The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1– 3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical -2- quench ⁄MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygendependent degradation domain of hypoxia-inducible factor-a with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow⁄ UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mo¨ ssbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2- oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.

Vitamin K is a fat-soluble vitamin involved in blood coagulation and bone metabolism. The detection and monitoring of vitamin K homologues in rheumatoid arthritis (RA) patients is a challenging problem due to the smaller concentrations of vitamin K and the presence of several interfering medications. Therefore, this study aimed to develop a new highly sensitive and selective chemiluminescence (CL) method designated to quantify vitamin K homologues in plasma of RA patients including phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4, vitamin K2) and menaquinone-7 (MK-7, vitamin K2). The method was based on the unique photochemical properties of vitamin K homologues that were exploited for selective luminol CL reaction. The correlation coefficients of 0.998 or more were obtained in the concentration ranges of 0.1–100 ngmL−1 vitamin K homologues. The detection limits were 0.03–0.1 ngmL−1 in human plasma for vitamin K homologues. The developed HPLC-CL system was successfully applied for selective determination of vitamin K homologues in plasma of RA patients. The developed method may provide a useful tool for monitoring vitamin K homologues in different clinical studies such as RA, osteoporosis and hepatocellular carcinoma in which vitamin K is intervented.

Hairy root cultures of Echium rauwolfii were obtained by infection of sterile apical shoots with Agrobacterium rhizogenes. The linear increase in fresh weight was found to be parallel to the alkaloids production. The transformed cultures were exposed to different elicitors, such as methyl jasmonate (MJ), quercetin and salicylic acid in order to increase their productivity. Pyrrolizidine alkaloids were quantitatively determined by HPLC. Estimation of total alkaloids was achieved by peak area calculations. MJ at a concentration of 100 mM induced the accumulation of total alkaloids about 19-fold compared to the untreated control. The flavonoid quercetin (Q) at a concentration of 50 mM enhanced the pyrrolizidine accumulation approximately 6-fold. The induction effect of both MJ and Q can be suppressed by pre-incubation of hairy root cultures with salicylic acid

A solid dispersion (SD) approach has been performed to enhance the dissolution rate of insoluble drugs by improving their aqueous solubility. An insoluble drug, nimesulide (Nims), was chosen as a model for non-steroidal anti-inflammatory drugs. It was dispersed in a water-soluble carrier poloxamer 407. Different methods were employed to prepare such dispersion, namely: Solvent method (SM), Melting method (MM) and Kneading method (KM) in different drug: carrier ratios. Nims solid dispersions were characterized for their physicochemical properties using scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and the powder X-ray diffractometry (XRD). In addition to dissolution studies, the results revealed that Nims was converted to its amorphous state. The dissolution rate of the prepared solid dispersion systems was determined in phosphate buffer pH 7.4. The solubility of drug from different systems was also determined in water. This study revealed that, the presence of the hydrophilic carrier allows improving the drug solubility. The dissolution rate was enhanced in the following order KM > MM > SM. The enhancement of dissolution rate may be due to increase in wettability, dispersibillity as well as particle size reduction of the drug.

A solid dispersion (SD) approach has been performed to enhance the dissolution rate of insoluble drugs by improving their aqueous solubility. An insoluble drug, nimesulide (Nims), was chosen as a model for non-steroidal anti-inflammatory drugs. It was dispersed in a water-soluble carrier poloxamer 407. Different methods were employed to prepare such dispersion, namely: Solvent method (SM), Melting method (MM) and Kneading method (KM) in different drug: carrier ratios. Nims solid dispersions were characterized for their physicochemical properties using scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and the powder X-ray diffractometry (XRD). In addition to dissolution studies, the results revealed that Nims was converted to its amorphous state. The dissolution rate of the prepared solid dispersion systems was determined in phosphate buffer pH 7.4. The solubility of drug from different systems was also determined in water. This study revealed that, the presence of the hydrophilic carrier allows improving the drug solubility. The dissolution rate was enhanced in the following order KM > MM > SM. The enhancement of dissolution rate may be due to increase in wettability, dispersibillity as well as particle size reduction of the drug.

Surface modification of liposomal nanocarriers with a novel polymer-lectin conjugate was proposed for enhancing the systemic uptake of encapsulated peptide and protein therapeutics after oral administration. Wheat germ agglutinin (WGA) was covalently attached to carbopol (CP) using the carbodiimide method. The prepared WGA-CP conjugate retained the biological cell binding activity of WGA without any evidence of cytotoxicity to Caco-2 monolayers. Cationic liposomes in the size range of 100 nm were prepared by the lipid film hydration method followed by probe sonication and surface modification with negatively charged WGA-CP. The uptake of WGA-CP liposomes by Caco-2 cells was significantly higher than non-modified or CP liposomes. The uptake was dependent on the surface concentration of WGA, temperature, and incubation period, and was significantly inhibited in the presence of chlorpromazine and 10-fold excess of free WGA. These results suggest the involvement of active transport mechanism for the cellular uptake of the modified liposomes, mediated mainly by binding of WGA to its specific cell membrane receptors. Dual channel confocal microscopy confirmed the simultaneous association and internalization of the polymer conjugate and the liposomal carrier by Caco-2 cells and intestinal membrane of rats. In addition, the pharmacological efficacy of calcitonin, a model peptide drug, was enhanced by more than 20 and 3 folds following peroral administration of calcitonin-loaded WGA-CP liposomes as compared to non-modified and CP liposomes, respectively.

Chitosan nanoparticles (CS NPs) have been commonly regarded as potential carriers for the mucosal delivery of therapeutic peptides because of their biocompatibility, bioadhesion and permeation enhancing properties. However, they have limited colloidal stability and readily dissociate and dissolve in the acidic gastric conditions. In the current study, CS NPs were formulated by ionic cross-linking with hydroxypropyl methylcellulose phthalate (HPMCP) as a pH-sensitive polymer and evaluated for the oral delivery of insulin. In vitro results revealed a superior acid stability of CS/HPMCP NPs with a significant control over insulin release and degradation in simulated acidic conditions with or without pepsin. Furthermore, fluorescently-labeled CS/HPMCP NPs showed a 2- to 4-fold improvement in the intestinal mucoadhesion and penetration compared to CS/TPP NPs as evidenced by quantitative fluorescence analysis and confocal microscopy. After s.c. injection to rats, no significant difference in the hypoglycemic effect of insulin solution or insulin-loaded CS/HPMCP NPs was observed, confirming the physico-chemical stability and biological activity of the entrapped peptide. Following peroral administration, CS/HPMCP NPs increased the hypoglycemic effect of insulin by more than 9.8 and 2.8 folds as compared to oral insulin solution and insulin-loaded CS/tripolyphosphate (TPP) NPs, respectively.

A simple, reproducible and efficient dual separation mode high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of antihypertensive drug combinations including; hydrochlorothiazide (HCTZ), valsartan (VAL), amiloride (AML) and captopril (CAP). The newly developed PlatinumTM column, which provides a dual-mode separation with its polar and non-polar sites, was used for rapid separation of these co-administered drugs. Good resolution was obtained when PlatinumTM column was used compared with C18 column. Additionally, simple isocraticmodewith mobile phase containing methanol and 0.02 mole L-1 phosphate buffer adjusted to pH 3.0 (45:55, v/v) was used for separation. The flow rate was 0.5mLmin-1 and effluent was monitored at 270 nm. All the investigated drugs were completely separated within less than 6 min. The linearity range obtained for the developed HPLC method was 0.5–100 μgmL-1 with detection limits of 0.13–1.2 μgmL-1 for all the studied drugs. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for -2- intended applications. The method was successfully used for determination of the studied drugs in pure form and pharmaceutical dosage forms without prior need for separation. The method is valuable for quality control laboratories for simultaneous determination of these co-administered antihypertensive drugs in binary, ternary and quaternary mixtures.