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Antinociceptive effect induced by intraperitoneal administration of vitamin K2 (menatetrenone) in ICR mice
Life Sciences 68 (2000) 91–97
Pharmacology letters Accelerated communication
Antinociceptive effect induced by intraperitoneal administration of vitamin K2 (menatetrenone) in ICR mice Kenji Onoderaa,*, Hisashi Shinodaa, Ko Zushidab, Kentaro Takib, Junzo Kameib a
Department of Pharmacology, School of Dentistry, Tohoku University, Aoba-ku, Sendai 980-8575, Japan Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan
b
(Submitted March 17, 2000; accepted May 11, 2000; received in final form July 5, 2000)
Abstract The antinociceptive effect of vitamin K2 (menatetrenone) in mice was examined using tail-flick and formalin test. Menatetrenone at doses of 10, 50 and 100 mg/kg, i.p. produced a dose-dependent and significant inhibition of the tail-flick response in mice. Menatetrenone (50 and 100 mg/kg, i.p.) had no significant effect on the duration of the first phase of the formalin-induced flinching. However, menatetrenone (100 mg/kg, i.p.) significantly inhibited the second phase of the formalin-induced flinching. I.p. administration of menatetrenone (100 mg/kg) significantly reduced the duration of nociceptive responses induced by i.t. injection of bradykinin, but not of substance P, prostaglandin E2 or N-methyl-D-aspartate (NMDA). These present data suggest that i.p. pretreatment with menatetrenone produced dose-dependent antinociceptive effect in mice. This effect may be, at least in part, mediated by the inhibition of bradykinin dependent nociceptive transmission in the spinal cord. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Vitamin K2 (menatetrenone); Antinociception; Bradykinin; Prostaglandin E2
Introduction Menatetrenone, vitamin K2 with 4 isoprene units, has not only high g-carboxylation activity in hypoprothrombinaemic rats [1], but also has prevented bone-loss induced by ovariectomy [2] or by predonisolone administration in rats [3]. In clinics, menatetrenone is used for * Corresponding author. Department of Pharmacology, Tohoku University, School of Dentistry, Seiryo-machi 4-1, Sendai 980-8575, Japan. Tel.: 181-22-717-8311; fax: 181-22-717-8313. E-mail address: [email protected] (K. Onodera) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 1 7 -6
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preventing postmenopausal osteoporosis [4] and has been effective to treat of patients with osteoporosis [5, 6]. Thus, menatetrenone, a known treatment for vitamin K deficiency (marketed since 1984) has been accepted for clinical application in the treatment of osteoporotic osteopenia from 1995 in Japan [7]. Interestingly, Yamaji et al. [8] have reported that female osteoporosis patients whose pain had not been relieved by vitamin D3, elcatonin, non-steroid anti-inflammatory drugs and physical therapy showed significant decrease in pain scores at 2 to 4 weeks after treatment with menatetrenone. Furthermore, they also reported that bone mass measured by computed X-ray densitometer was significant increased at 4 and 8 months after treatment of menatetrenone with improving pain scores [8]. These results indicate that menatetrenone may be useful in the treatment with osteoporosis patients suffering from pain. However, potency and its possible mechanism of menatetrenone-induced antinociception were still unclear. Thus, in this study, we investigated the antinociceptive effect of menatetrenone in mice using tail-flick and formalin test.
Methods Animals Six-weeks old male ICR mice (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan) weighing 26–30 g were used. They had free access to food and water in an animal room that was maintained at 24 6 1 8C with a 12-h light-dark cycle. This study was carried out in accordance with the Declaration of Helsinki and/or with the guiding principles for the care and use of laboratory animals approved by the Japanese Pharmacological Society. Tail-flick test The antinociceptive effect was evaluated by recording the latency in the tail-flick test using radiant heat as a stimulus. The tail of mice was blackened using indian ink and exposed to the focused beam of light from a preheated 500W projection bulb. The heat intensity was set by adjusting the source voltage of the bulb to 50 V. When withdrawal response occurred, the stimulus was terminated and the response latency measured automatically. A cut off latency of 15 s was used to prevent injury to the tail. Changes in tail flick latency (Dt (s)) was calculated for each animal according to the formula: Dt (s) 5 post-drug latency - pre-drug latency. Antinociceptive effect was measured 30, 60, 90 and 120 min after administration of menatetrenone. Formalin-induced flinching response The experiment was performed according to the method described by Shibata et al. [9]. Each mouse was acclimated to an acrylic observation chamber (32 3 23 3 17 cm3) for at least 5 min before the injection of formalin. Twenty-five ml of a 0.5% solution of formalin in 0.9% saline were administered into the dorsal surface of the right hindpaw. Immediately after the injection, each animal was returned to the observation chamber and its flinching response was recorded for 30 min. The mouse licked and bit the injected paw, and these responses were distinct and easily observed. The accumulated response time (s), i.e., the duration of
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licking and biting of the injected paw, was measured for each 5-min block. The accumulation response time during the initial two blocks and during on and after third blocks was regarded as the first-phase and second-phase response, respectively. Menatetrenone was injected 30 min before the injection of formalin. Algogenic mediator-induced nociceptive responses The experiment was performed according to the method described by Hylden and Wilcox [10]. Each mouse was acclimated to an acrylic observation chamber (39326324 cm3) for at least 5 min before the injection of an algogenic mediator. Intrathecal injection was performed by the method described by Hylden and Wilcox [11]. Each i.t. injection was administered using a 30-gauge needle directly through the intact skin between the L5 and L6 vertebrae. Drugs were given in a volume of 5 ml/mouse. Immediately following the injection, the mice were placed in the observation chamber. The cumulative duration (s) of biting, paw licking and scratching episodes was measured for 10 min after the injection of substance P or N-methylD-aspartate (NMDA), and 30 min after the injection of bradykinin or prostaglandin E2. Menatetrenone was injected 30 min before the injection of substance P, bradykinin, prostaglandin E2 or NMDA. The doses of algogenic mediators were based on our previous report [12]. Drugs Menatetrenone (Kaytwo N Injection) and its vehicle were generously supplied by Eisai Co., Ltd., Tokyo, Japan. Vehicle of menatetrenone, a mixture of glycerine, D-sorbitol, and purified soybean lecithin was the same composition of Kaytwo N Injection except menatetrenone. Menatetrenone and its vehicle were intraperitoneally (i.p.) administered. Substance P and bradykinin were purchased from Peptide Institute Inc., Osaka, Japan. N-methyl-Daspartate was purchased from Sigma Co., St. Louis, MO. U.S.A. Prostaglandin E2 was purchased from Calbiochem-Novabiochem International, La Jolla, CA, USA. Prostaglandin E2 was stored in ethanol solutions at 220 8C. For injection, an aliquot of the desired stock prostaglandin E2 solution was put into a borosilicate tube and the ethanol was removed by evaporation to dryness under nitrogen gas. Sterile saline was then added to dissolve the prostaglandin E2. Statistics The data are expressed as the mean 6 S.E.M. The statistical significance of differences was assessed with the Bonferroni/Dunn test. Results Fig. 1 shows the time course of the antinociception produced by menatetrenone (10–100 mg/kg, i.p.) in mice. The reduction in the tail-flick latency reached its peak 30 min after injection, gradually declined and returned to the preinjection level 120 min after menatetrenone injection. Thus, a time interval of 30 min after administration of menatetrenone was chosen for experiments designed to quantify its effect. As shown in Fig. 2, menatetrenone at doses of 10–100 mg/kg, i.p. produced a dose-dependent and significant inhibition of the tail-flick response in mice.
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Figure 1. Time course of menatetrenone on the tail-flick latency in mice. The effects of menatetrenone (10 mg/ kg, closed circle; 50 mg/kg, open triangle; 100 mg/kg, closed square) and vehicle (open circle) were measured 30, 60, 90 and 120 min after i.p. injection. Dt (s) 5 post-drug latency - pre-drug latency. Each point represents the mean with S.E. for 10 mice in each group.
In mice, s.c. injection of 0.5% formalin into the hindpaw caused an acute, immediate flinching response, i.e., licking and biting, which lasted about 5 min in the first-phase. In the second-phase, flinching response reappeared and lasted about for 20 min. Menatetrenone (50 and 100 mg/kg, i.p.) had no significant effect on the duration of the first phase of the formalininduced flinching (Fig. 2). However, menatetrenone (50 and 100 mg/kg, i.p.) dose-dependently inhibited the second phase of the formalin-induced flinching (Fig. 2). Indeed, 100 mg/kg of menatetrenone significantly reduced the duration of the second phase of the formalininduced flinching (Fig. 2). Intrathecal (i.t.) injection of either substance P (0.1 mg), bradykinin (1.0 mg), prostaglandin E2 (0.1 mg) or NMDA (0.1 mg) elicited a behavioral syndrome consisting of reciprocal hindlimb scratching directed towards caudal parts of the body, and biting or licking of the hind legs in mice. I.p. administration of menatetrenone (100 mg/kg) significantly reduced the duration of nociceptive responses induced by i.t. injection of bradykinin (Fig. 3A). However,
Figure 2. Effect of menatetrenone (50 and 100 mg/kg, i.p.) on the total duration of the response during the first (0–10 min) and second (10–30 min) phases of formalin-induced flinching responses in mice. Menatetrenone was administered i.p. 30 min before the injection of formalin. Each column represents the mean with S.E.M for 10 mice in each group. * P,0.05 vs. vehicle-treated group (Vehi, open column).
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Figure 3. Effects of menatetrenone (dotted column) on the bradykinin-, substance P-, prostaglandin E2 (PGE2)- or NMDA-induced nociceptive response in mice. Menatetrenone (100 mg/kg) was administered i.p. 30 min before the i.t. injection of bradykinin, substance P, PGE2 or NMDA. Each column represents the mean with S.E. (n5910). * P,0.05 vs. vehicle (open column)-treated group.
menatetrenone (100 mg/kg, i.p.) had no significant effect on the duration of substance P-, prostaglandin E2- or NMDA-induced nociceptive responses (Fig. 3B, C and D). Discussion In the present study, i.p. administration of menatetrenone reduced the tail-flick response in a dose-dependent manner in mice. This study also demonstrated that the second phase, but not the first phase of the formalin-induced flinching response is significantly and dose-dependently reduced by i.p. treatment with menatetrenone. Hunskaar and Hole [13] noted that the first phase of the formalin-induced nociceptive response may represent a direct effect on nociceptors, whereas the second phase may represent an enhanced response of sensitized dorsal horn neurons resulting from low-level neuronal input due to peripheral inflammatory insult. Moreover, it has been suggested that substance P participates in the short-lasting pain in the first phase of the formalin-induced nociceptive response, whereas somatostatin, bradykinin and prostaglandins participate in the prolonged and inflammatory pain in the second phase of the formalin-induced nociceptive response [14, 15]. Furthermore, it is well known that the stimulation of NMDA receptor in the spinal cord is also involved in the second phase of formalininduced nociceptive response [e.g., 16]. In addition, Kuraishi et al. [17] and Morton et al. [18] suggested that application of noxious thermal stimuli specifically increases the release of somatostatin, but not substance P. Taken together, these results suggest that menatetrenoneinduced antinociception may account from an inhibitory effect on the nociceptive transmission which mediated by an inflammatory mediators, such as somatostatin, bradykinin and prostaglandins, or NMDA. Indeed, in the present study, we observed that menatetrenone sig-
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nificantly reduced the duration of bradykinin-, but not substance P-, prostaglandin E2- or NMDA-induced nociceptive responses in mice. Thus, it is unlikely that menatetrenoneinduced antinociception may account from inhibition of prostaglandin E2, substance P and NMDA in mice. Nevertheless, since previous studies showed that menatetrenone inhibited prostaglandin E2 synthesis in vitro [19, 20], further research will be needed to clarify whether decrease in synthesis and/or release of prostaglandin E2 may be involved in the mechanism of menatetrenone-induced antinociception. Finally, although the detail mechanism of inhibition of bradykinin-induced nociceptive transmission is not well known, the results of present study clearly showed that menatetrenone induced antinociception and it may cause through the inhibition bradykinin dependent nociceptive transmission in the spinal cord. Accordingly, menatetrenone could induce antinociception not only related to its effect on bone remodeling.
References 1. Akiyama Y, Hara K, Matsumoto A, Takahashi S, Tajima T. Comparison of intestinal absorption of vitamin K2 (menaquinone) homologues and their effects on blood coagulation in rats with hypoprothrombinaemia. Biochemical Pharmacolology 1995; 49 (12):1801–7. 2. Akiyama Y, Hara K, Ohkawa I, Tajima T. Effects of menatetrenone on bone loss induced by ovariectomy in rats. Japanese Journal of Pharmacolology 1993; 62 (2):145–53. 3. Hara K, Akiyama Y, Ohkawa Y, Tajima T. Effects of menatetrenone on prednisolone-induced bone loss in rats Bone 1993; 14 (6):813–8. 4. Knapen MH, Hamulyak H, Vermeer C. The effect of vitamin K supplementation on circulating osteocalcin (bone Gla protein) and urinary calcium excretion. Annals Internal Medicine 1989; 111 (12):1001–5. 5. Iwamoto H, Kosha S, Noguchi S, Murakami M, Fujino T, Douchi T, Nagata Y. A longitudinal study of the effect of vitamin K2 on bone mineral density in postmenopausal women a comparative study with vitamin D3 and estrogen-progestin therapy. Maturitas 1999; 31 (2): 161–4. 6. Sato Y, Honda Y, Kuno H, Oizumi K. Menatetrenone ameliorates osteopenia in disuse-affected limbs of vitamin D- and K-deficient stroke patients. Bone 1998; 23 (3):291–6. 7. Prous JR. The Year’s Drug News. Barcelona: Prous Sciences SA, 1999. 8. Yajima A, Yajima T, Yajima K. The effects of Menatetrenone (Vitamin K2) on the pain management and the maintenance of bone mass in elderly woman with osteoprosis. Journal of the Japan Society of Pain Clinicians 1998; 5 (1):1–4. 9. Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: characteristic biphasic pain response. Pain 1989; 38 (3):347–52. 10. Hylden JKL, Wilcox GL. Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice. Brain Research 1981; 217 (1): 212–5. 11. Hylden JKL, Wilcox GL. Intrathecal morphine in mice: a new technique. European Journal of Pharmacology 1980; 67 (2–3):313–6. 12. Kamei J, Kashiwazaki T, Hitosugi H, Nagase H. Algogenic mediator-induced nociceptive response in diabetic mice. European Journal of Pharmacology 1999; 369 (3):319–23. 13. Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987; 30 (1):103–14. 14. Haley JE, Dickenson AH, Schachter M. Electrophysiological evidence for a role of bradykinin in chemical nociception in the rat. Neuroscience Letter 1989; 97 (1–2):198–202. 15. Ohkubo T, Shibata M, Takahashi H, Inoki R. Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord. Journal of Pharmacology and Experimental Therapeutics 1990; 252 (3):1261–8.
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16. Fisher K, Coderre TJ. The contribution of metabotropic glutamate receptors (mGluRs) to formalin-induced nociception. Pain 1996; 68 (2–3):255–63. 17. Kuraishi Y, Hirota N, Sato Y, Hino Y, Satoh M, Takagi H. Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn. Brain Research 1985; 325 (1–2):294–8. 18. Morton CR, Hutchison WD, Hendry IA, Duggan AW. Somatostatin: evidence for a role in thermal nociception. Brain Research 1989; 488 (1–2):89–96. 19. Hara K, Akiyama Y, Tajima T, Shiraki M. Menatetrenone inhibits bone resorption partly through inhibition of PGE2 synthesis in vitro. Journal of Bone and Mineral Research 1993; 8(5):535–42. 20. Koshihara Y, Hoshi K, Shiraki M. Vitamin K2 (menatetrenone) inhibits prostaglandin synthesis in cultured human osteoblast-like periosteal cells by inhibiting prostaglandin H synthase activity. Biochemical Pharmacology 1993; 46 (8):1355–62.
Pharmacology letters Accelerated communication
Antinociceptive effect induced by intraperitoneal administration of vitamin K2 (menatetrenone) in ICR mice Kenji Onoderaa,*, Hisashi Shinodaa, Ko Zushidab, Kentaro Takib, Junzo Kameib a
Department of Pharmacology, School of Dentistry, Tohoku University, Aoba-ku, Sendai 980-8575, Japan Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan
b
(Submitted March 17, 2000; accepted May 11, 2000; received in final form July 5, 2000)
Abstract The antinociceptive effect of vitamin K2 (menatetrenone) in mice was examined using tail-flick and formalin test. Menatetrenone at doses of 10, 50 and 100 mg/kg, i.p. produced a dose-dependent and significant inhibition of the tail-flick response in mice. Menatetrenone (50 and 100 mg/kg, i.p.) had no significant effect on the duration of the first phase of the formalin-induced flinching. However, menatetrenone (100 mg/kg, i.p.) significantly inhibited the second phase of the formalin-induced flinching. I.p. administration of menatetrenone (100 mg/kg) significantly reduced the duration of nociceptive responses induced by i.t. injection of bradykinin, but not of substance P, prostaglandin E2 or N-methyl-D-aspartate (NMDA). These present data suggest that i.p. pretreatment with menatetrenone produced dose-dependent antinociceptive effect in mice. This effect may be, at least in part, mediated by the inhibition of bradykinin dependent nociceptive transmission in the spinal cord. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Vitamin K2 (menatetrenone); Antinociception; Bradykinin; Prostaglandin E2
Introduction Menatetrenone, vitamin K2 with 4 isoprene units, has not only high g-carboxylation activity in hypoprothrombinaemic rats [1], but also has prevented bone-loss induced by ovariectomy [2] or by predonisolone administration in rats [3]. In clinics, menatetrenone is used for * Corresponding author. Department of Pharmacology, Tohoku University, School of Dentistry, Seiryo-machi 4-1, Sendai 980-8575, Japan. Tel.: 181-22-717-8311; fax: 181-22-717-8313. E-mail address: [email protected] (K. Onodera) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 1 7 -6
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preventing postmenopausal osteoporosis [4] and has been effective to treat of patients with osteoporosis [5, 6]. Thus, menatetrenone, a known treatment for vitamin K deficiency (marketed since 1984) has been accepted for clinical application in the treatment of osteoporotic osteopenia from 1995 in Japan [7]. Interestingly, Yamaji et al. [8] have reported that female osteoporosis patients whose pain had not been relieved by vitamin D3, elcatonin, non-steroid anti-inflammatory drugs and physical therapy showed significant decrease in pain scores at 2 to 4 weeks after treatment with menatetrenone. Furthermore, they also reported that bone mass measured by computed X-ray densitometer was significant increased at 4 and 8 months after treatment of menatetrenone with improving pain scores [8]. These results indicate that menatetrenone may be useful in the treatment with osteoporosis patients suffering from pain. However, potency and its possible mechanism of menatetrenone-induced antinociception were still unclear. Thus, in this study, we investigated the antinociceptive effect of menatetrenone in mice using tail-flick and formalin test.
Methods Animals Six-weeks old male ICR mice (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan) weighing 26–30 g were used. They had free access to food and water in an animal room that was maintained at 24 6 1 8C with a 12-h light-dark cycle. This study was carried out in accordance with the Declaration of Helsinki and/or with the guiding principles for the care and use of laboratory animals approved by the Japanese Pharmacological Society. Tail-flick test The antinociceptive effect was evaluated by recording the latency in the tail-flick test using radiant heat as a stimulus. The tail of mice was blackened using indian ink and exposed to the focused beam of light from a preheated 500W projection bulb. The heat intensity was set by adjusting the source voltage of the bulb to 50 V. When withdrawal response occurred, the stimulus was terminated and the response latency measured automatically. A cut off latency of 15 s was used to prevent injury to the tail. Changes in tail flick latency (Dt (s)) was calculated for each animal according to the formula: Dt (s) 5 post-drug latency - pre-drug latency. Antinociceptive effect was measured 30, 60, 90 and 120 min after administration of menatetrenone. Formalin-induced flinching response The experiment was performed according to the method described by Shibata et al. [9]. Each mouse was acclimated to an acrylic observation chamber (32 3 23 3 17 cm3) for at least 5 min before the injection of formalin. Twenty-five ml of a 0.5% solution of formalin in 0.9% saline were administered into the dorsal surface of the right hindpaw. Immediately after the injection, each animal was returned to the observation chamber and its flinching response was recorded for 30 min. The mouse licked and bit the injected paw, and these responses were distinct and easily observed. The accumulated response time (s), i.e., the duration of
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93
licking and biting of the injected paw, was measured for each 5-min block. The accumulation response time during the initial two blocks and during on and after third blocks was regarded as the first-phase and second-phase response, respectively. Menatetrenone was injected 30 min before the injection of formalin. Algogenic mediator-induced nociceptive responses The experiment was performed according to the method described by Hylden and Wilcox [10]. Each mouse was acclimated to an acrylic observation chamber (39326324 cm3) for at least 5 min before the injection of an algogenic mediator. Intrathecal injection was performed by the method described by Hylden and Wilcox [11]. Each i.t. injection was administered using a 30-gauge needle directly through the intact skin between the L5 and L6 vertebrae. Drugs were given in a volume of 5 ml/mouse. Immediately following the injection, the mice were placed in the observation chamber. The cumulative duration (s) of biting, paw licking and scratching episodes was measured for 10 min after the injection of substance P or N-methylD-aspartate (NMDA), and 30 min after the injection of bradykinin or prostaglandin E2. Menatetrenone was injected 30 min before the injection of substance P, bradykinin, prostaglandin E2 or NMDA. The doses of algogenic mediators were based on our previous report [12]. Drugs Menatetrenone (Kaytwo N Injection) and its vehicle were generously supplied by Eisai Co., Ltd., Tokyo, Japan. Vehicle of menatetrenone, a mixture of glycerine, D-sorbitol, and purified soybean lecithin was the same composition of Kaytwo N Injection except menatetrenone. Menatetrenone and its vehicle were intraperitoneally (i.p.) administered. Substance P and bradykinin were purchased from Peptide Institute Inc., Osaka, Japan. N-methyl-Daspartate was purchased from Sigma Co., St. Louis, MO. U.S.A. Prostaglandin E2 was purchased from Calbiochem-Novabiochem International, La Jolla, CA, USA. Prostaglandin E2 was stored in ethanol solutions at 220 8C. For injection, an aliquot of the desired stock prostaglandin E2 solution was put into a borosilicate tube and the ethanol was removed by evaporation to dryness under nitrogen gas. Sterile saline was then added to dissolve the prostaglandin E2. Statistics The data are expressed as the mean 6 S.E.M. The statistical significance of differences was assessed with the Bonferroni/Dunn test. Results Fig. 1 shows the time course of the antinociception produced by menatetrenone (10–100 mg/kg, i.p.) in mice. The reduction in the tail-flick latency reached its peak 30 min after injection, gradually declined and returned to the preinjection level 120 min after menatetrenone injection. Thus, a time interval of 30 min after administration of menatetrenone was chosen for experiments designed to quantify its effect. As shown in Fig. 2, menatetrenone at doses of 10–100 mg/kg, i.p. produced a dose-dependent and significant inhibition of the tail-flick response in mice.
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Figure 1. Time course of menatetrenone on the tail-flick latency in mice. The effects of menatetrenone (10 mg/ kg, closed circle; 50 mg/kg, open triangle; 100 mg/kg, closed square) and vehicle (open circle) were measured 30, 60, 90 and 120 min after i.p. injection. Dt (s) 5 post-drug latency - pre-drug latency. Each point represents the mean with S.E. for 10 mice in each group.
In mice, s.c. injection of 0.5% formalin into the hindpaw caused an acute, immediate flinching response, i.e., licking and biting, which lasted about 5 min in the first-phase. In the second-phase, flinching response reappeared and lasted about for 20 min. Menatetrenone (50 and 100 mg/kg, i.p.) had no significant effect on the duration of the first phase of the formalininduced flinching (Fig. 2). However, menatetrenone (50 and 100 mg/kg, i.p.) dose-dependently inhibited the second phase of the formalin-induced flinching (Fig. 2). Indeed, 100 mg/kg of menatetrenone significantly reduced the duration of the second phase of the formalininduced flinching (Fig. 2). Intrathecal (i.t.) injection of either substance P (0.1 mg), bradykinin (1.0 mg), prostaglandin E2 (0.1 mg) or NMDA (0.1 mg) elicited a behavioral syndrome consisting of reciprocal hindlimb scratching directed towards caudal parts of the body, and biting or licking of the hind legs in mice. I.p. administration of menatetrenone (100 mg/kg) significantly reduced the duration of nociceptive responses induced by i.t. injection of bradykinin (Fig. 3A). However,
Figure 2. Effect of menatetrenone (50 and 100 mg/kg, i.p.) on the total duration of the response during the first (0–10 min) and second (10–30 min) phases of formalin-induced flinching responses in mice. Menatetrenone was administered i.p. 30 min before the injection of formalin. Each column represents the mean with S.E.M for 10 mice in each group. * P,0.05 vs. vehicle-treated group (Vehi, open column).
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Figure 3. Effects of menatetrenone (dotted column) on the bradykinin-, substance P-, prostaglandin E2 (PGE2)- or NMDA-induced nociceptive response in mice. Menatetrenone (100 mg/kg) was administered i.p. 30 min before the i.t. injection of bradykinin, substance P, PGE2 or NMDA. Each column represents the mean with S.E. (n5910). * P,0.05 vs. vehicle (open column)-treated group.
menatetrenone (100 mg/kg, i.p.) had no significant effect on the duration of substance P-, prostaglandin E2- or NMDA-induced nociceptive responses (Fig. 3B, C and D). Discussion In the present study, i.p. administration of menatetrenone reduced the tail-flick response in a dose-dependent manner in mice. This study also demonstrated that the second phase, but not the first phase of the formalin-induced flinching response is significantly and dose-dependently reduced by i.p. treatment with menatetrenone. Hunskaar and Hole [13] noted that the first phase of the formalin-induced nociceptive response may represent a direct effect on nociceptors, whereas the second phase may represent an enhanced response of sensitized dorsal horn neurons resulting from low-level neuronal input due to peripheral inflammatory insult. Moreover, it has been suggested that substance P participates in the short-lasting pain in the first phase of the formalin-induced nociceptive response, whereas somatostatin, bradykinin and prostaglandins participate in the prolonged and inflammatory pain in the second phase of the formalin-induced nociceptive response [14, 15]. Furthermore, it is well known that the stimulation of NMDA receptor in the spinal cord is also involved in the second phase of formalininduced nociceptive response [e.g., 16]. In addition, Kuraishi et al. [17] and Morton et al. [18] suggested that application of noxious thermal stimuli specifically increases the release of somatostatin, but not substance P. Taken together, these results suggest that menatetrenoneinduced antinociception may account from an inhibitory effect on the nociceptive transmission which mediated by an inflammatory mediators, such as somatostatin, bradykinin and prostaglandins, or NMDA. Indeed, in the present study, we observed that menatetrenone sig-
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nificantly reduced the duration of bradykinin-, but not substance P-, prostaglandin E2- or NMDA-induced nociceptive responses in mice. Thus, it is unlikely that menatetrenoneinduced antinociception may account from inhibition of prostaglandin E2, substance P and NMDA in mice. Nevertheless, since previous studies showed that menatetrenone inhibited prostaglandin E2 synthesis in vitro [19, 20], further research will be needed to clarify whether decrease in synthesis and/or release of prostaglandin E2 may be involved in the mechanism of menatetrenone-induced antinociception. Finally, although the detail mechanism of inhibition of bradykinin-induced nociceptive transmission is not well known, the results of present study clearly showed that menatetrenone induced antinociception and it may cause through the inhibition bradykinin dependent nociceptive transmission in the spinal cord. Accordingly, menatetrenone could induce antinociception not only related to its effect on bone remodeling.
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