Dimeric Pentapeptide and Tetrapeptide Enkephalins: New Tools for the Study of Delta Opioid Receptors

Abstract

When a dimeric ligand can react bivalently, one would expect an increase in affinity, selectivity, and possibly biological activity. On this premise, we have synthesized and characterized two series of dimers, viz.: Dimeric Pentapeptide Enkephalin (DPE_n = (H-Tyr-D-Ala-Gly-Phe-Leu-NH-)₂ · (CH₂)_n, and Dimeric Tetrapeptide Enkephalin (DTE_n) = (H-Tyr-D-Ala-Gly-Phe-NH)₂ · (CH₂)_n, with n = 2, 4, …, 12. These dimers display affinity, activity, and δ/μ selectivity which vary systematically with chain length (n). DPE₂ shows a seven-fold increase in affinity for the δ receptor of whole brain and NG108-15 cells, relative to monomer, while its activity for the μ receptor is similar to enkephalin monomers. DTE₁₂ shows a dramatic increase in δ selectivity relative to its monomer. The association rate constant for ³H-DPE₂ is increased two-fold and its dissociation rate constant is significantly reduced, relative to monomer. DPE₂ shows a loss of affinity in the presence of Na⁺ or Mn⁺⁺, while GTP unexpectedly increases its affinity under some conditions. DPE₂ shows equal potency with the agonist [D-Ala², D-Leu⁵] enkephalin in assays measuring their inhibitory effect on prostaglandin E₁-stimulated cAMP production by NG108-15 cells. DPE₂ is very potent in the mouse vas deferens assay; DTE₁₂ shows substantially less activity. These results suggest that δ opiate receptors may be closely clustered in the cell membrane, and provide new approaches to the development of δ and μ receptor ligands.     

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.     

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.     

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.     

Selective inhibition of [D-ALA2, GLU4]deltrophin antinociception by supraspinal,but not spinal,administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the δ opioid receptor (DOR), which have been termed δ₁ and δ₂. These proposed DOR subtypes are thought to be activated by [D-Pen², D- Pen⁵]enkephalin (DPDPE, δ₁) and [D-Ala², Glu⁴]deltorphin (δ₂). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala², Glu⁴]deltorphin, but not of [D-Ala², NMPhe⁴, Gly-ol]enkephalin (DAMGO, μ agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonist selective for δ, μ and κ receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala², Glu⁴deltorphin but not of i.th. DAMGO or U69, 593 (κ agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, seletively inhibited the anitinociceptive response to i.c.v. [D-Ala², Glu⁴]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as δ₂ and further, suggest that this δ receptor subtype may play a major role in eliciting spinal δ-mediated antinociception.     

δ antagonist and κ agonist activity of naltriben: Evidence for differential κ interaction with the δ1 and δ2 opioid receptor subtypes

The selective δ₂ receptor antagonist Naltriben (NTB) has played an important role in the identification of subtypes of the δ opioid receptor, termed δ₁ and δ₂, and their role in antinociception. However, the majority of these studies have been conducted in the mouse. The present study determined the opioid receptor selectivity of subcutaneously (s.c.) administered NTB in the rat. Five minute pretreatment with 1 mg/kg s.c. NTB antagonized the increase in TFL produced by i.t. administration of equieffective doses of the δ₂ receptor agonist [D-Ala², Glu⁴]deltorphin (DELT) or the δ₁ receptor agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), but did not antagonize the μ receptor agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO). These data confirm previous reports that NTB is a selective δ opioid receptor antagonist. However, this dose of NTB antagonized DELT and DPDPE to an equivalent extent, suggesting that its selectivity for the δ₂ receptor is not maintained after s.c. administration in the rat. A lower dose of NTB (0.56 mg/kg s.c.) was ineffective. When the dose of NTB was increased to 3 mg/kg s.c. the antagonism of DELT and of DPDPE was unexpectedly lost. Pretreatment with the κ receptor antagonist nor-binaltorphimine (nor-BNI) partially restored the antagonism of DELT, but not DPDPE by this dose of NTB and did not modify the antagonism of DAMGO by NTB. These data suggest that high doses of NTB have κ receptor agonist-like activity and support the proposal that κ opioid agonists diminish the actions of δ receptor antagonists. They also suggest that nor-BNI-sensitive κ opioid receptors interact with δ₂, but not δ₁ opioid receptors in the spinal cord.     

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells

The binding of the enkephalin dimer [d-Ala², Leu⁵-NH-CH₂-]₂ (DPE₂) is characterized by (1) its high affinity for receptors on NG108-15 hybrid cells, the affinity constantK=4.7×10⁹ M^–1 is up to 8-fold that of monomers (0.6 to 1.0×10⁹ M^–1), and (2) a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE₂ binds with a 2-fold higher rate, k₁=6.3×10⁷ M^–1min^–1, than monomers (2.4 to 3.8×10⁷ M^–1min^–1), and dissociates at a slower rate than monomers. Dissociation of DPE₂ was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE₂ binds by cross-linking two subunits of one receptor.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

Studies of inhibition of the luteinizing hormone-releasing hormone by an irreversible inhibitor at the receptor site

[Leu², Leu³, D-Ala⁶]-LHRH is an analog of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (LHRH) and inhibits the release of LH and FSH induced by LHRH. This analog and inhibitor has been modified with the objective of developing an active-site-directed irreversible inhibitor. The modification consisted of replacing < Glu¹ with Chl¹ which is the moiety of chlorambucil (a nitrogen mustard). The Chl analog inhibited the release of LH and FSH by LHRH after addition prior to LHRH and after three changes of the incubation medium; in contrast, [Leu², Leu³, D-Ala⁶]-LHRH and [des-His²]-LHRH only inhibit release when added together with LHRH. The Chl analog released LH and FSH but not TSH or GH, indicating that its agonist and antagonist activities could be specific at the receptor site for LHRH.     

Treatment with Antisense Oligodeoxynucleotide to a Conserved Sequence of Opioid Receptors Inhibits Antinociceptive Effects of Delta Subtype Selective Ligands

Abstract

Previous work has suggested the existence of subtypes of the delta opioid receptor (DOR) which have been termed δ₁ and δ₂. [D-Ala², Glu⁴]deltorphin has been suggested to selectively elicit antinociception via the δ₂ receptor while [D-Pen², D-Pen⁵]enkephalin (DPDPE) is thought to act via the δ₁ receptor. Treatment with an antisense oligodeoxynucleotide (oligo) directed towards the N-terminal portion of the cloned DOR has been demonstrated to selectively inhibit the antinociceptive actions of [D-Ala², Glu⁴]deltorphin, but not of DPDPE, suggesting that the cloned DOR corresponds to that pharmacologically defined as δ₂. Here, an antisense oligo (or a mismatch sequence) was designed to target a conserved region of the cloned μ δ and opioid receptor. These oligos were employed in order to determine whether the antinociceptive effects of [DAla², Glu⁴]deltorphin, as well as DPDPE, could be inhibited. The data indicate that the antinociceptive actions of both ligands were inhibited by treatment with this antisense, but not with the mismatch oligo. Taken together, the results of the treatments with oligos directed towards the N-terminal portion of the cloned DOR and with that directed to the conserved region of the opioid receptors suggest that (a) DPDPE effects are mediated by a subtype of the DOR which shares a domain common to the cloned opioid receptors, and (b) the N-terminal region differs between these putative DOR subtypes.     

Cyclic enkephalin analogs containing a cystine bridge

Two conformationally constrained enkephalin analogs were synthesized by substitution of cysteines in positions 2 and 5 and oxidative disulfide bond formation. In the guinea pig ileum assay the obtained cyclic analogs, [D-Cys²-L-Cys⁵]enkephalinamide and [D-Cys²-D-Cys⁵]enkephalinamide, showed potency ratios of 37.9 ± 0.8 and 73.3 ± 0.9, respectively, relative to [Met⁵]enkephalin. The extremely high potency of the analogs was shown to be a consequence of the conformational restrictions introduced by cyclization. Rat brain membrane binding studies with [³H]naloxone and [³H](D-Ala², D-Leu⁵)enkephalin as radiolabels revealed a moderate preference of both analogs for μ-receptors over δ-receptors. Furthermore, the cystine-containing analogs were shown to be highly resistant to enzymatic degradation.     

Synthesis and opioid activities of stereoisomers and other D-amino acid analogs of methionine-enkephalin

A series of D-amino acid-substituted analogs of the opiate peptide, methionine⁵-enkephalin, were synthesized by solid-phase methods and tested for their abilities to inhibit electrically-evoked contractions of mouse vasa deferentia and to compete with tritiated enkephalin for opiate receptors on particulate fractions isolated from homogenates of rat brain. [D-Ala²]-enkephalin and [D-Ala²]-enkephalin amide were found to be the most potent peptides in both assay systems, being about 1000% active in the vas deferens bioassay and 120% and 150% active, respectively, in the stereospecific binding test relative to methionine⁵-enkephalin itself. In comparison, [D-Met⁵]-, [D-Tyr¹]-, [D-Leu²]-, [D-Phe²]-, [D-Ala³]-, and [D-Phe⁴]-enkephalin had not more than 10% activity. The stabilization of the β-bend conformation of methionine⁵-enkephalin by the substitution of D-alanine in position 2 of the peptide chain may contribute to the high activities of the [D-Ala²]-analogs.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: Synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [d -Pen², d -Pen⁵]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [d -DFAB², Met⁵-NH₂]enkephalin showed μ and δ agonist potencies comparable to those of natural [Leu⁵]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While l -amino acid substitution in position 3 of DPDPE usually resulted in higher δ agonist potency than d -amino acid substitution, [d -DFAB³]DPDPE turned out to be a more potent δ agonist than [l -DFAB³]DPDPE. Furthermore, [d -DFAB³]DPDPE showed over 100-fold higher δ agonist potency than [d -Abu³]DPDPE (Abu=2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a δ opioid receptor subsite. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

D-Ala2, deltaZPhe4-methionine enkephalin amide, a dehydropeptide hormone.

The enkephalin analog, D-Ala², Δ^zPhe⁴ amide, has been prepared and shown to be five times as active as D-Ala²-methionine enkephalin amide $\text{in}\text{vitro}$ and the dehydrophenylalanine moiety conferred complete stability to chymotrypsin on the peptide.     

Radioligands for Probing Opioid Receptors

Abstract

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains β-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu⁵] enkephalin, three [Met⁵] enkephalins, one [Met⁵] enkephalyl-Arg-Arg-Val-NH₂ (metorphamide or adrenorphin), one [Met⁵] enkephalyl-Arg-Gly-Leu and one [Met⁵] enkephalyl-Arg-Phe. [Leu⁵] enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to α- and β-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are μ, δ and ?. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.     

Interaction of synthetic peptide octarphin with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of nonopioid β-endorphin receptor, was prepared with specific activity 28 Ci/mmol. The binding of [³H]octarphin to T and B lymphocytes isolated from the blood of donors was studied. It was found that [³H]octarphin binds both to T and B cells with high affinity: K _d = 3.0 ± 0.2 and 3.2 ± 0.3 nM, respectively. The specific binding of [³H]octarphin to T and B lymphocytes was competitively inhibited by unlabeled β-endorphin (K _i = 1.9 ± 0.2 and 2.2 ± 0.3 nM, respectively) and was not inhibited by unlabeled naloxone, [Met⁵]enkephalin, [Leu⁵]enkephalin, α-endorphin, and γ-endorphin. Thus, T and B lymphocytes of human blood possess a nonopioid β-endorphin receptor whose binding is provided by the fragment 12–19 (the octarphin sequence).     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.