Doxepin inhibits paclitaxel neuropathic pain

Doxepin reverses the expression and development of paclitaxel-induced neuropathic pain

Abstract

Anticancer drugs such as paclitaxel frequently produce peripheral neurotoxicity which leads to sensory function, mild paresthesia, dose reduction and discontinuation of treatment. This study was designed to evaluate the effect of acute and chronic doxepin administration on the development and expression of neuropathic pain during cancer chemotherapy.

Neuropathic pain was induced by paclitaxel (2 mg/kg, i.p., once daily from the first day to the 5^th day) injection in the mice that resulted in mechanical and cold allodynia.

Acute doxepin injections (10 and 15 mg/kg i.p.) on the 11^th day reduced tactile and cold hypersensitivity significantly compared to the control group.

When doxepin (2.5, 5 and 10 mg/kg i.p.) administered daily from the 1^st day to the 5^th day, cold and mechanical allodynia development significantly has been inhibited.

Besides doxepin (5 and 10 mg/kg i.p.) administration from the 6^th day, once the model had been fully established, to 10^th day significantly inhibit cold and mechanical allodynia expression.

Because of concerns related to decrease chemotherapy efficacy we also have evaluated paclitaxel cytotoxicity effect in combine with doxepin. Our results showed that doxepin even at high concentrations (1 and 10 µg/ml) didn’t interfere with cytotoxic effect of paclitaxel (0.05µg/ml).

These results indicate that doxepin, when administered during chemotherapy, can prevent the development and expression of paclitaxel induced neuropathic pain.

Key words: Paclitaxel, Neuropathic pain, Mice, Doxepin

Introduction

Anticancer drugs frequently produce peripheral neuropathy, which is clinically significant and may lead to dose reduction or discontinuation of treatment [1]. Paclitaxel is one of the most effective and frequently used chemotherapy for treatment of breast cancer [2]. However, paclitaxel produces peripheral neurotoxicity that leads to the sensory abnormalities and neuropathic pain during the treatment and it is even persisting after paclitaxel therapy. In some cases, neuropathic pain would impair patient’s quality of life and their daily activity [3].

Paclitaxel affects microtubules interrupts mitochondrial function and DNA synthesis. These effects on peripheral nerves may lead to spontaneous activity of fibers   [4].

Nowadays the etiology and underlying mechanisms of such pain are poorly understood and the existing treatments including anticonvulsant agents, local anesthetics, and opioids are often ineffective [5, 6].

Chronic pain can cause depression in these patients. Tricyclic antidepressants (TCAs) is the most common treatment methods for chronic pain treatment [7-10].

Doxepin belongs to the TCA that inhibits serotonin and noradrenaline reuptake [11].

The analgesic property of doxepin like the other antidepressants has been reported (12). Topical application of doxepin produces analgesia. Mika et al have reported that doxepin and amitriptyline attenuated the symptoms of neuropathic pain in the mice subjected to chronic constriction injury (CCI), chronic administration of doxepin or amitriptyline also produced allodynia and hyperalgesia in naïve mice [13]. On the other hand Wrzosek et al have reported that doxepin was effective in reducing thermal hyperalgesia and mechanical allodynia [14].

Advantages of doxepin therapy include its lack of adverse interactions with prescription and non-prescription drugs taken and the high degree of safety seen in patients with concomitant cardiovascular and other physical disorders. Thus, doxepin appears to be an excellent choice in the long-term maintenance outpatient treatment of chronic depression [15].

Research has also suggested an interaction between antidepressants and opioid receptors. Activation of the endogenous opioid mechanisms by serotonergic and/or noradrenergic pathways is believed to be involved in the antinociceptive effect of antidepressants [16, 17].

Chronic antidepressant administration can modify opioid receptor densities and increase endogenous opioid levels in certain brain regions [18].

The anti-nociceptive effects of doxepin and it’s mechanisms of action in chemotherapy induced neuropathic pain have not been well understood.

This study was designed to determine the effect of acute and chronic doxepin administration and its related mechanisms in alleviating symptoms of paclitaxel-induced neuropathic pain in mice.

To identify the role of opioid system in the anti-nociceptive effects of doxepin in the neuropathic pain, we examined the effect of naloxone (an opioid receptor antagonist) on the anti-nociceptive effects of doxepin in paclitaxel induced neuropathic pain. Also because of concerns related to decrease chemotherapy efficacy we also have evaluated paclitaxel cytotoxicity effect in combine with doxepin.

2. Materials and Methods

2.1. Animals

NMRI male mice weighing 20-30 g were used. They were housed under a 12-hour light/dark cycle and free access to food and water. The animals were randomly assigned to different treatment groups (n=6-8 in each group). All studies were conducted in accordance with the guidelines for the care of laboratory animals of Ethics Committee of Isfahan University of Medical Sciences and followed the European Commission Directive (86/609/EEC) for animal experiments.

2.2. Chemicals

Paclitaxel was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).  Paclitaxel was dissolved in ethanol-cremophor-saline (5:5:90, v/v/v). Doxepin was purchased from Razak Laboratory (Tehran, Iran). Naloxone was obtained from Touliddarou (Tehran, Iran). They all dissolved in normal saline 0.9%.

2.3. General procedures for drug treatments

To induce the neuropathic pain, paclitaxel (2 mg/kg) intra peritoneally (i.p) was injected on the 1^st day and given daily for 4 additional days with cumulative dose of 10 mg/kg (19). Sham group received only vehicle.

To assess the effects of doxepin on the development (chronic effect) of neuropathic pain, Doxepin (2.5, 5 and 10 mg/kg i.p.) were administered daily from the 1^st day to the 5^th day in the different groups of mice. Also doxepin was injected (2.5, 5 and 10 mg/kg) once daily from the 6^th day until the 10^th day. Mechanical and cold allodynia were assessed on the 11^th day after the first paclitaxel injections [20].

To determine the effect of doxepin on the expression (acute effect) of paclitaxel-induced neuropathic pain, it was injected 30 min before pain assessment on the 11^thday.

2.4. Behavioral tests of neuropathic pain

Animals were placed on top of an aluminum mesh table. They were allowed to adapt for 15 minute. For assessment the cold allodynia, acetone drop was applied via a needle to the plantar surface of each hind paw for three times with 30 seconds interval. The time spent for licking the paw was recorded. The frequency of licking was calculated and expressed as a percentage with the following formula (number of trial attending the hind-paw /total number of trial) × 100. For assessment the mechanical allodynia, von Frey filaments (Steeling, Wood Dale, IL, USA) ranging from 0.16 to 6 g were used as previously described [17]. Bending force was applied to the plantar skin of the right hind paw, and each application was held for 6 seconds, using the up–down method to determine threshold sensitivity.

2.5. Evaluation of the role of opioid receptors

To identify the role of opioid system in the anti-nociceptive effects of doxepin, animals were treated by naloxone (1 mg/kg i.p.) on the 11^thday. Naloxone was injected 10 min before behavioral tests in different groups of mice. In these cases doxepin (15 mg/kg i.p.) was administrated form the 6^thday to the 10^th day.

2.6. In vitro cytotoxic activity

Human breast cancer cell lines, MCF-7 ( ER⁺ ) and MDA-MB231 (estrogen receptor negative [ER⁻]) were purchased from the National Cell Bank of Pasteur Institute of Iran. Cell lines were maintained at 37°C and 5% CO₂. The different cell populations were cultured in RPMI (Biosera, France) containing 10% FBS (Gibco, USA).

In vitro cytotoxicity assay was initiated by separately plating (180 μl) of two cell line (5×10⁴ cells/  ml of media) in 96-well micro plates and incubating for 24 h (37°C, air humidified 5% CO2). After 24h, doxepin and paclitaxel were added together to the 96-well micro plate to obtain 1 and 10 μg/ml and 0.05 μg/ml concentrations, respectively.  They also added to the plates separately with the mentioned doses. Wells containing 180 μl of the cell suspension and 20 μl of DMSO (1%) were considered as negative control while the blank wells contained only 200 μl of the RPMI medium. The micro-plates were further incubated for 48 h. Each well was then treated with 20 μl of MTT solution for 3 h.  Afterwards, the media in each well was replaced with 200 μl DMSO to dissolve the blue insoluble formazan crystals. The metabolic activity in each well was determined by a rapid colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Plates were read by using an enzyme-linked immunosorbent assay (ELISA) plate reader at 540 nm. The cell viability was determined by the following formula 1 and was compared with untreated control [21].

formula 1:

2.7. Data analysis

Data are presented as mean ± standard deviation. Data were compared by one-way analysis of variance (ANOVA) followed by Fisher LSD post-hoc test for multiple comparisons. Time-course analysis of behavioral data was compared by repeated measures ANOVA for each experimental group. Two-way analysis of variance (ANOVA) were used to compare the antagonistic effect of naloxone.

3. Results

3.1. Time-course of paclitaxel-induced cold and mechanical allodynia

As shown in Figure1A and 2A mice, which were treated with paclitaxel, had become much more sensitive to acetone application in paw withdrawal frequency (F_4,29=12.67) and duration of paw licking (F_4,29=8.071) in comparison with control group (p<0.001). Paclitaxel injection led to a significant decrease of paw withdrawal threshold in comparison with control group as shown in Figure 3 (F_4,29=4.521) (p<0.01).

3.2. Effect of doxepin on the expression of cold allodynia

Acute administration of doxepin (10 and 15 mg/kg i.p.) 30 minute before acetone test on the 11^thday significantly blocked paw withdrawal frequency (F_4,29=12.67) and duration of paw licking (F_4,29=8.071) (Figure 1A and 2A) (p<0.001).

3.3. Effect of doxepin on the expression of mechanical allodynia

As shown in Figure 3A, the statistically significant difference was observed in the animals treated with acute administration of doxepin (10 and 15 mg/kg i.p.) on the 11^th day in comparison with control group (F_4,29=4.521) (p<0.01).

3.4. Effect of doxepin on the development of cold allodynia

Chronic treatment with doxepin (5 and 10 mg/kg) from the 1^st day until the 6^th day produced significant anti nociceptive effects against paw withdrawal frequency (F_4,29=15.44) and duration of paw licking (F_4,29=14.423) compare to control group (Fig. 1B and 2B) (p<0.001). Also administration of doxepin (5 and 10 mg/kg i.p.) from the 6^th day until the 10^th day reduced paw withdrawal frequency (F_4,29=6.303) and duration of paw licking (F_4,29=6.785) (Fig.1C and 2C) (p<0.001).

3.5. Effect of doxepin on the development of mechanical allodynia

Chronic administration of doxepin (5 and 10 mg/kg i.p.) from 1^st day until the 6^th day (F_4,29=10.515) and also from 6^th day (F_4,29=6.776), after establishment of neuropathic pain to the 10th day noticeably reduced paw withdrawal threshold compare to control group (Fig. 3B and C) (p<0.01).

3.6. Effects of naloxone on doxepin-induced effects

Naloxone (1 mg/kg i.p.) did not induce any significant change in the nociceptive thresholds.

Figures 4A, B and C show the effect of naloxone (1 mg/kg i.p.) on the anti-nociceptive effect of doxepin injection (15 mg/kg i.p.). Naloxone could not change anti allodynic effects of doxepin on the paw withdrawal frequency (F_3,21=16.356) (Fig 4A), duration of paw licking (F_3,21=5.954) (Fig. 4B) and mechanical allodynia (F_3,21=5.213) (Fig. 4C).

3.7. Cytotoxicity effect of doxepin

The cytotoxicity effect of doxepin on MCF-7 and MDA cell lines was evaluated through micro-culture tetrazolium assay (MTT). Results of the cytotoxicity evaluation against MCF-7 and MDA cell lines are shown in figure 5. Doxepin (1 and 10 µg/ml) exhibited no significant activity against MCF-7 and MDA cell lines. On the contrary paclitaxel (0.05 µg/ml) exhibited significant activity against both cell lines. Also doxepin did not reverse the cytotoxicity effect of paclitaxel (Figures 5A and B).

4. Discussion

Chemotherapy-induced peripheral neuropathy is a relatively common and serious consequence of cancer treatment and, since it is often the main reason for reduction or discontinuation of therapy, it may limit the application of lifesaving agents. We found that systemic administration of doxepin inhibited paclitaxel-induced neuropathic pain.

Considering our results, acute doxepin (10 and 15 mg/kg i.p.) administration inhibited the expression of paclitaxel-induced mechanical and cold allodynia in mice. The repeated co administration of doxepin (5 and 10 mg/kg i.p.) and paclitaxel prevented the development of both kinds of allodynia. When doxepin (5 and 10 mg/kg i.p.) administration was initiated at the 6^th day, once the model had been fully established, and given daily for 4 additional days, mechanical and cold allodynia significantly have been blocked. Results from doxepin cytotoxicity evaluation indicated lack of cytotoxic property and didn’t interfere with cytotoxic effect of paclitaxel.

Note that considerable adverse effects of doxepin such as dizziness, drowsiness or ataxia were not observed. This result is consistent with previous studies that suggested doxepin did not decrease motor performance at low doses (i.e. <30 mg/kg) [13, 22].

Since pain is a complex neurobiological phenomenon with a diversity of neurochemical factors contributing to the both peripheral and central pain-signaling mechanisms its possible that various mechanisms are involved in the anti-nociceptive effect of doxepin.

The earliest focus, with regard to the mechanism of action, was the ability of doxepin to inhibit biogenic amine reuptake [23]. Doxepin inhibits the reuptake of serotonin (5-HT) and, to a lesser extent, noradrenaline (NE).

Given that both 5-HT and NE are involved in the nociception, it is possible that antidepressants that act by modulating these neurotransmitters produced antinociceptive effects. Although studies suggest that antidepressants that target both 5-HT and NE neurotransmission are more effective than serotonergic agents in relieving various types of chronic pain.

Research has also suggested an interaction between antidepressants and opioid receptors. Activation of the endogenous opioid mechanisms by serotonergic and/or noradrenergic pathways is believed to be involved in the antinociceptive effect of antidepressants [16, 17].

Chronic antidepressant administration can modify opioid receptor densities and increase endogenous opioid levels in certain brain regions [18]. It appears that antidepressants may interact both directly and indirectly with endogenous opioid systems to produce analgesia [24].

For example, a study evaluating venlafaxine and mirtazapine found that both agents significantly potentiated antinociceptive effects through interactions with multiple opioid receptors. Other investigations have demonstrated inhibitory effects of naloxone an opioid antagonist on the analgesic effects of antidepressants [8]. Additional data suggest that dual-acting antidepressants (e.g., TCAs) may produce analgesic effects as a result of both direct and indirect interaction with opioid systems [25].

In the present study, we have investigated whether opioid-related mechanisms contribute to the antinociceptive effects of doxepin in paclitaxel induced neuropathic pain. Our results showed that naloxone as an opioid antagonist could not reverse the antinociceptive effects of doxepin.

These results demonstrate that doxepin, which is TCAs, differs from the other tricyclic antidepressants in it antinociception mechanisms.

Interestingly it’ apparent that TCAs exhibit diverse pharmacological properties and this may account for differing specific pharmacological profiles between agents.

The differential involvement of myelinated and unmyelinated peripheral nerve fibers in the pathogenesis of each sign of pain has been reported. Mechanical allodynia is evoked by inputs from myelinated fibers (mainly Aβ-fibers, but also Aβ-fibers), whereas heat hyperalgesia is evoked mainly by unmyelinated C-fibers, and both A – and C-fibers are recruited to mediate cold allodynia [26].

It is well known that voltage gated Na⁺ channels play a critical role in neuronal function and in the development of chemotherapy-induced peripheral neurotoxicity (CINP) [27]. Doxepin inhibits sodium channels which may participate in it’s antinociception effects [28]. However further studies are needed to validate this hypothesis.

5. Conclusions

These results indicate that doxepin, when administered during chemotherapy or ever after that can prevent the expression and development of neuropathic pain. This effect is not inhibited by naloxone which, interestingly, distinguishes doxepin from the other TCAs. Further investigations are required to clarify the possible mechanisms of doxepin in attenuation of neuropathic pain.

Also we have showed that doxepin didn’t interfere with cytotoxic effect of paclitaxel. These results suggest a potential use of doxepin in the management of neuropathic pain during chemotherapy with paclitaxel. However, further pre-clinical and clinical studies are needed to validate this effect and establish the effective doses of doxepin in human, when prescribe for pain.

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Fig .1 Paw withdrawal frequency. (A), Effect of acute treatment with doxepin (5, 10 and 15 mg/kg i.p.) on the paw withdrawal frequency. (B), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 1^st day to the 6^th day. (C), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 6^th day to the 10^th day. The results are expressed as Mean ± S.E.M., ###P<0.001 versus sham group, *P<0.05, **P< 0.01 and ***P<0.001 versus control (mice were treated with paclitaxel 2mg/kg i.p.) group, n=6 in all groups.

Fig. 2 Duration of paw licking.(A), Effect of acute treatment with doxepin (5, 10 and 15 mg/kg i.p.) on the duration of paw licking. (B), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 1^st day to the 6^th day. (C), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 6^th day to the 10^th day. The results are expressed as Mean ± S.E.M., ###P<0.001 versus sham group, **P< 0.01 and ***P<0.001 versus control (mice were treated with paclitaxel 2mg/kg i.p.) group, n=6 in all groups.

Fig3. Mechanical allodynia, (A) Effect of acute treatment with doxepin (5, 10 and 15 mg/kg i.p.) on the paw withdrawal threshold. (B), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 1^st day to the 6^th day. (C), Effect of chronic treatment with doxepin (2.5, 5 and 10 mg/kg i.p.) from 6^th day to the 10^th day. The results are expressed as Mean ± S.E.M., ##P<0.01 versus sham group, *P<0.05, **P< 0.01 and ***P<0.001 versus control (mice were treated with paclitaxel 2mg/kg i.p.) group, n=6 in all groups.

Fig.4. (A) The effect of naloxone (1mg/kg i.p.) on the antiallodynic effect of acute treatment with doxepin (15 mg/kg i.p.) on the paw withdrawal frequency. (B) The effect of naloxone (1mg/kg i.p.) on the antiallodynic effect of acute treatment with doxepin (15 mg/kg i.p.) on the duration of paw licking. (C) The effect of naloxone (1mg/kg i.p.) on the anti allodynic effect of acute treatment with doxepin (15 mg/kg i.p.) on the paw withdrawal threshold. The results are expressed as Mean ± S.E.M., n=6 in all groups.^##P<0.01, ^###P<0.001 versus sham group,**P<0.01,  ***P<0.001 versus control (mice were treated with paclitaxel 2mg/kg i.p.) group.

Fig 5.Cytotoxicity effect of doxepin, (A) Evaluation the cytotoxicity effect of doxepin (1 and 10 µg/ml) alone and in combination with paclitaxel (0.05 µg/ml) on MDA cell lines., (B)Evaluationthe cytotoxicity effect of doxepin (1 and 10 µg/ml) alone and in combination with paclitaxel (0.05 µg/ml) on MCF-7 cell lines, ***P<0.001 versus control (vehicle treated cells) group, Dox 1+ Pac 0.05 (cells were treated with doxepin 1µg/ml in combination with paclitaxel 0.05µg/ml), Dox 10 + Pac 0.05 (cells were treated with doxepin 10µg/ml in combination with paclitaxel 0.05µg/ml).