Analgesic effects of the non-nitrogen-containing bisphosphonates etidronate and clodronate, independent of anti-resorptive effects on bone

European Journal of Pharmacology 699 (2013) 14–22

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Analgesic effects of the non-nitrogen-containing bisphosphonates etidronate and clodronate, independent of anti-resorptive effects on bone Siyoung Kim a,b, Masahiro Seiryu b, Satoru Okada a, Toshinobu Kuroishi a, Teruko Takano-Yamamoto b, Shunji Sugawara a, Yasuo Endo a,n a b

Department of Molecular Regulation, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan Departments of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2012 Received in revised form 14 November 2012 Accepted 14 November 2012 Available online 28 November 2012

Nitrogen-containing bisphosphonates (NBPs) have greater anti-bone-resorptive effects than nonnitrogen-containing bisphosphonates (non-NBPs). Hence, NBPs are the current first-choice drug for osteoporosis. However, NBPs carry a risk of osteonecrosis of jaws. Some animal and human studies suggest that non-NBPs may have anti-bone-resorptive effect-independent analgesic effects, but there has been no detailed comparison between NBPs and non-NBPs. Here, we compared the analgesic effects of several non-NBPs and NBPs, using (a) writhing responses induced in mice by intraperitoneal injection of 1% acetic acid, (b) acetic acid-induced neuronal expression of c-Fos, (c) acetic acid-induced elevation of blood corticosterone, and (d) hindpaw-licking/biting responses induced by intraplantar injection of capsaicin. Among the NBPs and non-NBPs tested, only etidronate and clodronate displayed clear analgesic effects, with various routes of administration (including the oral one) being effective. However, they were ineffective when intraperitoneally injected simultaneously with acetic acid. Intracerebroventricular administration of etidronate or clodronate, but not of minodronate (an NBP), was also effective. The effective doses of etidronate and clodronate were much lower in writhing-highresponder strains of mice. Etidronate and clodronate reduced acetic acid-induced c-Fos expression in the brain and spinal cord, and also the acetic acid-induced corticosterone increase in the blood. Etidronate and clodronate each displayed an analgesic effect in the capsaicin test. Etidronate and clodronate displayed their analgesic effects at doses lower than those inducing anti-bone-resorptive effects. These results suggest that etidronate and clodronate exert potent, anti-bone-resorptive effectindependent analgesic effects, possibly via an interaction with neurons, and that they warrant reappraisal as safe drugs for osteoporosis. & 2012 Elsevier B.V. All rights reserved.

Keywords: Bisphosphonate Etidronate Clodronate Pain Analgesic effect

1. Introduction The bisphosphonates (BPs) (Fig. 1) bind strongly to bone hydroxyapatite, and the bone-bound BPs exert anti-boneresorptive effects via their cytotoxic effects on osteoclasts (Rogers et al., 2000; Roelofs et al., 2006). Because nitrogencontaining bisphosphonates (NBPs) have greater anti-boneresorptive effects than non-nitrogen-containing bisphosphonates (non-NBPs) (Fig. 1), NBPs are the current first-choice drugs for diseases involving enhanced bone resorption. However, attached to NBPs is the risk that they might cause osteonecrosis of jaws (Marx et al., 2005; Ruggiero et al., 2004, 2009; Woo et al., 2006). Indeed, in Japan hundreds of cases of osteonecrosis of jaws have been reported, including patients treated with oral NBPs (Urade,

n

Corresponding author. Fax: þ81 22 717 8322. E-mail address: [email protected] (Y. Endo).

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.11.031

2010). It is noteworthy that in contrast, clear evidence is lacking that non-NBPs (e.g., etidronate and clodronate) cause such necrotic side effects. In fact, cases of osteonecrosis of jaws are very few among patients treated with these non-NBPs (Cre´pin et al., 2010), despite their coming into use much earlier than NBPs (e.g., etidronate was approved in 1977 in the USA and in 1990 in Japan). Severe bone pain can result from bone metastases (Costa et al., 2006; Yoneda et al., 2011), and recent studies have led to NBPs being used against such metastatic pain (Costa et al., 2006; Von Moos et al., 2008). However, it should be noted that NBPs may cause painful trigeminal neuropathy via osteonecrosis of jaws (Zadik et al., 2012). Interestingly, Fujita et al. (2009) reported that in patients with osteoporosis and/or osteoarthritis, the analgesic effect of etidronate (a non-NBP) was greater than that of either alendronate or risedronate (both NBPs). In some animal studies (a) BPs have been shown to exhibit analgesic effects that are not associated with their anti-bone-resorptive effects

S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

O O

P P O

O

OH OH

R1 R2

OH OH

C

P

HO

C

H3C

P P O

OH OH OH OH

Etidronate (Eti) 1 HO H N 2

C

OH OH

O

O

O Cl Cl

C

OH OH

P P

H H

OH OH

O

P OH OH O

Alendronate (Ale) 1,000

C

P P O

Clodronate (Clo) 3

O OH P OH

Non-NBPs: Eti, Clo, Med, Oxi NBPs: Ale, Ris, Min Relative potencies of ABREs

BPs

Pyrophosphoric acid O

OH OH

P

15

O

OH OH

HO

OH OH

H

Medronate (Med) 1

P

OH OH OH OH

Oxidronate (Oxi) 3 O HO

C N

P

O

O OH P OH

HO

C

P OH O OH

N

C

OH P OH P OH O OH

Risedronate (Ris) 3,300 Minodronate (Min) 10,000

Fig. 1. Structures of BPs (showing only those tested in this study). Drug-name abbreviations, followed by relative anti-bone-resorptive activities, are also shown.

(Goicoechea et al., 1999; Bonabello et al., 2001, 2003; Walker et al., 2002; Carvalho et al., 2006; Bianch et al., 2008; Kakimoto et al., 2008), and (b) clodronate (a non-NBP) has been found to have a more powerful analgesic effect than either pamidronate or alendronate (both NBPs) (Bonabello et al., 2001, 2003). However, those animal studies differed among themselves in animal species, experimental systems, BPs, routes of administration of BPs, and timing of BP administration, and the existing evidence is insufficient as regards comparisons of analgesic potencies among BPs, especially between NBPs and non-NBPs. The writhing (abdominal constriction) response – induced in mice by intraperitoneally injecting dilute acetic acid – and the hindpaw-licking/biting response – induced in mice by intraplantar injection of capsaicin – are widely used for evaluating the analgesic effects of test materials (Koster et al., 1959; Vinegar et al., 1979; Sakurada et al., 2011). Here, using these methods, we compared the analgesic effects of various BPs used clinically against osteoporosis. We paid special attention to the effects of non-NBPs, especially etidronate, because it is the only non-NBP used clinically in Japan, and because little is known from animal experiments concerning such an anti-bone resorptive effectindependent analgesic effect of etidronate. In addition, we examined the effects of BPs on the two molecular markers for pain, c-Fos protein expression in neurons (Morgan and Curran, 1989) and the blood level of corticosterone (Yarushkina, 2008), which have not been examined in previous studies.

2. Materials and methods 2.1. Mice BALB/c mice were bred in our laboratory. C57BL/6, C3H/HeN, ICR, and ddY mice were purchased from SLC (Shizuoka, Japan). IL-1 knockout (IL-1-KO) mice (BALB/c background; deficient in both IL-1a and IL-1b), TNF-a KO mice (BALB/c background), and Triple-KO mice (TKO, BALB/c background; deficient in IL-1a, IL-1b, and TNF-a) were established from original IL-1a KO, IL-1b KO, and TNF-a KO mice (Horai et al., 1998; Tagawa et al., 1997). Histamine-H1-receptor (H1R) KO mice (C57BL/6 background) and histidine decarboxylase (HDC) KO mice (C57BL/6 background) were established as previously described (Ohtsu et al., 2001, 2002; Inoue et al., 1996). The experiments were performed in accordance with IASP guidelines for the study of pain in animals (Zimmermann, 1983). All experiments complied

with the Guidelines for Care and Use of Laboratory Animals in Tohoku University and were approved by the Committee on Animal Research of Tohoku University. 2.2. Reagents Acetic acid, capsaicin, and etidronate were purchased from Wako Pure Chemical Industries (Osaka, Japan). Oxidronate was synthesized (Quimby et al., 1967). Medronate and clodronate were from Sigma (St. Louis, MO, USA), alendronate and risedronate from LKT Laboratories, Inc. (St. Paul, MN, USA), and minodronate from Chengdu D-Innovation Pharmaceutical Co., Ltd. (Chengdu, China). The above drugs (except capsaicin) were dissolved in sterile saline, with the pH of the solutions being adjusted to 7 with NaOH. For oral administration, drugs were dissolved in distilled water without adjusting pH. BPs were administered intravenously (i.v.), subcutaneously (s.c.), orally (p.o.), intraperitoneally (i.p.), or intracerebroventricularly (i.c.v.). The injection volume was 0.1 ml/10 g body weight for all except i.c.v. (10 ml/head). Capsaicin was dissolved in dimethyl sulfoxide, and this stock solution (5 mmol/ml) was diluted to 0.25 mmol/ml with sterile saline on the day of the experiment. Experimental protocols are described in the text or relevant figure legend. 2.3. Evaluation of anti-nociceptive or analgesic effects of BPs BPs were given via various routes before the delivery of nociceptive stimuli (acetic acid or capsaicin) as described below. Experimental protocols are described either in the text or in the relevant figure legend. Writhing test: Writhing was induced by i.p. injection of 1% (vol/vol) acetic acid (0.1 ml/10 g body weight). The number of writhing movements was counted in the period from 5 to 25 min after acetic acid injection. Capsaicin test: ddY mice (22–26 g at the time of the test) were used for assessing antinociception by the capsaicin test (Sakurada et al., 2011). To reduce variability, each mouse was acclimatized to an acrylic observation chamber (22.0  15.0  12.5 cm) for approximately 1 h before the injection of capsaicin. Each mouse was injected with 20 ml of a capsaicin solution (0.25 mmol/ml) (i.e., 1.5 mg/paw) beneath the skin of the plantar surface of the right hindpaw using a 50 ml Hamilton microsyringe with a 26-gauge needle. The injection was completed as quickly as possible, with only minimal animal restraint. Following capsaicin

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injection, the animals were immediately placed in the test box for a 5-min observation period. Licking/biting behavior induced by the injection was observed as an indicator of the nociceptive response. The accumulated response-time (in seconds) spent in licking/biting the capsaicin-injected paw was recorded for a period of 5 min immediately after the injection of capsaicin.

2.7. Data analysis Experimental values are given as mean 7standard deviation (S.D.). The statistical significance of differences was evaluated using a Bonferroni multiple-comparison test after an analysis of variance (ANOVA), P values less than 0.05 being considered significant.

2.4. Estimation of the anti-bone-resorptive effects of BPs A clear sclerotic band (tentatively called the BP-band) is detectable in tibias by radiography a few weeks after a single injection of a BP into mice, reflecting an inhibition of bone resorption (Monma et al., 2004; Funayama et al., 2005; Yu et al., 2005; Oizumi et al., 2009). Hence, we estimated the anti-boneresorptive effects of NBPs by using the BP-band as a marker. Briefly, each NBP solution was s.c.-injected into young (5-weekold) male mice. The mice were decapitated two weeks later, and tibias were removed and subjected to X-ray analysis for the detection of the BP-band, as previously described (Oizumi et al., 2009). The tibias were also subjected to micro-computed tomography (micro-CT) analysis for the quantification of the BP-band using a CT apparatus for experimental animals (LATHETATM, LCT-200; ALOKA Corp., Tokyo, Japan) and OnDeman3D Application software (Cybermed Co., Seoul, Korea). The area of each BPband was measured in the vertical section of the tibia displaying its maximal length.

2.5. Immunostaining of c-Fos After injection of acetic acid (detailed experimental protocols are described in the relevant figure legend), mice (BALB/c) were sacrificed by decapitation, and the medulla oblongata was removed. The vertebral column was transected at the level of the cauda equina. Then, the spinal cord was propelled out from the vertebral column by applying hydraulic pressure to the caudal opening, ice-cold saline being injected through an 18-gauge needle attached to a 10 ml syringe. The brain stem and spinal cord were then immersed overnight in 4% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4 1C and then for 3 days in 20% sucrose in 0.02 M phosphate-buffered saline at 4 1C. After freezing the tissues in a Yamato Electro Freeze MC-802A, sections (50-mm thick) were cut from the brain stem and spinal cord and subjected to immunostaining using anti-c-Fos antibody (Santa Cruz Biotechnology, Inc.) by a peroxidase–antiperoxidase method (Sugimoto et al., 1998). For brain-stem analysis, four sections were selected from the region within 500 mm around obex, and the numbers of c-Fos-positive neurons in the gracile and cuneate nuclei of the medullary dorsal horn were counted. For spinal-cord analysis, four sections were selected from the region around L6 (i.e., L5 to S1), and the numbers of c-Fos-positive neurons in the dorsal horn of the spinal cord were counted. In each section, the stained dots that could be clearly distinguished from the surrounding tissue were counted by an investigator blinded to the experimental protocol.

3. Results 3.1. Evaluation of safe doses of non-NBPs In the present study, in addition to etidronate and clodronate, we examined two other non-NBPs, oxidronate and medronate. Oxidronate and medronate are not used against osteoporosis but as carriers of 99mTc for bone scintigraphy. Before the experiments proper, we estimated the safe doses when the non-NBPs were given by various routes (Table 1). In all the following experiments, we used doses lower than those doses.

3.2. Effects of s.c.-injected etidronate and clodronate We first examined the effects of s.c.-injected etidronate and clodronate (30 mg/kg) when given at various pre-medication times. Etidronate significantly reduced the number of writhing movements only when injected at 0.5 to 2 h before the acetic acid injection (Fig. 2A). In contrast, the effect of clodronate was significant at all the pre-medication times tested (0.5 h to 2 days). For either of these drugs, the anti-nociceptive effect appeared to be maximal when the drug was injected 0.5–2 h before the acetic acid injection, but clodronate seemed to be effective for longer than etidronate. Next, we examined the effects of various BPs, including other non-NBPs (oxidronate and medronate) and NBPs (minodronate, risedronate, and alendronate). In this experiment, various doses of BPs were s.c.-injected 1 h before an acetic acid injection. As shown in Fig. 2B, etidronate and clodronate displayed dose-dependent anti-nociceptive effects, but neither of the other two non-NBPs, oxidronate or medronate, displayed such an effect even at or near its maximal safe dose (see inset Table in Fig. 1). Strangely, among the three NBPs tested, minodronate alone reduced acetic acidinduced writhing, and then only at 10 mg/kg (Fig. 2B). As described below, NBPs exert clear anti-bone-resorptive effects at much lower doses than non-NBPs. Therefore, we tested the effects of low doses of these NBPs on acetic acid-induced writhing. However, we could not detect anti-nociceptive effects at either 0.1 or 1 mg/kg of the NBPs (data not shown). These results suggest that among the BPs tested here, only etidronate and clodronate have clear anti-nociceptive effects against acetic acid-induced writhing. Table 1 Maximal doses of non-NBPs at which no abnormal or non-healthy behavior or activity was observed within 2 days of subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (i.p.), oral (p.o.), or intracerebroventricular (i.c.v.) injection.

2.6. Measurement of corticosterone in blood Blood was collected directly into a tube by decapitation, and the samples were left to coagulate for 2 h at room temperature, then centrifuged for 15 min at 2000g. The serum was stored at 20 1C. The corticosterone in the stored serum was measured using an ELISA kit (Stressgen, Michigan, USA) as described by the manufacturer.

Eti Clo Med Oxi

S.c.

I.v.

I.p.

P.o

I.c.v.

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

(mg/mouse)

60 60 45 15

30 60 25 15

50 60 20 25

1000 1000 nd nd

0.01 0.01 nd 0.005

nd: not determined.

S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

t Eti or Clo (30 mg/kg, s.c.)

1h 1% AA Saline or BPS (s.c.)

1% AA

No. of Writhings 0

10

20

***

1h

30

*** ***

2h 4h

Eti Clo

**

ns

2d

Oxi Med

20

ns **

** ns **

**

ns ns ns

5 10 30 10 15 30 10 30

ns NBPs Ris

Saline: n = 40 Others n = 6-11 Ale

30

*

15 30

Min *

10

7.5 15 30

Non- Clo NBPs

** ns *

1d

0

Saline 3.75 7.5 Eti 15 30

***

***

No. of Writhings

BPs (mg/kg)

C 0.5 h

17

**

ns ns ns ns ns ns

Saline : n = 12

Others : n = 6

Fig. 2. Writhing response to Eti and Clo. (A) Effects of timing of administration. Saline or the indicated dose of etidronate (Eti) or clodronate (Clo) was subcutaneously (s.c.)-injected, then 1% acetic acid (AA) was injected at the indicated time. Since there was no significant difference among the saline-injected groups, the results were combined and the mean 7 S.D. used as the control (C) for this experiment. (B) Effects of various doses of BPs. Saline or a BP at the indicated dose was s.c.-injected 1 h before AA injection. *Po 0.05, **P o 0.01, and ***P o 0.001 vs. Saline or control. ns: not significant.

S or BP (i.v.)

30 min

DW or BP (p.o.)

1% AA

0 (S) Eti

Clo

5

10

15

20

25

30

n=6 n=6 n=6

3.75 7.5 15

n=6 n=6 n=6

BP (mg/kg) 0

0 (DW)

n=9 3.75 7.5 15

ns **

1% AA

No. of Writhings

No. of Writhings BP (mg/kg) 0

1h

Eti

* ns

* *

Clo

5

10

15

20

25

30

n=9

250 n = 5 500 n = 10 1000 n = 8 250 n = 6 500 n = 9 1000 n = 10

ns **

* ns **

*

Fig. 3. Anti-nociceptive effects of intravenous (i.v.) and oral (p.o.) administrations of etidronate and clodronate. (A) Effects of i.v. administration. Saline (S), etidronate (Eti), or clodronate (Clo) was IV-injected at the indicated doses. Thirty minutes later, 1% acetic acid (AA) was injected. (B) Effects of p.o. administration. Distilled water (DW), Eti, or Clo was p.o.-administered at the indicated doses. One hour later, 1% AA was injected. Symbols indicating P values are as in Fig. 2.

3.3. Effects of i.v. and p.o. administrations of etidronate and clodronate The experiments described above were performed using s.c. injection of BPs, a route used only in animal experiments. Thus, we also examined clinically relevant routes (intravenous and oral routes) to inform the possible transfer of experimental data to clinical applications. We found that i.v.-injected etidronate and clodronate were each effective at reducing acetic acid-induced writhing at 7.5 mg/kg (Fig. 3A), and they were effective at 500 mg/kg when given p.o. (Fig. 3B). 3.4. Writhing in various strains of mice and effects of etidronate and clodronate in writhing-high-responder mice The data described above were obtained from BALB/c mice. To examine the antinociceptive effects of etidronate and clodronate

in other strains of mice, we first compared acetic acid-induced writhing in various mouse strains (Fig. 4A). We found that H1RKO and C3H/HeN mice are writhing-high-responder strains. We anticipated that the antinociceptive effects of etidronate and clodronate would be more clearly evident in these mice than in BALB/c mice. In these two high-responder strains, s.c.-injected etidronate and clodronate significantly reduced the writhing at 2 mg/kg (Fig. 4B and C) even though 3.75 mg/kg etidronate and 7.5 mg/kg clodronate had been ineffective in BALB/c mice (Fig. 2B). In addition, although p.o.-administered etidronate and clodronate were not effective at 250 mg/kg in BALB/c mice (Fig. 3C), they were effective at 125 mg/kg in H1R-KO mice (Fig. 4D). In BALB/c mice, 30 mg/kg etidronate had had no antiwrithing effect when s.c.-injected 2 days before acetic acid injection (Fig. 2A). However, in H1R-KO mice, etidronate at this dose was effective 2 days after its s.c. injection, as was clodronate at the same dose (data not shown). These results suggest that

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S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

No. of Writhings 0

20

BALB/c IL-1-KO TNF-KO

40

60

In C3H/HeN 1h S or BP (s.c.) 1% AA No. of writhings

80

BALB/c n = 12 others n = 5 ~ 8

0

Male Female

TKO

20

40

60

Saline Eti 2 mg/kg 4 Clo 2 mg/kg 4

C57BL HDC-KO H1R-KO

n=6

*** ** *** ***

C3H/HeN ICR ddY In H1R-KO 1h S or BP (p.o.)

In H1R-KO 1h S or BP (s.c.) 1% AA

No. of Writhings

No. of Writhings 0

20

Saline

n = 18

Eti 2 mg/kg 4

n=6 n=6

Clo 2 mg/kg 4

n=6 n=6

40

1% AA

0

60

10 20 30 40 50

Saline * * *** ***

n=6

ns

Eti 62.5 mg/kg 125 250 Clo 62.5 mg/kg 125 250

* *** ns ***

** n=5~6

Fig. 4. Writhing responses in various mouse strains and effects of etidronate (Eti) and clodronate (Clo) on writhing-high-responder mice. (A) Writhing responses in various mouse strains. 1% acetic acid (AA) was injected in various strains of male and/or female mice, and their writhing responses recorded. ((B) and (C)) Effects of subcutaneously (s.c.) injected Eti and Clo in H1R-KO and C3H/HeN mice. The indicated dose of Eti or Clo was s.c.-injected. One hour later, 1% AA was injected. (D) Effects of orally (p.o.) administered Eti and Clo in H1R-KO mice. The indicated dose of Eti or Clo was p.o.-administered to H1R-KO mice. One hour later, 1% AA was injected. Symbols indicating P values are as in Fig. 2.

etidronate and clodronate are effective at much lower doses in writhing-high-responder mice than in BALB/c mice, although the relevance of this to humans remains to be established.

3.5. Effects of i.p. and i.c.v. injections of BPs To explore mechanism(s) underlying the antinociceptive effects of etidronate and clodronate, we performed the following experiments. As shown in Fig. 5A, when etidronate was i.p.injected simultaneously with acetic acid, even at 15 mg/kg [the effective dose when it was s.c.-injected (Fig. 3)], it did not reduce acetic acid -induced writhing. However, when either etidronate or clodronate was i.p.-injected at this dose 1 h before an acetic acid injection, the acetic acid-induced writhing was significantly reduced (Fig. 5B). These results suggest that etidronate and clodronate may reduce pain conduction, but not pain initiation. Next, to test whether etidronate and clodronate might exert their anti-nociceptive effects via direct actions on neurons, a small dose of either of these drugs (0.01 mg/head) was i.c.v.injected, and 1 h later 1% acetic acid was i.p.-injected. This i.c.v. dose corresponds to 0.5 mg/kg, which is below the doses that induced analgesic effects when injected s.c. or i.v. (see Fig. 2B and Fig. 3B). Fig. 5C shows that when given i.c.v. (as above) etidronate and clodronate, but not minodronate, were each effective at reducing the writhing response, supporting the idea that etidronate and clodronate may exert their anti-nociceptive effects via direct interaction with neurons.

3.6. Effects of etidronate and clodronate on acetic acid-induced c-Fos expression in spinal cord and brain and on acetic acid-induced elevation of serum corticosterone To examine further how etidronate and clodronate exert antinociceptive effects, we tested their effects on acetic acid-induced c-Fos expression in neurons. c-Fos expression was nearly maximal within 15 min of acetic acid injection (data not shown). As shown in Fig. 6A and B, when s.c.-injected at 1 h before acetic acid, etidronate and clodronate each reduced c-Fos expression in both the lumbar spinal dorsal horn and the medulla oblongata, when these expressions were measured at 15 min after the acetic acid injection. Acetic acid injection also elevated the serum level of corticosterone, which peaked around 15 to 30 min after the acetic acid injection (data not shown). S.c.-injected saline by itself increased the serum corticosterone, which peaked at around 15 min and returned to its basal level at 30 min after the injection (data not shown). The acetic acid -induced elevation of serum corticosterone measured at 30 min after the acetic acid injection was reduced by clodronate, but only tended to be reduced by etidronate (Fig. 6C). 3.7. Effects of etidronate and clodronate on capsaicin-induced responses The capsaicin test in mice is an additional model widely used in pain studies (Sakurada et al., 2011). Therefore, we compared

S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

Saline or Eti 2% AA

1:1 mixture

Saline or BP 1 h 1% AA (0.01 mg/head, i.c.v.) No. of Writhings

i.p. (0.1 ml/10 g body weight) 0

No. of Writhings 0

5

Saline

10

15

ns

n=4

Eti 3 mg/ml (15 mg/kg)

10

15

20

25

Eti

n=6

Clo

n=6

Min

n=6

** **

ns

ns

n=7

1h

S or Eti 15 mg/kg, i.p.

Saline

5

Saline n = 7

25

n=7

Eti 1 mg/ml (5 mg/kg)

No. of Writhings 5 10 15 20

0

20

19

1% AA

25

n=6

Eti

n=6

Clo

n=6

** *

Fig. 5. Effects of intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) injections of etidronate (Eti), clodronate (Clo), or minodronate (Min). (A) Effects of concomitant i.p. administration of AA and Eti. An equivolume mixture of either saline or Eti (1 or 3 mg/ml) with 2% acetic acid (AA) was i.p.-injected (0.1 ml/10 g body weight). This made the concentration of AA 1% and the dose of Eti as shown in parenthesis. Then, writhing responses were recorded. (B) Effects of i.p. injection of Eti at 1 h before AA injection. Saline or Eti (15 mg/kg) was i.p.-injected. One hour later, 1% AA was injected. (C) Effects of i.c.v. injection. Eti, Clo, or Min (each at 0.01 mg/head) was i.c.v.-injected (see Methods). One hour later, 1% AA was i.p.-injected to induce writhing. Symbols indicating P values are as in Fig. 2.

1h

BP (30 mg/kg, s.c.) or S No injection

S

15 (A & B) or 30 min (C) S or 1% AA Sampling S

S

Eti

AA

AA

Clo

AA

Spinal cord

700µm

Brain stem

300µm

Number of Fos-positive neurons in spinal cord 0

No injection S S S AA AA Eti AA Clo

10

20

30

Serum corticosterone (ng/ml)

in brain stem 40 0

10

20

30

n = 16

40

0

1000

2000

3000

n = 5~10 ns

*** ***

*** ***

*

Fig. 6. Effects of etidronate (Eti) and clodronate (Clo) on acetic acid-induced c-Fos expression in spinal cord and brain and on acetic acid-induced elevation of blood corticosterone. (A) C-Fos expression. Saline (S), Eti (30 mg/kg), or Clo (30 mg/kg) was subcutaneously (s.c.) injected. One hour later, 1% acetic acid (AA) was injected. Mice were decapitated 15 min after the AA injection. Spinal cords and brains were removed for immunostaining of c-Fos protein (see Methods) (right panels). (B) Quantification of c-Fos expression. c-Fos-positive neurons (seen as points) were counted per section of spinal cord (both right and left lumbar spinal dorsal horns) and brain stem (medulla oblongata). (C) Corticosterone levels in blood. S, Eti (30 mg/kg), or Clo (30 mg/kg) was s.c.-injected. One hour later, 1% AA was injected. Blood was collected directly into a tube by decapitation after the AA injection for measurement of serum corticosterone. In the analysis of c-Fos-positive neurons, 4 sections from each of 4 mice in a given group were stained as described in Methods. Counting was done on the right side. Thus, c-Fos-positive neurons were counted in a total of 16 sections per group. Mean and SD values were subjected to statistical analysis. Symbols indicating P values are as in Fig. 2.

20

S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

S or BP (s.c.)

1h

capsaicin

Licking/biting response (sec) 0

20

Saline

n=7

Eti 30 mg/kg

n=6

40

60

80

***

Clo 30 mg/kg n = 6 Min 10 mg/kg n = 6 Ale 10 mg/kg n = 6

***

inducing anti-bone-resorptive effects, as estimated by the BP-band formation seen after a single s.c. injection of a BP. As described in Introduction, the anti-bone-resorptive effects of NBPs are much higher than those of etidronate and clodronate, and NBPs are the current first-choice drugs for osteoporosis. However, NBPs carry a risk of inflammatory and necrotic side effects. Taking these points into consideration, we discuss the above findings in the following paragraphs. 4.2. Difference between NBPs and non-NBPs

ns ns

Fig. 7. Effects of BPs on capsaicin-induced pain responses. Etidronate (Eti), clodronate (Clo), minodronate (Min), or alendronate (Ale) at the indicated dose was subcutaneously (s.c.) injected, and 1 h later capsaicin was injected (see Methods). Symbols indicating P values are as in Fig. 2.

the effects of non-NBPs (etidronate and clodronate) and NBPs (alendronate and minodronate) on the hindpaw-licking/biting responses induced in mice by an injection of capsaicin into a hindpaw sole. In this experiment, ddY mice were used because they are the strain widely used for the capsaicin test in Japan. As shown in Fig. 7, the non-NBPs etidronate and clodronate each reduced the capsaicin-induced responses at the subcutaneous dose found to be effective in the writhing test in BALB/c mice (see Figs. 2 and 6). However, the NBPs minodronate and alendronate, which were ineffective in the writhing test, were not effective in the capsaicin test, either.

3.8. Anti-bone-resorptive effects of BPs Finally, to compare doses between anti-nociceptive effects and anti-bone-resorptive effects, we examined the anti-boneresorptive effects of the BPs used in the present study. It should be noted that the anti-bone-resorptive effects of alendronate, risedronate, and minodronate are 1000-fold, 3300-fold, and 10,000-fold greater than that of etidronate (Fig. 1). As shown in Fig. 8A, when injected s.c. at 10 mg/kg, clodronate formed a faint BP-band, but etidronate did not. In contrast, alendronate, risedronate, and minodronate formed clear BP-bands at 0.01 or 0.1 mg/kg. The rank order of their BP-band-forming abilities was minodronate4risedronate4alendronate, the same as the order of their anti-bone-resorptive effects (see Fig. 1). These results, together with those described in the previous section, indicate that etidronate and clodronate have anti-nociceptive effects at doses similar to or lower than those inducing anti-boneresorptive effects, while NBPs do not exhibit anti-nociceptive effects even at doses high enough for their anti-bone-resorptive effects.

4. Discussion 4.1. Summary of the findings The present findings may be summarized as follows. (i) Among the BPs tested, only etidronate and clodronate displayed clear analgesic effects, with various routes of administration being effective. (ii) In writhing-high-responder strains of mice, etidronate and clodronate were effective at much lower doses than in writhingnormal-responder strains. (iii) Etidronate and clodronate may exert their anti-bone-resorptive effect-independent analgesic effects via an interaction with neurons. (iv) Etidronate and clodronate displayed their analgesic effects at doses similar to or lower than those

In addition to osteonecrosis of jaws, NBPs have such inflammatory side effects as influenza-like fever, increase in acute-phase proteins, gastrointestinal disturbance, and ophthalmic inflammation (Adami et al., 1987; Siris, 1993; Macarol and Frauenfelder, 1994; Sauty et al., 1996; Thie´baud et al., 1997; Munns et al., 2004). The mechanism underlying these side effects in humans remains to be elucidated, but an important point to note is that convincing evidence is lacking of non-NBPs (etidronate and clodronate) having such side effects. In mice, all the NBPs tested so far have had inflammatory and necrotic side effects (Endo et al., 1993, 1999; Oizumi et al., 2009). For example, a single intraperitoneal injection of an NBP induces a variety of inflammatory reactions (including a prolonged induction of the histamine-forming enzyme, pleural exudation, an increase in granulocytic cells, and splenomegaly) (Endo et al., 1999), together with changes in hematopoiesis (Nakamura et al., 1999). Further, when injected topically, NBPs induce necrosis at the injection site (Schenk et al., 1986; Oizumi et al., 2009). In contrast, the nonNBPs clodronate and etidronate are neither inflammatory nor necrotic. On the contrary, they antagonize the inflammatory and necrotic effects of NBPs in mice (Endo et al., 1999; Funayama et al., 2005; Oizumi et al., 2009, 2010), and the order of this antagonizing effect is clodronate4etidronate. Also noteworthy is a reported contrast between the effects of NBPs (exacerbating) and non-NBPs (suppressing) on collagen-induced arthritis in mice (Nakamura et al., 1996). The above clinical experience and animal studies suggest that the non-NBPs etidronate and clodronate have properties that are not possessed by NBPs. 4.3. Possible use of etidronate and clodronate as safe drugs for osteoporosis Pain is a source of serious distress in patients with osteoporosis or osteoarthritis, and NBPs by themselves can reportedly cause musculoskeletal pain (Papapetrou, 2009; Bock et al., 2007). We found here that in mice, the non-NBPs etidronate and clodronate displayed analgesic effects at doses similar to or lower than those exerting anti-bone-resorptive effects (Fig. 8), whereas NBPs had no detectable anti-nociceptive effects, except for 10 mg/kg of minodronate (Fig. 2B and Fig. 7). The doses of NBPs tested in the present study, when i.p. injected into mice, cause various inflammatory side effects, as described above (see the references cited in the above section). In addition, NBPs, like non-steroidal anti-inflammatory drugs, directly cause gastric ulcer in rats (Amagase et al., 2011). In contrast, etidronate and clodronate lack such a gastric side effect. To allow for their weak anti-boneresorptive effects, the clinical doses of etidronate and clodronate (e.g., etidronate 200–1000 mg/day) are much larger than those of NBPs (1–5 mg/day). Thus, at the currently employed clinical doses, etidronate and clodronate may be effective at displaying their analgesic effects. Indeed, Fujita et al. (2009) reported that in patients with osteoporosis and/or osteoarthritis, the analgesic effect of etidronate was greater than those of alendronate and risedronate. Consequently, in addition to the use of etidronate and clodronate as antagonizing agents against the inflammatory

S. Kim et al. / European Journal of Pharmacology 699 (2013) 14–22

Ale 0.01 mg/kg

Ale 0.01 mg/kg

Ale 0.1 mg/kg

Eti 10 mg/kg

Ris 0.01 mg/kg

Ris 0.1 mg/kg

Eti 10 mg/kg

Clo 10 mg/kg

Min 0.01 mg/kg

Min 0.1 mg/kg

Clo 10 mg/kg

BPs (s.c.)

2w BP-band

21

Ale 0.1 mg/kg

Ris 0.1 mg/kg

Ris 0.01 mg/kg

Min 0.01 mg/kg

Min 0.1 mg/kg

Area of BP-band (mm2)

Dose (mg/kg)

0

Eti

10

Clo

10

0.2

0.4

0.6

0.8

1

Ale 0.01 0.1 Ris 0.01 0.1 Min 0.01 0.1 Fig. 8. Anti-bone-resorptive effects of BPs used in this study. Etidronate (Eti), clodronate (Clo), alendronate (Ale), risedronate (Ris), or minodronate (Min) was subcutaneously (s.c.) injected at the indicated doses into young (5-week-old) BALB/c mice. Two weeks later, their tibias were subjected to BP-band analysis (see Methods). (A) X-ray analysis for the detection of BP-bands. (B) Sections of tibia figured by a micro-CT apparatus. (C) Quantification of the BP-bands by means of the micro-CT apparatus (see Methods).

and necrotic side effects of NBPs, the present findings of their analgesic effects may encourage inquiry into the potential use of etidronate and clodronate as safe and useful drugs against osteoporosis (instead of NBPs).

4.4. Mechanism underlying the analgesic effects of etidronate and clodronate The present results suggest that the analgesic effects of etidronate and clodronate may be due to direct actions on neurons. As yet, however, we have no available data concerning the molecular mechanism underlying these analgesic effects. NBPs have cytotoxic effects on various types of cells, as well as on osteoclasts, via intracellular inhibition of farnesyl pyrophosphate synthase (an enzyme involved in cholesterol biosynthesis), leading to the accumulation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate (Rogers et al., 2000; Roelofs et al., 2006). Interestingly, Bang et al. (2011 and 2012) found that isopentenyl pyrophosphate and dimethylallyl pyrophosphate (both pyrophosphate derivatives) modify the functions of the transient receptor potential ion channels (TRPs) involved in sensing and transducing noxious stimuli for the transmission of appropriate signals to the brain. In the present study, although we detected no clear analgesic or nociceptive effects of NBPs, we speculate that etidronate and clodronate (both pyrophosphate analogs) may exert their analgesic effects via TRPs. However, until we perform experiments to test that hypothesis, it remains in the realm of speculation, and it is unclear why NBPs were not effective at reducing the nociceptive responses examined in the present study.

5. Conclusion In mice, the non-NBPs etidronate and clodronate display potent anti-nociceptive or analgesic effects that are independent of their anti-bone-resorptive effects, possibly via an interaction with neurons. This encourages us to suggest the reappraisal of etidronate and clodronate as anti-osteoporosis drugs with a low risk of inflammatory and necrotic side effects.

Acknowledgments This work was supported by grants from the Japan Society for the Promotion of Science (21390529, 20592318, and 21890019). We are grateful to Professor Koichi Tan-no (Department of Pharmacology, Tohoku Pharmaceutical University, Sendai, Japan) for teaching us the method used to remove spinal cords, to Professor Shinobu Sakurada (Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan) for teaching us the method used for the capsaicin test, and to Dr. Robert Timms for editing the manuscript. References Adami, S., Bhalla, A.K., Dorizzi, R., Montesani, F., Rosini, S., Salvagno, G., Lo Cascio, V., 1987. The acute-phase response after bisphosphonate administration. Calcif. Tissue Int. 41, 326–331. Amagase, K., Inaba, A., Senata, T., Ishikawa, Y., Nukui, K., Murakami, T., Takeuchi, K., 2011. Gastric ulcerogenic and healing impairment effects of risedronate, a nitrogen-containing bisphosphonate in rat. Comparison with alendronate and minodronate. J. Physiol. Pharmacol. 62, 609–618. Bang, S., Yoo, S., Yang, T., Cho, H., Hwang, S.W., 2011. Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPVA1 ion channels. Pain 152, 1156–1164.

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