Nevena Pencheva

Aminoderivatives of cycloalkanespirohydantoins: synthesis and biological activity

3-Aminocycloalkanespiro-5-hydantoins were synthesized and their biological activity was studied. In contrast to hydantoins, these compounds failed to induce either anticonvulsive effects in the central nervous system or inhibitory effects on cholinergic contractions in the enteric nervous system. However, they exerted well pronounced, atropinsensitive, contractile effects on the guinea-pig ileum longitudinal muscle preparations. Structure-activity relationships established allow the assumption that: (i) the reduction of the ring size in the molecule of the spirohydantoins leads to an increase in the potency of the respective analogue to induce contractile effect; (ii) the introduction of -NH2 in position 3 increases the ability of all the compounds studied to exert contractions; (iii) the enlargement of the ring leads to: (1) an increase of the degree of desensitization of the preparations; and (2) a decrease (except 1a) of the potency of the analogues to exert contractile effects.

Activity profiles of dalargin and its analogues in μ‐, δ‐ and κ‐opioid receptor selective bioassays

To elucidate the structural features ensuring action of [D‐Ala2, Leu5]‐enkephalyl‐Arg (dalargin), a series of dalargin analogues were tested for their effectiveness in depressing electrically‐evoked contractions of the guinea‐pig myenteric plexus‐longitudinal muscle preparations (μ‐ and κ‐opioid receptors) and the vasa deferentia of the hamster (δ‐opioid receptors), mouse (μ‐, δ‐ and κ‐opioid receptors), rat (similar to μ‐opioid receptors) and rabbit (κ‐opioid receptors). The naloxone KB values in the myenteric plexus were also obtained. [L‐Ala2]‐dalargin was 19 times less potent than dalargin, and its pharmacological activity was peptidase‐sensitive. The ratio of δ‐activity to μ‐activity for [L‐Ala2]‐dalargin was 6.78, and KB was 7.9 nM. This emphasizes the role that D‐configuration of Ala2 plays in determining the active folding of dalargin molecule as well as in conferring resistance to peptidases. [Met5]‐dalargin was equipotent to dalargin in the myenteric plexus, but was more potent in the vasa deferentia of hamster and mouse (KB=5.5 nM). Leu5 and the interdependence of Leu5 and D‐Ala2 are of importance for the selectivity of dalargin for μ‐opioid receptors. Dalarginamide was more potent and selective for μ‐opioid receptors than dalargin, whilst dalarginethylamide, though equipotent to dalarginamide in the myenteric plexus, was more potent at δ‐opioid receptors (KB=5.0 nM). [D‐Phe4]‐dalarginamide and N‐Me‐[D‐Phe4]‐dalarginamide were inactive indicating the contribution of L‐configuration of Phe4 to the pharmacological potency of dalargin. N‐Me‐[L‐Phe4]‐dalarginamide possessed the highest potency and selectivity for μ‐opioid receptors (the ratio of δ‐activity to μ‐activity was 0.00053; KB=2.6 nM). The CONH2 terminus combined with the N‐methylation of L‐Phe4 increased the potency and selectivity of dalargin for μ‐opioid receptors.

GABAA and GABAB receptor-mediated effects on the spontaneous activity of the longitudinal layer in cat terminal ileum

1. GABA and GABAergic agonists-muscimol and (+/-)baclofen changed the spontaneous mechanical activity in isolated cat terminal ileum. 2. GABA at doses ranging from 5 microM to 2 mM produced concentration-dependent biphasic responses consisting of a transient relaxation followed by contractions with a tonic and a phasic components. 3. The GABA-induced relaxation was sensitive to bicuculline and picrotoxinin and was mimicked by muscimol, while the GABA-induced contractions were insensitive to bicuculline and picrotoxinin and were mimicked by (+/-)baclofen. Specific cross desensitization occurred between GABA and muscimol or GABA and (+/-)baclofen. 4. The bicuculline-sensitive relaxation induced by GABA and muscimol was abolished by atropine or tetrodotoxin (TTX), while the bicuculline-insensitive contractions induced by GABA and (+/-)baclofen were not antagonized by atropine or TTX, though they were slightly suppressed. 5. The GABA effects in the longitudinal layer of cat terminal ileum were mediated by the following receptors: -GABAA prejunctional receptors whose activation causes relaxation, probably through an inhibitory action on cholinergic neurons; -GABAB prejunctional receptors whose activation cause contractions; -GABAB postjunctional receptors located on the smooth muscle membrane whose activation induces tonic and phasic contractions.

Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine

Naloxone-dependent effects of Met-enkephalin (10(-8) M) on the spontaneous and electrically induced mechanical activities were studied in longitudinal and circular preparations isolated from the cat duodenum, jejunum and ileum. Met-Enkephalin changed the spontaneous activity of all preparations tested with the exception of the circular preparations from the ileum. Met-Enkephalin-induced responses of the longitudinal preparations from the ileum were abolished by treatment with tetrodotoxin (10(-7) M), while the responses of both longitudinal and circular preparations from the duodenum and jejunum were only partially depressed, being resistant to tetrodotoxin components. The latter were most pronounced in the duodenum. The neurogenic electrically induced (0.5 msec, 5 Hz, 150 pulses) responses of all the preparations consisted mainly of contractile components which were significantly and naloxone-dependently reduced by Met-enkephalin (10(-8) M). The contractile components of the responses, which were reduced by Met-enkephalin, were entirely abolished by atropine (3 x 10(-6) M). Both Met-enkephalin and atropine inhibitory effects on the neurogenic responses were more pronounced in the ileum. Met-Enkephalin was found in nerve fibers of the myenteric plexus distributed mainly among the circular muscle. Single immunoreactive nerve fibers were observed in the longitudinal muscle layer of the duodenum but not in the jejunum and ileum. The distribution of Met-enkephalin-like immunoreactivity along the small intestine did not show significant differences among the three intestinal regions tested. The results obtained suggest that Met-enkephalin can modulate the mechanical activity of the cat small intestine, inhibiting cholinergic transmission and/or activating smooth muscle opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the contractile responses to carbachol and in the inhibitory effects of verapamil and nitrendipine on isolated smooth muscle preparations from rats subchronically exposed to Co2+ and Ni2+

Male Wistar rats were exposed to subtoxic doses of Co2+ or Ni2+, receiving Co(NO3)2 or NiSO4 with drinking water for 30 days. No significant differences in the body weight and no visible changes in the behaviour of the controls and experimental animals were established. Cumulative concentration-effect curves for carbachol were obtained in ileum and trachea isolated from control and Co2+- or Ni2+-treated rats. The effect of the Ca2+ antagonists on the carbachol-induced contractions was studied by adding increasing concentrations of verapamil or nitrendipine to the bath solution 20 min prior to carbachol. The results showed that exposure of rats to subtoxic doses of Co(NO3)2 or NiSO4 altered the contractile responses to carbachol. The changes in the pD2 values and the shift to the left of the concentration-effect curves suggest a higher sensitivity to carbachol in preparations from the ileum of Co2+- or Ni2+-exposed rats. The tracheal strips isolated from control and heavy metal-treated rats showed a less potent sensitiveness to carbachol as compared to the ileal segments. An opposite tendency for decreased cholinergic reactivity was observed in tracheal strips from Co2+- and Ni2+-treated animals. The inhibitory effect of the Ca2+-antagonists on the contractility of ileal preparations from Co2+-treated rats increased at all concentrations of verapamil and at the highest concentration of nitrendipine, but decreased at lower concentrations of nitrendipine. The effect of verapamil on the preparations from Ni2+-exposed rats was unchanged or even decreased at higher verapamil concentrations. The inhibitory effect of nitrendipine on preparations from Ni2+-exposed rats decreased at the lowest concentration but increased at the highest concentration of the blocker. In Co2+- or Ni2+-treated tracheal preparations verapamil inhibited the contractions induced by low and medium concentrations of carbachol but increased the maximal contractile responses to high concentrations of carbachol.

Biphasic GABA-A receptor-mediated effect on the spontaneous activity of the circular layer in cat terminal ileum

1. The GABA and GABA-A receptor agonist muscimol changed the spontaneous mechanical activity of a circular layer isolated from cat terminal ileum, while the selective GABA-B receptor agonist (+/-)baclofen had no effect. 2. GABA at doses ranging from 1 microM to 2 mM elicited concentration-dependent biphasic responses which consisted of a relaxation followed by contraction, with a tonic and a phasic component. The EC50 values, calculated at 95% confidence limits (CL), were 94.9 microM (83.5-109.8 microM) and 66.0 microM (51.2-75.5 microM) for the relaxation and contractile phases, respectively. 3. The GABA-induced biphasic responses were sensitive to bicuculline and picrotoxinin and were entirely mimicked by muscimol. Bicuculline competitively antagonized the effects of GABA and gave closely similar pA2 values for both phases of these responses--inhibitory and stimulatory. Cross-desensitization occurred only between GABA and muscimol and not between (+/-)baclofen and GABA, or (+/-)baclofen and muscimol. 4. Both bicuculline-sensitive phases evoked by GABA and muscimol were abolished by tetrodotoxin or atropine, but were unaffected by guanethidine or naloxone. 5. The present results suggested that the biphasic GABA effect on the mechanical activity of the circular layer in cat terminal ileum was mediated by prejunctional GABA-A receptors, most probably through an action on the cholinergic pathway.

Dalargin and [Cys-(O2NH2)]2 analogues of enkephalins and their selectivity for μ opioid receptors

1. Effects of the enkephalins Met-enk (M) and Leu-enk (L), of two newly synthesized analogues--[Cys-(O2NH2)]2-Met-enk (CM) and [Cys-(O2NH2)]2-Leu-enk (CL)--and of a hexapeptide--D-Ala2-Leu5-Arg6 (Dalargin; DL) on the spontaneous and electrically stimulated activity were examined with respect to their selectivity for the mu opioid receptors in the longitudinal layer of guinea pig ileum. 2. M and CM exerted relaxing and contractile effects on the spontaneous contractile activity while L, CL and DL produced only relaxation. The order of potency towards the relaxatory phase was DL > M > CM > L > CL and towards the contractile phase CM > M. 3. The effects of enkephalins on the spontaneous activity were naloxone and TTX sensitive except for the contractile phase of M and CM which persisted in the presence of TTX. NO was not involved in the neurotransmission of the relaxatory responses, while the blockade of alpha and beta adrenoceptors showed the participation of adrenergic mechanisms. Relaxation and contraction induced by enkephalins could not be directly attributed to cholinergic neurotransmission. 4. The naloxone-sensitive and concentration-dependent inhibitory effects of enkephalins and their analogues on the electrically stimulated cholinergic contractions were established. The order of the relative potency of opioids was: DL-3.8; M-1.0; L-0.4; CM-0.01; CL-0.005. 5. These data indicated that the D-Ala2 substitution and lengthening of the peptide chain by Arg6 in the molecule of L increased the potency at the mu opiate receptors, while the substitution in position 2 with Cys-(O2NH2) in the molecule of M and L yielded a less potent and selective mu agonists.

GABAB receptor-mediated contractile effects resistant to tetrodotoxin in isolated cat ileum

The effects of gamma-aminobutyric acid (GABA) and GABAergic drugs were studied on longitudinal strips from cat terminal ileum prepared after removing the myenteric plexus. GABA and baclofen exerted concentration-dependent contractile effects. Muscimol was ineffective, and bicuculline did not antagonize the effect of GABA. The complete elimination of the neural input to the smooth muscle cells by tetrodotoxin failed to prevent the action of GABA and baclofen. Pharmacological analyses of the effects indicated the existence of GABAB receptors on the smooth muscle cells in the longitudinal layer of cat terminal ileum.

[Cys(O2NH2)2]enkephalin analogues and dalargin: Selectivity for δ-opioid receptors

Abstract To investigate the structure-activity relationships for potent and selective action of enkephalins at the δ-opioid receptors, two newly synthesized analogues, [Cys(O 2 NH 2 ) 2 ,Leu 5 ]enkephalin and [Cys(O 2 NH 2 ) 2 ,Met 5 ]enkephalin and the hexapeptide [ d -Ala 2 ,Leu 5 ]enkephalyl-Arg (dalargin) were tested and compared with [Leu 5 ]enkephalin and [Met 5 ]enkephalin, for their effectiveness to inhibit electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective δ-opioid receptors) and the guinea pig ileum (μ- and κ-opioid receptors). The mouse vas deferens assays included evaluation of the effects of opioid agonists on the first, purinergic, and the second, adrenergic, components of electrically evoked biphasic responses (10 Hz and 20 Hz) and on ATP- or noradrenaline-evoked, tetrodotoxin-resistant responses. The opioids tested inhibited in a similar manner: (i) the purinergic and the adrenergic components of the electrically evoked contractions; and (ii) the ATP- and noradrenaline-induced postjunctional responses of the mouse vas deferens. Extremely low IC 50 values (of 2–5 orders) were found for [Cys(O 2 NH 2 ) 2 ,Leu 5 ]enkephalin, whose relative potency was between 239 and 1316 times higher than that of [Leu 5 ]enkephalin. The order of potency for the other peptides in this tissue was: [Cys(O 2 NH 2 ) 2 ,Met 5 ]enkephalin > [Leu 5 ]enkephalin > dalargin > [Met 5 ]enkephalin. The highest IC 50 values in the guinea pig ileum assays, indicating the lowest affinity for μ-/κ-opioid receptors, were obtained for the cysteine sulfonamide analogues, while dalargin showed a potency four times higher than that of [Met 5 ]enkephalin. The order of potency in this tissue was: dalargin > [Met 5 ]enkephalin > [Leu 5 ]enkephalin > [Cys(O 2 NH 2 ) 2 ,Met 5 ]enkephalin > [Cys(O 2 NH 2 ) 2 ,Leu 5 ]enkephalin. The ratio, IC 50 in guinea pig ileum: IC 50 in mouse vas deferens, indicating selectivity of the respective peptide for δ-opioid receptors, was extremely high for [Cys(O 2 NH 2 ) 2 ,Leu 5 ]enkephalin and especially for the adrenergic component of the responses. This ratio for [Cys(O 2 NH 2 ) 2 ,Met 5 ]enkephalin was higher than the ratios for dalargin, [Leu 5 ]enkephalin and [Met 5 ]enkephalin, which were about 3 orders of magnitude lower. The results suggest that incorporation of hydrophilic Cys(O 2 NH 2 ) in the enkephalin molecule greatly increases the potency and selectivity of the analogues at δ-opioid receptors, while both d -Ala 2 substitution and lengthening of the peptide chain by Arg 6 in the molecule of [Leu 5 ]enkephalin decrease them.

Opioid effects of short enkephalin fragments containing the Gly-Phe sequence on contractile responses of guinea pig ileum

1. Effects of the fragments H-Gly-Phe-OH, H-Gly-Phe-NH2 or H-Gly-Phe-OMe on the electrically stimulated cholinergic contractions of the longitudinal layer in isolated guinea pig ileum and on the Morphine-, Met-enkephalin- or Leu-enkephalin-induced inhibition of these contractions were analyzed for opioid activity in respect to Gly-Phe sequence. 2. H-Gly-Phe-OH or H-Gly-Phe-NH2 exerted no effects, while H-Gly-Phe-OMe applied cumulatively (1 pM-1 mM), concentration-dependently reduced the contractions to electrical stimulation, the IC50 value being 1.96 +/- 0.06 microM. Naloxone (1-5 microM) did not reverse the H-Gly-Phe-OMe effects. 3. H-Gly-Phe-OMe at single concentrations (1-10 microM) significantly decreased the maximum inhibition produced by cumulatively added (0.1 nM-100 microM) morphine, Met-enkephalin or Leu-enkephalin. The regression lines for the opioids were shifted to the right but not always in a parallel fashion; the IC50 values were higher as compared to the controls and lower as compared to the IC50 values after naloxone. 4. The pA2 value for H-Gly-Phe-OMe with respect to morphine (6.43 +/- 0.14) did not differ from that to Met-enkephalin (6.68 +/- 0.35) or Leu-enkephalin (9.06 +/- 0.98); the slope of the pA2 plot to morphine was near unity. 5. These data indicated that H-Gly-Phe-OMe exerted predominantly a potent non-competitive opioid antagonistic effect suggesting that short enkephalin fragments containing the Gly-Phe sequence might possess an opioid activity.