Long-term analgesic effect of clodronate in rodents

Bone 33 (2003) 567–574

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Long-term analgesic effect of clodronate in rodents A. Bonabello,a,* M.R. Galmozzi,a R. Canaparo,b L. Serpe,b and G.P. Zarab b

a Research Department, SPA-Societa’ Prodotti Antibiotici S.p.A., Milan, Italy Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Turin, Italy

Received 21 February 2003; revised 13 June 2003; accepted 18 June 2003

Abstract Several studies have shown that treatment with bisphosphonates can reduce the pain associated with different painful diseases. In a previous study we demonstrated that in mice two bisphosponates, clodronate and pamidronate, had an antinociceptive effect under acute conditions not related to bone processes, after in vein (iv) or intracerebroventricular (icv) injection. The present study tested the time-dependent antinociceptive action of clodronate and pamidronate in comparison with that of acetylsalicylic acid (ASA) and morphine after iv and icv injection using the tail-flick test in acute and chronic treatment. The effects of clodronate on other measures of animal behaviour were also evaluated. In the tail-flick test, administration of clodronate iv produced an antinociceptive effect that was greater than that of ASA and statistically significant up to 16 h; pamidronate iv showed a significant antinociceptive effect for only 6 h. Clodronate and pamidronate icv showed an increase in tail-flick latency time that was significant and lasted for 16 and 6 h, respectively, while morphine produced an antinociceptive effect for 24 h. In the test we found significant differences between male and female mice in the latency time values but not in the length of the analgesic effect. In the chronic treatment paradigm, clodronate produced a significant increase of the tail-flick latency after the first injection. The analgesic effect increased up to 50% after 5 days of treatment. Significant analgesic effects were still present after 3, 7, and 14 days from the end of treatment. Clodronate did not produce any significant behavioural effects in the Rota-rod test, pentobarbital-induced sleeping time, and locomotor activity cage. These data indicate that clodronate presents a central and peripheral prolonged antinociceptive effect, without any behavioural side effects. © 2003 Elsevier Inc. All rights reserved. Keywords: Visceral pain; Somatic pain; Bisphosphonates; Clodronate

Introduction Bisphosphonates such as clodronate (dicloromethylene-1, 1-bisphosphonate) and pamidronate (3-amino-1-hydroxypropylidenebisphosphonate) are synthetic, nonhydrolyzable analogues of pyrophosphate that contain C–C–P rather than P–O–P bonds. These compounds have been known to chemists since the 19th century, with the first synthesis dating back to 1865 [1]. After the discovery that they can effectively control calcium phosphate formation and dissolution in vitro, as well as mineralization and bone resorption in vivo [2– 4], they became a major class of

* Corresponding author. SPA-Societa’ Prodotti Antibiotici-S.p.A., Via Biella 8, Milan, Italy. Fax: ⫹39-02-8132983. E-mail address: [email protected] (A. Bonabello). 8756-3282/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S8756-3282(03)00229-1

antiresorptive drugs for the treatment of postmenopausal and corticosteroid-induced osteoporosis [5,6]. During the past 10 years the bisphosphonates have become the treatment of choice of tumour-induced hypercalcemia [7,8] and Paget’s disease [9,10]. More recently, the bisphosphonates have been used to treat osteoporosis in some countries [11,12]. Bisphosphonates can be considered an important therapeutic tool, in association with other therapeutic modalities (radiotherapy, radioisotopes such as 89Sr, palliative chemotherapy, appropriate use of analgesics), in the treatment of metastatic bone disease, with marked osteolysis, such as multiple myeloma and breast cancer [13]. In these clinical circumstances, bisphosphonates have been shown to be able to reduce skeletal complications and pain [14,15]. Liposome-encapsulated clodronate is a useful “antimacrophage” agent for the elimination of macrophage popula-

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tions in vivo. Indeed, in patients or animal models of arthritis, rheumatoid arthritis, uveitis, allergic encephalomyelitis, and immune thrombocytopenic purpura, liposomeencapsulated clodronate has been shown to effectively deplete the number of macrophages and reduce inflammation in vivo [16 –20]. Moreover, another bisphosphonate, alendronate, administered iv or orally, is able to reduce pain in patients with bone metastases due to prostate carcinoma [21]. The same drug, given iv, induced a reduction in spontaneous pain associated with reflex sympathetic dystrophy syndrome [22] (RSDS). Recently a short treatment with clodronate or pamidronate reduced the pain complicating RSDS for a long period after the end of treatment [23]. The lack of relationship between pain decrease, measured by the Huskinsson visual analogue scale (VAS), and urinary escretion of the bone resorption marker crosslinked type I collagen Ntx suggests that clodronate does not improve analgesia through the inhibitory effect on osteoclasts [24]. Alendronate, injected intraperitoneally (ip) in mice, is able to reduce visceral pain induced by acetic acid administered into the abdomen, but has no effect on the inflammatory pain following formalin injected in the plantar surface of the rat hind paw [25]. In a recent study our laboratory studied several bisphosphonates and observed that in mice clodronate and pamidronate showed an antinoceptive effect in acute conditions not related to bone process, after iv or icv injection [26]. The aim of the present work was to study the timedependent analgesic effect in mice of disodium clodronate and pamidronate using the tail-flick test, in comparison with that of morphine and acetylsalicylic acid (ASA). Furthermore, we evaluated the effects of clodronate on animal behaviour measured by the Rota-rod test, the pentobarbitalinduced sleeping time assay, and the locomotor activity cage.

Materials and methods Experiments were carried out following the Guiding Principles for the Care and Use of Laboratory Animals, the Recommendation from the Declaration of Helsinki, and European Community legislation No. 86/605. The protocol was reviewed and approved by the Local Animal Committee. Animals Mice (strain CD1) of either sex, weighing 20 to 30 g, were used in all experiments (Harlan Italy srl, San Pietro al Natisone, Udine). The animals were housed in temperatureand humidity-controlled rooms with a 12-h light/dark cycle and allowed free access to food and water. The animals were randomly assigned to different drug/doses tests; the

control animal groups received the vehicle treatment while the other animal groups received the test drugs. Drugs The following drugs were used for the experiments: clodronate (Difosfonal) (SPA, Societa` Prodotti Antibiotici S.p.A., Milan, Italy); pamidronate (Aredia) (Novartis Pharma, Bern, Switzerland); acetylsalicylic acid (Flectadol) (Sanofi Winthrop, Milan, Italy); morphine, (S.A.L.A.R.S., Societa` Azionaria Laboratorio Alcaloidi Rifornimenti Sanitari, Como, Italy). All compounds were dissolved in saline solution (0.9% sodium chloride). All doses are expressed as the drug-free base. Tail-flick Somatic pain was assessed by the D’Amour and Smith [27] tail-flick method, as modified by Dewey [28]. The mice (20 –25 g) were individually loosely wrapped in a thin cotton towel with head and tail exposed. Each animal was then placed on a platform with the tail positioned in a special shallow groove and a light beam was focused at the tail from above, approximately 2.5 cm from the tip. Movement of the tail from the groove allows the light beam to hit a sensor, formerly covered by the tail, which then automatically switched off the beam and stops the timer. The duration of the time required for the tail response after exposure to the thermal stimulus was considered the tail response latency time. The maximum time allowed was 15 s in order to prevent tissue damage. The mice, 10 males or females per group per time, were tested once to determine the predose tail response latency following which they were then dosed and again tested 15 and 60 min and 3, 6, 16, and 24 h later. For each animal we calculated the difference between postdose and predose latency time (⌬ time). The following drugs were administered iv into the tail vein: clodronate at a dose of 30 mg/kg and pamidronate at a dose of 1.25 mg/kg. The effect of iv administration of ASA (30 mg/kg) was used in order to have a positive control of the antinociception. To estimate the central action of the drugs we evaluated the antinociceptive effect produced by icv injection of clodronate, pamidronate, and morphine in conscious mice under a light anaesthesia (ethyl ether). The animal was grasped firmly by the loose skin behind the head. Using a modified microsyringe (0.025 ml), a 25-gauge needle, with a sleeve of PE 20 tubing to control the depth of the injection, was inserted perpendicularly through the skull into the brain and 0.01 ml of solution was injected [29]. The injection was administered at a site about 2 mm rostral and 2 mm lateral to the bregma at a depth of 2 mm. The doses of drugs used were clodronate, 0.70 ␮g/ mouse, pamidronate, 0.50 ␮g/mouse; morphine, 0.15 ␮g/

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mouse. The analgesic effect was tested 15 and 60 min and 3, 6, 16, and 24 h after drug administration in groups of 10 male or female animals. To study the effect of chronic drug treatment on the length of the analgesic activity, we treated a group of 15 male mice with 0.625 mg/kg clodronate iv for 5 consecutive days; the tail-flick test was performed after the first and fifth treatments (30 min after the drug injection) and 3, 7, 14, and 21 days after the end of the treatment. All doses used were selected on the basis of our previous published study [30]; in particular we used high doses of clodronate, ASA, and morphine for the acute study in order to evaluate the duration of the effects. For the chronic treatment we used a dose very similar to the dose used in human clinical settings. Behaviour on the Rota-rod The Rota-rod Trendmills (Ugo Basile Italy, Model No. 7600) were used to evaluate possible side effects of the drugs on animals’ motor coordination. Mice were trained by placing them in the test room in the morning of the first day and allowing them to get accustomed for at least 1 h. They remained in this room until the experiment was completed. The mice were trained for 3 days to run on the rod (3 cm in diameter) rotating at a rate of 10 rpm. The animals which remained on the Rota-rod for more than 300 s were previously selected. Ten male animals were used for 100 mg/kg clodronate iv. Each of them was placed on the Rota-rod 30 min after the administration of the drug or the appropriate vehicle [31]. Effect on pentobarbital-induced sleeping time Sedation was assessed by prolongation of pentobarbitalinduced sleeping time after administration of 100 mg/kg clodronate iv or appropriate vehicle. The sleeping time was defined as the time elapsed between the disappearance and recovery of the righting reflex measured with a chronometer and expressed in minutes. For the barbiturate-induced sleep, the animals received a single iv injection of pentobarbital sodium (46 mg/kg). Active drug or vehicle was administered simultaneously with the pentobarbital injections. The experiment was conducted in groups of 10 male mice [32]. Effect on spontaneous locomotor activity Two groups of 10 mice each were used for a test. Mice were placed in a plastic cage (31 ⫻ 36 ⫻ 17.5 cm) and habituated for 30 min. The locomotor activity was measured 30 min after administration of 100 mg/kg clodronate or vehicle at 10-min intervals for 1 h using the Activity Cage (Ugo Basile, Italy, Type 7401).

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Statistics The data obtained in all groups were analysed using a Kruskal–Wallis one-way analysis of variance followed by the distribution-free multiple comparisons and a Wilcoxon rank test (SPSS 6.1 for Windows). We compared drug vs control groups. Differences were considered statistically significant when P ⬍ 0.05.

Results Tail-flick test In the tail-flick test saline administration did not statistically affect the tail response latency time. As a positive control ASA iv induced a statistically significant antinociceptive effect (P ⬍ 0.05) that lasted 6 h. Administration of clodronate iv produced an antinociceptive effect that was statistically significant (P ⬍ 0.05) up to 16 h; pamidronate iv showed a significant antinociceptive effect for only 6 h (P ⬍ 0.05) (Figs. 1A and 2A). No difference was found between male and female mice concerning the duration of the analgesic effect. After icv administration morphine produced antinociceptive effect for 24 h (P ⬍ 0.05). Clodronate and pamidronate showed an increase in tail-flick latency time that was significant and lasted for 16 and 6 h, respectively (Figs. 1B and 2B). Also in this test no differences were observed between male and female mice. In the chronic treatment 0.625 mg/kg clodronate iv produced a 22% significant increase of the tail-flick latency after the first injection in comparison with control animals. The analgesic effect increased up to 40% after 5 days of treatment. A significant analgesic effect was still present after 3, 7, and 14 days from the end of treatment (Fig. 3). Behavioural tests Behaviour on the Rota-rod Clodronate (100 mg/kg iv), 30 min before the test, did not affect the Rota-rod performance and thus failed to show a significant sedative/ataxic action. Effect on pentobarbital-induced sleeping time Clodronate (100 mg/kg iv) did not produce any significant increase in the pentobarbital-induced sleeping time [values expressed as mean ⫾ SEM: 72.2 ⫾ 8.31 min vs 60.4 ⫾ 14.90 min; n ⫽ 10 per group]. Effect on spontaneous locomotor activity Clodronate (100 mg/kg) did not significantly modify the locomotor activity when the group of mice was challenged with the drug in comparison with the vehicle throughout the entire evaluation period.

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Fig. 1. A: Tail-flick latency time–response curves of clodronate (■, n ⫽ 10), pamidronate (䊐, n ⫽ 10), and acetylsalicylic acid (Œ, n ⫽ 10) after iv injection in the tail-flick test. Vehicle (}, n ⫽ 10). Female mice were tested 15 and 60 min and 3, 6, 16, and 24 h after drug administration. Each point represents the mean ⫾ SEM. *P ⬍ 0.05, compared with time of control group. B: Tail-flick latency time–response curves of clodronate (■, n ⫽ 10), pamidronate (䊐, n ⫽ 10), and morphine (Œ, n ⫽ 10) after icv injection in the tail-flick test. Vehicle (}, n ⫽ 10). Female mice were tested 15 and 60 min and 3, 6, 16, and 24 h after drug administration. Each point represents the mean ⫾ SEM. *P ⬍ 0.05, compared with time of control group.

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Fig. 2. A: Tail-flick latency time–response curves of clodronate (■, n ⫽ 10), pamidronate (䊐, n ⫽ 10), and acetylsalicylic acid (Œ, n ⫽ 10) after iv injection in the tail-flick test. Vehicle (}, n ⫽ 10). Male mice were tested 15 and 60 min and 3, 6, 16, and 24 h after drug administration. Each point represents the mean ⫾ SEM. *P ⬍ 0.05, compared with the control group. B: Tail-flick latency time–response curves of clodronate (■, n ⫽ 10), pamidronate (䊐, n ⫽ 10), and morphine (Œ, n ⫽ 10) after icv injection in the tail-flick test. Vehicle (}, n ⫽ 10). Male mice were tested 15 and 60 min and 3, 6, 16, and 24 h after drug administration. Each point represents the mean ⫾ SEM. *P ⬍ 0.05, compared with the control group.

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Fig. 3. Tail-flick latency time after chronic treatment with 0.625 mg/kg clodronate iv (■, n ⫽ 15) and vehicle (䊐, n ⫽ 15) for 5 days. Mice were tested at 30 min and 5 days during the treatment and at 3, 7, 14, and 21 days after the end of the treatment. *P ⬍ 0.05, compared with the control group.

Discussion Clodronate is a bisphosphonate that has shown efficacy when given intravenously in the management of a variety of painful skeletal disorders associated with enhanced bone resorption, such as Paget’s disease, hypercalcemia of malignancy, osteolitic bone metastases, and multiple myeloma [33–35]. Although the effects of the bisphosphonates on calcium homeostasis are well known, their mechanism of action has not yet been completely clarified. The bisphosphonates bind to bone minerals with a high affinity, because they alter the surface charge of hydroxyapatite by exerting a marked chemicophysical effect on the physiology of hydroxyapatite crystals. The calcium ⫻ phosphate product is macroscopically altered, which inhibits further crystal development. This chemical interaction may account for the effect of bisphosphonates on bone mineralization and the incapacity of osteoclasts to recognise the bone sites capable of mineral binding [36,37]. Since accelerated osteolysis and inflammatory response are critical in development of bone pain, several authors suggest that substances such as the bisphosphonates could have an effect on bone pain by inhibiting the osteoclast process and exerting a possible anti-inflammatory effect [13]. In a recent paper Goicoechea et al. [25] observed that alendronate produced a dose- and time-dependent analgesic effect in the abdominal constriction test in mice but did not have an analgesic effect on the formalin test. In previous research we showed that clodronate and pamidronate iv produced a significant dose response analgesic effect in rodents undergoing the tail-flick test whereas etidronate and alendronate had no such effect. After icv administration clodronate and pamidronate were less potent than morphine in the tail-flick test, but clodronate was more potent than

pamidronate in the abdominal constriction test after iv administration. Both bisphosphonates were more than 10-fold more potent than ASA in the same experiment [26]. In the present experiment the analgesic effect of clodronate and pamidronate was confirmed. This effect lasted 16 h for clodronate after iv or icv administration with no difference in duration between male and female mice. We also showed that chronic ip treatment increases the analgesic effect which lasts for at least 2 weeks after treatment ends. The clodronate analgesic effect did not affect animal behaviour and locomotor activity when using a dose about 100 times higher than the common therapeutic dose in patients. We used this high dose because in acute treatment we injected the animals with a dose of clodronate that is around 30 times higher than what is used in human clinical settings, and we wanted to evaluate any possible central toxic effect on mouse behaviour (motor coordination, sedation, and motor activity) affecting nociceptive perception of the animal. With the chronic ip treatment we used clodronate with doses similar to those used in human patients. These data confirm the clinical observations in RSDS patients: in a double-blind placebo-controlled study, 300 mg clodronate administered iv for 10 days significantly reduced the pain in patients assessed by visual analogue scale (VAS). The clodronate analgesic effect lasted not less than 40 days. According to the authors the absence of a relationship between decreased VAS and urinary excretion of type I collagene crosslinked N-telopeptide (a sensitive and specific bone resorption marker) would suggest that the clodronate pain-decreasing effect is unrelated to the inhibitory effect on osteoclast-mediated bone resorption [24]. Similar results were observed in RSDS patients using pamidronate. The drug was administered iv at daily doses of 60 mg over a period of 3 consecutive days. Total pain disappearance was obtained in 58.6% of the patients after 15 days and in

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86.2% after 45 days. No significant difference between the sexes was observed [23]. The prolonged effect of pamidronate was also observed in clinical studies by Cortet et al. and Maillefert et al. [38,39]. Prolonged treatment with 100 mg clodronate intramuscularly for 6 days a week for 2 months produced a reduction of VAS test in patients with rheumatoid arthritis [40]. Due to the poor correlation between animal results and human studies the present data cannot be used to predict the human response to the drug, but can suggest possible mechanisms for the analgesic effect of clodronate. Recently Liu et al. [41] studied the inflammatory response, the Wallerian degeneration, and the macrophage recruitment following nerve injury in a neuropatic pain model in rats. The author showed that treatment of nerveinjured animals with liposome-incapsulated clodronate decreased macrophage recruitment, alleviated thermal hyperalgesia, and reduced degeneration of both myelinated and unmyelinated axons. Similar results were observed using different disease models like immune throbocytopenia purpura in mouse [20] and endotoxin-induced uveitis in rats [18]. In these experiments clodronate was specifically targeted into the macrophages; the liposomes, in fact, are normally phagocytosed by the mononuclear phagocyte system. Drugs encapsulated in liposomes can, therefore, be targeted to monocytes and macrophages, without affecting other cell types. The main mechanism of the antinociceptive effect of bisphosphonates (central and/or peripheral) cannot be precisely determined from the data with these tests. Clodronate is able to inhibit the release of proinflammatory cytokinin (IL-1, IL-6, and tumour necrosis factor-␣). Moreover, clodronate also inhibits the production of lactic acid, PGE2 synthesis, and interstitial collagenase activation. Clodronate could induce rapid and prolonged pain relief by interrupting the action of those substances, which are possibly involved in the sensitisation of afferent nerve fibers and pain modulation [42]. It is unlikely that the peripheral antinoceptive effect is mediated through a central mechanism because the bisphosphonates are highly water-soluble compounds which cross lipid membranes poorly. At least in rabbits, the brain concentration of clodronate, pamidronate, and etidronate 24 h after iv administration is negligible [43]. In these experiments clodronate injected at high doses did not have any significant effect on animal behaviour and locomotor activity. In a previous study we showed that clodronate had an antinociceptive effect in the writhing and tail-flick tests in mice and in the Randall–Selitto test in rats, only when the drug was injected by the iv route; after oral administration clodronate did not produce a statistically significant antinociceptive effect [30] and this is probably due to the very low bioavailability of the bisphosphonates. Based on the observation that bisphosphonates change the ionic composition of the aqueous layer surrounding the hydroxyapatite crystals, by altering the calcium ⫻ phos-

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phate product [13] and changing the calcium homeostastis in patients [44], we can speculate on a possible central antinociceptive action of the bisphosphonate involving the Ca2⫹ mechanism [45]. Ca2⫹ seems to play an important role in the endogenous regulation of pain sensitivity [46]. It is known that Ca2⫹ influx is crucial for the release of neurotransmitters and other substances implicated in nociception and inflammation, such as substance P, vasoactive intestinal peptide, neuropeptide Y, prostaglandins, serotonin, and kinines. Several animal studies provide evidence that Ca2⫹ channel blockers, centrally and peripherally, have a pharmacological role of antinociception under acute conditions [47– 49] and clinical studies have shown that morphine-induced antinociception can be potentiated by Ca2⫹ channel blockers [50]. In conclusion we have shown that clodronate and pamidronate have a pharmacological role in the modulation of antinociception under acute conditions not related to osteolysis or acute inflammatory bone process. These effects last for not less than 16 h regardless of the animal’s sex. Chronic treatment with clodronate produces and analgesic effect lasting not less than 2 weeks from the end of treatment. High clodronate doses did not produce side effects, such as loss of motor control or activity levels, in the animals treated.

Acknowledgments We thank Mr. Gabriele Buffa and Mr. Claudio Rossi for technical assistance and Mrs. Franca Costi for general assistance.

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