Abstract
Background
Acute detoxification may lead to withdrawal syndrome. The syndrome is sufficiently aversive in those who are morphine-dependents and thus it hinders abstinence. The opioids are most often used clinically to lighten this syndrome. Here, we evaluated the effects of tricyclic antidepressants (TCAs) in treating physical dependence to opioids upon acute detoxification in mice.
Materials and methods
Adult NRL mice were rendered physically dependent on morphine by increasing daily doses of subcutaneous morphine for 3 days and precipitated withdrawal jumping by subcutaneous naloxone on Day 4. The mice were then assigned to receive intramuscular saline or one of the five TCAs 1 hour prior to creating naloxone-precipitated withdrawal. The withdrawal jumping frequency in 30 minutes was counted after naloxone-precipitated withdrawal.
Results
Our results (the newest findings) based on the equimolar dose (30 μmol/kg), showed that the severity of physical dependence on morphine could be attenuated with less intramuscular TCAs [percent maximal possible effect (MPE) < 50). Amoxapine, protriptyline, amitriptyline, clomipramine, and trimipramine produced a mean percent MPE (43, 18, 37, 45, and 36, respectively); amoxapine, protriptyline, amitriptyline, and clomipramine, also produced a dose-related effect on attenuating the severity of morphine dependence, but not trimipramine. A higher dose had a stronger effect.
Conclusion
Amoxapine, trimipramine, protriptyline, amitriptyline, and clomipramine could have a lightening effect on physical dependence on morphine.
Keywords
morphine; substance withdrawal syndrome; tricyclic antidepressants;
1. Introduction
The hazard of opioid abuse is serious worldwide and creates increasing health and criminal problems. According to a report of the Ministry of Justice in Taiwan, opioids were the major substances of abuse in Taiwan from 2000 to 2007.1 The total number of opioid addicts has decreased annually, but >80% of abusers were repeat offenders in 2008–2011,2 signifying the rampancy of substance addiction.
Until now, the opioids have most frequently been used clinically for treating morphine withdrawal syndrome, which manifests unpleasant autonomic stimulation, and emotional problems (e.g., anxiety, depression, dysphoria, craving, and insomnia). After acute detoxification, the withdrawal syndrome could extend and remain for several months with psychological problems such as sleeplessness, emotional insecurity, and depression. It is hard to withstand the unpleasant withdrawal syndrome, therefore, addicts often give up the treatment and become repeat offenders. There are many reasons for an abstainer to become a repeat offender; one of which is psychological in origin. There may have been a high comorbidity between depression and drug addiction beside neurochemical similarities in depression and drug dependence.3 Tricyclic antidepressants (TCAs) that are used primarily clinically for mood disorders may attenuate morphine withdrawal symptoms.
TCAs potently block the serotonin transporter and the norepinephrine transporter, and therefore, could act as serotonin reuptake inhibitors and norepinephrine reuptake inhibitors.3 TCAs increase both synaptic concentrations of serotonin or norepinephrine and therefore enhance neurotransmission.4
Although a few studies have indicated that TCAs could attenuate withdrawal symptoms,5, 6 not all TCAs have been investigated, and the effects of TCAs on opioid dependence and withdrawal remain incompletely understood. The present study investigated the influence of TCAs on attenuating morphine withdrawal jumping. The chosen five TCAs, amoxapine, trimipramine, protriptyline, amitriptyline, and clomipramine with different aminergic profiles have not been reported previously, to the best of our knowledge.
2. Materials and methods
Adult male NRL mice purchased from the National Laboratory Animal Center, Taiwan weighed 25–30 g at the start of the study. They were housed in groups of three per cage for at least 1 week in a climate-controlled room maintained at 20 ± 1°C with approximately 50 ± 10% relative humidity. Lighting was on a 12-hour light/dark cycle (lights on at 07:00 hours), with food and water available ad libitum except during testing. All tests were performed in accordance with the recommendations and policies of the International Association for the Study of Pain, and the protocol was approved by the Animal Investigation Committee of Chi-Mei Medical Center.
Morphine HCl was purchased from Food and Drug Administration, Department of Health, Executive Yuan, R.O.C. (Taiwan); naloxone HCl, and TCAs (amoxapine, trimipramine meleate, protriptyline HCl, amitriptyline HCl, and clomipramine HCl) from Sigma–Aldrich (St Louis, MO, USA). All drugs were prepared in 0.9% saline. Morphine and naloxone were injected subcutaneously, whereas TCAs were injected intramuscularly. The site for subcutaneous injection was 2 cm below the neck at the midline of the back, whereas that for intramuscular injection was the biceps femoris or semitendinosus of the right hind leg. The volume of injection was 3 mL/kg for all the medications.
In Study 1 for inducing morphine dependence and expressing morphine withdrawal, physical dependence on morphine was induced three times daily by subcutaneous morphine injections (at 09:00 hours, 13:00 hours, and 17:00 hours) for 3 days, with daily increment of the dose (i.e., 20 mg/kg on Day 1, 40 mg/kg on Day 2, and 80 mg/kg on Day 3). The severity of morphine dependence was assessed by measuring the withdrawal jumping after combining a single subcutaneous injection of morphine 40 mg/kg 1 hour later with naloxone 50 mg/kg (at 10:00 hours).7, 8
This morphine–naloxone injection has been widely used as a standard to precipitate dependence on morphine, and the jumping response has been the most reliable withdrawal sign in mice.7, 8 Immediately after naloxone injection, the tested mouse was placed in an individual Plexiglas observation cage (30 cm high × 11 cm long × 11 cm wide). The jumping response was recorded, which was defined as the simultaneous removal of all four paws from the horizontal surface over the next 30 minutes.7, 8 All mice received only one naloxone injection during the whole test. The number of mice subjected to naloxone injection at each time point was three.
In Study 2, for the single dose test of TCAs in morphine-dependent mice, the physical dependence was induced by daily subcutaneous morphine injection according to the dosing schedule demonstrated in Study 1. On the morning (09:00 hours) of Day 4, mice further received 30 μmol/kg TCAs (amoxapine, trimipramine, protriptyline, amitriptyline, or clomipramine) injection intramuscularly in the left hind leg (3 mL/kg). Following the administration of TCAs, the withdrawal jumping was measured by the morphine–naloxone injection described in Study 1. The control group with saline injection received only one naloxone injection during the whole test. The number of mice that received naloxone injection at each time point was three.
In Study 3 for the dose–response (DR) test of TCAs in morphine-dependent mice, the dosing regimens for inducing physical dependence and withdrawal jumping were the same as those in Study 2. On the morning (09:00 hours) of Day 4, the mice received one of the dosage forms of intramuscular TCAs. In Study 3, mice received amoxapine 0.3 μmol/kg, 3 μmol/kg, 30 μmol/kg, or 60 μmol/kg; trimipramine 0.0003 μmol/kg, 0.03 μmol/kg, 3 μmol/kg, 30 μmol/kg, or 300 μmol/kg; protriptyline 0.003 μmol/kg, 0.01 μmol/kg, 0.03 μmol/kg, or 30 μmol/kg; amitriptyline 3 μmol/kg, 30 μmol/kg, or 300 μmol/kg; or clomipramine 3 μmol/kg, 30 μmol/kg, or 300 μmol/kg.
In the regression analysis (version 1.27, Pharm Tools Pro, McCary Group, Wynnewood, PA, USA), the DR curves of TCAs were constructed.9 After fitting, the ED50 (50% effective dose) values from the DR curves were calculated.
3. Results
In Study 1, physical dependence on morphine in mice was induced by daily subcutaneous injection of morphine. The number jumping in the control group with saline injection was 118 [100% maximal positive effect (MPE)].
In Study 2, based on the equimolar dose (30 μmol/kg), the severity of physical dependence on morphine was attenuated by intramuscular TCAs (% MPE < 50). Amoxapine, protriptyline, amitriptyline, clomipramine, and trimipramine produced a mean % MPE (43, 18, 37, 45, and 36, respectively) (Fig. 1).
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In Study 3, with the higher dose (≥30 μmol/kg), amoxapine, protriptyline, amitriptyline, and clomipramine attenuated the severity of morphine dependence more strongly, but not trimipramine (Fig. 2).
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The ranking of drug potency was trimipramine > protriptyline > amoxapine > amitriptyline > clomipramine on an ED50 basis (Table 1).
4. Discussion
TCAs have been widely used in treating major mood disorders. Recent studies have further demonstrated that TCAs could attenuate naloxone-precipitated withdrawal (NPW) jumping, and trimipramine, one of the TCAs, had a potent effect based on ED50. However, under the dosing regimen of trimipramine injections, the number jumping decreased gradually and reached a plateau between doses of 0.0003 μmol/kg and 3 μmol/kg. Trimipramine could not significantly attenuate jumping with increased dosage. Regarding this, TCAs not only could help addicts manage withdrawal, but also could control the mood symptoms after maintenance therapy. These results were consistent with previous TCA data.5, 6
These five TCAs can be divided into secondary amine drugs (i.e., amoxapine and protriptyline) and tertiary amine drugs (i.e., amitriptyline, clomipramine, and trimipramine) in chemical structure. In this study, we found that at ED50 the attenuation of morphine withdrawal jumping by the secondary amine drugs was lower than by tertiary amine drugs, and the slope of the regression equation for the secondary amine drugs was greater than that of the tertiary amine drugs, except for trimipramine. The ED50 of trimipramine was very small, but the slope of the regression equation was greater than for the others. Trimipramine is a tertiary amine drug, but it is not a potent norepinephrine and serotonin uptake inhibitor. This means that the severity of reducing morphine withdrawal jumping may involve the potency of norepinephrine and serotonin uptake activity.
TCAs have many functions, namely as serotonin reuptake inhibitors, norepinephrine reuptake inhibitors, α1-adrenergic antagonists, and anticholinergic–antimuscarinic agents.3 The mechanism of TCAs in depression blocks serotonin transporter to inhibit reuptake of serotonin and increase synaptic concentrations of serotonin.4 Considerable evidence recommends that altered noradrenergic and serotonergic functions could attenuate opioid withdrawal symptoms.10, 11 Therefore, enhancing neurotransmission through increasing the serotonin concentration suggests that the direct effect on serotonin synapses might be involved in the action of TCAs in physical dependence on morphine.12, 13, 14
Some reports have indicated that several different serotonin receptor agonists or antagonists can decrease NPW signs15, 16; serotonin is an important factor in producing some withdrawal symptoms.17 TCAs might decrease NPW signs due to modifying serotonin receptors.
By contrast, TCAs increase serotonin contents, as reported previously in the medial prefrontal cortex of sham-treated rats during opiate withdrawal.13 Thus, TCAs might enhance the inhibitory influences of serotonin on dopamine neurons and then attenuate the stimulatory effects of morphine on these neurons by elevating extracellular levels of serotonin.18 Opioid receptors also have been widely studied in morphine dependence. Some relations between TCAs and opioid receptors have been reported, including those between the former and antidepressants, and could regulate and activate opioid systems,19 through both a direct opioid receptor interaction and an indirect action through enhanced release of opioid peptides to interact with μ- and δ-opioid receptors as agonists.20, 21 If TCAs attenuate the morphine-induced activation of dopamine release, the rewarding effect of morphine would be reduced, resulting in less morphine self-administration,18 which might be another possible role for TCAs.17, 22
In summary, based on the equimolar dose (30 μmol/kg), the severity of physical dependence on morphine could be attenuated by intramuscular TCAs (% MPE < 50). Amoxapine, protriptyline, amitriptyline, clomipramine, and trimipramine produced a mean % MPE of 43, 18, 37, 45, and 36, respectively. With a higher dose, amoxapine, protriptyline, amitriptyline, and clomipramine could strongly attenuate the severity of morphine dependence, except for trimipramine, all of which could potently attenuate morphine withdrawal jumping based on ED50. Our results suggest that TCAs attenuate morphine withdrawal jumping by increasing the synaptic concentrations of neurotransmitters via modifying serotonin receptors.
Acknowledgments
This work was supported by the National Science Council, Taiwan (NSC 96-2314-B-041-002-MY2).