Dose-escalation study of tabalumab with bortezomib and dexamethasone in Japanese patients with multiple myeloma.

B-cell activating factor (BAFF) promotes the survival and adhesion of multiple myeloma (MM) cells. Tabalumab (LY2127399) is an anti-BAFF monoclonal antibody. This phase 1, multicenter, open-label, nonrandomized, dose-escalation study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy of tabalumab in combination with bortezomib and dexamethasone in Japanese patients with relapsed or refractory MM (RRMM). Sixteen patients received intravenous i.v. tabalumab 100 mg (Cohort 1, n = 4) or i.v. tabalumab 300 mg (Cohort 2, n = 12) in combination with oral dexamethasone 20 mg/day and i.v. or s.c. bortezomib 1.3 mg/m(2) . All patients had treatment-emergent adverse events (TEAE) possibly related to study treatment; the most common TEAE were thrombocytopenia (81.3%), lymphopenia (43.8%) and increased alanine aminotransferase (43.8%). Two (20.0%) dose-limiting toxicities were observed, both in Cohort 2 (tabalumab 300 mg), which was below the predefined cutoff for tolerability (<33%). The pharmacokinetics of tabalumab were similar when bortezomib was coadministered i.v. versus s.c. The overall response rate was 56.3%, suggesting that the combined treatment was effective. In conclusion, combined treatment with these three agents was well tolerated in this population of Japanese patients with RRMM. The study was registered at www.clinicaltrials.gov (NCT01556438).

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents.

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.

Antiallodynic effect of etidronate, a bisphosphonate, in rats with adjuvant-induced arthritis: involvement of ATP-sensitive K+ channels.

Bisphosphonates, pyrophosphate analogues, known as inhibitors of bone resorption, appear to cause analgesia in certain clinical painful situations. To detect clinically relevant analgesic property of etidronate, a non-aminobisphosphonate, we examined and characterized its antiallodynic effect in the rat with adjuvant-induced arthritis, in comparison with alendronate, an aminobisphosphonate, as determined by the von Frey test. Repeated systemic administration of etidronate at 10-40 mg/kg/day suppressed the adjuvant-induced mechanical allodynia in rat hindpaw, an effect reaching a plateau in approximately 10 days. Systemic or intraplantar (i.pl.) administration of ATP-sensitive K+ (K+ ATP) channel inhibitors, glibenclamide and/or tolbutamide, completely reversed the antiallodynic effect of etidronate within 1h in the arthritic rats, without affecting the nociceptive scores in naïve or arthritic animals that had not received etidronate. Alendronate, administered repeatedly, also revealed similar glibenclamide-reversible antiallodynic effect. In contrast, the antiallodynic effect of repeated systemic indomethacin was resistant to i.pl. glibenclamide in the arthritic rats. Repeated administration of etidronate or alendronate only slightly attenuated the adjuvant-evoked hindpaw edema. Among K+ ATP channel subunits, mRNAs for Kir6.1, SUR1, SUR2A and SUR2B were abundant in rat dorsal root ganglia, while Kir6.2 mRNA was poor. Our data demonstrate that repeated etidronate as well as alendronate exhibits antiallodynic activity in arthritic rats, which might be clinically relevant, and suggest involvement of K+ ATP channels in the underlying mechanisms.

Suppression of pancreatitis-related allodynia/hyperalgesia by proteinase-activated receptor-2 in mice.

1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH(2), coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH(2) moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.

Distinct activity of peptide mimetic intracellular ligands (pepducins) for proteinase-activated receptor-1 in multiple cells/tissues.

Proteinase-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, can be activated not only by PAR1-activating peptides (PAR1APs) based on the N-terminal cryptic tethered ligand sequence but also by an N-palmitoylated (Pal) peptide, Pal-RCLSSSAVANRSKKSRALF-amide (P1pal-19), based on the intracellular loop 3 of PAR1, designated pepducin, in human platelets or PAR1-transfected cells. The present article evaluated the actions of P1pal-19 and also the shorter peptide, Pal-RCLSSSAVANRS-amide (P1pal-12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal-19 as well as a PAR1AP, TFLLR-amide, evoked cytosolic Ca(2+) mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal-19 and TFLLR-amide, but not a PAR2-activating peptide, SLIGRL-amide, caused delayed prostaglandin E(2) formation in RGM1 cells. P1pal-19, like TFLLR-amide, produced endothelial NO-dependent relaxation in rat aorta and epithelial prostanoid-dependent relaxation in mouse bronchus. The P1pal-19-induced relaxation remained constant even after desensitization of PAR1 with TFLLR-amide in either tissue. P1pal-19 failed to mimic the contractile effects of TFLLR-amide in the endothelium-denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal-12 partially inhibited the vasorelaxation caused by TFLLR-amide and P1pal-19, but not SLIGRL-amide, in the rat aorta. Our data thus indicate that P1pal-19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal-12 may act as a PAR1 antagonist in the vascular endothelium.

Colonic hyperalgesia triggered by proteinase-activated receptor-2 in mice: involvement of endogenous bradykinin.

Intracolonic (i.col.) administration of the PAR2-activating peptide (PAR2AP) SLIGRL-NH2 slowly develops visceral hypersensitivity to i.col. capsaicin in ddY mice. Thus, we further analyzed roles of PAR2 in colonic hypersensitivity, using the novel potent PAR2AP, 2-furoyl-LIGRL-NH2 and PAR2-knockout (KO) mice. In ddY mice, i.col. 2-furoyl-LIGRL-NH2 produced delayed (6 h later) facilitation of capsaicin-evoked visceral nociception, an effect being much more potent than SLIGRL-NH2. Such effects were mimicked by i.col. trypsin. In wild-type (WT), but not PAR2-KO, mice of C57BL/6 background, i.col. PAR2 agonists caused delayed facilitation of sensitivity to capsaicin. The PAR2-triggered visceral hypersensitivity was abolished by a bradykinin B2 receptor antagonist, HOE-140. Our data thus provide ultimate evidence for role of PAR2 in colonic hypersensitivity, and suggest involvement of the bradykinin-B2 pathway.

Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery.

OBJECTIVE: Protease-activated receptors (PARs) 1 and 2 are expressed in various blood vessels including rat aorta, modulating vascular tone. We investigated the roles of PAR-1 and PAR-2 in vasomotor modulation in rat superior mesenteric artery. METHODS AND RESULTS: Effects of the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide) and the PAR-1-activating peptide Thr-Phe-Leu-Leu-Arg-amide (TFLLR-amide) on isometric tension were examined in isolated rat superior mesenteric artery or aorta. Both SLIGRL-amide and TFLLR-amide caused relaxation in the precontracted rat aortic rings. The latter peptide, but not the former, produced contraction in the resting rings. NG-nitro-L-arginine methyl ester (L-NAME), but not apamin/charybdotoxin known to block the endothelium-derived hyperpolarizing factor (EDHF) pathway, abolished the relaxation and facilitated the contraction. In the precontracted rat superior mesenteric artery, SLIGRL-amide, but not TFLLR-amide, elicited endothelium-dependent relaxation, which was only partially inhibited by L-NAME with and without indomethacin. The residual relaxation was abolished by apamin/charybdotoxin. Carbenoxolone, a gap junction inhibitor, significantly attenuated the SLIGRL-amide-evoked, EDHF-dependent relaxation, although neither 17-octadecynoic acid, a P450 epoxygenase inhibitor, nor catalase, a hydrogen peroxide scavenger, revealed inhibitory effects. The residual response resistant to carbenoxolone was unaffected by ouabain/BaCl2. In the resting artery, TFLLR-amide, but not SLIGRL-amide, produced only slight contraction, which was dramatically facilitated by combination of L-NAME and apamin/charybdotoxin or by removal of the endothelium. CONCLUSIONS: Our data suggest that, in rat superior mesenteric artery, endothelial PAR-2, upon activation, causes relaxation via both NO and EDHF pathways, and that activation of muscular PAR-1 exhibits potential contractile activity that is largely masked by NO and EDHFs pathways triggered by endothelial PAR-1. Gap junctions might be involved in the EDHF mechanisms in this artery.

Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric artery via multiple mechanisms.

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca2+-activated K+ channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.

Rhodanese, but not cystathionine-gamma-lyase, is associated with dextran sulfate sodium-evoked colitis in mice: a sign of impaired colonic sulfide detoxification?

Clinical studies suggest that colonic luminal hydrogen sulfide (H(2)S), produced by sulfate-reducing bacteria or through other pathways, might be involved in the pathogenesis of inflammatory bowel disease (IBD). Nonetheless, this hypothesis has been poorly investigated by basic studies using laboratory animals. We thus focused on two enzymes, cystathionine-gamma-lyase (CSE) that generates H(2)S from l-cysteine, and rhodanese that directly or indirectly detoxifies H(2)S, particularly in relation to the colitis induced by dextran sulfate sodium (DSS) in mice. CSE was a major H(2)S-forming enzyme in colonic and renal homogenates from mice and rats, and the rhodanese activity was also detectable in both tissues. Colitis-related symptoms including decreased body weight gain, diarrhea, hematochezia and shortening of colon length were observed in the mice drinking DSS. Those symptoms were not or only slightly attenuated by repeated administration of a CSE inhibitor. CSE activity and protein levels in the colonic tissue did not notably change in the mice with colitis. In contrast, the activity and protein/mRNA levels of rhodanese in the colon, but not kidney, significantly decreased nearly in parallel with the development of colitis, followed by elevation of rhodanese activity in red blood cells (RBCs). These data show that rhodanese, but not CSE, is associated with DSS-induced colitis in mice, leading to a hypothesis that impaired detoxification of H(2)S due to down-regulation or suppression of colonic rhodanese is involved in IBD. The delayed enhancement of rhodanese activity in RBCs, a possible compensative event, might be available as a disease marker for IBD.

Hydrogen sulfide as a novel nociceptive messenger.

Hydrogen sulfide (H(2)S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H(2)S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H(2)S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H(2)S-evoked hyperalgesia was blocked by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T-type Ca(2+) channel inhibitors. L-Cysteine, an endogenous source for H(2)S, given i.pl., also elicited hyperalgesia, an effect being abolished by DL-propargylglycine (PPG) and beta-cyanoalanine (BCA), inhibitors of cystathionine-gamma-lyase, a H(2)S synthesizing enzyme. PPG and/or BCA partially inhibited the hyperalgesia induced by i.pl. lipopolysaccharide, an effect being reversed by i.pl. NaHS. In the patch-clamp study using undifferentiated NG108-15 cells that express T-type, but not other types, of Ca(2+) channels, NaHS enhanced the currents through the T-type channels, an effect being blocked by DTNB. Thus, H(2)S appears to function as a novel nociceptive messenger through sensitization of T-type Ca(2+) channels in the peripheral tissues, particularly during inflammation.

Proteinase-activated receptor-2-mediated relaxation in mouse tracheal and bronchial smooth muscle: signal transduction mechanisms and distinct agonist sensitivity.

We characterized the tracheal and bronchial relaxation caused by proteinase-activated receptor-2 (PAR-2) activation in ddY mice and/or in wild-type and PAR-2-knockout mice of C57BL/6 background. Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and Thr-Phe-Leu-Leu-Arg-amide, PAR-2- and PAR-1-activating peptides, respectively, caused relaxation in the isolated ddY mouse trachea and main bronchus. The relaxation was abolished by specific inhibitors of cyclooxygenase (COX)-1, COX-2, mitogen-activated protein kinase kinase (MEK), and p38 MAP kinase. The MEK and p38 MAP kinase inhibitors did not affect prostaglandin E(2)-induced relaxation. Inhibitors of cytosolic Ca(2+)-dependent phospholipase A(2) (PLA), Ca(2+)-independent PLA(2), diacylglycerol lipase, tyrosine kinase, and protein kinase C exhibited no or only minor inhibitory effects on the PAR-mediated relaxation. Trypsin, a PAR-2 activator, and 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide, a potent PAR-2-activating peptide, in addition to SLIGRL-NH(2), caused airway relaxation in wild-type C57BL/6 mice, as in ddY mice. In PAR-2-knockout mice, the peptide effects were absent and the potency of trypsin decreased. Desensitization of PAR-2 and/or PAR-1 greatly suppressed the relaxant effect of trypsin. The bronchial and tracheal tissues displayed distinct sensitivities toward trypsin and the PAR-2-activating peptides. Our data indicate an involvement of both COX-1 and COX-2, and the MEK-extracellular signal-regulated kinase and p38 MAP kinase signaling pathways in the PAR-2- and PAR-1-triggered relaxation of mouse airway tissue, and substantiate a role for PAR-2 in regulating both the trachea and bronchial responsiveness in the mouse lung.

Potent and metabolically stable agonists for protease-activated receptor-2: evaluation of activity in multiple assay systems in vitro and in vivo.

To develop potent and metabolically stable agonists for protease-activated receptor-2 (PAR-2), we prepared 2-furoylated (2f) derivatives of native PAR-2-activating peptides, 2f-LIGKV-OH, 2f-LIGRL-OH, 2f-LIGKV-NH(2), and 2f-LIGRL-NH(2), and systematically evaluated their activity in PAR-2-responsive cell lines and tissues. In both HCT-15 cells and NCTC2544 cells overexpressing PAR-2, all furoylated peptides increased cytosolic Ca(2+) levels with a greater potency than the corresponding native peptides, although a similar maximum response was recorded. The absolute potency of each peptide was greater in NCTC2544, possibly due to a higher level of receptor expression. Furthermore, the difference in potency between the 2-furoylated peptides and the native peptides was enhanced when evaluated in the rat superior mesenteric artery and further increased when measuring PAR-2-mediated salivation in ddY mice in vivo. The potency of 2f-LIGRL-NH(2), the most powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca(2+) signaling assays, 517 in the vasorelaxation assay, and 1100 in the salivation assay. Amastatin, an aminopeptidase inhibitor, augmented salivation caused by native peptides, but not furoylated peptides. The PAR-2-activating peptides, including the furoylated derivatives, also produced salivation in the wild-type C57BL/6 mice, but not the PAR-2-deficient mice. Our data thus demonstrate that substitution of the N-terminal serine with a furoyl group in native PAR-2-activating peptides dramatically enhances the agonistic activity and decreases degradation by aminopeptidase, leading to development of 2f-LIGRL-NH(2), the most potent peptide. Furthermore, the data from PAR-2-deficient mice provide ultimate evidence for involvement of PAR-2 in salivation and the selective nature of the 2-furoylated peptides.