Laparoendoscopic two-site myomectomy (LETS-M) using conventional laparoscopic instruments and the glove-port technique.

PURPOSE: To evaluate the perioperative outcome of laparoendoscopic two-site myomectomy (LETS-M). METHODS: The medical records of 204 women receiving LETS-M in a tertiary referral center, including 183 surgeries performed by the experienced surgeon and 21 surgeries performed by 3 well-supervised trainees were retrospectively reviewed. RESULTS: The age of the participants was 39.3 ± 6.4 years. The mean diameter of the largest myoma and the mean number of myomas were 8.5 ± 2.2 cm and 1.7 ± 1.1, respectively. Thirty-one (15%) operations removed more than 2 myomas larger than 5 cm in diameter. The mean weight of the myomas was 281.1 ± 183.1 g. The operation time was 97.6 ± 40.2 min, and the intraoperative blood loss was 99.3 ± 115.2 mL. There were 3 (1%) cases of excessive blood loss (more than 500 mL) and 2 (1%) of postoperative hematoma. The only significant difference between the experienced surgeon and trainees was the operation time (92.3 ± 32.2 min vs. 141.2 ± 54 min, p < .001), while the myoma number, myoma diameter, myoma weight, and intraoperative blood loss were not significantly different. The operation time did not differ among different myoma locations. In multivariate analysis, virginity, myoma number, more than 2 large myomas, and myoma size were independent variables for longer operation times. No patient experienced any major complications. CONCLUSION: LETS-M using conventional laparoscopic equipment is a minimally invasive surgical method that is safe, effective, and easy to learn for managing uterine myoma. It is useful to achieve a favorable perioperative outcome with acceptable operation time.

Role of Amino Acid Arginine and Nitric Oxide in Mechanisms of Cytoprotective Effect of Non-Opiate Leu-Enkephalin Analogue In Vitro.

Incubation of primary culture of pulmonary fibroblasts with non-opiate analogue of leuenkephalin (NALE; Phe-D-Ala-Gly-Phe-Leu-Arg, 0.1 µM) reduced generation of superoxide anion-radical (by 20.7%) and decreased the number of p53+ cells (by 40.2%) induced by exposure to H2O2 (60 µM). The cytoprotective effect of NALE was potentiated by NO synthase inhibitor L-NAME (1 mM): the number of p53+ cells decreased by 65.3% and morphometric parameters of the cell nuclei and nucleoli were improved. Incubation of pulmonary fibroblasts culture with peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly, 0.1 µM) also significantly reduced the damaging effect of H2O2: the number of p53+ cells decreased by 73.5%, the area of cell nuclei returned to normal, and generation of superoxide anion-radical decreased by 18.4%. These results indicate that C-terminal amino acid Arg and activation of NO synthase are not involved in the direct cytoprotective effect of NALE.

Beta-endorphin inhibits the inflammatory response of bovine endometrial cells through δ opioid receptor in vitro.

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma ß-endorphins increase during bovine uterine inflammation. However, the effect of ß-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of ß-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P < 0.05). By using Western blot and qPCR techniques, we found that ß-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P < 0.05). The co-treatment of ß-endorphins and opioid antagonists showed that the anti-inflammatory effect of ß-endorphins could be blocked (P < 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the µ opioid receptor antagonist CTAP (P > 0.05). In conclusion, ß-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.

δ Opioid Receptor Inverse Agonists and their In Vivo Pharmacological Effects.

The discovery of δ opioid receptor inverse agonist activity induced by ICI-174,864, which was previously reported as an δ opioid receptor antagonist, opened the door for the investigation of inverse agonism/constitutive activity of the receptors. Various peptidic or non-peptidic δ opioid receptor inverse agonists have since been developed. Compared with the reports dealing with in vitro inverse agonist activities of novel compounds or known compounds as antagonists, there have been almost no publications describing the in vivo pharmacological effects induced by a δ opioid receptor inverse agonist. After the observation of anorectic effects with the δ opioid receptor antagonism was discussed in the early 2000s, the short-term memory improving effects and antitussive effects have been very recently reported as possible pharmacological effects induced by a δ opioid receptor inverse agonist. In this review, we will survey the developed δ opioid receptor inverse agonists and summarize the possible in vivo pharmacological effects by δ opioid receptor inverse agonists. Moreover, we will discuss important issues involved in the investigation of the in vivo pharmacological effects produced by a δ opioid receptor inverse agonist.

Hepatoprotective Effect of Neonatal Administration of Non-Opioid Leu-Enkephalin Analogue in Adult Albino Rats Subjected to Antenatal Hypoxia.

We studied the effects of neonatal administration of non-opioid leu-enkephalin analogue peptide NALE) on the morphological parameters of the liver and redox status of 60-dayold albino rats subjected to antenatal hypoxia. In animals subjected to antenatal hypoxia, a decrease body weight and reduction of the area of hepatocytes and total area of their nucleoli were observed; these changes were accompanied by activation of free-radical processes and impairment of antioxidant defense in the liver and blood serum. Intraperitoneal administration of NALE (100 µg/kg, daily) during postnatal days 2-6 normalized body weight, hepatocyte area, and total area of their nucleoli, significantly reduced the intensity of ROS generation, and improved antioxidant protection in the liver and blood serum in 60-day-old animals subjected to antenatal hypoxia.

N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties.

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of ß-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.

Synthesis and Opioid Activity of Tyr1 -ψ[(Z)CF=CH]-Gly2 and Tyr1 -ψ[(S)/(R)-CF3 CH-NH]-Gly2 Leu-enkephalin Fluorinated Peptidomimetics.

We describe the design, synthesis, and opioid activity of fluoroalkene (Tyr1 -ψ[(Z)CF=CH]-Gly2 ) and trifluoroethylamine (Tyr1 -ψ[(S)/(R)-CF3 CH-NH]-Gly2 ) analogues of the endogenous opioid neuropeptide, Leu-enkephalin. The fluoroalkene peptidomimetic exhibited low nanomolar functional activity (5.0±2 nm and 60±15 nm for δ- and µ-opioid receptors, respectively) with a µ/δ-selectivity ratio that mimics that of the natural peptide. However, the trifluoroethylamine peptidomimetics, irrespective of stereochemistry, did not activate the opioid receptors, which suggest that bulky CF3 substituents are not tolerated at this position.

Effects of Opioid Peptides on Changes in Lipid Metabolism in Rats Subjected to Swimming Stress.

Blood levels of nonesterified fatty acids, total cholesterol, triglycerides, and LDL increased in rats subjected to forced swimming stress. Administration of opioid peptides dynorphin A(1-13), DSLET, or DAGO reduced stress-induced disturbances in lipid metabolism. Dynorphin A(1-13) and DAGO produced the most pronounced effects and prevented an increase in concentrations of nonesterified fatty acids, triglycerides, total cholesterol, and LDL as soon as 39 h after treatment. Only DSLET increased HDL content in the plasma of stressed rats. The observed effects can be explained by the stress-limiting effects of opioids, e.g. attenuation of the effect of catecholamines on the adipose tissue and inhibition of the generation LPO products suppressing activity of the cholesterol metabolizing enzyme.

Synthesis of Gly-ψ[(Z)CF═CH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic.

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

[THE INFLUENCE OF OPIOID PEPTIDES DSLET AND DAGO ON LIPID PEROXIDATION AND THE ACTIVITY OF ANTIOXIDANT ENZYMES IN THE LIVER TISSUE OF RATS IN THE STRESS OF DIFFERENT DURATION].

It was established that immobilization stress of different duration (3, 6 or 12 hours) causes the activation of lipid peroxidation in liver tissue of rats 39 hours after stressor action. Activation of lipid peroxidation within 7 days was observed in animals exposed to 6 or 12 hours stress. The activation of superoxide dismutase and catalase activity was revealed in rats after 3 hours immobility, but 12 hours stress was accompanied by superoxide dismutase inhibition. Agonists of different opioid receptors were shown to afford the antioxidant effects inhibiting the accumulation of malondialdehyde and acylhydroperoxides in the liver tissue and stimulating the antioxidant enzyme activity. Opioid peptides had a stimulatory effect on superoxide dismutase activity and less one on catalase activity in animal exposed to immobilization of different duration. A selective agonist of opioid delta-receptors DSLET manifested more expressed antioxidant effect, causing the inhibition of LPO metabolites production and the stimulation of superoxide dismutase and catalase activity.

[THE INFLUENCE OF OPIOID PEPTIDES ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYME ACTIVITY IN RATS AFTER SWIMMING STRESS].

It was established in experiments on rats, that injection of opioid peptides DAGO (a selective igonist of opioid mu-receptors), DSLET (a selective agonist of opioid delta-receptors) or dynorpiin A (1-13) (a selective agonist of opioid kappa-receptors) decreased the stress-induced activatin of lipid peroxidation in liver tissue and plasma. A selective agonist of opioid mu-receptors) AGO manifested the most expressed activity. The using of investigating peptides caused the increase of superoxiddismutase activity in liver tissue. The reinforcement of catalase activity was )bserved in DSLET or dynorphin A (1-13). DAGO decreased its activity. The peptide effects of lifferent directions oncatalase activity in plasma were established. These effects can be explained y the stress-limiting action of peptides in entire organism, the peculiarities of opioid receptors spreading in liver tissue and by the influence of preceded load with non-complete oxidized sub stances after intensive swimming on the opioid receptor affinity.

Naltrindole derivatives with fluorinated ethyl substituents on the 17-nitrogen as δ opioid receptor inverse agonists.

We synthesized derivatives of the δ opioid receptor (DOR) antagonists naltrindole (NTI) and compound 1 that were modified with small alkyl or fluorinated ethyl substituents on the 17-nitrogen. Although the derivatives showed decreased binding affinities for the opioid receptors, their selectivities for the DOR were higher than the parent compounds NTI and compound 1. Surprisingly, 17-fluoroethyl NTI derivatives exerted DOR inverse agonistic activities. The DOR inverse agonism of compounds 4c-e was less efficacious but significant, as compared with a standard DOR inverse agonist ICI-174864. On the other hand, compound 1 and its derivatives with small alkyl or monofluoroethyl substituents were partial agonists, but the derivatives having di- or trifluoroethyl group showed neither agonistic nor inverse agonistic activities.

Chitosan amphiphile coating of peptide nanofibres reduces liver uptake and delivers the peptide to the brain on intravenous administration.

The clinical development of neuropeptides has been limited by a combination of the short plasma half-life of these drugs and their ultimate failure to permeate the blood brain barrier. Peptide nanofibres have been used to deliver peptides across the blood brain barrier and in this work we demonstrate that the polymer coating of peptide nanofibres further enhances peptide delivery to the brain via the intravenous route. Leucine(5)-enkephalin (LENK) nanofibres formed from the LENK ester prodrug - tyrosinyl(1)palmitate-leucine(5)-enkephalin (TPLENK) were coated with the polymer - N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) and injected intravenously. Peptide brain delivery was enhanced because the GCPQ coating on the peptide prodrug nanofibres, specifically enables the peptide prodrug to escape liver uptake, avoid enzymatic degradation to non-active sequences and thus enjoy a longer plasma half life. Plasma half-life is increased 520%, liver AUC0-4 decreased by 54% and brain AUC0-4 increased by 47% as a result of the GCPQ coating. The increased brain levels of the GCPQ coated peptide prodrug nanofibres result in the pharmacological activity of the parent drug (LENK) being significantly increased. LENK itself is inactive on intravenous injection.

The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases.

Synthesis and analgesic activity of alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin.

We report the design and the parallel solid phase synthesis of linear and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The described peptidomimetics represent different unique scaffolds that distribute in the space the peptidyl side chains of amino acids essential for biological activity and mimic the bioactive conformation of the Leu-enkephalin peptide. All the compounds were screened in competitive radioligand binding assays to determine their affinities for µ-(MOR), and κ-(KOR) opioid receptors. A reduced analog of Leu-enkephalin TPI1879-26 with activity Ki=60 nM for the mu receptor was identified.

Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo-medullary pathway may represent the anatomical substrate by which hcrt-1/L-Enk neurons function in the coordination of autonomic-cardiovascular responses during different behavioral states.

δ opioid receptor antagonist, ICI 174,864, is suitable for the early treatment of uncontrolled hemorrhagic shock in rats.

BACKGROUND: Fluid resuscitation is the essential step for early treatment of traumatic hemorrhagic shock. However, its implementation is greatly limited before hospital or during evacuation. The authors investigated whether δ opioid receptor antagonist ICI 174,864 was suitable for the early treatment of traumatic hemorrhagic shock. METHODS: With uncontrolled hemorrhagic-shock rats, the antishock effects of six dosages of ICI 174,864 (0.1, 0.3, 0.5, 1, 3, and 5 mg/kg) infused with or without a small volume of lactated Ringer's solution (LR) before bleeding controlled or bleeding cessation at different times were observed. RESULTS: ICI 174,864 (0.1-3 mg/kg) with or without 1/4 volume of LR infusion showed dose-dependent increase in the mean arterial blood pressure, and significantly prolonged the survival time and 8-h survival rate, as compared with ICI 174,864 plus 1/2 volume of LR infusion. The best effect was shown with 3 mg/kg of ICI 174,864. Bleeding cessation at 1, 2, or 3 h during infusion of ICI 174,864 (3 mg/kg) plus 1/4 volume of LR improved subsequent treatment (70% 24-h survival rate vs. 50 and 10% 24-h survival rate in hypotensive resuscitation and LR group, respectively). There was significant improvement in hemodynamic parameters, oxygen delivery, and tissue perfusion of hemorrhagic-shock rats with 3 mg/kg of ICI 174,864 plus 1/4 volume of LR infusion. CONCLUSION: δ Opioid receptor antagonist ICI 174,864 alone or with small volume of fluid infusion has good beneficial effect on uncontrolled hemorrhagic shock. Its early application can "buy" time for subsequent treatment of traumatic shock.

Preparation and evaluation at the delta opioid receptor of a series of linear leu-enkephalin analogues obtained by systematic replacement of the amides.

Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.

Effect of opioid peptides on the content of LPO products and antioxidant enzyme activity in the liver of rats after restraint stress.

Administration of opioid peptides dynorphin A (1-13) and DSLET was followed by a decrease in the stress-induced activation of LPO and increase in SOD activity in the liver tissue of rats. DAGO produced a similar, but less pronounced effect. The observed changes can be related to a specific distribution of opioid receptors in the liver tissue and stress-limiting influence of these peptides in the whole body.