Mariana Spetea

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.

Peripheral versus central antinociceptive actions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone in acute and inflammatory pain in the rat.

Opioid analgesics with restricted access to the central nervous system represent a new approach to the treatment of severe pain with an improved safety profile. The objective of this study was to investigate the peripheral and central components of the antinociceptive actions of the 6-amino acid conjugates (glycine, alanine, and phenylalanine) of 14-O-methyloxymorphone. Their antinociceptive activities were compared with those of the centrally penetrating μ-opioid agonists morphine, fentanyl, and 14-O-methyloxymorphone. In the tail-flick test in rats, the 6-amino acid conjugates were 45- to 1170-fold more potent than morphine after i.c.v. administration and 19- to 209-fold after s.c. administration. They showed potencies similar to fentanyl after s.c. administration and were more potent after i.c.v. application. The time course of action was different between s.c. and i.c.v. administration, with significant long-lasting effects after i.c.v. administration. Systemic administration of the peripherally selective opioid antagonist naloxone methiodide antagonized the effects after s.c. but not after i.c.v. administration in the tail-flick test. Subcutaneous 6-amino acid derivatives also elicited antihyperalgesic effects in the formalin test in rats, which were reversed by systemically administered naloxone methiodide. Although morphine exerts its analgesic effects by central and peripheral mechanisms, the investigated new opioids interact primarily with peripheral opioid receptors after s.c. administration. The present data indicate that the 6-amino acid conjugates of 14-O-methyloxymorphone have limited access to the central nervous system and can mediate antinociception at peripheral sites. Also, they might find clinical application when the central actions of opioids are unwanted.

Modulation of basal and stress‐induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L‐822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self‐regulating role of SP, we used a selective neurokinin‐1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress‐evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L‐822429 represents a potent and selective rat NK1 receptor antagonist. Intra‐amygdaloid administration of L‐822429 via inverse microdialysis enhanced basal, but attenuated swim stress‐induced SP release, while the low‐affinity enantiomer of L‐822429 had no effect. Using light and electron microscopy, synaptic contacts between SP‐containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self‐regulatory capacity of SP‐mediated neurotransmission that differs in the effect on basal and stress‐induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor‐mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Specific activation of the μ 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 μ opioid receptor, an effect reversed by the μ receptor antagonist CTAP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2), but they had no influence on cells stably expressing the δ opioid receptor. To further establish the selectivity of these peptides for the μ opioid receptor, brain preparations of mice lacking the μ 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 μreceptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]GTPγS) 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 μ opioid receptors.

Alteration in endogenous opioid systems due to chronic inflammatory pain conditions

The influence of chronic arthritic pain on two endogenous opioid peptides, dynorphin B and [Met5]enkephalin-Arg6-Phe7, and multiple opioid receptors in discrete brain, lumbar spinal cord and pituitary pools was investigated. Using radioimmunoassay and receptor binding assay, we examined the changes in regional opioid peptide levels and opioid receptor activity due to chronic inflammation in adjuvant arthritic rats. At 4 weeks post-inoculation, increased levels of immunoreactive dynorphin B and [Met5]enkephalin-Arg6-Phe7 were measured in tissues of arthritic rats compared with controls. No significant changes in mu-, delta- or kappa-opioid receptors were seen after chronic inflammation. Taken together, these results indicate that in chronic arthritis, opioid receptor changes do not follow the peptide alterations of pro-dynorphin and pro-enkephalin systems. Thus, dynamic modification and modulation of nociceptive information takes place during chronic inflammation. This supports the key role of the central nervous system in chronic inflammatory pain conditions.

Morphinans and isoquinolines: Acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors

Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors.

The µ Opioid Receptor and Ligands Acting at the µ Opioid Receptor, as Therapeutics and Potential Therapeutics

Although the µ opioid receptor (MOR) was pharmacologically and biochemically identified in binding studies forty years ago, its structure, function, and true complexity only have emerged after its cloning in 1993. Continuous efforts from many laboratories have greatly advanced our understanding of MORs, ranging from their anatomic distribution to cellular and molecular mechanisms, and from cell lines to in vivo systems. The MOR is recognized as the main target for effective pain relief, but its involvement in many other physiological functions has also been recognized. This review provides a synopsis on the history of research on MORs and ligands acting at the MOR with the focus on their clinical and potential use as therapeutic drugs, or as valuable research tools. Since the elucidation of the chemical structure of morphine and the characterization of endogenous opioid peptides, research has stimulated the development of new generations of MOR ligands with distinct pharmacological profiles (agonist, antagonist, mixed agonist/antagonist and partial agonist) or site of action (central/peripheral). Discovery of therapeutically useful morphine-like drugs and innovative drugs with new scaffolds, with several outstanding representatives, is discussed. Extensive efforts on modifications of endogenous peptides to attain stable and MOR selective analogs are overviewed with stimulating results for the development of peptide-based pharmaceuticals. With pharmacophore modeling as an important tool in drug discovery, application of modern computational methodologies for the development of morphinans as new MOR ligands is described. Moreover, the crystal structure of the MOR available today will enable the application of structure-based approaches to design better drugs for the management of pain, addiction and other human diseases, where MORs play a key role.

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

The anti‐nociceptive effects of contralateral administration of κ‐opioid agonist U‐50,488H were investigated in rats. 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. The results showed that the inflammatory effect of 1% carrageenan peaked after 6 h with bilateral decreases in withdrawal latencies and ipsilateral oedema formation. 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. 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. 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. 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 κ‐opioid receptors and, possibly, spinal cord mechanisms, suggesting a new treatment approach for acute inflammatory conditions.

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, δ-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.

In vitro and in vivo pharmacological profile of the 5-benzyl analogue of 14-methoxymetopon, a novel μ opioid analgesic with reduced propensity to alter motor function

Opioids are the most effective analgesics for pain management, and efficient pain control is a therapeutic priority. Herein, we describe the synthesis and pharmacological activities of the 5-benzyl analogue of the μ opioid analgesic 14-methoxymetopon (14-MM). The result of the replacement of the 5-methyl in 14-MM with a benzyl group on in vitro opioid receptor binding and functional profiles, and in vivo behavioural properties, i.e. nociception and motor activity, was investigated. In rodent brain membranes, the 5-benzyl derivative showed high affinity at the μ opioid receptor and decreased interaction with δ and κ receptors, hence displaying a similar binding profile as 14-MM. It displayed potent agonist activity in vitro and in vivo. In in vitro guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding assay, it activated G-proteins in rat brain membranes through a μ opioid receptor-mediated mechanism having significantly enhanced potency compared to DAMGO (d-Ala2,Me-Phe4,Gly-ol5]enkephalin), and to the μ opioid agonist morphinans 14-MM, 14-O-methyloxymorphone (14-OMO) and morphine. In vivo, the 5-benzyl analogue of 14-MM elicited dose-dependent and naloxone-sensitive antinociceptive effects in hot-plate and tail-flick tests in mice after subcutaneous (s.c.) administration. Its analgesic potency was comparable to 14-MM, and was 50-fold higher than that of morphine. Contrary to morphine, 14-MM and 14-OMO, no motor dysfunction was produced by the new opioid in the mouse rotarod test at any of the tested doses. In summary, the 5-benzyl analogue of 14-MM emerged as a novel potent μ opioid antinociceptive agent with reduced propensity to cause unwanted motor impairment.