Bioequivalence assessment of two Ticagrelor formulations under fasting condition in healthy Pakistani subjects.

Objective: To investigate the Bioequivalence of Anplag® 90mg (Ticagrelor) tablet and Brilinta® 90 mg (Ticagrelor) tablet under fasting conditions in healthy Pakistani subjects. Method: This was an open-label, cross-over, randomized, single-dose, two-period, single-center Bioequivalence Study conducted at Center of Bioequivalence Studies and Clinical Research (CBSCR), ICCBS, University of Karachi, Karachi, Pakistan from September 2020 to January 2021. This was an open-label, randomized, single-dose, two-period, cross-over Bioequivalence Study. After randomization, a single dose of Ticagrelor 90mg tablet (test or reference drug) were administered orally in 1:1 ratio to each subject under fasting conditions. Seven days washout period was kept between the two periods in order to avoid carry over. Blood samples were then taken up to 48th hours post-dose. Point estimates and 90% confidence intervals (CI) for the ratio of the log-transformed values were calculated. Bioequivalence assessment of both, the reference and the test drugs were based on the primary Pharmacokinetic PK metrics including peak maximum concentration (Cmax), area under the curve (AUC) from zero to last quantifiable concentration (AUClast), and AUC from zero to infinity (AUCtotal) after log-transformation of data with ANOVA. In this bioequivalence study, the primary pharmacokinetic parameters were assessed for both Ticagrelor and its Active Metabolite (AR-C124910XX). Safety endpoints were evaluated by monitoring adverse events (AEs). Results: The 90% Confidence Intervals (CIs) of the Geometric Mean Ratio for primary PK parameters including Cmax, AUClast, and AUCtotal all were within the accepted bioequivalence range of 80%- 125%. In the current study, no serious adverse events were reported. Conclusion: Our results showed that the two tested formulations of Ticagrelor tablets were bioequivalent and well tolerated.Trial Registration: ClinicalTrials.gov Identifier: NCT04941196.

CD5 molecule-like and transthyretin as putative biomarkers of chronic myeloid leukemia - an insight from the proteomic analysis of human plasma.

Better and sensitive biomarkers are needed to help understand the mechanism of disease onset, progression, prognosis and monitoring of the therapeutic response. Aim of this study was to identify the candidate circulating markers of chronic-phase chronic myeloid leukemia (CP-CML) manifestations, having potential to develop into predictive- or monitoring-biomarkers. A proteomic approach, two-dimensional gel electrophoresis in conjunction with mass spectrometry (2DE-MS), was employed for this purpose. Based on the spot intensity measurements, six proteins were found to be consistently dysregulated in CP-CML subjects compared to the healthy controls [false discovery rate (FDR) threshold ≤0.05]. These were identified as α-1-antichymotrypsin, α-1-antitrypsin, CD5 molecule-like, stress-induced phosphoprotein 1, vitamin D binding protein isoform 1 and transthyretin by MS analysis [PMF score ≥79; data accessible via ProteomeXchange with identifier PXD002757]. Quantitative ELISA, used for validation of candidate proteins both in the pre-treated and nilotinib-treated CP-CML cases, demonstrate that CD5 molecule-like, transthyretin and alpha-1-antitrypsin may serve as useful predictive markers and aid in monitoring the response of TKI-based therapy (ANOVA p < 0.0001). Two of the circulating marker proteins, identified in this study, had not previously been associated with chronic- or acute-phase myeloid leukemia. Exploration of their probable association with CP-CML, in a larger study cohort, may add to our understanding of the disease mechanism besides developing clinically useful biomarkers in future.

Glycosylated Alpha-1-acid glycoprotein 1 as a potential lung cancer serum biomarker.

Presently existing screening approaches for lung cancer are not being proving sufficient and sensitive, so a study was conducted to identify disease related biomarker proteins for diagnostic applications. A total of 100 lung cancer patients (88 non-small cell lung cancer and 12 small cell lung cancer) and 50 healthy controls were included in this study. Serum samples of patients and healthy controls were subjected to a series of proteomic approaches and as a result of two dimensional gel electrophoresis, a ∼ 43 kDa protein was found to be differentially expressed compared to healthy controls. Quantitative profiling of two dimensional gels by Dymension software analysis displayed 3.58 fold increased expression of ∼ 43 kDa protein in squamous cell carcinoma and 2.92 fold in case of adenocarcinoma. Mass spectrometric analysis resulted in identification of 8 differentially expressed proteins, out of which human Alpha-1-acid glycoprotein 1 was targeted for further validations. This candidate protein exhibited N-linked glycosylation at five amino acid residues; 33, 56, 72, 93, and 103 with significant score of 0.66, 0.78, 0.78, 0.53 and 0.66, respectively. Sandwich ELISA quantified high serum levels of Alpha-1-acid glycoprotein 1 in squamous cell carcinoma (2.93 g/l ± 1.22) and adenocarcinoma (2.39 g/l ± 1.13) when compared with healthy controls (0.83 g/l ± 0.21). One-way ANOVA analysis predicted highly significant variation of Alpha-1-acid glycoprotein 1, among all the study types (F-value 65.37, p-value 0.000). This study may prove as a non-invasive, cost effective and sensitive scheme for diagnosis of lung cancer, by passing the expensive and painful screening procedures.

Mass spectrometric identification, characterization and validation of the haptoglobin ß-chain protein as a lung cancer serum biomarker.

Lung cancer is the major contributor to overall cancer-related mortality. Biomarkers are important in early detection and prognosis, in addition to developing treatment regimes, which may improve the patient survival rates. Biomarkers may also assist in investigating the in depth metabolic pathways and in establishing a set of therapeutic agents leading to early detection of the disease. The present study was designed to identify and confirm a lung cancer protein biomarker and to correlate the differential expression of the protein to a particular histological disease type. A total of 100 lung cancer patients and 50 healthy controls were included in the present study and were categorized into the two main histological types of lung cancer; non­small cell lung cancer (NSCLC; n=88) and small cell lung cancer (SCLC; n=12). NSCLC was further subclassified into three histological types; adenocarcinoma (n=34), squamous cell carcinoma (n=48) and large cell carcinoma (n=6). The patient and control serum samples underwent sodium dodecyl sulphate polyacrylamide gel electrophoresis characterization followed by two­dimensional gel electrophoresis. Following mass spectrometry, human haptoglobin was identified with a mass of ~42­46 kDa and an isoelectric point (pI) of ~5.5­6.2. The experimental mass of the protein was found to be 45.8 kDa with a pI of 6.13. The matrix­assisted laser desorption/ionization time­of­flight/time­of­flight data exhibited spectral peaks of 1146.134, 1724.191, 1345.339 and 2210.319 m/z and Mascot search analysis identified these peaks as haptoglobin (accession no. P00738; Mascot score 87; sequence coverage 23%). This protein was significantly overexpressed in squamous cell carcinoma and adenocarcinoma, as compared with the control. The present study described differentially expressed human haptoglobin as a lung cancer serum protein biomarker, which may serve as a diagnostic and therapeutic target and set a standard criteria for the evaluation of histological types of lung cancer compared with other disease types.

Comparison of plasma from healthy nonsmokers, smokers, and lung cancer patients: pattern-based differentiation profiling of low molecular weight proteins and peptides by magnetic bead technology with MALDI-TOF MS.

CONTEXT: Smoking is the major contributor of lung cancer (LC), which accounts for millions of death. OBJECTIVE: This study focused on the correlation between the proteomic profiling of LC patients, and healthy nonsmokers and smokers. METHOD: Pattern-based peptide profiling of 186 plasma samples was performed through reversed-phase chromatography-18 magnetic bead fractionation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and resulted data were evaluated statistically by ClinProTool. RESULTS: Marker peaks at m/z 1760, 5773, 5851, 2940, and 7172 were found with an excellent statistical figure. CONCLUSION: Selected marker peaks can be served as a differentiated tool of LC patients with high sensitivity and specificity.

Structural characterization of metalloprotease vibriolysin of cholera pathogen Vibrio cholerae.

Vibriolysin is among several zinc metalloproteases produced by Vibrio cholerae. It is involved in the molecular pathogenicity of cholera. Here, we cloned and expressed full-length vibriolysin gene from V. cholerae. Electrophoretic and mass spectrometric data showed that the N-terminal pro-peptide was removed from pro-vibriolysin generating a 45-kDa segment containing the metalloprotease plus the C-terminal domains, and the 35 kDa metalloprotease. The 35 kDa metalloprotease segment of vibriolysin was purified to homogeneity using ion-exchange and gel filtration chromatography. Circular dichroism (CD) analysis of vibriolysin indicated α+ß secondary structure, similar to other closely related metalloproteases of known structure. Positive dichroic absorption maxima in near-UV CD spectrum provided evidence for bound metal atom(s). Dynamic Light Scattering (DLS) measurements at different pHs were also performed to establish the aggregational properties of purified vibriolysin in solution. The results of DLS studies revealed that vibriolysin exists as a homomer with a hydrodynamic radius of 56.7 nm ± 2% under physiological conditions and remains catalytic when BSA was used as a protein substrate. While, extreme acidic (pH 3.5-5.4; R(H) = 65-239 nm) and alkaline (pH 8.5-9.4; R(H) = 57-72 nm) buffering conditions induced further aggregation of vibriolysin, without any trace of the monomeric state in solution.