A phase I/II trial of cisplatin, docetaxel and ifosfamide in advanced or recurrent non-small cell lung cancer.

This trial was initiated to evaluate the toxicity and activity of combination chemotherapy employing cisplatin (CDDP), docetaxel (DCT) and ifosfamide (IFX) in non-small cell lung cancer (NSCLC), and to determine the maximum tolerated dose (MTD) of IFX. Chemotherapy-naive patients with advanced or recurrent NSCLC received 60 mg/m(2) DCT followed after a 3-h interval by 60 mg/m(2) CDDP on chemotherapy day 1, and IFX at an escalating dose with mesna protection on days 2-4. The chemotherapy was repeated every 3 weeks. Granulocyte colony-stimulating factor (GCSF) was administered in the event of grade 3 leukopenia/neutropenia. The patients tolerated the treatment well up to level 4 of IFX dosing 1.5 g/day, but not the IFX dose at level 6 (2.0 g/day). Additional patients were enrolled in level 5 (IFX 1.7 g/day) to evaluate the toxicity of the drugs around the MTD. Level 5 was also judged as having exceeded the MTD, with febrile neutropenia and hepatic toxicity being observed as the dose-limiting toxicities. No toxicity-related deaths occurred. The majority of the chemotherapy courses were supported by GCSF administration. A total of 33 eligible patients were entered into the trial; the overall response rate was 10/33 or 30% among all eligible cases, and the rate for patients treated with the MTD or higher (levels 4-6) was 8/24, or 33% (90% confidence limit: 18-52%). The MTD of IFX was 1.5 g/m(2) administered for 3 days in this triplet combination. The clinical activity does not seem to justify the toxicity profile.

Deposition of oxidized low-density lipoprotein and collagenosis occur coincidentally in human coronary stenosis: an immunohistochemical study of atherectomy.

BACKGROUND: Coronary stenosis involves lipid accumulation, fibrosis and cell proliferation. OBJECTIVE: To clarify the role of oxidized low-density lipoprotein (LDL) in coronary stenosis by examining atherectomized coronary lesions from patients with primary stenosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). METHODS: Atherectomized coronary tissue from 28 patients with primary stenosis and restenosis at 4.3 +/- 1.0 months after PTCA were examined using morphometrical and immunohistochemical techniques. RESULTS: Serum lipids in all of the patients were within the normal range and no differences were noted between the two groups. There were no differences in the mean cross-sectional areas of whole specimens obtained from each group, and sclerotic lesions with atheroma or calcification were found to a similar extent in both groups. However, the restenosis group had a significantly greater area (6-fold) of immature smooth-muscle-rich lesions than the primary stenosis group, although there was no difference in lipid-laden foam-cell containing lesions. In foam-cell-containing lesions, apolipoprotein B was accumulated extracellularly, while oxidized LDL was primarily deposited intracellularly in lipid-laden foam cells. However, no deposition of apolipoprotein B, oxidized LDL or lipids was noted in smooth-muscle-rich lesions. Proline hydroxylase, a key enzyme for collagen synthesis, was detected in most of the foam-cell-containing lesions, but not in smooth-muscle-rich lesions. CONCLUSIONS: Atherectomized lesions from patients with coronary stenosis contained smooth-muscle-rich lesions in restenosis and lipid-laden cellular lesions in both stenosis and restenosis, in which the deposition of oxidized LDL and increased collagen synthesis occur coincidentally. Therefore, the mechanism of atherogenesis may involve coronary stenosis regardless of the occurrence of restenosis after PTCA therapy.

Deleterious effect of short-term exposure to Coca-Cola on rats.

Semiacute toxicity of cola fluid, Coca-Cola, conducting for approximately a month in rats, was studied. A trend of cariogenicity of Coca-Cola was strongly indicated. When the fluid was given ad libitum, decarbonized Coca-Cola and carbohydrate solution, consisted 8% of glucose and 3.5% of sugar and then adjusted pH to 2.4 with oxalic acid, were consumed 2 to 3 times greater than the control (water). A hyperuresis was observed as the result of great consumption of liquid, but no liver nor kidney degeneration was observed by histopathological examination. The diet consumption of the groups of Coca-Cola and carbohydrate solution was approximately a half of the control, water. However, when a complete diet was given, no physiological difference in time was observed, except in diarrhea and depression of hair gloss in Coca-Cola group.

Innervation pattern of substance P- and calcitonin gene-related peptide-immunoreactive nerves of the cerebral arteries in the quail.

The pattern of cerebrovascular substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) innervation was investigated in the quail. SP- and CGRP-IR nerves were relatively a few in the rostral part of the anterior circulation, and very scanty or lacking in its caudal part and the whole of the posterior circulation. A significant finding was that the anterior circulation in the majority of individuals is furnished with a varying proportion of SP-IR nerves with or without CGRP immunoreactivity. There was a good correlation in the expression of CGRP immunoreactivity between SP-IR cells in the ophthalmic division of the trigeminal ganglion and SP-IR nerves supplying the major cerebral arteries. In the quail, SP- and CGRP-IR fiber bundles are usually present in the internal ethmoidal artery (IEA). From these and other findings, it is most probable that cerebral perivascular SP- and CGRP-IR nerves are mainly derived from the same categories of neurons in the primary sensory ganglion via the IEA. The close association of varicose SP-IR axons to the nerve cells in the pial arteries suggests that these intrinsic neurons may play some vasocontrolling roles through the modulatory effect of their pericellular SP-IR axons.

[Phase I clinical trial design of anticancer agents--a Fibonacci and a modified Fibonacci sequence].

A Phase I clinical trial of an anticancer agent is the first evaluation in humans, and it is an important step in drug development. From the ethical point of view, the goal is to escalate to the maximum tolerated dose quickly, yet safely, to minimize the likelihood of treating patients at doses that are too low or high. It is expected that the contradictions between safety and efficacy in the Phase I clinical trials will be solved by developing methods. The modified Fibonacci sequence has been generally adopted for dose escalation, although it includes some problems. It is necessary to recognize that the method used for Phase I clinical trials for anticancer agents remains unsatisfactory, and that it is also necessary to develop more ethical and scientific methods.

[A summary report of response evaluation criteria in solid tumors (RECIST criteria)].

The World Health Organization definitions for objective tumor response published in the 1979 WHO Handbook have been the most commonly used criteria around the world. However, some problems developed with the use of WHO criteria. These issues led to various modifications to the WHO criteria, resulting in a situation in which response criteria were no longer comparable among research organizations or researchers. However, because the efficacy of novel therapies and drugs is estimated according to tumor regression, it is very important to unify the definitions of objective tumor response. A revised version of the WHO criteria, Response Evaluation Criteria in Solid Tumors (RECIST criteria), was published in 1999 to deal with these issues. However, some problems concerning the evaluation of non-cytotoxic drugs and unidimensional measurements remain in the new criteria.

[Problems and prospects for combined chemotherapy with 5-fluorouracil and low-dose cisplatin].

Recently, the efficacy of combined chemotherapy with 5-fluorouracil and low-dose cisplatin for advanced gastrointestinal cancer has been reported in Japan. However, this method has not become established, because the underlying logic is unclear and the quality of its clinical trials has been unsatisfactory. We suggest the mechanism for this depends not only on the action of CDDP as a modulator of 5-FU, but also on a mutual biochemical modulation in which 5-FU acts as a modulator of CDDP and enhances the effect of CDDP. We expect that numerous phase II studies and the accumulation of reliable data will lead to improvements.

[Interferon therapy after ablation of Kent bundle for a patient with chronic hepatitis type B complicated with WPW syndrome].

Cardiotoxicity of interferon-alpha or gamma, such as fatal arrhythmia and myocardial infarction, has been reported. Therefore cardiotoxicity of interferon should be seriously considered before administration for patients with a pre-existing heart disease. We treated a patient with chronic active hepatitis type B, coexisted with Wolff-Parkinson-White syndrome, who has had frequent attacks of paroxysmal atrial fibrillation. To prevent the occurrence of fatal arrhythmia with an interferon therapy in this patient, we performed radiofrequency catheter ablation of the Kent bundle. After the successful ablation, we could safely administered recombinant interferon alpha-2b for chronic hepatitis type B.

Low-cost catalysts for the control of indoor CO and PM emissions from solid fuel combustion.

Cu-Mn based mixed oxide type low-cost catalysts have been prepared in supported form using mesoporous Al(2)O(3), TiO(2) and ZrO(2) supports. These supports have been prepared by templating method using a natural biopolymer, chitosan. The synthesized catalysts have been characterized by XRD, BET-SA, SEM, O(2)-TPD and TG investigations. The catalytic activity for CO as well as PM oxidation was studied, in a view of their possible applications in the control of emissions from solid fuel combustion of rural cook-stoves. The trend observed for the catalytic activity of the synthesized catalysts for CO oxidation was ZrO(2)>TiO(2)>Al(2)O(3) while for PM oxidation it was observed to be TiO(2)>ZrO(2)>Al(2)O(3). The effect of CO(2), SO(2) and H(2)O on CO oxidation activity was also investigated, and despite partial deactivation, the catalysts show good CO oxidation activity. An effective regeneration treatment was attempted by heating the partially deactivated catalysts in presence of oxygen. Redox properties of TiO(2) and ZrO(2) and their structure appeared to be responsible for their promotional activity for CO and PM oxidation reactions. These unordered mesoporous materials could be useful for such reactions where mass transfer is more important than shape and size selectivity.

[Atherectomy in patients with arteriosclerosis obliterans: angiographic follow-up study of 186 lesions in 146 patients].

Atherectomy (ATE) is a new catheter-mediated technique for the removal of atheroma in patients with arteriosclerosis obliterans (ASO). ATE was performed using 7-10 Fr. Simpson Peripheral Athero Track catheters at 186 sites in 146 patients, whose lesions involved 49 common iliac arteries, 66 external iliac arteries, 68 femoral arteries and three other arteries. The initial success rate was 94.1%. The mean percent of the diameter stenosis was reduced from 79.0 +/- 1.2% (mean +/- SD) to 22.7 +/- 1.0%. There were two cases of perforation that required surgical treatment (1.1%). The complication rate was 4.4%. The 0.5-, 1-, 2- and 3-year patency rates were 87.6%, 79.6%, 62.5% and 62.5%, respectively. The rate of long-term patency in each segment of arterial lesions revealed that the patency rate in the common iliac artery was significantly higher than the rates in the external iliac artery (p < 0.05) and femoral artery (p < 0.01). The patency rates for long lesions (> or = 2.0 cm) and occluded lesions were significantly (p < 0.01) lower than those for short lesions (< 2.0 cm). Diabetic patients had a higher re-stenosis rate than nondiabetic patients (p < 0.05). In conclusion, ATE is an effective new method for the treatment of patients with ASO.

Association of 5' CpG demethylation and altered chromatin structure in the promoter region with transcriptional activation of the multidrug resistance 1 gene in human cancer cells.

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. However, the precise mechanism underlying such transcriptional activation of MDR1 remains unclear. The relation between methylation status of CpG sites in the MDR1 promoter region and transcriptional activation of MDR1 has now been investigated. The P-glycoprotein-overexpressing, multidrug-resistant KB/VJ300 and KB-C1 cells, which were established from human cancer KB3-1 cells, were examined; MDR1 is transcriptionally activated but not amplified in KB/VJ300 cells, whereas it is amplified in KB-C1 cells. Determination of the methylation status revealed that the MDR1 promoter region was hypomethylated in KB/VJ300 and KB-C1 cells, but hypermethylated in KB3-1 cells. Prior treatment of KB3-1 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in a 90-fold increase in the frequency of vincristine-resistance. Of three lines, KB/CdR-1, KB/CdR-2, and KB/CdR-3, established from KB3-1 cells after exposure to 5-aza-2'-deoxycytidine, MspI/HpaII sites in the MDR1 promoter region were hypomethylated in KB/CdR-1 and KB/CdR-2 cells, but not in KB/CdR-3 cells. MDR1 mRNA expression was detected in KB/CdR-1 and KB/CdR-2 cells, but not in KB/CdR-3 cells. The binding of YB-1 and Sp1, transcription factors implicated in MDR1 expression, in the MDR1 promoter was not affected by the methylation status of a neighboring CpG sites. The MDR1 promoter region in KB/VJ300 cells showed an increased sensitivity to DNase I compared with that in KB3-1 cells, suggesting an altered chromatin structure. The methylation status of the promoter region may plays an important role in MDR1 overexpression and in acquisition of the P-glycoprotein-mediated multidrug resistance phenotype.

Retrovirus insertion and transcriptional activation of the multidrug-resistance gene in leukemias treated by a chemotherapeutic agent in vivo.

To understand the molecular basis for multidrug-resistant (MDR) cancer cells in vivo, this study analyzed molecular changes of the mdr1a gene region in leukemia cells in mice during continuous treatment with vincristine. An inverse insertion of murine leukemia retrovirus (MuLV) into the 5'-flanking region of the mdr1a gene was found. This insertion was concomitantly accompanied by up-regulation of the mdr1a gene and the loss of chemosensitivity. Deletion of long-terminal repeat (LTR) sequences dramatically decreased the mdr1a promoter-driven reporter activity. The MuLV LTR insertion appears to exert its enhancer activity on mdr1a transcription during the appearance of MDR leukemia cells. Two mechanisms were postulated to explain the mdr1a gene activation by retrovirus insertion during in vivo chemotreatment: de novo insertion of MuLV induced by vincristine treatment and selection of a small fraction of pre-existing cells carrying MuLV insertion during vincristine treatment. No rearranged sequence was detected by polymerase chain reaction in parental cells. This result argued for the first mechanism. The randomly altered distribution of MuLV during repetitive chemotreatment might also be consistent with this hypothesis. On the other hand, the retrovirus insertion was detected at the same site of the mdr1a promoter region in 2 independent experiments, which suggests the second mechanism. It should be noted that in vivo chemotreatment using vincristine could generate the mdr1a-overexpressing cells through retrovirus insertion and the enhancer effect of the LTR.

Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells.

Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed.

A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs. A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones. The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes. Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others.

Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias.

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear. We recently described an inverse correlation between the methylation status of CpG sites at the promoter region and expression of the MDR1 gene in malignant cell lines. In this study, we expanded this analysis to 42 clinical AML samples. We adapted a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for gene expression and a quantitative PCR after digestion by Hpa II for methylation status of the MDR1 gene. We observed a statistically significant inverse correlation between methylation and MDR1 expression in clinical samples. The hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients.

Localization of 67 exons on a YAC contig spanning 1.5 Mb around the multidrug resistance gene region of human chromosome 7q21.1.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) was previously assembled across 1.5 Mb of the multidrug resistance (MDR) gene (PGY1 and PGY3) region of human chromosome 7q21.1. This region of the human genome has now been subjected to exon amplification to detect the presence of additional genes. Exon trapping was performed directly on the YACs. Sixty-seven gene fragments were isolated and characterized by sequence analysis and comparison with public databases. The localization of these exons in the 1.5-Mb region was determined by hybridization to YAC clones, and they were localized in 11 subregions of YAC contigs. The exon collection includes 21 exons that were identical to known cDNA sequences of PGY1, PGY3, sorcin (SRI), the cDNA similar to the delta subunit of the human amiloride-sensitive Na- channel (SCNED), and 4 cDNAs with unknown function; 43 exons that showed homology/similarity to known cDNA sequences of mouse DMP1, rat COT, mouse and human NADHD, human MDC, 3 cDNAs encoding possible membrane proteins, and 21 other cDNAs; and 3 exons that shared no homology/similarity with any sequence in public databases. The nucleotide sequences of all the PGY1 and PGY3 exons were identical to the corresponding cDNA sequences previously determined, and these exons were localized to the expected positions on the appropriate YAC clones. No other member of the MDR gene family thus appeared to be present in the 1.5-Mb region. The integrated physical and exon maps should prove valuable for both fine mapping and determination of a complete gene map of this segment of the genome.

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

A dose escalation study of paclitaxel and carboplatin in untreated Japanese patients with advanced non-small cell lung cancer.

BACKGROUND: The combination of paclitaxel (225 mg/m(2), 3-h infusion) and carboplatin (area under the curve 6) is widely used for non-small cell lung cancer in the USA. In Japan, however, the recommended dose for single-use paclitaxel in 3-h infusion is 210 mg/m(2) and the optimal dose of this agent in combination with carboplatin has not been established. This dose escalation study was designed to determine the maximum tolerated dose of paclitaxel in 3-h infusion plus carboplatin at a fixed dose of area under the curve 6 for Japanese patients with advanced, untreated non-small cell lung cancer. METHODS: Between October 1999 and May 2000, 19 patients were enrolled and 18 of these patients were evaluable for toxicity. Chemotherapy consisted of carboplatin area under the curve 6 and an escalated dose of paclitaxel on day 1 every 3-4 weeks. The initial dose of paclitaxel was 175 mg/m(2) and was increased by 25 mg/m(2) at each dose level. RESULTS: Neutropenia was the major toxicity observed, but was not dose related. Febrile neutropenia was not observed. No grade 3 or more peripheral neuropathy, myalgia or arthralgia was reported. The maximum tolerated dose was not determined even at the highest paclitaxel dose level (225 mg/m(2)) in this study. Partial responses were observed in six of the 19 patients (31.6%). CONCLUSION: We conclude that paclitaxel at 225 mg/m(2) in 3-h infusion and carboplatin area under the curve 6 can safely be given to Japanese patients with non-small cell lung cancer.

A phase I/II study of cisplatin and vinorelbine chemotherapy in patients with advanced non-small cell lung cancer.

BACKGROUND: A combination of cisplatin and vinorelbine chemotherapy is effective in cases of advanced non-small cell lung cancer, but the optimum administration schedule for both drugs has not yet been defined. The aim of this study was to determine the maximum dose of vinorelbine that can be tolerated while receiving a fixed dose of cisplatin every 3 weeks and to observe the response in Japanese patients with advanced non-small cell lung cancer who had not previously received chemotherapy. METHODS: Cisplatin was given at a dose of 80 mg/m2 on day 1. Vinorelbine was administered on days 1 and 8 at a starting dose of 25 mg/m2 that was then increased by 5 mg/m2 increments. This treatment was repeated every 3 weeks. RESULTS: Twenty-one patients received a total of 54 chemotherapy cycles consisting of three different vinorelbine dosages. Toxicity and efficacy were evaluated in all of the patients. The main dose-limiting toxicity was neutropenia. Grades 3-4 leukopenia and neutropenia were observed in 57% and 86% of all cycles, respectively. These conditions were reversible and did not result in death from toxicity. The most severe non-hematological toxicity symptom was a grade 3 infection and reaction at the site of injection. The maximum tolerated dose of vinorelbine was 35 mg/m2. The objective response was noted in one of six patients at dose level 1, in four of 12 patients at dose level 2 and in two of three patients at dose level 3. CONCLUSION: The recommended doses were 80 mg/m2 for cisplatin and 30 mg/m2 for vinorelbine. The combination of cisplatin and vinorelbine repeated every 3 weeks is well tolerated and has shown promising anti-tumor activity against non-small cell lung cancer.