Stability of some novel thymidine, 5-bromo-2'-deoxyuridine and 3'-azido-2'-3'-dideoxythymidine analogues.

In the search of new prodrugs effective against herpes simplex virus series of thymidine, 5-bromo-2'-deoxyuridine esters with amino acid and peptide chains and 3'-azido-2',3'-dideoxythymidine derivatives have been synthesized and evaluated for antiviral activity. The chemical stability of some of them containing different residues was studied at pH 1 and 7.4 and temperature of 37 degrees C. An HPLC method was developed for quantification of the unchanged ester concentration. It was proved that esters with simple aliphatic straight side chain (containing alanyl-, glycyl-, or glycyl-glycyl-glycyl-residues) are relatively stable both at acidic and neutral media, 37 degrees C. Some of them undergo negligible hydrolysis with half lifes ranging between 6 and 23 h. In contrast, more complex esters with branched side chain (valyl-), with phenyl residue (phenylalanyl-), as well as containing thiazol ring are rather unstable especially at acidic conditions and undergo rapid hydrolysis resulting in the respective chemical precursor. The stability of the former group esters outlines them as suitable candidates for prodrugs: with higher lipophilicity facilitating po absorption, satisfying chemical stability and possibility to release the active moiety following enzymatic hydrolysis.

Binding of kebuzone to human serum albumin studied by high performance liquid affinity chromatography.

The binding of the anti-inflammatory agent kebuzone (CAS 853-34-9) to human serum albumin (HSA) was examined by means of high performance liquid affinity chromatography. Phenylbutazone and diazepam, the binding properties of which were an object of our previous work, were used as markers for the major supposed binding sites on HSA. It was established that kebuzone binds to the high-affinity phenylbutazone binding sites as well as to the low-affinity diazepam binding sites. The estimated values of the equilibrium affinity constants were 5.9 x 10(4) l/mol and 1.7 x 10(3) l/mol, respectively, suggesting a low binding affinity for kebuzone. A stepwise mechanism of the binding process could be proposed: primary at phenylbutazone sites, and secondary at the diazepam sites. The binding behavior of kebuzone was very similar to that of the structurally related compound phenylbutazone. The observed lower affinity is well explained in view of the lower lipophilicity of kebuzone.

Protein binding of piroxicam studied by means of affinity chromatography and circular dichroism.

The protein binding of piroxicam, a widely used non-steroidal anti-inflammatory drug has been investigated by high-performance liquid affinity chromatography, with phenylbutazone and diazepam used as markers for binding-site characterization, and by circular dichroism titration. It was found that piroxicam binds to high-affinity phenylbutazone-binding sites and to high-affinity diazepam-binding sites. No binding to the low-affinity sites of either marker was established. High values of the primary (high-affinity) binding constants corresponding to both types of binding site were obtained by means of a mathematical method cited in the literature. The circular dichroic spectra of piroxicam were studied at a given albumin concentration and various drug concentrations. A new Cotton effect was observed and was ascribed to the binding of piroxicam to the protein molecule. The values of differential molar ellipticity (delta theta) were treated by a new mathematical procedure for analysis of the data obtained. A high affinity constant was calculated for one class of binding site. Its value is in good agreement with the values obtained by affinity chromatography. These results reveal that circular dichroism is an acceptable method for investigation of protein binding.

Protein binding of some nonsteroidal anti-inflammatory drugs studied by high-performance liquid affinity chromatography.

The protein binding of indomethacin, sulindak and diclofenac sodium is studied in the presence of some competitors: phenylbutazon and diazepam. A high-performance liquid affinity chromatography based on chiral stationary phases with immobilized human serum albumin is used. The competition of the markers and the drugs for two major high- and low-affinity binding sites is investigated. Using a mathematical procedure proposed by the same authors in a previous work the affinity constants of the binding drugs and markers for both types of site are calculated. An analogous behaviour is established for the three drugs-they have nearly the same affinity for the primary binding sites marked by phenylbutazon and diazepam and only one type of low-affinity site (diazepam-binding sites) is involved in binding. That can be explained assuming an overlapping sites.

Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography.

A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites.

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography.

A novel mathematical approach for investigation of drug-human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.

Pharmacokinetics of galanthamine hydrobromide after single subcutaneous and oral dosage in humans.

Galanthamine hydrobromide (Nivalin, dose 10 mg) was given subcutaneously and orally to 8 volunteers. Galanthamine and its metabolites were quantified in plasma and urine by reversed-phase HPLC. An unusual two-stage absorption and biexponential drug decline were observed. Galanthamine plasma peaks (1.24 micrograms/ml after subcutaneous and 1.15 micrograms/ml after oral doses) were reached 2 h following administration, the t1/2(beta) values being 5.70 and 5.26 h, respectively. Minor epigalanthamine and galanthaminone plasma levels were detected. An almost complete urinary recovery of galanthamine and its metabolites was obtained within 72 h. The plasma AUC, Cmax, tmax and ka suggest that the subcutaneous and oral Nivalin formulations are bioequivalent.

In vitro metabolism of galanthamine hydrobromide (Nivalin) by rat and rabbit liver homogenate.

The metabolism of galanthamine hydrobromide (Nivalin) was investigated in rat and rabbit liver homogenates. Experiments were carried out varying several parameters of incubation: substrate (galanthamine hydrobromide, galanthamine, galanthaminone and epigalanthamine), cofactor enrichment (NADPH, NADP/G-6-P, NAD), pH (7.4 and 9.3), time of incubation. Substrates and metabolites were identified and quantitatively determined by GC/MS. In vitro metabolism in rat liver homogenate was negligible. The experiments with rabbit liver homogenate indicated, that galanthamine was actively metabolised the major metabolites being the oxidised product - galanthaminone, and the isomer of galanthamine - epigalantamine. The experimental results show that the metabolism of galanthamine is substrate and product stereoselective.