Marijana Zovko

Macromolecular prodrugs. IX. Synthesis of polymer-fenoprofen conjugates.

Synthesis of several polymer-fenoprofen conjugates is described. Fenoprofen was first chemically modified into benzotriazolide 2 and amino acid amide derivatives: glycine fenoprofenamide (3a) and beta-alanine fenoprofenamide (3b) and their benzotriazolides 6a and 6b. Compounds 2 and 6 readily reacted with polyhydroxy aspartamide-type polymers, i.e. poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)] (PHEA) and poly[alpha,beta-(N-3-hydroxypropyl-DL-aspartamide)] (PHPA) forming conjugates 5, 8a,b and 9a,b, respectively. Conjugate 11 was obtained by partial aminolysis of poly-DL-(2,5-dioxo-1,3-pyrrolidinediyl) (PSI) with 2-aminoethyl fenoprofenamide (3c), followed by total aminolysis with 2-hydroxyethylamine. The synthesised polymer-drug conjugates differed in type of covalent bounding, type and/or length of spacer and drug-loading.

Fenoprofen and ketoprofen amides as potential antitumor agents.

Following numerous experimental observations that various non‐steroidal anti‐inflammatory drugs have antitumor potentials, a series of fenoprofenamides (1a–g) and ketoprofenamides (2a–c) was tested on proliferation of different human tumor cell lines and normal human fibroblasts in vitro. Fenoprofen and ketoprofen showed modest antiproliferative activity, whereas the growth inhibitory activity of the tested amides clearly demonstrates that the substituents linked by an amide bond are essential for the significantly stronger cytostatic activity, probably because of a greater lipophilicity and/or better cell uptake. Additionally, it was shown that the most active derivatives (1d and 2a) induced cell cycle arrest at the G1 phase, as well as apoptosis.

Macromolecular prodrugs: X. Kinetics of fenoprofen release from PHEA-fenoprofen conjugate

The kinetics of fenoprofen release from poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)]-fenoprofen conjugate (PHEA-Fen) in aqueous buffer solutions (pH 10 and 1.1), simulated gastric (SGF) and intestinal fluids (SIF) was studied. In borate buffer pH 10, the following rate constants were obtained: k=0.2659 (t=60 degrees C) and k=0.0177 h(-1) (t=37 degrees C) and in glycine buffer solution pH 1.1 k=0.0036 h(-1). In SGF and SIF fenoprofen release did not occur in significant extend within 12 h. The hydrolysis of the ester bond between the polymeric carrier and fenoprofen followed the pseudo first-order kinetics, with activation energy indicative for the breakage of a sigma bond (E(a)=100.6 kJ mol(-1)). The concentration of the released fenoprofen was determined by high performance liquid chromatography (HPLC).

Piroxicam benzoate--synthesis, HPLC determination, and hydrolysis.

Abstract An improved method of piroxicam benzoate synthesis was described, and an isocratic reversed-phase high-performance liquid chromatography method for its determination was developed and fully validated. The method was found to be specific, precise (relative standard deviation 0.3%), accurate (mean recovery 99.9%), and robust. Limit of detection was estimated at 0.055 µg mL−1 and limit of quantification at 0.185 µg mL−1. The kinetics of piroxicam benzoate hydrolysis in aqueous buffer solutions (pH 1.1 and 10), simulated gastric and intestinal fluids was studied. The hydrolysis followed first-order kinetics. The following rate constants were obtained at pH 10: k = 1.8 × 10−3 hr−1 at 37°C and k = 3.4 × 10−2 hr−1 at 60°C. In acidic media, no significant hydrolysis was observed after 24 hr. During the 24-hr period in simulated intestinal fluid, only 10.9% of the starting ester was hydrolyzed.

Mucoadhesive and antioxidant activity of low-thiosulfinate Allium extracts

The purpose of this study was to evaluate the composition, antioxidant and mucoadhesive properties of extracts of onions that are used in human nutrition: onion (Allium cepa L.), garlic (Allium sativum L.), leek (Allium porrum L.) and bear garlic (Allium ursinum L.), with reduced content of thiosulfinates, important antioxidant constituents. Extracts were prepared by blending fresh bulb or leaves of onions with aqueous or 20 % ethanolic solution, pH =3, which inactivates allinase, followed by freeze-drying. Total phenols, thiosulfinates, thiols as well as sugars were determined. For investigation of antioxidant activity two tests were employed: scavenging activity for DPPH free radical and reductive power assay. Among the extracts, aqueous extracts of garlic bulbs was richest in thiosulfinates (93.7 micromol/g, expressed as allicin) and thiols (48.6 micromol/g, expressed as cystein). Although thiosulfinate content was greatly reduced, the extracts demonstrated notable antioxidant activity in both tests. The most active DPPH radical scavenger was the ethanolic extract of leek leaf with EC50 of 2.1 mg/ml. The same extract exhibited the strongest reducing power. However, its activity was significantly lower than the activity of positive controls: butylated hydroxyanisol and ascorbic acid. In general, extracts made of leaves were stronger antioxidants than extracts made of bulbs. There was also positive correlation (r2 = 0, 70) between antioxidant activity and thiol content in the extracts which implicates that these compounds may play an important role in overall antioxidant activity of onions. To determine mucoadhesive properties of prepared extracts tensile studies were performed and work of adhesion values were measured. The results clearly showed that all the extracts tested had mucoadhesive characteristics. The most pronounced mucoadhesive properties were determined for aqueous extract of bulbs of A. sativum and A. ursinum.

Chromatographic fingerprinting of Berberis croatica vs. B. vulgaris (Common and Croatian barberry) using HPLC

In Croatia, two indigenous species of the Berberis L. genus, common barberry (B. vulgaris L.) and Croatian barberry (B. croatica Horvat), occur. While common barberry is a well known species growing in many parts of Europe, Croatian barberry is endemic to the illyric-balcanic region (Croatia, Bosnia and Herzegovina, Montenegro, and Macedonia) [1]. As a contribution to chemotaxonomy, differences of RP-HPLC chromatograms of B. croatica and B. vulgaris were investigated. Herbal samples were collected at six different locations (three locations per species) in Croatia. Methanolic extracts of roots, twigs, leaves and fruit were analysed with regard to their berberine content and chromatographic fingerprints. The HPLC conditions were: isocratic (TEA-adjusted 0.02 mol L-1 H3PO4 (pH 4.82)– acetonitrile (75:25)) and gradient elution (H2O/HCOOH, 9:1 followed by acetonitrile/HCOOH). Signals were detected at 205.4, 254.2 and 330.2 nm. Cluster analysis (UPGMA) was employed for evaluation of fingerprints. The average quantity of berberine in extracts of B. vulgaris was higher than in extracts of B. croatica. In extracts of roots it ranged from 7.85 to 12.19% and from 2.44 to 7.29%, for B. vulgaris and B. croatica, respectively. The quantity of berberine in extracts of twigs varied from 0.82 to 1.53% and from 0.48 to 0.63%, for B. vulgaris and B. croatica, respectively. Even though differences in the individual chromatograms of two species could be noted and UPGMA tended to group samples of the same species together, cluster analyses of fingerprints did not reveal important differences between the two species. This finding indicates that common and Croatian barberry are probably very closely related.

Comparison of antimicrobial activity of traditionally used herbal uroantiseptics against clinical isolates of uro-genital pathogens

In present study, we compared the antimicrobial activity of ethanolic extracts of four traditionally used drugs in the treatment of urogenital infections (Ericaceae flos, Myrtilli folium, Uvae ursi folium, Vitis ideae folium) with arbutin, hydrochinone, and commonly used uroantiseptic norfloxacin. The activity against clinical isolates of the most common urinary pathogens was evaluated against gram-negative (Escherichia coli p-fimbriae positive-strains, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa) and gram-positive bacterial species (Enterococcus faecalis, Staphylococcus aureus), as well as against the yeast Candida albicans, using hole plate diffusion and broth dilution methods. In general, the activity of the extracts was species-dependent. In hole plate diffusion method, the extracts of Uvae ursi folium and Myrtilli folium showed stronger bactericidal activity than the rest of the extracts with average inhibition zones of 28.47 and 25.47 mm, respectively. In the same method none of the extracts showed noticeable antifungal activity against Candida albicans. Using broth dilution method minimal inhibitory concentrations (MICs) were determined. All the investigated bacterial species were sensitive to the extracts with MICs below or equal to 10.42 mg/mL while the MIC values for the isolates of C. albicans yeast were between 8.33 and 25 mg/mL. The highest activity was demonstrated by Uvae ursi folium extract against E. faecalis (MIC 2.60 mg/mL) and P. aeruginosa strains (3.13 mg/mL). The antimicrobial activity of the extracts was lower than the activity of hydrochinone and norfloxacin, but higher than arbutin. Statistical analysis did not reveal clear correlation between content of arbutin, methylarbutin, polyphenols or tannin in extracts and in vitro MIC values. However, the extracts with higher amount of investigated substances tended to be more active in both methods. The results of the study confirm the justifiability of use of these drugs in treatment of urinary infections caused by investigated bacterial species.