Potent, Efficacious, and Stable Cyclic Opioid Peptides with Long Lasting Antinociceptive Effect after Peripheral Administration.

Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for µ-, δ-, and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands, MACE3 demonstrated a better activity than biphalin after intravenous administration, and its corresponding analogue incorporating the hydrazine linker (MACE2) was able to induce longer lasting analgesia following subcutaneous administration. An analogue of MACE2 containing 2,6-dimethyl-l-tyrosine (MACE4) showed the best potency and in vivo antinociceptive activity of this series.

Nociceptin Receptors in Alcohol Use Disorders: A Positron Emission Tomography Study Using [11C]NOP-1A.

BACKGROUND: The neuropeptide transmitter nociceptin, which binds to the nociceptin/orphanin FQ peptide (NOP) receptor, is a core component of the brain's antistress system. Nociceptin exerts its antistress effect by counteracting the functions of corticotropin-releasing factor, the primary stress-mediating neuropeptide in the brain. Basic investigations support a role for medications that target nociceptin receptors in the treatment of alcohol use disorders. Thus, it is of high interest to measure the in vivo status of NOP receptors in individuals with alcohol use disorders. METHODS: Here, we used [11C]NOP-1A and positron emission tomography to measure the in vivo binding to NOP receptors in 15 alcohol-dependent humans as identified by DSM-IV and 15 healthy control subjects matched for age, sex, and smoking status. Alcohol-dependent individuals with no comorbid psychiatric, medical, or drug abuse disorders were scanned following 2 weeks of outpatient monitored abstinence (confirmed with three times per week urine alcohol metabolite testing). [11C]NOP-1A distribution volume in regions of interest (including the amygdala, hippocampus, and midbrain, striatal, and prefrontal cortical subdivisions) was measured with kinetic analysis using the arterial input function. RESULTS: Regional [11C]NOP-1A distribution volume in alcohol dependence was not significantly different compared with healthy control subjects. No relationship between [11C]NOP-1A distribution volume and other clinical measures (including duration and severity of alcohol abuse, craving, and anxiety or depressive symptoms) were significant after correction for the multiple hypotheses tested. CONCLUSIONS: The results of this study do not support alterations in the binding to NOP receptors in alcohol dependence. However, this finding does not necessarily rule out alterations in nociceptin transmission in alcohol dependence.

Pharmacokinetics and pharmacodynamics of dermorphin in the horse.

Dermorphin is a µ-opioid receptor-binding peptide that causes both central and peripheral effects following intravenous administration to rats, dogs, and humans and has been identified in postrace horse samples. Ten horses were intravenously and/or intramuscularly administered dermorphin (9.3 ± 1.0 µg/kg), and plasma concentration vs. time data were evaluated using compartmental and noncompartmental analyses. Data from intravenous administrations fit a 2-compartment model best with distribution and elimination half-lives (harmonic mean ± pseudo SD) of 0.09 ± 0.02 and 0.76 ± 0.22 h, respectively. Data from intramuscular administrations fit a noncompartmental model best with a terminal elimination half-life of 0.68 ± 0.24 (h). Bioavailability following intramuscular administration was variable (47-100%, n = 3). The percentage of dermorphin excreted in urine was 5.0 (3.7-10.6) %. Excitation accompanied by an increased heart rate followed intravenous administration only and subsided after 5 min. A plot of the mean change in heart rate vs. the plasma concentration of dermorphin fit a hyperbolic equation (simple Emax model), and an EC(50) of 21.1 ± 8.8 ng/mL was calculated. Dermorphin was detected in plasma for 12 h and in urine for 48 or 72 h following intravenous or intramuscular administration, respectively.

Retest imaging of [11C]NOP-1A binding to nociceptin/orphanin FQ peptide (NOP) receptors in the brain of healthy humans.

[(11)C]NOP-1A is a novel high-affinity PET ligand for imaging nociceptin/orphanin FQ peptide (NOP) receptors. Here, we report reproducibility and reliability measures of binding parameter estimates for [(11)C]NOP-1A binding in the brain of healthy humans. After intravenous injection of [(11)C]NOP-1A, PET scans were conducted twice on eleven healthy volunteers on the same (10/11 subjects) or different (1/11 subjects) days. Subjects underwent serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (VT; a measure of receptor density) was determined by compartmental (one- and two-tissue) modeling in large regions and by simpler regression methods (graphical Logan and bilinear MA1) in both large regions and voxel data. Retest variability and intraclass correlation coefficient (ICC) of VT were determined as measures of reproducibility and reliability respectively. Regional [(11)C]NOP-1A uptake in the brain was high, with a peak radioactivity concentration of 4-7 SUV (standardized uptake value) and a rank order of putamen>cingulate cortex>cerebellum. Brain time-activity curves fitted well in 10 of 11 subjects by unconstrained two-tissue compartmental model. The retest variability of VT was moderately good across brain regions except cerebellum, and was similar across different modeling methods, averaging 12% for large regions and 14% for voxel-based methods. The retest reliability of VT was also moderately good in most brain regions, except thalamus and cerebellum, and was similar across different modeling methods averaging 0.46 for large regions and 0.48 for voxels having gray matter probability >20%. The lowest retest variability and highest retest reliability of VT were achieved by compartmental modeling for large regions, and by the parametric Logan method for voxel-based methods. Moderately good reproducibility and reliability measures of VT for [(11)C]NOP-1A make it a useful PET ligand for comparing NOP receptor binding between different subject groups or under different conditions in the same subject.

Increased agonist affinity at the µ-opioid receptor induced by prolonged agonist exposure.

Prolonged exposure to high-efficacy agonists results in desensitization of the µ-opioid receptor (MOR). Desensitized receptors are thought to be unable to couple to G-proteins, preventing downstream signaling; however, the changes to the receptor itself are not well characterized. In the current study, confocal imaging was used to determine whether desensitizing conditions cause a change in agonist-receptor interactions. Using rapid solution exchange, the binding kinetics of fluorescently labeled opioid agonist, dermorphin Alexa594 (derm A594), to MORs was measured in live cells. The affinity of derm A594 binding increased after prolonged treatment of cells with multiple agonists that are known to cause receptor desensitization. In contrast, binding of a fluorescent antagonist, naltrexamine Alexa594, was unaffected by similar agonist pretreatment. The increased affinity of derm A594 for the receptor was long-lived and partially reversed after a 45 min wash. Treatment of the cells with pertussis toxin did not alter the increase in affinity of the derm A594 for MOR. Likewise, the affinity of derm A594 for MORs expressed in mouse embryonic fibroblasts derived from arrestin 1 and 2 knock-out animals increased after treatment of the cells with the desensitization protocol. Thus, opioid receptors were "imprinted" with a memory of prior agonist exposure that was independent of G-protein activation or arrestin binding that altered subsequent agonist-receptor interactions. The increased affinity suggests that acute desensitization results in a long-lasting but reversible conformational change in the receptor.

[Physiological effects induced by dermorphin synthetic analogue--opilong].

The effects of repeated opilong injections in a dose of 50 microg/kg/day on subsequent learning of Wistar rats have been studied. The substance caused significant anxiolytic and analgesic effects, as the majority of animals could be learned (90% against 40% in control group) despite of painful stimulus preceding to education. Opilong in a small dose displaced a relation of excitatory-inhibit processes to significant prevalence of excitation although the substance was already absent in an organism for a long time. Raised peripheral sensitivity in all rats, provoked by opilong, correlated with CNS hyper excitability, expressed in stressful, neurotic psychoemotional reactions and in the form of active avoidance. The biochemical blood analysis in opilong-induced rats demonstrated the attributes of prethrombosis in the form of fibrinolysis depression and hypercoagulation. A view is expressed, that the neuromediator brain systems can be the basic point of opilong action, that are responsible for the excitatory-inhibit conditions of CNS functioning referred on maintenance of conditioned field stability.

CNS penetration of the opioid glycopeptide MMP-2200: a microdialysis study.

Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson's disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood-brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/µ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10 mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80 min). The detected level at the end of the experiment (around 250 pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood-brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS.

Tapentadol retard en el dolor crónico intenso / No disponible

Desde la introducción en España de la primera morfina de administración oral, en el año 1988, se ha ido evolucionando en la biodisponibilidad de fármacos opioides potentes no administrados por vía endovenosa. Las formulaciones que permiten la absorción de estos fármacos por la piel o por la mucosa oral han supuesto un antes y un después en la administración de medicación analgésica. Posteriormente, las nuevas formas de liberación inmediata o liberación controlada han acabado de perfilar la seguridad y eficacia de estos nuevos fármacos opioides. Tapentadol retard es un fármaco analgésico de acción central de última generación, aprobado tanto por la EMA (“European Medicines Agency”) en 2010 como recientemente por la Agencia Española de Medicamentos y Productos Sanitarios (“AEMPS”) para el tratamiento del dolor crónico intenso. La sinergia de sus dos mecanismos de acción, el agonismo μ-opioide y la inhibición de la recaptación de noradrenalina permite abordar el dolor desde diferentes mecanismos fisiológicos. Tapentadol retard se absorbe rápidamente tras su administración oral y es excretado casi exclusivamente vía renal. Así como la eficacia analgésica de otros opioides, como codeína y tramadol dependen de su actividad metabólica, la analgesia producida por tapentadol se debe a la acción de la molécula principal, sin metabolitos farmacológicamente activos. Los trabajos analizados en esta revisión demuestran la eficacia de tapentadol retard en el tratamiento del dolor de moderado a intenso en pacientes con dolor lumbar crónico, dolor crónico por artrosis de rodilla o cadera y también en pacientes con dolor por neuropatía diabética. En aspectos de seguridad, este fármaco presenta un perfil de tolerabilidad superior al de los analgésicos opioides actuales, lo cual le confiere un potencial importante en el tratamiento de dolor por procesos crónicos. En esta revisión bibliográfica se hace análisis del mecanismo de acción de tapentadol retard, de su farmacocinética y farmacodinamia, eficacia y seguridad y de su intercambiabilidad respecto a analgésicos opioides (AU)

Since the first orally administered morphine was introduced in Spain back in 1988 the bioavailability of potent opioid drugs to be administered via a route other than the intravenous one has increasingly evolved. Formulations allowing these drugs to be absorbed via the skin or oral mucosa have made a major difference in the administration of analgesic medication. Later still, novel immediate-release or controlled-release forms have finished off the positive efficacy and safety profile of these new opioid drugs. Tapentadol retard is a latest-generation, central-acting pain killer that was approved by the EMA (European Medicines Agency) in 2010 and recently by Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) for the treatment of severe chronic pain. The synergy between its two mechanisms of action -- μ-opioid agonism and noradrenalineuptake inhibition -- allows approaching pain from two different physiological aspects. Tapentadol retard is rapidly absorbed following oral administration, and excreted almost exclusively by the kidneys. While the analgesic efficacy of other opioids, including codeine and tramadol, depend upon their metabolic actions, the analgesia resulting from tapentadol administration derives from its primary molecule’s action, with no active metabolites involved. The papers analyzed in this review show the efficacy of tapentadol retard in the management of moderate to severe pain in patients with chronic low-back pain, chronic knee or hip osteoarthritis, and diabetic neuropathy. Regarding safety, this drug has a superior tolerability profile when compared to current opioid analgesics, which confers a significant potential for the treatment of pain resulting from chronic conditions. This literature review analyzes tapentadol retard’s mechanism of action, pharmacokinetics, pharmacodynamics, efficacy, and safety, as well as its exchangeability for opioid pain killers (AU)

Factors that restrict the intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part I. Role of efflux transporters in the intestinal mucosa.

The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In the Caco-2 cell model, the high P(app,BL-to-AP)/P(app,AP-to-BL) ratios of AOA-DADLE, CA-DADLE, and OMCA-DADLE (71-117) were significantly decreased by including known P-gp inhibitors, GF-12098, cyclosporine (CyA), or PSC-833, in the incubation media, suggesting that P-gp is restricting the AP-to-BL permeation of these cyclic prodrugs. In the in situ perfused rat ileum model, AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to exhibit very low permeation into the mesenteric blood (P(B) = 0.40, 0.56 and 0.42 x 10(-7) cm/s, respectively). PSC-833 was found to increase significantly the P(B) values for all three prodrugs. In contrast, CyA and GF-12918 were either inactive or substantially less active than PSC-833 in increasing the P(B) values of these prodrugs. These data suggest that, while P-gp plays a role, other factors (e.g., substrate activity for other efflux transporters and/or for metabolic enzymes) may contribute to restricting the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the rat intestinal mucosa.

Factors that restrict intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part II. Role of metabolic enzymes in the intestinal mucosa.

The objective of this study was to determine the relative importance of metabolism by cytochrome P450 (CYP) enzymes versus efflux by P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to be rapidly metabolized by rat liver microsomes and human CYP-3A4 and to a lesser extent by esterases. Using an in situ perfused rat ileum model, ketoconazole, a CYP 3A inhibitor, was shown to have no effect (AOA-DADLE) or a slight enhancing effect (OMCA-DADLE, twofold; CA-DADLE, threefold) on their intestinal mucosal permeation. In contrast, inclusion of PSC-833, a P-gp inhibitor, in the perfusate significantly enhanced (7-16-fold) the permeation of the three cyclic prodrugs. Since PSC-833 was found to be a weak inhibitor of CYP 3A4 and to have no inhibitory effects on esterases, phenol sulfotransferases, and glucuronyltransferases, it is suggested PSC-833 enhances intestinal mucosal permeation of these cyclic prodrugs by inhibiting their polarized efflux and not by inhibiting their metabolism. Furthermore, efflux transporters (e.g., P-gp), not metabolic enzymes (e.g., CYP 3A, esterases), restrict the permeation of peptide prodrugs across the rat intestinal mucosa.

Functional identification of a novel transport system for endogenous and synthetic opioid peptides in the rabbit conjunctival epithelial cell line CJVE.

PURPOSE: To investigate whether conjunctival epithelial cells express transport processes for opioid peptides. METHODS: We monitored the uptake of [(3)H]deltorphin II and [(3)H]DADLE, two hydrolysis-resistant synthetic opioid peptides, in the rabbit conjunctival epithelial cell line CJVE and elucidated the characteristics of the uptake process. RESULTS: CJVE cells express robust uptake activity for deltorphin II and DADLE. Both opioid peptides compete with each other for transport. Several endogenous and synthetic opioid peptides, but not non-peptide opioid antagonists, are recognized by the transport process. Though various peptides inhibit the uptake of deltorphin II and DADLE in a similar manner, the uptake of deltorphin II is partly Na(+)-dependent whereas that of DADLE mostly Na(+)-independent. The transport process shows high affinity for many endogenous/synthetic opioid peptides. Functional features reveal that this transport process may be distinct from the opioid peptide transport system described in the retinal pigment epithelial cell line ARPE-19 and also from the organic anion transporting polypeptides, which are known to transport opioid peptides. CONCLUSIONS: CJVE cells express a novel, hitherto unknown transport process for endogenous/synthetic opioid peptides. This new transport process may offer an effective delivery route for opioid peptide drugs to the posterior segment of the eye.

Blood-brain barrier penetration by two dermorphin tetrapeptide analogues: role of lipophilicity vs structural flexibility.

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

[Synthesis, pharmacokinetics, and metabolism of the C-terminal tripeptide of dermorphin and its diastereomer].

A tritium-labeled C-terminal fragment of dermorphin (H-Tyr-13,4-3HJPro-Ser-NH2) and its isomer (H-Tyr-D-[3,4-3H]Pro-Ser-NH2) with molar radioactivity of 35 Ci/mmol were synthesized, and their pharmacokinetics and metabolism in rat organs were studied after their intramuscular injections. The tripeptides were detected in the blood only for 5 min after the injection, and maximum contents of both compounds (approximately 5% of the total amount of the injected label) were registered in the kidneys after 20 min. Both stereomers were shown to penetrate into the brain. We failed to detect any radioactive metabolite, except proline, due to rapid proteolytic degradation of these peptides.

Endomorphin analogs.

Opiate alkaloids, such as morphine, are powerful analgesic agents that are the drugs of choice for the treatment of severe pain. The pharmacological effects of opiates are mediated through the binding and activation of membrane-bound opioid receptors that are found in the central and peripheral nervous systems. Opioid receptors have been classified into three different types, mu, delta and kappa, and are activated by the specific ligands. It has been demonstrated that the most potent antinociceptive effects are mediated by the mu-receptor. However, until 1997 no endogenous ligand for this receptor was known. The identification of endomorphins opened a new era in the research of the mu-opioid system. They are the first reported brain peptides that label mu-receptor with high affinity and selectivity and therefore are proposed as the endogenous mu-opioid receptor ligands. Morphine and endomorphins act as agonists at the same mu-opioid receptor, but the latter are thought to inhibit pain without some of the undesired side-effects of plant opiates. This observation encouraged extensive studies on the possible use of endomorphin analogs as analgesics instead of morphine. This review summarizes a decade of research on structure-activity relationship studies of endomorphin analogs, aimed at obtaining compounds with increased bioavailability, in particular with better barrier penetration and resistance against enzymatic degradation. Chemical modifications that led to obtaining potent and selective agonists and antagonists based on the structure of endomorphins are discussed.

Coupling of ORL1 (NOP) receptor to G proteins is decreased in the nucleus accumbens of anxious relative to non-anxious mice.

We studied the involvement of endogenous ORL1 (NOP) receptors in the anxiety state. In mice selected as "anxious" and "non-anxious", ORL1 (NOP) receptor has been analysed by means of two autoradiographic approaches: [3H]nociceptin binding and nociceptin-stimulated [35S]GTPgammaS binding. We show that differences in anxiety state are associated with differences in G protein coupling efficiency of ORL1 (NOP) receptor in the nucleus accumbens, without any change in the density of the receptors.

Effects of intrathecally administered nociceptin/orphanin FQ in monkeys: behavioral and mass spectrometric studies.

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide that is an endogenous ligand for the N/OFQ peptide (NOP) receptor. The aim of this study was to investigate the behavioral responses of N/OFQ and its major fragment N/OFQ(2-17) in monkeys following i.t. administration. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to quantify the amounts of N/OFQ and N/OFQ(2-17) in the cerebrospinal fluid at specific time points when effects of i.t. N/OFQ were sustained and disappeared. Intrathecal administration of N/OFQ dose dependently (10-100 nmol) produced long-lasting antinociception against a noxious stimulus, 50 degrees C water, and did not elicit itch/scratching responses in monkeys. Subcutaneous pretreatment with a selective NOP receptor antagonist, (+)J-113397 [(1-[3R,4R)-1-cyclooctymethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3,-dihydro-2H-benzimidazol-2-one] (0.1 mg/kg), completely blocked i.t. N/OFQ (100 nmol)-induced antinociception. In contrast, a classic opioid receptor antagonist, naltrexone (0.01 and 1 mg/kg), failed to reverse i.t. N/OFQ-induced antinociception. MALDI-TOF-MS showed that the amount of N/OFQ(2-17) was 4-fold higher than that of N/OFQ at 1.5 h after i.t. administration of 100 nmol N/OFQ. Intrathecal N/OFQ-induced antinociception disappeared at 4.5 h, which corresponded to nearly undetectable cerebrospinal fluid levels of N/OFQ. No other metabolite of N/OFQ was detected at appreciable levels at either the 1.5- or 4.5-h time points. Although significant amounts of N/OFQ(2-17) were detected at the 1.5- and 4.5-h time points, 100 nmol N/OFQ(2-17) i.t. was inactive in changing the monkeys' nociceptive threshold. These results provide the first functional evidence of spinal N/OFQ-induced antinociception in primates and indicate that activation of spinal NOP receptors may be a potential target for spinal analgesics.

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides.

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.

Targeted drug delivery crossing cytoplasmic membranes of intended cells via ligand-grafted sterically stabilized liposomes.

In this study, we tested whether sterically stabilized liposomes (SSL) with surface ligands specific for the mu opioid receptor (MOR) can actively target MOR-expressing cells. Dermorphin, a selective MOR agonist, was conjugated to DSPE-PEG(3400) to obtain DSPE-PEG(3400)-dermorphin. Dermorphin-grafted SSL (dermorphin-SSL) was prepared by thin-film rehydration-extrusion and post-insertion method. DSPE-PEG(3400)-dermorphin and dermorphin-SSL retained the affinity to MOR as determined by receptor binding assay using [(3)H]DAMGO, whereas plain SSL without surface ligands showed no binding to the receptor. Cellular uptake of cholesteryl BODIPY encapsulated dermorphin-SSL was studied by microplate spectrofluorometry as well as fluorescent and confocal microscopy. Significant fluorescence signal was observed inside CHO-hMOR cells after the treatment with dermorphin-SSL, indicative of MOR-mediated endocytosis. In contrast, no uptake of dermorphin-SSL was found in naive CHO cells or CHO-hDOR cells that lack MOR. Taken together, these results demonstrate that dermorphin-SSL delivery system is capable of targeting intracellular components of MOR-expressing cells. Such a system may be applied to carry pharmaceutical agents to achieve region-specific delivery of analgesics and/or to attenuate side effects associated with opioids.

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration.

The stabilities of DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), the capped derivative Ac-DADLE-NH2, and the oxymethyl-coumarinic acid (OMCA)-based cyclic prodrug of DADLE and [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH) were determined at 37 degrees C in rat and guinea pig liver microsomes in the presence and absence of paraoxon, an esterase B inhibitor, and ketoconazole, a CYP3A4 inhibitor. These studies showed that the order of stability in microsomes was: DADLE >> Ac-DADLE-NH2 > OMCA-DADLE = OMCA-[D-Ala2,Leu5]-Enk. While paraoxon produced no significant effect on the stability of the studied compounds in liver microsomes, ketoconazole inhibited the metabolism, suggesting that the capped peptide and the cyclic prodrugs are substrates for cytochrome P450 enzymes. For pharmacokinetic studies, the cyclic prodrugs of DADLE and [D-Ala2,Leu5]-Enk were administered i.v. to rats and guinea pigs. Various biological fluids and tissue (brain, bile, and blood) were collected and analyzed for the free peptide and the prodrugs by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS-MS). These studies showed that the conversion of the cyclic prodrugs to the respective linear peptides (i.e., DADLE and [D-Ala2,Leu5]-Enk) was rapid in rat and guinea pig. In terms of drug elimination, only trace amounts of OMCA-DADLE and OMCA-[D-Ala2,Leu5]-Enk were recovered in guinea pig bile (3.3% and 0.82%, respectively), while significant amounts were recovered in rat bile (38.1% and 51.7%, respectively). Brain uptake of the cyclic prodrugs in guinea pigs compared to previously determined brain uptake of OMCA-DADLE in rats was also significantly different. For OMCA-DADLE, the brain levels of the cyclic prodrug and DADLE in guinea pigs were approximately 80 and 8.5 times greater, respectively, than the levels observed in rat brain. The brain-to-plasma prodrug concentration ratios in guinea pigs (>or= 0.6) were significantly higher than the ratio observed in rats (0.01). These species differences are most likely due to the different substrate specificities of the efflux transporters that facilitate liver clearance of these prodrugs and limit their permeation into the brain.

Melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by increasing the release of beta-endorphin an endogenous opioid.

The occurrence of systematic diurnal variations in pain thresholds has been demonstrated in human. Salivary melatonin levels change following acute pain when other factors that could explain the change have been removed or controlled. Melatonin-induced analgesia is blocked by naloxone or pinealectomy. By using selective radioligands [3H]-DAMGO, [3H]-DPDPE, [3-U69593, and 3H]-nociceptin, we have shown that the bovine pinealocytes contain delta and mu, but not kappa or ORL1 opioid receptor subtypes. In the present study, by using melatonin receptor agonists (6-chloromelatonin or 2-iodo-N-butanoyl-5-methoxytryptamine) or melatonin receptor antagonist (2-phenylmelatonin), we have shown that these agents do not compete with opioid receptor subtypes. However, we observed a time-dependent release of beta-endorphin an endogenous opioid peptide, by melatonin from mouse pituitary cells in culture. Hence, it is suggested that melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by binding to its own receptors and increasing the release of beta-endorphin.