highest differences for EP AuNPs and ELFEMF 50 mT/30 min in comparison to control cells. The highest content of cells arrested in G2/M phase was observed in ELFEMF-treated cells for 30 min both at 25 or 50 mT, while the cells treated with EP AuNPs or ELFEMF 50 mT/15 min showed highest ratios of apoptotic cells. HE staining of electroporated cells and cells exposed to ELFEMF's low and higher frequencies for different times showed nuclear pleomorphic cells. Numerous apoptotic bodies were observed in the irregular cell membrane of neoplastic and necrotic cells with mixed euchromatin and heterochromatin. CONCLUSIONS: Our observations indicate that treatment of Hep-2 laryngeal cancer cells with ELFEMF for 30 min at 25-50 mT and EP Au-NPs can cause cell damage inducing apoptosis and cell cycle arrest.

HCV/PCR positivity in bile and doudenal aspiration of fascioliasis and/or HCV patients.

In this study, three bile aspirates taken from 10 fascioliasis patients (30.0%) showed HCV positivity by PCR/RNA. Also, four duodenal aspirates (66.7%) taken from six HCV/PCR-RNA positive patients and three duodenal aspirates (20%) taken from 15 pure fascioliasis patients showed HCV positivity by PCR/ RNA. This is the first time to demonstrate HCV/PCR-RNA in the bile and duodenal aspirates of fascioliasis patients and in the duodenal aspirates of HCV patients. So, PCR can be used for the detection of HCV in the bile and/or duodenal aspirates of HCV suspected patient. On the other hand, this outcome results may incriminate HCV infection as a concomitant with fascioliasis or incriminate fascioliasis as paving the way to HCV.

The liver profile in patients with hepatitis C virus and/or fascioliasis.

In this study, twenty HCV/PCR-RNA positive patients with neither infection nor infestation were 15 males and five females with ages ranging between 17-78 years old. Liver function tests: S. albumin was decreased in 15 patients (75%), total protein decreased in 3 patients (15%), total bilirubin increased in 7 patients (35%), AST/ALT increased in 3 patients (15%). Globulin value increased in 15 patients (75%). A/G ratio decreased in 12 (60%). Ten normal individuals (five males & five females), gave normal findings. However, a 19 years-old female had non significant elevation (0.26 mg/dl) in direct bilirubin. Liver function tests: one patient had increased AST/ALT, Globulin value decreesed (-0.2) in another one (10%) and A/G ratio increased (+0.3 to +0.6) in three (30%) individuals. Twenty randomly selected patients (15 males and five females) HCV/3rd generation ELISA showed ALT elevation in 17 (85%), AST in all (100%), S. albumin decreased in 9 (45%), and increased in 1 (5%). Total protein decreased in 3 (15%), total bilirubin increased in 7 (35%) and direct bilirubin in 4 (20%), AST/ALT value increased in 3 patients (15%), Globulin value increased in 15 patients (75%). A/G ratio decreased in 12 (60%). Three patients had schistosomiasis, one 30 years old male had increased AST/ALT, normal globulin, increased A/G and positive HBs-Ag. The second one was a 33-year-old male had normal AST/ALT, normal globulin, increased A/G and positive HBs-Ag. The third patient was a 19-year-old female with normal AST/ALT and normal globulin, increased A/G and positive HBs-Ag. Two patients had fascioliasis, one was a 20-year-old male with increased AST/ALT, globulin normal, A/G normal and negative HBs-Ag. The second one was a 26-year-old female with normal AST/ALT and normal globulin, increased A/G and positive HBs-Ag. The other four positive HBs-Ag patients were parasite-free. Also, the other HCV/ELISA positive were negative for HBs-Ag. HCV/ELISA may have cross-reacted with HBs-Ag and/or with elevated ALT and gave false positive HCV. The 3rd generation ELISA in detection of HCV was not as sensitive as PCR/RNA. Out of 41 fascioliasis patients (26 males & 15 females), 14 were positive HCV/ELISA (34.1%), but only six were positive HCV/PCR (14.6%). ALT increased in 18 fascioliasis patients (43.9%), AST in 23 patients (56.1%) but, albumin decreased in 7 patients (17.1%), total protein decreased in 5 (12.2%), but total bilirubin increased in 14 (34.1%) and direct bilirubin increased in 2 (4.9%). Liver function tests of 14 fascioliasis and ELISA positive HCV showed AST/ALT increased in 6 (42.9%), globulin increased in 3 (21.4%) and decreased in 6 (42.8%). A/G decreased in 4 (28.6%) and increased in 8 (57.2%). Liver function tests of pure 27 fascioliasis patients showed that AST /ALT increased in 8 (29.6%), globulin increased in one patient (3.7%) but decreased in 10 (37.0%) and A/G ratio increased in 13 (48.1%).

The clinical picture of hepatitis C virus as a concomitant infection with fascioliasis.

Hepatitis C and human fascioliasis are two of the most important public health problems locally and internationally. Each one has its own complications regarding spreading to man, clinical picture, laboratory and serologic diagnosis, treatment and prevention. Concomitant human infection with both magnified the complications. Clinically, both diseases (HCV and fascioliasis) have their own broad signs and symptoms. In concomitant infection, the clinical pictures of both showed some variations. The most common feature in fascioliasis and HCV patients was easy fatigability in 78.6% and the lowest was 7.18% for each of tympanic abdomen, tender colon, tender right upper quadrant, itching, arthritis, epi-gastric pain, and right quadrant pain. The eosinophilia % ranged from 5-24 and the haemoglobin ranged from 7-11.1 gm/dl. In patients with fascioliasis alone, the most come feature was pallor in 96.15% and the lowest was 3.85% for each of splenomegaly, ascites and itching. Eosinophilia % ranged from 1 to 22 and haemoglobin ranged from 6-12 gm/dl. In general, the double infection with both HCV and fascioliasis magnified the laboratory and clinical pictures of such patients.

The liver function profile in PCR-RNA Egyptian HCV-patients and normal controls.

No doubt, Hepatitis C virus (HCV) is a real health problem worldwide. The liver function tests (S.ALT, S.AST, Albumin, Total Protein, Total Bilirubin and Direct Bilirubin) were evaluated in 20 PCR-RNA positive HCV-patients and 10 cross matched apparently healthy population. All the HCV-patients and controls were free from liver helminthes. The results showed that in the HCV-patients, there was elevation in the level of S.ALT (17/20 or 85%), S.AST (20/20 or 100%), Total Bilirubin (7/20 or 35%), and (4/20 or 20%). Besides, there was neither a correlation between sexes nor the degrees of viraemia and the elevation of these four parameters. However, serum levels of Albumin, and Total Protein were within the normal range. On the other hand, in the controls the levels of the six tests were within the normal range. Nevertheless, only one control subject who had positive HBs-Ag, showed elevated Total Bilirulin and Direct Bilirubin. Consequently, these tests are indicative as useful and dependable markers in the non-invasive diagnosis of the hepatitis C virus (HCV).