Preparation and pharmaceutical properties of Hangeshashinto oral ointment and its safety and efficacy in Syrian hamsters with 5-fluorouracil-induced oral mucositis.

Chemotherapy-induced oral mucositis (COM) is a common adverse effect of cancer chemotherapy. Several clinical studies reported that repetitive use of mouthwashes containing 2.5-6.25% Hangeshashinto (HST), a Kampo formula, relieves COM, but the effect is insufficient. To solve this problem, we produced an oral ointment of 12% HST extract (considered quantitatively equivalent to 20% commercially available HST), which will increase the local concentrations of its active ingredients and prolong the contact time with COM. In this study, we evaluated the pharmaceutical properties (spreadability and stability) of HST oral ointment. In addition, its safety (oral mucosal irritation) and therapeutic effects on 5-fluorouracil-induced oral mucositis were evaluated in male Syrian hamsters. The HST ointment showed good spreadability and stability for more than 8 weeks at 4 °C. In the oral mucosal irritation test, topical application of HST ointment (0.2 g) three times per day for 14 days had no adverse effect on the oral mucosa of hamsters. In hamsters treated with 5-fluorouracil (60 mg/kg) twice, COM was induced by a submucosal injection of 5% acetic acid into the cheek pouch. When HST ointment (50 µg) was topically applied to the mucositis area once per day for 12 days, the area and macroscopic score of mucositis were significantly decreased, and the depth of the wound tended to be reduced compared with the lactose ointment-treated control animals. These findings suggest that HST oral ointment shows good properties in spreadability, stability, and safety, and elicits a therapeutic effect in an animal model of COM.

Cilostazol is an effective causal therapy for preventing paclitaxel-induced peripheral neuropathy by suppression of Schwann cell dedifferentiation.

Chemotherapy-induced peripheral neuropathy (CIPN) can lead to discontinuation of chemotherapy and is consequently a serious impediment to effective cancer treatment. Due to our limited understanding of mechanisms underlying the pathogenesis of CIPN, no causal therapy has been approved for relief of this condition. We previously demonstrated that taxanes (paclitaxel and docetaxel) induce Schwann cell dedifferentiation, characterized by increased expression of p75 and galectin-3, ultimately leading to demyelination. These changes appear to be responsible for CIPN pathogenesis. This study was designed to identify a novel candidate therapeutic for CIPN with the ability to suppress paclitaxel-induced Schwann cell dedifferentiation. Given that elevation of cyclic adenosine monophosphate (cAMP) signaling participates in Schwann cell differentiation, we performed immunocytochemical screening of phosphodiesterase (PDE) inhibitors. We found that the PDE3 inhibitor cilostazol strongly promoted differentiation of primary cultures of rat Schwann cells via a mechanism involving cAMP/exchange protein directly activated by cAMP (Epac) signaling. Co-treatment with cilostazol prevented paclitaxel-induced dedifferentiation of Schwann cell cultures and demyelination in a mixed culture of Schwann cells and dorsal root ganglia neurons. Notably, continuous oral administration of cilostazol suppressed Schwann cell dedifferentiation within the sciatic nerve and the development of mechanical hypersensitivity in a mouse model of paclitaxel-related CIPN. Importantly, cilostazol potentiated, rather than inhibited, the anti-cancer effect of paclitaxel on the human breast cancer cell line MDA-MB-231. These findings highlight the potential utility of cilostazol as a causal therapeutic that avoids the development of paclitaxel-related CIPN without compromising anti-cancer properties.

Pronociceptive Roles of Schwann Cell-Derived Galectin-3 in Taxane-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency (Galectin-3 -/- mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.

Schwann cell-derived CXCL1 contributes to human immunodeficiency virus type 1 gp120-induced neuropathic pain by modulating macrophage infiltration in mice.

The neuroinflammatory responses to human immunodeficiency virus type 1 (HIV-1) coat proteins, such as glycoprotein 120 (gp120), are considered to be responsible for the HIV-associated distal sensory neuropathy. Accumulating evidences suggest that T-cell line tropic X4 gp120 increases macrophage infiltration into the peripheral nerves, and thereby induces neuroinflammation leading to pain. However, the mechanisms underlying X4 gp120-induced macrophage recruitment to the peripheral nervous systems remain unclear. Here, we demonstrated that perineural application of X4 gp120 from HIV-1 strains IIIB and MN elicited mechanical hypersensitivity and spontaneous pain-like behaviors in mice. Furthermore, flow cytometry and immunohistochemical studies revealed increased infiltration of bone marrow-derived macrophages into the parenchyma of sciatic nerves and dorsal root ganglia (DRG) 7 days after gp120 IIIB or MN application. Chemical deletion of circulating macrophages using clodronate liposomes markedly suppressed gp120 IIIB-induced pain-like behaviors. In in vitro cell infiltration analysis, RAW 264.7 cell (a murine macrophage cell line) was chemoattracted to conditioned medium from gp120 IIIB- or MN-treated cultured Schwann cells, but not to conditioned medium from these gp120-treated DRG neurons, suggesting possible involvement of Schwann cell-derived soluble factors in macrophage infiltration. We identified using a gene expression array that CXCL1, a chemoattractant of macrophages and neutrophils, was increased in gp120 IIIB-treated cultured Schwann cells. Similar to gp120 IIIB or MN, perineural application of recombinant CXCL1 elicited pain-like behaviors accompanied by macrophage infiltration to the peripheral nerves. Furthermore, the repeated injection of CXCR2 (receptor for CXCL1) antagonist or CXCL1 neutralizing antibody prevented both pain-like behaviors and macrophage infiltration in gp120 IIIB-treated mice. Thus, the present study newly defines that Schwann cell-derived CXCL1, secreted in response to X4 gp120 exposure, is responsible for macrophage infiltration into peripheral nerves, and is thereby associated with pain-like behaviors in mice. We propose herein that communication between Schwann cells and macrophages may play a prominent role in the induction of X4 HIV-1-associated pain.

Improvement of peripheral vascular impairment by a phosphodiesterase type 5 inhibitor tadalafil prevents oxaliplatin-induced peripheral neuropathy in mice.

Oxaliplatin, a platinum-based chemotherapeutic drug, frequently induces peripheral neuropathy. Accumulating evidences suggest a possible relationship between peripheral vascular impairment and peripheral neuropathy. In this study, we investigated the effects of vasodilators on cumulative peripheral neuropathy induced by repeated injections of oxaliplatin (10 mg/kg) once a week for 8 weeks in mice. Single injections of vasodilators, including a phosphodiesterase type 5 inhibitor tadalafil acutely alleviated oxaliplatin-induced cold hypersensitivity, while tadalafil had no effect on the mechanical hypersensitivity. By contrast, long-term administration of tadalafil (0.1% in chow diets) during the oxaliplatin injection period reduced the oxaliplatin-induced decreases in skin temperature and blood flow without affecting platinum concentrations in blood, sciatic nerves, and dorsal root ganglion. The long-term administration significantly suppressed cold, mechanical, and electrical current hypersensitivities as well as thermal hypoesthesia. Furthermore, it prevented the decreases in sensory nerve conductance velocity and the number of endoneurial microvessels, and axon degeneration in the sciatic nerves. In vitro studies confirmed that tadalafil does not interfere with the cytotoxicity of oxaliplatin against human cancer cell lines. Altogether, these results suggest that improvement of peripheral vascular impairment by tadalafil could alleviate and prevent oxaliplatin-induced peripheral neuropathy.

Taxanes and platinum derivatives impair Schwann cells via distinct mechanisms.

Impairment of peripheral neurons by anti-cancer agents, including taxanes and platinum derivatives, has been considered to be a major cause of chemotherapy-induced peripheral neuropathy (CIPN), however, the precise underlying mechanisms are not fully understood. Here, we examined the direct effects of anti-cancer agents on Schwann cells. Exposure of primary cultured rat Schwann cells to paclitaxel (0.01 µM), cisplatin (1 µM), or oxaliplatin (3 µM) for 48 h induced cytotoxicity and reduced myelin basic protein expression at concentrations lower than those required to induce neurotoxicity in cultured rat dorsal root ganglion (DRG) neurons. Similarly, these anti-cancer drugs disrupted myelin formation in Schwann cell/DRG neuron co-cultures without affecting nerve axons. Cisplatin and oxaliplatin, but not paclitaxel, caused mitochondrial dysfunction in cultured Schwann cells. By contrast, paclitaxel led to dedifferentiation of Schwann cells into an immature state, characterized by increased expression of p75 and galectin-3. Consistent with in vitro findings, repeated injection of paclitaxel increased expression of p75 and galectin-3 in Schwann cells within the mouse sciatic nerve. These results suggest that taxanes and platinum derivatives impair Schwan cells by inducing dedifferentiation and mitochondrial dysfunction, respectively, which may be important in the development of CIPN in conjunction with their direct impairment in peripheral neurons.

Effect of three fluoride compounds on the growth of oral normal and tumor cells.

AIM: Comparative study of the growth inhibition by different types of fluoride compounds used in dentistry has been limited. We investigated the effects of sodium fluoride (NaF), diammine silver fluoride [Ag(NH3)2F] and 5-fluorouracil (5-FU) on the growth of eleven human normal and tumor cells in total. MATERIALS AND METHODS: Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was evaluated by caspase-3 activation and DNA fragmentation. Fluoride was determined using a fluoride-specific electrode. RESULTS: All compounds had little or no growth stimulating effect (hormesis) on all cells. Ag(NH3)2F exhibited the highest cytotoxicity towards both normal and tumor cells. 5-FU had the selective cytostatic activity towards oral squamous cell carcinoma cell lines, whereas NaF was selectively cytotoxic towards glioblastoma cell lines. None of the compounds induced internucleosomal DNA fragmentation and only 5-FU induced slight activation of caspase-3 in an oral squamous cell carcinoma cell line (HSC-2). Cytotoxicity of fluoride compounds was not reduced by superoxide dismutase and catalase, reducing the possibility of the involvement of reactive oxygen species in the mechanism of action. Approximately 0.01-0.09% initially added NaF was recovered from the cells, whereas the cellular uptake of Ag(NH3)2F and 5-FU was below the detection limit. CONCLUSION: Cytotoxicity of fluoride compounds may not be directly linked to their tumor specificity nor to their apoptosis-inducing activity.

Prospective randomized study of two laparotomy incisions for gastrectomy: midline incision versus transverse incision.

BACKGROUND: We performed a randomized study to evaluate the differences between upper midline incision and transverse incision for gastrectomy. METHODS: Patients undergoing distal gastrectomy or total gastrectomy for gastric cancer were randomly allocated to have either an upper midline incision or a transverse incision. The times taken to open and close the abdominal cavity, the number of doses of postoperative analgesics, and the incidence of postoperative pneumonia, wound infection, and intestinal obstruction were compared between the patients having the two incisions. RESULTS: Times for both opening and closing the abdominal cavity were longer with a transverse incision, in both the distal gastrectomy group and total gastrectomy group. In the patients in whom continuous epidural analgesia was used postoperatively, the number of additional doses of analgesics was smaller in the transverse-incision group after distal gastrectomy. The incidence of postoperative pneumonia was lower in the transverse-incision group after distal gastrectomy. The number of patients with postoperative intestinal obstruction was smaller in the transverse-incision group than in the midline-incision group after distal gastrectomy. In contrast to distal gastrectomy, there was no significant difference in the number of doses of postoperative analgesics, incidence of postoperative pneumonia, or incidence of postoperative intestinal obstruction between the two study groups after total gastrectomy. CONCLUSION: A transverse incision for distal gastrectomy may be more beneficial than an upper midline incision in attenuating postoperative wound pain, decreasing the incidence of postoperative pneumonia, and preventing postoperative intestinal obstruction.

Usefulness of immunomodulators for maturation of dendritic cells.

Biological response modifiers (BRMs) augment the cytotoxic activity of various effector cells by the induction of multiple cytokines and suppression of immunosuppressive factors. BRMs are used extensively in adjuvant therapy for gastric cancer in Japan. In dendritic cell (DC)-based vaccine therapy, the quality of DCs is important in inducing strong antitumor immunity. A good manufacturing practice (GMP) grade agent for DCs maturation is desirable for safety. Here we report the effects of two BRMs, OK432 and PSK, which are GMP grade agents for the functional maturation of DCs. OK432 and PSK were examined in vitro, and compared with lipopolysaccharide (LPS) and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6 and PGE2). In the immunophenotypical analysis, the expression of CD80 and CD83 of DCs stimulated with OK-432 increased significantly compared with PSK and medium, and this up-regulation was the same as levels of DCs stimulated with cytokine cocktail. DCs stimulated with OK-432 showed significantly higher production of IL-12 and Th1-type cytokines (IL-2 and IFN-gamma) compared with DCs stimulated with LPS or cytokine cocktail. OK-432 stimulated DCs could induce the significantly high level of cytotoxic T cell activity compared with PSK-stimulated or unstimulated DCs. These results suggest that OK432 is a GMP-grade reagent that promotes functional maturation of DCs and could be applied in DC-based vaccinations.