Investigation of artificial biomembrane systems in biopartitioning micellar chromatography by method of mathematical design.

An attempt to create and study an artificial membrane system was realized via biopartitioning micellar liquid chromatography. Towards this end the known formula of membrane permeability (on the basis of Fick's diffusion equation) was modified so that membrane permeability may be estimated in terms of chromatographic characteristics. The two-factoral experiments on the basis of mathematical design of second order were carried out. The regression equations are derived which describe the dependence of membrane permeability on the concentration of polyoxyethylene (23) lauryl ether in the mobile phase and its flow-rate for compounds with biomedical significance. Some regularities were revealed, which characterize the permeability of compounds of the different nature through membranes. The extremal dependence (with passing through minimum) of permeability on the concentration of non-ionic surfactant was observed for anionic compounds. The increasing character of permeability in relation with flow-rate of mobile phase was recognized for cationic samples. Both dependences were basically fulfilled for zwitterionic compounds.

[Effect of inhibitors of sodium-calcium exchangers on acetylcholine-induced hyperpolarization of endothelial cells in rat aorta]. / Vplyv blokatoriv natrii-kal'tsiievoho obminnyka na atsetylkholinovu hiperpoliaryzatsiiu endotelial'nykh klityn aorty shchuriv.

The effect of selective sodium-calcium exchanger blockers on acetylcholine-induced hyperpolarization of endothelial cells from excised rat aorta was investigated. Acetylcholine administration induced sustained endothelial hyperpolarization. In the presence of 100 microM benzamil, the blocker of forward and reversed mode of the exchanger, acetylcholine induced only short-lived hyperpolarization. When benzamil was introduced during the plateau phase of hyperpolarization the amplitude of hyperpolarization significantly and reversibly decreased. KB-R7943 (20 microM), the blocker of reversed sodium-calcium exchanger, exerted the similar effect. Benzamil-mediated inhibition of sustained hyperpolarization was observed when recordings were commenced from isolated endothelium. The results obtained suggest that reversed mode of sodium-calcium exchanger is activated in intact endothelial cells following stimulation by acetylcholine.

Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells.

The metabolism and action of trans-anethole (anethole) and the estrogen-like activity of the compound and its metabolites were studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with anethole (0.25-2.0mM) caused a concentration- and time-dependent cell death accompanied by losses of cellular ATP and adenine nucleotide pools. Anethole at a weakly toxic level (0.5mM) was metabolized to 4-methoxycinnamic acid (4MCA), 4-hydroxy-1-propenylbenzene (4OHPB), and the monosulfate conjugate of 4OHPB; the levels of 4OHPB sulfate and 4MCA reached approximately 20 and 200 microM within 2 hr, respectively, whereas that of free unconjugated 4OHPB was less than approximately 0.5 microM. At a moderately toxic concentration (1.0mM), unconjugated 4OHPB reached approximately 10 microM, followed by abrupt loss of 3'-phosphoadenosine 5'-phosphosulphate (PAPS). Based on cell viability and adenine nucleotide levels, 4OHPB was more toxic than anethole and 4MCA. The addition of 2,6-dichloro-4-nitrophenol (50 microM), an inhibitor of sulfotransferase, enhanced the anethole-induced cytotoxicity associated with losses of ATP, PAPS, and 4OHPB sulfate, and symmetrically increased the unconjugated 4OHPB concentration. 4OHPB as well as diethylstilbestrol (DES) and bisphenol A (BPA), which are known xenoestrogenic compounds, competitively displaced 17beta-estradiol bound to the estrogen receptor alpha in a concentration-dependent manner; IC(50) values of these compounds were approximately 1 x 10(-5), 1 x 10(-8) and 5 x 10(-5)M, respectively. 4OHPB also caused a concentration (10(-8) to 10(-6)M)-dependent proliferation of MCF-7 cells, whereas neither anethole nor 4MCA (10(-9) to 10(-5)M) affected cell proliferation. However, at higher concentrations (>10(-4)M), 4OHPB rather than anethole and 4MCA was cytotoxic. These results suggest that the biotransformation of anethole induces a cytotoxic effect at higher concentrations in rat hepatocytes and an estrogenic effect at lower concentrations in MCF-7 cells based on the concentrations of the hydroxylated intermediate, 4OHPB.

Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse--pentobarbital example.

Internal standards are commonly used for the quantitative determination of drugs of abuse and their metabolites (drug/metabolite) in biological fluids and tissues by the selective ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS) procedure. Analogs of drugs/metabolites that are labeled with three or more deuterium atoms (isotopic analog) at appropriate positions are considered to be the most effective internal standards for these applications. Before a specific deuterated analog can be adopted as an internal standard in a GC/MS assay, the mass spectrum of the compound or its derivative must be evaluated along with the corresponding spectrum from the parent drug/metabolite. There should be an adequate number of sufficiently high-mass ions (typically three for the drug/metabolite and two for the isotopic analog) that can be attributed to each analyte, and these ions should be sufficiently free of interference from the other analyte of the pair (cross-contribution). Interferences may be caused by the presence of an isotopic impurity in the deuterated analog (extrinsic factor) or may be due to the ion fragmentation characteristics of the compound (intrinsic factor). The extrinsic factor may be corrected by the manufacturer with different synthetic methods and purification procedures, while the intrinsic factor may be partially or wholly corrected through the use of different chemical derivatives (sample preparation stage) or different ionization (GC/MS assay stage) procedures. In this study, pentobarbital/d5-pentobarbital is used as the exemplar analyte/deuterated analog pair to illustrate the ion selection and evaluation procedures. Full-scan mass spectra were employed for preliminary ion selection. SIM data were then used to calculate the extent, if any, of cross-contributions.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the diastereomers of pentobarbital N-glucosides excreted in human urine.

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (1'RS,5RS)-1-(beta-D-Glucopyranosyl)pentobarbital ((1'RS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (1'RS,5R)-PTBG and (1'RS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/MS). A HPLC method was developed for detecting and quantifying (1'RS,5R)-PTBG, (1'RS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6-6.2% of the pentobarbital dose was excreted as (1'RS,5S)-PTBG over 60 hours. (1'RS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (1'R,5R)-PTBG and (1'S,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.

Anticonvulsive activity of a barbiturate derivative HB-7 in a model of allylglycine-induced epilepsy in cats.

With the aim of characterizing further the anticonvulsive activity of the newly-synthesized barbituric derivative 2-hydroxylamino-5-ethyl-5-sec. penthylbarbituric acid (HB-7), we studied its effects on allylglycine-induced epileptiform electrocorticogram (ECoG) in cats under acute conditions. Pentobarbital (PB) was used for comparison. Allylglycine (AG) was administered in a dose of 100 mg/kg i.v. to all cats. The control animals were treated with AG only. HB-7 and PB were applied in doses of 20 mg/kg i.p., about 60 min after AG. Intermittent light stimulation (ILS) at flash rate of 8/s and 25/s was applied to some animals. The ILS-stimulated animals were also divided in three groups: controls and animals treated with HB-7 or PB in the same dose. AG led to the appearance of paroxysmal ECoG activity: single and multiple spikes and spike-waves appeared first, to be followed later by generalized seizure activity. No AG-induced convulsions appeared in the cats treated with HB-7, but the AG-induced spikes and spike-waves remained unchanged. The effect of PB on the AG-induced paroxysmal ECoG activity was similar to that of HB-7, but the addition of PB provoked the appearance of slow waves and spindles, mixed with spikes and spike-waves. Although the paroxysmal character of the activity was more pronounced in the ILS-stimulated animals than in the group without ILS, HB-7 and PB (albeit to a lesser extent) inhibited the AG-induced discharges.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of 2-hydroxylamino-5-ethyl-5-sec-pentylbarbituric acid for anxyolitic action.

Comparative tests were performed on male albino rats to study the activity of the original hydroxylamine barbiturate HB-7, with phenobarbital (PhB) and pentobarbital (PB) using the conflict situation test (after Vogel et al., 1971) with thirsty rats (deprived of water for 48 hours). The three barbiturates studied (HB-7, PhB and PB) were administered intraperitoneally in four doses (5, 10, 15 and 20 mg/kg). Two parameters were registered: number of punished responses (electrical shocks punishing attempts to drink) and the number of non-punished responses (approaching the drinking tube) for the 5-min period of observation. In a second group of experiments the same test was used to study the effect of picrotoxin and of its combinations with the effective doses of HB-7, PhB and PB. The three barbiturates tested were found to have an anxiolytic effect, i.e. to increase the number of attempts to drink water, in the higher doses used. Picrotoxin causes a decrease in the number of punished responses (anxiomimetic effect). When HB-7, PhB and PB are combined with picrotoxin, the latter eliminates their anxiolytic action. The conclusion reached is that the anxiolytic action of the barbiturates tested is probably connected with an effect on the GABA-ergic transmitter system in the central nervous system.

[The effect of the newly synthesized barbiturate HB-7, phenobarbital and pentobarbital on the GABA content of the brain in rats]. / Vliianie na novosinteziraniia barbiturat HB-7, fenobarbitala i pentobarbitala vurkhu sudurzhanieto na GAMK v mozuk na plukh.

The content of GABA in the following regions of the brain: cerebellum, left and right hemisphere was studied on white male rats. The determination was made by the method of Sutton and Simmonds (1974). One control and 7 experimental groups were investigated. All examined substances were administered intraperitoneally and the animals were decapitated at their peak activity. The effect of newly synthesized hydroxyl amine barbiturate HB-7, administered in several doses, was studied, comparing it with the action of pentobarbital (PB) and of phenobarbital (PB). A considerable increase in the content of barbiturates was established in the cerebellum of rats under the influence of the applied barbiturates while the content of GABA in both large hemispheres was less affected by the administered compounds. A conclusion is made that a single administration of barbiturates affects the content of GABA in some cerebral structures of the rat.

N,N'-diallylpentobarbital (DAPB) metabolites and their effects on pentobarbital-induced sleep and hepatic drug-metabolizing enzymes.

1. The biological half-life (t 1/2) of N,N'-diallylpentobarbital (DAPB) in brain after i.p. injection to mouse was 96 min (first phase) and 11 h (second phase). The t 1/2 values in plasma were 102 min and 9.4 h, respectively, after i.p. injection. After intracerebroventricular (i.c.v.) administration, the t 1/2 values in brain and plasma were 18 and 120 min, and 42 and 177 min, respectively. 2. Following i.p. administration of 2-14C-DAPB (80 mg/kg), 58% of the 14C was excreted in the urine in 72 h. Several urinary metabolites were identified by g.l.c.-mass spectrometry, DAPB was metabolized by three major pathways, i.e., omega-1 hydroxylation, epoxide-diol pathway and N-deallylation. 3. The effects of DAPB and its metabolites on pentobarbital (PB)-induced sleep were examined after i.p., i.v. and i.c.v. administration. Metabolite 1 [M-1; (omega-1)-hydroxy-DAPB], an active metabolite, exhibited the most potent prolonging effect. 4. M-1 and other metabolites, as well as unchanged DAPB, showed significant inhibitory effects on mouse hepatic drug-metabolizing enzymes.

[Study of three barbiturate compounds using the Porsolt test]. / Izsledvane na tri barbiturovi suedineniia po testa na Porsolt.

The effects of newly synthetized hydroxylominic barbiturate NV-7, phenobarbital and pentobarbital, administered intraperitoneally, were studied on mice by the test of Porsolt. The obtained results were compared with the results, obtained by the test of rota-rod. There was prolongation of the time for immobility of the animals e.g. the effect of "despair" of the three examined barbiturates. The question is discussed whether the observed effect is due to the axyolytic action, established in the preceding experiments, or to the sedative and anticonvulsive actions of barbiturates, investigated before.

Antihypoxic effect of the hydroxylamine derivative HB-7.

The antihypoxic effect of the newly-synthesized hydroxylamine barbiturate HB-7, compared with the effects of phenobarbital and pentobarbital, was studied in experiments on albino mice, using the following models of hypoxia: hypobaric, anoxic, asphyctic and haemic. A dose-effect dependence was established when the survival of the mice was prolonged under the effects of the barbiturates tested, applying the asphyctic and haemic hypoxy tests. The latter two tests were also used to determine the average effective doses (ED60) of the barbiturates. HB-7 was superior in its action to phenobarbital and pentobarbital, manifesting higher protective indices (PI-LD50: ED50): for HB-7 PI is 4.2, for phenobarbital--3.2 and for pentobarbital--2.5, using the haemic hypoxy test. It is concluded that HB-7, i.e. 2-hydroxylamine-5-ethyl-5-sec, pentylbarbituric acid, manifests a marked protective effect in the hypoxy tests used, exceeding in some cases the effects of phenobarbital and pentobarbital.

Anticonvulsive effect of 2-hydroxylamine-5-ethyl-5-sec-pentyl barbituric acid (HB-7) in two experimental models of epilepsy.

The effects of barbiturate HB-7 (2-hydroxylamine-5-ethyl-5-sec-pentylbarbituric acid) injected intraperitoneally compared to the effects of pentobarbital and/or phenobarbital were investigated in three series of experiments on cats: normal electrocorticogram (ECoG); pentylentetrazol-induced generalized paroxysmal ECoG; and paroxysmal ECoG activity induced by topical application of penicillin on the exposed cortical surface. In the first series, HB-7 led to the appearance of ECoG spindles of the same type as those elicited by pentobarbital. In the second series (pentylentetrazol-treated group), HB-7 increased the two thresholds: one associated with the appearance of isolated paroxysmal spike-wave complexes and the other corresponding to the appearance of generalized paroxysmal ECoG activity. In the third series, HB-7 led to an inhibition of penicillin spikes, both in the focus itself and in the other cortical areas where they propagated. The possibility for using HB-7 as an antiepileptic drug was discussed.

GABA-ergic mechanisms in the anticonvulsive activity of newly synthesized barbiturates. II. Correlation of HB-7 with GABA-ergic agents.

The effect of the newly synthesized barbiturate HB-7 (2-hydroxylamino-5-ethyl-5-sec. pentylbarbituric acid) was investigated in experiments on mice in comparison with the effect of pentobarbital (PB), as well as their correlations with agents potentiating GABA-ergic transmission: GABA, aminooxyacetic acid, nipecotic acid, di-n-propylacetate (depakin, DPA), muscimol and baclofen. After the test applied (electroshock convulsions) 50 per cent of the animals in the control group had a tonic convulsion. HB-7 and all the other agents were administered in threshold doses. GABA-ergic agents were found to have a different effect on the anticonvulsive action of HB-7 and of pentobarbital. The anticonvulsive effect of HB-7 was potentiated when it was applied in combination with baclofen and AOAA, and partially with depakin (DPA). The anticonvulsive action of pentobarbital is potentiated when it is administered in combination with DPA and AOAA. The results obtained suggest that the GABA-ergic transmission in the brain participates in the anticonvulsive effects of the barbiturates investigated (HB-7 and PB).

Study of the participation of the dopaminergic transmission system in the effects of two newly synthesized barbiturates.

The effects of two newly synthesized barbiturates--HB-7 (2-hydroxylamino-5-ethyl-5-sec. pentylbarbituric acid) and HB-2 (2-hydroxylamino-5-ethyl-5-propylbarbituric acid--on models of apomorphine stereotypy and haloperidol catalepsy were investigated in experiments on male rats and mice. The test drugs used were phenobarbital (PB)--in stereotypy and catalepsy, and diphenylhydantoin (DPH)--in catalepsy. HB-7 changes the stereotypy considerably: it developed faster and disappeared faster than in the control group. Phenobarbital had a weaker effect on the intensity and duration of the stereotypy. Catalepsy was induced with haloperidol in doses of 1 and 2 mg/kg. HB-7 and HB-2 weakened haloperidol catalepsy, compared with PB and DPH. The faster occurrence of apomorphine stereotypy and the weakening of the haloperidol-induced catalepsy under the effect of the two hydroxyl-amine derivatives of barbituric acid suggest an effect on the cerebral dopaminergic transmission. This effect may be mediated through the effect of HB-7 on DA-ergic transmission or through the GABA-ergic transmission in the brain structures responsible for these behavioural reactions.

[Hepatotropic action of benzobamil in CCl4 poisoning]. / Gepatotropnoe deistvie benzobamila pri intoksikatsii CCl4.

The administration of an antiepileptic drug--benzobamilum-to rats with CCl4-induced hepatitis prevents the development of liver parenchyma necrosis, promotes the retention of normal enzyme activity in hepatocytes, stimulates the excretory and antitoxic liver functions. The drug has antioxidant properties, inhibits the production of lysophosphatidylcholine and the reduction in phosphatidylcholine content in the liver homogenates but fails to intensify CCl4-induced steatosis.

Mechanism of N,N'-diallylpentobarbital potentiation of pentobarbital-induced sleep in mice.

Potentiation mechanism of pentobarbital (PB)-induced sleep by N,N'-diallylpentobarbital (DAPB) was studied in mice. DAPB significantly prolonged the PB [40 mg/kg, intraperitoneal (i.p.)]-induced sleeping time by two routes of administration [intravenous (i.v.) and intracerebroventricular (i.c.v.)], nevertheless DAPB alone was devoid of hypnotic activity even by both routes of administration (i.v. and i.c.v.). In addition, DAPB (160 and 320 mg/kg, i.p.) significantly prolonged the sleeping time induced by i.c.v. injection of PB (200 micrograms/mouse). The brain PB half-life (T1/2) of DAPB (80 mg/kg, i.p.) treated group (9.0 h) was 13-fold longer than that of the control (0.7 h). The plasma PB half-life (T1/2) of DAPB treated group (15.2 h) was longer than that of the control (0.6 h). Moreover, DAPB significantly decreased the activities of ethylmorphine (EM) N-demethylase and aniline hydroxylase, and the content of cytochrome P-450 in mouse liver microsomes. The inhibitory effect of DAPB (40 mg/kg, i.p.) on the mouse hepatic drug-metabolizing enzymes was shown til 6 h after administration. DAPB exhibited non-competitive inhibition on the EM N-demethylase activity in vitro. These results indicate that DAPB prolongs the PB-induced sleeping time by both its depressant action to the central nervous system (CNS) and inhibitory effect on the hepatic drug-metabolizing enzymes.

N,N'-diallylpentobarbital: antagonism of barbital in mice and rats.

N,N'-Diallylpentobarbital (DAPB) antagonized barbital (B)-induced sleep in mice and rats. DAPB [80 mg/kg, intraperitoneal (i.p.)] reduced the barbital (350 mg/kg, i.p.)-induced sleeping time to 40% of the control in mice. Twenty, 40 and 80 mg/kg, i.p. of DAPB reduced barbital-induced sleeping time when administered 60 min prior to injection of barbital. DAPB (80 mg/kg, i.p.) also shortened barbital (250 mg/kg, i.p.)-induced sleeping time to about 70% of the control in rats. The result indicates that DAPB is an antagonist against hypnotic activity of barbital.

[Effect of anticonvulsant agents, inducers of microsomal oxidation, on the B-link of immunity and on the natural cytotoxicity of lymphoid organ cells in rats]. / Vliianie protivosudorozhnykh sredstv--induktorov mikrosomal'nogo okisleniia na B-zveno immuniteta i estestvennuiu tsitotoksichnost' kletok limfoidnykh organov krys.

Administration of phenobarbital, benzonal and benzobamil in a dose of 1/20 of LD50 to rats was shown to be followed by phase changes in the system of microsomal oxidation of the liver--activation in the first days after administration with the subsequent (in 1-3 months) decrease of the activity. The drugs activate the specific link of immunity directed at detoxication of the administered substances: increase the level of specific antibody-forming cells in the spleen and antibodies in the peripheral blood. Benzonal and bezobamil enhance the killing activity of cells of the thymus, phenobarbital and benzonal suppress the natural cytotoxicity of cells of the spleen and peritoneal exudate.