Abstract
Background and aims
We have previously reported that sinomenine, an alkaloid isolated from the root of the plant Sinomenium acutum, had antinociceptive effect in rodent models of acute inflammatory or neuropathic pain. As a traditional medicine, sinomenine is used in China to treat rheumatoid arthritis (RA).
Methods
In the present study, we evaluated the potential antinociceptive effect of sinomenine in a mouse model of RA, collagen type II antibody (CII Ab) induced arthritis (CAIA) after acute and chronic administration.
Results
As single administration, sinomenine at 40 or 80 mg/kg significantly reduced mechanical hypersensitivity both at the time of peak joint inflammation (days 11–19 after CII Ab injection) or during the post-inflammatory phase (days 35–54). No tolerance to the effect of 80 mg/kg sinomenine was observed during repeated injection twice a day for 5 days from day 11 to day 19 or from day 49 to day 53 after CII Ab injection in CAIA mice.
Conclusions
We have shown that sinomenine is effective in alleviating localized and spread hypersensitivities in CAIA mice both during acute inflammation and in post-inflammatory phase. Further, repeated sinomenine administration has elevated the baseline mechanical threshold without producing tolerance.
Implications
Sinomenine may be clinically useful to treat chronic pain in RA, including wide-spread pain which appears to be a difficult clinical problem despite the improvement in the acute treatment of RA by disease modifying agents.
1 Introduction
Pain is a major clinical feature of rheumatoid arthritis (RA). Not only has pain been suggested to be an important patient-reported outcome in RA [1,2], but also the severity of pain has strong impact on the quality of life of patients. Although pain is usually considered as a marker for inflammation, recent studies have shown that in many RA patients chronic pain continue to be a major problem even after the remission of inflammation [2]. Furthermore, many RA patients also suffer from chronic wide-spread pain in a fashion similar to fibromyalgia [3].
Non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen are first line pain therapies in RA and weak opioids are also sometimes used [2]). In both cases, analgesia in a substantial portion of patients remains unsatisfactory and long-term application of these drugs is limited by side effects and in the case of opioids, the development of tolerance [4]. Disease-modifying anti-rheumatic drugs such as methotrexate are known to reduce acute symptoms in RA including pain, but their efficacy against the development of chronic pain is less clear [1,3,5].
The root of the climbing plant Qingteng (Sinomenium acutum) has long been used in East Asia as a remedy for disease conditions similar to rheumatism as recorded in for example the 16th century book, Bencao Gangmu (Compendium of Materia Medica) [6]. The major active component in Qingteng has been identified as sinomenine, a morphinan derivative alkaloid that is structurally similar to dextromethorphan. We have recently shown that sinomenine possesses a wide spectrum of analgesic properties towards different experimental pain conditions in rodents, including acute inflammatory pain by carrageenan or neuropathic pain after peripheral/central nervous system injury [7]. In the present study, we examined the potential antinociceptive effect of single or repeated sinomenine administration against experimental arthritic pain. The collagen antibody induced arthritis (CAIA) model was used, which is a mouse model of RA based on the injection of a cocktail of monoclonal antibodies directed against type II collagen followed by immunizing the animals with lipopolysaccharide (LPS) [8]. In this model the local joint pathology resembles that observed in RA patients and pain-like responses, mainly manifested as localized and/or spread mechanical hypersensitivity, can be detected both during the acute inflammatory phase or chronic post-inflammatory phase [9].
2 Material and methods
2.1 Animals
All experiments were approved and conducted strictly followed regulations of the regional research ethics committee. Female CBA mice (Harlan, Horst, The Netherlands), weighing 25–30 g were used. The animals were housed 6 per cage with a constant room temperature at 22°C in a 12:12 light-dark cycle and ad libitum access to food and water.
2.2 CAIA in mice
In CBA mice, as described previously [9], CAIA was induced by intravenous (i.v.) injection of anti-CII arthritogenic antibody cocktail (0.15 ml, Chondrex, USA), which contains 5 monoclonal antibodies on day 0. Then, immune reaction was triggered by intraperitoneal (i.p.) injection of 35 μg LPS, (serotype O55:B5; Sigma) diluted in 100 μl of physiologic saline on day 5. Inflammation in the joints was examined and evaluated by visual inspection after antibody cocktail injection. The scoring was based on joint inflammation in each paw, being defined by swelling and redness [8]. Briefly, each inflamed toe gave one point, an inflamed wrist or ankle gave five points, resulting in a score of between 0 and 15 for each paw and between 0 and 60 for each mouse.
2.3 Assessments of mechanical hypersensitivity
Baseline mechanical sensitivity in the hind paw, neck and flank areas were measured five times at three day intervals before the collagen antibody injection. Animals with baseline threshold below 50% of the average value were excluded. After collagen type II antibody injection, mechanical threshold was tested for 54 days, always at the same time during the day. For testing of paw withdraw threshold, the mice were placed in plastic cages with a metal mesh floor. After habituation for 1 h the plantar surface of the hind paw was stimulated with a set of calibrated von Fray hairs (Marstock, Denmark). The up-down method [10] was used to calculate the force that caused paw withdrawal in 50% of trials. For testing spread mechanical hypersensitivity the mice were gently restrained in a standing position, and the flanks and upper back were stimulated using von Frey hairs (Stoelting, Chicago, IL, USA). Stimuli were applied 5–10 times at each intensity at 1 s-1. The stimulus which induced consistent avoiding or offensive behaviours (>60% respond rate) was considered as responding threshold. The cut-off value was 4g on the paws and 100 g on the flanks and back.
2.4 Assessments of side effects
To assess if mice develop motor deficiencies, severe allergy or sedation after sinomenine application, we performed an open field test in naive mice. The open field arena is 50 cm × 50 cm with 25 grids (the area of one grid is 10 cm × 10 cm), in which mice (without any previous experience in the open field test) were allowed to move freely for 5 min. The total travel distance (quantified by the number of passed grids), number of rearing behaviours and duration of passivity (time when animal showed no movement) were measured. After the open field test, rectal temperature was monitored by a thermometer (FHC, Bowdoin, ME, USA).
2.5 Drugs
For preparation of injecting solutions, sinomenine (standard substance was obtained from The National Institute for Food and Drug Control, Beijing, China) was first dissolved with DMSO (Sigma-Aldrich), then mixed with Cremophor EL oil (Sigma-Aldrich) and saline by a vortex mixer (Bibby Scientific, UK) using the volume rate of 1:4:5. Any further dilution was made with saline. Sinonemine was injected subcutaneously (s.c.), into the loose skin over the neck. Dose response curves for sinomenine were acquired in the acute phase (days 11–19 after CII antibody injection) and chronic phase (days 36–54 after CII antibody injection).
2.6 Statistics
The experiments were conducted blindly. Data were presented as mean ±SEM, and were analyzed by ANOVA (with or without repeated measurements) followed by Fisher’s PLSD post hoc test, Wilcoxon signed rank test, and Mann–Whitney U test. P < 0.05 was considered to be statistically significant.
3 Result
3.1 The dose-dependent effect of sinomenine against mechanical hypersensitivity of the hind paw
During the first 3 h after drug administration in the inflammatory phase of CAIA (days 11–19 after CII antibody injection), a single dose of 40 and 80 mg/kg s.c. sinomenine dose-dependently reduced mechanical hypersensitivity in the hind paws (Fig. 1A). In the post-inflammatory phase during days 35–54 post CII antibody, sinomenine also had a similar effect as during peak inflammation (Fig. 1B).
The effect of saline or various doses of sinomenine injected s.c. on hind paw withdrawal threshold to mechanical stimulation during acute inflammation (at one day during days 11–19, A) and post inflammation (at one day during days 36–54, B) in CAIA mice. Arrow indicates drug application which is just prior to behavioural testing at time 0 h. The threshold at time 0 h represents hyperalgesic value. Increased threshold represents an attenuated/alleviated hyperalgesia. N = 6–11 mice in each group. Data are presented as mean±SEM. ANOVA with repeated measures indicated a significant general difference betweenthegroups (F = 11.70, P < 0.0001 for A, and F = 14.27, P < 0.0001 for B). Fisher’s PLSD post hoc test indicated that the effect of sinomenine at 40 or 80 mg/kg is significantly different from the saline group. *P < 0.05, **P < 0.01, post-drug time points were compared with pre-drug baselines using Wilcoxon signed rank test. #P < 0.05, ##P < 0.01, post-drug time points were compared with corresponding saline controls using Mann-Whitney U test.
3.2 The dose-dependent effect of sinomenine against spread mechanical hypersensitivity
As we have previously observed, mice subjected to CAIA developed, in addition to localized mechanical hypersensitivity of the paws, a spread mechanical hypersensitivity primarily at the neck and flanks. A single dose of 40 or 80 mg/kg sinomenine also significantly alleviated the spread mechanical hypersensitivity during the first 3–4 h after drug administration both in the inflammatory and post-inflammatory phases of CAIA (Fig. 2A and B).
The effect of saline or various doses of sinomenine on response threshold tomechanical stimulation at the neck/flank region during acute inflammation (at oneday during days 11–19, A) and post inflammation (at one day during days 36–54, B) inCAIA mice. Arrow indicates drug application which is just prior to behavioural test-ing at time 0 h. The threshold at time 0 h represents hyperalgesic value. Increasedthreshold represent an attenuated/alleviated hyperalgesia. N = 6–11 mice in eachgroup. Data are presented as mean ± SEM. ANOVA with repeated measures indi-cated a significant general difference between the groups (F = 7.00, P < 0.01 for A, and F = 2.68, P < 0.05 for B). Fisher’s PLSD post hoc test indicated that the effectof sinomenine at 40 or 80 mg/kg is significantly different from the saline group. *P < 0.05, **P < 0.01, post-drug time points were compared with pre-drug baselinesusing Wilcoxon signed rank test (A, B). #P < 0.05, ##P < 0.01, post-drug time pointswere compared with corresponding saline controls using Mann–Whitney U test.
3.3 No side effects were observed following single-dose sinomenine administration
For detecting potential side effects produce by single-dose sinomenine, we applied open field test for 5 min in naive mice and naive mice injected with saline or 80 mg/kg sinomenine (1 h prior to test). There was no increase of the duration of passivity (Fig. 3A), which is the sign for allergy or sedation, after sinomenine or saline application. In addition, locomotor activities quantified by number of passed grids (Fig. 3B) and number of rearing behaviours (Fig. 3C) were also not changed following sinomenine administration. Further, rectal temperature (which can be affected by severe allergy), was also similar in the sinomenine treated group compared to naive or saline treated animals (Fig. 3D).
3.4 Effect of repeated administration of sinomenine
Repeated injection of 80 mg/kg sinomenine 2 times/day for 5 days during days 11–15 post CII antibody administration (inflammatory phase), had no effect on the arthritis scores in the CAIA model in comparison to saline treated animals (Fig. 3A). During the post-inflammatory phase, the arthritis in CAIA animals slowly dissipated from day 30 to day 54, repeated sinomenine at days 49–53 had no effect on the arthritis scores (Fig. 3A).
Sinomenine administered 2 times/day for 5 days during the peak of inflammation significantly alleviated the mechanical hypersensitivities in the hind paws (Fig. 3B) and in the neck/flank region (Fig. 3C). Baseline mechanical hypersensitivity was significantly increased from the second day after the start of repeated sinomenine treatment, and remained significantly elevated for 3 days after the cessation of sinomenine treatment (Fig. 3B and C).
Effect of saline or 80 mg/kg sinomenine on duration of passivity (A), moved distance (B), number of rearing behaviours (C) and rectal temperature (D), in naive mice during open field test for 5 min. N = 5–6 mice. Data are presented as mean±SEM. One-way ANOVA indicated there is no significant difference between groups in (A-D; P > 0.05).
During the post-inflammatory phase, repeated sinomenine administration (at days 49–53 post CII antibody administration, 80 mg/kg, 2 times/day) significantly alleviated mechanical hypersensitivity both of the hind paws and on the neck/back regions (Fig. 3A and B). Baseline mechanical hypersensitivity was significantly increased from the second day after the onset of repeated sinomenine treatment for the hind paw, but only on day 5 for the spread hypersensitivity (Fig. 3B and C). The effect persisted for at least one day after the cessation of sinomenine treatment as the experiments were terminated on day 54 according to a predetermined schedule (Fig. 3B and C).
No side effects were observed during repeated sinomenine treatments during both the inflammatory and post-inflammatory phases (Fig. 4).
Effect of 80 mg/kg sinomenine injected 2 times/day for 5 days on the development of arthritic scores (A), mechanical hypersensitivity of the hind paw (B) and spread mechanical hypersensitivity (C) in CAIA mice. N = 6 mice per group. Data presented as mean±SEM. In (A) there is no significant difference between sinomenine and saline treated groups in the arthritic scores. In (Band C) sinomenine upon repeated treatment reduced mechanical hypersensitivitiy (#P < 0.05) compared to corresponding pre-drug value on each day of the treatment (B, C), and *P < 0.05, compared to the baseline value of days 11 and 49 (starting time of repeated sinomenine treatment) using Wilcoxon signed rank test.
4 Discussion
As previously observed [9], experimental arthritis using the CAIA model generated hyperalgesic response (mechanical hypersensitivity to paws) both during the acute inflammatory phase and during the post-inflammatory phase. Furthermore, in CAIA mice there is a spread mechanical hypersensitivity in the neck/back region which can be observed both acutely and post inflammation. In the present study, we were able to demonstrate that administration of sinomenine effectively and dose-dependently alleviated the localized and spread mechanical hypersensitivity during both phases without producing side effects such as motor deficiency, severe allergy or sedation. This supports our previous conclusion that sinomenine is a novel analgesic with a wide spectrum of activities against different types of pain [7]. Furthermore, repeated administration of sinomenine during the peak of inflammation did not change the arthritic scores, despite producing marked analgesia. Thus, it is likely that the analgesic mechanism of sinomenine is independent from possible anti-inflammatory action of the compound.
Sinomenine is used in China and Japan as an anti-rheumatic drug [11]. It has been reported that in collagen induced arthritis (CIA) mice, treatment with sinomenine decreased the incidence and severity arthritis [12]. Clinical research has also indicated that compared with NSAIDs, sinomenine was more effective in ameliorating morning stiffness, painful joints and erythrocyte sedimentation rate in RA patients [13]. In the present study, however, we did not find that repeated sinomenine (2 times/day for 5 days) reduced acute inflammation (arthritis score) in the CAIA mice. This could be due to several factors, such as dose, timing of the treatment and models used. In contrast to the CIA model, which requires T-cell activation, the CAIA model, by directly injecting antibodies against the type II collagen to trigger arthritis, bypasses this step. Thus, the anti-rheumatic effect of sinomenine may be related to inhibition of T-cell activation. It is also possible that some of the attribution of sinomenine as an anti-rheumatic may be derived from its analgesic effect against arthritic pain.
No tolerance was seen to the analgesic effect of sinomenine following repeated administration. Rather, there was a significant increase in pre-drug response threshold which lasted beyond the duration of drug treatments. Lack of tolerance to the effect of sinomenine was similarly noted in rodent models of neuropathic pain [14]. It has also been shown that long-term pretreatment with sinomenine may delay the analgesic tolerance to morphine [15]. Sinomenine has a relatively short half-life in the plasma of rodents with no accumulation [16,17]. It is thus unlikely that this effect of repeated sinomenine administration is due to an accumulation of the drug. The metabolites of sinomenine are present in at least three forms [18]. It is unclear whether these metabolites are responsible for the effect of repeated sinomenine since pharmacological properties of these metabolites are unknown. Finally, the effects of repeated sinomenine may reflect lasting, but not permanent, changes in the nervous system resulting from repeated drug treatment.
The mechanism for the effect of sinomenine in the CAIA model is not clear. Sinomenine is not an opioid and the antinociceptive effect of sinomenine in neuropathic pain is not mediated by naloxone sensitive opioid receptors [7]. Sinomenine can interact with neuro-immune crosstalk by suppressing microglia activation [19,20], reduce inflammation and hyperactivity in the central nervous system. Previously, microglial activation was found in the spinal cord of CAIA but not control mice [9], which suggests the presence of microglia mediated central sensitization in this model. Thus, it is possible that down-regulation of microglial activities in the spinal cord by sinomenine can be responsible for the reduction pain-related behaviour in the CAIA model. Moreover, sinomenine can modulate the synthesis of factors considered important for RA induced inflammation and pain, such as TNF, prostaglandin E2, INF-γ, reactive oxygen species, NO, NF-kB, p38MAPK and metal-loproteinases [12,19,21,22,23]. Suggesting that, it is also conceivable that the analgesic mechanisms of sinomenine can be mediated by these factors.
5 Conclusions
In conclusion, the present results have shown that sinomenine is effective in alleviating localized and spread hypersensitivities in CAIA mice both during acute inflammation and in post-inflammatory phase. Further, repeated sinomenine administration has raised the baseline mechanical threshold without producing tolerance.
6 Implications
The present results indicate that sinomenine by itself or in combination with other established drugs, may be clinically useful in management of chronic pain in RA conditions, including widespread pain which appears to be a difficult clinical problem despite the improvement in the acute treatment of RA by disease modifying agents.
Highlights
Sinomenine is effective against mechanical allodynia in mice with experimental RA.
Sinomenine also alleviated spread pain behaviours in these mice.
Repeated sinomenine achieved better analgesic efficacy without tolerance.
Sinomenine may be clinically useful to treat chronic pain in RA.
DOI of refers to article: http://dx.doi.org/10.1016/j.sjpain.2015.01.002.
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Conflict of interest We declare that there is no conflict of interest.
Acknowledgements
This study was supported by Swedish Science Council (project 12168), the Swedish Foundation for Strategic Research and research funds of the Karolinska Institutet.
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