Efficacy of non-steroidal anti-inflammatory drugs on zoledronic acid-induced acute-phase reactions: randomized, open-label, Japanese OZ study.

INTRODUCTION: Zoledronic acid infusion is used to treat osteoporosis but patients, especially Japanese patients, often experience acute-phase reactions (APRs). In this multicenter, randomized, open-label, parallel-group study, we examined the efficacy of the most commonly used non-steroidal anti-inflammatory drug loxoprofen in Japan in reducing the incidence rate of zoledronic acid-induced APRs and body temperature, and investigated risk/protective factors for APRs in this population. MATERIALS AND METHODS: Patients aged ≥ 60 years with primary osteoporosis (n = 368) were allocated randomly to zoledronic acid plus loxoprofen (ZOL + LOX) or zoledronic acid alone (ZOL). All patients received 5-mg zoledronic acid infusion on day 1, and patients in the ZOL + LOX group also received 120 mg and 180 mg of oral loxoprofen on days 1 and 2, respectively. Adverse events and body temperature were recorded during the 7-day observation period. RESULTS: The incidence rates of APRs were 34.4% (64/186 patients) and 47.8% (87/182 patients) in the ZOL + LOX and ZOL groups, respectively (P = 0.0109). The proportions of patients with increased body temperature (≥ 1 °C and ≥ 37.5 °C) were similar in both groups (P = 0.1186). Past bisphosphonate users had a significantly lower incidence rate of APRs than treatment-naïve patients (odds ratio 0.444, 95% confidence interval 0.285-0.692, P = 0.0003). CONCLUSIONS: Zoledronic acid-induced APRs appeared to be suppressed by loxoprofen. Known risk/protective factors, including prior osteoporosis treatment, were applicable to Japanese patients.

Influence on the bone mineral density and bone metabolism marker after the interruption and reinitiation of monthly minodronate therapy in postmenopausal women with osteoporosis.

OBJECTIVES: The purpose of this study was to investigate the influences of interruption and reinitiation of monthly minodronate therapy on the bone mineral density (BMD) and bone metabolism markers in postmenopausal women with osteoporosis. METHODS: Study patients were included if they had been administered monthly minodronate therapy for ≥6 months, interrupted the therapy, and reinitiated the therapy for ≥12 months. The BMD and bone metabolism markers were assessed at 4 time points: initiation, interruption, reinitiation and 1 year after reinitiation of therapy. RESULTS: A total of 23 patients were enrolled. The mean monthly minodronate treatment period was 23.8 ±â€¯12.9 months following a mean interruption period of 11.9 ±â€¯5.4 months. Once increased by monthly minodronate treatment for 2 years on average, the BMD of lumbar spine and radius did not significantly decrease even after an interruption for 1 year on average. However, the BMD of the femoral neck did decrease after interruption. The BMD of the lumbar spine and radius increased further after 1 year of monthly minodronate retreatment. The BMD of the femoral neck did not change. Once decreased after the treatment for an average of 2 years followed by an interruption for 1 year, bone metabolism markers increased gradually but did not recover to baseline levels. A potent suppressive effect on bone resorption was noted. The change rate was greater for the bone formation marker procollagen 1 N-terminal propeptide. CONCLUSIONS: Monthly minodronate treatment increases BMD and reduces bone metabolism markers. The effect lessens after treatment interruptions, and can be restored by retreatment.

Possible involvement of TRPV1 and TRPV4 in nociceptive stimulation- induced nocifensive behavior and neuroendocrine response in mice.

Members of the transient receptor potential (TRP) family of ion channels play important roles in inflammation and pain. Here, we showed that both TRPV1 and TRPV4 might contribute to biphasic nocifensive behavior and neuroendocrine response following a formalin test. We subcutaneously injected saline, formalin, or the TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) into one hindpaw of wild-type (WT), TRPV1-deficient (Trpv1(-/-)), and TRPV4-deficient (Trpv4(-/-)) mice to investigate nocifensive behaviors (phase I [0-10 min] and phase II [10-60 min]) and Fos expression in the dorsal horn of the spinal cord and other brain regions related to pain, in the paraventricular nucleus (PVN), paraventricular nucleus of the thalamus, the medial habenular nucleus, the medial nucleus of the amygdala and capsular part of the central amygdala. Subcutaneous (s.c.) injection of formalin caused less nocifensive behavior in Trpv1(-/-) and Trpv4(-/-) mice than in WT mice during phase I. In phase II, however, formalin induced less nocifensive behavior only in the Trpv1(-/-) mice, but not in the Trpv4(-/-) mice, relative to WT mice. The number of Fos-like immunoreactive (LI) neurons in laminae I-II of the dorsal horn increased in all types of mice 90 min after s.c. injection of formalin; however, there was no difference in the other regions between saline- and formalin-treated mice. Furthermore, s.c. injection of 4α-PDD did not induce nociceptive behavior nor influence the number of Fos-LI neurons in the all above mentioned regions in any of the mice. These results suggest that TRPV4-mediated nociceptive information from the peripheral tissue excluding the spinal pathway might be involved the formalin behavioral response during phase I. Only TRPV1 might regulate the formalin behavioral response in peripheral neuron.

A role of nesfatin-1/NucB2 in dehydration-induced anorexia.

Nesfatin-1/NucB2, an anorexigenic molecule, is expressed mainly in the hypothalamus, particularly in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). Nesfatin-1/NucB2 is also expressed in the subfornical organ (SFO). Because the SON and PVN are involved in body fluid regulation, nesfatin-1/NucB2 may be involved in dehydration-induced anorexia. To clarify the effects of endogenous nesfatin-1/NucB2, we studied changes in nesfatin-1/NucB2 mRNA levels in the SFO, SON, and PVN in adult male Wistar rats after exposure to osmotic stimuli by using in situ hybridization histochemistry. Significant increases in nesfatin-1/NucB2 mRNA levels, ∼2- to 3-fold compared with control, were observed in the SFO, SON, and PVN following water deprivation for 48 h, consumption of 2% NaCl hypertonic saline in drinking water for 5 days, and polyethylene glycol-induced hypovolemia. In addition, nesfatin-1/NucB2 expression was increased in response to water deprivation in a time-dependent manner. These changes in nesfatin-1/NucB2 mRNA expression were positively correlated with plasma sodium concentration, plasma osmolality, and total protein levels in all of the examined nuclei. Immunohistochemistry for nesfatin-1/NucB2 revealed that nesfatin-1/NucB2 protein levels were also increased after 48 h of dehydration and attenuated by 24 h of rehydration. Moreover, intracerebroventricular administration of nesfatin-1/NucB2-neutralizing antibody after 48 h of water deprivation resulted in a significant increase in food intake compared with administration of vehicle alone. These results suggested that nesfatin-1/NucB2 is a crucial peptide in dehydration-induced anorexia.

Effects of food deprivation on the hypothalamic feeding-regulating peptides gene expressions in serotonin depleted rats.

We examined the effects of serotonin (5-HT) depletion induced by peripheral injection of 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) on the expression of feeding-regulating peptides expressions by using in situ hybridization histochemistry in adult male Wistar rats. PCPA pretreatment had no significant effect on basal levels of oxytocin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), neuropeptide-Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin in the hypothalamus. Food deprivation for 48 h caused a significant decrease in CRH, TRH, POMC, and CART, and a significant increase in NPY, AgRP and MCH. After PCPA treatment, POMC and CART did not decrease despite food deprivation. NPY was significantly increased by food deprivation with PCPA, but was attenuated compared to food deprivation without PCPA. These results suggest that the serotonergic system in the hypothalamus may be involved in the gene expression of POMC, CART, and NPY related to feeding behavior.

The gene expression of the hypothalamic feeding-regulating peptides in cisplatin-induced anorexic rats.

Cisplatin has been widely used; however, various disadvantageous side effects afflict patients. Rikkunshito (RKT), a traditional Japanese herbal medicine, has been widely prescribed in Japan to improve anorexia; but the mechanisms are unknown. Here we studied whether RKT could improve anorexia induced by cisplatin and changes in feeding-regulating peptides in the hypothalamus in rats. Adult male rats were divided into 4 groups: water+saline (WS), water+cisplatin (WC), RKT+saline (RS), and RKT+cisplatin (RC) groups. Water or RKT (1g/kg) was intragastrically administered for 4 days, from day -1 to day 2, and saline or cisplatin (6mg/kg) was intraperitoneally (i.p.) administered at day 0. After i.p. administration, cumulative food intake, water intake, urine volume and body weight were measured. The rats were then decapitated, followed by removal of the brain, and feeding-regulating peptides in the hypothalamus were measured by in situ hybridization histochemistry. In the three-day measurements, there were no significant changes in cumulative water intake and urine volume. The body weight and cumulative food intake in WC significantly decreased compared to WS, whereas these were not observed in RC. Pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC) in WC significantly increased, and neuropeptide Y (NPY) in the ARC decreased compared to WS, whereas those in RS and RC were comparable to WS. These results suggest that RKT may have therapeutic potential for anorexia induced by cisplatin.

[Visualization of the response in the central nervous system after nociceptive stimulation using transgenic animals].

Physiological response to acute and chronic nociceptive stimulation are important for living organisms. In our laboratory, we generated transgenic rats expressing the arginine vasopressin (AVP) and enhanced green fluorescent protein (eGFP) fusion gene, and the c-fos and monomeric red fluorescent protein 1 (mRFP1) fusion gene in the central nervous system. We made it possible to visualize the pain response in the living cells. Using these transgenic rats, the aim of our studies is the elucidation of the physiological role of AVP after nociceptive stimulation and the pathophysiology of work-related pain. We describe the previous findings of nociceptive response, using these transgenic animals.

Expression of the c-fos-monomeric red fluorescent protein 1 fusion gene in the spinal cord and the hypothalamic paraventricular nucleus in transgenic rats after nociceptive stimulation.

We generated transgenic rats expressing the c-fos and monomeric red fluorescent protein 1 (mRFP1) fusion gene in the central nervous system after adequate stimulation. In the present study, the time-course of the induction patterns of mRFP1 fluorescence in the spinal cord and the paraventricular nucleus (PVN) was compared with that of Fos-like immunoreactivity (LI) within 24 h after subcutaneous (s.c.) injection of 0.9% saline and 5% formalin in both hind paws. Control rats were not treated. In the control and saline/formalin injected rats, scattered mRFP1 fluorescence in the spinal cord and the PVN was observed at 0 min, though there was little Fos-LI in the same region. The mRFP1 fluorescence in the spinal cord and the PVN was increased at 3h after formalin. On the other hand, the changes of Fos-LI in the spinal cord and the PVN were relatively shorter than those of the mRFP1 fluorescence after formalin. These results suggest that the c-fos-mRFP1 fusion gene expression is slightly upregulated in normal conditions and nociceptive stimulation-induced induction of the fusion gene may be maintained longer than the endogenous c-fos gene expression in the spinal cord and the PVN. Next, nocifensive behavior and mRFP1 fluorescence and Fos-LI in the spinal cord and the PVN after s.c. injection of formalin, 4α-phorbol 12,13-didecanoate (4α-PDD) and saline were compared. Although the 4α-PDD injected rats seldom displayed nocifensive behaviors like s.c. saline injection, 4α-PDD injection caused mRFP1 fluorescence and Fos-LI significantly in the spinal cord and the PVN unlike s.c. saline injection.

Induction of arginine vasopressin-enhanced green fluorescent protein expression in the locus coeruleus following kainic acid-induced seizures in rats.

Seizure causes autonomic, neuroendocrine and stress responses. We examined the effects of kainic acid (KA)-induced seizures on the expression of the arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) in the locus coeruleus (LC), an area known to contain noradrenergic cells, in AVP-eGFP transgenic male and female rats, with the rationale to identify stressors which induce AVP synthesis in the LC. Subcutaneous (s.c.) administration of KA caused a progressive development of seizure behavior within 24 h. AVP-eGFP fluorescence in the LC was detected 6, 24, and 48 h and 1 week after administration of KA (12 mg/kg). From a nearly undetectable level, it reached a maximum at 48 h after s.c. administration of KA and returned to the basal levels after 2 weeks. AVP-eGFP fluorescence in the LC after s.c. administration of KA was significantly reduced by the pretreatment with MK-801 (nonselective N-methyl-D-aspartate (NMDA) receptor antagonist). In the KA-administered rats, immunohistochemistry for tyrosine hydroxylase (TH) revealed that the eGFP fluorescence was co-localized with TH-immuno-reactivity in the LC. These results suggest that the synthesis of AVP-eGFP is potentially up-regulated in noradrenergic neurons in the LC after KA-induced seizures through the activation of NMDA receptors.

Upregulation of arginine vasopressin synthesis in the rat hypothalamus after kainic acid-induced seizures.

We examined the effects of kainic acid (KA)-induced seizures on arginine vasopressin (AVP) gene expression in the paraventricular (PVN) and the supraoptic nuclei (SON) of normal rats using in situ hybridization histochemistry. We also investigated the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene after KA-induced seizures in transgenic rats. AVP heteronuclear (hn) RNA levels in the PVN and the SON were significantly increased at 3h and 24h after subcutaneous (s.c.) administration of KA in normal rats. AVP mRNA levels in the PVN and the SON did not change significantly at 3h, 24h and 1 week after s.c. administration of KA in normal rats. In KA-administered transgenic rats, AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON were significantly stronger compared to vehicle-administered transgenic rats. By pretreatment with MK-801 (nonselective N-methyl-D-aspartate, NMDA, receptor antagonist), AVP-eGFP transgenic rats after administration of KA did not show preconvulsive symptoms or convulsions and AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON of these rats was significantly reduced. These results suggested that KA-induced increases in AVP transcripts and AVP were prevented by MK801 because seizure activity was prevented or reduced.

Possible contribution of pannexin channel to ATP-induced currents in vitro in vasopressin neurons isolated from the rat supraoptic nucleus.

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activity of these neurons. ATP plays a crucial role in the regulation of SON MNCs by activating the purinergic P2X and P2Y receptors. Recent reports of interaction between P2X receptors and pannexin channels have provided new insights into the physiology of the central nervous system; however, the function of pannexin channels has not been assessed in AVP neurons. In the present study, we examined the possible contribution of the pannexin channel in ATP-induced responses in SON AVP neurons. We used the whole-cell patch-clamp technique in isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein transgene. The ATP-induced current was inhibited in a concentration-dependent manner by pannexin channel blockers carbenoxolone and mefloquine, whereas the connexin channel blockers flufenamic acid and lanthanum had no effect. Multi-cell reverse transcriptase-polymerase chain reaction experiments confirmed the existence of pannexin-1 mRNA in AVP neurons. The involvement of the ATP-activated transient receptor potential vanilloid and acid-sensing ion channels was excluded. These results suggest that pannexin channels in SON AVP neurons are involved in the regulatory mechanisms of neuronal activity.

Similar changes of hypothalamic feeding-regulating peptides mRNAs and plasma leptin levels in PTHrP-, LIF-secreting tumors-induced cachectic rats and adjuvant arthritic rats.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in malignancy. However, it is difficult to explain the mechanism of anorexia/cachexia with PTHrP secretion in detail. Previously, we demonstrated that the expressions of orexigenic peptides increased and anorexigenic peptides decreased under cachectic conditions in rats carrying tumors secreting PTHrP. In this study, we investigated whether such changes in the expression of hypothalamic feeding-regulating peptides can be solely attributed to PTHrP or are a general response under cachectic conditions. Cachectic syndromes were induced in rats by: (i) inoculation of human lung cancer LC-6 cells that secreted PTHrP, (ii) inoculation of human melanoma SEKI cells that secrete not PTHrP but LIF1, (iii) injection of heat-killed Mycobacterium leading to arthritis (AA) and (iv) oral administration of a high dose of 1α,25(OH)(2)D(3) that resulted in hypercalcemia. The LC-6-bearing rats and AA rats were treated with or without anti-PTHrP antibody and indomethacin, respectively, and the expression of the hypothalamic feeding-regulating peptide mRNAs were examined by in situ hybridization histochemistry. The orexigenic peptide mRNAs, such as neuropeptide Y and agouti-related protein, were significantly increased, and that of anorexigenic peptide mRNAs, such as proopiomelanocortin, cocaine- and amphetamine-regulated transcript and corticotropin-releasing hormone were significantly decreased when they developed cachectic syndromes and AA. A high dose of 1α,25(OH)(2)D(3) caused hypercalcemia and body weight loss but did not affect the expression of hypothalamic feeding-regulating peptide mRNAs. The expressions of the hypothalamic feeding-regulating peptides change commonly in different chronic cachectic models without relating to serum calcium levels.

Modulators of BK and SK channels alter electrical activity in vitro in single vasopressin neurons isolated from the rat supraoptic nucleus.

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activities of the MNCs. Ca(2+)-activated K(+) channels, such as the BK and SK channels, are K(+)-selective ion channels that are activated in response to increased intracellular calcium concentrations. Intrinsic affinities for Ca(2+) permit these channels to exert a negative feedback effect on cellular excitability. In the present study, we used the whole-cell patch-clamp technique to examine the effects of BK or SK channel modulators on neuronal activity in single isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Application of BK or SK channel activators abolished the action potentials and induced hyperpolarization. In contrast, the number of action potentials was significantly increased after application of BK or SK channel blockers. Our results suggest that BK and SK channels in AVP neurons may play a role in the regulatory mechanisms of neural activity.