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To describe the treatment patterns of nivolumab and ipilimumab in Japan, a retrospective observational study was conducted in melanoma patients who received nivolumab and ipilimumab sequentially. Patients who received nivolumab and ipilimumab in combination were excluded from this study. Efficacy was evaluated by the Response Evaluation Criteria in Solid Tumors (RECIST) in terms of the overall response rate (ORR), progression-free survival (PFS), and disease control rate (DCR). Overall survival (OS) was also evaluated. Safety was assessed by the Common Terminology Criteria for Adverse Events (CTCAE). The treatment for all 68 patients enrolled involved switching from nivolumab to ipilimumab in 61 patients and switching from ipilimumab to nivolumab in seven patients. Switching occurred because of progressive disease in 55 patients and adverse events in eight patients. The median number of ipilimumab doses was three. Ipilimumab treatment achieved an ORR and DCR of 4.9% and 21.3%, respectively, and the median OS from start of ipilimumab was 7.0 months. During the study period, no new safety signals were noted. Independent factors which were indicative of poor prognosis for PFS were high neutrophil-to-lymphocyte ratio (NLR) and high C-reactive protein (CRP) levels before ipilimumab treatment. An evaluation over a washout period indicated that no significant relationship existed with efficacy or safety. For the sequential administration of nivolumab and ipilimumab in Japanese melanoma patients, switch from nivolumab to ipilimumab was common, and the major reason for switching was progressive disease. The major prognostic factors for ipilimumab PFS after nivolumab were NLR and CRP before ipilimumab treatment.

Prostaglandin E2 (PGE2) is essential for fracture healing. Systemic administration of EP4 ligands such as PGE2 and other synthetic EP4 agonists appears to transduce anabolic signals by binding to receptor EP4. Therefore, the present study was designed to test whether administration of EP4 agonist accelerates the healing of drill-hole injury in the femoral diaphysis. After surgery, a total of 128 Wistar rats, at the age of 12 weeks, were assigned to basal control (n = 8), and three groups with respective doses of 0 (vehicle control), 10 (low-dose), and 30 (high-dose) μg/kg body weight of the agent were subcutaneously injected twice a day. Femoral specimens were obtained at 0, 5, 7, 14, 21, and 28 days. In EP4 agonist-treated groups, the total bone volume of the regenerating bone in the defect did not significantly differ, but the regenerated cortical bone volume measured by histomorphometry and cortical bone mineral content (Ct. BMC) by pQCT dose-dependently increased at 14 and 21 days compared to the control. In the high-dose group, the value of osteoclast surface significantly increased compared with that in the control at 14 days. Expression levels of osteocalcin and TRAP mRNAs in the injured tissue increased at 14 days. Expression levels of EP4, BMP-2, and RANKL mRNAs increased at 7 days in the high-dose group. The bone mineral values of the lumbar bone at 28 days, measured by DXA, did not differ in the three groups. These data indicated that systemic administration of EP4 agonist ONO-4819.CD accelerated cortical bone healing after drill-hole injury by upregulating the local turnover of the regenerating bone.

To investigate the mechanism of bone formation by EP4 activation, an osmotic pump was implanted subcutaneously into the backs of rats and an EP4 receptor agonist was administered at a dose of 100 ng/kg/min for up to 28 days. The histology of the femur (including bone marrow) and the serum and/or urinary bone metabolism parameters were examined on Day 0, 1, 3, 5, 7, 14, and Day 28. In EP4 receptor agonist treated-rats, increase of osteoclasts in the metaphysis was observed on Day 1 and the number of osteoblast showed an increase from Day 3. In addition, cancellous bone and endosteal bone formation were observed in the metaphysis and diaphysis from Day 5 and this peaked on Day 28. Serum alkaline phosphatase activity showed a transient decrease on Day 1, but thereafter showed an increase. The Gla-type osteocalcin level showed an increase from Day 1. Moreover, Gla/Glu osteocalcin ratio showed an increase on Day 5. The urinary excretion of deoxypyridinoline increased on Day 3, showed a transient decrease on Day 5, and increased once again from Day 7. These results indicate that EP4 receptor agonist-induced bone formation is related to an increase of osteoclasts at the initial stage and a subsequent increase of osteoblasts.

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E 2 promotes both bone resorption and bone formation. By infusing PGE 2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE 2 induced expression of core-binding factor α1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

The constituent amino acids of plusbacins A1-A4 were determined to be two moles of L-trans-3-hydroxyproline and one mole each of D-threo-beta-hydroxyaspartic acid, L-threo-beta-hydroxyaspartic acid, D-allo-threonine, D-serine, D-alanine and L-arginine. In plusbacins B1-B4, one mole of L-trans-3-hydroxyproline is replaced by L-proline. The fatty acid residue of A1 and B1 was determined to be 3-hydroxy-tetradecanoic acid, for A2 and B2 to be 3-hydroxy-isopentadecanoic acid, for A3 and B3 to be 3-hydroxy-isohexadecanoic acid, and for A4 and B4 to be 3-hydroxy-hexadecanoic acid. A lactone linkage was suggested to reside between L-threo-beta-hydroxyaspartic acid and 3-hydroxy-fatty acid residues by degradation experiments. The amino acid sequences of plusbacins A2 and B2 were confirmed by Edman degradation of their deacylated products, and supported by mass spectrometric studies. From the above, structures of all components of plusbacins were concluded.

New antibiotics, plusbacins A1-A4 and B1-B4, were isolated from the culture broth of a strain of Pseudomonas sp. These antibiotics were isolated as a complex by column chromatographies on Diaion HP-20 and silica gel, and then separated by HPLC. They are amphoteric in nature. The hydrochlorides are obtained as colorless powders, soluble in methanol and alkaline water. From their physico-chemical properties, these antibiotics are presumed to be acyloctapeptides containing a lactone linkage, and their differences occur in amino acid and fatty acid residues. The antibiotics are active against Gram-positive bacteria in vitro and in vivo.

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with arachidonate at concentrations of less than 20 μ m induced slight stimulation of Superoxide anion (O − 2 ) production with little enhancement of the phosphorylation of the 46K protein(s). The stimulation of the phosphorylation of those protein(s) has been observed in parallel with an activation of NADPH oxidase in our previous studies (N. Okamura et al. (1984) Arch. Biochem. Biophys. 228 , 270–277; T. Ohtsuka et al. (1986) Biochim. Biophys. Acta 888 , 332–337; T. Ohtsuka et al. (1987) J. Biochem . 101 , 897–903). On the other hand, the phosphorylation of the same protein(s) was increased by the treatment of PMNL with 10 μ m 1-oleoyl-2-acetylglycerol (OAG), a permeable diacylglycerol, with little change in O − 2 production. Treatment of PMNL with a combination of such low concentrations of arachidonate and OAG, induced marked increase in O − 2 production in accordance with the increase in the phosphorylation of 46K protein(s) which was probably due to OAG action. Thus, it is likely that this protein phosphorylation is a prerequisite or regulatory to the stimulation of the O − 2 production by arachidonate in PMNL.

The phosphorylation of 46K protein(s), which was generally observed in parallel with an activation of NADPH oxidase of guinea pig polymorphonuclear leukocytes (PMNL) in our previous studies (N. Okamura et al. (1984) Arch. Biochem. Biophys. 228 , 270–277; T. Ohtsuka et al. (1987) J. Biochem. 101 , 897–903), was increased by treatment with 1-oleoyl-2-acetylglycerol (OAG), even at low concentrations at which both superoxide anion (O − 2 ) production and arachidonate release were little induced. On the other hand, exposure of PMNL to low concentrations of a calcium ionophore, A23187, stimulated arachidonate release without causing substantial O − 2 production and increase in phosphorylation of 46K protein(s). Simultaneous addition of the above-mentioned suboptimal concentrations of both OAG and A23187 markedly enhanced O − 2 production in PMNL. This enhancing effect may be ascribable to the increase in the phosphorylation of 46K protein(s) and arachidonate release, which are induced by the addition of OAG and A23187, respectively. Another arachidonate-releasing agent, N -formyl-methionyl-leucyl-phenylalanine (FMLP), also stimulated O − 2 production in accordance with an increase in the arachidonate release and protein phosphorylation. Simultaneous addition of OAG significantly enhanced the FMLP-induced O − 2 production, presumably by maintaining the 46K protein phosphorylation which would facilitate the effect of arachidonate released by FMLP. Thus, the present results suggest that phosphorylation of 46K protein(s) and arachidonate release synergistically induce O − 2 production in PMNL, although either of them alone hardly induces the production.

New antibiotics, resorcinomycins A and B, were isolated from the culture broth of a streptomycete strain identified as Streptoverticillium roseoverticillatum. The antibiotics are water-soluble amphoteric substances, positive to SAKAGUCHI'S reagent. The molecular formulas C14H20N4O5 and C13H18N4O5 for A and B were indicated by elemental analysis and secondary ion MS. The structures of these antibiotics were determined by 1H and 13C NMR spectrometry and some chemical evidences to be N-[(S)-alpha-guanidino-3,5-dihydroxy-4-isopropylphenylacetyl]glyci ne and N-[(S)-alpha-guanidino-3,5-dihydroxy-4-ethylphenylacetyl]-glycine, respectively.

NADPH oxidase, a complex enzyme system in the cell membrane responsible for the bactericidal function of polymorphonuclear leukocytes through the production of superoxide anion, was facilely released by mild treatment with a press. At the pressure where almost all NADPH oxidase activity was released, releases of the activities of lactate dehydrogenase, 5'-nucleotidase, lysozyme, and N-acetyl-beta-glucosaminidase, and of the amount of total protein were negligible. This method can be useful for the elucidation of NADPH oxidase.

Production of superoxide anion (O-2) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1, 2-benzisoselenazol-3 (2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O-2 production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

Three new antibiotics, verticillins A (Ia), B (XVIII), and C have been isolated from Verticillium sp. Stereo-structures of verticillins A and B, which are derivatives of bi-(3,11 a-epidithio-1.4-dioxopyrazino[1′,2′ : 1,5]pyrrolo-[2,3-b]indol-10b-yl), have been elucidated by chemical and physicochemical methods. Verticillin C is thought to be an epitrithio-analogue of verticillin B.