Formulation development and optimization of Lamivudine 300 mg and Tenofovir Disoproxil Fumarate (TDF) 300 mg FDC tablets by D-optimal mixture design.

The usage of fixed dose combination (FDC) tablets of Lamivudine and Tenofovir Disoproxil Fumarate (TDF) is increasing due to increased incidences of HIV/Hepatitis B and HIV/TB co-infections. This is likely to increase the financial crisis due to limited resources for funding procurement of ready-made products from the pharmaceuticals manufacturing leading countries. Therefore, production of local oral tablets containing Lamivudine and TDF FDC is inevitable. Lamivudine 300 mg/TDF 300 mg tablets were developed and optimized by D-optimal mixture design and produced by direct compression technique. Twenty trial formulations with independent variables, including PVP-CL 1-12.00%, PVP-K30 1-10.00%, starch-1500 2.5-12.5% and Avicel-PH102 2-19.25% were prepared by direct compression technique. The formulations were assessed on assay, dissolution, friability, weight variation and disintegration time. It was found that assay ranged from 98.13-101.95% for Lamivudine, 98.25-102.84 for TDF, both were within the in-house assay specification of 95 to 105%. Dissolution at single point was above 80% for Lamivudine 93.96-100.55% and 95.85-103.15% for TDF, disintegration time was between 1.92-66.33 min and friability 0.06-12.56%. Out of twenty formulation trials, eight formulations had all parameters in proven acceptable range. On optimization, one formulation with independent variables, PVP-CL 5.67%, PVP-K30 1.00%, Starch-1500 5.76% was selected. The optimized formulation was comparable to the reference product on the market with similarity factor (f2) and difference factor (f1) within the acceptable range for both Lamivudine and TDF.

Spectroscopic and electronic structure studies probing covalency contributions to C-H bond activation and transition-state stabilization in xanthine oxidase.

A detailed electron paramagnetic resonance (EPR) and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed and serves as a basis for developing a valence-bond description of C-H activation and transition-state (TS) stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde-inhibited XO have been analyzed in order to provide information regarding the relationship between the g, (95,97)Mo hyperfine (A(Mo)), and (13)C hyperfine (A(C)) tensors. Analysis of the EPR spectra has allowed for greater insight into the electronic origin of key delocalizations within the Mo-O(eq)-C fragment and how these contribute to C-H bond activation/cleavage and TS stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo═S π → C-H σ* (ΔE = 24.3 kcal mol(-1)) and C-H σ → Mo═S π* (ΔE = 20.0 kcal mol(-1)) back-donation are important in activating the substrate C-H bond for cleavage. Additional contributions to C-H activation derive from O(eq) lp → C-H σ* (lp = lone pair; ΔE = 8.2 kcal mol(-1)) and S lp → C-H σ* (ΔE = 13.2 kcal mol(-1)) stabilizing interactions. The O(eq)-donor ligand that derives from water is part of the Mo-O(eq)-C fragment probed in the EPR spectra of inhibited XO, and the observation of O(eq) lp → C-H σ* back-donation indicates a key role for O(eq) in activating the substrate C-H bond for cleavage. We also show that the O(eq) donor plays an even more important role in TS stabilization. We find that O(eq) → Mo + C charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal mol(-1)) and the Mo-O(eq)-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-O(eq)-C delocalization at the TS allows for the TS to be described in valence-bond terms as a resonance hybrid of the reactant (R) and product (P) valence-bond wave functions.

Mutation in the flavin mononucleotide domain modulates magnetic circular dichroism spectra of the iNOS ferric cyano complex in a substrate-specific manner.

We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.

Spectroscopic characterization of YedY: the role of sulfur coordination in a Mo(V) sulfite oxidase family enzyme form.

Electronic paramagnetic resonance (EPR), electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, Arabidopsis thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from Starkeya novella. Low-energy dithiolene --> Mo and cysteine thiolate --> Mo charge-transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. In particular, a more acute O(oxo)-Mo-S(Cys)-C dihedral angle results in increased cysteine thiolate S --> Mo charge transfer and a large g(1) in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active-site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.