Ahmed H. Bakheit

Fluorescence spectroscopic and molecular docking studies of the binding interaction between the new anaplastic lymphoma kinase inhibitor crizotinib and bovine serum albumin.

Binding of the recently introduced anti-cancer drug, crizotinib (CRB) with the bovine serum albumin (BSA) was comprehensively studied with the aid of fluorescence and UV-Vis spectroscopic as well as molecular docking techniques. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the CRB and BSA with binding constants of 104Lmol-1. BSA conformational changes were investigated using three dimensional (3D) and synchronous fluorescence measurements. Moreover, the results of site marker competitive experiments and molecular docking, it could be deduced that CRB was inserted into the subdomain IIA (site I) of BSA yielding a more stabilized system. This was further confirmed with the molecular docking results which revealed that CRB is located in the active site residues Try149, Glu152, Ser191, Arg194, Arg198, Trp213, Arg217, Arg256, His287, Ala290, Glu291, Ser343, Asp450 within a radius of 6Å. Combining the molecular docking studies and the computed thermodynamic parameters, it can be inferred that hydrophobic and electrostatic interactions are the major binding forces involved in formation of the CRB-BSA complex.

Comparative ANNs with different input layers and GA-PLS study for simultaneous spectrofluorimetric determination of melatonin and pyridoxine HCl in the presence of melatonin’s main impurity.

Melatonin (MLT) has many health implications, therefore it is important to develop specific analytical methods for the determination of MLT in the presence of its main impurity, N-{2-[1-({3-[2-(acetylamino)ethyl]-5-methoxy-1H-indol-2-yl}methyl)-5-methoxy-1H-indol-3-yl]ethyl}acetaamide (DMLT) and pyridoxine HCl (PNH) as a co-formulated drug. This work describes simple, sensitive, and reliable four multivariate calibration methods, namely artificial neural network preceded by genetic algorithm (GA-ANN), principal component analysis (PCA-ANN) and wavelet transform procedures (WT-ANN) as well as partial least squares preceded by genetic algorithm (GA-PLS) for the spectrofluorimetric determination of MLT and PNH in the presence of DMLT. Analytical performance of the proposed methods was statistically validated with respect to linearity, accuracy, precision and specificity. The proposed methods were successfully applied for the assay of MLT in laboratory prepared mixtures containing up to 15% of DMLT and in commercial MLT tablets with recoveries of no less than 99.00%. No interference was observed from common pharmaceutical additives and the results compared favorably with those obtained by a reference method.

Micellar enhanced synchronous spectrofluorimetric method for determination of dasatinib in tablets, human plasma and urine: application to in vitro drug release and content uniformity test

A highly sensitive and simple spectrofluorimetric method has been developed and validated for the determination of dasatinib (DSB) in its pharmaceutical formulations, spiked human plasma and urine. The suggested method depended on studying the fluorescence spectral behavior of DSB in Cremophor EL (Cr EL) micellar system using synchronous scan technique (Δλ = 50 nm). In aqueous solution, the fluorescence intensity of DSB was markedly enhanced in the presence of Cr EL. The fluorescence–concentration plot was rectilinear over the range 25–500 ng mL−1, with lower detection limit of 2.70 ng mL−1. The proposed method was successfully applied to the assay of commercial tablets, spiked human plasma and urine samples as well as content uniformity test. The application of the proposed method was extended to test the in vitro drug release of DSB tablets, according to United States Pharmacopeia (USP) guidelines.

Chapter One – Azithromycin

Azithromycin is an azalide, a subclass of macrolide antibiotics. It is derived from erythromycin, with a methyl-substituted nitrogen atom incorporated into the lactone ring, thus making the lactone ring 15-membered. It prevents bacteria from growing by interfering with their protein synthesis. It binds to the 50S subunit of the bacterial ribosome and thus inhibits translation of mRNA. Azithromycin is used to treat or prevent certain bacterial infections, most often those causing middle ear infections, strep throat, pneumonia, typhoid, bronchitis, and sinusitis. In recent years, it has been used primarily to prevent bacterial infections in infants and those with weaker immune systems. It is also effective against certain sexually transmitted infections, such as nongonococcal urethritis, chlamydia, and cervicitis. Recent studies have indicated it also to be effective against late-onset asthma, but these findings are controversial and not widely accepted. The present study gives a comprehensive profile of azithromycin, including detailed physico-chemical properties, nomenclature, formulae, methods of preparation, and methods of analysis (including compendial, electrochemical, spectroscopic, and chromatographic methods of analysis). Developed validated stability-indicating (HPLC and biodiffusion assay methods under accelerated acidic, alkaline, and oxidative conditions, in addition to effect of different types of light, temperature, and pH. Detailed clinical applications also presented (mechanism of action, ADME profile, clinical uses and doses, side effects, and drug interactions). Each of the above stages includes appropriate figures and tables. More than 80 references were given as a proof of the above-mentioned studies.

Micellar Enhanced Spectrofluorimetric Method for the Determination of Ponatinib in Human Plasma and Urine via Cremophor RH 40 as Sensing Agent

An impressively simple and precise spectrofluorimetric procedure was established and validated for ponatinib (PTB) quantitation in biological fluids such as human plasma and human urine. This method depends on examining the fluorescence characteristics of PTB in a micellar system of Cremophor RH 40 (Cr RH 40). Cr RH 40 enhanced the intrinsic fluorescence of PTB distinctly in aqueous water. The fluorescence spectra of PTB was recorded at 457 nm following its excitation at 305 nm. Maximum fluorescence intensity was attained by addition of 0.7 mL of Cr RH 40 and one mL of phosphate buffer to PTB aliquots and then dilution with distilled water. There is a linear relationship between the fluorescence intensity of PTB and its concentration over the range 5–120 ngmL−1, with limit of detection and limit of quantification equal to 0.905 ngmL−1 and 2.742 ngmL−1, respectively. The accuracy and the precisions of the proposed method were checked and gave adequate results. The adopted method was applied with a great success for PTB quantitation in different biological matrices (spiked human plasma and urine) giving high recovery values.

A Stability-Indicating HPLC-DAD Method for Determination of Stiripentol: Development, Validation, Kinetics, Structure Elucidation and Application to Commercial Dosage Form

A rapid, simple, sensitive, and accurate isocratic reversed-phase stability-indicating high performance liquid chromatography method has been developed and validated for the determination of stiripentol and its degradation product in its bulk form and pharmaceutical dosage form. Chromatographic separation was achieved on a Symmetry C18 column and quantification was achieved using photodiode array detector (DAD). The method was validated in accordance with the ICH requirements showing specificity, linearity (r 2 = 0.9996, range of 1–25 μg/mL), precision (relative standard deviation lower than 2%), accuracy (mean recovery 100.08 ± 1.73), limits of detection and quantitation (LOD = 0.024 and LOQ = 0.081 μg/mL), and robustness. Stiripentol was subjected to various stress conditions and it has shown marked stability under alkaline hydrolytic stress conditions, thermal, oxidative, and photolytic conditions. Stiripentol degraded only under acidic conditions, forming a single degradation product which was well resolved from the pure drug with significantly different retention time values. This degradation product was characterized by 1H-NMR and 13C-NMR spectroscopy as well as ion trap mass spectrometry. The results demonstrated that the method would have a great value when applied in quality control and stability studies for stiripentol.

Spectrophotometric and molecular modelling studies on in vitro interaction of tyrosine kinase inhibitor linifanib with bovine serum albumin

Linifanib (LNF) possess antitumor activity and acts by inhibiting receptor tyrosine kinase VEGF and PDGF. The interaction of BSA with the drug can provide valuable information regarding the pharmacokinetic and pharmacodynamics behavior of drug. In our study the spectrophotometric methods and molecular docking studies were executed to understand the interaction behavior of BSA and LNF. BSA has an intrinsic fluorescence and that fluorescence was quenched by LNF. This quenching process was studied at three different temperatures of 288, 300and 308 K. The interaction between LNF and BSA was due to static quenching because the Ksv (Stern-Volmer constant) at 288 K was higher than at 300 and 308 K. Kq (quenching rate constant) behaved in a similar fashion as the Ksv. Several other parameters like binding constants, number of binding sites and binding energy in addition to molecular docking studies were also used to evaluate the interaction process. A decrease in the binding constants was observed with increasing temperatures and the binding site number approximated unity. The decreasing binding constant indicates LNF–BSA complex stability. The site mark competition experiment confirmed the binding site for LNF was located on site II of BSA. UV–visible studies along with synchronous fluorescence confirm a small change in the conformation of BSA upon interaction with LNF. The thermodynamic analysis provided the values for free energy ΔG0, ΔH0 and ΔS0. The ΔG0 at the 288, 300 and 308 K ranged in between -21.5 to -23.3 kJ mol-1, whereas the calculated values of ΔH (-55.91 kJ mol-1) and ΔS0 (-111.74 J mol-1·K-1). The experimental and molecular docking results suggest that the interaction between LNF and BSA was spontaneous and they exhibited hydrogen bonding and van der Waals force between them.

Biophysical and In Silico Studies of the Interaction between the Anti-Viral Agents Acyclovir and Penciclovir, and Human Serum Albumin

Acyclovir (ACV) and penciclovir (PNV) have been commonly used during the last few decades as potent antiviral agents, especially for the treatment of herpes virus infections. In the present research their binding properties with human serum albumin (HSA) were studied using different advanced spectroscopic and in-silico methods. The interactions between ACV/PNV and HSA at the three investigated temperatures revealed a static type of binding. Extraction of the thermodynamic parameters of the ACV-HSA and PNV-HSA systems from the measured spectrofluorimetric data demonstrated spontaneous interactions with an enthalpy change (∆H0) of −1.79 ± 0.29 and −4.47 ± 0.51 kJ·mol−1 for ACV and PNV, respectively. The entropy change (∆S0) of 79.40 ± 0.95 and 69.95 ± 1.69 J·mol−1·K−1 for ACV and PNV, respectively, hence supported a potential contribution of electrostatic binding forces to the ACV-HSA and PNV-HSA systems. Putative binding of ACV/PNV to HSA, using previously reported site markers, showed that ACV/PNV were bound to HSA within subdomains IIA and IIIA (Sudlow sites I and II). Further confirmation was obtained through molecular docking studies of ACV-HSA and PNV-HSA binding, which confirmed the binding site of ACV/PNV with the most stable configurations of ACV/PNV within the HSA. These ACV/PNV conformers were shown to have free energies of −25.61 and −22.01 kJ·mol−1 for ACV within the HSA sites I and II and −22.97 and −26.53 kJ·mol−1 for PNV in HSA sites I and II, with hydrogen bonding and electrostatic forces being the main binding forces in such conformers.

A new method to determine the new C-Met inhibitor “Cabozantinib” in dosage form and human plasma via micelle-enhanced spectrofluorimetry

A highly sensitive and simple micelle-enhanced spectrofluorimetric method was developed for the determination of cabozantinib (CBZ) in its pharmaceutical formulation and spiked human plasma without any derivatization. The proposed method is based on the investigation of the fluorescence spectral behavior of CBZ in a Cremophor RH 40 (Cr RH 40) micellar system. In aqueous solution, the fluorescence intensity of CBZ was greatly enhanced (nine fold) in the presence of Cr RH 40. The fluorescence intensity was measured at 343 nm after excitation at 244 nm. The fluorescence–concentration plot was linear over the range 25–800 ng mL−1, with a lower detection limit of 13.34 ng mL−1. The proposed method was successfully applied to the determination of CBZ in laboratory-prepared dosage form and spiked human plasma. Recovery values of CBZ with the current method were 99.68 ± 0.88, 100.53 ± 0.51 and 100.44 ± 3.91% for pure powder, lab-prepared dosage form and spiked human plasma, respectively. The results were statistically compared with those obtained by the reported chromatographic method and were found to be in good agreement.

A highly sensitive fluorimetric method for determination of lenalidomide in its bulk form and capsules via derivatization with fluorescamine

BackgroundLenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable clinical activity in treatment of multiple myeloma disease via a multiple-pathways mechanism. The strong evidences-based clinical success of LND in patients has led to its recent approval by US-FDA under the trade name of Revlimid® capsules by Celgene Corporation. Fluorimetry is a convenient technique for pharmaceutical quality control, however there was a fluorimetric method for determination of LND in its bulk and capsules.ResultsA novel highly sensitive and simple fluorimetric method has been developed and validated for the determination of lenalidmide (LND) in its bulk and dosage forms (capsules). The method was based on nucleophilic substitution reaction of LND with fluorescamine (FLC) in aqueous medium to form a highly fluorescent derivative that was measured at 494 nm after excitation at 381 nm. The factors affecting the reaction were carefully studied and optimized. The kinetics of the reaction was investigated, and the reaction mechanism was postulated. Under the optimized conditions, linear relationship with good correlation coefficient (0.9999) was found between the fluorescence intensity and LND concentration in the range of 25–300 ng/mL. The limits of detection and quantitation for the method were 2.9 and 8.7 ng/mL, respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 1.4%. The proposed method was successfully applied to the determination of LND in its bulk form and pharmaceutical capsules with good accuracy; the recovery values were 97.8–101.4 ± 1.08–2.75%.ConclusionsThe proposed method is selective and involved simple procedures. In conclusion, the method is practical and valuable for routine application in quality control laboratories for determination of LND.