Usefulness of the Kohlrausch-Williams-Watts Stretched Exponential Function to Describe Protein Aggregation in Lyophilized Formulations and the Temperature Dependence Near the Glass Transition Temperature

Purpose. We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing mean aggregation time (_a) and stretched exponential constant (_a) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate () varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. Methods. Protein aggregation in lyophilized bovine serum -globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80°C, was followed by size-exclusion chromatography. Results. Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The _a and _a parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the _a parameter of these formulations exhibited continuous temperature dependence. The parameter , as calculated from _a and _a, reflected differences in values between the two excipients. Conclusions. The results indicate that the parameter _a is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in _a were observed, knowledge regarding the _aand _a parameters allows the rate of protein aggregation to be predicted. The parameter was found to be useful in comparing the protein aggregation behavior of formulations having different _a and _a values.     

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.     

Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors

Summary Effects of dynorphin-(1–13), Leu⁵-enkephalin,d-Ala²,d-Leu⁵-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S₁, S₂). Drugs were administered before S₂. Bremazocine 10 g/kg+2g/kg/h and 100 g/kg+20 g/kg/h, dynorphin 1 and 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 10 and 30 g/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 g/kg/min) was given twice for 3 min each (N₁, N₂). Drugs were administered before N₂. Bremazocine 100 g/kg+20 g/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 30 g/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 g/kg/min and DADLE 10g/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 g/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 g/kg/min or DADLE 10 g/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure. They act at peripheral sites, presumably prejunctional opioid receptors at postganglionic sympathetic axons. Dynorphin on the one hand, and Leu⁵-enkephalin and DADLE on the other hand, appear to act at different receptors, dynorphin probably at a - and DADLE and Leu⁵-enkephalin at a -receptor.     

The Solution Conformations of Lidocaine Analogues

IR and ¹H NMR studies in CDCl₃ and CCl₄ of a series of tertiary aminoxylidides with the amino group in the 2 to 6 position of the acyl chain are described. Lidocaine, diethylaminoaceto-2,6-xylidide, forms an intramolecular five-membered ring hydrogen-bonded monomer at all concentrations in both solvents. -Diethyl-amino-propiono-2,6-xylidide forms an intramolecular six-membered ring hydrogen-bonded monomer in CDCl₃ and CCl₄ but a trans intermolecularly associated species is the major form present at high concentrations in CCl₄. The longer-chain homologues are mixtures of nonassociated trans and cis monomers at low concentrations but associated trans forms predominate at high concentrations. Evidence for the presence of a hydrogen-bonded seven-membered ring intramolecular monomer in CDCl₃ for -diethylaminobutyro-2,6-xylidide is presented. The relationship between the molecular conformation and the partition coefficient is discussed.     

Isoprenaline-induced increase in mRNA levels of inhibitory G-protein α-subunits in rat heart

Summary Long-term -adrenergic stimulation has been shown to desensitize the -adrenoceptor/adenylyl cyclase signalling pathway at both the receptor and the G-protein level. To further elucidate the cellular mechanism of G-protein regulation we investigated the influence of prolonged infusion of isoprenaline (2.4 mg/kg·d) on myocardial mRNA levels of different G-protein -subunits in rats. For comparison rats were treated with triiodothyronine (T₃; 0.5 mg/kg·d) which induces cardiac hypertrophy like isoprenaline but has different effects on the adenylyl cyclase system. Isoprenaline- and T₃-treated animals developed an increase in heart/body weight ratio of 41±3% and 27±4%, respectively (P<0.05). Isoprenaline increased myocardial total RNA concentration by 39±6% (P<0.05). Hybridization with ³²P-labeled rat cDNAs demonstrated an expression rank order of G_s-mRNA>G_i-2-mRNA>G_i–3-mRNA and no detectable expression of G_i–1-mRNA in rat myocardium. mRNA levels of G_s G_i–2 and G_i–3 were 36.9±1.28, 10.7±1.07 and 3.7±0.19 pg/g total RNA, respectively. Isoprenaline increased G_i–2 ^– and G_i–3-mRNA concentrations per g total RNA by 49±18% and 27±710, respectively (P<0.05). This effect was abolished by simultaneously administered propranolol (9.9 mg/kg·d), indicating a,-adrenoceptor-mediated mechanism. In contrast, T₃-induced cardiac hypertrophy was not accompanied by changes in G_i-mRNA expression. G_sa-mRNA levels were unaffected by either treatment.In conclusion, long-term stimulation with isoprenaline in vivo induces a -adrenoceptor-mediated increase in myocardial G_i–2 ^– and G_i–3-mRNA without affecting G_s-mRNA. These results suggest that similar increases in myocardial G_i–2-mRNA in end-stage human heart failure may be at least partly explained by increased -adrenergic stimulation due to increased sympathetic activity.Parts of this work were presented at the wintermeeting of the Deutsche Gesellschaft fur Pharmakologie und Toxikologie in Hannover, 1990 (Eschenhagen et al.), Naunyn-Schmiedebergs Arch Pharmacol 342 (Suppl):R8. The work was supported by the Deutsche Forschungsgemcinschaft Send offprint requests to: T. Eschenhagen at the above address     

Cyclic nucleotide phosphodiesterases of human and rat gastric mucosa

Summary Cyclic adenosine-3,5-monophosphate (cAMP) phosphodiesterases (PDE) were partially purified from human and rat gastric mucosa. Drugs known to affect the cyclic nucleotide system and/or gastric secretion were tested for effects on the PDE-activities from both species.In rat gastric mucosa PDE-activity can be detected in the 100 000×g sediment (K _m =8.3 M; V _max=3.2 nmoles cAMP hydrolyzed/mg protein x min) and the cytoplasma (K _m =5.6 M; V _max=2.6 nmoles cAMP hydrolyzed/mg protein x min).The most effective inhibitors of the particle-bound activity are papaverine (K _i =4 M, non-competitive) and 3-isobutyl-1-methylxanthine (K _i=14 M, competitive). There was only a modest competitive inhibition by theophylline (K _i =495 M). PDE-activity in the cytoplasma was inhibited competitively by these three drugs (papaverine: K _i =6.5 M; 3-isobutyl-1-methylxanthine: K _i =37 M; theophylline: K _i =152 M.In human gastric mucosa PDE-activity can be detected in the particular fraction (K _m =23.9 M; V _max=1.2 nmoles cAMP hydrolyzed/mg protein x min), and the soluble fraction (K _m =12.1 M; V _max=2.4 nmoles cAMP hydrolyzed/mg protein x min).PDE-activity in the 100 000×g sediment was inhibited by papaverine (K _i =5.6 M, non-competitive), 3-isobutyl-1-methylxanthine (K _i =16 M, non-competitive), theophylline (K _i =165 M, non-competitive), and N₆-2-O-dibutyryl-cAMP (K _i =746 M, competitive).Inhibition in the 100 000×g supernatant was noncompetitive with 3-isobutyl-1-methylxanthine (K _i =7.1 M and papaverine (K _i =8.5 M), but competitive with N₆-2-O-dibutyryl-cAMP (K _i =170 M), and theophylline (K _i =225 M). This study indicates that PDE-activities of the two species are qualitatively similar, but quantitative differences exist.     

β 1- andβ 2-adrenoceptor binding and functional response in right and left atria of rat heart

Summary The properties of ₁- and ₂-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to-adrenoceptor agonists were examined. [¹²⁵I](-)-pindolol (¹²⁵IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek ₁'s for association in right and left atria were 6.5×10⁹ l/mol-min and 2.3×10⁹ l/mol-min respectively, while thek _–1's for dissociation were 0.20 min^–1 and 0.17 min^–1. The kinetically determinedK _D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK _D's determined from Scatchard analysis of saturation isotherms of specific¹²⁵IPIN binding. Inhibition of¹²⁵IPIN binding by-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition by ₁- and ₂-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by ₁-selective (practolol, atenolol and metoprolol) and ₂-selective (ICI 118,551) antagonists gave estimates of the proportion of ₁- and ₂-adrenoceptors present in rat atria. Right atria contained 67±4.2% ₂-adrenoceptors and 33±4.2% ₂-adrenoceptor, while left atria contained 67±2.8% ₁- and 33±2.8% ₂-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by-adrenoceptor agonists were also measured. pA₂ values for non-subtype selective-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK _D values determined for specific¹²⁵IPIN binding. pA₂ values for ₁- and ₂-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK _D values of these drugs in binding to ₁-adrenoceptors, but not with the pK _D values in binding to ₂-adrenoceptors. Dose-response curves for stimulation of both rate and force by the ₂-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of ₁-adrenoceptors with metoprolol than by selective blockade of ₂-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating ₁-adrenoceptors. These results suggest that ₁- and ₂-adrenoceptors coexist in both left and right atria of rat heart in approximately a 21 ratio, however only ₁-adrenoceptors mediate the chronotropic and inotropic effects of-adrenoceptor agonists.Supported by a grant from the American Heart Association — Georgia Affiliate     

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.     

Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

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.     

Presynaptic α2-autoreceptors in mouse heart atria: evidence for the α2D subtype

Presynaptic ₂-autoreceptors in mouse atria were characterized in terms of the _2A, _2B, _2C and _2D subtypes. Segments of the atria were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC_30% values, i.e. concentrations that increased previously autoinhibited release of ³H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pK_d values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s).The pK_d values correlated well with the pEC_30% values (r = 0.98; P<0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native ₂ radioligand binding sites, (2) radioligand binding sites in COS cells transfected with ₂ subtype genes, and (3) previously classified presynaptic ₂-autoreceptors — all taken from the literature — indicated that the mouse atrial autoreceptors corresponded to the _2D subtype. For example, the pK_d values at mouse atrial autoreceptors correlated closely with pK_d values at native _2D binding sites in the bovine pineal gland (r = 0.96; P<0.001); with pK_d values at _2D binding sites in COS cells transfected with the rat _2D gene (r= 0.85; P<0.01); and with pK_d values at guinea-pig cerebral and atrial and mouse cerebral _2D-autoreceptors (r=0.96–0.98; P<0.001). The antagonist pK_d values at mouse atrial autoreceptors correlated less with pK_d values at _2A, _2B, and _2C sites.It is concluded that the presynaptic ₂-autoreceptors in mouse atria are _2D This identification supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D pair of orthologous ₂-adrenoceptors.     

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

Summary Four groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (K _I, nM) were determined against the binding of the -ligand, [³H]-[d-Ala ² ,MePhe ⁴ , Gly-ol⁵]enkephalin, of the -ligand, [³H]-[d-Ala ² ,d-Leu ⁵]enkephalin and of the -ligand, [³H]-(±)-ethylketazocine after suppression of - and -binding by 100 nM of the unlabelled -ligand and 100 nM of the unlabelled -ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to -binding but phenazocine and particularly etorphine had also high affinities to the - and -binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, -binding and -binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the -binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective -antagonist, Mr 2266 having high affinities to the - and -binding sites and diprenorphine having considerable affinities to the -, - and -binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the - and -binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.     

Quantitative Structure–Pharmacokinetics Relationships: II. A Mechanistically Based Model to Evaluate the Relationship Between Tissue Distribution Parameters and Compound Lipophilicity

The tissue-to-unbound plasma distribution coefficients (Kpus) of 14 rat tissues after iv administration of nine 5-n-alkyl-5-ethyl barbituric acids, determined in a previous study, were used to identify a model of the relationship between tissue distribution and lipophilicity of the homologs, expressed in terms of their octanol to water partition ratio, P. Based on mechanistic considerations and assumptions, the parameter model was expressed asKpu= f_W,[l + a(nP_l,)P^b], where f_W, is the tissue water content, (nP_l, ) is the binding capacity of the tissue, n is the number of the binding sites, a and b are the parameters of the relationship Ka = aP^b and Ka is the binding association constant of each tissue. The parameter model was linearized and fitted to the predetermined Kpu values, yielding correlation coefficients ranging between .940 and .997. The predictive performance of the parameter model was evaluated using a leave-one-out procedure with subsequent computation of the mean prediction error (ME = measurement of the prediction bias) and the square root of the mean squared prediction error (RMSE = measurement of the prediction accuracy). The ME varied between –22.48 and 61.14%, indicating a slight tendency for overpredicting. The RMSE was between 24.73 and 102% for the individual tissues across the different homologs; and between 28.33 and 85.2% for the individual homologs across the different tissues. The apparently high Kpu prediction errors, when translated through the low sensitivity of the barbiturate whole-body physiologically based pharmacokinetic model, established previously, leads to predicted tissue concentration–time profiles within 5 to 20% of the original ones. Therefore, it is concluded, that the identified mechanistically based model is a good predictor of the tissue-to-unbound Kpus in the rat tissues.     

EEG,heart rate and mood change (“high”) after Cannabis

Fourteen experienced marijuana users smoked marijuana, hashish, ⁹-THC, and placebo. EEG, ECG and ratings of subjective feelings of high and pleasantness were recorded. EEGs were processed by period analysis.In EEG, marijuana and ⁹-THC increased the amount of alpha activity, and the three Cannabis preparations decreased the amount of beta activity. The average frequency of alpha activity was decreased by 0.15–0.20 c/sec after marijuana, hashish and ⁹-THC. The peak EEG effect occurred during the first 10 min after smoking; most of the changes disappeared after 40 min. Heart rate was increased by all the three drugs, and the effect persisted for the entire observation period (50 min).Feelings of high were elicited by each Cannabis preparation. This was not true of the pleasantness of the experience: only marijuana and hashish were perceived as more pleasant than placebo. Intensity of high increased with the amount of alpha activity, and decreased with the average alpha frequency. Pleasantness was unrelated to the EEG.The high showed a linear increase with heart rate, whereas pleasantness of the experience was an inverted U-function of heart rate.Aided, in part, by MH 13358, 18172 and by a contract with the New York State Narcotic Addiction Control Commission.     

Effect of epinine on tension of human renal arteries

Background: The present study aimed to characterize the effects of epinine, the active metabolite of ibopamine on tension development in human renal arteries.Methods and results: Experiments were performed on isolated human renal arteries rings obtained during surgery due to kidney tumors (n = 12). Epinine concentration-dependently relaxed isolated precontracted (PGF₂) human renal artery rings (P < 0.05) in the presence of phentolamine, as effectively (epinine – 30 +/– 4 mN, dopamine – 31 +/– 5 mN) and with the same potency as dopamine (epinine EC₅₀ 0.7 mol/l (0.4–1.2 mol/l), dopamine 0.5 mol/l (0.2–1.7 mol/l)). This effect was antagonized by the specific D₁-receptor-antagonist SCH 23390. Effective -adrenoceptor antagonistic concentrations of propranolol did not affect epinine-induced vasorelaxation. In the absence of -and -adrenoceptor-antagonists the potency of epinine to contract renal artery rings was significantly higher compared to dopamine indicating a higher affinity of epinine to -adrenoceptors.Conclusion: The present study provides evidence for direct vasodilatory effects of epinine via activation of D₁-receptors on human renal arteries.     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

Stimulation of prostaglandin E2-synthesis by noradrenaline in primary cell cultures from rabbit splenic pulpa is mediated by atypical α-adrenoceptors

Summary In primary cell cultures originating from rabbit splenic pulpa the effects of various adrenoceptor agonists on prostaglandin (PG)-synthesis were studied. The cells-microscopically identified as fibroblasts-released PGs into the medium: especially PGE₂ besides small amounts of PGF₂ and PGD₂.Noradrenaline increased dose-dependently the amount of PGs released into the medium. Besides noradrenaline, only the catecholamines adrenaline and -methylnoradrenaline strongly activated PG-synthesis. Other -adrenoceptor agonists like the phenylethylamine and imidazoline derivatives were only weak agonists or completely ineffective. All adrenoceptor agonists without intrinsic activity in these cells antagonized the noradrenaline effect on PG-synthesis, the imidazolines being more potent antagonists than the phenylethylamines.The -adrenoceptor agonist isoprenaline stimulated PG-synthesis at high concentrations only. The effects of both noradrenaline and isoprenaline were inhibited by low concentrations of phentolamine and phenoxybenzamine, but not by propranolol. The preferential ₂-adrenoceptor antagonists yohimbine and rauwolscine were about 50 times more potent in blocking the noradrenaline effect on PG-synthesis than the more ₁-specific antagonist corynanthine. However, prazosin, another ₁-adrenoceptor antagonist, was about equipotent with yohimbine.It is concluded that noradrenaline elicits PG-synthesis in rabbit splenic fibroblasts via -adrenoceptor stimulation. The -adrenoceptor involved has properties which are different from those reported so far for ₁- or ₁-adrenoceptors.     

Analysis of the Compression Mechanics of Pharmaceutical Agglomerates of Different Porosity and Composition Using the Adams and Kawakita Equations

Purpose. To analyze the mechanics of some pharmaceuticalagglomerates during uniaxial confined compression by using compressionparameters derived from the Heckel, Kawakita and Adams equations, and tostudy the influence of these compression parameters on the tablet-formingability of agglomerates. Methods. Force and displacement data sampled during in-diecompression of agglomerates was used to calculate compression parametersaccording to the Heckel ( _y ), Kawakita(1/b and a), and Adams (₀)equations. Mechanical strength of single agglomerates as well as the airpermeability and tensile strength of tablets prepared from them were alsodetermined. Results. _y from the Heckelequation did not differ between agglomerates of different porosity. Both1/b and ₀ varied with agglomerate porosityand composition. These two compression parameters were linearly related toeach other. No general correlation was found between 1/b and₀ and the strength of single agglomerates. The twoparameters were related to the intergranular pore structure and tensilestrength of tablets formed from the agglomerates. Conclusions. 1/b and ₀ maybe interpreted as measures of the agglomerate shear strength during uniaxialconfined compression, and as such they may be used as indicators of thetabletting performance of the agglomerates.     

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [³H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline (0.05 M), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 LM-3 mM), 2-methylthio-ATP (3–100 M), as well as DMPP (10 and 100 M) induced a significant overflow of tritium. The EC₅₀-value of ATP was 20 M. Both the ATP-induced and the DMPP-induced tritium overflow was Ca²⁺-dependent and sensitive to tetrodotoxin (0.3 M) and -conotoxin (0.1 M); in addition, it was inhibited by the ₂-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 M). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P₂-purinoceptor antagonists suramin (300 M) and reactive blue 2 (3 M); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 M) and markedly inhibited by hexamethonium (100 M). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [³H]-choline.The results suggest that ATP at molar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.     

P2-purinoceptor antagonists discriminate three contraction-mediating receptors for ATP in rat vas deferens

The sites of action at which ATP elicits contraction of the rat vas deferens were studied by means of the P₂-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), suramin and reactive blue 2.Increasing concentrations of PPADS (up to 1 mM), suramin (up to 1 mM) and reactive blue 2 (up to 320 M) reduced and eventually abolished contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP 3 M with IC₅₀ values of 2.1, 10.1 and 27.0 M, respectively. In contrast, PPADS and suramin caused only a partial inhibition of contractions elicited by ATP 1 mM, maximal reduction by about 40%, IC₅₀ values 1.3 and 5.0 M, respectively; reactive blue 2 did not change ATP-induced contractions. In tissues exposed to PPADS 320 M throughout, increasing concentrations of reactive blue 2 or suramin decreased contractions elicited by ATP 1 MM, IC₅₀ values 2.6 and 14.5 M, respectively. In tissues exposed to suramin 320 M throughout, increasing concentrations of PPADS decreased contractions elicited by ATP 1 mM, IC₅₀ 37.9 M, whereas reactive blue 2 slightly enhanced these contractions. In tissues exposed to reactive blue 2 100 M throughout, increasing concentrations of PPADS reduced contractions elicited by ATP 1 MM, IC₅₀ 26.6 M, whereas suramin caused no change. Pre-exposure to ,-methylene ATP 1 M to desensitize P_2X-purinoceptors reduced the response to ATP 1 mM by 91% in otherwise untreated tissues, but did not reduce the response to ATP 1 mM in tissues exposed throughout to PPADS 320 M, suramin 320 M or reactive blue 2 100 M. Neither PPADS nor suramin nor reactive blue 2 altered contractions elicited by KCl 35 mM. The P₁-purinoceptor antagonist 8-(p-sulfophenyl) theophylline 100 M did not change contractions elicited by ,-methylene ATP 3 M or ATP 1 mM.It is concluded that ATP 1 mM elicits contraction of the rat vas deferens through three sites: P_2X-purinoceptors which are blocked by PPADS, suramin and reactive blue 2; P_2Y-purinoceptors blocked by reactive blue 2 and suramin but resistant to PPADS; and non-P_2X-non-P_2Y-purinoceptors blocked by PPADS but resistant to inhibition by suramin and reactive blue 2. Correspondence to: R. Bültmann at the above address