Design, synthesis, and activity evaluation of novel erythropoietin mimetic peptides.

The approval of the erythropoietin (EPO) mimetic peptide drug peginesatide in 2012 was a breakthrough for the treatment of secondary anemia. However, due to severe allergic reactions, peginesatide was recalled a year later. In this study, 12 novel peptides were designed and synthesized by substituting specific amino acids of the monomeric peptide in peginesatide, with the aim of obtaining new EPO mimetic peptides with higher activities and lower side effects than the parent compound. Their cell proliferation activities were evaluated, and the structure-activity relationships were analyzed. Five compounds had equal cell proliferation activity to the control peptide. Among them, one compound showed a higher in vivo activity than the control peptide, with no obvious side effects.

The effect of chronic thienorphine administration on long-term potentiation and synaptic structure in rat hippocampus.

Thienorphine is a new nonselective partial agonist of opioid receptors, which is currently under a Phase II clinical trial in China as a new treatment for opioid dependence. In this study, we compared the effect of thienorphine with morphine on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of the rat dentate gyrus (DG). Furthermore, the effect of thienorphine on the synaptic structure of the CA1 hippocampal region and the expression of synaptophysin was investigated. Results indicated interesting differences between thienorphine and morphine on the modulation of hippocampal synaptic plasticity. Chronic thienorphine treatment facilitated LTP in the LPP-DG cell synapses more than chronic morphine treatment. Morphometric measurement and analysis showed that chronic thienorphine administration decreased the length of the active zone and reduced the thickness of CA1 postsynaptic densities compared with the saline group (control), but were elevated compared with the morphine group. Furthermore, the expression of hippocampal synaptophysin was increased with chronic thienorphine administration but reduced with chronic morphine treatment. Taken together, our study clearly demonstrates that chronic thienorphine treatment enhances LTP, modulates hippocampal synaptic structure, and increases the expression of hippocampal synaptophysin. Therefore, further study is warranted to investigate thienorphine as a new treatment for opioid dependence.

The antinociceptive effects of intracerebroventricular administration of Chicago sky blue 6B, a vesicular glutamate transporter inhibitor.

Accumulating evidence suggests that vesicular glutamate transporters (VGLUTs), which control the storage and release of glutamate, may play a role in pain processing. Chicago sky blue 6B (CSB6B), which is structurally related to glutamate, is a competitive VGLUT inhibitor without affecting plasma membrane transporters. The present study was designed to investigate the antinociceptive effects of CSB6B in a number of pain models. The hot-plate test was used as an acute thermal pain test. Inflammatory pain was evaluated using acetic acid writhing, formalin, and complete Freund's adjuvant tests. Intracerebroventricular administration of CSB6B did not affect acute thermal pain responses in 50 or 55°C hot plate tests. However, CSB6B attenuated acetic acid-induced writhing in a dose-dependent and time-dependent manner. In addition, CSB6B reduced licking/biting behavior during the second phase, but not during the first phase, following an intraplantar injection of formalin. In the complete Freund's adjuvant test, a significant attenuation of thermal hyperalgesia was also observed in CSB6B-treated mice. At antinociceptive doses, CSB6B did not affect mice spontaneous locomotor activity. The present study shows that pharmacological inhibition of VGLUT activity was sufficient to attenuate experimental inflammatory pain and suggests that regulation of VGLUTs might be a novel therapeutic strategy for the treatment of pain.

Pharmacological mechanisms underlying the antinociceptive and tolerance effects of the 6,14-bridged oripavine compound 030418.

AIM: To investigate possible pharmacological mechanisms underlying the antinociceptive effect of and tolerance to N-methyl-7α-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydronororipavine (030418), a derivative of thienorphine. METHODS: The binding affinity and efficacy of 030418 were determined using receptor binding and guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) assays in CHO-µ, CHO-κ, CHO-δ, and CHO-ORL1 cell membranes. The analgesic activity of and tolerance to 030418 were evaluated in thermal nociceptive tests in mice. The effects of 030418 on opioid receptors were further investigated using in vivo pharmacological antagonist blockade and in vitro tissue preparations. RESULTS: The compound 030418 displayed high binding affinity to all subtypes of opioid receptors with K(i) values in the nanomolar range. In [(35)S]GTPγS binding assay, the maximal stimulation of 030418 to µ-, κ-, δ-receptors and the ORL1 receptor was 89%, 86%, 67% and 91%, respectively. In hot-plate test, the antinociceptive effect of 030418 was more potent and longer than morphine. The nonselective opioid receptor antagonist naloxone could completely block 030418-induced antinociception, while both the µ-opioid receptor antagonist ß-FNA and the κ-opioid receptor antagonist nor-BNI attenuated 030418-induced antinociception. In contrast, the ORL1 receptor antagonist J-113397 enhanced the antinociceptive effect of 030418. Additionally, chronic treatment with 030418 resulted in a dramatic development of tolerance that could not be effectively prevented by J-113397. In guinea pig ileum preparation, the existing action of 030418 could be removed with difficulty after prolonged washing. CONCLUSION: The compound 030418 is a novel agonist of opioid receptors with high efficiency, long-lasting effect and liability to tolerance, which may be closely correlated with the methyl group at the N(17) position and the high hydrophobicity of the C(7)-thiophene group in its chemical structure.

[Primary investigation on heterodimerization of kappa-opioid receptor and ORL1 receptor].

This study investigates whether kappa-opioid receptor and ORL1 receptor may interact to form a heterodimer. In immunofluorescence and co-immunoprecipitation experiments, differentially epitope-tagged receptors, colocalization and heterodimerization of kappa-opioid receptor and ORL1 receptor were used and examined in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. The results show that fluorescence of both kappa-opioid receptor and ORL1 receptor were overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified to have the ability to co-immunoprecipitate ORL1-receptors with kappa-opioid receptor and vice versa. In the current study, further evidence was provided for the direct interaction of two subtypes of opioid receptors, kappa-opioid receptor and ORL1-receptor, to form the heterodimerization. The finding represents the novel pharmacological mechanism for modulation of opioid receptor function as well as diversity of G protein-coupled receptors.

Inhibition of endothelin-1 and hypoxia-induced pulmonary pressor responses in the rat by a novel selective endothelin-A receptor antagonist, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe.

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.

[Structure-activity relationships analysis of thienorphine and its derivatives].

Thienorphine is a chemically-new opioid developed in Beijing Institute of Pharmacology and Toxicology. To elucidate the chemical basis for the unique pharmacological effects of thienorphine, 15 derivatives were synthesized according to combinatorial chemistry and the structure-activity relationships of these compounds were studied. It is demonstrated that thienorphine is a potent long-acting partial agonist. N-Cyclopropylmethyl is responsible for the antagonist effect of thienorphine. More importantly, thiophene at the end of side chain is most likely the pharmacophore accounts for the long-lasting effect of thienorphine. Change of the connection of thiophene and the side chain does not result in changes in the antinociceptive activity.

[Pharmacokinetics of 6beta-naltrexol after single and multiple intramuscular injections in Beagle dogs].

The pharmacokinetics of 6beta-naltrexol (6beta-NOL) following single intramuscular administration and multiple intramuscular injection once per day for seven days was studied in 4 Beagle dogs. Plasma concentration of 6beta-NOL in dogs was analyzed by a combination of high performance liquid chromatography (HPLC) and electrochemical detection with naloxone (NLX) as internal standard. After single intramuscular injection of 0.2 mg x kg(-1) 6beta-NOL, the plasma concentration-time curve of the drug was found to fit to a two compartment model with first-order absorption. The main parameters of single dosing were as follows: t1/2alpha was (0.26 +/- 0.23) h, t1/2beta was (4.77 +/- 1.65) h, C(max) was (81.65 +/- 5.61) ng x mL(-1), t(peak) was (0.27 +/- 0.07) h, CL(s) was (1.20 +/- 0.06) L x kg(-1) x h(-1), V/F(c) was (1.94 +/- 0.15) L x kg(-1), and AUC(0-t) was (166.82 +/- 7.68) ng x h x mL(-1), separately. After multiple intramuscular injection of 0.2 mg x kg(-1) 6beta-NOL once per day for seven days, the plasma concentration-time curve of the drug fitted to a two compartment model with first-order absorption too. The main parameters of the last dosing were as follows: t1/2alpha was (0.19 +/- 0.18) h, t1/2beta was (5.79 +/- 1.50) h, C(max) was (79.82 +/- 10.5) ng x mL(-1), t(peak) was (0.18 +/- 0.08) h, CL(s) was (1.12 +/- 0.07) L x kg(-1) x h(-1), V/F(c) was (2.10 +/- 0.27) L x kg(-1), and AUC(0-t) was (173.23 +/- 9.49) ng x h x mL(-1), separately. The difference of the parameters between the first and the last dosing was not significant, showing that the plasma kinetics of 6beta-naltrexol was not changed after multiple administrations. In the course of multiple administration, the peak and valley concentration of plasma 6beta-naltrexol were (79.03 +/- 10.3) and (1.50 +/- 0.93) ng x mL(-1), respectively. No clear adverse events were noted during this study. These results showed that plasma 6beta-naltrexol fits to a two compartment model with first-order absorption in dog after intramuscular administration and their pharmacokinetic parameters were reported. There was no remarkable change on plasma pharmacokinetics of 6beta-naltrexol after multiple intramuscular administrations.

[Detection of blood dissemination during the operation of lung cancer and its significance].

OBJECTIVE: To evaluate the possibility of dissemination of lung cancer cells through blood during the operation for lung cancer. METHODS: The blood samples were taken from 52 patients with non-small cell lung cancer (NSCLC) and 5 patients with benign lung diseases at four different intervals during the operation. The transcription of carcinoembryonic antigen (CEA) messenger ribonucleic acid was assayed by means of nested reverse transcriptase polymerase chain reaction (RT-PCR). A549 (a human adenocarcinoma cell line) served as positive control. The sensitivity has been tested using quantificationally diluted A549 cells. RESULTS: The CEA mRNA positive rates of all four time spots are as follows: 31% (16/52) at beginning of the operation (sample taken from peripheral vein), 54% (28/52) at ligating the pulmonary vein (peripheral vein), 54% (28/52) at ligating the pulmonary vein (pulmonary vein) and 54% (28/52) at 1 hour after ligating the pulmonary vein (peripheral vein). There is no relationship between the tumor identity and the positive rate of CEA mRNA. The positive rate of CEA mRNA is higher in patients with centrally located lung cancer than that in patients with peripherally located lung cancer, similar phenomenon is also found between patients with advanced lung cancer and the patients with early stage of lung cancer. No negative control samples was found to be positive for CEA mRNA, the sensitivity of our test was 1 x 10(-6). CONCLUSIONS: The cancer cell dissemination during operation was demonstrated indirectly in our study, the time of pulmonary vein ligation (later or earlier) may affect the quantity of tumor cells released into circulation. Patients with lung cancer of central type and late TNM stage have more possibility of cancer cell dissemination during operation. More effective means may be needed to avoid the dissemination of cancer cells.

[Significance of microvascular density and vascular endothelial growth factor in breast cancer].

OBJECTIVE: To study the significance of vascular endothelial growth factor (VEGF) and microvascular density (MVD) expression in breast cancer. METHODS: The expression of MVD and VEGF was studied in 81 patients with primary breast cancer by SP immunohistochemical technique. RESULTS: There were 49 patients with high VEGF expression (60.5%) and 35 patients with high MVD expression (43.2%). There was significant correlation between VEGF or MVD expression and TNM stage, but not age, tumor size, ER expression or lymph node status. VEGF was significantly related with MVD expression. Univariate analysis showed that patients with low expression of VEGF and MVD had longer disease free survival (DFS) and overall survival (OS). Grouping univariate analysis showed that VEGF had significant correlation with the DFS of all grouped patients and the OS of patients with LN(+) or stage III lesions. MVD had significant correlation with the DFS and OS of LN(+) patients and the DFS of stage III lesion patients. Multivariate analysis showed that MVD was a risk predictor because of its positive statistic value, the higher expression, the shorter survival time of the patients. CONCLUSION: Both VEGF and MVD are useful prognostic factors in breast cancer. MVD appears to be a strong and independent biologic marker for breast cancer prognosis.

[Comparison of the antagonistic effects of 6 beta-naltrexol and naltrexone against morphine analgesia].

AIM: To compare the antagonistic effects of 6 beta-naltrexol and naltrexone against morphine analgesia. METHODS: The effects of 6 beta-naltrexol and naltrexone against morphine analgesia were observed in mouse heat radiant tail-flick assay and in mouse (55 +/- 1) degrees C hot plate test. The displacement of 6 beta-naltrexol and naltrexone on binding to CHO-mu receptor was observed by radioligand binding study. RESULTS: 6 beta-naltrexol antagonized morphine analgesia but the potency was (6.1 +/- 1.7)% that of naltrexone. The effective duration of 6 beta-naltrexol was 3-4 times that of naltrexone and the peak time of the response was about 0.5-1 h after s.c. equivalent efficacy dose (ED95) in two models. Like naltrexone, 6 beta-naltrexol was effective by oral administration and the potency ratio of p.o./s.c. was 1/3. As an antagonist to opioid receptor, the displacement of 6 beta-naltrexol was about 12.5% that of naltrexone, which was almost in agreement with the efficacies against morphine analgesia in mouse. CONCLUSION: Compared with naltrexone, 6 beta-naltrexol was less potent but duration was longer.

RASSF1A suppresses proliferation of cervical cancer cells.

BACKGROUND: This study aimed to explore the effects of ras association domain family 1 A (RASSF1A) on proliferation and apoptosis of human cervical cancer cell line Hela cells. MATERIALS AND METHODS: RASSF1A was cloned into the pcDNA3.1(+) vector to generate pcDNA3.1(+)-RASSF1A plasmid for transfection into Hela cells. Changes in the proliferation and apoptosis of cultured Hela cells were examined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium chloride assay and flow cytometry. A protein array was used to analyze the expression of apoptotic factors. RESULTS: Plasmid pcDNA3.1(+)-RASSF1A was generated and transfected into Hela cells to stably express RASSF1A in Hela cells. RASSF1A transfection was effective in inhibiting the proliferation of Hela cells up to 52.4%, as compared to cells transfected with an empty plasmid. RASSF1A expression also successfully induced apoptosis in human cervical cells with an apoptosis rate of 20.5%. More importantly, protein array results showed that RASSF1 A transfection induced overexpression of p21 and caspase 8, while decreasing the expression of survivin in Hela cells. CONCLUSIONS: RASSF1A expression was effective in suppressing the proliferation and increasing apoptosis of Hela cells, and may be a potential therapy for cervical cancer in clinic.

Inhibition of vesicular glutamate transporters contributes to attenuate methamphetamine-induced conditioned place preference in rats.

Accumulating evidence suggests that glutamatergic system plays a crucial role in methamphetamine (METH) addiction. In the glutamatergic transmission, vesicular glutamate transporters (VGLUTs) are responsible for transporting glutamate into synaptic vesicles and affect the glutamate concentrations in the synaptic cleft. It is well documented that VGLUTs play an essential role in pathophysiology of several psychiatric and neurological diseases, however, whether VGLUTs also have a role in addiction caused by psychostimulant drugs is still unknown. The present study was underwent to investigate the effect of inhibition of VGLUTs on METH-induced induce conditioned place preference in rats. Rats were induced to conditioned place preference with METH (0.5, 1.0 and 2.0mg/kg) by intraperitoneal injection. Intracerebroventricular administration of 1.0 or 5.0µg Chicago sky blue 6B (CSB6B), a VGLUTs inhibitor, and 2.5h prior to METH was to observe its effect on METH-induced conditioned place preference in rats. The rats receiving METH showed stronger place preference at the dose of 1.0mg/kg than that of other doses. The intracerebroventricular administration of CSB6B (1.0, 5.0µg) 2.5h prior to the exposure to METH attenuated the acquisition of METH-induced conditioned place preference, while CSB6B itself had no effect on place preference. These results indicate that VGLUTs are involved in the effect of METH-induced conditioned place preference and may be a new target against METH addiction.

Multiple mechanisms underlying the long duration of action of thienorphine, a novel partial opioid agonist for the treatment of addiction.

AIMS: It is considered that a long-acting therapy would be advantageous in the treatment of addiction. In a search for novel buprenorphine analogues, thienorphine was demonstrated to be an extremely long-acting orally active partial opioid agonist. This study explored the mechanisms underlying the long-lasting effects of thienorphine. METHODS: The binding kinetics of [(3) H]thienorphine were measured in membrane preparations expressing cloned rat opioid receptors. Flow cytometric analysis was used to determine the effect of thienorphine on the surface opioid receptor number. The long-lasting effects of thienorphine were also confirmed at the tissue level and in vivo. RESULTS: At 37°C, [(3) H]thienorphine showed rapid association with µ- and κ-opioid receptors, while its dissociation was sluggish and biphasic (K-1 = 0.21 min(-1) , K-2 = 0.0078 min(-1) for the µ-receptor; K-1 = 0.17 min(-1) , K-2 = 0.0042 min(-1) for the κ-receptor). Treatment with thienorphine for 24, 48, and 72 h downregulated surface µ-receptor in a dose- and time-dependent manner. The inhibitory effect of thienorphine on guinea pig ileum persisted for more than 120 min after prolonged washing. In vivo, thienorphine exhibited significant antagonism of morphine-induced antinociception for more than 7 days. CONCLUSIONS: These results indicate that multiple factors, including persistent receptor occupation and enhanced receptor downregulation, may contribute to the long-lasting effects of thienorphine that would be beneficial for its application in addiction treatment.

Up-regulation of monoamine oxidase activity in the striatum of rats following chronic treatment of thienorphine.

Thienorphine is a new, non-selective partial agonist of opioid receptors. In our previous study using microdialysis, thienorphine persistently increased the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of rats, without affecting the level of dopamine. This finding suggested that monoamine oxidase (MAO) activity in the striatum of rats increased by chronic thienorphine treatment. In the present study, we investigated whether chronic treatment of thienorphine affected MAO activity in the striatum of rats compared with morphine. Rats were treated subcutaneously (three times/day) with either saline (control), morphine, thienorphine, or a pre-treatment of thienorphine followed by morphine, for 3 or 5 continuous days. A 20-min naloxone challenge given to a sub-group of animals from each group occurred at the end of their 5-day treatment. The results showed that repeated administration of thienorphine significantly elevated MAO activity in the striatum, thus restoring MAO activity, which was inhibited by chronic morphine treatment. These results suggest that increased activity of MAO in the striatum may accelerate the metabolism of dopamine, leading to the elevations of DOPAC and HVA. Furthermore, these findings implicate the involvement of MAO in the pharmacological characteristics of thienorphine.

CHP1002, a novel andrographolide derivative, inhibits pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW264.7 macrophages via up-regulation of heme oxygenase-1 expression.

Andrographolides, a type of diterpene lactone, are widely known to have anti-inflammatory and anti-oxidative properties. CHP1002, a synthetic derivative of andrographolide, has similar anti-inflammatory action in mouse ear swelling test and rat paw edema test. In the present study, the mechanism of anti-inflammatory effects of CHP1002 was investigated in RAW264.7 macrophages. CHP1002 potently suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CHP1002 reduced the production of iNOS-derived nitric oxide (NO) and COX-2-derived prostaglandin E2 (PGE2). CHP1002 induced heme oxygenase-1 (HO-1) expression via activation of extracellular signal-regulated kinase (ERK) and NF-E2 related factor 2 transcription factor (Nrf2). Down-regulation of LPS-induced iNOS and COX-2 expressions was partially reversed by the HO-1 inhibitor zinc protoporphyrin (ZnPP). In addition, CHP1002 significantly attenuated LPS-induced TNF-α, IL-1ß and IL-6 production. CHP1002 effectively induced HO-1 and was capable of inhibiting some macrophage-derived pro-inflammatory mediators, which may be closely correlated with its anti-inflammatory action.

Chicago sky blue 6B, a vesicular glutamate transporters inhibitor, attenuates methamphetamine-induced hyperactivity and behavioral sensitization in mice.

Several lines of evidence demonstrate that glutamatergic system plays an important role in drug addiction. The present study was designed to investigate the effects of Chicago sky blue 6B (CSB6B), a vesicular glutamate transporters (VGLUTs) inhibitor, on methamphetamine (METH)-induced behaviors in mice. Mice were induced behavioral sensitization to METH by subcutaneous injection of 1mg/kg METH once daily for 7 days and then challenged with 1mg/kg METH in 14th day. Intracerebroventricular administration of CSB6B (7.5µg) 2.5h prior to METH was to observe its effects on METH -induced behavioral sensitization. Our results showed that the expressions of behavioral sensitization were significantly attenuated by intracerebroventricular administration of CSB6B 2.5h prior to METH either during the development period or before methamphetamine challenge in mice, while CSB6B itself had no effect on locomotor activity. Meanwhile, pretreatment of CSB6B also attenuated hyperactivity caused by a single injection of METH in mice. These results demonstrated that CSB6B, a VGLUTs inhibitor, attenuated acute METH-induced hyperactivity and chronic METH-induced behavioral sensitization, which indicated that VGLUTs were involved in the effect of chronic METH-induced behavioral sensitization and may be a new target against the addiction of METH.

Effect of thienorphine on the isolated uterine strips from pregnant rats.

Opioid dependence is a serious worldwide health problem. Buprenorphine was used as an alternative to methadone for the treatment of opioid dependence, especially for pregnant women. Thienorphine was a partial opioid agonist with long-lasting antinociceptive effect and high oral bioavailability compared with its analogue buprenorphine. Till now, there was still no research about the effect of thienorphine on the isolated uterine muscles. This study examined the effects of thienorphine on the isolated rat oestrus and pregnant uterine strips. Area under the curve (AUC), amplitude and frequency were studied. Thienorphine induced a concentration-dependent decrease in the frequency and amplitude of the contraction on the isolated oestrus and pregnant uterine strips. Thienorphine exhibited less inhibition on the contractile amplitude of the isolated uterine strips from pregnant rats with the IC50 of 54.11 ± 7.41 µΜ, compared with buprenorphine (IC50, 19.42 ± 2.34 µΜ). In addition, thienorphine also exhibited less inhibition on the contractile frequency of the isolated uterine strips from pregnant rats, with the IC50 of 70.68 ± 12.44 µΜ, compared with buprenorphine (IC50, 19.20 ± 3.87 µΜ). On the isolated uterine muscle from pregnant rats, the AUC was decreased by thienorphine but was less potent than buprenorphine, the IC50 was 37.31 ± 7.43 µΜ for thienorphine and 13.52 ± 2.03 µΜ for buprenorphine. Thienorphine exhibited longer duration on the isolated rat pregnant uterine strips than buprenorphine. Thienorphine has less influence and longer duration on the isolated rat uterine muscles during pregnancy, which may be a new useful candidate for the opioid dependent pregnant women.

Effect of thienorphine on intestinal transit and isolated guinea-pig ileum contraction.

AIM: To evaluate the effect of thienorphine on small intestinal transit in vivo and on guinea-pig ileum (GPI) contraction in vitro. METHODS: The effects of thienorphine on intestinal transit were examined in mice and in isolated GPI. Buprenorphine and morphine served as controls. The distance traveled by the head of the charchol and the total length of the intestine were measured in vivo. Gastrointestinal transit was expressed as a percentage of the distance traveled by the head of the marker relative to the total length of the small intestine. The isolated GPI preparations were connected to an isotonic force transducer and equilibrated for at least 1 h before exposure to drugs. Acetylcholine was used for muscle stimulation. RESULTS: Thienorphine (0.005-1.0 mg/kg, ig) or buprenorphine (0.005-1.0 mg/kg, sc) dose-dependently significantly inhibited gut transit compared with saline. Thienorphine inhibited gut transit less than buprenorphine. The maximum inhibition by thienorphine on the intestinal transit was 50%-60%, whereas the maximum inhibition by morphine on gut transit was about 100%. Thienorphine also exhibited less inhibition on acetylcholine-induced contraction of GPI, with a maximum inhibition of 65%, compared with 93% inhibition by buprenorphine and 100% inhibition by morphine. Thienorphine induced a concentration-dependent decrease in the basal tonus of spontaneous movement of the GPI, the effect of which was weaker than that with buprenorphine. The duration of the effect of thienorphine on the GPI was longer than that with buprenorphine. CONCLUSION: Thienorphine had less influence, but a longer duration of action on GPI contraction and moderately inhibited intestinal transit.

Effects of thienorphine on release of dopamine and noradrenalin: an in vivo microdialysis study in rats.

Thenorphine is a new potent long-acting partial opioid agonist. In present study, the effect of thienorphine on noradrenalin (NA) in the locus coeruleus (LC) and dopamine (DA) and its metabolites in the nucleus acumbens (NAc) and the striatum were examined in freely moving rats during acute and chronic thienorphine treatment followed by naloxone-precipitated withdrawal using the in vivo microdialysis technique. Acute thienorphine (1.0mg/kg, s.c.) treatment had no effect on the level of NA in the LC and the level of DA in the NAc and the striatum. Chronic thienorphine (1.0mg/kg, s.c.) third per day for continued 5 days treatment followed by naloxone-precipitated (5.0mg/kg, i.p.) had not alter the extracellular NA level in the LC and the extracellular level of DA in the NAc and the striatum, but significantly increased the level of DOPAC in the striatum. These changes are thought to reflect a direct effect of thienorphine on release of NA and DA. Thus thienorphine deserves further study as a new treatment for opioid dependence.