Stereoselective binding of the enantiomers of four closely related N-methyl-barbiturates to human, bovine, and rat serum albumin.

Albumin binding for the enantiomers of four closely related N-methyl-5-phenyl-5-alkyl-barbiturates 1-4 was investigated for three different mammalian species by means of equilibrium dialysis. Lipid solubility (n-heptane/phosphate buffer distribution coefficient) increased stepwise by a factor of 56 from 1 to 4. Bovine serum albumin: The (S)-(+)-enantiomers of 1-4 were bound in a higher percentage than the (R)-(-)-enantiomers; lengthening of the aliphatic side-chain increased the binding extent in both enantiomeric groups. Human serum albumin: Binding of (S)-(+)-1 and (S)-(+)-4 was higher than that of the (R)-(-)-enantiomers; with (S)-(+)-2 and (S)-(+)-3 it was much lower than that of the corresponding (R)-(-)-enantiomers. Lengthening of the aliphatic side chain increased the binding extent of the (S)-(+)- as well as the (R)-(-)-enantiomers, but with two exceptions: 1. The (S)-(+)-1 binding exceeded that of the (S)-(+)-2 by a factor of nearly two. 2. The binding extent of (R)-(-)-4 was not further increased in comparison to (R)-(-)-3. Rat serum albumin: (S)-(+)-1 and (S)-(+)-2 were bound in a lower percentage than the (R)-(-)-enantiomers, both 3-enantiomers showing an equal binding extent; (S)-(+)-4 was bound to a slightly greater extent than the (R)-(-)-4. In the group of the (S)-(+)-enantiomers, the binding extent increased from 1 to 4, whereas in that of the (R)-(-)-enantiomers only between 1 and 4. Structural differences between the serum albumins of three mammalian species possibly cause the enantioselective binding pattern found for the enantiomers of 1-4, and are responsible for the finding that the binding extent in some cases did not correlate with the lipid solubility of the compounds.

Influence of albumin concentration, albumin species, pH and temperature on the stereoselectively different binding of (S)-(+)- and (R)-(-)-1-methyl-5-phenyl-5-propyl-barbiturate to serum albumin.

Using normal conditions (phosphate buffer pH 7.4, room temperature) and 1% human serum albumin (HSA), (R)-(-)-1-methyl-5-phenyl-5-propyl-barbiturate = (R)-(-)-1) as compared to (S)-(+)-1 was preferentially bound to albumin: the difference in the enantioselective binding was large (factor of 2) with low substrate concentrations and much smaller (factor of 1.3) with high substrate concentrations (range 5-620 micrograms/ml); with (S)-(+)-1 the decrease of the percent binding values observed with increasing concentrations was only moderately marked. The stereoselective binding difference ((R)-(-)- being greater than (S)-(+)-1) was always present independent of the albumin concentration (range 0.5-10%), the pH value (range 5-10) and the temperature (range 5-50 degrees C). Using 20 micrograms/ml as substrate concentration the stereospecific binding difference was large in solutions with low HSA concentration, in acidic and neutral pH range, and in experiments with low temperature. In contrast to HSA, bovine as well as rabbit serum albumin bound (S)-(+)-1 to a higher percentage than (R)-(-)-1; using rat serum albumin an enantioselective binding difference was not obtained. Taking into account for each of three mammalian albumins the respective number of binding classes and sites in each class, the affinity constant and total binding constant, it becomes evident that they bind (S)-(+)- and (R)-(-)-1 quantitatively differently in a species-specific manner.

Propofol binding to human blood proteins.

The binding of the intravenous anesthetic propofol (CAS 2078-54-8) to human native plasma, hemoglobin and serum albumin was studied by means of equilibrium dialysis. Propofol binding to plasma over the large concentration range from 0.04 to 150 micrograms/ml was independent on the substrate concentration and amounted 97.4-98.6%. Serum albumin and hemoglobin also showed a marked binding for propofol. A 4% solution of albumin bound 88.7% and hemoglobin 86.2% of the anesthetic. In studies with constant protein concentration and variation of the propofol concentration a decrease of the percentage bound with increasing substrate concentration was seen for hemoglobin, indicating saturable binding sites. The opposite was found for the interaction between propofol and albumin: increase of the binding extent with increasing substrate concentration.

[1,5-Benzodiazepines. 2. Diametrically opposite central nervous system actions of the two stereoisomers of 3,3-dialkyl-1,5-benzodiazepine-2,4-diones]. / 1,5-Benzodiazepine, 2. Mitt.: Entgegengesetzte zentralnervöse Wirkungen der Enantiomere von zwei 3,3-Dialkyl-1,5-benzodiazepin-2,4-dionen.

I.p. applicated S(+)-1, R(-)-1 und rac. 1 prolonged hexobarbital sleeping in rats. The rac. 8-chloro compound 3 given i.p. produced no prolongation. Determination of rac. 1 in serum and tissues of rats 30 min after i.p. administration of 50 mg/kg showed that rac. 1 was detectable in serum and brain, yet its concentration was below the limit of determination. I.v. applicated, the enantiomers of 1 and 3 showed diametrically opposite CNS-effects: The S(+)-enantiomers were convulsively active as pentetrazol, whereas the R(-)-enantiomers were CNS depressant active prolonging hexobarbital sleeping time dose-dependently. High doses of diazepam antagonized dose-dependently the convulsive action of S(+)-1 supporting the hypothesis that this enantiomer acted as a strong inverse agonist, whereas R(-)-1 produced weak agonistic activity at the benzodiazepine binding site of the GABA-receptor.--Enantioselective differences for the binding of the 1-enantiomers to human serum albumin were found, too. R(-)-1 was bound to a greater extent than S(+)-1.

HPLC assay for atropine in serum and protein solutions after in vitro addition of the tropane alkaloid.

A simple and sensitive method for the determination of atropine in serum and protein solutions is presented. Atropine and procaine (internal standard) were extracted with diethylether from protein containing solutions after alkalinization. The residue of the evaporized ether phase, redissolved in diluted sulfuric acid, was injected directly onto a LiChrosorbR column. Atropine, procaine and serum constituents were separated by HPLC using an ion-pair containing mobile phase. The limit for the detection of atropine was 200 ng/ml. The method was used for the determination of atropine in a binding study. Percent binding values to human serum, human serum albumin and human alpha 1-acid glycoprotein were evaluated by equilibrium dialysis.

Stereoselective differences in the binding of N-methylated barbiturates to human serum albumin.

The binding to human serum albumin (HSA) of a homologous series of N-methylated chiral barbiturates was studied by means of equilibrium dialysis. The length of the aliphatic side chain at C-5 of the barbiturate ring was variable, and the compounds used were: (+)-(S)- and (-)-(R)-5-methyl-(A), -5-ethyl-(B), -5-propyl-(C) and -5-butyl-(D)-1-methyl-5-phenyl-barbiturate. Binding parameters (numbers of binding classes and of binding sites in each class, affinity constant, total binding constant) were obtained from the Scatchard plot of the percent binding values. For both enantiomers of A and D as well as for (-)-(R)-B 2 classes were obtained; (+)-(S)-B and both enantiomers of C had only 1 class. The total binding constant (K) indicated a more than twofold higher binding of (-)-(R)-B (2.64 x 10(3).mol-1) and C (5.75 x 10(3).mol-1) compared with the corresponding (+)-(S)-enantiomer (1.02 and 2.00 x 10(3).mol-1, respectively); in the case of D the (+)-(S)-enantiomer was preferentially bound (K = 10.14 vs. 5.40 x 10(3).mol-1 for the (-)-(R)-enantiomer). The percent binding values of (+)-(S)-A were higher than those of (-)-(R)-A; however, the K-values of the A-enantiomers were almost identical.

[Cyclic ureas. 2. Racemates and enantiomers of imidazolidin-2-ones: synthesis, configuration and sedative-hypnotic action]. / Racemate und Enantiomere von Imidazolidin-2-onen Synthese, Konfiguration und sedativ-hypnotische Wirkung.

The racemates and the enantiomers of the imidazolidin-2-ones 2a and 2b, which can be considered as cyclic ureas, are obtained from the racemates and the enantiomers of the hydantoins 1a and 1b by reduction with LiAlH4/AlCl3. The enantiomers that are dextrorotating in ethanol possess S-configuration. In a study with Wistar-rats, 2a and 2b show sedative-hypnotic activity. The enantiomers exhibit marked enantioselective differences in their potency.

Rapid and sensitive pre-column extraction high-performance liquid chromatographic assay for propofol in biological fluids.

A completely automated high-performance liquid chromatographic system is described for the determination of the phenolic anaesthetic propofol. The method is based on pre-column extraction in a closed system allowing direct injection of biological samples without any sample pretreatment. The assay is sensitive (limit of quantification is 5 ng/ml serum), reliable (the variability within a series is 2%) and rapid (results are available after 6 min).

[Cyclic ureas. 1. Racemates and enantiomers of hexahydropyrimidine-2-ones: synthesis, configuration and sedative-hypnotic action]. / Zyklische Harnstoffe, 1. Mitt.: Racemate und Enantiomere von Hexahydropyrimidin-2-onen: Synthese, Konfiguration und sedativ-hypnotische Wirkung.

The racemates and the enantiomers of the hexahydropyrimidin-2-ones 2a-2e which can be regarded as cyclic ureas are obtained from the racemates and the enantiomers of the barbiturates 1a-1e by reduction with LiAlH4/AlCl3. The enantiomers of 2a-2d, dextrorotatory in ethanol, possess S-configuration. In a study with rats all cyclic ureas synthesized showed sedative-hypnotic activity. Some of the enantiomers exhibited marked enantioselecive differences in their potency.

Uptake and effects of thiopental in isolated heart preparations of the rat in the presence of halothane.

The uptake of thiopental (CAS 76-75-5) (0.13-0.27 mmol.l-1) into tissue of rat hearts (Langendorff's preparation) was studied in the presence of halothane (CAS 151-67-7). Up to a thiopental concentration of 0.19 mmol.l-1 in the perfusion medium its concentration in heart tissue was significantly increased vs. control when halothane (0.8 vol%) simultaneously was present; using 0.13 mmol.l-1 thiopental and 0.8, 1.5 or 2.0 vol% halothane this increase amounted +12%, +29% and +43%, respectively. Frequency of spontaneously beating rat hearts decreased in the presence of increasing thiopental concentrations. 0.8 vol% halothane (without thiopental) did not influence heart rate; in the presence of thiopental it attenuated heart rate reduction. This attenuation was absent in hearts of rats pretreated with reserpine. 1.5 vol% halothane (itself also without influence on heart rate) increased the negative chronotropic action of thiopental. In isolated right and left atria of rat hearts frequency and contractility decreased concentration-dependently in the presence of thiopental; simultaneously present halothane additionally increased this negative chronotropic and negative inotropic effect of thiopental.

[Effect of the volatile anesthetics halothane, enflurane and isoflurane on liver circulation in the human]. / Einfluss der volatilen Anästhetika Halothan, Enfluran und Isofluran auf die Leberdurchblutung beim Menschen.

In 40 patients with normal liver function total hepatic blood flow (HBF) was determined by the indocyanine-green clearance method simultaneously with haemodynamic parameters, including cardiac output by means of the noninvasive thoracic electrical bioimpedance method. Furthermore, the influence of halothane, enflurane or isoflurane on HBF and the interaction with haemodynamic parameters was studied. HBF and the cardiocirculatory parameters were determined under normal conditions (waking state) and the 40 patients were then divided into 4 groups (each n = 10). After standardised induction of anaesthesia (0.3 mg/kg etomidate and 2 micrograms/kg fentanyl) and tracheal intubation (1.5 mg/kg suxamethonium chloride) an inhalation anaesthesia in O2/air under control of normal end tidal carbon dioxide concentration was performed by intermittent positive pressure ventilation. Anaesthesia was maintained in the 4 groups either with 1 MAC halothane, 1 MAC enflurane, 1 MAC isoflurane or 1.3 MAC isoflurane. The measurements were repeated at a steady of the desired end expiratory concentration of the respective volatile anaesthetic. All three anaesthetics produced a significant and comparable decrease of cardiac output and arterial blood pressure. Differences between halothane, enflurane and isoflurane in respect of haemodynamic parameters were only minimal. Contrariwise, marked differences could be seen in the effects of the anaesthetics on HBF. In the presence of halothane and enflurane HBF dropped to 58% and 56% resp. of the control value, whereas during isoflurane anaesthesia HBF remained unchanged. Furthermore, only during halothane anaesthesia a significant correlation between arterial blood pressure and HBF could be observed indicating a loss of autoregulation of the hepatic blood flow.

Interactions between thiopental and volatile anesthetics (halothane and isoflurane) in isolated heart preparations of the rat.

Thiopental uptake into heart muscle tissue was studied in spontaneously beating rat hearts (Langendorff preparation, 0.13-0.27 mmol/l thiopental in the perfusion fluid). Up to 0.19 mmol/l the concentration of thiopental in heart muscle tissue was increased vs. control when halothane (0.8 vol%) was present. Using a constant thiopental concentration (0.13 mmol/l) and 0.8, 1.5 or 2.0 vol% halothane +12%, +29% or +43% more thiopental was taken up into heart muscle tissue compared to the control. This increased uptake was not seen in the presence of 1.2 and 2.0 vol% isoflurane. Frequency of right rat atria was decreased by increasing thiopental concentrations (0.02-0.23 mmol/l in the incubation medium). Halothane (0.8 and 1.5 vol%) and isoflurane (1.0 and 2.0 vol%) alone had no influence on frequency of right atria. Both volatile anesthetics additionally increased the negative chronotropic action of thiopental when the corresponding higher concentration was applied. Contractile force of left rat atria was decreased concentration-dependently by thiopental (0.02-0.23 mmol/l). Halothane and isoflurane alone decreased contractility. Dependent on the concentration used, both volatile anesthetics further increased the negative inotropic action of thiopental, yet preferentially at higher barbiturate concentrations.

Increase of thiopental concentration in rat tissues due to anesthesia with isoflurane.

Thiopental distribution was studied in rats (30 mg/kg i.v.) anesthetized simultaneously with 1.25 "rat"-MAC isoflurane. The thiopental concentration in serum and several tissues was determined UV-photometrically at 305 nm after extraction and TLC. In the serum of rats anesthetized with isoflurane the thiopental concentration was significantly increased to +39----+74% in comparison to controls during 30 min following the barbiturate injection. Also in liver, brain, heart, kidney, lung and spleen of rats anesthetized with isoflurane the thiopental concentration was significantly increased at 3 and 10 min; at 30 min the difference vs. control had vanished in brain, heart, lung and spleen. Obviously, thiopental was transiently "trapped" during the early distribution phase to a considerable amount in these vessel-rich tissues when anesthesia with isoflurane was simultaneously performed; this pharmacokinetic interaction might be explained at least to some extent hemodynamically; in many tissues regional blood flow is reduced during anesthesia with isoflurane; thereby the "washout" of thiopental from the tissues and the redistribution are delayed.

Cardiac output and liver blood flow in humans: effect of the volatile anesthetic halothane.

In 40 men with normal circulatory and liver function, from whom 10 were undergoing general anesthesia with halothane for minor orthopedic surgery, the relationship between hemodynamic parameters and total hepatic blood flow (HBF) was investigated. Cardiac output (CO) was measured noninvasively by means of the thoracic electrical bioimpedance method, systemic arterial blood pressure (BPsys, BPdia, mean arterial pressure) by an automated oscillometric device and HBF by indocyanine green clearance. In the subjects without halothane anesthesia no relationship was found between BP and HBF, but a significant correlation could be seen between CO and HBF, whereby the fraction of HBF decreased with increasing CO. In contrast, in the presence of halothane the systemic arterial blood pressure correlated with the HBF, indicating a loss of autoregulation of the liver circulation.

Increased binding of thiopental in tissue homogenates of the rat in the presence of a volatile anesthetic.

Thiopental (CAS 76-75-5) binding (0.4 mmol.l-1) in tissue homogenate of rats (liver, brain, heart, kidney, lung, spleen and skeletal muscle) was studied by equilibrium dialysis. Percentage of thiopental bound was relatively low in homogenate of brain, lung, spleen and skeletal muscle (14-19%); it was much higher in that of liver, heart and kidney (24-27%). Simultaneously present halothane (11.8 mmol.l-1) increased the percentage of thiopental bound in the homogenate of all tissues investigated at least to a factor of 1.4 (spleen) and maximally of 2.4 (brain). The same phenomenon of an increased thiopental binding in tissue homogenate was found in the presence of 10.3 mmol.l-1 enflurane (except skeletal muscle) and 10.2 mmol.l-1 isoflurane (except kidney, spleen and skeletal muscle), yet to a significantly lower extent in the presence of these halogenated ethers as compared with halothane.

Increase of thiopental concentration in tissues of the rat due to an anesthesia with halothane.

In rats anesthetized with halothane (CAS 151-67-7) (1.5 vol%) and rats without any further treatment (control) the early distribution phase of thiopental (CAS 76-75-5) (i.v. 30 mg/kg) was studied. In serum and 8 tissues thiopental concentration (T) was determined using ultraviolet detection at 305 nm after extraction and TLC. In rats anesthetized with halothane, T in serum was significantly higher during the 30 min following the thiopental injection (at least +27% and maximally +51%) as compared to the control (same dose), and several pharmacokinetic parameters (e.g. central volume of distribution) were found to be changed thereby; furthermore, at 3, 10 and 30 min T was significantly increased in liver, brain, heart, lung and spleen; in kidney and skeletal muscle a rise of T was also seen, however, it occurred later (after 10 and 30 min). T in fat tissue increased time-dependently; a T-difference in adipose tissue between both groups was not observed. Thiopental was "trapped" during the early distribution phase to a considerable extent in the vessel-rich tissues of rats simultaneously anesthetized with halothane; this pharmacokinetic interaction might be explained hemodynamically: in many tissues regional blood flow is reduced by halothane; thereby a delayed "washout" of thiopental from the vessel-rich tissues could take place and redistribution would be delayed; additional factors as e.g. an increased binding of thiopental at tissue proteins could also play a role. An unusually high T was found at least temporarily in myocardial tissue due to this interaction between the two anesthetics.

pH-dependent interaction of halothane upon thiopental binding to human serum albumin.

At pH 7.4 the binding of thiopental to human serum albumin (HSA) was increased in the presence of halothane. In order to obtain information about the mechanism of this interaction, in vitro binding experiments by means of equilibrium dialysis were carried out at different pH values. At pH 4.97 the binding of thiopental to HSA 1% was low (23% bound) and not influenced by halothane. An increase of thiopental binding caused by halothane could be seen at pH 7.4 (55% bound vs. 41% in the control = without halothane) and at pH 8.23 (62% vs. 54%). At pH 10.15 an opposite interaction was found: in the presence of halothane thiopental binding was considerably decreased (36.2% vs. 47.0% in the control). Evaluation of the binding parameters of experiments using increasing substrate concentrations (Scatchard plot) revealed quite different changes of the two classes of binding sites of HSA for thiopental. It is assumed that halothane causes reversible conformational changes of the albumin molecule resulting in altered binding characteristics for thiopental.

Absorption and metabolism of procaine by the rat small intestine.

The aim of this study was to obtain information about the absorption of procaine in the rat small intestine (Fisher-Parsons preparation). In the range from 0.25-10 mmol.l-1 procaine in the luminal perfusate, much more of the unchanged drug was absorbed in segments of the ileum than of the duodenum and jejunum. Besides procaine, two metabolites, p-aminobenzoic acid (PABA) and acetylated p-aminobenzoic acid (AABA), formed in the intestinal mucosa, appeared in the absorbate. With increasing substrate concentration in the perfusate the PABA in the absorbate increased considerably in all three segments; from 0.75 mmol.l-1 procaine upwards the PABA produced was highest in the jejunum. AABA formed in the mucosa and measured in the absorbate did not increase in the same manner with increasing substrate concentration; in the absorbate of jejunal segments the amount of AABA was significantly higher than in duodenal and ileal segments. Taking into account that in rats the microclimate of the ileum differs considerably from that of the upper part of the small intestine, the marked difference observed in the absorption of procaine between ileal segments on the one side, and duodenal and jejunal segments on the other, can be explained on the basis of the "non-ionic diffusion" theory.

[Stereoselective differences of central nervous system-depressive action of a homologous series of 3-alkyl-thalidomide analysis]. / Stereoselektive Unterschiede der zentralnervös-depressorischen Wirkung in einer homologen Reihe von 3-Alkyl-Thalidomid-Analoga.

The racemates and the enantiomers of the 3-alkyl-thalidomide analogues 2a-c were synthesized via the lactams 1a-c. The absolute configuration of 1a-c and 2a-c was elucidated, their enantiomeric purity established. From racem. 2a 1,3-dimethylthalidomide (3) was synthesized. By means of the hexobarbital sleeping time prolongating effect in rats it was shown that racem. 2a and 3 were less CNS-depressant active than thalidomide; 3 caused a more prolonged sleeping time than 2a. Both enantiomers of 2a (i.p. 200 mg/kg) exceeded in their sleeping time prolongation action racem. 2a (same dose), whereby the (S)-enantiomer was to a factor of 1.8 more effective than the (R). With both enantiomers of 2a time and dose dependency of the effect were investigated. The enantiomers of 2b + c and 1a-c had also a prolongating effect on the hexobarbital sleeping time. Independently of the alkyl group the (S)-enantiomers were always more active than the respective (R)-enantiomers. (S)-1b (200 mg/kg) caused a long lasting loss of the righting reflexes as "own effect". Stereoselective differences between the enantiomers of 2a and 2b were observed only when a dose of 200 mg/kg was applied, whereas the higher activity of the (S)-enantiomers of 2c, 1b + c was obtained already with a lower dosage range.

Thiopental binding to human serum albumin in the presence of halothane.

In vitro thiopental binding (substrate concentration 0.04.10(-3) M = 10 micrograms/ml) to 1% human serum albumin (HSA) increased significantly from 40.2% (= control) to 47.3% in the presence of 1.18.10(-3) M = 2.84 vol% halothane. A 4-fold higher halothane concentration (4.71.10(-3) M) had an even greater effect with an increase in the thiopental fraction bound to 55.5%. With a constant HSA concentration (1% or 5%) and thiopental concentrations in the range 0.01-1.5.10(-3) M or 0.01-0.38.10(-3) M, respectively, the halothane effect (increase in thiopental binding) was always evident, as well as in other experiments with constant thiopental concentration (0.04.10(-3) M) and variation in the HSA concentration (0.5-10%). Two classes of binding sites for thiopental were apparent at the HSA molecule. In the control experiments the following binding parameters were found: n1 = 0.01, k1 = 181.10(3) M-1; n2 = 45.73, k2 = 0.08.10(3) M-1, K = 5.47.10(3) M-1. In the presence of halothane the binding parameters changed as follows: n1 = 0.14, k1 = 29.4.10(3) M-1; n2 = 11.68, k2 = 0.42.10(3) M-1, K = 9.02.10(3) M-1.