Phase I Dose-Escalation Study of Ramucirumab in Chinese Patients with Advanced Solid Tumors.

LESSONS LEARNED: Ramucirumab was well tolerated in Chinese patients with advanced solid tumors, and adverse events were manageable in this study.Pharmacokinetics characteristics in Chinese patients were similar to those in other populations. Immunogenicity was not detected.No efficacy conclusion could be drawn, and further randomized studies are warranted. BACKGROUND: This single-arm, nonrandomized, open-label, dose-escalation, phase I study was designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of ramucirumab in Chinese patients with advanced solid tumors that were resistant to standard therapy or no standard therapy was available. METHODS: Dose escalation was a 3 + 3 design, with expansion in Cohorts 2 and 3 for PK. Ramucirumab was given intravenously at three different dosages: 6 mg/kg every 2 weeks, 10 mg/kg every 3 weeks, and 8 mg/kg every 2 weeks. Safety analyses included all patients. PK, immunogenicity, and antitumor activity were also assessed. RESULTS: Among 28 patients treated, 2 experienced dose-limiting toxicity, possibly related to ramucirumab. No maximum tolerated dose was determined. All patients experienced at least one treatment-emergent adverse event. Grade ≥3 adverse event was reported for 53.6% (n = 15) of patients. PK analyses indicated that ramucirumab had low clearance, small volume of distribution, and long half-life in Chinese patients, as in other populations. Immunogenicity was not detected. No patient had complete/partial response, and 64.3% (n = 18) had stable disease with a median duration of 5.55 months (95% confidence interval: 3.38-7.13 months). CONCLUSION: Ramucirumab appeared to be well tolerated in Chinese patients with advanced solid tumors. PK characteristics in Chinese patients were similar to those in other populations.

Effects of dexmedetomidine on insulin secretion from rat pancreatic ß cells.

PURPOSE: Dexmedetomidine acts as a selective α2-adrenergic receptor agonist and an imidazoline receptor agonist, both of which are known to affect insulin secretion. Here, we investigated the effects of clinically relevant concentrations of dexmedetomidine on insulin secretion under in vivo conditions. Furthermore, its underlying mechanisms were examined using isolated islets in vitro. METHODS: For the in vivo oral glucose tolerance test (OGTT), male Sprague-Dawley rats were randomly allocated to one of three groups (n = 7 in each group): two groups infused with dexmedetomidine at a low (group L) or a high (group H) dose, and one control group infused with the same amount of saline (group C). For the in vitro perifusion study, insulin released from isolated islets was measured during stepwise changes in glucose. Dexmedetomidine (0.1-100 µM) was added to the chamber. RESULTS: During the OGTT test, the insulin levels in group H were significantly lower than those in group C at 30, 60, and 90 min after glucose load. On the other hand, insulin levels in group L were comparable to those of group C at all time points. In the perfusion study, dexmedetomidine inhibited glucose-stimulated insulin secretion in a concentration-dependent manner. When co-treated with yohimbine, an α2-adrenoceptor blocker, dexmedetomidine adversely increased glucose-induced insulin secretion. However, co-treatment with idazoxan, an antagonist for α2-adrenergic and imidazoline receptors, completely abolished the action of dexmedetomidine. CONCLUSIONS: Dexmedetomidine had no effect on insulin secretion at sedative dose, whereas it significantly inhibited insulin secretion at supraclinical high concentrations mainly via the α2-adrenoceptor.

Efficacy and safety of pemetrexed/cisplatin versus gemcitabine/cisplatin as first-line treatment in Chinese patients with advanced nonsquamous non-small cell lung cancer.

OBJECTIVES: Retrospective subgroup analysis in JMDB study indicates that the between-arm differences in overall survival (OS) in the East Asian subgroup were consistent with those observed in the entire JMDB study population. This bridging study (JMIL) further evaluated the efficacy and safety of first-line pemetrexed/cisplatin (PC) versus gemcitabine/cisplatin (GC) in Chinese patients with nonsquamous non-small cell lung cancer (NSCLC). The primary endpoint of this local registration trial was designed to compare OS in the combined dataset, consisting of Chinese patients in JMIL and 1252 nonsquamous patients in JMDB. MATERIALS AND METHODS: Chinese patients with stage IIIB/IV nonsquamous NSCLC were randomly assigned (1:1) to 6 cycles maximum (21 days/cycle) of pemetrexed 500mg/m(2)+cisplatin 75mg/m(2) (day 1), or gemcitabine 1250mg/m(2) (days 1 and 8)+cisplatin 75mg/m(2) (day 1). RESULTS: In JMIL, 256 Chinese patients were randomized (PC, n=126; GC, n=130). Patient baseline characteristics were balanced between treatment arms. In the combined dataset, PC was superior to GC in prolonging OS, with adjusted hazard ratio (HR) of 0.87 (95% CI: 0.77-0.98, p=0.023) and median OS of 11.76 versus 10.94 months. In the JMIL-only population, no significant OS difference observed between treatment arms (adjusted HR=1.03 [95% CI: 0.77-1.39, p=0.822]; unadjusted HR=0.996 [95% CI: 0.74-1.33, p=0.980]), nor for other secondary efficacy endpoints. Significantly fewer patients in the PC arm experienced drug-related grade 3/4 toxicities, 54 (43.2%) versus 71 (55.9%) for GC (p=0.045), with significantly lower rates of leukocytopenia, thrombocytopenia, and fatigue. CONCLUSION: This study showed that in the combined population, OS of PC was superior to GC, while in the Chinese-only population, no significant difference was observed; a better safety and risk/benefit profile was found in the PC arm. A PC regimen should be considered as a standard of care in Chinese nonsquamous NSCLC patients in a first-line setting.

Postoperative pain impairs subsequent performance on a spatial memory task via effects on N-methyl-D-aspartate receptor in aged rats.

AIMS: Pain may be associated with postoperative cognitive dysfunction (POCD); however, this relationship remains under investigated. Therefore, we examined the impact of postoperative pain on cognitive functions in aged animals. MAIN METHODS: Rats were allocated to the following groups: control (C), 1.2 % isoflurane for 2 hours alone (I), I with laparotomy (IL), IL with analgesia using local ropivacaine (IL+R), and IL with analgesia using systemic morphine (IL+M). Pain was assessed by rat grimace scale (RGS). Spatial memory was evaluated using a radial maze from postoperative days (POD) 3 to 14. NMDA receptor (NR) 2 subunits in hippocampus were measured by ELISA. Finally, effects of memantine, a low-affinity uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, on postoperative cognitive performance were tested. KEY FINDINGS: Postoperative RGS was increased in Group IL, but not in other groups. The number of memory errors in Group I were comparable to that in Group C, whereas errors in Group IL were increased. Importantly, in Group IL+R and IL+M, cognitive impairment was not found. The memory errors were positively correlated with the levels of NMDA receptor 2 subunits in hippocampus. Prophylactic treatment with memantine could prevent the development of memory deficits observed in Group IL without an analgesic effect. SIGNIFICANCE: Postoperative pain contributes to the development of memory deficits after anesthesia and surgery via up-regulation of hippocampal NMDA receptors. Our findings suggest that postoperative pain management may be important for the prevention of POCD in elderly patients.

Activation of ATP-sensitive potassium channels by nicorandil is preserved in aged vascular smooth muscle cells in rats.

Nicorandil, an ATP-sensitive potassium (KATP) channel opener having the properties of a nitrate, causes vasodilation, particularly of coronary arteries, and has been reported to reduce the frequency of perioperative cardiac events. We previously demonstrated that isoflurane could activate vascular KATP channels through an intracellular signaling pathway, but that this isoflurane-induced channel opening is suppressed by aging. Here, we investigated whether advanced age modifies nicorandil-induced activation of vascular KATP channels. We used a cell-attached patch-clamp configuration to test the effects of nicorandil on KATP channel activity in vascular smooth muscle cells (VSMCs) obtained from 12- to 15-week-old (adult) and 24- to 25-month-old (aged) male Wistar rats. Bath application of nicorandil (0.1-100 µM) activated KATP channels to a level similar to that observed in VSMCs from the arteries of both adult and aged rats. Furthermore, concomitant bath application of nicorandil in the aged group dose-dependently ameliorated the age-related reduction in isoflurane-induced vascular KATP channel activation. Our findings indicate that nicorandil could be used effectively in elderly patients to directly activate vascular KATP channels during the perioperative period.

Anti-inflammatory effect of three iridoids in human neutrophils.

To verify the anti-inflammatory potency of iridoids, three iridoids (two natural, loganic acid: LA; geniposide: GE; and an artefact, 7(S)-n-butyl morroniside: BM) were investigated in vitro on the inhibition of superoxide generation in human neutrophils. All compounds showed inhibitory effect on fMLP-induced superoxide generation in a concentration-dependent manner with the following order: BM>LA>GE. BM exhibits potent inhibitory activity on superoxide anion induced by PMA, while LA and GE showed weak effect. When AA was used as stimulus, the generation of superoxide anion was suppressed by BM in a concentration-dependent manner. LA and GE exhibit both sides effect on superoxide generation.

Influence of hematopoietic stem cell-derived hepatocytes on liver regeneration after sex-mismatched liver transplantation in humans.

BACKGROUND: Presence of hematopoietic stem-cell-derived hepatocytes after clinical liver transplantation was demonstrated repeatedly. The relevance of this controversial mechanism of regeneration was discussed. Regarding frequency, the demonstrated results were divergent. In the present study, we propose to investigate the influence of growth and regeneration on the frequency of hematopoietic stem-cell-derived hepatocytes in transplanted organs. MATERIAL AND METHOD: Paraffin-embedded liver specimens, obtained as clinically indicated, from female grafts transplanted into male recipients were investigated. The presence of Y-chromosome in hepatocytes, detected by fluorescence in situ hybridization (FISH), was the indicator for recipient origin. Slides were evaluated by assessing the relative number of Y-chromosome containing hepatocytes within 50 images representing an average of 775 hepatocytes. RESULTS: In only 9 out of 81 specimens, single Y-chromosome positive hepatocytes were detected, resulting in a maximal frequency of 0.64%. Six positive specimens were obtained from full-size liver grafts and one from a partial liver graft. In the pediatric group, two positive samples were found. By staining additional sections from the nine positive specimens, no additional positive hepatocytes were detected suggesting an even lower frequency within the whole sample. We did not find any accumulation of Y-chromosome positive cells in any of the individually analyzed patient groups. CONCLUSION: Transdifferentiation of hematopoietic stem cells is an extremely rare event in liver growth and regeneration after transplantation. Due to the low number of positive events, the biological relevance seems questionable.

Biophysical and pharmacological properties of glucagon-like peptide-1 in rats under isoflurane anesthesia.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) increases insulin secretion and has an important role in maintaining glucose homeostasis. In this study, we evaluated the biophysical and pharmacological properties of GLP-1 by performing in vivo and in vitro experiments to determine the applicability of GLP-1 in glycemic control in rats under isoflurane anesthesia. METHODS: Levels of portal GLP-1, insulin, and glucose and dipeptidyl peptidase-4 activity were measured in the basal fasting state and after gastric glucose load before, during, and after exposure to 30% O(2) in air (control) or 1.4% isoflurane in a mixture of 30% O(2) and air. The direct effects of isoflurane on GLP-1 secretion were assessed in human enteroendocrine NCI-H716 cells. Insulin release from isolated pancreatic islets was measured using a radioimmunoassay. Single pancreatic ß-cell membrane potentials were recorded using whole-cell current-clamp patches perforated by ß-escin. RESULTS: In fasting rats, inhalation of isoflurane led to a decrease in the basal levels of GLP-1 but did not affect insulin and glucose levels. Levels of GLP-1, insulin, and glucose increased after gastric administration of glucose in control rats. However, isoflurane attenuated the glucose-induced increase in GLP-1 and insulin levels and increased plasma glucose levels. In contrast, isoflurane did not affect dipeptidyl peptidase-4 activity before or after gastric glucose loading. Isoflurane (0.35 mM) inhibited GLP-1 release in NCI-H716 cells; this finding was similar to that observed in in vivo studies. In perifusion experiments, isoflurane (0.35 mM) inhibited glucose-induced insulin release, whereas exogenous GLP-1 (10 nM) enhanced insulin release. Importantly, combined administration of isoflurane and GLP-1 enhanced both phases of glucose-induced insulin release to an extent similar to that achieved with GLP-1 alone. Whole-cell patches showed that exposure to GLP-1 (10 nM) led to nearly complete restoration of glucose-stimulated depolarization that had been suppressed by isoflurane (0.35 mM). CONCLUSIONS: GLP-1 secretion is impaired during isoflurane anesthesia. However, our study showed that the insulinotropic action of GLP-1 was not affected by isoflurane. Furthermore, exposure to GLP-1 increased the membrane activity of pancreatic ß-cells, preventing isoflurane-induced impairment of glucose-induced insulin secretion. These results support the hypothesis that GLP-1-based therapy may be a useful approach for achieving intraoperative glycemic control.

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels.

AIMS: Dexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K(+) (K(ATP)) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular K(ATP)-channel activity at the single-channel level. MAIN METHODS: We used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular K(ATP) channels, recombinant K(ATP) channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels. KEY FINDINGS: Dexmedetomidine was observed to inhibit the native rat vascular K(ATP) channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 K(ATP) channels as well as Kir6.2ΔC36 channels with equivalent potency. SIGNIFICANCE: These results indicate that dexmedetomidine directly inhibits K(ATP) channels through the Kir6.0 subunit.

Inhibitory effects of secoiridoids from the roots of Gentiana straminea on stimulus-induced superoxide generation, phosphorylation and translocation of cytosolic compounds to plasma membrane in human neutrophils.

Gentiana straminea Maxim. has been used widely as a traditional Chinese medicine for the treatment of rheumarthritis, icterepatitis, constipation, pain and hypertension. Five secoiridoids, gentiopicroside (GTP), 6'-O-(2-hydroxy-3-O-ß-D-glucopyranosyl-benzoyl)-sweroside (HGBS), 6'-O-ß-D-glucosylgentiopicroside (GGTP), sweroside (SW) and swertiamarin (STM) were isolated from the roots of G. straminea. The effect of these secoiridoids on stimulus-induced superoxide generation in human neutrophils was assayed by measuring the reduction of ferricytochrome c. Tyrosyl or serine/threonine phosphorylation of neutrophil proteins, and translocation of the cytosolic compounds to the cell membrane were also investigated using specific monoclonal antibodies. The five secoiridoids used in the present experiment suppressed N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration dependent manner. GTP and HGBS also suppressed phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA)-induced superoxide generation. However, the other three secoiridoids showed no effect on PMA- and AA-induced superoxide generation. fMLP-, PMA- and AA-induced tyrosyl or serine/threonine phosphorylation and translocation of the cytosolic proteins to the cell membrane were suppressed in parallel with the suppression of the stimulus-induced superoxide generation.

The involvement of the neurosteroid allopregnanolone in the antihyperalgesic effect of paroxetine in a rat model of neuropathic pain.

Paroxetine increases the levels of neurosteroids, such as allopregnanolone (AP), that influence the excitability of the central nervous system by positive allosteric modulation of γ-aminobutyric acid type A receptors. Here, we investigated the role of AP synthesis on the paroxetine-induced antihyperalgesic effect in a rat model of neuropathic pain induced by lumbar spinal nerve ligation (SNL). Subcutaneous administration of paroxetine in SNL rats, dose-dependently decreased the probability of hyperalgesic response and increased AP levels in the spine but not in either brain or serum. Concomitant treatment with an inhibitor of the AP-synthesizing enzyme, finasteride, attenuated the paroxetine-induced antihyperalgesic effect as well as the paroxetine-induced increase in spinal AP levels. Intrathecal injection of exogenous AP mimicked the analgesic effects of paroxetine in vehicle-treated SNL rats, whereas no additional analgesic effects were observed in paroxetine-treated SNL rats. Our findings suggest that the antihyperalgesic effect of paroxetine in a rat neuropathic pain model is AP-mediated. These results also suggest that pharmacological-based therapies targeting AP synthesis might be a promising treatment for neuropathic pain.

Role of the neurosteroid allopregnanolone in the hyperalgesic behavior induced by painful nerve injury in rats.

The neurosteroid allopregnanolone (AP) influences the excitability of the central nervous system by acting as a positive allosteric modulator of γ-aminobutyric acid type A (GABA(A)) receptors. Here, we investigated the role of AP and its therapeutic potential in rats that showed hyperalgesic behavior after undergoing spinal nerve ligation (SNL). AP levels measured in the spinal cord and brain of rats that underwent SNL were greater than the corresponding levels in control animals. More importantly, spinal AP levels in hyperalgesic rats were lower than those in the rats that did not develop hyperalgesia following SNL; in contrast, brain AP levels were comparable among these groups. No differences in serum AP levels were observed among the groups. In addition, intrathecal exogenous administration of AP showed the antihyperalgesic effects in hyperalgesic rats after SNL. These findings suggest that changes in spinal AP biosynthesis are involved in the pathogenesis of neuropathic pain following peripheral nerve injury, and pharmacological manipulation of this phenomenon may provide a potential therapeutic target for neuropathic pain.

Lapatinib plus capecitabine in treating HER2-positive advanced breast cancer: efficacy, safety, and biomarker results from Chinese patients.

Overexpression of human epidermal growth factor receptor-2 (HER2) in metastatic breast cancer (MBC) is associated with poor prognosis. This single-arm open-label trial (EGF109491; NCT00508274) was designed to confirm the efficacy and safety of lapatinib in combination with capecitabine in 52 heavily pretreated Chinese patients with HER2-positive MBC. The primary endpoint was clinical benefit rate (CBR). Secondary endpoints included progression-free survival (PFS), time to response (TTR), duration of response (DoR), central nervous system (CNS) as first site of relapse, and safety. The results showed that there were 23 patients with partial responses and 7 patients with stable disease, resulting in a CBR of 57.7%. The median PFS was 6.34 months (95% confidence interval, 4.93-9.82 months). The median TTR and DoR were 4.07 months (range, 0.03-14.78 months) and 6.93 months (range, 1.45-9.72 months), respectively. Thirteen (25.0%) patients had new lesions as disease progression. Among them, 2 (3.8%) patients had CNS disease reported as the first relapse. The most common toxicities were palmar-plantar erythrodysesthesia (59.6%), diarrhea (48.1%), rash (48.1%), hyperbilirubinemia (34.6%), and fatigue (30.8%). Exploratory analyses of oncogenic mutations of PIK3CA suggested that of 38 patients providing a tumor sample, baseline PIK3CA mutation status was not associated with CBR (P = 0.639) or PFS (P = 0.989). These data confirm that the lapatinib plus capecitabine combination is an effective and well-tolerated treatment option for Chinese women with heavily pretreated MBC, irrespective of PIK3CA status.

Effects of aging on isoflurane-induced and protein kinase A-mediated activation of ATP-sensitive potassium channels in cultured rat aortic vascular smooth muscle cells.

Isoflurane activates protein kinase A (PKA) in vascular smooth muscle cells (VSMCs), which in turn activates ATP-sensitive potassium (K(ATP)) channels and causes vasodilation. The present study was undertaken to examine whether advanced age influences the effect of isoflurane on K(ATP) channel activity in cultured VSMCs. We used VSMCs obtained from 12- to 15-week-old (adult) and 24- to 25-month-old (aged) male Wistar rats. Electrophysiological experiments were performed using cell-attached and inside-out patch-clamp techniques to monitor the K(ATP) channel activity. Application of isoflurane or forskolin to the bath solution in cell-attached recordings induced a significant increase in K(ATP) channel activity in the VSMCs from the adult group. However, K(ATP) channel opening induced by isoflurane, but not forskolin, was significantly suppressed by aging. On the other hand, cell-free recordings showed similar pharmacologic sensitivity to the K(ATP) channel opener pinacidil, inward rectification, and unitary conductance (40­45 pS) between groups. In addition, direct K(ATP) channel activation by c-PKA in the inside-out patches was similar in both groups. Furthermore, increasing PKA activation in cell-attached patches by CPT-cAMP restored isoflurane's effects in the aged group. These results suggest that aging decreases isoflurane-induced PKA activation, resulting in attenuation of K(ATP) channel opening.

Effects of sulfur amino acids on tyrosyl or serine/threonine phosphorylation and translocation of cytosolic compounds to cell membrane in stimulus-treated human neutrophils.

We investigated the effects of various sulfur amino acids on the phosphorylation of proteins and the translocation of cytosolic compounds to cell membrane in stimulus-treated human neutrophils using specific monoclonal antibodies. D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced tyrosyl phosphorylation of proteins and the translocation of p47phox, p67phox, and rac to the cell membrane in a concentration-dependent manner. L-cystathionine, NAc-L-cysteine and carboxymethylcysteine suppressed the tyrosyl phophorylation and translocation of cytosolic compounds to the cell membrane. L-cystathionine, L-cysteine and NAc-L-cysteine suppressed PMA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane. L-cysteine, NAc-L-cysteine and D,L-homocysteine enhanced AA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane, but L-cystathionine had opposite effects. These results indicated that the effects of sulfur amino acids on tyrosyl or serine/threonine phosphorylation and the translocation of p47phox, p67phox, and rac to the cell membrane in the stimulus-treated human neutrophils were in parallel with those of the stimulus-induced superoxide generation reported in previous paper. L-cysteine, D,L-homocysteine and L-cystathionine weakly inhibited lipid peroxidation, but the other sulfur amino acids tested had no effect.

Activity of prolidase isoenzymes in the rat brain: subcellular and regional distribution during development.

Prolidase deficiency is characterized by chronic ulcerative dermatitis, mental retardation, and frequent infections. In the present study we examined the characteristics of rat brain prolidase isoenzymes. Prolidase isoenzymes (PD I and PD II) were isolated from the rat brain using DEAE cellulose column chromatography. PD I showed higher activity against seryl-proline and alanyl-proline, while PD II was particularly active against methionyl-proline. Prolidase activity in the whole brain and in the different brain regions showed higher activity against methionyl-proline and seryl-proline. PD II activity was highest in the hippocampus, followed by the cerebellum, cerebral cortex, caudatum, and the midbrain. The most rapid changes in the activities of PD I and PD II occurred perinatally, with a peak at three days before birth and a nadir at two days after birth, which then gradually increased until 21 days. N-benzyloxycarbonyl-l-proline inhibited PD I activity against various substrates in a dose-dependent manner. In contrast, there was no inhibition of PD II activity against methionyl-proline at low concentrations. In summary, these data suggest that maintenance of levels of proline, other amino acids and peptides containing proline in the rat brain is regulated by prolidase isoenzymes. The age-related alterations in PD I and PD II also may help to elucidate the fundation of prolidase isoenzymes in brain nervous system.

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency.

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by D: -methionine. L: -Methionine and D: ,L: -methionine slightly enhanced the activity at low concentration, but N-acetyl-L: -methionine had no effect. D: -Ethionine, L: -ethionine, and D: ,L: -ethionine also enhanced the activity of prolidase I. D: ,L: -Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM: . The activity of prolidase II against methionylproline was enhanced by D: -methionine, D: ,L: -methionine, and L: -methionine, but N-acetyl-L: -methionine had no effect. D: -Ethionine and D: ,L: -ethionine strongly enhanced the activity of prolidase II compared with L: -ethionine; D: ,L: -homocysteine weakly enhanced the activity. D: ,L: -Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K (m) values were changed by adding sulfur-containing amino acids, but V (max) values were unchanged.

Effect of seven tricyclic diterpenoids from needles of Taxus media var. Hicksii on stimulus-induced superoxide generation, tyrosyl or serine/threonine phosphorylation and translocation of cytosolic compounds to the cell membrane in human neutrophils.

Taxol has been widely used as an anticancer drug for ovarian, breast, lung and prostate cancer. Some kinds of Taxus plants are widely distributed in the Northeast Asia region. We have isolated seven tricyclic diterpenoids, taxinine, taxagifine, 5-O-cinnamoyltaxacin I triacetate, 5-decinnamoyltaxinine J, 5-cinnamoyl-9-acetyltaxicin I, taxacin and taxol from the needles of Taxus media var. Hicksii, and investigated their effects on stimulus-induced superoxide generation and translocation of cytosolic compounds to the cell membrane in human neutrophils. Six tricyclic diterpenoids used in this experiment suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and arachidonic acid (AA) in a concentration-dependent manner. Taxinine significantly suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA). The compounds also suppressed fMLP- and AA-induced tyrosyl or PMA-induced serine/threonine phosphorylation, and translocation of cytosolic compounds, p47 (phox), p67 (phox) and Rac to the cell membrane in parallel with the suppression of the stimulus-induced superoxide generation.

Antiperoxidation activity of triterpenoids from rhizome of Anemone raddeana.

Four triterpenoid compounds hederacolchiside E (1), hederasaponin B (2), raddeanoside 20 (3) and raddeanoside 21 (4) were isolated from ethanol extracts of rhizome of Anemone raddeana Regel. The effects of these triterpenoids on superoxide generation, tyrosyl phosphorylation of proteins and translocation of cytosolic compounds, such as p47(phox), p67(phox) and Rac to the cell membrane in human neutrophils was investigated. The superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was slightly suppressed by hederasaponin B, raddeanoside 20 and raddeanoside 21 in a concentration dependent manner. The superoxide generation induced by arachidonic acid (AA) was suppressed by hederasaponin B and raddeanoside 21 significantly. fMLP- and AA-induced tyrosyl phosphorylation and translocation of the cytosolic proteins: p47(phox), p67(phox), and Rac to the cell membrane were suppressed in parallel with the suppression of stimulus-induced superoxide generation.

Combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model.

The essential role of multispecific immune responses for the control of hepatitis B virus (HBV) infection implies the need of multimodal therapeutic strategies for chronic HBV infection, including antiviral chemotherapy and immunomodulation. This hypothesis was tested in the woodchuck model by a combination of lamivudine pretreatment and subsequent immunizations of woodchucks chronically infected with woodchuck hepatitis virus. The immunizations were performed with DNA vaccines or antigen-antibody immune complexes (IC)/DNA vaccines. Immunizations with IC/DNA vaccines led to an anti-woodchuck hepatitis virus surface antibody response and significant reductions of viral load and antigenemia, suggesting that such a strategy may be effective against chronic HBV infection.