Application of Kampo medicines for the palliation of cancer cachexia.

Cancer cachexia results in the discontinuation of aggressive cancer therapy, and halting its progression has a significant effect on the survival rate and quality of life of patients with cancer. Currently, there are few therapies to control or slow down the progression of cancer cachexia. Although traditional Japanese Kampo medicine is widely used to support aggressive cancer therapy, the relevant scientific evidence is limited. Additionally, Kampo medicines are based on historical experience. In recent years, there have been widespread attempts to prove the efficacy of Kampo medicines through basic research, and an increasing number of studies have clarified the mechanism of action of Kampo medicines at the molecular level. It has been proposed that the improvement of cancer cachexia by Kampo medicines might involve enhancement of feeding via the central nervous system, improvement of protein maintenance in the skeletal muscle, and suppression of inflammatory cytokine production. In particular, among Kampo medicines, tonifying formulae, called "hozai" in Japanese, have been shown to be effective in alleviating cancer cachexia. In this review, we summarize the recent progress of basic and clinical research in Kampo medicines on cancer cachexia, and introduce Kampo medicines that are expected to be attractive supportive cancer medication.

Neoline, an active ingredient of the processed aconite root in Goshajinkigan formulation, targets Nav1.7 to ameliorate mechanical hyperalgesia in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Goshajinkigan (GJG), a traditional Japanese Kampo formula, has been shown to exhibit several pharmacological actions, including antinociceptive effects. Processed aconite root (PA), which is considered to be an active ingredient of GJG, has also been demonstrated to have an ameliorative effect on pain, such as diabetic peripheral neuropathic pain. We recently identified neoline as the active ingredient of both GJG and PA that is responsible for its effects against oxaliplatin-induced neuropathic pain in mice. AIM OF THE STUDY: In the present study, we investigated whether GJG, PA, and neoline could inhibit Nav1.7 voltage-gated sodium channel (VGSC) current and whether neoline could ameliorate mechanical hyperalgesia in diabetic mice. MATERIALS AND METHODS: To assess the electrophysiological properties of GJG extract formulation, powdered PA, and neoline on Nav1.7 VGSCs, whole-cell patch clamp recording was performed using human HEK293 cells expressing Nav1.7 VGSCs. In addition, the ameliorative effects of neoline on diabetic peripheral neuropathic pain were evaluated using the von Frey test in streptozotocin (STZ)-induced diabetic model mice. RESULTS: GJG extract formulation significantly inhibited Nav1.7 VGSC peak current. Powdered PA also inhibited Nav1.7 VGSC peak current. Like GJG and PA, neoline could inhibit Nav1.7 VGSC current. When diabetic mice were treated with neoline by intraperitoneal acute administration, the mechanical threshold was increased in diabetic mice, but not in non-diabetic mice, in a behavioral study. CONCLUSION: These results suggest that neoline might be a novel active ingredient of GJG and PA that is one of responsible ingredients for ameliorating mechanical hyperalgesia in diabetes via the inhibition of Nav1.7 VGSC current at least.

Neoline is the active ingredient of processed aconite root against murine peripheral neuropathic pain model, and its pharmacokinetics in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Processed aconite root (PA), the root of Aconitum carmichaeli (Ranunculaceae), is a crude drug used in traditional Chinese or Japanese kampo medicine to treat pain associated with coldness. In our previous study, PA and its active ingredient, neoline, alleviated oxaliplatin-induced peripheral neuropathy in mice. AIM OF THE STUDY: The present study investigated the effects of PA on a murine peripheral neuropathy model induced by intraperitoneal injection of paclitaxel and partial ligation of the sciatic nerve (Seltzer model), and identified its active ingredients. MATERIALS AND METHODS: PA powder (1 g/kg/day) was orally administered, and either neoline or benzoylmesaconine (10 mg/kg/day) was subcutaneously injected into the murine model. Mechanical hyperalgesia was evaluated via the von Frey filament method. PA extract was orally administered to rats; blood samples were chronologically collected, and the plasma concentrations of Aconitum alkaloids were measured. The contents of Aconitum alkaloids in commercial PA products were also measured. RESULTS: PA extract and neoline significantly attenuated the mechanical hyperalgesia induced by either paclitaxel or partial ligation of the sciatic nerve in mice. In the plasma samples of rats treated with PA extract, higher concentrations of benzoylmesaconine and neoline were apparent among Aconitum alkaloids. The contents of benzoylmesaconine and neoline varied among PA products with different processing procedures. Subcutaneous injection of benzoylmesaconine did not attenuate the hyperalgesia induced by each paclitaxel, partial ligation of the sciatic nerve, or oxaliplatin in mice. CONCLUSIONS: The present results indicate that PA and its active ingredient, neoline, are promising agents for the alleviation of neuropathic pain. Neoline can be used as a marker compound to determine the quality of the PA products for the treatment of neuropathic pain.

B-ring-homo-tonghaosu, isolated from Chrysanthemum morifolium capitulum, acts as a peroxisome proliferator-activated receptor-γ agonist.

The capitula of Chrysanthemum morifolium and C. indicum are used to prepare Chrysanthemi Flos in traditional Japanese Kampo medicine. In our previous study, we reported on the agonistic effect of methanol extract of C. indicum capitulum on peroxisome proliferator-activated receptor (PPAR)-γ. We further isolated (E)-tonghaosu from C. indicum capitulum as one of the active ingredients. In the present study, we aimed to evaluate the PPAR-γ agonistic activity of a methanol extract of C. morifolium capitulum (MCM) in which (E)-tonghaosu could not be detected. MCM exhibited PPAR-γ agonistic activity in a concentration-dependent manner, and at a dose of 100 µg/ml, it showed similar activity to pioglitazone (30 µM), a standard PPAR-γ agonist. Through activity-guided fractionation, we isolated two geometric isomers, (E)- (1) and (Z)-B-ring-homo-tonghaosu (2), as the active ingredients of MCM. Both compounds exerted concentration-dependent PPAR-γ agonistic effects, and 1 had higher activity than 2. At 1.4 µM, 1 had similar activity to pioglitazone (30 µM), which was achieved by 2 at a concentration of 140 µM. Thus, 1 has the potential to become a lead compound for the drug discovery of PPAR-γ agonists. We compared the activities and the contents of (E)-, (Z)-tonghaosu, 1, and 2 among 13 commercial samples of Chrysanthemi Flos, including those derived from both C. morifolium and C. indicum. Their PPAR-γ agonistic activities were not related to the contents of these compounds. 1 and 2 were detected in the samples derived from both species but (E)- and (Z)-tonghaosu were not detected in the samples derived from C. morifolium; hence (E)- and (Z)-tonghaosu can serve as marker compounds to identify the capitula of C. indicum in Chrysanthemi Flos samples.

Effect of Ninjin'yoeito on the Loss of Skeletal Muscle Function in Cancer-Bearing Mice.

Ninjin'yoeito (NYT), a traditional Japanese Kampo medicine formula, is used as a remedy for conditions, and physical weakness. Cancer cachexia is seen in advanced cancer patients and is defined by an ongoing loss of skeletal-muscle mass that leads to progressive functional impairment. In the present study, we examined the hypothesis whether NYT improves the functional loss of skeletal muscle cancer cachexia. Male C57/BL 6J mice with B16BF6 melanoma tumor showed decreased expression of myosin heavy chain (MHC) in the gastrocnemius muscle. Moreover, the expression of SOCS3 and phosphorylated STAT3 and AMPK was increased, and the expression of phosphorylated 4E-BP1 was decreased in the gastrocnemius muscle of tumor-bearing mice. These data suggested that amino acid metabolism was altered in tumor-bearing mice, which were normalized by the NYT intervention. The present study showed that NYT might be a novel therapeutic option for the treatment of sarcopenia occurring cancer cachexia.

Effect of ninjin'yoeito and ginseng extracts on oxaliplatin-induced neuropathies in mice.

Oxaliplatin (L-OHP) is a platinum-based anticancer agent used to treat various types of cancer. It frequently causes acute and chronic peripheral neuropathies, such as cold allodynia and mechanical hyperalgesia. Ninjin'yoeito (NYT) is a formula used in traditional Japanese Kampo medicine to improve recovery from diseases and other medical disorders. We previously reported that treatment with a boiling-water extract of NYT prevented L-OHP-induced damage to neurite-like outgrowths from differentiated PC12 cells. The objectives of the present study were to evaluate the in vivo effects of NYT on L-OHP-induced neuropathic pain in mice and identify the active ingredients in NYT. Treatment with NYT extract significantly ameliorated both cold allodynia and mechanical hyperalgesia induced by L-OHP. While L-OHP treatment suppressed neurite outgrowths from primary dorsal root ganglion cells in vitro, NYT extract blocked this suppression in a concentration-dependent manner. Among the herbal components of NYT, the extract of ginseng (Panax ginseng roots) showed a protective effect against neurite damage induced by L-OHP, and one of its active ingredients was identified as ginsenoside Rg3. Ginseng extract partially relieved L-OHP-induced neuropathic pain in mice. Our results suggest that NYT could be an attractive agent for treating L-OHP-induced neuropathic pain, and that the active ingredient of NYT may be ginseng.

Possible Involvement of Insulin Resistance in the Progression of Cancer Cachexia in Mice.

Malnutrition is a common problem among cancer patients, affecting up to 85% of patients with certain cancers. In severe cases, malnutrition can progress to cachexia, a specific form of malnutrition characterized by loss of lean body mass and muscle wasting. Although this muscle wasting might be a product of enhanced protein degradation, the precise mechanisms of cancer cachexia are not fully elucidated. Based on basic and clinical research, glucose intolerance and insulin resistance have been postulated to be associated with cancer cachexia. Since insulin in the skeletal muscle inhibits protein degradation and promotes protein synthesis, insulin resistance could be a possible cause of cancer cachexia. Therefore, we investigated the involvement of insulin resistance in the development of cancer cachexia in tumor-bearing mice. The signaling protein in the insulin cascade was attenuated in the skeletal muscle and hypothalamus from tumor-bearing mice. We identified Chrysanthemum morifolium RAMAT., known as Kikuka, as a peroxisome proliferator-activated receptor γ (PPARγ) ligand. Treatment with Kikuka attenuates the skeletal muscle changes in tumor-bearing mice. These results suggest that this natural PPARγ activator might be an attractive candidate for the treatment of cancer cachexia. In the symposium, we presented the PPARγ activator-induced improvement of cancer cachexia.

Processed aconite root and its active ingredient neoline may alleviate oxaliplatin-induced peripheral neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Processed aconite root (PA, the root of Aconitum carmichaeli, Ranunculaceae) is a crude drug used in traditional Chinese or Japanese kampo medicine to generate heat in the body and to treat pain associated with coldness. Oxaliplatin (L-OHP) is a platinum-based anticancer drug that frequently causes acute and chronic peripheral neuropathies, including cold and mechanical hyperalgesia. AIM OF THE STUDY: We investigated the effects of PA on L-OHP-induced peripheral neuropathies and identified the active ingredient within PA extract. MATERIALS AND METHODS: L-OHP was intraperitoneally injected into mice, and PA boiled water extract was orally administered. Cold and mechanical hyperalgesia were evaluated using the acetone test and the von Frey filament method, respectively. Dorsal root ganglion (DRG) neurons were isolated from normal mice and cultured with L-OHP with or without PA extract. Cell viability and neurite elongation were evaluated. RESULTS: PA extract significantly attenuated cold and mechanical hyperalgesia induced by L-OHP in mice. In cultured DRG neurons, L-OHP reduced cell viability and neurite elongation in a dose-dependent manner. Treatment with PA extract significantly alleviated the L-OHP-induced reduction of neurite elongation, while the cytotoxicity of L-OHP was not affected. Using activity-guided fractionation, we isolated neoline from PA extract as the active ingredient. Neoline significantly alleviated L-OHP-induced reduction of neurite elongation in cultured DRG neurons in a concentration-dependent manner. Moreover, subcutaneous injection of neoline attenuated cold and mechanical hyperalgesia in L-OHP-treated mice. PA extract and neoline did not show sedation and motor impairment. CONCLUSIONS: The present study indicates that PA and its active ingredient neoline are promising agents to alleviate L-OHP-induced neuropathic pain.

Ninjin'yoeito and ginseng extract prevent oxaliplatin-induced neurodegeneration in PC12 cells.

Ninjin'yoeito (NYT) is a formula of Japanese traditional kampo medicine composed of 12 crude drugs, and is designed to improve the decline in constitution after recovery from disease, fatigue, anemia, anorexia, perspiration during sleep, cold limbs, slight fever, chills, persistent cough, malaise, mental disequilibrium, insomnia, and constipation. Oxaliplatin (L-OHP) is a platinum-based anticancer drug used to treat colorectal, pancreatic, and stomach cancers. However, it often causes acute and chronic peripheral neuropathies including cold allodynia and mechanical hyperalgesia. In this study, we investigated the preventive effects of NYT on neuronal degeneration caused by L-OHP using PC12 cells, which are derived from the rat adrenal medulla and differentiate into nerve-like cells after exposure to nerve growth factor. L-OHP treatment decreased the elongation of neurite-like projection outgrowths in differentiated PC12 cells. When PC12 cells were treated with NYT hot water extract, neurodegeneration caused by L-OHP was significantly prevented in a concentration-dependent manner. Among the 12 crude drugs composing NYT, the extract of Ginseng (the root of Panax ginseng) exhibited the strongest preventive effects on neurodegeneration in differentiated PC12 cells. By activity-guided fractionation, we found that the fraction containing ginsenosides displayed preventive activity and, among several ginsenosides, ginsenoside F2 exhibited significant preventive effects on L-OHP-induced decreases in neurite-like outgrowths in differentiated PC12 cells. These results suggest that NYT and ginseng are promising agents for preventing L-OHP-induced neuropathies and present ginsenoside F2 as one of the active ingredients in ginseng.

Olanzapine induces glucose intolerance through the activation of AMPK in the mouse hypothalamus.

Treatment with atypical antipsychotic drugs is known to increase the risk of glucose intolerance and diabetes. However, the mechanism of this effect is unclear. Since central adenosine 5'-monophosphate-activated protein kinase (AMPK) plays an important role in regulating nutrient homeostasis, the present study was performed to examine the involvement of central AMPK in the glucose intolerance induced by olanzapine, an atypical antipsychotic drug, in mice. Acute intraperitoneal treatment with olanzapine dose-dependently increased blood glucose levels in the glucose tolerance test. Intracerebroventricular administration of olanzapine also increased blood glucose levels in the glucose tolerance test. The glucose intolerance induced by both intraperitoneal and intracerebroventricular treatment with olanzapine was significantly attenuated by intracerebroventricular pretreatment with the AMPK inhibitor compound C. Intracerebroventricular treatment with the AMPK activator AICAR increased blood glucose levels in the glucose tolerance test, and this increase was inhibited by compound C. Moreover, the hypothalamic level of phosphorylated AMPK after glucose injection was significantly increased by intracerebroventricular pretreatment with olanzapine. Olanzapine did not affect plasma glucagon and insulin levels. Our results indicate that acute treatment with olanzapine causes glucose intolerance through the activation of hypothalamic AMPK. The present study suggests that the inhibition of central AMPK activity may have a therapeutic effect on the metabolic disturbance induced by atypical antipsychotic drugs.

Involvement of the benzodiazepine system in the anxiolytic-like effect of Yokukansan (Yi-gan san).

The traditional Japanese Kampo medicine Yokukansan (YKS, Yi-gan san in Chinese) has been demonstrated to improve the behavioral and psychological symptoms of dementia (BPSD), such as anxiety, hallucinations, agitation and irritability. The aim of this study was to elucidate the mechanism of the anxiolytic-like effects of YKS and Chotoko, which is an active component of YKS. Oral treatment with YKS (300 and 1000 mg/kg) significantly increased the number of head-dipping behaviors in mice in the hole-board test. Head-dipping behavior in mice was also significantly increased by treatment with Chotoko (50 and 100mg/kg, p.o.). In addition, oral treatment with the water-extracted fractions from YKS (YKS-W; 250 and 500 mg/kg, p.o.) and Chotoko (Chotoko-W; 10 and 30 mg/kg) significantly increased the number of head-dipping behaviors in mice. On the other hand, treatment with the methanol-extracted fraction of YKS (YKS-Met; 15 and 30 mg/kg, p.o.) did not affect head-dipping behavior. The total distance and number of rearing behaviors were not affected by treatment with any of these drugs. The increase in the number of head-dipping behaviors by treatment with YKS-W (500 mg/kg, p.o.) and Chotoko-W (30 mg/kg, p.o.) was inhibited by pretreatment with the benzodiazepine receptor antagonist flumazenil (1mg/kg, i.v.). In the elevated plus-maze test, the percentage of time spent in open arms was increased in YKS (1000 mg, p.o.) treatment. Based on these results, we suggest that YKS produces an anxiolytic-like effect mediated by the benzodiazepine system. Chotoko is an effective component of YKS for producing an anxiolytic-like effect. The effective compound(s) should be contained, at least in part, in the water-soluble fraction of YKS.

Beacon/ubiquitin-like 5-immunoreactivity in the hypothalamus and pituitary of the mouse.

Beacon is a 73-amino acid peptide encoded by a novel gene in the hypothalamus of Israeli sand rat Psammomys obesus. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical techniques were used to investigate the presence of beacon mRNA and the distribution of beacon-immunoreactivity (irBC) in the hypothalamus of ICR mice. RT-PCR experiments revealed beacon mRNA in the mouse hypothalamus. Using a rabbit polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73), irBC was detected in the mouse hypothalamus and pituitary. In the hypothalamus, irBC was concentrated in perikarya of the supraoptic (SO), paraventricular (PVH) and accessory neurosecretory nuclei and in cell processes of the median eminence and pituitary stalk. In the pituitary, irBC was noted mainly in the posterior lobe. Double-labeling the hypothalamic sections with guinea-pig vasopressin-antiserum or mouse monoclonal oxytocin-antibody and beacon-antiserum revealed that <30% of vasopressin-immunoreactive neurons and nearly all oxytocin-immunoreactive neurons in the PVH and SO were irBC. The result shows the presence of beacon mRNA in the mouse hypothalamus, and the distribution of irBC is distinctively different from that reported in the hypothalamus of Psammomys obesus, but similar to that of the Sprague-Dawley rats described in our earlier study. More interestingly, Blast search uncovered a 73-amino acid peptide, human ubiquitin-like 5, which has the same exact sequence as beacon. Thus, irBC observed in the mouse brain could be that of ubiquitin-like 5.

Apelin-immunoreactivity in the rat hypothalamus and pituitary.

With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.