Phase II study of irinotecan, leucovorin, 5-fluorouracil and tegafur/uracil for metastatic colorectal cancer.

Irinotecan combined with continuous-infusion 5-fluorouracil (5FU) has been shown to be an effective and tolerable regimen in the treatment of metastatic colorectal cancer (MCRC). Tegafur/uracil (UFT) during 5FU infusion enhances plasma 5FU concentration, mimics continuous 5FU infusion and delivers the drug to target tumor cells. We conducted a phase II trial of four-agent combined therapy for MCRC, giving patients (pts) intravenous irinotecan (30 mg/m2 on day 1), leucovorin (LV, 200 mg/m2 on day 1 and 2), 5FU (300 mg/m2 on day 1 and 2), and UFT (400 mg/day for 14 days). The main endpoint was the objective tumor response rate. Sixteen pts with a good performance status were enrolled from February 2001 to May 2002. The response rate was 19% (3 partial responses), and 13 pts had stable disease. The median time to progression was 5.2 months, and the median survival time was 20.2 months. Considering the low toxicity and reasonable cost, this regimen deserves further investigation.

Tea catechins prevent the development of atherosclerosis in apoprotein E-deficient mice.

Green tea contains various antioxidative flavan-3ols (tea catechins), such as (-)-epigallocatechin gallate (EGCg, the major catechin), which exert potent inhibitory effects on LDL oxidation in vitro and ex vivo in humans. In this study, the antiatherogenic effects of tea catechins were examined in atherosclerosis-susceptible C57BL/6J, apoprotein (apo)E-deficient mice. Male apoE-deficient mice (10 wk old) were fed an atherogenic diet for 14 wk; during that time, one group (tea) was supplied drinking water supplemented with green tea extract (0.8 g/L), and another group (control) was offered the vehicle only. The tea extract consisted of the following (g/100 g): EGCg, 58.4; (-)-epigallocatechin (EGC), 11.7; (-)-epicatechin (EC), 6.6; (-)-gallocatechingallate (GCg), 1.6; (-)-epicatechin gallate (ECg), 0.5; and caffeine, 0.4. The estimated actual intake of tea catechin was 1.7 mg/(d. mouse). Tea ingestion did not influence plasma cholesterol or triglyceride concentrations. Plasma lipid peroxides were reduced in the tea group at wk 8, suggesting that the in vivo oxidative state is improved by tea ingestion. Atheromatous areas in the aorta from the arch to the femoral bifurcation and aortic weights were both significantly attenuated by 23% in the tea group compared with the control group. Aortic cholesterol and triglyceride contents were 27 and 50% lower, respectively, in the tea group than in the control group. These results suggest that chronic ingestion of tea extract prevents the development of atherosclerosis without changing the plasma lipid level in apoE-deficient mice, probably through the potent antioxidative activity of the tea.

Differential interaction of tubulin isotypes with the antimitotic compound IKP-104.

The tubulin molecule is a heterodimer composed of two polypeptide chains, designated alpha and beta; both alpha and beta exist in numerous isotypic forms, which differ in their assembly and drug binding properties. 2-(4-Fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP-104) is an antimitotic compound which inhibits polymerization and induces depolymerization of microtubules [Mizuhashi, F., et al. (1992) Jpn. J. Cancer Res. 83, 211]. Since the previous work was undertaken with isotypically unfractionated tubulin, we have investigated the interactions of IKP-104 with the isotypically purified tubulin dimers (alpha beta(II), alpha beta(III), and alpha beta(IV)). We find that IKP-104 binds to alpha beta(II) and alpha beta(III) at two classes of binding sites. However, affinities for each class of site are much weaker for alpha beta(III) than for alpha beta(II). Interestingly, the low-affinity site on alpha beta(IV) was not detectable. Its high-affinity site was weaker than those of either alpha beta(II) or alpha beta(III). In a pattern consistent with these results, IKP-104 inhibited assembly better with alpha beta(II) than with the other two dimers. Higher concentrations of IKP-104 induced formation of spiral aggregates from alpha beta(II) and alpha beta(III) but not from alpha beta(IV). Our results suggest that the interaction of IKP-104 with tubulin isotypes is very complex: alpha beta(II) and alpha beta(III) differ quantitatively in their interaction with IKP-104, and alpha beta(IV)'s interaction differs both quantitatively and qualitatively from those of the other two dimers.

Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans.

Oxidation of low density lipoprotein (LDL) plays crucial roles in atherogenesis. We previously reported that green tea polyphenols (flavan 3-ols), especially epigallocatechingallate (EGCg) and epicatechingallate, exerted potent inhibitory effects on LDL oxidation in vitro. To examine whether intake of green tea polyphenols renders LDL resistant to ex vivo oxidation in humans, 22 male volunteers aged between 22 and 32 years were recruited and assigned the same dietary regimen for 2 weeks. After a 1-week baseline period, they were equally divided into two groups: control and tea. The tea group ingested 300 mg of green tea polyphenol extract twice daily for 1 week. Plasma EGCg concentration at the end of the experiment was 56 nmol/L on average (56% in free form) in the tea group; no EGCg was detected before the experiment. Plasma concentrations of lipids, ascorbate, alpha-tocopherol, and lipid peroxides did not change before and after the experiment in either group, but beta-carotene was higher in the tea group (P< 0.01 by paired Student'st-test). LDL (0.1 mg/mL) was incubated with 5 microM Cu(2+) and the oxidation was measured by absorbance at 234 nm. The lag time was significantly prolonged by 13.7 min in the tea group (P < 0.05 by paired Student'st-test, before versus after), whereas such a change was not observed in the control group. These results suggest that daily consumption of seven to eight cups (approximately 100 mL each cup) of green tea may increase resistance of LDL to in vivo oxidation, leading to reduction in the risk of cardiovascular diseases.

[Role of national insurance pharmacies in community complete home care networks--home TPN and home care supported by local national insurance pharmacies].

The reforms in the medical system and introduction of home care insurance have brought great changes to national health insurance pharmacies. In April 1998, Dr. Hirai became new director of the Chiba Togane Hospital. The development of a community complete medical system was included in a restructuring of the hospital, and various reforms were begun. A system covering all aspects of the medical/pharmaceutical field was started in August 1998. For its part, the Sanbu-gun Pharmacists Association began accepting prescriptions outside the hospital, and regular meetings for the exchange of knowledge were held with members of the physicians and pharmacists associations. After building a relationship of trust in this way, a community complete home treatment system was begun in July 1999 with Togane Hospital functioning as its backup support hospital. To date, home TPN terminal care has been provided in cases of terminal cancer, incurable neurological diseases, and for the very aged and patients with cerebrovascular impairments. Any general pharmacy in the region can participate in the program to fill prescriptions for TPN, provided that the pharmacist him or herself so wishes, establishes a clean bench at the pharmacy, and undergoes training at Togane Hospital on the preparation of i.v. medicines in order to function responsibly in this capacity. These pharmacies are called satellite pharmacies, and at present there are four of them located with a good balance within the region. These satellite pharmacies prepare liquid medications for TPN, including narcotics, and oral medicines following the prescriptions written by the physician from the hospital who is serving as the primary home treatment physician. The pharmacy also delivers the medicines to the home of the patient. The pharmacist checks the status of remaining TPN liquids and oral medicines and informs the primary home physician and support hospital by e-mail or fax, so that everyone shares the same information. The success or failure of home TPN from a general pharmacy depends on the formation of a digital network in order to share information using the Internet and a back-up system for unconditional support by the support hospital in times of patient emergency. In our region, these conditions have been fulfilled.

Lewy body-type degeneration in cardiac plexus in Parkinson's and incidental Lewy body diseases.

Heart tissues of patients with PD or incidental Lewy body (LB) disease (ILBD) were examined by light and electron microscopy. LBs and alpha-synuclein-positive neurites were identified in the hearts from 9 of 11 patients with PD and from 7 of 7 patients with ILBD. LBs were present in both tyrosine hydroxylase-positive and -negative nerve processes, which are nerves of extrinsic sympathetic and intrinsic origin, respectively. These findings provide histologic evidence that the postganglionic sympathetic and intrinsic neurons in the heart are involved in the PD disease process.

Increases in 4-hydroxynonenal and hexanal in bone marrow of rats subjected to total body X-ray irradiation: association with antioxidant vitamins.

Radiation-induced lipid peroxidation and its association with antioxidant vitamins in the bone marrow (BM), of rats subjected to total body irradiation (TBI) of X-rays at a dose of 3 Gy was investigated. The concentration of vitamin C in the BM decreased at 4 h, and reached about 2% of the control level at 24 h after irradiation. The concentration of vitamin E in the BM also decreased to 43% at 24 h. Corresponding to the decrease in vitamin E concentration, the concentration of 4-hydroxynonenal (HNE) in the BM increased 2.5-fold at 24 h. Similarly, increases in the concentrations of hexanal and thiobarbituric acid-reactive substances (TBA-RS) were detected in the BM. In the plasma, these parameters of lipid peroxidation were unchanged up to 48 h, but were increased at 96 h after irradiation. Four days of vitamin E administration to rats (p.o. 460 mg/kg body weight) prior to the 3 Gy X-irradiation increased the vitamin E concentration in the BM to 1.3-fold the control level, but did not attenuate the increases in HNE and hexanal in the BM. The slight accumulation of vitamin E in the BM as a result of the vitamin E treatment may be partly related to this lack of vitamin E effect.

Loss of 123I-MIBG uptake by the heart in Parkinson's disease: assessment of cardiac sympathetic denervation and diagnostic value.

UNLABELLED: Myocardial imaging with 123I-metaiodobenzylguanidine (MIBG) was performed on 35 patients with Parkinson's disease and 24 control subjects to evaluate cardiac sympathetic function in patients with Parkinson's disease, verify this phenomenon and examine whether myocardial MIBG uptake and clearance are correlated with the clinical severity of Parkinson's disease. METHODS: We studied 35 patients with Parkinson's disease and 24 control subjects with other central nervous system diseases. The latter group consisted of 12 subjects with other neurodegenerative disorders (4 with spinocerebellar degeneration, 2 with amyotrophic lateral sclerosis, 3 with progressive supranuclear palsy and 3 with corticobasal degeneration and 12 patients with cerebral infarction (CI), 6 with vascular parkinsonism and 6 without it. Early and delayed images of the anterior view were obtained 15 min and 4 h after injection of 123I-MIBG, respectively. MIBG uptake was quantified by calculating a heart-to-mediastinum count (H/M) ratio. RESULTS: The H/M ratio was markedly reduced in the patients with Parkinson's disease (II to V on the Hoehn and Yahr scale) compared with the control subjects. None of the subjects with neurodegenerative diseases showed a marked decrease in myocardial MIBG uptake nor did any subject with CI. CONCLUSION: Our findings indicate that, in Parkinson's disease, a reduction in myocardial MIBG uptake is a very common, specific phenomenon that can be used to detect cardiac autonomic dysfunction to diagnose Parkinson's disease, particularly in patients without typical signs and symptoms.

Effects of phosphatidylcholine/phosphatidylethanolamine composition in cholesteryl ester-micellar substrates on neutral cholesterol esterase activity.

The effect of phospholipid composition in cholesteryl ester (CE)-micellar substrates on neutral cholesterol esterase (N-CEase) activity was examined. N-CEase preparation was incubated with micelles composed of cholesteryl-[1-14C]-oleate, sodium taurocholate, and phosphatidylcholine (PC)/phosphatidylethanolamine (PE) at varying ratios (%PE:0 = PC only, 17, 33, 50, 66, 83). The activity increased dependently with the increase in PE content; the activity with the micelles containing the highest ratio of PE was 2.5-fold compared with the micelles consisting of PC only. Vmax with the micelles of 83, 66, and 50% PE was 3.1-, 2.7-, and 1.9-fold, respectively, compared with the micelles of PC only. Each micellar preparation was chromatographed through a Superose 6 column by the FPLC system. In 66 and 83% PE-containing micelles, PC, PE, CE, and part of sodium taurocholate eluted completely together in a single peak, whereas in micelles with 33 and 50% PE they eluted loosely together. The micelles with PC only or 17% PE formed PC-micelles without including CE and PE. It is concluded that PE plays a critical role in the formation of CE micelles with PC, and in the interaction with N-CEase. The CE-micelles with 66-83% PE serve as substrates for sensitive and reproducible N-CEase assay.

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay.

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)- -(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104's acceleration of decay. We found that podophyllotoxin abolished IKP104's accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole's effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin.

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuri et al., 1998, J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.

IKP104-induced decay of tubulin: role of the A-ring binding site of colchicine.

Tubulin, the major subunit protein of microtubules, has a tendency to lose its ability to assemble or to interact with ligands in a time-dependent process known as decay. Decay involves the increase in exposure of sulfhydryl groups and hydrophobic areas. The antimitotic drug IKP104 [2-(4-fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone] accelerates the decay of tubulin [Ludueña et al. (1995) Biochemistry 34, 15751-15759]. In the presence of colchicine, however, IKP104 stabilizes tubulin against decay. We have shown that the stability and the acceleration of the decay of tubulin are mediated respectively by the high- and low-affinity binding site(s) of IKP104 [Chaudhuri et al. (1998) J. Protein Chem. 17, 303-309]. To better understand the mechanism by which colchicine protects tubulin from IKP104-induced decay, we examined the effect of colchicine and its analogues on this process. We found that IKP104 unfolds tubulin in a process involving a specific domain where colchicine interacts, although the binding sites of these two drugs are distinctly different. 2-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropolone (MTPT), the bicyclic analogue of colchicine that lacks the B-ring, can also protect tubulin from IKP104-induced decay. An A-ring analogue of colchicine, 3,4,5-trimethoxybenzaldehyde (TMB), can also stop IKP104-induced unfolding of tubulin significantly. Interestingly, the C-ring analogue of colchicine, tropolone methyl ether (TME), does not prevent this process. Our results thus suggest that neither the B-ring nor the C-ring binding regions of colchicine are involved in the IKP104-induced decay and that the A-ring binding site of colchicine on tubulin plays a crucial role in IKP104-induced decay.

[Studies on antioxidant effects of Hypsizigus marmoreus. II. Effects of Hypsizigus marmoreus for antioxidant activities of tumor-bearing mice].

The in vivo effect of Hypsizigus marmoreus on the plasma antioxidant status of tumor-bearing mice was examined. Female DBA/2 mice treated with subcutaneous injection of 3-methylcholanthrene were fed on a basal diet (CE-2) or CE-2 containing 5% fruit bodies of the mushroom for 76 weeks. Antioxidant activities (AOA) of mice with tumor were significantly higher than those of mice without tumor. The high levels of AOA were attributable to the increase of high molecular weight AOA in the plasma. A similar increase in plasma AOA was also observed in sarcoma-180 solid tumor-bearing mice. The mushroom feeding exhibited a potent antitumor effect and caused a significant decrease in lipid peroxide levels (as thiobarbituric acid reactive substances; TBARS). These results suggest that the increase of AOA in the plasma of tumor-bearing mice is one of the mechanism of cancer preventive effects and also suggest that Hypsizigus marmoreus might play a role in decreasing TBARS by controlling AOA induction.

[Intra-arterial preventive chemotherapy for residual liver after resection of hepatic metastasis from colorectal cancer].

We treated 18 cases with intra-hepatic arterial infusion chemotherapy after resection of hepatic metastasis from colorectal cancer (June 1991-September 1997). Eight cases were H1, 7 were H2, and 3 were H3. Hepatic lobectomy was done in 3 cases, lobectomy + partial resection in 2 cases, and partial resection in 13 cases. All cases received high-dose intermittent 5-FU infusion (WHF = 5-FU 1,000 mg/m2/5 hrs/w) on an outpatient basis. The total frequency of WHF was 4-54 times (average 29), and total 5-FU doses ranged from 6.0 to 81.0 g (average 40 g). The 1- and 5-year cumulative survival rates were 100% and 77.5% in all patients 100% and 87.5% in H1 group and 100% and 64.3% in H2 + H3 group, respectively. There was no significant difference of survival between the H1 and H1 + H3 groups. The 1- and 5-year recurrence rates in residual liver were 5.9% and 14.4%, respectively. One of 2 cases with residual liver recurrence was resected for metastasis again, and the patient is now in a disease-free state. WHF after resection of hepatic metastasis from colorectal cancer has a preventive effect for their survival, not only in H1 group but also in H2 + H3 group.

Tubulin stability and decay: mediation by two distinct classes of IKP104-binding sites.

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751-15759]. Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both alpha and beta subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the alpha and beta subunits of alphabeta tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl titer or the exposure of hydrophobic areas of this subtilisin-treated tubulin (alpha(s)beta(s)). Moreover, IKP104 lowers the sulfhydryl titer of alpha(s)beta(s) tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of alpha(s)beta(s) tubulin. The cleavage at the C-terminal ends of both alpha and beta modulates the effect of IKP104 on the beta subunit, but not on the alpha subunit. Fluorometric binding data analysis suggests that IKP104 binds to the alpha(s)beta(s) tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for alpha and beta and the fluorometric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of alpha and beta and the effect of the high-affinity site is restricted largely to the alpha subunit, while the low-affinity-site binding is modulated by the C-terminal domain of beta. It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.

Modification of enzyme sulfhydryl groups suppresses UV-induced mutagenesis depending on the nucleotide excision repair system in Escherichia coli B/r WP2.

S-Methyl methanethiosulfonate (MMTS), which was isolated from cauliflower (Brassica oleracea var. botrytis) homogenate as a potent bio-antimutagen, has been used as an enzyme-sulfhydryl (SH) temporary blocking agent in modification studies of enzyme activities. We examined whether 23 kinds of MMTS-related compounds have a suppressing effect on UV mutagenesis in Escherichia coli B/r WP2. Disulfide derivatives of diphenyl, 2.2'-dipyridine and 4.4'-dipyridine, and N-ethyl maleimide (NEM), which temporarily or tightly block sulfhydryl (SH)-groups, showed similar suppressing effect in E. coli B/r WP2, but not in WP2s hcr- (uvrA-) in the range of nanomolar/plate as MMTS previously did. Cystamine sulfate, methyl methylsulfinylmethyl sulfide and S-methyl-L-cysteinesulfoxide moderately suppressed, and diallyl disulfide and glutathione (oxidized form) weakly suppressed UV mutagenesis in E. coli B/r WP2 in the range of micromolar/plate. MMTS and phorone, a glutathione (GSH)-depleting agent, lowered the intracellular GSH level in E. coli B/r WP2, but phorone did not inhibit UV-induced mutation. These results indicate that the target for SH-modification is enzyme-SHs but not GSH, and that the direct or indirect modification of enzyme-activity by SH-blocking might be involved in the antimutagenesis through a pathway associated with the DNA-excision repair system.