Recent advances in the development of 14-alkoxy substituted morphinans as potent and safer opioid analgesics.

Morphine and other opioid morphinans produce analgesia primarily through µ opioid receptors (MORs), which mediate beneficial but also non-beneficial actions. There is a continued search for efficacious opioid analgesics with reduced complications. The cornerstone in the development of 14-alkoxymorphinans as novel analgesic drugs was the synthesis of the highly potent MOR agonist 14-O-methyloxymorphone. This opioid showed high antinociceptive potency but also the adverse effects associated with morphine type compounds. Further developments represent the introduction of a methyl and benzyl group at position 5 of 14-O-methyloxymorphone leading to the strong opioid analgesics 14-methoxymetopon and its 5-benzyl analogue, which exhibited less pronounced side effects than morphine although interacting selectively with MORs. Introduction of arylalkyl substituents such as phenylpropoxy in position 14 led to a series of extremely potent antinociceptive agents with enhanced affinities at all three opioid receptor types. During the past years, medicinal chemistry and opioid research focused increasingly on exploring the therapeutic potential of peripheral opioid receptors by peripheralization of opioids in order to minimize the occurrence of centrally-mediated side effects. Strategies to reduce penetration to the central nervous system (CNS) include chemical modifications that increase hydrophilicity. Zwitterionic 6-amino acid conjugates of 14-Oalkyloxymorphones were developed in an effort to obtain opioid agonists that have limited access to the CNS. These compounds show high antinociceptive potency by interacting with peripheral MORs. Opioid drugs with peripheral site of action represent an important target for the treatment of severe and chronic pain without the adverse actions of centrally acting opioids.

Anti-inflammatory effects of contralateral administration of the kappa-opioid agonist U-50,488H in rats with unilaterally induced adjuvant arthritis.

OBJECTIVE: The effect of repeated contralateral treatment with the kappa-opioid receptor agonist U-50,488H {trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide methanesulphonate} was investigated in rats with unilaterally induced adjuvant arthritis. METHODS: Arthritis was induced by injection of Mycobacterium butyricum into the right hindpaw. Inflammatory parameters, nociceptive behaviour and cartilage turnover were evaluated up to 21 days after induction of arthritis. RESULTS: Contralateral treatment with 0.3 mg U-50,488H into the left hindpaw twice per week reduced the hindpaw oedema, ankle joint inflammation, pain behaviour to mechanical stimuli and severity score of inflammation in the hindpaws of both sides as well as the systemic spread of inflammation to other areas, e.g. tail and/or forepaws, compared with saline-treated animals. Moreover, a significant decrease in the levels of cartilage oligomeric matrix protein was found in animals treated with U-50,488H, suggesting reduction of cartilage damage. The anti-inflammatory and chondroprotective effects of U-50,488H were abolished by administration of the peripheral opioid receptor antagonist naloxone methiodide. CONCLUSIONS: This is the first report demonstrating that repeated contralateral administration of a kappa-opioid receptor agonist diminishes the development of a symmetrical joint disorder.

Binding, pharmacological and immunological profiles of the delta-selective opioid receptor antagonist HS 378.

HS 378 is a recently developed indolomorphinan with high selectivity and antagonist potency at the delta-opioid receptor. The present study was performed to characterize the opioid binding properties and pharmacological and immunological activity of HS 378 and to compare them with those of two well-known delta-opioid receptor antagonists, naltrindole (NTI) and naltriben (NTB). In vitro opioid receptor binding profiles were determined in rat brain homogenates. HS 378 showed 4.7- and 2.4-fold higher mu/delta selectivity compared to NTI and NTB, respectively. In the [35S]GTPgammaS functional assay carried out in cell lines expressing cloned human opioid receptors, HS 378 was found to be a pure delta-opioid receptor antagonist. In vitro, exposure of HS 378 resulted in an apparent dose-related suppression of concanavalin A induced rat T-lymphocyte proliferation with an IC50 value of 0.54 microM. NTI showed also immunosuppression with an IC50 value of 6.93 microM, whereas NTB had no effect. The IC50 of HS 378 was 13 times lower than that of NTI and 8 times higher than that of cyclosporin A. Taken together, our findings indicate that the small molecule HS 378 has properties that may be of therapeutic value in the setting of human inflammatory diseases.

An opioid system in connective tissue: a study of achilles tendon in the rat.

The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, delta-opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [3H]-naloxone (Kd 7.01 +/- 0.98 nM; Bmax 23.52 +/- 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery.

Bone reinnervation after fracture: a study in the rat.

Reinnervation after tibial fracture in the rat was studied by analyzing the occurrence of growth-associated protein 43 (GAP-43), a marker for regenerating nerve fibers, and protein gene product 9.5 (PGP-9.5), a marker for mature nerve fibers, by immunohistochemistry. At 3 days postfracture, GAP-43--immunoreactive nerve fibers were first observed in the fracture hematoma and periosteum. At 7 days postfracture, abundant sprouting of GAP-43--positive fibers was seen in the callus, hyperplastic periosteum, and edge of fibrocartilage. In the latter region, the nerve fibers were nonvascular, showing dense ramifications and terminal sprouting close to chondroid cells. At 14 days and 21 days postfracture, many GAP-43--positive fibers were still sprouting into the fibrocartilage and new woven bone. Fine varicose GAP-43--positive fibers also were present in the bone marrow. In contrast to GAP-43, PGP-9.5-positive nerve fibers were observed only occasionally at 3 days postfracture but gradually increased in number from day 14 to 21. Our study shows that intense nerve regeneration occurs in early fracture healing partly unrelated to neovascularization. Considering that neuronal mediators have been shown to participate in local bone formation and resorption, the nerve regeneration observed may prove to be essential for delivery of neuronal mediators required for normal callus formation and/or neovascularization.

Contralateral but not systemic administration of the kappa-opioid agonist U-50,488H induces anti-nociception in acute hindpaw inflammation in rats.

1. The anti-nociceptive effects of contralateral administration of kappa-opioid agonist U-50,488H were investigated in rats. 2. Inflammation was induced by unilateral injection of 1% carrageenan into the right hindpaw. Prior to carrageenan injection, U-50,488H or saline was administered into the left hindpaw. Withdrawal responses to mechanical and heat stimulation and oedema levels were evaluated at 3, 6 and 24 h post-carrageenan injection. 3. The results showed that the inflammatory effect of 1% carrageenan peaked after 6 h with bilateral decreases in withdrawal latencies and ipsilateral oedema formation. 4. Contralateral treatment with 0.01, 0.05, 0.3 and 2 mg of U-50,488H attenuated nociceptive reflexes to mechanical stimulation on the inflamed side at 6 h. The anti-nociceptive effect of contralateral treatment was dose-dependent at 3 and 24 h. The hindpaw withdrawal latencies to heat stimulation were prolonged at 3 and 24 h after contralateral treatment with 0.3 mg U-50,488H. No effect on inflammatory oedema formation was observed, except for a decrease at 3 h after treatment with 2 mg of U-50,488H. 5. Sciatic nerve denervation on the contralateral side abolished the anti-nociceptive effects of U-50,488H (0.3 and 2 mg). In contrast, contralateral injection of 1 mg morphine prolonged paw latencies in denervated rats. 6. Both co-administration of the peripherally selective opioid antagonist naloxone methiodide with 0.3 mg U-50,488H, and alternatively, systemic administration of 0.3 mg U-50,488H reversed the anti-nociceptive effects induced by contralateral injection of U-50,488H. 7. Taken together, our findings indicate that the contralateral administration of U-50,488H attenuates nociceptive behaviour resulting from acute inflammation. The effect is mediated via peripheral neuronal kappa-opioid receptors and, possibly, spinal cord mechanisms, suggesting a new treatment approach for acute inflammatory conditions.

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

In vitro binding and signaling profile of the novel mu opioid receptor agonist endomorphin 2 in rat brain membranes.

The recently discovered endogenous mu receptor selective endomorphin 2 was prepared in tritiated form by a catalytic dehalogenation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), and used for in vitro labelling of rat brain membranes. The binding was saturable, stereospecific and of high affinity (Kd: 0.97 and 1.12 nM based on kinetic and equilibrium binding studies, respectively). The maximal number of binding sites (Bmax) was found to be 114.8 fmol/mg protein. [3H]Endomorphin 2 was displaced by mu-receptor selective specific peptides and heterocyclic compounds with high affinity, whereas kappa and delta receptor specific ligands were much less potent. The Ki values of endomorphin 1 and 2 in inhibiting [3H]naloxone binding increased by 15-fold in the presence of 100 mM NaCl which indicates the agonist property of these peptides. Endomorphins stimulated [35S]GTPgammaS binding and inhibited adenylyl cyclase activity which also provides evidence for the agonist character of endomorphins.

Affinity profiles of novel delta-receptor selective benzofuran derivatives of non-peptide opioids.

Highly selective heterocyclic opioid ligands with potent delta-antagonist activity have been developed on the basis of the "message-address" concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the mu-opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the delta-opioid receptor, much less potency toward the mu-binding site, and were the least effective at the kappa-site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers delta-receptor selectivity. The Na+ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dose-dependent.

Interaction of agonist peptide [3H]Tyr-D-Ala-Phe-Phe-NH2 with mu-opioid receptor in rat brain and CHO-mu/1 cell line.

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.

Synthesis and binding characteristics of the highly selective radiolabelled deltorphin analogues containing 2-aminotetralin-2-carboxylic acid in position 3.

Following the description of [3H]Ile5,6deltorphin II, when it was reported that changes in hydrophobicity at positions 5 and 6 give rise to analogues with increased delta-receptor affinity and selectivity, new conformationally restricted deltorphin analogues were designed. A synthetic amino acid, 2-aminotetralin-2-carboxylic acid (Atc), was introduced at position 3 instead of Phe in Ile5,6deltorphin I and II, and the resultant compounds were prepared in tritiated form. Opioid binding sites specific for [3H]S-Atc3,Ile5,6deltorphin I and [3H]R-Atc3,Ile5,6deltorphin II were characterized in rat brain membranes. Their binding was saturable, stereoselective and inhibited by delta-selective ligands with high potency. They labelled single class of opioid sites at 35 degrees C with high affinity (Kd approximately 0.3 nM), Bmax values of 130 fmol/mg protein, and very low non-specific binding was observed. Both tritiated deltorphin analogues showed delta-receptor specificity in rat brain, therefore they could represent excellent new radioligands for investigating the complexity of the opioid receptor systems.