LH-releasing activity of potent LH-RH analogs in vitro

The LH-releasing activity of eight superactive analogs of LH-RH was measured in pituitary cells in primary culture. Introduction of the C-terminal ethylamide modification into [D-Ala⁶]- and [D-Leu⁶]-LH-RH (two peptides already 3 times more active than LH-RH) increases their activities 10-fold. [D-Phe⁶]- and [D-Trp⁶]-LH-RH are 90 and 100 times more active than LH-RH, respectively. The ethylamide derivatives of these two compounds are however approximately six times less active than the parent peptides.     

Supernormal insulin: [D-PheB24]-insulin with increased affinity for insulin receptors

[D-Phe^B24]- and [D-Phe^B25]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe^B24]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe^B24]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe^B24]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.     

Some analogs of luteinizing hormone releasing hormone (LH-RH) having intense ovulation-inducing activity

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly¹⁰-[Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[α-aminoisobutyric acid⁶]-LH-RH-ethylamide, des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Ile⁵, D-Ala⁶]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide, were 50 times or more active than the original molecule.     

Luteinizing hormone-releasing hormone analogs with increased anti-ovulatory activity

A series of LH-RH antagonist analogs has been developed in which inhibitory activities have been increased to a potentially clinically useful level. The new peptides, which are typified by [N-acetyl-D-p-Cl-Phe^1,2, D-Trp³, D-Phe⁶,D-Ala¹⁰]-LH-RH and [N-acetyl-D-Trp^1,3,D-p-Cl-Phe²,D-Phe⁶, D-Ala¹⁰]-LH-RH, most importantly contain new modification to positions 1, 2 and 10, and induce full blockade of ovulation at single doses as low as 10 μg per rat (50 μg/kg). Various ring substituents on D-Trp or D-Phe in position 1 or other D-amino acid replacements in position 10 did not significantly improve anti-ovulatory activity. Incorporation of N-Me-Leu in position 7 was slightly detrimental to activity.     

The direction of opiodid agonists towards mu-, delta- and epsilon-receptors in the vas deferens of the mouse and the rat

Chronic treatment of mice with specific opioids results in the development of tolerance of particular opiate receptors in the mouse vas deferens (MVD). Accordingly, the infusion of animals with the specific δ-receptor ligand [D-Ala²,D-Leu⁵]-enkephalin (DADL) or the potent μ-agonist sufentanyl (SUF) produces MVD highly tolerant to δ- and μ-agonists, respectively. Investigating a series of opioids in these preparations provides unequivocal evidence for the simultaneous existence of δ- and μ-receptors in the MVD. Thus, the possibility exists to obtain vasa deferentia, which almost exclusively contain either μ- or δ-opiate receptors. In combination with the rat vas deferens (RVD), a supposedly selective ε-receptor preparation, useful tools are provided for the classification of opioids according to their preference for the μ-, δ- and ε-type of the opiate receptors.     

Preliminary ligand binding data for subtypes of the delta opioid receptor in rat brain membranes

Delta opioid binding sites were assayed using [³H][D-ala²,D-leu⁵]enkephalin and rat brain membranes depleted of μ binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5 -isothiocyanatobenzimidazole-HCI. [D-Pen², D-Pen⁵]enkephalin (DPDPE), [D-Pen²,L-Pen⁵]enkephalin, [D-Ala²]deltorphin-I and [D-Ala²]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 μM 5'-guanylyimidodiphosphate in the assay. Quantitative analysis of [D-ala²,D-leu⁵]enkephalin, DPDPE and [D-Ala²]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala²,D-leu⁵]enkephalin had equal affinity for both sites, DPDPE and [D-Ala²]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating δ receptor subtyes which mediate antinociception.     

Comparison of the behavioral effects of β-endorphin and enkephalin analogs

The behavioral effects of β-endorphin, enkephalin analogs, morphine and etorphine were briefly compared. In the tail-flick test in mice and in the wet shake test in rats, β-endorphin and D-Ala²-D-Leu⁵-enkephalin had equal antinociceptive activity; D-Ala² -Met-enkephalinamide and D-Leu⁵-enkephalin were less active. The order of activity of the enkephalin analogs and opiate alkaloids for stimulating locomotor activity in mice paralleled their analgesic activities; β-endorphin, however, had only minimal stimulatory actions. Morphine sulfate, 50 μg injected into the periaqueductal gray, produced hyperactivity but this effect was not observed with etorphine or opioid peptides. By contrast, “wet dog” shakes was observed with the opioid peptides but not with either opiate alkaloid. These heterogenous behavioral responses, which were all antagonized by naloxone, indicate that multiple types of receptors mediate the effects of opiates in the central nervous system.     

Gramicidin S analogs with a D-Ala, Gly, or L-Ala residue in place of the D-Phe residue: molecular conformations and interactions with phospholipid membrane

The proton nmr and CD spectra of gramicidin S (GS) cyclic-(Val^1,1′-Orn^2,2′-Leu^3,3′-D-Phe^4,4′-Pro^5,5′)₂ and of GS analogs—namely, [D-Ala^4,4′]-GS, [Gly^4,4′]-GS, and [L-Ala^4,4′]-GS—were analyzed. The molecular conformation of [D-Ala^4,4′]-GS is similar to that of GS, with the trans form about the D-Ala-Pro peptide bond. The molecular conformation of [Gly^4,4′]-GS depends on the solvent composition of dimethylsulfoxide-d₆/trifluoroethanol (DMSO)-d₆/TFE and DMSO-d₆/H₂O as well as the solute concentration. In DMSO-d₆ solution, [Gly^4,4′]-GS forms the GS-type conformation of the monomer at lower concentration. At higher concentration, the GS-type conformer is converted to the other one that forms molecular aggregates. The cis form about the X-Pro peptide bonds is found for [Gly^4,4′]-GS and [L-Ala^4,4′]-GS in DMSO-d₆ and for [L-Ala^4,4′]-GS in TFE solution. The large temperature dependences of α-proton chemical shifts of [L-Ala^4,4′]-GS in DMSO-d₆ solution indicate that the conformer equilibrium changes with temperature. The GS-type conformation is not formed in [L-Ala^4,4′]-GS. The two active peptide analogs, [D-Ala^4,4′]-GS and [Gly^4,4′]-GS, interact with the phospholipid membrane, taking the GS-type conformation. By contrast, an inactive analog, [L-Ala^4,4′]-GS, does not interact with phospholipid membrane. The activities of GS analogs are found to correlate to the formation of the GS-type conformation upon binding with phospholipid membrane.     

The metabolic stability of the enkephalins.

[Met⁵] and [Leu⁵]-enkephalins inhibit ³H-naloxone binding to brain opiate receptors much more effectively at 4°C than at 25°C. At 25°C several protease inhibitors potentiate the action of the enkephalins. Bacitracin is the most effective of these. In the presence of 100 μg/ml of bacitracin, the potencies of [Met⁵]- and [Leu⁵]-enkephalins are similar to those at 4°C. Some protease inhibitors, such as TLCK and TAME, are effective by themselves in inhibiting the binding of ³H-naloxone. Enkephalins with D-amino acids in the 2-position are equally effective at 0°C and at 25°C and their action is not potentiated by bacitracin. In particular, [D-Ala²]-enkephalins do not seem to be significantly degraded by the membrane enzymes which destroy the natural enkephalins.     

Unsulfated C-terminal 7-peptide of cholecystokinin: a new ligand of the opiate receptor.

On the basis of structural and conformational similarities between the C-terminal 7-peptide of cholecystokinin (pancreozymin) (CCK-(27–33)) and the active enkephalin analog [Trp⁴,Met⁵]-enkephalin, the affinity of CCK-(27–33) for the opiate receptor was determined. With unsulfated CCK-(27–33) half-maximal inhibition of stereospecific binding of [³H]-naloxone in a rat brain membrane preparation was observed at a 200 times higher concentration than that required with [Met⁵]-enkephalin. Sulfated CCK-(27–33) did not bind at concentrations up to 4 × 10^?5M. In the bioassay based on inhibition of electrically evoked contractions of guinea pig ileum similar potency ratios were observed and the effect of CCK-(27–33) was shown to be naloxone-reversible. These findings are of interest in view of the recently demonstrated presence of CCK-fragments in the brain.     

On the importance of position one of ovulation inhibitors, as based on studies on [D-Phe2, Pro3, D-Phe6]-LHRH.

Substitution of cyclopentylcarbonyl-(Cpc) for 1 in the effective and potent antiovulatory inhibitor, [D-Phe², Pro³, D-Phe⁶]-LHRH (I) retained the $\text{in vitro}$ potency. We know of no other inhibitor of the luteinizing hormone releasing hormone (LHRH) with a modification at position 1, which is as potent $\text{in vitro}$. This result agrees with the concept of the role of 1, D-Phe², Pro³, D-Phe⁶]-LHRH did not inhibit ovulation in rats at the same dosage as did I; this result is under study to circumvent. Des-Gly¹⁰-[D-Phe², Pro³, D-Phe⁶]-LHRH ethylamide and [Glu¹, D-Phe², Pro³, D-Phe⁶]-LHRH were significantly less active $\text{in vitro}$ than I.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Dehydro-enkephalins. IV. Discriminative recognition of delta and mu opiate receptors by enkephalin analogs

We have studied the receptor binding activities of $\text{C}$-terminal free and amidated enkephalins with and without the dehydrophenylalanine⁴ residue. For the selective labeling of so-called δ and μ opiate receptors, specific tracers were used at low concentrations in rat brain membranes and neuroblastoma cells containing pure δ receptors. $\text{C}$-Terminal free enkephalins are five to eight times more potent in the assay for δ receptors than in μ assay, while the amides are almost equipotent in both assays. The presence of a C-terminal carboxyl group is a determining factor for selective activity. [D-Ala², ΔPhe⁴, Met⁵]-enkephalin amide is very potent in all of the binding assays examined, and, in particular, twice as active as the saturated amide and the $\text{C}$-terminal free enkephalin in the δ assay. We suggest that the steric arrangement of the dehydrophenylalanine residue in position 4 is very important to the enhanced interaction with the δ receptors.     

Differential effects of GTP and cations on binding of labeled dimeric and monomeric enkephalins to neuroblastoma-glioma cell delta opiate receptors

Binding of radio-labeled enkephalin monomers [D-Ala²,Met⁵]Enkephalin Amide (DAMEA) and [D-Ala²,D-Leu⁵]Enkephalin (DADLE) and a dimer of [D-Ala²,Leu] Enkephalin Amide (DPE₂) to neuroblastoma-glioma (NG108-15) cells was examined in the presence and absence of GTP and/or cations. We found that: (1) binding occurs to a single class of homogeneous and non-interacting membane sites; (2) the affinity of the enkephalin dimer is reduced 50% in the presence of Mn²⁺ and 65% in the presence of both Mn²⁺ and GTP; (3) GTP alone either increases or does not change affinity of DPE₂; (4) Na⁺ and GTP significantly decrease the affinities of monomers, but not that of the dimer; and (5) a higher concentration (0.1 mM) of GTP increases the binding of DPE₂ but significantly decreases binding of monomers. Conclusion: Changes in binding of a dimeric enkephalin by Na⁺, Mn²⁺ and GTP are significantly and qualitatively different than those occurring for monomers.     

Enkephalin dimers: Regulation of cyclic AMP levels in NG108-15 cells

Intracellular cyclic AMP levels were determined for dimeric and monomeric enkephalins interacting with PGE₁-stimulated NG108-15 cells. The dimeric pentapeptide enkephalin (DPE₂), [D-Ala², Leu⁵ -NH-CH₂]₂, displaying very high affinity (K = 4.2 ± 0.3 nM^?1) for the δ-opiate receptor, inhibited cyclic AMP production by 70%. Its IC₅₀-value was between 0.1 and 0.2 nM, similar to that of the potent δ-agonist [D-Ala², D-Leu⁵] enkephalin (DADLE) with K = 1.0 ± 0.1 nM^?1. [D-Ala², Leu⁵] enkephalin amide (DALEA), which is the monomer of DPE₂, showed an IC₅₀ = 4 nM. The dimeric tetrapeptide enkephalin (DTE₁₂), [D-Ala², des-Leu⁵-NH-(CH₂)₆]₂ and its monomer [D-Ala², desLeu⁵] enkephalin amide (DAPEA) showed IC₅₀ = 2 and 20 nM, respectively. These results indicate that the DPE₂ and DTE₁₂ enkephalin dimers are potent δ-agonists.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

D-pGlu1,D-Phe2,D-Trp3,6]-LRF. A potent luteinizing hormone releasing factor antagonist in vitro and inhibitor of ovulation in the rat.

The decapeptide D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂ was synthesized in gram quantities by solid phase techniques on a methyl benzhydrylamine resin. This peptide was purified by ion exchange and partition chromatographies. It was fully characterized by amino acid analysis, tlc in four systems, optical rotation and high pressure liquid chromatography (reversed phase) using a triethyl ammonium phosphate buffer. This peptide was found to inhibit ovulation by 100% in the rat at a subcutaneous dose of 250μg in corn oil when administered at noon on the day of proestrus. Furthermore, it was found to be long acting since 1.5mg blocked ovulation in 9 out of 10 rats when administered as early as 19 hrs before the afternoon of proestrus. [D-pGlu¹, D-Phe², D-Trp^3,6]-LRF reduces the amount of LH normally secreted in response to LRF by 50% at a molar ratio (IDR₅₀) of 3/1 ([antagonist])/[LRF]). Three other analogs, equally well-characterized and with the D-pGlu¹ modification, i.e. [D-pGlu¹, D-Phe², Pro³, D-Trp⁶]-LRF, [D-pGlu¹, D-Phe², Phe³, D-Trp⁶]-LRF and [D-pGlu¹, D-Phe^2,6, D-Trp³]-LRF were found to be somewhat less potent ($\text{IDR}{}_{50}\text{=}\text{150}\text{1}\text{,}\text{60}\text{1}\text{,}\text{5}\text{1}$) respectively.     

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10.

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10 were synthesised and tested in androgen-sterilised constant-oestrus rats. The agonist, [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin, induced ovulation at a dose of 6ng/rat i.v., 10μg/rat p.o. and was at least five times as potent as [D-Ser(Bu^t)⁶, des-Gly-NH₂¹⁰, Pro-ethylamide⁹]-luliberin. [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin (1μg/rat) also prevented HCG-induced increases in ovarian and uterine weight in immature rats and was a highly potent antitumour agent when given to rats bearing DMBA-induced mammary tumours. The antagonist, [D-Phe², D-Phe⁶, Azgly¹⁰]-luliberin at a dose of 15μg/rat completely inhibited ovulation induced by luliberin (0.5μg/rat), whereas [D-Phe², D-Phe⁶]-luliberin lost activity below 125μg/rat.     

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, β-casomorphins, [D-Ala², D-Leu⁵] enkephalin and Sandoz peptide, FK 33–824, were examined by intracerebroventricular administration in rats. Their relative potencies $\text{in vivo}$ were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [³H]naloxone binding in the absence and presence of sodium ions for morphine (μ) receptors and against ¹²⁵I-[D-Ala², D-Leu⁵] enkephalin binding for enkephalin (δ) receptors. A good correlation between analgesic activity and morphine (μ) receptor but not enkephin (δ) receptor binding affinity was obtained. These data extend the hypothesis that morphine (δ) receptors mediate the major portion of the analgesic activity of opioids.     

Modulation of Binding at Opioid Receptors by Monoand Divalent Cations and by Cholinergic Compounds

Abstract

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH₄Cl and KCl, inhibit the equilibrium binding (25°C) of the selective μ-agonist [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin, the selective δ-agonist [³H]-[D-Pen²,D-Pen⁵]enkephalin and the selective δ-agonist [³H]-dynorphin A (1-9). Choline chloride inhibits the binding of the μ- and δ-agonists but not of the δ-agonist; the choline derivative, methacholine, inhibits also the binding of the δ-agonist. Binding of the δ-agonist is potentiated by CaCl₂, MgCl₂ and MnCl₂; these salts inhibit binding of the δ-agonist. As far as binding of the μ-agonist is concerned, MgCl₂ and MnCl₂ may potentiate or inhibit whereas CaCl₂ is only inhibitory. The binding of the μ-antagonist [³H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the μ-type; the binding of the μ-agonist [³H]-[D-Ala², MePhe⁴, Gly-ol⁵]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the μ-antagonists [³H]-naloxone and [³H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the δ-type; they are particularly effective for assays of K-receptors when the selective K-agonist [³H]-dynorphin A (1-9) is used as ligand.