Š. Bezek

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.

Protection of the vascular endothelium in experimental situations.

Protection of the vascular endothelium in experimental situations One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo.

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.

EFFECT OF DIETARY INTAKE OF VITAMIN A OR E ON THE LEVEL OF DNA DAMAGE, CHROMOSOMAL ABERRATIONS AND MICRONUCLEI INDUCED IN FRESHLY ISOLATED RAT HEPATOCYTES BY DIFFERENT CARCINOGENS

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo[c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

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.

Hepatobiliary Disposition of 3′-Azido-3′-deoxythymidine (AZT) in the Rat: Effect of Phenobarbitone Induction*

Abstract— Isolated liver with a recirculating perfusate was used to study 3′‐azido‐3′‐deoxythymidine (AZT) disposition in phenobarbitone‐pretreated rats at 68 μm AZT concentration in the reservoir. Clearance of AZT in the livers obtained from control animals was 0·42 ± 0·01 (mean ± s.d.) mL min−1/10 g liver. Over the study period of 105 min, 12·7 ± 2·6% of the dose was excreted in bile and of this 95% was recovered as 3′‐azido‐3′‐deoxy‐5′‐O‐β‐d‐glucopyranuronosylthymidine (GAZT). The amount of GAZT found in the perfusate after 105 min of liver perfusion was < 1% of the AZT dose introduced into the reservoir. Phenobarbitone pretreatment of rats resulted in a 5·5‐fold increase of AZT clearance. In addition, the area under the perfusate concentration‐time curve (AUC0–105 min) for 3′‐amino‐3′‐deoxythymidine (AMT) and for a catabolite of unknown structure was increased 3‐ and 10‐fold, respectively, and the amount of AZT dose excreted in the bile was nearly doubled. Thus phenobarbitone was capable of stimulating both detoxification of AZT to GAZT and bioactivation of AZT to AMT, a catabolite known to be highly toxic to human bone marrow cells. This induction was the result of enhancement of AZT catabolism rather than its transport into the cells, since on incubation of AZT (0–250 μm) with rat isolated hepatocytes, a linear relationship between concentration and amount taken up by the cells was shown. In addition, the rate of AZT uptake was not influenced by KCN, dinitrophenol, or temperature, which is consistent with a simple diffusion of AZT through the hepatocellular membrane. Rats dosed intraduodenally with [3H]GAZT excreted 5·8 ± 3·3% of the GAZT dose in bile within 4 h after administration. This suggests that enterohepatic recycling is involved in AZT disposition in the rat.

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.