Male guinea-pigs (300-400 g) and male Kunming mice (18-22 g) were obtained from Beijing Animal Center (Beijing, China). Animals were housed in a temperature-controlled room (25 °C ± 1 °C) and maintained on a 12-h/12-h light/dark cycle. Animals had free access to food and water. Animal care and procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and this study was approved by the Animal Care Committee of Beijing Institute of Pharmacology and Toxicology.

Thienorphine HCl and buprenorphine HCl were synthesized in our institute[4]. Naloxone and acetylcholine (Ach) were purchased from Sigma (St. Louis, MO, United States). NaCl, KCl, CaCl₂, KH₂PO₄, NaHCO₃, MgSO₄, glucose and charcoal were provided by Beijing Chemical Plant. Morphine was produced by Qinghai Pharmaceutical Factory (Xining, China).

Mice were randomly divided into 29 groups with 10 animals in each group. The groups were treated with saline, thienorphine (0.005, 0.05, 0.5, 1.0, 2.5, 5.0, 10 and 20 mg/kg, ig), buprenorphine (0.005, 0.05, 0.5, 1.0, 2.5, 5.0, 10 and 20 mg/kg, sc), morphine (0.5, 1.0, 2.5, 5.0, 10 and 20 mg/kg, sc), and naloxone (0.1, 0.5, 1.0, 5.0, 10 and 20 mg/kg, sc), respectively. Charcoal, used as a marker, was administered orally to mice at 0.3 mL (5 g of charcoal in 100 mL of 0.5% methylcellulose). The mice were subcutaneously treated with saline (10 mL/kg), a single dose of buprenorphine, morphine or naloxone 15 min before administration of the marker. Thienorphine was administered intragastrically 30 min before the marker. At 15 min after charcoal administration, the mice were sacrificed by cervical dislocation, the abdomen was dissected and the intestine removed from the pyloric junction to the cecal end. The distance traveled by the head of the marker and the total length of the intestine were measured. 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.

To determine the effects of an opioid receptor antagonist on the inhibition of intestinal transit by thienorphine (0.5 mg/kg), buprenorphine (0.5 mg/kg) and morphine (10 mg/kg), the animals were pretreated with the non-selective opioid antagonist, naloxone (10 mg/kg, sc), 15 min prior to the administration of these chemicals.

All experiments were performed on isolated ileum from male guinea-pigs weighing 300-400 g. The animals were stunned and decapitated, and the ileum was quickly isolated about 10 cm from the ileo-cecal junction. The myenteric plexus-longitudinal muscle (MPLM) was prepared using the method of Rang[10]. A glass rod was inserted into the lumen of an intestinal segment and the MPLM was removed by rubbing with a cotton swab soaked in Krebs’ solution. The preparations (2.0-2.5 cm length) were suspended under 1.0 g tension in a 10 mL organ bath containing Krebs’ solution (KCl 4.69 mmol/L, CaCl₂ 2.52 mmol/L, KH₂PO₄ 1.18 mmol/L, MgSO₄ 1.22 mmol/L, NaHCO₃ 25.0 mmol/L, NaCl 118.06 mmol/L, Glucose 10.0 mmol/L, pH = 7.4), at 37 °C and bubbled with 95% O₂ and 5% CO₂. The preparations were connected to an isotonic force transducer linked to eight channel organ baths (Medlab6, Meiyi Ltd., Nanjing, China). All the tissues were stimulated by Ach[11,12]. Only the tissue preparations which responded to Ach (1 μmol/L) and produced contractions of more than 1.5 g tension were used. Preparations were equilibrated for at least 1 h with washes every 15 min before exposure to drugs. At the start of each experiment, a maximum response to Ach (1 μmol/L) was obtained in each tissue to confirm its suitability. After washing the preparation, the opioid agonists or antagonist were added to the organ bath for 10 min after which the second contraction with Ach was obtained. Maximal phasic responses were calculated as a percentage of the primary Ach-induced contraction, which was taken as 100% in each experiment. Each experiment was repeated with at least four separate tissue preparations obtained from different animals.

To determine the effects of naloxone, thienorphine or buprenorphine on the inhibition by morphine on MPLM preparations of GPI, the MPLM preparations were treated with morphine (2.4 mmol/L) for 10 min, and then washed twice with Krebs’ solution within 30 min. Before the second morphine (2.4 mmol/L) application, the preparations were treated with naloxone (0.5 mmol/L), thienorphine (32 μmol/L) or buprenorphine (32 μmol/L) for 10 min in the organ baths.

To study the influence of thienorphine on the recovery of GPI contraction, thienorphine (0.1 mmol/L), morphine (3.2 mmol/L), buprenorphine (0.1 mmol/L) or naloxone (1.0 mmol/L) were added to the organ baths containing the tissue preparations. After 10 min treatment, the preparations were washed with Krebs’ solution. The recovery of GPI contraction was evaluated based on stimulation with Ach (1 μmol/L) at different times during the prolonged washing process, and the recovery time-course curves were generated based on GPI contraction.

Statistical and curve-fitting analysis were performed using PRISM 5.0 (GraphPad Software Inc., La Jolla, CA, United States). The data were expressed as mean ± SE. Student’s t test was used to compare single treatment means with control means. Analysis of variance followed by Newman-Keuls post hoc test was used for analysis of multiple treatment means. P < 0.05 was considered statistically significant.

Charcoal (0.3 mL, charcoal in 100 mL 0.5% methylcellulose) did not induce diarrhea in the mice. Stools colored by the marker were of the same form as normal stools, but could easily be distinguished by their black color. Gastrointestinal transit over 15 min was approximately 50% in the saline treated mice. Morphine (0.05-20.0 mg/kg, sc) significantly inhibited gut transit (Figure 2). Thienorphine (0.005-1.0 mg/kg, ig) dose-dependently inhibited gut transit, however, the inhibitory effect was not as strong as that of buprenorphine (0.005-1.0 mg/kg, sc). At higher doses, both thienorphine (1.0-20.0 mg/kg, ig) and buprenorphine (1.0-20.0 mg/kg, sc) inhibited intestinal transit by about 50%-60%. Naloxone (0.5-20.0 mg/kg, sc) had no effect on gut transit compared with the saline treated group. However, naloxone (10.0 mg/kg, sc) antagonized the inhibitory effect of thienorphine (0.5 mg/kg, ig), buprenorphine (0.5 mg/kg, sc) and morphine (10.0 mg/kg, sc) on gastrointestinal transit when it was administered 15 min prior to these chemicals (Figure 3).

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Figure 2 Effects of thienorphine, buprenorphine, morphine and naloxone on gastrointestinal propulsive activity in Kunming mice. Gastrointestinal transit was expressed as % of the distance traveled by an orally administered marker relative to the total length of the small intestine over 15 min after marker administration. Each column and vertical bar represent the mean ± SE of 9-10 mice. Thie: Thienorphine; Bup: Buprenorphine; Mor: Morphine; NLX: Naloxone.

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Figure 3 Effects of naloxone on the gastrointestinal propulsive activity in Kunming mice treated with thienorphine (0. 5 mg/kg), buprenorphine (0.5 mg/kg), or morphine (10 mg/kg). Gastrointestinal transit was expressed as % of the distance traveled by an orally administered marker relative to the total length of the small intestine over 15 min after marker administration. Each column and vertical bar represent the mean ± SE of 9-10 mice. ^aP < 0.05, ^bP < 0.001 vs intestinal transit without naloxone treatment. Thie: Thienorphine; Bup: Buprenorphine; Mor: Morphine.

The GPI displayed regular spontaneous rhythmic contractions following the equilibration period of 45-60 min. A contraction lasted without fading for up to several hours in the control state (Figure 4A). Thienorphine (0.32-32.0 μmol/L) or buprenorphine (0.32-32.0 μmol/L) decreased the basal tonus of GPI contraction in a concentration-dependent manner. The basal tonus of GPI was decreased by 10.0 μmol/L of thienorphine or buprenorphine (Figure 4B and C). The maximum decrease in the basal tonus was 0.02 g by thienorphine (32.0 μmol/L) and 0.03 g by buprenorphine (32.0 μmol/L), with no difference between thienorphine and buprenorphine (Figure 5A). In comparison, morphine (0.4-3.2 mmol/L) concentration-dependently increased the basal tonus and spontaneous movement of GPI, and the maximum increase in basal tonus was about 0.65 g at 2.4 mmol/L (Figure 5B). The basal tonus and spontaneous movement of GPI was increased after the application of morphine 1.6 mmol/L (Figure 4E). Naloxone did not influence the basal contractile tonus, but increased the spontaneous movement of GPI at higher concentrations (Figure 4D).

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Figure 4 Typical trace of thienorphine, buprenorphine, naloxone and morphine on guinea-pig ileum. A: Spontaneous movement of the guinea-pig ileum without chemical; B: The basal tonus decreased after buprenorphine (10.0 μmol/L) treatment; C: The basal tonus decreased after thienorphine (10.0 μmol/L) treatment; D: Spontaneous movement increased after naloxone (0.5 mmol/L) treatment; E: The basal tonus and spontaneous movement increased after morphine (1.6 mmol/L) treatment. The arrow indicates the addition of chemicals. Con: Control; Thie: Thienorphine; Bup: Buprenorphine; Mor: Morphine; NLX: Naloxone.

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Figure 5 Effects of thienorphine, buprenorphine and morphine on the basal tonus of spontaneous movement of the guinea-pig ileum. Each point represents the mean ± SE (n = 5 or 6 preparations). A: Thienorphine (Thie) or buprenorphine (Bup) concentration-dependently decreased the basal tonus of guinea-pig ileum (GPI); B: Morphine (Mor) concentration-dependently increased the basal tonus of GPI; C: Naloxone (NLX, 0.50 mmol/L), Thie (32.0 μmol/L) or Bup (32.0 μmol/L) antagonized the elevation of basal tonus of GPI induced by Mor (2.4 mmol/L). ^aP < 0.05, ^bP < 0.001 vs the basal tonus before chemical application.

Morphine (2.4 mmol/L) increased the basal tonus of GPI from 0.60-0.75 g (Figure 5C). After a 30 min resting period in Krebs’ solution, naloxone (0.5 mmol/L), thienorphine (32.0 μmol/L) or buprenorphine (32.0 μmol/L) was added to the organ baths, the increases in basal tonus following the second addition of morphine (2.4 mmol/L) were all significantly inhibited by about 50%-60% (Figure 5C). Therefore, thienorphine, as well as buprenorphine, showed a potent antagonizing effect against morphine.

The effects of thienorphine, buprenorphine, morphine and naloxone on GPI contraction are shown in Figure 6. All three drugs inhibited ileal muscle contraction induced by Ach (1 μmol/L) in a concentration-dependent manner. Thienorphine exhibited a moderate inhibition of 65% (P < 0.01) at the highest concentration (100 μmol/L). However, buprenorphine exhibited a highly significant inhibition rate of 93% (P < 0.01) at 100 μmol/L. Morphine also showed a significant inhibition rate of 100% (P < 0.01) at the highest dose of 6.4 mmol/L. Naloxone exhibited an inhibition rate of 70% (P < 0.01) at the highest concentration of 1.0 mmol/L.

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Figure 6 Effects of thienorphine, buprenorphine, morphine and naloxone on Ach-induced guinea-pig ileum contraction. Values represent mean ± SE for 4-6 preparations. Thie: Thienorphine; Bup: Buprenorphine; Mor: Morphine; NLX: Naloxone.

When GPI was treated with a single concentration of thienorphine 100 μmol/L, buprenorphine 100 μmol/L, morphine 3.2 mmol/L or naloxone 1.0 mmol/L, the contractile amplitude response to Ach (1 μmol/L) was 27.53% ± 4.21%, 9.60% ± 2.43%, 21.78% ± 4.78% and 31.75% ± 5.08% with respect to the amplitude before treatment, respectively. The ileal muscle strip was washed with Krebs’ solution. During the washing process, the ileal muscle contractile activity response to Ach (1 μmol/L) recovered more slowly in the thienorphine treated group than in the buprenorphine treated group. In the morphine and naloxone treated groups, the response of ileal muscle to Ach (1 μmol/L) recovered completely within 15 min. These results suggested that the effects of thienorphine on GPI lasted longer than those of the other opioids. At 60 min after the washout, the contractile amplitude of the ileal muscle after buprenorphine application recovered to 92.85% ± 3.89% with respect to the amplitude before treatment, whereas in the thienorphine treated preparation, the contractile amplitude remained at 51.25% ± 10.81%, which was significantly lower than that of the buprenorphine treated groups (P < 0.01). At 180 min after the washout, the response to Ach (1 μmol/L) of ileal muscle treated with thienorphine recovered to 85.25% ± 9.97% (Figure 7).

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Figure 7 Time course of the amplitude inhibited by thienorphine (100 μmol/L), buprenorphine (100 μmol/L), morphine (3. 2 mmol/L) or naloxone (1.0 mmol/L) on guinea-pig ileum contraction induced by Ach (1 μmol/L) during the prolonged washing process. The data are expressed as % amplitude with respect to the amplitude before chemical application, and each point represents the mean ± SE (n = 4). Thie: Thienorphine; Bup: Buprenorphine; Mor: Morphine; NLX: Naloxone.

Thienorphine, the new analog of buprenorphine, had several advantages over buprenorphine and may have wider application in the treatment of pain and opioid dependence. However, the utility of opioids is limited by a number of well-known side effects, including tolerance, physical dependence, respiratory depression and gastrointestinal effects.

Opioids exhibit their effects through μ, κ and δ receptors both in the central and peripheral nervous system. Opiates can influence the autonomic outflow to the gut through their effects on the nervous system and have a direct effect on the bowel, and therefore induce changes in gastrointestinal motility and propulsion.

Thienorphine has several advantages over buprenorphine, such as stronger antinociceptive effects, potent rate-decreasing effects and better oral bioavailability than buprenorphine. Therefore, thienorphine, the new analog of buprenorphine, may have wider application in the treatment of pain and opioid dependence. This study reported on thienorphine, a new partial agonist of opioid receptors, which has fewer effects on intestinal transit and isolated guinea-pig ileum contraction.

Thienorphine has less influence and a longer duration of action on intestinal motility, and may be a new candidate for the treatment of pain and opioid dependence.

In vivo intestinal transit tests and isolated guinea-pig ileum assays are well reported methods used to study the effects of chemicals on smooth muscle contraction.

This is an interesting and important paper, which used well known tests for studying the effect of a new drug on motility. Thienorphine had moderate inhibition on intestinal transit, less inhibition on the contractile amplitude and longer duration of action on the guinea-pig isolated ileum. Thienorphine has less influence and longer duration on motility which may be a new useful candidate for the treatment of pain and opioid dependence.