T. Trnovec

Direct high-performance liquid chromatographic determination of (R)- and (S)-propranolol in rat microdialysate using on-line column switching procedures.

Two different column-switching HPLC systems (CSWs), employing restricted access material for initial pretreatment of biological samples, were developed for the determination of propranolol enantiomers in microdialysate. CSW 1 was a single-pump set-up based on an initial sample clean-up step with a RP-18 ADS precolumn coupled with an ovomucoid analytical column for direct drug enantioseparation. For the two-pump column set-up (CSW 2), a teicoplanin analytical column was applied for the enantioselective assay after initial sample pretreatment using a RP-8 ADS precolumn. The inter-day precision of the CSW 1 ranged from 0.5 to 5.1% for (R)-propranolol and from 5.1 to 10.5% for (S)-propranolol. The limit of detection (LOD) was set at 10 ng/ml and 15 ng/ml for (R)- and (S)-propranolol, respectively. Inter-day relative standard deviation values of the CSW 2 ranged from 1.1 to 9.9% for (R)-propranolol and from 1.3 to 9.6% for (S)-propranolol. The LOD of the method was 3.0 ng/ml for (R)-propranolol and 2.5 ng/ml for (S)-propranolol. Both approaches were successfully applied for stereoselective monitoring of unbound propranolol levels in rat microdialysates.

Effects of ionizing radiation on the blood brain barrier permeability to pharmacologically active substances

Ionizing radiation can impair the integrity of the blood brain barrier (BBB). Data on early and late damage after brain irradiation are usually reported separately, yet a gradual transition between these two types has become evident. Signs appearing within 3 weeks after irradiation are considered to be early manifestations. The mechanism of radiation-effected integrity impairment of the BBB is discussed in relation to changes in morphological structures forming the BBB, the endothelium of intracerebral vessels, and in the surrounding astrocytes. Alterations in the function of the BBB are manifested in the endothelium by changes in the ultra-structural location of the activity of phosphatases and by the activation of pinocytotic vesicular transport, and in astrocyte cytoplasm by glycogen deposition. The changes in ultrastructure were critically surveyed with regard to increasing doses of radiation to the brain in the range of 5 Gy to 960 Gy. The qualitative as well as the semiquantitative and quantitative observations on the passage of substances across the damaged BBB were treated separately. Qualitative changes are based mainly on findings of extravasation of vital stains and of labelled proteins. The quantitative studies established differences in radiation-induced changes in the permeability of the BBB depending on the structure and physico-chemical properties of the barrier penetrating tracers. Indirect evaluation of radiation-induced BBB changes is based on studies of pharmacological effects of substances acting on the CNS. In conclusion, radiation impairs significantly the integrity of the BBB following single irradiation of the brain with a dose exceeding 10-15 Gy. The response of the BBB to ionizing radiation is dependent both on the dose to which the brain is exposed and on specific properties of the tracer. Either an increase or a decrease of BBB permeability, or both, occurring in a certain time sequence, was observed. The mechanism of hyperpermeability after irradiation is not fully understood, but the activation of vesicular transport offers one possible explanation. Even less understood is the mechanism of decreased permeability. The response of the BBB to ionizing radiation is most probably nonspecific and its nature may be assumed to be similar to its responses to other physical or chemical noxious factors.

Radiochemical assay of stability of14C-cytostasan solutions during preparation and storage

Cytostasan, 5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazolyl-2-butyric acid, is an antineoplastic agent which degrades spontaneously in water solutions yielding two hydrolysis products, monohydroxy- and dihydroxycytostasan. We developed a stability-indicating radiochemical assay based on ion-pair extraction to investigate the stability of solutions of14C-cytostasan under conditions that might be expected when the drug is being prepared and stored for pharmacokinetic studies in animals. The possibility of using the distribution coefficient of14C-cytostasan as an indicator of stability was investigated in the extraction system benzene-dicarbolide of cobalt-0.5N HClO4. The mechanism of extraction is believed to be that of ion-pair forming process between the hydrophobic anion and the protonized cytostasan. Since no extraction of hydroxy derivates was observed the value of the distribution coefficient of the parent drug appears to be a suitable indicator of the stability of14C-cytostasan solutions.

Ion-pair extraction of pentacaine from biological materials

Simple and rapid extraction method for quantitative and selective isolation of the new local anesthetic pentacaine from biological materials is proposed. The technique of ion-pair formation was found to be more effective than usual access using the extraction of the nonionized species. The extraction yield of the unchanged molecule3H-pentacaine after double extraction and single scrubbing was found to be more than 90%. The radiochemical purity was over 90%. The method appears suitable for pharmacokinetic studies in the animal body.

Determination of Ethimizol and Two of Its Metabolites in Serum by High-Pressure Liquid Chromatography

A liquid chromatographic assay has been developed for the determination of ethimizol and two of its metabolites in serum. The analysed compounds are preseparated from serum by a micro-column packed with an octadecylsilanized silica gel. Components of the micro-column eluate are analysed on a silica-gel-packed column by means of a high-performance liquid chromatograph fitted with a photometric detector. At 262 nm the detection limit of the injected amount of ethimizol is about 5 ng. The mass spectra of two ethimizol metabolites isolated from rat serum are presented. The suitability of the developed assay for pharmacokinetic studies of ethimizol is demonstrated.

Fluorimetric assay of stobadine in serum of dogs

In the present paper a specific fluorimetric assay is now described for the determination of stobadine in serum of dogs. The assay involves the first step of the radiochemical method. The comparison of the proposed fluorimetric assay and the previously developed radiochemical method demonstrates that both methods yielder similar serum-time curves after both routes of administration (oral or intravenous) of stobadine

Irradiation of the head by 60Co opens the blood-brain barrier for drugs in rats.

The passage of 6 model drugs; acetylsalicylic acid, chloramphenicol, ethimizol, carbisocaine, heptacaine, and diazepam, through the blood-brain barrier, was determined in unirradiated control rats and in animals 1, 3, and 7 days after irradiation of the head only with a dose of 25 Gy from a60Co source. The brain uptake index (BUI), which compares the uptake of the test substance with that of3H2O 5 s after their injection into the common carotid artery, was significantly increased in comparison with unirradiated controls 7 days after irradiation, for all substances tested except for ethimizol. For acetylsalicylic acid and chloramphenicol it was also significantly increased in the other time intervals. The less lipophilic substances showed a greater relative increase of BUI than the more lipophilic ones.

Pharmacokinetics of Gentamicin Administered Intratracheally to Rats

The pharmacokinetics of intratracheally instilled and intravenously injected gentamicin were compared in the rat and analyzed by a one-compartment open model. The maximum concentration of gentamicin in plasma occurred within 10 min after intratracheal instillation. Considerable amounts of gentamicin were absorbed from lungs after intratracheal instillation, as shown by its concentrations in plasma and elimination in urine. The data suggest that the absorption of gentamicin from the pulmonary system would be sufficient to maintain therapeutic levels of this agent in plasma.

Pharmacokinetics of carbisocaine in rats and mice

Summary[14C]carbisocaine, N-(2-(2-[1-14C]-heptyloxyphenylcarbamoyloxy)-propyl)-diethylammonium chloride was administered to male Wistar rats, weighing 180–210 g IV in doses of 0.425, 1.425, 2.425 or 4.425 mg/kg or orally in a dose of 2.425 mg/kg. Extraction of carbisocaine from alkaline media into n-heptane was used for assessment of the unchanged drug in plasma, organs and excreta in predetermined time intervals. The two-way analysis of variance confirmed the insignificant effect of subject variability of experimental animals (p > 0.05) on plasma data after IV administration. Plasma data following the IV administration were approximated by a linear open two-compartment model with elimination from the central compartment. Regression analysis indicated linearity between carbisocaine plasma AUC and the IV administered dose within the range tested. The following model parameters were obtained: elimination half-life 161.2±37.5 min, total body clearance 59.5 ml/min/kg, distribution volume in steady state 5616.2 ml/kg and mean residence time 96.7 min. The systemic availability of the orally given carbisocaine was 45.2%, assessed by AUCpo/AUCiv (0–360 min). The brain uptake index of carbisocaine in relation to3H2O was 57.7±3.9%. Whole body autoradiographs of mice injected with [14C] carbisocaine documented accumulation of14C in gall and urinary bladder and in the gut contents and the effective placental barrier against carbisocaine and its metabolites.

Dose dependent pharmacokinetics of a xanthine-related nootropic drug, ethimizol, in rats

Pharmacokinetics of the respiratory analeptic ethimizol, 4,5-bis(methylcarbamoyl)-1-ethylimidazole, with recently recognized nootropic properties, its metabolite 4-carbamoyl-5-methylcarbamoyl-1-ethylimidazole and of two metabolites of unknown structure was studied in plasma of rats after intravenous administration of the [2-14C]-labelled compound in 5 doses: 1.1, 3.3, 10, 20, and 30 mg/kg. The apparently monoexponential time course of dose normalized ethimizol plasma concentrations were not superimposable and the derived pharmacokinetic parameters were dose-dependent. The elimination rate constant and the initial volume of distribution decreased with increasing dose. The AUC versus dose relationship displayed disproportionate increases with increasing dose. Plasma ethimizol failed to obey the Michaelis-Menten kinetics and in an attempt to rationalize the observed nonlinearity a complete competitive product inhibition was suggested. Similar to the parent drug, the three metabolites exhibited a distinctly dose-dependent behavior as could be deduced from their dose-normalized concentration-time curves.