[D-Thr2, Thz5]- and [D-Met2, Thz5]-enkephalinamides: Potent analgesics by intravenous injection

Two enkephalin analogs, [D-Met², Thz⁵]-enkephalinamide and [D-Thr², Thz⁵]-enkephalinamide, have been synthesized by the solid-phase method. When injected centrally, [D-Thr², Thz⁵]-enkephalinamide is 3.5 times more potent than the [D-Met², Thz⁵] analog. However, the two are equipotent and 4.2–4.8 times more potent than morphine when injected intravenously.     

D-Thr2, Thz5]-enkephalinamide: a potent analgesic by subcutaneous and oral administration

[D-Thr², Thz⁵]-enkephalinamide administered orally or subcutaneously inhibited the tail-flick response to heat stimuli in mice. On a molar basis, this peptide was found to be 1.5–1.7 times more potent than morphine by oral administration.     

Behavioral effects and in vivo degradation of intraventricularly administered dynorphin-(1-13) and D-Ala2-dynorphin-(1-11) in rats

Lateral intraventricular (LV) or cerebral aqueduct (CA) administration of the opioid peptide, dynorphin-(1-13), induced catalepsy and analgesia in rats. Onset was earlier and duration shorter than with morphine or β_c-endorphin. The dose required to induce analgesia was reduced at least tenfold when dynorphin-(1-13) was administered into CA rather than LV. An analogue, D-Ala²-dynorphin-(1-11), was more stable than dynorphin-(1-13) in brain, and produced a comparable degree of catalepsy and even more profound analgesia at one-tenth the dose. These effects of dynorphin-(1-13) and D-Ala²-dynorphin-(1-11) were significantly antagonized by naloxone pretreatment. Rats treated with dynorphin-(1-13) or a high dose of D-Ala²-dynorphin-(1-11) exhibited bizarre postures immediately following LV administration, with limb rigidity and “barrel-rolling”. These effects were not blocked by naloxone.     

A comparison of the analgesic and behavioral effects of [D-Ala2] Met-enkepha-linamide and morphine in the mesencephalic reticular formation of rats.

[D-Ala²]Met-enkephalinamide (DALA) injected intracerebrally (IC) at low doses into specific sites of the mesencephalic reticular formation (MRF), produced a profound, long-lasting analgesia that was blocked by naloxone, a specific opiate antagonist. Morphine was only half as potent as DALA because morphine, injected IC at similar sites in the MRF, yielded a comparable analgesia only when injected at twice the dose. The analgesic effects of morphine were also antagonized by naloxene. Both DALA and morphine produced specific behavioral effects. Naloxone blocked the behavioral effects of DALA, but not those produced by morphine.     

beta-Endorphin administration increases hippocampal acetylcholine levels.

The contents of acetylcholine and choline were determined in rat cortex, striatum, and hippocampus following intraventricular injection of β-endorphin or D-Ala²-enkephalinamide, a synthetic enkephalin analog, in doses known to produce analgesia in experimental animals. These opiate polypeptides produced significant increases in acetylcholine levels in the hippocampus, a subcortical structure rich in cholinergic terminals. The acetylcholine content of the hippocampus (but not the cortex or striatum) was significantly elevated 15, 30, and 60 minutes after a single intraventricular injection of β-endorphin (10 μg/brain) or D-Ala²-enkephalinamide (10 μg/brain). Peak alterations in regional acetylcholine concentrations and in analgetic effectiveness both occurred 30 minutes after peptide administration. Choline concentrations were unchanged by any of the experimental treatments. Naloxone hydrochloride (1 mg/kg, subcutaneously) affected neither brain acetylcholine concentrations, nor the response latencies of rats placed on a hot-plate; it did, however, antagonize the changes in these parameters caused by β-endorphin or D-Ala²-enkephalinamide. These data suggest that endorphins may normally regulate the physiologic activity of some cholinergic neurons.     

Analgesic effects of enkephalin analogs in rats

The analgesic effects of intracerebroventricular injections of Met-enkelphalin and five of its analogs in a dose of 10 μg each were quantified with a hot plate test in rats. Two analogs showed analgesic effect. ?D-Ala₂, Met⁵⊥-enkephalinamide and short-lasting analgesic effect. ?D-Ala², Met⁵⊥-enkelphalinamide had a weak (DALA) had a striking and long-lasting analgesic effect. However, sulfation of tyrosine residue totally abolished the analgesic action of DALA. The analgesic effect of DALA was not affected by preinjection of its sulfated analog.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Blockade of morphine supersensitivity by an antisense oligodeoxynucleotide targeting the delta opioid receptor (DOR-1)

An antisense oligodeoxynucleotide (ODN) targeting 20 bases of the coding sequence of the cloned delta opioid receptor (DOR-1), a mismatched ODN (different from the antisense ODN at 4 bases) or saline was administered to 3 groups of CD-1 mice implanted with naltrexone pellets (7.5 mg) for 7 days. Morphine supersensitivity (i.e., increased potency as defined by decreased morphine ED₅₀ values) was observed 24 h after pellet removal (day 8) in mice treated with saline or mismatch ODN, but not in antisense ODN treated mice. Antisense ODN alone had no effect on basal nociceptive thresholds or morphine analgesia but reduced the analgesic potency of the delta₂ opioid agonist [D-Ala²]deltorphin II. These data suggest that the delta₂ opioid receptor system participates in the adaptive changes contributing to increased morphine potency following chronic naltrexone treatment.     

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}$.     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

Electrophysiological effects of enkephalin analogs in rat cortex.

The microiontophoretic potencies of methionine-enkephalin (ME) analogs with similar charges but different potencies in the $\text{in vitro}$ assays were studied with respect to spontaneous and evoked activity of rat cerebral cortex neurons (CX). ME slowed discharge in 65% of the 62 CX tested; activity evoked by acetylcholine (ACh) or glutamate, as well as spontaneous discharge, was affected. The vast majority of responsive cells were deeper than 500 μ and excited by ACh. Antidromic spikes were elicited in many of these neurons by pyramidal tract stimulation. The electrophysiological effects of D-Ala² ME amide, a more potent analog $\text{in vitro}$, was similar to ME when tested on the same parietal CX, however, D-Ala² ME amide was much more potent than ME on frontal CX. On the other hand N-acetyl D-Ala²-Lys³ ME amide, which has low potency $\text{in vitro}$, had little effect on CX which were readily depressed by equivalent doses of ME or the D-Ala² analog. The depressions induced by ME or the D-Ala² derivative were readily and reversibly antagonized by prior iontophoretic administration of naloxone. It is concluded that opiate receptor interactions may play an important role in electrophysiological responses to iontophoretically administered ME and that the deeper cholineceptive cortical cells, possibly pyramidal neurons, may be preferentially affected.     

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.     

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.     

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.     

Double-enkephalins—Synthesis,activity on guinea-pig ileum,and analgesic effect

We have synthesized enkephalin analogues in which C-terminal methionine or leucine residues are replaced by a second active fragment of the enkephalin analogue. Synthesis of two compounds is described: in one, two fragments of a D-Ala²-enkephalin analogue are connected by a -NH-NH-bridge, and in the other, three methylene groups are incorporated between the amino groups. The first compound is a very potent inhibitor of electrically induced contractions of guinea-pig ileum and produces a strong analgesia when administered intraperitoneally in mice. The second compound is less active on the ileum and fails to produce analgesia after systemic injection. The double-enkephalins may interact with μ-receptors.     

Dissimilar efficacy of opioids to produce μ-mediated analgesia: Role of Gx/z and G12 transducer proteins

Intracerebroventricular (i.c.v.) administration to mice of IgGs raised against α subunits of G₁₂ or G_x/z transducer proteins lessened the activation of low Km GTPase induced by morphine, DAMGO and DADLE in P₂ membranes from mouse periaqueductal grey matter (PAG). In mice injected with anti G₁₂α, DADLE, DPDPE and [D-Ala²] Deltorphin II, but not β-endorphin-(1-31), antagonized the analgesic activity of morphine. Conversely, following anti G_x/zα, morphine antagonized the antinociceptive potency of DADLE. It is concluded that opioids display diverse efficacy at μ-G_i2 and μ-G_x/z complexes to produce supraspinal analgesia in mice.     

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.     

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.     

A behavioral role for enkephalins in regulating locomotor activity in the insect Leucophaea maderae: evidence for high affinity kappa-like opioid binding sites

D'-Ala-2 met-5-enkephalinamide application to the cerebral ganglia of Leucophaea maderae results in a decrease in locomotor activity. The opiate antagonist, naloxone, can block this effect as well as the depressant effect of morphine on locomotor activity. D-Ala-2,leu-5-enkephalinamide and dynorphin enhance locomotor activity following their topical application to the cerebral ganglia. This effect also can be antagonized by concomitant naloxone treatment. Benzomorphans were the most potent ligands tested in their ability to displace [3H]D'-ala-2,met-5-enkephalinamide whereas mu and delta ligands were by comparison less potent. These results suggest the presence of kappa-like opioid receptors in Leucophaea cerebral ganglia. The kappa ligands also are potent in enhancing locomotor activity in addition to being weakly antagonized by naloxone. Again, these results indicate the presence of multiple-opiate receptor types in invertebrates.     

Cardiovascular effects of peptides related to the enkephalins and β-casomorphin

In the urethane-anesthetized rat, intravenous injections of morphine produced a short-lasting fall in heart rate and blood pressure. The fall in heart rate (which is vagal in origin) was used here to bioassay peptides related to the enkephalins [-enk] and β-casomorphin [β-C, H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH]. A > 10% decrease in heart rate was used as a quantal index of response and the intravenous ED50 [μmol/kg] were estimated as: methionine-enk, 1.3 ± .24; leucine-enk, 1.5 ± .20; [D-Ala², D-Leu⁵]-enk, 0.45 ± .05; [D-Ala², NMePhe⁴, Met(0)⁵-ol]-enk, 0.0011 ± .0002; H-Tyr-Arg-OH, > 2.0; β-C (1–7), > 2.0; β-C(1–5), > 2.0; β-C(1–4), > 4.0; β-C(1–3), > 2.0; morphine sulfate, 0.11 ± .03; and human β-endorphin,, 0.07 ± .01. One β-C derivative, H-Tyr-Pro-Phe-Pro-NH₂ [β-C(1–4)-NH₂], was active at 0.32 ± .08. Naloxone pretreatment blocked the bradycardia produced by the enkephalins and β-C(1–4)-NH₂. The bioassay described here, based on heart rate, may prove to be useful for the rapid detection and estimation of the $\text{in vivo}$ pharmacological activities of new opioid peptides.