Preparation of Heptakis(2,6-di-O-ethyl)-β-cyclodextrin and Its Nuclear Magnetic Resonance Spectroscopic Characterization

Heptakis(2,6-di-O-ethyl)--cyclodextrin (DE--CyD) was prepared and its ¹H and ¹³C nuclear magnetic resonance (NMR) signals in DMSO-d₆ were unequivocally assigned by two-dimensional COSY and ROESY. The results on ¹H coupling constants indicated that all ethylated glucose units are in a ⁴C₁ chair conformation. The average spin-lattice relaxation times (T ₁) of ring carbons of DE--CyD were only slightly shorter, and their standard deviations from the mean T _l value were larger, than those of -cyclodextrin (-CyD) and heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), suggesting the presence of slightly irregular internal motion in the ethylated glucose units. The temperature dependence of chemical shift of DE--CyD in DMSO-d₆ suggested that the C3 hydroxyl protons may participate as proton donor in the intramolecular hydrogen bond to the C2 ethoxyl groups of neighboring glucose, and the intramolecular hydrogen bond of DE- and DM--CyDs is much stronger than that of -CyD, suggesting the stable macrocyclic ring structure of DE--CyD.     

Absorption Enhancing Effect of Cyclodextrins on Intranasally Administered Insulin in Rats

The absorption enhancing effect of -, -, and -cyclodextrin (CD), dimethyl--cyclodextrin (DMCD), and hydroxypropyl--cyclodextrin (HPCD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DMCD to the insulin solution resulted in a high bioavailability, 108.9 ± 36.4% (mean ± SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% -CD gave rise to an insulin bioavailability of 27.7 ± 11.5% (mean ± SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the -CD-containing solution as compared to the DMCD preparation. The other CDs, HPCD (5%), -CD (1.8%), and -CD (5%), did not have significant effects on nasal insulin absorption. DMCD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DMCD is a potent enhancer of nasal insulin absorption in rats.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Evaluation of Cytotoxicity of Various Ophthalmic Drugs, Eye Drop Excipients and Cyclodextrins in an Immortalized Human Corneal Epithelial Cell Line

Purpose. An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/ toxicity by pharmaceuticals. Methods. Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitrocytotoxicity tests, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. Results. Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA₂EDTA) > poly-vinyl alcohol (PVA) > methylparaben; and for CDs: -CD > dimethyl--cyclodextrin (DM--CD) > sulfobutyl ether (-cyclodextrin ((SBE)_7m--CD) hydroxypropyl--cyclodextrin (HP--CD) > -CD. In consideration of the in vivoclinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure times (30 and 60 min). Conclusions. Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivocorneal toxicity of ocularly applied compounds.     

Enhancement of the Antiinflammatory Effect of Ethyl 4-Biphenylyl Acetate in Ointment by β-Cyclodextrin Derivatives: Increased Absorption and Localized Activation of the Prodrug in Rats

Ethyl 4-biphenylyl acetate (EBA) is a prodrug of the antiinflammatory 4-biphenylyl acetic acid (BPAA). The inclusion complexes of EBA with -cyclodextrin (-CyD), heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), and 2-hydroxypropyl--cyclodextrin (HP--CyD) at a molar ratio of 1:2 (EBA:cyclodextrin) were prepared and used to make hydrophilic antiinflammatory ointments. The in vitro release of EBA from the ointments was enhanced by complexation in the order of -CyD < DM--CyD HP--CyD. The improvement correlated with the improved solubility and not with the decreased diffusibility observed to occur upon the complexation of EBA. In vivo the complexation with cyclodextrin derivatives increased both the release of EBA from the vehicle and its conversion in the underlying tissue to BPAA, but the total of EBA and BPAA in the tissue was decreased. In vitro studies confirmed that the effects of cyclodextrin derivatives on the conversion were exerted indirectly. The combination of the enhanced release and of the enhanced prodrug hydrolysis by esterases in the site where the antiinflammatory action is required resulted in increased therapeutic effects. In the model of carrageenan-induced acute edema in rat paw, the complexation improved the therapeutic effects over those of EBA alone in the order of -CyD < DM--CyD < HP--CyD. HP--CyD may be a particularly useful cyclodextrin derivative since it improves the topical availability and does not irritate tissues.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

Effect of ethanol on the metabolism of lithocholic acid in rat liver

Summary Lithocholic acid 24-C¹⁴ is converted by 20000x g-supernatant of rat liver homogenate into several products of higher polarity. 3-6-dihydroxy-5-cholanic acid is the main metabolite, whereas 7-hydroxylation occurs only to a small extent. Besides of the hydroxylations conjugation with taurine and the formation of a 3-sulfate ester of lithocholic acid can be demonstrated. Addition of ethanol to the enzymatic system results in an inhibition of the formation of 3,6-dihydroxy-5-cholanic acid, whereas no effect can be observed with respect to the formation of the other products. This inhibition is present also in 20000x g-supernatant obtained from rats pretreated with ethanol 100 min before being preparation from ethanol-pretreated rats amounts to 68% of the controls. The chrome P-450 mediated oxidative mechanism, which has been shown to be involved in microsomal 6-hydroxylation of bile acids.This work was supported by the Deutsche Forschungsgemeinschaft.     

A pharmacokinetic model-independent approach for estimating dose required to give desired steady-state trough concentrations of drug in plasma

A maintenance dose designed to give a desired minimum concentration of drug in plasma at steady-state can be determined in a model-independent manner assuming that concentration-time data needed for the calculation are obtained after absorption and distribution are complete. Using a few concentration-time points obtained after the first dose, numerical values of and Z, a parameter consisting of different pharmacokinetic parameters for different models, can be obtained. An administration interval () can be chosen based on . Using the values of , Z, and , a maintenance dose is calculated. This approach will allow calculation of a maintenance dose when drug is present in plasma at the time the first monitored dose is given.     

Stabilizing and Solubilizing Effects of Sulfobutyl Ether β-Cyclodextrin on Prostaglandin E1 Analogue

Purpose. Parent cyclodextrins are known to accelerate the degradations such as dehydration and isomerization of E-type prostaglandins in neutral and alkaline solutions. The objective of this study was to attempt the stabilization and solubilization of E₁-type prostaglandin analogue in aqueous solution by biocompatible cyclodextrin derivatives. Methods. The interaction of an E₁-type prostaglandin, methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)1-butenyl]-5-oxocyclopentyl]-5-thiaheptanoate (MEester) with cyclodextrins (CyDs) was studied by spectroscopies and the solubility method. The degradation of MEester was monitored by high-performance liquid chromatography. Results. ¹H-nuclear magnetic resonance spectroscopic studies indicated that MEester forms 1:1 inclusion complexes with -, -, and -CyDs in solutions, where -CyD interacts with the -side chain containing methyl ester moiety of the drug, whereas - and -CyDs preferentially include around the five-membered ring and both side chains of the drug. Parent -CyD and hydrophilic derivatives, such as 2-hydoxypropyl-- and --CyDs, sulfobutyl ether -CyD (SBE--CyD) and maltosyl -CyD showed higher solubilizing abilities against MEester over parent - and -CyDs. SBE--CyD and 2,6-dimethyl--CyD (DM--CyD) significantly decelerated the degradation of MEester, particularly the base-catalyzed dehydration, in neutral and alkaline solutions, whereas other CyDs accelerated the degradation. The acid-catalyzed degradation of MEester (pH < 3) was decelerated by the addition of CyDs, especially -CyD. Conclusions. SBE--CyD with low hemolytic activity and low toxicity is useful as a pharmaceutical carrier for the preparation of injectable MEester, because of its higher stabilizing and solubilizing effects on MEester. Furthermore, SBE--CyD can be useful as a stabilizing agent for drugs, that are subject to base-catalyzed degradations, probably because of the electric repulsion between anionic charges of the sulfobutyl moiety and catalytic anionic species such as hydroxide ion.     

Evidence for two separate vasoconstriction-mediating nucleotide receptors,both distinct from the P2X-receptor,in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

Summary Uridine 5-triphosphate- (UTP-) and adenosine 5-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5-O-(3-thio)triphosphate (ATPS), and ,-imido adenosine 5-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, , -methylene-ATP and ,-methylene-ATP up to 10^–3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP UMP = CTP; that of the purine nucleotides was: ATPS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = , -methylene-ATP = ,-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to ,-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10^–3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10^–3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10^–3 to 2 × 10^–3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P₂-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P₂-receptor which is distinct from the known P₂-subtypes.Send offprint requests to I. v. Kügelgen at the above address     

Formulation of Vaccine Adjuvant Muramyldipeptides (MDP). 1. Characterization of Amorphous and Crystalline Forms of a Muramyldipeptide Analogue

A relatively nonhygroscopic crystalline form of the glycopeptide, N-acetylmuramyl-L--aminobu-tyryl-D-isoglutamine (I), containing approximately one molecule of water was prepared from amorphous material. The crystalline material, consisting of a mixture of the and anomers, exhibited better physical and chemical stability than the lyophilized amorphous material. The /-anomer ratios of I in both the crystalline and the amorphous state were approximately equal but different from that in solution.     

Effect of prednisolone and ketotifen onβ 2-adrenoceptors in asthmatic patients receivingβ 2-bronchodilators

Summary In 13 patients with bronchial asthma, who were on ₂-adrenergic bronchodilator therapy, the effects of prednisolone and ketotifen on lymphocyte ₂-adrenoceptor density and -responsiveness were investigated. The mean lymphocyte ₂-adrenoceptor density and -responsiveness was significantly lower than in healthy controls, presumably due to the long-term ₂-adrenergic bronchodilator treatment. Both prednisolone 100 mg i.v. and ketotifen 1 mg b.d.p.o. for 6 days rapidly improved lymphocyte ₂-adrenoceptor function. 16 h after prednisolone and about 6 days after the first dose of ketotifen lymphocyte ₂-adrenoceptor density and-responsiveness had risen to values within the range in normal volunteers.The improvement of lymphocyte ₂-adrenoceptor function was accompanied by a significant increase in peak expiratory flow rate before and after inhalation of salbutamol.It is concluded that prednisolone and ketotifen may act beneficially on the recovery of ₂-adrenoceptor responsiveness to ₂-adrenergic bronchodilators in tolerant asthmatic patients.     

Vergleich der enteralen Resorbierbarkeit vonβ-Acetyldigoxin und Digoxin am Meerschweinchen

Zusammenfassung Vergleichende Untersuchungen anwachen Meerschweinchen über die enterale Resorption von Digoxin und -Acetyldigoxin (-AD) ergaben, daß -AD nach peroraler Gabe der LD 50 (2,62 mg/kg) innerhalb von 45–90 min zum Tod der Versuchstiere führt. Für Digoxin liegt die perorale LD 50 wesentlich höher (13,3 mg/kg), und die Zeitspanne bis zum Eintritt des Todes variiert von 1 Std bis zu 4 Tagen.Versuche annarkotisierten Meerschweinchen wiesen darauf hin, daß -Acetyldigoxin im Ileum am besten resorbiert wird; aus der Zeit bis zum Wirkungseintritt sowie den Angaben über die i.v. Toxicität wird geschlossen, daß -Acetyldigoxin enteral rascher und vollständiger resorbiert wird als Digoxin.

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Summary Comparative studies on enteral resorption of digoxine and -acetic digoxine (-AD) were carried out in not narcotized guinea-pigs. -AD, after application of the oral LD 50 (2,62 mg/kg), proved to be lethal within 45–90 min. For digoxine the oral LD 50 is considerably higher (13,3 mg/kg), times of death were found between 1 hour and 4 days.Studies with narcotized guinea-pigs showed, that -AD is optimally resorbed in the ileum. From the time between application and response and from their intravenous toxicity it can be concluded, that the enteral resorption of -acetic digoxine takes place more quickly and more completely than of digoxine.

     

(±)[125Iodo]cyanopindolol,a new ligand for β-adrenoceptors: Identification and quantitation of subclasses of β-adrenoceptors in guinea pig

Summary (±)[¹²⁵Iodo]cyanopindolol (ICYP) is a radioligand which binds with an extraordinarily high affinity and specificity to -adrenoceptors. In contrast to (±)[¹²⁵Iodo]-hydroxybenzylpindolol (IHYP), the new ligand has neither affinity to - nor to 5-HT-receptors. The dissociation constants of ICYP for -adrenoceptors in various tissues range from 27 to 40 pM, thereby exceeding the affinity of IHYP by a factor of 3.ICYP does not discriminate between ₁– and ₂–. Therefore, the densities of the two receptor subtypes can be determined from competition curves of ICYP by drugs previously found to show in vitro selectivity for ₁–adrenoceptors.The guinea pig left ventricle contains only ₁– adrenoceptors, whereas in the lung tissue, the ratio of ₁– to ₂–adrenoceptors is 1 to 4. The calculated affinities of five ₁– selective antagonists for ₁–adrenoceptors were nearly identical in the ventricle and the lung.Kinetic studies of ICYP binding to guinea pig lung membranes indicated that the dissociation reaction consists of two components, a fast process (t _1/2=9 min) and a slower process (t _1/2=8.8 h). A mathematical treatment revealed two possibilities of interpretation: 1. Two forms of the receptor exist which are interconvertible. 2. The (+)- and (–)-enantiomers of ICYP dissociate with different rate constants.The low dissociation constant of ICYP in combination with its high specific radioactivity (2175 Ci mmole^–1) allows binding studies to be carried out with small protein and ligand concentrations, e.g. 3 g protein per assay in guinea pig lung membranes.Abbreviations CYP (±)cyanopindolol, [(±)4-(3-tert-butylamino-2-hydroxypropoxy)-1H-indole-2-carbonitrile] - ICYP (±)-3-[¹²⁵iodo]-cyanopindolol - HYP (±)hydroxybenzylpindolol; IHYP, (±)[¹²⁵iodo]-hydroxybenzylpindolol - ³H-DHA (–)-[³H]dihydroalprenolol Part of this work has been presented in Mainz, March 1980, at the Spring Session of the German Pharmacological Society, and in Brussels, June 1980, at the 4th International Conference on Cyclic Nucleotides     

Differential modulation by muscimol and baclofen on antinociception induced by morphine, β-endorphin,d-Pen2,5-enkephalin and U50,488H administered intracerebroventricularly in the mouse

The present study was designed to investigate the modulatory effects of stimulation of GABA_A and GABA_B receptors at supraspinal sites on antinociception induced by supraspinally administered -, -, -, and -opioid receptor agonists. The effects of the GABA_A and GABA_B receptor agonists, muscimol and baclofen respectively, on the antinociception induced by morphine (a -receptor agonist), -endorphin (an -receptor agonist), D-Pen^2,5-enkephalin (DPDPE, a -receptor agonist) and U50,488H ({trans-3,4-di-chloroN-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeocetamide}; a -receptor agonist) injected intracerebroventricularly (i.c.v.) were studied. The anti-nociception was assayed using the tail-flick and hot-plate tests. Muscimol at doses of 25–200 ng, administered i.c.v. alone did not affect the latencies of tail-flick and hot-plate thresholds, but attenuated dose-dependently the inhibition of the tail-flick and hot-plate responses induced by i.c.v. administered morphine (2 g), -endorphin (1 g), DPDPE (10 g), and U50,488H (60 g). Baclofen (1.25–10 ng) administered i.c.v. alone did not affect the latencies of the tail-flick and hot-plate responses, but attenuated dose-dependently the inhibition of the tail-flick and hot-plate responses induced by -endorphin and U50,488H, without affecting morphine-or DPDPE-induced responses. Our results indicate that activation of GABA_A receptors at the supraspinal sites by i.c.v. injection of muscimol antagonizes antinociception induced by supraspinally administered -, -, -, and -opioid receptor agonists. On the other hand, activation of GABA_B receptors at supraspinal sites by i.c.v. baclofen antagonizes antinociception induced by i.c.v. administered - and -opioid agonists, but not - or -opioid agonists.     

Radioimmunoassay of β-endorphin basal and stimulated levels in extracted rat plasma

Summary A sensitive radioimmunoassay for -endorphin is described. Antibodies against human -endorphin which exhibit a high avidity for the C-terminal of the peptide were raised in rabbits following the injection of thyroglobulin-coupled human -endorphin (h-E) as immunogen. Methionineenkephalin, - -endorphin, as well as ACTH peptides did not cause interference in the radioimmunoassay. -Lipotropin, however, showed a 50% cross-reactivity. The sensitivity of the assay is 25 pg/0.5 ml tube volume for -endorphin. -Endorphin was extracted with a high recovery from the rat plasma using silicic acid and -endorphin levels as low as 100 pg/ml could be measured.Basal levels of -endorphin-like immunoreactivity in plasma of rats were about 400 pg/ml. -Endorphin levels in adrenalectomized rats and in animals chronically treated with the cortisol synthesis blocker metyrapone were found to be markedly increased (about 7-fold). Exposure of the rats to electrically induced foot-shocks caused a similar increase of immunoreactive -endorphin in plasma. A significant increase was also seen after insulin injection.     

The pharmacokinetics of amygdalin

Amygdalin (d-mandelonitrile--d-gentiobioside) is a cyanogenic glycoside claimed to show anti-cancer activity, sold under the incorrect name Laetrile. For a sensible discussion of its alleged activity and its established toxicity it is necessary that its fate in the organism is known. The pharmacokinetics of amygdalin have been investigated in the Beagle dog after both intravenous and oral administration. The excretion of amygdalin has also been studied in the rat. Amygdalin concentrations were determined by high performance liquid chromatography in plasma ultrafiltrate and urine. The pharmacokinetics of amygdalin after intravenous administration were compared with those of diatrizoate, a model substance for extracellular volume and glomerular filtration. The amygdalin clearance is significantly larger than that of diatrizoate. The volumes of distribution of both substance are the same. After oral administration only a few percents of the amygdalin dose are systemically available. A part of the oral dose is recovered from the urine as prunasin (d-mandelonitrile--d-glucoside).Part of this investigation has been presented as a poster at the First European Congress of Biopharmaceutics and Pharmacokinetics at Clermont-Ferrand, April 1st–3rd, 1981.This investigation has been carried out at the request of the Medicines Inspection, Leidschendam, The Netherlands     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

The significance ofβ-adrenoceptor down regulation in the desipramine action in the forced swimming test

Summary The present studies were undertaken to clarify whether central-adrenoceptor down regulation is responsible for the greater effect of chronic treatment with desipramine (DMI) compared with acute treatment in the forced swimming test in rats. Repetitive administration of DMI activated the rat behaviour pattern and consequently reduced the duration of immobility. The degree of activation depended on the length of treatment, i.e. no effect when given in a single dose, moderate effect when given subchronically (3 doses) and marked activation after chronic (31 doses) treatment. Chronic treatment with DMI also produced a decrease in³H-dihydroalprenolol (³H-DHA) binding site in the cerebral cortex. Acute stimulation of brain-adrenoceptors by intracerebroventricular (i.c.v.) isoprenaline significantly, though partially, attenuated the behavioural effect of chronic DMI by ₁-adrenoceptor-related mechanisms. Similarly, chronic i.c.v. co-administration of atenolol or practolol, ₁-adrenoceptor antagonists, together with DMI attenuated both-adrenoceptor down regulation and the behavioural activation by chronic DMI. On the other hand, chronic i.c.v administration of isoprenaline, supposedly leading to down regulation of-adrenoceptors, facilitated the activating behavioural effect of DMI, as a single dose became effective. Changes, however, in³H-DHA binding parameters in the cerebral cortex were not observed after chronic isoprenaline. These results suggest that down regulation of-adrenoceptors in brain is reponsible, at least in part, for the marked activatory effect of chronic DMI in the forced swimming test, possibly by reducing an inhibitory function of ₁-adrenoceptor mediated mechanisms.