High mobility group box1 (HMGB1), a nuclear protein, once acetylated by histone acetyltransferase, is released into the extracellular space, and causes pain signals, thereby contributing to pathological pain. Repeated intracolonic administration of butyrate, known to inhibit histone deacetylase (HDAC), produces colonic hypersensitivity in rodents, being widely used as models for irritable bowel syndrome (IBS). Thus, we asked whether HMGB1 would participate in the butyrate-induced colonic hypersensitivity in mice, and analyzed the underlying mechanisms. Repeated butyrate treatment caused colonic hypersensitivity to distension and intraluminal sulfide, a functional enhancer of Cav3.2 channels, in mice, which was prevented by repeated treatment with an anti-HMGB1-neutralizing antibody, thrombomodulin alfa (TMα) capable of causing thrombin-dependent degradation of HMGB1, antagonists for RAGE, TLR4 and CXCR4, membrane receptors of HMGB1, liposomal clodronate, a macrophage depletor, and ethyl pyruvate capable of inhibiting HMGB1 release from macrophages. Butyrate treatment increased the number of Iba1-positive macrophages, but not S100B-positive enteric glial cells (EGCs), and the rate of cytosolic/whole cell HMGB1 levels in both types of cells in the colonic mucosa. In macrophage-like RAW264.7 cells and EGC-like CRL-2690 cells, butyrate as well as trichostatin A, a well-known HDAC inhibitor, at the same concentrations that increased histone acetylation, evoked cytoplasmic translocation and extracellular release of nuclear HMGB1. Together, butyrate is considered to cause HMGB1 release from macrophages and EGCs most probably by inhibiting HDAC, resulting in colonic hypersensitivity in mice. HMGB1 and its membrane receptors might serve as drug targets for colonic hypersensitivity in IBS patients.

We tested the hypothesis that Cav3.2 T-type Ca2+ channels, which can be rebooted by sulfides from Zn2+ inhibition under physiological conditions, and sulfide-generating enzymes including cystathionine-β-synthase (CBS) would participate in the colitis-related visceral pain in mice treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The visceral hypersensitivity following TNBS-induced colitis was abolished by an inhibitor or genetic deletion of Cav3.2 and by a CBS inhibitor, and accompanied by CBS upregulation in the colon. Our data thus suggest that the enhanced activity of Cav3.2 brought about by sulfides generated by upregulated CBS is involved in the colitis-related visceral hypersensitivity.

Hormone therapy, especially androgen deprivation therapy (ADT), is effective against prostate cancer (PC), whereas long-term ADT is a risk for metabolic/cardiovascular disorders including diabetes (DM), hypertension (HT) and dyslipidemia (DL), and might result in progression to castration-resistant prostate cancer (CRPC). We thus conducted a multicenter retrospective cohort study to ask whether CRPC progression would be associated positively with HT, DM or DL and negatively with statins prescribed for treatment of DL. In this study, 1,112 nonmetastatic PC patients undergoing ADT were enrolled. Univariate statistical analyses clearly showed significant association of HT or DM developing after ADT onset, though not preexisting HT or DM, with early CRPC progression. On the other hand, preexisting DL or statin use, but not newly developed DL or started statin prescriptions following ADT, was negatively associated with CRPC progression. Multivariate analysis revealed significant independent association of the newly developed DM or HT, or preexisting statin use with CRPC progression [adjusted hazard ratios (95% confidence intervals): 3.85 (1.65-8.98), p = 0.002; 2.75 (1.36-5.59), p = 0.005; 0.25 (0.09-0.72), p = 0.010, respectively]. Together, ADT-related development of HT or DM and preexisting statin use are considered to have positive and negative impact on CRPC progression, respectively.

Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Cav3.2 T-type Ca2+ channels by eliminating Zn2+ bound to the channels, thereby promoting somatic and visceral pain. Cav3.2 is under inhibition by Zn2+ in physiological conditions, so that sulfides function to reboot Cav3.2 from Zn2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Cav3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Cav3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain.

Abstract Given possible involvement of the central and peripheral angiotensin system in pain processing, we conducted clinical and preclinical studies to test whether pharmacological inhibition of the angiotensin system would prevent diabetic peripheral neuropathy (DPN) accompanying type 2 diabetes mellitus (T2DM). In the preclinical study, the nociceptive sensitivity was determined in leptin-deficient ob/ob mice, a T2DM model. A clinical retrospective cohort study was conducted, using the medical records of T2DM patients receiving antihypertensives at three hospitals for nearly a decade. In the ob/ob mice, daily treatment with perindopril, an angiotensin-converting enzyme inhibitor (ACEI), or telmisartan, an angiotensin receptor blocker (ARB), but not amlodipine, an L-type calcium channel blocker (CaB), significantly inhibited DPN development without affecting the hyperglycemia. In the clinical study, the enrolled 7464 patients were divided into three groups receiving ACEIs, ARBs and the others (non-ACEI, non-ARB antihypertensives). Bonferroni’s test indicated significantly later DPN development in the ARB and ACEI groups than the others group. The multivariate Cox proportional analysis detected significant negative association of the prescription of ACEIs or ARBs and β-blockers, but not CaBs or diuretics, with DPN development. Thus, our study suggests that pharmacological inhibition of the angiotensin system is beneficial to prevent DPN accompanying T2DM.

[This corrects the article DOI: 10.1371/journal.pone.0261473.].

Community pharmacists may play a key role in promoting deprescribing of potential inappropriate medications (PIMs) that are highly prevalent among community-dwelling elderly with dementia. To characterize PIMs categories that need a special attention for dementia patients, in the present study, we analyzed the anonymized pharmacy claims data of patients aged 65 years and older (n = 333869) who visited nationwide 905 community-based pharmacies of Sugi Pharmacy Co., Ltd. during December 1-31, 2019. A dementia group was defined as patients who received typical dementia medications marketed in Japan, i.e., donepezil, galantamine, memantine or rivastigmine, and a non-dementia group was defined as patients who received no such medications. After propensity score matching on the basis of patients' age, gender and home healthcare insurance usage, the data of 11486 patients in each group were subjected to logistic regression analyses, to identify PIMs categories particularly important for dementia patients. Univariate analysis indicated that the proportions of dementia patients who received 1 and 2≤ of PIMs were significantly (p < 0.001) greater than those of non-dementia patients (odds ratios were 1.35 and 1.47, respectively). Multivariate analyses identified 5 categories of PIMs that were significantly more frequently prescribed in dementia patients, i.e., 'H2 blockers,' 'drugs for overactive bladder,' 'anti-diabetes drugs' and 'sulpiride' listed as PIMs categories for non-specific cases (adjusted odds ratios (aORs): 1.29, 1.91, 1.17, and 1.38, respectively), in addition to 'antipsychotics' listed only for dementia patients (aOR: 4.29). These results provide useful information to establish strategies for pharmacist-led deprescribing of PIMs in dementia patients.

Cav3.2, a T-type calcium channel (T-channel) family member, is expressed in the nociceptors and spinal cord, and its activity is largely suppressed by zinc under physiological conditions. In rats, intrathecal and intraplantar administration of a zinc chelator, TPEN, caused T-channel-dependent mechanical hyperalgesia, and the intraplantar, but not intrathecal, TPEN induced Cav3.2 upregulation in the dorsal root ganglion. In mice, intraplantar TPEN also caused mechanical allodynia, which was abolished by T-channel inhibitors or Cav3.2 gene deletion. Together, spinal and peripheral zinc deficiency appears to enhance Cav3.2 activity in the spinal postsynaptic neurons and nociceptors, respectively, thereby promoting pain.

Poly-trans-[ (2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Irregular regeneration or inappropriate remodeling of the axons of the primary afferent neurons after peripheral nerve trauma could be associated with the development of neuropathic pain. We analyzed the molecular mechanisms for the neuritogenesis and neurite outgrowth caused by prostaglandin E2 (PGE2) in mouse dorsal root ganglion (DRG) neurons, and evaluated their opioid modulation. PGE2 in combination with IBMX, a phosphodiesterase inhibitor, caused neuritogenesis/neurite outgrowth in DRG cells, an effect abolished by a prostanoid EP4, but not EP2, receptor antagonist, and inhibitors of adenylyl cyclase or protein kinase A (PKA). Blockers of T-type Ca2+ channels (T-channels), that are responsible for window currents involving the sustained low-level Ca2+ entry at voltages near the resting membrane potentials and can be functionally upregulated by PKA, inhibited the neuritogenesis/neurite outgrowth caused by PGE2/IBMX or dibutylyl cyclic AMP, a PKA activator, in DRG neurons, an inhibitory effect mimicked by ZnCl2 and ascorbic acid that block Cav3.2, but not Cav3.1 or Cav3.3, T-channels. Morphine and DAMGO, μ-opioid receptor (MOR) agonists, suppressed the neuritogenesis and/or neurite outgrowth induced by PGE2/IBMX in DRG neurons and also DRG neuron-like ND7/23 cells, an effect reversed by naloxone or β-funaltrexamine, a selective MOR antagonist. Our data suggest that the EP4 receptor/PKA/Cav3.2 pathway is involved in the PGE2-induced neuritogenesis/neurite outgrowth in DRG neurons, which can be suppressed by MOR stimulation. We propose that MOR agonists including morphine in the early phase after peripheral nerve trauma might delay the axonal regeneration of the primary afferent neurons but prevent the development of neuropathic pain.

Cav3.2 channels belong to the T-type calcium channel (T-channel) family, i.e., low voltage-activated calcium channels, and are abundantly expressed in the nociceptors, playing a principal role in the development of pathological pain. The channel activity of Cav3.2 is suppressed by zinc under physiological conditions. We thus tested whether dietary zinc deficiency would cause Cav3.2-dependent nociceptive hypersensitivity in mice. In the mice fed with zinc deficient diet for 2 weeks, plasma zinc levels declined by more than half, and mechanical allodynia developed. The dietary zinc deficiency-induced allodynia was restored by T-channel inhibitors or by Cav3.2 gene silencing. These data demonstrate that zinc deficiency induces Cav3.2-dependent nociceptive hypersensitivity in mice, thereby suggesting that pain experienced by patients with diseases accompanied by zinc deficiency (e.g., chronic kidney disease) might involve the increased Cav3.2 activity.

T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure-activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.

Oxaliplatin often induces peripheral neuropathy, a dose-limiting adverse reaction, and in rare cases leads to sinusoidal obstruction syndrome. We thus conducted a retrospective cohort study to examine the relationship between oxaliplatin-induced peripheral neuropathy (OIPN) and hepatic impairment, and then perform a fundamental study to analyze the underlying mechanisms. Analysis of medical records in cancer patients treated with oxaliplatin indicated that laboratory test parameters of hepatic impairment including AST, ALT and APRI (AST to platelet ratio index) moderately increased during oxaliplatin treatment, which was positively correlated with the severity of OIPN (grades 1-4), and associated with later incidence of survivors with OIPN grades ≥2. In mice, hepatic injury induced by CCl4 or ethanol accelerated OIPN in mice, an effect prevented by inactivation of high mobility group box 1 (HMGB1), known to participate in OIPN, by the neutralizing antibody or thrombomodulin alfa capable of promoting its thrombin-dependent degradation. Oxaliplatin also aggravated the hepatic injury in mice. CCl4 released HMGB1 from cultured hepatic parenchymal cells, and oxaliplatin at clinically achievable concentrations released HMGB1 from hepatic parenchymal and non-parenchymal cells. Our clinical and preclinical data suggest that the development of mild hepatic impairment during oxaliplatin treatment is associated with later aggravation of OIPN.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

【背景・目的】薬学部生は実務実習期間中，多くの医薬品を「商品名」で学ぶため，大学で習得した「一般名」の知識が実習後に低下する可能性が懸念される．そこで実務実習前後に「一般名」と「商品名」の理解度に関するテストを実施し，結果に影響を与える因子を解析した．【方法】近畿大学病院で実務実習に参加した学生64名を対象に，実習前，薬局実習後，病院実習後に「一般名」または「商品名」で表示された薬の薬理作用に対する知識を測るマークシート形式のテストを行った．【結果】実務実習により薬の「商品名」に関する知識が有意に上昇した一方，「一般名」に関する知識は変化しなかった．【考察】実務実習後，懸念されたほど薬の「一般名」に関する知識は低下していなかったが，実習期間中に「商品名」と「一般名」をリンクさせる学習を行うことが，大学で習得した知識を生かした調剤や処方解析を行うために役立つと考えられた．

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse reaction in cancer patients treated with several cytotoxic anticancer agents including paclitaxel. Duloxetine, an antidepressant known as a serotonin-noradrenalin reuptake inhibitor, is the only agent that has moderate evidence for the use to treat painful CIPN. The present retrospective cohort study aimed to analyze risk factors for paclitaxel-induced peripheral neuropathy (PIPN), and investigate ongoing prescription drug use for PIPN in Japan. Female breast and gynecologic cancer patients who underwent paclitaxel-based chemotherapy at a single center in Japan between January 2016 and December 2019 were enrolled in this study. Patients’ information obtained from electronic medical records were statistically analyzed to test possible risk factors on PIPN diagnosis. Patients’ age, total paclitaxel dose, the history of female hormone-related diseases, hypertension and body mass index (BMI), but not additional platinum agents, were significantly associated with increased PIPN diagnosis. Drugs prescribed for PIPN included duloxetine, pregabalin, mecobalamin and Goshajinkigan, a polyherbal medicine, regardless of poor evidence for their effectiveness against CIPN, and were greatly different between breast and gynecologic cancer patients diagnosed with PIPN at the departments of Surgery and Gynecology, respectively. Thus, older age, greater total paclitaxel dose, the history of estrogen-related diseases, hypertension and BMI are considered risk factors for PIPN in paclitaxel-based chemotherapy of female cancer patients. It appears an urgent need to establish a guideline of evidence-based pharmacotherapy for PIPN.

To identify risk factors for the prognosis of prostate cancer (PC), we retrospectively analyzed the impact of lifestyle-related disorders as well as PC characteristics at initial diagnosis on the progression to castration-resistant PC (CRPC) in PC patients undergoing hormone therapy. Of 648 PC patients, 230 who underwent hormone therapy and met inclusion criteria were enrolled in this study. CRPC developed in 48 patients (20.9%). Univariate analysis using Cox proportional hazard model indicated that newly developed diabetes mellitus (DM) following hormone therapy (postDM), but not preexisting DM, as well as PC characteristics at initial diagnosis including prostate-specific antigen (PSA) ≥ 18 were significantly associated with the progression to CRPC. A similar tendency was also observed in the relationship between newly developed hypertension following hormone therapy and CRPC progression. On the other hand, neither dyslipidemia nor hyperuricemia, regardless the onset timing, exhibited any association with CRPC progression. In multivariate analysis, postDM and PSA ≥ 18 were extracted as independent risk factors for CRPC progression (adjusted hazard ratios, 3.38 and 2.34; p values, 0.016 and 0.019, respectively). Kaplan–Meier analysis and log-rank test clearly indicated earlier progression to CRPC in PC patients who developed postDM or had relatively advanced initial PC characteristics including PSA ≥ 18. Together, the development of lifestyle-related disorders, particularly DM, following hormone therapy, as well as advanced PC characteristics at initial diagnosis is considered to predict earlier progression to CRPC and poor prognosis in PC patients undergoing hormone therapy.

Background: Accurate prognosis in terminal cancer patients is useful to improve their quality of life and also to decide the cessation of fluid administration. Nonetheless, few prognostic indicators are available for prediction of such a short-term life expectancy. Objectives: The present study aimed at evaluating the efficacy of C-reactive protein (CRP)/albumin (CRP/Alb) ratio, prognostic nutritional index (PNI), fibrosis-4 (FIB-4) index, and albumin-bilirubin (ALBI) score in identifying terminal cancer patients who have a life expectancy less than two weeks. Design: Retrospective study. Setting/Subjects: Of 483 patients who died between April 2019 and March 2020 at a single center in Japan, 102 who met the inclusion criteria were enrolled in this study. Measurements: CRP/Alb, PNI, FIB-4, and ALBI were calculated from the laboratory data collected 1-13, 14-27, 28-83, and 168-365 days before death and subjected to statistical analyses. Results: CRP/Alb, PNI, FIB-4, and ALBI values were significantly associated with the time before death during terminal 365 days. CRP/Alb ≥4.4, PNI <30, FIB-4 ≥ 9.4, and ALBI ≥ -1.26 were significantly associated with the transition from the first half to the second half of terminal four weeks. Of those prognostic indicators, three and four combinations provided significantly reliable estimation of a life expectancy less than two weeks. Conclusions: CRP/Alb, PNI, FIB-4, ALBI, and their combinations are considered to help identify cancer patients who have a life expectancy less than two weeks, which is useful to make appropriate end-stage treatment decisions, for example, cessation of artificial hydration therapy.

Given the role of macrophage-derived high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel, we analyzed the role of HMGB1 and macrophages in the CIPN caused by bortezomib, a proteasome-inhibiting chemotherapeutic agent used for the treatment of multiple myeloma. Repeated administration of bortezomib caused CIPN accompanied by early-stage macrophage accumulation in the dorsal root ganglion. This CIPN was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of accelerating thrombin-dependent degradation of HMGB1, antagonists of the receptor for advanced glycation end-products (RAGE) and C-X-C motif chemokine receptor 4 (CXCR4), known as HMGB1-targeted membrane receptors, or macrophage depletion with liposomal clodronate, as reported in a CIPN model caused by paclitaxel. In macrophage-like RAW264.7 cells, bortezomib as well as MG132, a well-known proteasome inhibitor, caused HMGB1 release, an effect inhibited by caspase inhibitors but not inhibitors of NF-κB and p38 MAP kinase, known to mediate paclitaxel-induced HMGB1 release from macrophages. Bortezomib increased cleaved products of caspase-8 and caused nuclear fragmentation or condensation in macrophages. Repeated treatment with the caspase inhibitor prevented CIPN caused by bortezomib in mice. Our findings suggest that bortezomib causes caspase-dependent release of HMGB1 from macrophages, leading to the development of CIPN via activation of RAGE and CXCR4.

A neuroimmune crosstalk is involved in somatic and visceral pathological pain including inflammatory and neuropathic components. Apart from microglia essential for spinal and supraspinal pain processing, the interaction of bone marrow-derived infiltrating macrophages and/or tissue-resident macrophages with the primary afferent neurons regulates pain signals in the peripheral tissue. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Peripheral nerve macrophages and intestinal resident macrophages, in addition to adult monocyte-derived infiltrating macrophages, secrete a variety of mediators, such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, high mobility group box 1 and bone morphogenic protein 2 (BMP2), that regulate the excitability of the primary afferents. Neuron-derived mediators including neuropeptides, ATP and macrophage-colony stimulating factor regulate the activity or polarization of diverse macrophages. Thus, macrophages have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain by interacting with neurons.

We performed clinical retrospective study in female cancer patients and fundamental experiments in mice, in order to clarify risk factors for paclitaxel-induced peripheral neuropathy (PIPN). In the clinical study, 131 of 189 female outpatients with cancer undergoing paclitaxel-based chemotherapy met inclusion criteria. Breast cancer survivors (n = 40) showed significantly higher overall PIPN (grades 1-4) incidence than non-breast cancer survivors (n = 91). Multivariate sub-analyses of breast cancer survivors showed that 57 years of age or older and endocrine therapy before paclitaxel treatment were significantly associated with severe PIPN (grades 2-4). The age limit was also significantly correlated with overall development of severe PIPN. In the preclinical study, female mice subjected to ovariectomy received repeated administration of paclitaxel, and mechanical nociceptive threshold was assessed by von Frey test. Ovariectomy aggravated PIPN in the mice, an effect prevented by repeated treatment with 17β-estradiol. Repeated administration of thrombomodulin alfa (TMα), known to prevent chemotherapy-induced peripheral neuropathy in rats and mice, also prevented the development of PIPN in the ovariectomized mice. Collectively, breast cancer survivors, particularly with postmenopausal estrogen decline and/or undergoing endocrine therapy, are considered a PIPN-prone subpopulation, and may require non-hormonal pharmacological intervention for PIPN in which TMα may serve as a major candidate.

T-Type Ca2+ channels (T-channels), particularly Cav3.2, are now considered as therapeutic targets for treatment of intractable pain including visceral pain. Among existing medicines, bepridil, a multi-channel blocker, used for treatment of arrhythmia and angina, and pimozide, a dopamine D2 receptor antagonist, known as a typical antipsychotic, have potent T-channel blocking activity. We thus tested whether bepridil and pimozide could suppress visceral pain in mice. Colonic and bladder pain were induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and systemic administration of cyclophosphamide (CPA), respectively. Referred hyperalgesia was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load with intracolonic water injection and spontaneous bladder pain were evaluated by observing nociceptive behaviors in conscious mice. The mice exhibited referred hyperalgesia and colonic hypersensitivity to distension on day 6 after TNBS treatment. Systemic administration of bepridil at 10-20 mg/kg or pimozide at 0.1-0.5 mg/kg strongly reduced the referred hyperalgesia on the TNBS-induced referred hyperalgesia and colonic hypersensitivity to distension. CPA treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia, which were reversed by bepridil at 10-20 mg/kg or pimozide at 0.5-1 mg/kg. Our data thus suggest that bepridil and pimozide, existing medicines capable of blocking T-channels, are useful for treatment of colonic and bladder pain, and serve as seeds for the development of new medicines for visceral pain treatment.

Chemotherapy-induced peripheral neuropathy (CIPN), one of major dose-limiting side effects of first-line chemotherapeutic agents such as paclitaxel, oxaliplatin, vincristine, and bortezomib is resistant to most of existing medicines. The molecular mechanisms of CIPN have not been fully understood. High mobility group box 1 (HMGB1), a nuclear protein, is a damage-associated molecular pattern protein now considered to function as a pro-nociceptive mediator once released to the extracellular space. Most interestingly, HMGB1 plays a key role in the development of CIPN. Soluble thrombomodulin (TMα), known to degrade HMGB1 in a thrombin-dependent manner, prevents CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine and in patients with colorectal cancer undergoing oxaliplatin-based chemotherapy. In this review, we describe the role of HMGB1 and its upstream/downstream mechanisms in the development of CIPN and show drug candidates that inhibit the HMGB1 pathway, possibly useful for prevention of CIPN.

Overexpression of Cav3.2 T-type Ca2+ channels in L4 dorsal root ganglion (DRG) participates in neuropathic pain after L5 spinal nerve cutting (L5SNC) in rats. The L5SNC-induced neuropathic pain also involves high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein, and its target, the receptor for advanced glycation end-products (RAGE). We thus studied the molecular mechanisms for the L5SNC-induced Cav3.2 overexpression as well as neuropathic pain in rats by focusing on; 1) possible involvement of early growth response 1 (Egr-1), known to regulate transcriptional expression of Cav3.2, and ubiquitin-specific protease 5 (USP5) that protects Cav3.2 from proteasomal degradation, and 2) possible role of HMGB1/RAGE as an upstream signal. Protein levels of Cav3.2 as well as Egr-1 in L4 DRG significantly increased in the early (day 6) and persistent (day 14) phases of neuropathy after L5SNC, while USP5 protein in L4 DRG did not increase on day 6, but day 14. An anti-HMGB1-neutralizing antibody or a low molecular weight heparin, a RAGE antagonist, prevented the development of neuropathic pain and upregulation of Egr-1 and Cav3.2 in L4 DRG after L5SNC. L5SNC increased macrophages accumulating in the sciatic nerves, and the cytoplasm/nuclear ratio of immunoreactive HMGB1 in those macrophages. Our findings suggest that L5SNC-induced Cav3.2 overexpression in L4 DRG and neuropathic pain involves Egr-1 upregulation downstream of the macrophage-derived HMGB1/RAGE pathway, and that the delayed upregulation of USP5 might contribute to the persistent Cav3.2 overexpression and neuropathy.

Given the role of Cav3.2 isoform among T-type Ca2+ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Cav3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Cav3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Cav3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Cav3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Cav3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na2S, known to enhance Cav3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Cav3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Cav3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.

OBJECTIVE: Bepridil prolongs the QT interval and can induce torsade de pointes. Although increased bepridil concentration may be a primary cause of prolonged QT, the relationship between serum bepridil concentration and prolonged QT remains unclear. We investigated the relationship between serum bepridil concentration and the corrected QT (QTc) interval in patients treated with bepridil. MATERIALS AND METHODS: A retrospective study was performed at the National Cerebral and Cardiovascular Center in Japan. Patients with atrial fibrillation who were treated with bepridil from January 2014 to December 2015 were enrolled in the study. Serum bepridil concentrations and electrocardiogram data collected more than 21 days after the initiation of bepridil were used for analysis. RESULTS: A total of 60 patients were included in this study. There was a significant difference in mean QTc interval before and after initiation of bepridil (p < 0.0001). A significant relationship was observed between bepridil dose (p = 0.014) or serum bepridil concentration (p < 0.001) and QTc interval. Additionally, a significant relationship was observed between serum bepridil concentration and ΔQTc (p = 0.034). In the study, 4 patients developed QTc prolongation ≥ 500 ms after the initiation of bepridil. Serum bepridil concentration in this group was significantly higher compared with the group that did not display prolonged QTc (973 ± 651 vs. 526 ± 310 ng/mL, p = 0.01). CONCLUSION: This study revealed that the QTc interval was significantly associated with serum bepridil concentration. Serum bepridil concentration beyond a therapeutic range may be a critical risk factor for developing QTc prolongation.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.

High-mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, facilitates pain signals as well as inflammation. Intraplantar or intraspinal application of HMGB1 elicits hyperalgesia/allodynia in rodents by activating the advanced glycosylation end-product specific receptor (receptor for advanced glycation end-products; RAGE) or Toll-like receptor 4 (TLR4). Endogenous HMGB1 derived from neurons, perineuronal cells or immune cells accumulating in the dorsal root ganglion or sensory nerves participates in somatic and visceral pain consisting of neuropathic and/or inflammatory components. Endothelial thrombomodulin (TM) and recombinant human soluble TM, TMα, markedly increase thrombin-dependent degradation of HMGB1, and systemic administration of TMα prevents and reverses various HMGB1-dependent pathological pain. Low MW compounds that directly inactivate HMGB1 or antagonize HMGB1-targeted receptors would be useful to reduce various forms of intractable pain. Thus, HMGB1 and its receptors are considered to serve as promising targets in developing novel agents to prevent or treat pathological pain.

TRPV1 is phosphorylated and functionally upregulated by protein kinases, and negatively regulated by phosphatases including calcineurin. Since the clinical use of calcineurin-inhibiting immunosuppressants is commonly associated with chronic diarrhea, we examined if tacrolimus, a calcineurin inhibitor, promotes TRPV1-dependent colonic hypersensitivity in mice. Intracolonic administration of capsaicin, a TRPV1 agonist, caused referred hyperalgesia in the lower abdomen, an effect prevented by capsazepine, a TRPV1 blocker. Tacrolimus accelerated the intracolonic capsaicin-induced referred hyperalgesia. Similarly, intracolonic capsaicin caused spinal ERK phosphorylation, a marker for nociceptor excitation, an effect promoted by tacrolimus. Thus, tacrolimus may aggravate TRPV1-related colonic pain accompanying irritable bowel syndrome.

Mohs paste is useful for controlling exudates from wounds and infections and is used to treat patients with inoperable skin tumors. Unfortunately, Mohs paste is difficult to preserve because its viscosity and stickiness increase dramatically immediately after preparation, resulting in decreased usability. In this study, the combined use of cryopreservation and kneading was shown to improve long-term storage of Mohs paste. At 25°C, Mohs pastes solidified rapidly, and viscosity reached approximately 700 Pa·s 5 h after preparation. In contrast, cryopreservation at -20°C attenuated hardening of Mohs pastes, and kneading also decreased viscosity. The viscosity of Mohs pastes cotreated with cryopreservation and kneading after 7 months of storage was <70 Pa·s. In addition, tissue invasion with these stored pastes was similar to freshly prepared Mohs paste. Results suggest that the combination of cryopreservation and kneading permits Mohs paste to be stored over extended periods, which may increase the utility of the paste for clinical use.

The percutaneous absorption of a fentanyl (FEN)-patch is affected by various external factors including the volume of sebum secretion, which causes changes in the skin surface environment. In this study, we prepared a lard-based sebum-like secretion (SLS), and applied it to investigate the effect of different skin surface conditions on the drug penetration of a FEN-patch. In vitro work to test drug release using the Franz diffusion cell indicated that drug release was significantly suppressed by treatment with 5% SLS, which is equivalent to the amount of daily human sebum secretion. Conversely, in ex vivo experiments using rat skin, the amount of FEN that accumulated in the skin tissue of the 5% SLS-treated rats was higher in comparison with the non-SLS treated group. Furthermore, in vivo experiments indicated that the plasma FEN concentration in rats treated with the FEN-patch was significantly increased by treatment with 5% SLS. These results suggest that the sebum affected the release, accumulation, and absorption of FEN from the FEN-patch, and the FEN concentration in the blood was reflected by the balance of the suppression of drug release and the enhancement of drug accumulation in the skin with SLS.

BACKGROUND: Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. METHODS: CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. RESULTS: Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. CONCLUSIONS: Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.

We tested whether genetic deletion of Cav3.2 T-type Ca2+ channels abolishes hydrogen sulfide (H2S)-mediated pain signals in mice. In Cav3.2-expressing HEK293 cells, Na2S, an H2S donor, at 100 μM clearly increased Ba2+ currents, as assessed by whole-cell patch-clamp recordings. In wild-type C57BL/6 mice, intraplantar and intracolonic administration of Na2S evoked mechanical allodynia and visceral nociceptive behavior, respectively, which were abolished by TTA-A2, a T-type Ca2+ channel blocker. In Cav3.2-knockout mice of a C57BL/6 background, Na2S caused neither somatic allodynia nor colonic nociception. Our study thus provides definitive evidence for an essential role of Cav3.2 in H2S-dependent somatic and colonic pain.

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion.

Objective and Background: Survival predictions by subjective evaluations are important for end-stage patients. However, subjective evaluations based on experience are difficult. Therefore, we investigated whether the Glasgow prognostic score (GPS), prognostic nutritional index (PNI), and C-reactive protein (CRP)/albumin ratio (CRP/Alb ratio) calculated from the laboratory values of objective evaluations are useful for predicting survival times in end-stage patients. Methods: We retrospectively investigated the age, sex, death cause, CRP levels, Alb levels, and lymphocyte counts in 363 cancer and noncancer patients who died in the 12-month period between April 2015 and March 2016. A multivariate analysis was performed to calculate GPS, PNI, and the CRP/Alb ratio from laboratory values and adjusted for confounding factors. Results: PNI and CRP/Alb ratio exhibited negative and positive correlations with survival days, respectively. All GPS, PNI, and CRP/Alb ratio were useful to predict two to four remaining weeks. Interestingly, CRP/Alb ratio, but not GPS or PNI, was higher in patients with predicted short-term survival of zero to two weeks than in that of two to four weeks (odds ratio 2.32; 95% confidence interval 1.61-3.34). Discussion: These results suggest that the CRP/Alb ratio is an independent factor that is beneficial to predict short-term survival of within two weeks.

Cav3.2 T-type Ca2+ channels are expressed in the primary afferents and play a pronociceptive role. The activity of Cav3.2 is enhanced by H2S, a gasotransmitter, and suppressed by ascorbic acid (vitamin C) through metal-catalyzed oxidation of the Zn2+-binding His191 in Cav3.2. Since rodents, but not humans, are capable of synthesizing ascorbic acid, the present study examined the role of ascorbic acid in nociceptive processing, using the mice lacking GNL/SMP30, an enzyme essential for ascorbic acid biosynthesis. Intraplantar and intracolonic administration of NaHS, an H2S donor, caused somatic allodynia and referred hyperalgesia, respectively, and repeated treatment with paclitaxel produced neuropathic allodynia in wild-type mice, all of which were suppressed by ascorbic acid or T-type Ca2+ channel blockers. Dietary ascorbic acid restriction caused dramatic decreases in plasma and tissue ascorbic acid levels in GNL/SMP30-knockout, but not wild-type, mice. The ascorbic acid restriction enhanced the somatic and visceral hypersensitivity following intraplantar and intracolonic NaHS, respectively, and paclitaxel-induced neuropathy in GNL/SMP30-knockout mice, while it had no such effect in wild-type mice. Together, our data unveil the critical role of ascorbic acid in regulating somatic and visceral pain sensitivity and support accumulating clinical evidence for the usefulness of ascorbic acid in pain management.

Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Cav3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Cav3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Cav3.2, but not Cav3.1 or 3.3, and silencing of Cav3.2 gene also suppressed BIPN. Protein levels of Cav3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Cav3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Cav3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Cav3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Cav3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Cav3.2 in nociceptors through inhibition of proteasomal degradation of Cav3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner.

Hydrogen sulfide (H2S), an endogenous gasotransmitter, is generated from L-cysteine by 3 distinct enzymes including cystathionine-γ-lyase (CSE), and targets multiple molecules, thereby playing various roles in health and disease. H2S triggers or accelerates somatic pain and visceral nociceptive signals in the pancreas, colon and bladder by enhancing the activity of Cav3.2 T-type calcium channels. H2S also activates TRPA1, which participates in H2S-induced somatic pain signaling. However, Cav3.2 predominantly mediates colonic nociception by H2S, because genetic deletion of TRPA1 does not reduce H2S-induced colonic pain. The functional upregulation of the CSE/H2S/Cav3.2 system is involved in neuropathic pain and visceral pain accompanying pancreatitis and cystitis. Cav3.2 also appears to participate in irritable bowel syndrome (IBS), although the role of endogenous H2S generation by CSE in IBS is still open to question. In this review, we describe how H2S regulates pain signals, particularly by interacting with Cav3.2.

Among voltage-gated Ca2+ channels, T-type Ca2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca2+ channels, Cav3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Cav3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Cav3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H2S that enhances the channel activity of Cav3.2. H2S generated by upregulated CSE appears to enhance the activity of upregulated Cav3.2 after the differentiation. The enhanced Cav3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Cav3.2 activity through asparagine (N)-linked glycosylation of Cav3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Cav3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Cav3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.

Chemotherapy-induced peripheral neuropathy (CIPN) considerably impairs cancer patients' QOL, and may lead to discontinuation of drug treatment of cancer. Currently, there is no effective strategy against CIPN. Therefore, it is an urgent issue to develop clinically available drugs that prevent or treat CIPN. We have shown that high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, plays an essential role in the development of CIPN. Most interestingly, thrombomodulin α, approved as a medicine for treatment of disseminated intravascular coagulation (DIC) in Japan, causes thrombin-dependent degradation of extracellular HMGB1 that is released in response to chemotherapeutics, and prevents CIPN. Thus, we expect that targeting HMGB1 or its receptors would lead to prevention of CIPN in cancer patients in near future.

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

Since 6-prenylnaringenin (6-PNG) was recently identified as a novel T-type calcium channel blocker with the IC50 value around 1 µM, a series of flavanone derivatives were designed, synthesized and subsequently evaluated for T-channel-blocking activity in HEK293 cells transfected with Cav3.2 T-type channels using a patch-clamp technique. As a result, several new flavanones blocked Cav3.2-dependent T-currents more potently than 6-PNG. In the synthesized compounds, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(2-hydroxyphenyl)chroman-4-one 8j, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11b, 6-(2-cyclopentylideneethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11d, and 6-(2-Cyclopentylethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 12c were more potent blocker than 6-PNG with the IC50 value of 0.39, 0.26, 0.46, and 0.50 µM, respectively. Among the above four derivatives, the compound 8j provided the best result in the in vivo experiments; i.e. systemic administration of 8j at the minimum dose completely restored neuropathic pain induced by partial sciatic nerve ligation in mice.

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (μM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

High mobility group box 1 (HMGB1), a nuclear protein, once released into the extracellular space under pathological conditions, plays a pronociceptive role in redox-dependent distinct active forms, all-thiol HMGB1 (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), that accelerate nociception through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4), respectively. Thrombomodulin (TM), an endothelial membrane protein, and soluble TM, known as TMα, promote thrombin-mediated activation of protein C and also sequester HMGB1, which might facilitate thrombin degradation of HMGB1. The present study aimed at clarifying the role of thrombin in TMα-induced suppression of peripheral HMGB1-dependent allodynia in mice. Thrombin-induced degradation of at-HMGB1 and ds-HMGB1 was accelerated by TMα in vitro. Intraplantar (i.pl.) injection of bovine thymus-derived HMGB1 in an unknown redox state, at-HMGB1, ds-HMGB1 or lipopolysaccharide (LPS), known to cause HMGB1 secretion, produced long-lasting mechanical allodynia in mice, as assessed by von Frey test. TMα, when preadministered i.pl., prevented the allodynia caused by bovine thymus-derived HMGB1, at-HMGB1, ds-HMGB1 or LPS, in a dose-dependent manner. The TMα-induced suppression of the allodynia following i.pl. at-HMGB1, ds-HMGB1 or LPS was abolished by systemic preadministration of argatroban, a thrombin-inhibiting agent, and accelerated by i.pl. co-administered thrombin. Our data clearly indicate that TMα is capable of promoting the thrombin-induced degradation of both at-HMGB1 and ds-HMGB1, and suppresses the allodynia caused by either HMGB1 in a thrombin-dependent manner. Considering the emerging role of HMGB1 in distinct pathological pain models, the present study suggests the therapeutic usefulness of TMα for treatment of intractable and/or persistent pain.

Hydrogen sulfide (H2S) formed by cystathionine-γ-lyase (CSE) enhances the activity of Cav3.2 T-type Ca2+ channels, contributing to the bladder pain accompanying hemorrhagic cystitis caused by systemic administration of cyclophosphamide (CPA) in mice. Given clinical and fundamental evidence for the involvement of the substance P/NK1 receptor systems in bladder pain syndrome (BPS)/interstitial cystitis (IC), we created an intravesical substance P-induced bladder pain model in mice and analyzed the possible involvement of the CSE/Cav3.2 pathway. Bladder pain/cystitis was induced by i.p. CPA or intravesical substance P in female mice. Bladder pain was evaluated by counting nociceptive behavior and by detecting referred hyperalgesia in the lower abdomen and hindpaw. The isolated bladder tissue was weighed to estimate bladder swelling and subjected to histological observation and Western blotting. Intravesical substance P caused profound referred hyperalgesia accompanied by little bladder swelling or edema 6-24 h after the administration, in contrast to i.p. CPA-induced nociceptive behavior/referred hyperalgesia with remarkable bladder swelling/edema and urothelial damage. The bladder pain and/or cystitis symptoms caused by substance P or CPA were prevented by the NK1 receptor antagonist. CSE in the bladder was upregulated by substance P or CPA, and the NK1 antagonist prevented the CPA-induced CSE upregulation. A CSE inhibitor, a T-type Ca2+ channel blocker and gene silencing of Cav3.2 abolished the intravesical substance P-induced referred hyperalgesia. The intravesical substance P-induced pain in mice is useful as a model for nonulcerative BPS, and involves the activation of the NK1 receptor/CSE/H2S/Cav3.2 cascade.

Hydrogen sulfide (H2 S) is generated from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE), and promotes nociception by targeting multiple molecules such as Cav 3.2 T-type Ca2+ channels. Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H2 S/Cav 3.2 pathway. Therefore, we investigated whether NF-κB, as an upstream signal of the CSE/H2 S system, contributes to bladder pain in mice with CPA-induced cystitis. Bladder pain-like nociceptive behaviour was observed in CPA-treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β-Cyano-l-alanine, a reversible selective CSE inhibitor, prevented CPA-induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF-κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor. PDTC and curcumin, which inhibits NF-κB signals, abolished CPA-induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA-induced activation of NF-κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H2 S/Cav 3.2 signaling. Therefore, NF-κB-inhibiting compounds including curcumin may be useful for the treatment of cystitis-related bladder pain.

Cav3.2 T-type Ca2+ channel activity is suppressed by zinc that binds to the extracellular histidine-191 of Cav3.2, and enhanced by H2S that interacts with zinc. Cav3.2 in nociceptors is upregulated in an activity-dependent manner. The enhanced Cav3.2 activity by H2S formed by the upregulated cystathionine-γ-lyase (CSE) is involved in the cyclophosphamide (CPA)-induced cystitis-related bladder pain in mice. We thus asked if zinc deficiency affects the cystitis-related bladder pain in mice by altering Cav3.2 function and/or expression. Dietary zinc deficiency for 2 weeks greatly decreased zinc concentrations in the plasma but not bladder tissue, and enhanced the bladder pain/referred hyperalgesia (BP/RH) following CPA at 200mg/kg, a subeffective dose, but not 400mg/kg, a maximal dose, an effect abolished by pharmacological blockade or gene silencing of Cav3.2. Acute zinc deficiency caused by systemic N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN), a zinc chelator, mimicked the dietary zinc deficiency-induced Cav3.2-dependent promotion of BP/RH following CPA at 200mg/kg. CPA at 400mg/kg alone or TPEN plus CPA at 200mg/kg caused Cav3.2 overexpression accompanied by upregulation of Egr-1 and USP5, known to promote transcriptional expression and reduce proteasomal degradation of Cav3.2, respectively, in the dorsal root ganglia (DRG). The CSE inhibitor, β-cyano-l-alanine, prevented the BP/RH and upregulation of Cav3.2, Egr-1 and USP5 in DRG following TPEN plus CPA at 200mg/kg. Together, zinc deficiency promotes bladder pain accompanying CPA-induced cystitis by enhancing function and expression of Cav3.2 in nociceptors, suggesting a novel therapeutic avenue for treatment of bladder pain, such as zinc supplementation.

Voltage-gated calcium channels (VGCCs) are classified into high-voltage-activated (HVA) channels and low-voltage-activated channels consisting of Cav3.1-3.3, known as T ("transient")-type VGCC. There is evidence that certain types of HVA channels are involved in neurogenic inflammation and inflammatory pain, in agreement with reports indicating the therapeutic effectiveness of gabapentinoids, ligands for the α2δ subunit of HVA, in treating not only neuropathic, but also inflammatory, pain. Among the Cav3 family members, Cav3.2 is abundantly expressed in the primary afferents, regulating both neuronal excitability at the peripheral terminals and spontaneous neurotransmitter release at the spinal terminals. The function and expression of Cav3.2 are modulated by a variety of inflammatory mediators including prostanoids and hydrogen sulfide (H2S), a gasotransmitter. The increased activity of Cav3.2 by H2S participates in colonic, bladder and pancreatic pain, and regulates visceral inflammation. Together, VGCCs are involved in inflammation and inflammatory pain, and Cav3.2 T-type VGCC is especially a promising therapeutic target for the treatment of visceral inflammatory pain in patients with irritable bowel syndrome, interstitial cystitis/bladder pain syndrome, pancreatitis, etc., in addition to neuropathic pain.

Thrombomodulin (TM), an endothelial protein with anti-coagulant activity, is composed of 5 domains, D1-D5. Recombinant human soluble TM (TMα) consisting of D1-D3, which is generated in CHO cells, suppresses inflammatory and nociceptive signals by inactivating high mobility group box 1 (HMGB1), one of damage-associated molecular patterns. TMα sequesters HMGB1 with the lectin-like D1 and promotes its degradation by thrombin binding to the EGF-like D2. We prepared TM's D123, D1 and D2 by the protein expression system of yeast, and evaluated their effects on HMGB1 degradation in vitro and on the allodynia caused by HMGB1 in distinct redox forms in mice in vivo. TMα and TM's D123, but not D1, promoted the thrombin-dependent degradation of all-thiol (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), an effect mimicked by TM's D2, though to a lesser extent. Intraplantar administration of TMα and TM's D123, but not D1, D2 or D1 plus D2, strongly prevented the mechanical allodynia caused by intraplantar at-HMGB1, ds-HMGB1 or lipopolysaccharide in mice. Our data suggest that, apart from the role of D3, TMα and TM's D123 require both lectin-like D1 capable of sequestering HMGB1 and EGF-like D2 responsible for thrombin-dependent degradation of HMGB1, in abolishing the allodynia caused by exogenous or endogenous HMGB1.

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely 'referred hyperalgesia', 6-24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

胃潰瘍の含亜鉛治療薬であるポラプレジンク(PZ)を水溶性のカルボキシビニルポリマー(CP)存在下で懸濁液として調製してその安定性について検討し、口腔粘膜炎の治療薬としての有効性を評価した。安定性試験では調製後28時間にわたり、そのpH、粘度、接着性およびPZ含量に変化は見られなかった。口腔粘膜炎の重症度分類に基づいた臨床評価では、PZ使用者(PZ群)の口腔粘膜炎の重症度が対照としたPZ非使用者(対照群)のそれに比べて、6週時(p=0.016)と7週時(p=0.018)のいずれでも有意に低かった。また、グレード3の重度口腔粘膜炎のPZ群と対照群における発生率は、6週時でそれぞれ15.0%と41.7%、7週時ではそれぞれ15.0%と33.3%であった。以上より、PZ-CPうがい薬は化学放射線療法で誘導される口腔粘膜炎の増悪を阻止し、その治癒を促進すると思われた。

がん化学療法の副作用として生じる神経障害性疼痛は，患者のQOL低下を招くだけでなく，化学療法の中止の原因にもなりうるので，その対策は急務である．神経障害性疼痛薬物療法の第一選択薬とされているプレガバリンは，高電位活性化型Ca²⁺チャネルのα₂δサブユニットを標的とした薬物であるが，プレガバリンが作用しないT型（低電位活性化型）Ca²⁺チャネルのうちCa_v3.2が神経障害性疼痛の病態に関与することが明らかとなり，T型Ca²⁺チャネル阻害薬が神経障害性疼痛の治療に応用できる可能性が示唆されている．Ca_v3.2は，内因性気体メディエーターである硫化水素やL-システインによって直接活性化され，またプロスタグランジンE₂によりプロテインキナーゼA依存的に活性化されるほか，生体内のZn²⁺やビタミンCによって機能が抑制される．本稿では，Ca_v3.2 T型Ca²⁺チャネルの分子機能調節機構を概説し，特にがん化学療法に伴う神経障害性疼痛の治療標的分子としての可能性について述べる．

Proteinase-activated receptor（PAR）は，特定のセリンプロテアーゼによって活性化されるGタンパク共役7回膜貫通型受容体であり，PAR1，PAR2，PAR3，PAR4の4つのファミリーメンバーが知られている．これらのPARは消化器系臓器全般に広く発現し，多様な役割を演じている．PAR2およびPAR1はいずれも胃粘膜において種々の機能の制御に関与し，第一義的には粘膜保護の方向に働いているが，その機能メカニズムは大きく異なっている．消化管平滑筋運動調節においてPARは抑制的な面と促進的な面を併せ持ち，その機能の特性やメカニズムは動物種や臓器部位によって大きく異なる．またPAR2は内臓痛および腸炎症の発現，さらに外分泌機能の調節にも関与している．このように，PARの消化管機能は複雑であるが，PARアゴニストあるいはアンタゴニストは，胃粘膜傷害のほか，消化管運動・内臓痛覚の異常に関連する過敏性腸症候群，炎症性腸疾患などの治療にも利用できる可能性があり，今後，特に臨床応用の面における研究の進歩が期待される．

Protease-activated receptor-2（PAR-2）は，トリプシン，トリプターゼなどの特定のセリンプロテアーゼの細胞への作用を媒介するGタンパク共役型受容体である．PAR-2は知覚神経C線維ニューロンに発現し，炎症性疼痛や熱痛覚過敏の発現に関与している．また，PAR-2は内臓痛の情報伝達・制御にも重要な役割を演じている．結腸内腔側に存在すると考えられるPAR-2の活性化により，遅発性の内臓痛覚過敏が誘起される．一方，PAR-2は膵臓では痛みに対して促進的な面と抑制的な面を併せ持つ．このように，PAR-2は痛みの情報伝達制御に関与する新たな生体内分子として，創薬の面からも興味がもたれている．

Protease-activated receptor-2（PAR-2）は，トリプシン，トリプターゼなどの特定のセリンプロテアーゼの細胞への作用を媒介するGタンパク共役型受容体である．PAR-2は知覚神経C線維ニューロンに発現し，炎症性疼痛や熱痛覚過敏の発現に関与している．また，PAR-2は内臓痛の情報伝達・制御にも重要な役割を演じている．結腸内腔側に存在すると考えられるPAR-2の活性化により，遅発性の内臓痛覚過敏が誘起される．一方，PAR-2は膵臓では痛みに対して促進的な面と抑制的な面を併せ持つ．このように，PAR-2は痛みの情報伝達制御に関与する新たな生体内分子として，創薬の面からも興味がもたれている．

2021年度は、ボルテゾミブ、パクリタキセル、オキサリプラチンなどの抗がん薬の副作用として生じる化学療法誘発性末梢神経障害（CIPN）、butyrateやTNBSなどにより誘発される過敏性腸症候群（IBS）モデルにおける内臓痛、２型糖尿病に伴う有痛性末梢神経障害に対して、遺伝子組換えヒト可溶性トロンボモジュリン（トロンボモジュリンアルファ；TMα）が予防的に作用することを検証あるいは証明した。一方、1型糖尿病の末梢神経障害に対してTMαは無効であった。また、抗凝固薬アルガトロバンは、上記痛みモデルにおけるTMαの効果を抑制した。また、TMαの抗CIPN効果にはトロンビン依存性のHMGB1不活性化作用に加えて、プロテインC（PC）およびthrombin-activatable fibrinolysis inhibitor (TAFI)の活性化が関与することを明らかにした。さらに活性化されたPC（APC）とTAFI（TAFIa）の標的分子はそれぞれproteinase-activated receptor 1 (PAR1)および補体アナフィラトキシンC5aであることも突き止めた。また、関西医科大学附属病院薬剤部との共同研究で実施した後ろ向きコホート研究により、2型糖尿病患者において有痛性末梢神経障害の発症率は、抗凝固薬投与患者の方が非投与群よりも有意に高いことを突き止め、内因性トロンビンが恐らく内皮トロンボモジュリン依存性に痛み抑制的に働いているとの仮説を支持する知見が得られた。このように、2021年度の研究により、本課題を進めるための基礎的知見が得られ、2022年度以降の実験計画を円滑に実行するための基盤を整備することができた。

本研究課題では、生体内で数種の合成酵素により産生される硫化水素（H2S）が、抗がん剤による化学療法誘起末梢神経障害（CIPN）の発症および抗がん剤抵抗性獲得に寄与するかを検討することを目的としている。実際に、抗がん剤によるCIPN発症と治療効果の減弱に内因性H2Sの寄与が明らかとなれば、H2S合成酵素阻害薬が抗がん剤との併用投与により、副作用であるCIPNの軽減と抗がん剤の治療効果増大の両面において高い有用性を示すことが期待できる。2020年度は、ヒト多発性骨髄腫（multiple myeloma, MM）由来KMS-11細胞と、MMの治療に使用される抗がん剤ボルテゾミブ（BTZ）に対して耐性を獲得したKMS-11/BTZ細胞を用いて、BTZ非存在下あるいは存在下における細胞増殖におよぼす内因性H2Sの関与について検討を行った。その結果、BTZ非存在下、存在下どちらにおいても、KMS-11およびKMS-11/BTZの細胞増殖はH2S合成酵素のcystathionine-β-synthase（CBS）の阻害薬により顕著に、cystathionine-γ-lyase（CSE）阻害薬により部分的に抑制されたが、CSE阻害薬の効果はKMS-11細胞で小さかった。これら阻害薬による増殖抑制効果はH2S供与体のNa2SあるいはGYY4137により一部減弱した。一方、別のH2S合成酵素である3-mercaptopyruvate sulfur transferase（3-MST）の阻害薬はこれら細胞の増殖にほとんど影響しなかった。これらの結果より、KMS-11およびKMS-11/BTZ細胞の増殖は内因性H2Sにより促進的に調節されており、その産生には主にCBSが寄与していることが示唆された。

RAGEを標的とする難治性疼痛治療薬のシーズを開発するため、神経障害性疼痛および内蔵痛の動物モデルを作成して既存のRAGEの有効性を確認した。ついで、中分子量ヘパリニルフェニルアラニン（MMWHI-F）がRAGEを選択的に遮断し難治性疼痛を抑制することを証明した。さらに、インシリコ解析により同定した低分子化合物がAGE-RAGE bindingを阻害することを証明し、新規RAGE拮抗薬開発のためのシーズになりうることを示唆した。以上、本研究によって高分子および低分子RAGE拮抗薬が痛みの治療に有用であることを明らかにした。

一次知覚神経のCav3.2 T型カルシウムチャネル（Tチャネル）は、体性痛、内臓痛、神経障害性疼痛などの痛みの発症に寄与することが知られている。本研究では始めに、種々の疼痛モデルを用いて、新規Tチャネル阻害薬RQ-00311651の鎮痛効果を検討し、この化合物が中枢抑制作用の少ない経口投与可能な鎮痛薬となりうることを報告した。また、我々が以前に報告している第5腰神経切断誘起神経障害性疼痛ラットにおける一次知覚神経のCav3.2発現増加に、転写因子Egr-1および脱ユビキチン化酵素USP5の発現増加を介したCav3.2の転写促進およびプロテアソーム分解抑制が関与することを明らかにした。

核内蛋白HMGB1はdamage-associated molecular patternsとして、病態時に細胞外に放出される。今回は、癌化学療法誘起神経障害性疼痛および内臓痛におけるHMGB1の役割を解析した。その結果、HMGB1は、RAGE、Toll-like receptor 4、CXCR4などの細胞膜受容体を介して癌化学療法誘起神経障害性疼痛や膵炎に伴う膵臓痛の発症および維持に関与することが明らかとなった。

ヒト胃癌由来AGS細胞において、生体内で硫化水素（H2S）産生に関わる酵素の1つであるcystathionine-gamma-lyase（CSE）により産生された内因性H2Sは、AGS細胞の増殖を促進的に調節していることが示唆された。この効果の一部には、H2SによるCav3.2 T型カルシウムチャネルの機能増強およびNF-kappaB系の活性化を介した抗アポトーシスタンパクBcl-2とBcl-xLの発現増加が関与している可能性が示唆された。これらの経路は胃癌治療の新たなターゲットとなることが期待される。

T型カルシムチャネルの３つのサブタイプのうち、Cav3.2は侵害受容ニューロンの末梢終末に豊富に発現し、痛みの情報伝達に重要な役割を果たしている。今回は、Cav3.2のリン酸化および脱リン酸化による機能調節とそれによる疼痛制御の分子メカニズムを解析した。その結果、プロスタグランジンEP4受容体刺激により活性化されるPKAが、足場タンパクAKAP150依存的にCav3.2をリン酸化し、チャネル機能を増強することで痛覚過敏を誘起すること、また、脱リン酸化酵素であるカルシニューリンはCav3.2の機能を抑制していることが明らかとなった。

胃癌患者の癌化した胃粘膜組織では、ヘリコバクター・ピロリ(H.pylori)感染が高い部位でproteinase-activated receptor-1(PAR1)発現量が増加している傾向が見られ、H.pylori感染による胃癌の発生や進行にとPAR1発現増加が関与する可能性が考えられた。一方、H.pyloriのもつプロテアーゼについて種々の細胞を用いて反応性を検討した結果、PAR1、PAR2、PAR4を活性化するプロテアーゼの存在は認められなかった。

硫化水素の疼痛情報伝達促進メカニズムにCav3.2 T型カルシウムチャネルが関与することを明らかにした。また、L5脊髄神経切断神経障害性疼痛モデルを作成し、痛覚過敏・アロディニアの保持に内因性硫化水素によるCav3.2の活性化が関与すること、さらに、本モデルではCav3.2の発現誘導がおこっていることを見出し、硫化水素/Cav3.2系が神経因性疼痛の新たな治療標的になりうることを明らかにした。

生体内新規ガス状メッセンジャーとして機能する可能性が示唆されている硫化水素の疼痛情報伝達制御における役割を検討し、以下の知見を得た。 1)硫化水素およびL-システインの投与による痛覚過敏について NaHSをラットの足底内投与することにより、機械的痛覚過敏が誘起されることを見出し、この効果の発現にはT型カルシウムチャネルが関与することを証明した。また、硫化水素の生体内originであるL-システインを足底内投与した場合にも痛覚過敏が認められ、これはL-システインからCSEによって産生される硫化水素によるものであるとの示唆が得られた。一方、NaHSの足底内投与によって、脊髄後角侵害受容ニューロンの活性化が起こることをFos蛋白の発現を指標として証明した。一方、ラットの脊髄内へNaHSを投与した場合にも痛覚過敏の発現を認めている。 2)NG108-15細胞およびマウス脊髄DRGニューロンにおけるT型カルシウムチャネル電流の硫化水素による増強 Whole cell patch clamp法により、NG108-15細胞およびマウス脊髄DRGニューロンにおけるバリウム電流を指標としてT型カルシウムチャネルを介する膜電流が硫化水素によって促進されることを証明した。 3)硫化水素のマウス結腸内投与による内臓痛発現について マウスの結腸内へNaHSを投与することにより内臓痛および関連痛覚過敏が発現するとともに、脊髄後角ではERKリン酸化が誘起されることを証明した。 以上、本研究により、生体内の硫化水素は、痛みの情報伝達における新たなメッセンジャーとして機能していることが明らかとなった。

G蛋白共役7回細胞膜貫通型受容体に属するProtease-activated receptor(PAR)は4つのファミリーメンバーからなる新しいタイプの受容体であり、炎症、組織損傷、体内出血などの際に活性化され、種々の生理機能を修飾している可能性が示唆されている。そこで、本研究課題においては、急性炎症時におけるPAR、特にPAR-2の役割、挙動について検討した。 はじめに、エンドトキシン(リポポリサッカライド)処置マウスにおいて、唾液腺のPAR-2の感受性がどのように変化するかを検討し、全身性炎症時には唾液腺PAR-2が内因性アゴニスト酵素により持続的に活性化される結果、脱感作状態になっていることが明らかとなった。敗血症ショック時には、PAR活性化作用を持つトロンビン、第VIIa, Xa因子が活性化され、さらに肥満細胞からトリプターゼが遊離される。これらの酵素による血管内皮PAR活性化による血管弛緩が敗血症による低血圧に関与する可能性が十分に考えられる。そこで、PAR活性化による血管の収縮・弛緩反応のメカニズムを解析した。さらに、カプサイシン感受性感覚神経末梢端に存在するとされるPARが炎症性内臓痛や耳下腺炎に伴う痛みの発現に関与する可能性についても検討した。本研究により、PAR、特にPAR-2が炎症あるいはそれに伴う痛み、循環異常など種々の機能の修飾に関与していることが示唆された。

臨床的に有効性が認められている大脳皮質第II次体性感覚野(SID電気刺激鎮痛を容易に検出し得るモデルの確立を目的として研究を行った。 1.ホルマリン誘発疹痛モデルにおけるSII電気刺激の鎮痛作用:ラットの大脳皮質SII領域に電気刺激用慢性電極を植込んだ後,実験を行った。ホルマリンを足底内に投与した場合に認められる疼痛行動および脊髄表層におけるFos発現は,SII電気刺激によって部分的に抑制される傾向が認められた。ついで,神経型N0合成酵素阻害薬である7・ニトロインダゾールとSII電気刺激を併用したところ,強い鎮痛効果と脊髄F0s発現の抑制が認められた。SII電気刺激によるこの効果はメチセルギドの脊髄内投与によって部分的に抑制されたことより,下降性セロトニン神経系の関与が示唆された。 2.プロテアーゼ受容体PAR-2を介する疹痛モデルの作成とその特徴:C-fiberに発現することが明らかとなったPAR-2を末梢側から刺激することにより,熱痛覚過敏,侵害受容行動,脊髄Fos発現が認められ,新しい疹痛モデルとなりうることを見出した。また,各種阻害薬を用いてその痛みの特徴を明らかにした。 3.PAR-2誘発疹痛モデルおよび外科的ニューロパチーモデルにおけるSII電気刺激の効果:PAR-2誘発落痛モデルおよび外科的手術により作成したニューロパチーモデルでSII刺激の効果を検討したが,明らかな鎮痛効果は検出することができなかった。今後さらにSII刺激鎮痛を検出しうる新たなモデルを検索していきたい。

G蛋白共役7回膜貫通型受容体の1ファミリーであるプロテアーゼ受容体PAR(protease-activated receptor)は、トロンビン、トリプシン、トリプターゼなどの特定のプロテアーゼによって活性化され、特に組織損傷や炎症状態において種々の生理的・病態生理学的役割を果たしている。本補助金研究においては、このPARが消化器系においてどのような生理機能に関与しているかを検討した。 はじめに、小腸平滑筋の運動性制御におけるPARの役割をin vitroにおいて調べたところ、PAR-1の活性化によりアパミン感受性弛緩反応とそれに続く収縮反応が誘発された。PAR-2活性化によっては弱い収縮反応のみが認められた。一方、PAR-4を刺激しても反応は全く見られなかった。次にPAR-1を介する収縮反応の細胞内情報伝達機構を検討したところ、この反応はナトリウム透過性の亢進、L型カルシウムチャネルの開口に大きく依存し、チロシンキナーゼ、蛋白キナーゼC,PI3キナーゼなども一部関与することが示唆された。 次に、唾液腺、膵臓においてPAR-2mRNAが多く発現していることを確認した。そこで、これらの臓器の外分泌機能の制御におけるPAR-2の役割を検討したところ、PAR-2の活性化により唾液腺、膵臓いずれの臓器においても外分泌反応が促進されることを見出した。 以上のように、本補助金研究により、PARが小腸、唾液腺、膵臓に存在し、種々の生理機能を修飾していることが明らかとなった。本研究成果は、PARが将来の創薬のための標的分子にもなりうることを強く示唆している。

臨床的に有効性が認められている大脳皮質第II次体性感覚野(S-II)電気刺激誘発鎮痛を検出しうるラットモデルを確立し,さらに痛みの情報伝達に関与するとされるNOの合成酵素阻害薬がS-II刺激誘発鎮痛にどのような影響を及ぼすかを検討した. 初年度(平成10年度)はまず,S-II刺激鎮痛を検出しうる動物モデルの確立を目的とし,機械的刺激,熱刺激および化学的刺激に対するラットの侵害受容反応におよぼす効果を検討した.S-II刺激は前2者ではほとんど効果を示さなかったが,ホルマリン誘発化学的侵害受容反応を抑制した.次いで,この抗侵害効果が神経型NO合成酵素阻害薬である7―ニトロインダゾールとの併用によって増強されることを見出した. 次年度(平成11年度)は,このS-II電気刺激と7-ニトロインダゾールの併用による抗侵害効果の発現機序について検討した.ラットの足底にホルマリンを投与すると,L4,L5脊髄後角表層にc-fosの発現が誘発された.これに対して,S-II電気刺激と7-ニトロインダゾール5mg/kgはそれぞれ単独では無効であったが,両者を併用するとc-fosの発現が有意に抑制された.また,ホルマリン疼痛法において認められる両者併用による抗侵害効果は,オピオイド拮抗薬であるナロキソンあるいはα―アドレナリン受容体拮抗薬であるフェントラミンの全身性投与によっては影響されなかったが,セロトニン拮抗薬であるメチセルギドの脊髄内投与により第1相における鎮痛効果が有意に抑制された. 以上より,S-II電気刺激は脊髄への下降性抑制神経系,特にセロトニン神経系を活性化することにより脊髄における痛みの情報伝達を抑制し鎮痛効果を発現する可能性が示唆された.

1.脳内での疼痛制御におけるL‐arginineの役割 一酸化窒素(NO)は,L‐arginine(L‐Arg)からNO合成酵素(NOS)により生成され,soluble guanylate cyclase (sGC)を活性化して細胞内cyclic GMP(cGMP)濃度を上昇させることによりその生理作用を発現する.一方,内因性鎮痛ペプチドkyotorphin(L‐tyrosyl‐L‐arginine,以下KTP)は,脳内でKTP合成酵素(KTP‐S)によってL‐ArgとL‐tyrosineから生合成され,Met‐enkephalin(Met‐Enk)の遊離を促進させることにより生理作用を発現する.著者らはL‐ArgがNOSおよびKTP‐Sの両酵素の基質である点に注目し,脳内での疼痛制御における役割を検討した.L‐Arg,NOS阻害薬およびsGC阻害薬はいずれも側脳室内投与により明らかな抗侵害作用を示した.L‐Argのこの効果はKTP‐Met‐Enk経路を促進することにより発現するのに対し,NOS阻害薬およびsGC阻害薬の効果はNO‐cGMP経路を抑制することにより発現することが明らかとなった.このことより,L‐Argは脳内で相反する2つの役割,すなわち,KTP系を介する抗侵害的な面とNO系を介する侵害的な面を有することが強く示唆された. 2.末梢での侵害受容におけるL‐arginineの役割 炎症時の侵害受容において,末梢組織中のNOが血管透過性を亢進させることにより侵害受容を促進させるとの見解と,nociceptorへの直接的な抑制作用により抗侵害的に作用するとの見解が対立した現状にある.著者らは,formalin誘発侵害反応モデルを用いてこの矛盾点の解明を試みた.その結果,末梢組織中でL‐Argより生成されるNOはその濃度に依存して侵害促進的な面と侵害抑制的な面を合わせ持つことが示唆された.