Choline: high-affinity uptake by rat brain synaptosomes.

Synaptosomes from rat brain accumulate choline by two kinetically distinct processes, a high-affinity uptake system [Michaelis constant (K(m)) = 1 x 10(-6)M], and a low-affinity system (K(m) = 9 x 10(-5)M). The high-affinity uptake system requires sodium, and is associated with considerable formation of acetylcholine. The low-affinity uptake system is less dependent on sodium, and does not appear to be associated with a marked degree of acetylcholine formation. The high-affinity choline uptake appears to represent selective choline accumulation by cholinergic neurons.

Placental beta-endorphin-like peptides.

Acid extract of human placental tissue contain, by both radioimmunoassay and radioreceptor assay, beta-endrophin-like material. Half of this material will not go through a 5000-dalton filter and on Sephadex G-200 has a molecular size between 25,00 and 50,000 daltons. Of the material going through a 5000-dalton ultrafilter, 80 percent is excluded on Sephadex G-25 and held back, very slightly, on Bio-Rad P6, indicating a molecular size of approximately 4500 to 4800 daltons. Thus, placenta appears to have macromolecular precursors from which a beta-endorphin-like material is released, with a size approximately 12 amino acids longer than half of the pituitary hormone.

Antidepressants and the muscarinic acetylcholine receptor.

Several tricyclic antidepressants have been assessed for their potency in binding to the muscarinic acetylcholine receptor of brain and intestine. Amitriptyline hydrochloride is about ten times as potent as imipramine hydrochloride. Dimethylated drugs are more potent than monomethylated ones. The relative anticholinergic activities of tricyclic antidepressants have implications for their use in patients who might be affected adversely by anticholinergic effects.

Decreased receptor-binding sites for kainic acid in brains of patients with Huntington's disease.

The specific binding of (3H)kainic acid (KA) to high- and low-affinity receptor sites was assayed in postmortem samples from the brains of patients affected with Huntington's disease (HD) and age-matched controls. Treatment of rat brain with conditions that closely mimic the temperature gradient occurring in postmortem human brain only slightly but not significantly decreased receptor binding by 12 hr after death. In HD brains, total specific binding of (3H)KA was reduced in the caudate nucleus by 51%, putamen by 77%, and frontal cortex by 47%. Specific binding to the high-affinity site was virtually undetectable in the caudate nucleus and was reduced by 90% in the putamen from HD brains. No significant alterations in specific binding of (3H)KA were noted in the insular or temporal cortex, hippocampus, or cerebellum. Thus, losses of KA receptor binding were mainly localized to those regions of the HD brain that are most severely affected by neuronal degeneration, and the high-affinity receptor site appeared more affected.

A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system.

Levels of the neuropeptides substance P, somatostatin, and neurotensin were measured by radioimmunoassay in regions of the rat and human central nervous system (CNS) in aging. Somatostatin levels were significantly lower only in the corpus striatum of older rats. Substance P levels and neurotensin levels were generally stable with aging as were levels of somatostatin in regions other than the corpus striatum. In post-mortem human CNS tissues, no significant negative correlations of levels of the three peptides were observed with time to refrigeration or time to freezer for the samples. In the human CNS, there were no significant age-related alterations in substance P levels in frontal cortex, thalamus, hypothalamus, caudate nucleus, globus pallidus, or substantia nigra. There was a significant age-related decrease in substance P levels in the human putamen. This age-related decrease was not present in tissues from victims of Huntington's disease nor was there any striking difference in substance P levels as a function of duration of the disease. There were no significant age-related changes in somatostatin levels in human frontal cortex, caudate nucleus, putamen, medial globus pallidus, or substantia nigra. Among these same regions, there was a significant age-related decrease in neurotensin levels only in the pars compacta and pars reticulata of the human nigra. These, results implicate neuropeptides in aging processes in certain regions of the CNS. There are differences between rats and humans with respect to neuropeptides in the aging process in the CNS. Deterioration of some neuropeptide pathways in and to human basal ganglia may be involved in the suspected functional deterioration of parts of the extrapyramidal system in aging.

Benzodiazepine receptor binding in young, mature and senescent rat brain and kidney.

Clinical reports have described age-altered pharmacological effects of anxiolytic drugs especially an increased susceptibility to their sedative actions. In order to test whether such changes may be due to age-related alterations in central benzodiazepine receptors, 3H-flunitrazepam binding was assayed in the frontal cortex and cerebellum of young, mature and senescent rats. The numbers of 3H-flunitrazepam binding sites and their affinity was determined by Scatchard analysis of saturation isotherms and the relative abundance of type I and type II benzodiazepine receptors was assessed by drug-inhibition studies using diazepam and the triazolopyridazine, CL 218,872. In addition, age related changes in the kidney and hippocampus of the Ro5-4864-sensitive benzodiazepine receptor were studied using 3H-Ro5-4864. No age-related alterations were noted in the binding characteristics of 3H-flunitrazepam. Furthermore, drug-inhibition of 3H-flunitrazepam binding by diazepam and CL 218,872 was nearly identical in young, mature and senescent rats, indicating that also the ratio of type I and type II receptors does not change with age. Binding of 3H-Ro5-4864 to membranes from rat hippocampus was not age-related. However, a significant decrease in 3H-Ro5-4864 binding to kidney membranes was demonstrated. Hence, central benzodiazepine receptors appear unaltered in the senescent rat model of aging. The clinical findings of an increased susceptibility to the sedative effects of benzodiazepines in the elderly may therefore be attributed to pharmacokinetic variables, or to events occurring secondarily to receptor activation.

Cerebrospinal fluid gamma-aminobutyric acid variations in neurological disorders.

Neuropathologically, Huntington's disease is characterized by a profound reduction in neuronal cells originating in the corpus striatum and globus pallidus. Since one of these cell types utilizes gamma-aminobutyric acid (GABA) as a neurotransmitter, it may be possible to differentially diagnose this disorder on the basis of the CSF content of this amino acid. In order to determine the validity of this hypothesis, cerebrospinal fluid GABA was analyzed, using a recently developed radioreceptor assay procedure and was found to be significantly reduced in patients diagnosed as having Huntington's disease and also lower in patients with Alzheimer's disease, though no difference was noted between Parkinson patients and control subjects. The results suggest that analysis of cerebrospinal fluid GABA may have diagnostic, and perhaps predictive, value in certain neurological disorders.

Synthesis using a Fmoc-based strategy and biological activities of some reduced peptide bond pseudopeptide analogues of dynorphin A1.

Eight analogues of Dyn A(1-11)-NH2 incorporating the enzymatically stable psi(CH2-NH) isosteric peptide bond replacement were synthesized and tested for binding affinity at the central opioid mu, delta, and kappa receptors in guinea pig brain (GPB) homogenates and for activity at the peripheral kappa (and mu) receptors in the guinea pig ileum (GPI). The peptidic analogues were synthesized by solid phase techniques using a Fmoc/tert-butyl strategy, and the psi(CH2-NH) bond, or reduced bond, was introduced via reductive alkylation of the N-terminal amino group of the growing peptide with a Fmoc-N(alpha)-protected amino aldehyde. The synthesis of Fmoc-N(alpha)-protected amino aldehydes also is described. Several other peptides have been previously synthesized incorporating this modification and showed for instance increased enzymatic stability and antagonist properties. Results obtained in the GPB show that modifications of the peptide bond in the address site (analogues 4-9) do not affect the binding at the kappa receptor and, with a few exceptions, at the mu and delta receptors. On the other hand, analogues 2 and 3, modified in the message segment of Dyn A(1-11)-NH2, show a decrease in binding affinity at all three receptors. In the GPI, the results are more varied as the influence of the peptide bond modification seems to be more important than in the GPB. Finally, selected analogues were tested with no indication for antagonist activity at the kappa peripheral receptor.

Conformationally restricted phenothiazine neuroleptics. 1. 3-(Dimethylamino)-1,2,3,4-tetrahydroazepino[3,2,1-kl]phenothiazine.

A rigid analogue of promazine, 3-(dimethylamino)-1,2,3,4-tetrahydroazepino[3,2,1-kl]phenothiazine (1), was prepared by reductive amination of the corresponding ketone 4. An X-ray crystallographic study revealed that the seven-membered ring of the hydrochloride salt of 1 exists as a half-chair-like form with the dimethylammonium group in an equatorial-like conformation. Compound 1 was approximately one-half as active as promazine as an inhibitor of [3H]spiperone binding in rat corpus striatal homogenates. In homogenates obtained from calf caudate tissue, however, 1 was only about one-twentieth as active as promazine as an inhibitor of [3H] spiperone binding. As a stimulator of homovanilic acid (HVA) synthesis in rat corpus striatum in vivo, it was about one-tenth as active as promazine.

Design and synthesis of conformationally constrained somatostatin analogues with high potency and specificity for mu opioid receptors.

A series of cyclic, conformationally constrained peptides related to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the mu opioid receptor. The following new peptides were prepared and tested for their mu opioid receptor potency and selectively in rat brain binding assays: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (2, CTOP); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (3, CTAP); D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 (4); D-Phe-Cys-Tyr-D-Trp-Lys-Val-Pen-Thr-NH2 (5); D-Phe-Cys-Tyr-D-Trp-Lys-Gly-Pen-Thr-NH2 (6); D-Phe-Cys-Tyr-Trp-Lys-Thr-Pen-Thr-NH2 (7); D-Tyr-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH (8); D-PhGly-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (9); and D-PhGly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (10). The most selective peptide, 2 (CTOP), displayed both high affinity (IC50 = 3.5 nM) and exceptional selectivity (IC50 delta/IC50 mu = 4,000) for mu opioid receptors. Furthermore, 2 exhibited very low affinity for somatostatin receptors in the rat brain (IC50 greater than 24,000 nM), with an IC50 somatostatin/IC50 mu receptor selectivity of 8,750. These conformationally constrained cyclic peptides should provide new insight into the structural and conformational requirements for the mu opioid receptor and the physiological role of this receptor.

Synthesis of highly mu and delta opioid receptor selective peptides containing a photoaffinity group.

A series of cyclic, conformationally constrained photolabile peptides related to the enkephalins and to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the delta and mu opioid receptors. The following new peptides were prepared and tested for their delta opioid receptor potency and selectivity in the guinea pig ileum assay, the mouse vas deferens assay, and the rat brain binding assay: H-Tyr-D-Pen-Gly-p-NH2Phe-D-Pen-OH (1, [p-NH2Phe4]DPDPE) and H-Tyr-D-Pen-Gly-p-N3Phe-D-Pen-OH (2, [p-N3Phe4]-DPDPE). The following new peptides were prepared and tested for their mu opioid receptor potency and selectivity in the same assays: H-D-Phe-Cys-p-NH2Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (3, [p-NH2Phe3]CTP) and D-Phe-Cys-p-N3Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (4, [p-N3Phe3]CTP). The delta selective photoaffinity peptide 2 displayed both high affinity (IC50 = 9.5 nM) and good selectivity (IC50 mu/IC50 delta = 1053) as an agonist at delta opioid receptors in bioassays, and 2 also displayed moderate affinity (33 nM) and excellent selectivity (IC50 mu/IC50 delta = 110) for rat brain delta opioid receptors. The mu selective photoaffinity peptide 4 displayed very weak affinity (8% contraction at 300 nM) at mu opioid receptors in bioassays, but good affinity (IC50 = 48.6 nM) and excellent selectivity (IC50 delta/IC50 mu = 412) for the rat brain mu opioid receptors. These conformationally constrained cyclic photoaffinity peptides may be useful tools to investigate the pharmacology of delta and mu opioid receptors.

Design, synthesis, and biological properties of highly potent cyclic dynorphin A analogues. Analogues cyclized between positions 5 and 11.

We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the kappa and mu receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11-NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5,D-Cys11] Dyn A1-11-NH2, c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs the mu and delta receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at kappa opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the kappa, mu, or delta central receptors.

Design of cyclic deltorphins and dermenkephalins with a disulfide bridge leads to analogues with high selectivity for delta-opioid receptors.

We earlier suggested that the low receptor selectivity observed for previously synthesized constrained analogues of deltorphin I (DT I) was the result of a reduction in the lipophilic surface of the C-terminal of the peptide. To confirm this prediction and to further test a previously proposed conformational model for bioactivity at delta opioid receptors, we have synthesized several new cyclic analogues with the general structure [D-Xaa2,Yaa5]deltorphin I and II in which Xaa2 is D-cysteine or D-penicillamine (D-Pen), and Yaa5 is an L- or D-penicillamine residue. Additional substitutions at positions 4, 6, and 7 also were examined. The analogues were tested for binding to mu- and delta-opioid receptors and in mouse vas deferens and guinea pig ileum biological assays. The introduction of a lipophilic L-Pen in position 5 and D-Cys or D-Pen in position 2 resulted in a highly delta-selective series of analogues, which fully confirmed our prediction. The cyclic analogues formula; see text: DT I are among the most delta-selective analogues described thus far.

Cyclic enkephalin analogs with exceptional potency at peripheral delta opioid receptors.

A series of super potent and delta-opioid-receptor-selective cyclic hexapeptides of the general formula [formula: see text] (where X is hydrogen or halogen) has been synthesized. The unsubstituted hexapeptide formula; see text: [Phe6]DPLCE) has extremely high potency at peripheral delta opioid receptors (IC50 value in the MVD assay is 0.016 nM) and in bioassays is the most selective compound in this series. The introduction of halogens in the phenyl ring of phenylalanine at position 4 led to significant changes in the selectivity and affinities at peripheral and central opioid receptors. In the binding studies, the most potent compound is the p-fluoro analog, whereas the most selective analog is the p-iodo-substituted peptide.

The use of topographical constraints in receptor mapping: investigation of the topographical requirements of the tryptophan 30 residue for receptor binding of Asp-Tyr-D-Phe-Gly-Trp-(N-Me)Nle-Asp-Phe-NH2 (SNF 9007), a cholecystokinin (26-33) analogue that binds to both CCK-B and delta-opioid receptors.

The cholecystokinin (26-33) [CCK (26-33)] octapeptide analog Asp-Tyr-D-Phe-Gly-Trp(N-Me)-Nle-Asp-Phe-NH2 (SNF 9007) is a potent and selective ligand for both the CCK-B and delta-opioid receptors. Pharmacological studies of SNF 9007 suggest a relationship between the ligand requirements of CCK-B and delta-opioid receptors, which further implies a possible structural relationship between these receptors. We have utilized topographical constrainment of the important Trp30 residue to investigate structural features of SNF 9007 that would distinguish between binding requirements in this region for the CCK-B and delta-opioid receptors. Thus, the four optically pure isomers of beta-MeTrp were substituted for L-Trp30 of SNF 9007. Receptor binding results suggest that the preferred topography of the Trp30 residue for CCK-B receptor binding may be the 2S,3S (erythro-L) configuration whereas for the delta-opioid receptor it may be the 2S,3R (threo-L) configuration. Molecular modeling studies of these ligands further support the recently revised receptor-bound model for CCK-B octapeptide ligands (Kolodziej et al. J. Med. Chem. 1995, 38, 137-149) and are in good agreement with the DPDPE-delta opioid receptor "template" model (Nikiforovich et al. Biopolymers 1991, 31, 941-955).

Cyclic enkephalin analogues with exceptional potency and selectivity for delta-opioid receptors.

Superpotent and highly delta-opioid receptor selective cyclic peptides of the general formula H-Tyr-c[D-Pen-Gly-Phe(p-X)-Pen]-Phe-OH (where X = hydrogen or halogen) have been synthesized. In the binding assays the most selective and most potent compound is the p-bromophenyl-alanine-4 analogue (IC50 value = 0.19 nM, selectivity ratio = 21,000 for delta vs mu). In the GPI and MVD bioassays the most selective and most potent analogue is the p-fluoro-substituted analogue Tyr-[D-Pen-Gly-Phe(p-F)-Pen]-Phe-OH. In the MVD assay it has an exceptionally low IC50 value of 0.016 nM and a delta vs mu selectivity ratio of 45,000.

Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen2,D-Pen5]enkephalin with delta opioid receptor selectivity.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5] enkephalin (DPDPE) was modified by 2' (CH3) and 3' (I, OCH3, NO2, NH2) ring substitutions and by beta-methyl conformationally constrained beta-methyltyrosine derivatives in the 1 position. The potency and selectivity of these analogues were evaluated by bioassay in the mouse vas deference (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay) assays and by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H][p-ClPhe4]DPDPE (delta ligand). The analogues showed highly variable potencies in the binding assays and in the bioassays. Aromatic ring substituents with positive Hammett constants had decreased potency, while substituents with negative Hammett constraints has increased potency for the opioid receptor. The most potent and most selective compound based on the binding was [2'-MeTyr1]DPDPE (IC50 = 0.89 nM and selectivity ratio 1310 in the binding assays). The 6-hydroxy-2-aminotetralin-2-carboxylic acid-containing analogue, [Hat1]DPDPE, also was highly potent and selective in both assays, demonstrating that significant modifications of tyrosine in enkephalins are possible with maintenance of high potency and delta opioid receptor selectivity. Of the beta-methyl-substituted Tyr1 analogues, [(2S,3R)-beta-MeTyr1]DPDPE was the most potent and the delta receptor selective. The results with substitution of beta-MeTyr or Hat instead of Tyr also demonstrate that topographical modification in a conformationally restricted ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity.

Design, synthesis, and biological activities of cyclic lactam peptide analogues of dynorphine A(1-11)-NH2.

We previously have reported four possible binding conformation of dynorphin A (Dyn A) for the central kappa opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an alpha-helical conformation in the cyclized address sequence. It was suggested that an alpha-helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and kappa opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize alpha-helical conformation. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH2 have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for kappa opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp(2) and Lys(6) in c[D-Asp(2),Lys(6)]Dyn A(1-11)-NH2 led to an analogue with pronounced potency and selectivity enhancement for the mu opioid receptor, whereas cyclization between D-Asp(3) and Lys(7) in c[D-Asp(3),Lys(7)]Dyn A(1-11)-NH2 led to a potent ligand (IC(50) 4.9 nM) with kappa receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the kappa receptor may have structural requirements that are distinct from those of mu and delta receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the kappa receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH2 may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between kappa, mu, and delta receptors.

Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific mu opioid receptor antagonists with greatly reduced binding at somatostatin receptors.

A series of conformationally restricted, cyclic octapeptides containing a conformationally stable tetrapeptide sequence related to somatostatin, -Tyr-D-Trp-Lys-Thr-, as a template, were designed and synthesized with the goal of developing highly potent and selective mu opioid antagonists with minimal or no somatostatin-like activity. Three distinct structures of the peptide became targets of chemical modifications and constraints; the N- and C-terminal amino acids and the cyclic 20-membered ring moiety. Based on the conformational analysis of active and inactive analogues of the parent peptide D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Pen7+ ++-Thr8-NH2, CTP (Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 44, 697-710), we designed analogues to include the tetrahydroisoquinolinecarboxylate (Tic) moiety as the N-terminal amino acid instead of D-Phe, since Tic can exist only as a gauche (-) or a gauche (+) conformer. In this series, the following peptides were synthesized and pharmacologically evaluated: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (TCTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (TCTOP), and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (TCTAP). In rat brain membrane opioid radioligand binding assays, all three peptides displayed high affinity for mu opioid receptors (IC50 = 1.2, 1.4, 1.2 nM, respectively), and exceptional mu vs delta opioid receptor selectivity: 7770, 11,396, and 1060, respectively. TCTOP and TCTAP also possess exceptional mu vs somatostatin receptor selectivity: 14,574 and 28,613, respectively. In the peripheral in vitro GPI bioassay, TCTP, TCTOP, and TCTAP were highly effective antagonists of the potent mu opioid receptor agonist PL017, with pA2 = 8.69 for TCTAP, 8.10 for TCTP, and 7.38 for TCTOP. Our results show that a 10-fold higher affinity and selectivity for mu opioid receptors (in both central and peripheral studies) over delta and somatostatin receptor was gained as a result of the D-Tic1 substitution. These three peptides, TCTP, TCTOP, and TCTAP, are the most potent and selective mu opioid antagonists known. CTP has been shown to possess prolonged biological action, much longer than that of naloxone. This renders these analogues potentially useful ligands for investigating the physiological functions of the mu opioid receptor. Analogues of TCTP in which the 20-membered disulfide ring was contracted by deletion of D-Trp4, and/or Lys5, and/or Thr6 led to compounds with greatly reduced potency at the mu opioid receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Design and synthesis of highly potent and selective cyclic dynorphin A analogues.

We have designed and synthesized several cyclic disulfide-containing peptide analogues of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogues exhibit unexpected selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogues has resulted in the kappa/mu opioid receptor ligands [Cys5,Cys11]Dyn A1-11-NH2 (1) and [Cys5,Cys11,D-Ala8]Dyn A1-11-NH2 (2), which possess high kappa and mu opioid receptor affinities centrally (guinea pig brain, GPB), but only weak activity at peripheral kappa and mu opioid receptors (guinea pig ileum, GPI). On the other hand, [Cys8,Cys13]Dyn A1-13-NH2 and [D-Cys8,D-Cys13]Dyn A1-13-NH2 (5) display high kappa potencies and selectivities at the peripheral (GPI) but not at the central (GPB) kappa opioid receptor. The lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays suggests the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral nervous systems.