Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

Viscoelastic Properties of Bioadhesive,Chlorhexidine-Containing Semi-Solids for Topical Application to the Oropharynx

Purpose. This study examined the viscoelastic properties of bioadhesive, chlorhexidine-containing semi-solid formulations, designed for topical application to the oropharynx. Methods. Oscillatory rheometry was performed using a Carri-Med CSL²-100 rheometer at 20.0 ± 0.1° C in conjunction with parallel plate geometry (2 cm diameter, 0.5 mm sample thickness). Samples were subjected to a constant strain (6.5 × 10^–3 rad) and defined viscoelastic parameters, namely storage modulus (G), loss modulus (G), loss tangent (tan ) and dynamic viscosity (), measured over a defined frequency range (0.01-1.0 Hz). Results. As the oscillatory frequency was increased, G G of all formulations increased, whereas both and tan significantly decreased. The magnitude of increase of G and G as a function of frequency was relatively small, indicating that, in general, the formulations were non-cross-linked elastic systems. Increasing concentrations of HEC, PVP and PC significantly increased G, G, yet decreased tan observations that may be attributed to the physical state of each polymer in the formulations. Formulation elasticity increased (i.e. tan decreased) as a result of increased entanglement of polymeric chains of dissolved components (i.e. HEC and PVP) and the restrained extension of swollen, cross-linked chains of PC. Additionally, in formulations where the saturation solubility of PVP was exceeded and/or insufficient 'free-water' was available for maximal swelling of PC, formulation elasticity increased as a result of the increasing mass of dispersed solid particles of PVP and/or PC. Formulation increased due to the attendent effects of polymer chain entanglement and polymer state on overall formulation viscosity. Conclusions. Following application to the oropharynx, the formulations will behave as elastic systems. Thus, these formulations would be expected to offer advantageous clinical properties, e.g., prolonged drug release, increased bioadhesion. However, it is noteworthy that the final choice of formulation for clinical evaluation will involve a compromise between viscoelastic characteristics and acceptable textural properties, e.g. ease of product application. This study has shown the applicability of oscillatory rheometry for both the characterisation and selection of candidate, topical bioadhesive formulations for clinical evaluation.     

The influence of hyperthyroidism on β-adrenoceptor-mediated relaxation of isolated small mesenteric arteries

We investigated the influence of hyperthyroidism on relaxant responses of small mesenteric resistance arteries to -adrenoceptor agonists and to compounds stimulating the corresponding second-messenger system. Hyperthyroidism was induced by feeding rats for 28 days with 5 mg/kg L-thyroxine (T4)-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by several biochemical/metabolic and haemodynamic parameters. Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for isoproterenol, noradrenaline and salbutamol as well as for forskolin, dibutyryl-cAMP and theophylline. We also determined concentration-effect curves to the -adrenoceptor agonists in the presence of ICI 118,551 and CGP 20712A (i.e., in the presence of a selective ₂- and ₁-adrenoceptor antagonist, respectively). Apparent pA₂-values were calculated to determine which -adrenoceptor subtype causes vasodilation. These experiments indicate that -adrenoceptor-mediated vasodilation involves both ₁- and ₂-adrenoceptors in mesenteric resistance vessels of both hyperthyroid and control rats. In our experiments hyperthyroidism has a sensitizing influence on vascular responses induced by the -adrenoceptor agonist isoproterenol and the selective ₂-adrenoceptor agonist salbutamol. Sensitization to isoproterenol was abolished in the presence of ICI 118,551, whereas it was emphasized in the presence of CGP 20712A. Although this was not fully supported by the results obtained with noradrenaline, these results indicate that the sensitization to -adrenoceptor agonists is probably limited to the ₂-adrenoceptor/G-protein complex and not associated with alterations of the corresponding second messenger system.     

Controlled Systemic Absorption and Increased Anesthetic Effect of Bupivacaine Following Epidural Administration of Bupivacaine-Hydroxypropyl-β-Cyclodextrin Complex

Purpose. To investigate the influence of complexation between bupivacaine and hydroxypropyl--cyclodextrin (HP--CD) on the systemic absorption and on the pharmacodynamic effect of bupivacaine following epidural administration in a rabbit model. Methods. Bupivacaine and bupivacaine-HP--CD complex were administered according to a randomized and cross-over design in six rabbits chronically instrumented with an epidural catheter. The plasma concentrations of bupivacaine and the duration and intensity of the motor blockade were evaluated. Results. Complexation with HP--CD led to a decrease in the maximum plasma concentration of bupivacaine. Individual absorption kinetics evaluated by Loo-Riegelman absorption analysis indicated that systemic absorption resulted from two parallel first-order processes. Only the faster absorption phase was slowed by complexation with HP--CD. The duration of the motor blockade was increased almost twice but the intensity was not modified. Conclusions. Complexation with HP--CD could be a promising drug delivery system to improve the therapeutic index of bupivacaine.     

Synthesis and Anti-inflammatory Effect of Chalcones and Related Compounds

Purpose. Mast cell and neutrophil degradations are the important players in inflammatory disorders. Combined with potent inhibition of chemical mediators released from mast cells and neutrophil degranulations, it could be a promising anti-inflammatory agent. 2,5-Dihydroxychalcone has been reported as a potent chemical mediator and cyclooxygenase inhibitor. In an effort to continually develop potent anti-inflammatory agents, a novel series of chalcone, 2- and 3-hydroxychalcones, 2,5-dihydroxychalcones and flavanones were continually synthesized to evaluate their inhibitory effects on the activation of mast cells and neutrophils and the inhibitory effect on phlogist-induced hind-paw edema in mice. Methods. A series of chalcones and related compounds were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and the anti-inflammatory activities of these synthetic compounds were studied on inhibitory effects on the activation of mast cells and neutrophils. Results. Some chalcones showed strong inhibitory effects on the release of -glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Almost all chalcones and 4-hydroxyflavanone exhibited potent inhibitory effects on the release of -glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Some chalcones showed potent inhibitory effects on superoxide formation of rat neutrophils stimulated with fMLP/cytochalasin B (CB) or phorbol myristate acetate (PMA). 2,3-Dihydroxy-, 2,5-dihydroxy-4-chloro-, and 2,5-dihydroxychalcone showed remarkable inhibitory effects on hind-paw edema induced by polymyxin B in normal as well as in adrenalectomized mice. Conclusions. These results indicated that the anti-inflammatory effects of these compounds were mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils.     

Subject-by-Formulation Interaction in Determinations of Individual Bioequivalence: Bias and Prevalence

Purpose. 1. To determine properties of the estimated variance component for the subject-by-formulation interaction (² _D) in investigations of individual bioequivalence (IBE), and 2. to evaluate the prevalence of interactions in replicate-design studies published by FDA. Methods. Four-period crossover studies evaluating IBE were simulated repeatedly. Generally, the true bioequivalence of the two formulations, including ² _D= 0, was assumed, ² _D was then estimated in a linear mixed-effect model by restricted maximum likelihood (REML). The same method was applied for estimating ² _D for the data sets of FDA. Results. 1._D estimated by REML was positively biased. The bias and dispersion of the estimated _Dincreased approximately linearly with the estimated within-subject standard deviation for the reference formulation (_WR). Only a small proportion of the estimated _D exceeded the estimated _WR. 2. Distributions of the estimated _D were evaluated. At _WR = 0.30, a level of estimated _D= 0.15 was exceeded, by random chance, with a probability of about 25%. 3. Importantly, the behaviour of the ² _D values estimated from the FDA data sets was similar to that exhibited by the simulated estimates of ² _D which were generated under the conditions of true bioequivalence. Conclusions. 1. _D estimated by REML is biased; the bias increases proportionately with the estimated _WR. Consequently, exceeding a fixed level of _D (e.g., 0.15) does not indicate substantial interaction. 2. The data sets of FDA are compatible with the hypothesis of ² _D = 0. Consequently, they do not demonstrate the prevalence of subject-by-formulation interaction. Therefore, it could be sufficient and reasonable to evaluate bioequivalence from 2-period crossover studies.     

Cytostatic and Apoptotic Effects of Paclitaxel in Human Ovarian Tumors

Purpose. The present study evaluated the cytostatic and apoptotic effects of a 24-hr paclitaxel treatment in ovarian tumors. Methods. Three-dimensional histocultures of surgical specimens from patients (n = 17) were used. The cytostatic effect was measured by inhibition of 96-hr cumulative DNA precursor incorporation and induction of apoptosis was determined by morphological changes. Results. Paclitaxel produced partial inhibition of DNA precursor incorporation in about 40% of tumors (maximum inhibition of 30%) and induced apoptosis in about 90% of tumors (maximum apoptotic index of 15%). In responsive tumors, maximum cytostatic and apoptotic effects were achieved at 1 M with no further enhancement by increasing the drug concentration to 10 M. In individual tumors, the apoptotic effect inversely correlated with cytostatic effect (r² = 0.27, p = 0.031), and the maximal apoptotic index correlated with the LI for the untreated controls (r² = 0.38, p < 0.01). More than 95% of apoptotic cells after paclitaxel treatment were labeled with DNA precursor. The incomplete cytostatic and apoptotic effects of paclitaxel and the link between DNA synthesis and apoptosis in ovarian tumors are similar to our previous findings in other human solid tumors. Conclusions. These findings suggest that (a) apoptosis is the major paclitaxel effect in advanced ovarian tumors, (b) tumor sensitivity to drug-induced cytostatic effect is opposite to sensitivity to apoptotic effect, (c) paclitaxel-induced apoptosis increases with increased cell proliferation and is completed after DNA synthesis, and (d) further increasing the dose to elevate plasma concentration beyond 1 M may not improve treatment outcome.     

Effects of (+)- and (±)-sotalol on repolarizing outward currents and pacemaker current in sheep cardiac Purkinje fibres

Summary This study was aimed to differentiate the action of (+)- and (±)-sotalol (10–1000 mol/l) on membrane currents which are active during the repolarization of cardiac action potentials Effects where studied in shortened sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique Action potentials were activated at a frequency of 0.25 Hz and membrane currents at 0.03 Hz or 0.05 Hz in most experiments.Out of the currents investigated the transient outward current (i_to) reacted most sensitively to (+)- and (±)-sotalol. I_to-amplitude was decreased on the average to 77% of reference at 10 mol/l and to 53% at 1000 mol/l (+)- or (±)-sotalol. The maximally available ito-current was decreased but the voltage-dependent control of inactivation was left nearly unchanged. The initial inwardly rectifying current (i_Ki), which propels the last repolarization phase of the action potential and controls resting potential to a large extent was reduced on the average to 93% of reference at 10 mol/l and to 62% at 1000 mol/l (+)- or (±)-sotalol. Time-dependent (delayed) outward current (i_K) was on the average not affected by (+)- or (±)-sotalol up to 100 mol/l and was decreased to 84% of reference current under the influence of 1000 mol/l. An initial outward current, which is activated at positive membrane potentials (i_inst) was not clearly affected by (+)- or (±)-sotalol at concentrations up to 1000 mol/l Pacemaker current (i_f) was not influenced by the drugs up to 100 mol/l. Only at 1000 mol/l was the amount of available i_f-current decreased to 79% of reference. (The potential-dependent control of activation was not affected) Time constants of time-dependent currents i_to, i_K and i_f did not change in concentrations up to 1000 mol/l of the drug.Action potential duration increased at (+)- or (±)-sotalol concentrations 10 mol/l and maximal prolongation was achieved at concentrations of 100–300 mol/l Resting potential remained nearly unchanged at these concentrations, but the membranes depolarized at 1000 mol/l. According to our data action potential prolongation in sheep Purkinje fibres under the influence of (+)- and (±)-sotalol correlates to the drug-induced block to i_to-current and inwardly rectifying i_K1-current.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

Factors in the estimated response of acne vulgaris to antibiotics

Summary The data of a double-blind cooperative trial of thiamphenicol (TP), tetracycline (TE) and placebo (PL) in a two-week oral treatment of acne vulgaris were subjected to a study of the observers' attitudes. The study provided no evidence that the PL-optimistic doctors judged either antibiotic more favorably than the PL-pessimistic. A technique for analyzing factorial experiments when the data are frequencies m unbalanced groups was used in a computerized form to evaluate the effects of the oral treatments as well as the interactions with the factors Sex, Severity and Topical treatment. The advantage of TP (63% satisfactory responses) over PL (34%) was proved beyond doubt, irrespective of any factors studied; the addition of a topical treatment is a useful cofactor only in the severe forms in female patients. The advantage of TE (50% overall satisfactory responses) over PL was detected in female patients only. This finding, as well as the potent action of TP, are discussed in view of the acne ameliorating mechanism/s of these antibiotics.     

Serum glycoside concentrations after single or repeated intravenous doses ofβ-methyl-digoxin and digoxin

Summary The aim of the present investigation was to estimate the ratio of the intravenous doses of-methyl-digoxin and digoxin required to produce identical serum glycoside concentrations in man.20 patients on intravenous maintenance therapy were changed from-methyl-digoxin to the identical dose of digoxin or vice versa. Each drug was given for 7 days. Serum concentrations 13% higher were found during administration of-methyl-digoxin. Assuming a half life of 60 h after with drawal, the dose of digoxin producing the same minimum serum concentration was estimated to be 1.16 times higher than that of-methyl-digoxin.18 healthy volunteers received 0.4 mg -methyldigoxin, and 23 the same dose of digoxin, as an intravenous infusion over 2 h. The serum concentrations and urinary glycoside excretion were measured over a period of 32 hrs. During the first hour after the infusion the serum concentration of digoxin declined more rapidly than that of-methyl-digoxin. Thereafter, the ratio of the serum concentrations did not change appreciably up to the end of the investigation. The area under the serum concentration/time curve was about 13% greater for-methyl-digoxin than for digoxin; this difference was not significant.The average renal clearance was 96±9 ml for-methyl-digoxin, 151±13 ml for digoxin. Since the total body clearance of digoxin is only about 1.16 times higher than that of-methyl-digoxin, the lower renal clearance of-methyl-digoxin must partly be compensated by higher extrarenal clearance.From the ratios of the areas under the serum concentration/time curves after single doses of -methyldigoxin and digoxin, and the minimum serum concentrations during maintenance therapy, it was concluded that the dose of digoxin to produce the same average serum concentrations would be about 1.15 times higher than that of-methyl-digoxin. In comparison with the large variations in individual dosage of digoxin and-methyl-digoxin, this difference is too small to be of practical importance.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Bedeutung der N- und α-Methylierung für die Affinität von Brenzcatechinaminen zu den adrenergischen Receptoren

Summary 1. N-methylation of dopamine yielding epinine means potentiation of the -adrenergic pressor action in the cat. It is only N-methylation which renders dopamine to a direct acting -sympathomimetic amine: the positive inotropic effect of epinine on the isolated electrically driven guinea pig auricle remained uninfluenced by pretreatment of the animals with reserpine, whereas the dose-response curve of dopamine was shifted to the right.The blood pressure lowering effect of epinine after phenoxybenzamine was—in contrast to that of dopamine—abolished by pronethalol.—N-methylation of noradrenaline doubled the affinity for the -receptors of the heart.2. Also by -methylation dopamine gains affinity to the adrenergic -receptors (heart and vessels): d--methyldopamine, an exclusively directly acting catecholamine, had a 40 times stronger inotropic action than dopamine on reserpinized auricles; the action of l--methyldopamine, a mainly indirectly acting amine, was only 4 times stronger than dopamine.-methylnoradrenaline, although having the same inotropic activity as noradrenaline, had a 10 times stronger depressor action than noradrenaline in the cat pretreated with phenoxybenzamine.By -methylation, the -adrenergic pressor effect of dopamine as well as that of noradrenaline was enhanced only in the range of lower doses since the dose-response curves of the -methylated compounds were generally less steep than those of the non-methylated catecholamines, indicating a lower intrinsic activity of the -methylated derivatives.3. The N- and -methylated catecholamines -methylepinine and -methyladrenaline resp. were blood pressure lowering agents per se, because both methylgroups additively enhanced the affinity to the vascular -receptors. -methylepinine was the most potent -sympathomimetic on the heart in the dopamine series (dopamine < d--methyldopamine epinine < dl--methylepinine). However, in the noradrenaline series the twofold methylated compound -methyladrenaline had the lowest positive inotropic action (d(–)-adrenaline > d(–)-noradrenaline (–)erythro--methylnoradrenaline > (–) erythro--methyladrenaline).4. From the results the following conclusions are drawn: The N- as well as the -methyl-group exerts and +I-effect on the ammonium group. Thereby, protonation will be increased which leads to an enhanced affinity of the resp. catecholamine to the adrenergic - and -receptor. Since the -CH₃ group—dependant upon its steric configuration—also causes a steric hindrance at the receptor site, the increase in affinity due to the +I-effect is partially neutralized. By that the lower intrinsic affinity of the -methylated compounds as indicated by the different slope of their pressor dose-response curves, is also readily explained. Furtheron it is intelligible why -methylation enhanced the -adrenergic activity in the less potent dopamine series (preponderance of the +I-effect), whereas it lowered the affinity to the cardiac -receptors in the noradrenaline series (preponderance of the steric hindrance).5. Although -methyladrenaline was the least potent -sympathomimetic of the noradrenaline series in the guinea pig heart, it was the most potent compound in lowering the cat's blood pressure. Therefore, it seems to be questionable whether the cardiac and the vascular -receptors and/or the mechanisms by which they induce the pharmacodynamic actions are identical.

Über einen Teil der Ergebnisse wurde auf der 7. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft in Mainz, 24.–27. April 1966, berichtet (Palm, Langeneckert u. Holtz, 1966).     

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. (lRS,5RS)-l--D-Glucopyranosyl) pentobarbital ((lRS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (lRS,5R)-PTBG and (lRS,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 (lRS,5R)-PTBG, (lRS,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 (lRS,5S)-PTBG over 60 hours. (lRS,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 (lR,5R)-PTBG and (lS,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.     

Antitumor Agents. 125. New 4β-Benzoylamino Derivatives of 4′-O-Demethyl-4-desoxypodophyllotoxin and 4β-Benzoyl Derivatives of 4′-O-Demethylpodophyllotoxin as Potent Inhibitors of Human DNA Topoisomerase II

A series of 4-benzoylamino (5-17) derivatives of 4-O-demethyl-4-desoxypodophyllotoxin and 4-benzoyl (18-20) derivatives of 4-O-demethyl podophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 5-13 and 15-17 are more potent than etoposide in causing DNA breakage, while compounds 9, 10, 13, 14, 16, and 20 are more active than etoposide in their inhibition of the human DNA topoisomerase II. The order for the enzyme inhibitory activity of the derivatives of 4-O-demethyl-4-desoxypodophyllotoxin is 4-arylamino > 4-benzylamino > 4-benzoylamino.     

Differential Effects of Sulfate and Sulfobutyl Ether of β-Cyclodextrin on Erythrocyte Membranes in Vitro

The hemolytic activity of -cyclodextrin (-CyD) on rabbit erythrocytes was reduced by the introduction of negatively-charged groups onto the hydroxyls of -CyD; the membrane disrupting abilities decreased in the order of -CyD > 2-hydroxypropyl--CyD (HP--CyD) > sulfobutyl--CyD (SB--CyD) >> -CyD sulfate (S--CyD). Under pre-hemolytic concentrations, both -CyD and SB--CyD induced shape changes of membrane invagination on the erythrocytes. In sharp contrast, S--CyD showed biphasic effect on the shape of the erythrocytes; i.e. the crenation at relatively low concentrations and the invagination at higher concentrations. The S--CyD-induced membrane crenation arose from a direct action on the membranes rather than cell metabolism-mediated effects. Unlike -CyD, S--CyD was found to bind to the erythrocytes and may be confined to the outer surface of the membrane bilayer, which may expand the exterior layer relative to the cytoplasmic half, thereby inducing the cells to crenate. On the other hand, the membrane invagination mediated by the three - CyDs was initiated by extracting specific membrane lipids from the cells, depending upon their inclusion abilities, subsequently leading to the lysis of the cells. These results indicate that SB--CyD and S--CyD interact with the erythrocyte membranes in a differential manner and possess lower membrane disrupting abilities than the parent -CyD and HP--CyD.     

Über die Wirkung von α-Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen

Zusammenfassung 1. Versuche über die Wirkung von -Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen haben ergeben, daß Noradrenalin im Herzen, sowie Dopamin und Noradrenalin im Gehirn durch äquimolare Mengen von -Methyl-Noradrenalin bzw. -Methyl-Dopamin ersetzt werden.2. Die Adrenalinverarmung der Nebennieren nach Vorbehandlung mit -Methyl-Dopa wird demgegenüber nur zu einem geringen Teil durch die methylierten Brenzcatechinamine -Methyl-Noradrenalin und -Methyl-Dopamin ausgeglichen. Ein weiteres Brenzcatechinamin konnte nicht nachgewiesen werden, so daß -Methyl-Adrenalin wahrscheinlich nicht gebildet wird.3. Im Meerschweinchenherzen gespeichertes -Methyl-Noradrenalin wird durch Reserpin in vivo schlechter freigesetzt als Noradrenalin.4. Das in isolierten Herzgranula (Sediment 100 000·g) gespeicherte -Methyl-Noradrenalin wird durch Segontin bei der Inkubation in vitro ebenfall schlechter freigesetzt als Noradrenalin aus normalen Herzgranula.5. Der Mechanismus der -Methyl-Dopa-Wirkung wird diskutiert.6. Es wird eine fluorimetrische Methode zur Bestimmung von -Methyl-Noradrenalin angegeben.

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Summary 1. The catecholamine content of guinea pig organs after administration of -methyldopa has been determined. The results show that the loss of noradrenaline from heart and that of noradrenaline and dopamine from brain is compensated by a stoichiometric uptake of -methylnoradrenaline and -methyldopamine respectively.2. On the contrary the loss of adrenaline from the suprarenal medulla induced by pretreatment with -methyldopa is not completely restored by the -methylated amines, -methyldopamine and -methylnoradrenaline. -methyladrenaline could not be detected even by paperchromatography.3. The release of the stored -methylnoradrenaline from guinea pig heart by reserpine is very small in comparison with that of noradrenaline.4. Furthermore only small amounts of the stored -methyl-noradrenaline are released from isolated granules of the guinea pig heart after incubation in vitro with segontin. Whereas the same amount of segontin depleted completely the noradrenaline content of the granules isolated from normal hearts.5. The mechanism of action of -methyldopa is discussed.6. A method for the fluorimetric determination of -methylnoradrenaline is described. It is at first separated from -methyldopamine, dopamine and histamine by column chromatography, condensed with o-phthaldialdehyd and the obtained fluorescense is measured by using an Aminco-Bowman spectrophotofluorimeter.

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Mit 6 Textabbildungen

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Herrn Dr. H. Blaschko zum 65. Geburtstag gewidmet.

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Über einen Teil der Ergebnisse wurde auf der Frühjahrstagung der Deutschen Pharmakologischen Ges. in Mainz, April 1964, berichtet. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 297 (1964).

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.     

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

Phase Transitions of Glycine in Frozen Aqueous Solutions and During Freeze-Drying

Purpose. To study the solid-state and phase transitions of glycine, (i) in frozen aqueous solutions, and (ii) during freeze-drying. Methods. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to analyze the frozen systems. In situ freeze-drying in the sample chamber of the diffractometer enabled characterization of phase transitions during freeze-drying. Results. Transitions in frozen systems. Rapid (20°C/min) or slow (2°C/min) cooling of aqueous solutions of glycine (15% w/w) to –70°C resulted in crystallization of -glycine. Annealing at –10°C led to an increase in the amount of the crystalline phase. When quench-cooled by immersing in liquid nitrogen, glycine formed an amorphous freeze-concentrate. On heating, crystallization of an unidentified phase of glycine occurred at \-65°C which disappeared at –55°C, and the peaks of -glycine appeared. Annealing caused a transition of - to the - form. The extent of this conversion was a function of the annealing temperature. Slower cooling rates and annealing in frozen solutions increased the crystalline -glycine content in the lyophile. Freeze-drying of quench-cooled solutions led to the formation of -glycine during primary drying resulting in a lyophile consisting of a mixture of - and -glycine. The primary drying temperature as well as the initial solute concentration significantly influenced the solid-state of freeze-dried glycine only in quench-cooled systems. Conclusions. The cooling rate, annealing conditions and the primary drying temperature influenced the solid-state composition of freeze-dried glycine.     

Plasma concentration and vascular effect of β-endorphin in spontaneously hypertensive and Wistar Kyoto rats

Summary In order to find out whether -endorphin (-E) is involved in the development of hypertension, we performed two series of experiments. Firstly, spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY) were submitted to ether stress. Plasma concentrations of -endorphin-like immunoreactivity (-EI), adrenocorticotropin (ACTH) and -melanotropin (-MSH) were measured by radioimmunoassay. The basal concentration of -EI was similar in WKY and SHR, whereas WKY had higher levels of ACTH and lower levels of -MSH than SHR. In both strains acute stress enhanced the plasma concentration of -EI to the same extent and with a similar time-course. The increase of plasma -El coincided with a rise in ACTH but not -MSH. Gel chromatography of -EI revealed that plasma extracts contain similar amounts of -lipotropin- (-LPH) and -E-sized immunoreactive components, and that acute stress elevated both forms of -El. Secondly, isolated tail arteries of SHR and WKY were perfused and field stimulated with two pulses at 1 Hz. -E depressed stimulation-evoked vasconstriction with the same potency in both strains. Thus, basal and stress-induced levels of -EI did not differ in SHR and WKY. Moreover, in the tail artery of both strains the sensitivity of presynaptic opioid receptors towards -E was almost identical. If the -E sensitivity of these receptors in other arteries of WKY and SHR is also similar, a major role of the circulating peptide in the development of hypertension is rather unlikely.This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 325) Send offprint requests to B. Bucher at the above address