Modified-release capsules containing sodium riboflavin 5'-phosphate.

INTRODUCTION: The focus of this work was to produce delayed-release capsules containing riboflavin (vitamin B2, as API) layered pellets. Riboflavin therapy is indicated in patients with a riboflavin deficiency, which usually occurs in conjunction with malabsorption, alcoholism or a protein-calorie deficiency and rarely as the sole vitamin deficiency. Riboflavin is readily absorbed from the upper gastrointestinal tract by a specific transport mechanism. The dissolution rate of coated capsules was controlled through the coating of the capsules and the thickness of the coating layer. METHODS: The core pellets (Cellet 300) were loaded with a 10% aqueous solution of sodium riboflavin 5'-phosphate by a layering technique in a coating pan. Hard capsules were filled with riboflavin layered pellets and coated with Eudragit NE polymer with different coating layer thicknesses. The dissolution was tested in gastric and intestinal fluids with the half-change method. The dissolution profiles were analyzed with the use of different mathematical models and an attempt was made to predict the optimum coating film thickness that ensures the required degree and rate of dissolution. RESULTS: A new solid dosage form was developed which can enhance the bioavailability of riboflavin. RRSBW distribution and the Chapman-Richards growth function were used to fit the dissolution profiles. Statistical analysis indicated that the best products were described by the Chapman-Richards equation. The results were utilized to create a theoretical model suitable for prediction of the optimum film thickness that ensures the required release of riboflavin.

Corneal absorption of a new riboflavin-nanostructured system for transepithelial collagen cross-linking.

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.

Enhancement of corneal permeation of riboflavin-5'-phosphate through vitamin E TPGS: a promising approach in corneal trans-epithelial cross linking treatment.

Corneal accumulation of riboflavin-5'-phosphate (riboflavin) is an essential step in the so called corneal cross-linking (CXL), an elective therapy for the treatment of progressive keratoconus, corneal ectasia and irregular astigmatism. CXL is usually performed after surgical debridement of corneal epithelium, since it impedes the stromal penetration of riboflavin in a relatively short time. d-Alpha-tocopheryl poly(ethylene glycol) 1000 succinate (VE-TPGS) is an effective permeation enhancer used to increase adsorption of drugs trough different biological barriers. Moreover, belonging to the group of tocopherol pro-drugs, VE-TPGS exerts a protective effect on biological membrane against free-radical damage. The aim of this work is the evaluation of VE-TPGS effects on riboflavin corneal permeability, and the assessment of its protective effect against free-radicals generated during CXL procedures. Different solutions containing riboflavin (0.125% w/w), dextran (20.0% w/w) and increasing concentration of VE-TPGS were tested. Corneal permeation was evaluated in vitro by the use of modified Franz-cell type diffusion cells and freshly excised porcine corneas as barrier. The effect of VE-TPGS on riboflavin corneal penetration was compared with a standard commercial solution of riboflavin in dextran at different times. Accumulation experiments were conducted both on epithelized and non-epithelized corneas. Moreover, epithelized porcine corneas, treated with the tested solutions, were subjected to an in vitro CXL procedure versus non-epithelized corneas, treated with a commercial solution of riboflavin. Differences were measured by means of corneal rigidity using Young's modulus. The photo-protective effect of tested solutions on corneal epithelium was, finally, evaluated. CXL treatment was applied, in vitro, on human explanted corneas and resulting morphology of corneal epithelium was investigated by scanning electron microscopy.

[Metabolic correction of the lipid-transport system in experimental diffuse purulent peritonitis].

The research was performed in 55 male chinchilla rabbits. For the first time the effect of metabolic preparations "citoflavin" and "neoton" of the protein-lipid spectrum of blood was studied in experimental diffuse purulent peritonitis. The development of diffuse purulent peritonitis caused negative changes in blood lipid-transport system which resulted in a decreased blood protein level and high density lipoproteins (HDL) and growth of triglycerides. In the HDL phospholipid spectrum the pathological changes are characterized by an increased lisophosphotide content and compensatory growth of the level of poliglycerophosphatides. A comparative analysis has shown that both preparations possess unidirectional action which is more pronounced in "citoflavin" than in "neoton".

Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium.

PURPOSE: To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma. METHODS: NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration. RESULTS: Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-µm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 µm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells. CONCLUSIONS: NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma.

Corneal distribution of riboflavin prior to collagen cross-linking.

PURPOSE: To evaluate the distribution of riboflavin in the corneal stroma, under varying concentrations and application time. MATERIALS AND METHODS: In 54 porcine eyes, the central corneal epithelium was removed, and 0.035, 0.1, or 0.2% riboflavin-5-phosphate (in 20% Dextran T-500) was applied for 10, 20, or 30 min (3 x 6 corneas in each of the 3 groups). Trephined corneal buttons were examined using confocal fluorescence microscopy. Stromal riboflavin distribution and concentration was determined by measuring riboflavin fluorescence in optical sections at 10 microm intervals through the entire cornea. The procedure was repeated in 7 human corneal donor grafts using 0.1% riboflavin-5-phosphate for 20 or 30 min. RESULTS: In porcine corneas, fluorescence intensity peaked within the first 50 microm followed by a steep decline to baseline. Increasing the riboflavin concentration from 0.1 to 0.2% did not increase stromal depth propagation, although a higher concentration in the anterior 200 microm was observed. Reducing the riboflavin application time from 30 to 20 min had no impact on corneal depth propagation or total riboflavin uptake. However, a 10-min further reduction of the application time caused a significantly reduced riboflavin uptake. In all human corneas, fluorescence peaked within the anterior 50 microm, followed by a steep decline to baseline over the next 200 microm; similar to the observations in porcine corneas. The human corneas imbibed more riboflavin compared to the porcine corneas. CONCLUSIONS: In human and porcine corneas, riboflavin does not appear to fully load the corneal stroma using the current clinical procedure. Instead, the uptake appears to be limited to the anterior approximately 200 microm. Changes in application time and riboflavin concentration have only little influence on stromal depth diffusion.

Absorption and emission spectroscopic characterisation of combined wildtype LOV1-LOV2 domain of phot from Chlamydomonas reinhardtii.

An absorption and emission spectroscopic characterisation of the combined wild-type LOV1-LOV2 domain string (abbreviated LOV1/2) of phot from the green alga Chlamydomonas reinhardtii is carried out at pH 8. A LOV1/2-MBP fusion protein (MBP=maltose binding protein) and LOV1/2 with a His-tag at the C-terminus (LOV1/2-His) expressed in an Escherichia coli strain are investigated. Blue-light photo-excitation generates a non-fluorescent intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm). The photo-cycle dynamics is studied by dark-state absorption and fluorescence measurement, by following the temporal absorption and emission changes under blue and violet light exposure, and by measuring the temporal absorption and fluorescence recovery after light exposure. The fluorescence quantum yield, phi(F), of the dark adapted samples is phi(F)(LOV1/2-His) approximately 0.15 and phi(F)(LOV1/2-MBP) approximately 0.17. A bi-exponential absorption recovery after light exposure with a fast (in the several 10-s range) and a slow component (in the near 10-min range) are resolved. The quantum yield of photo-adduct formation, phi(Ad), is extracted from excitation intensity dependent absorption measurements. It decreases somewhat with rising excitation intensity. The behaviour of the combined wildtype LOV1-LOV2 double domains is compared with the behaviour of the separate LOV1 and LOV2 domains.

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues.

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.