Participation of elderly patients in registration trials for oncology drug applications in Japan.

BACKGROUND: The objective of this study was to evaluate the age-based enrollment of cancer patients into registration trials of new drug applications or expanding the indications for use. MATERIALS AND METHODS: The data from 234 registration trials in Japan and overseas of 43 drugs, which were reviewed by the Pharmaceuticals and Medical Devices Agency and approved by the Ministry of Health, Labour and Welfare in Japan between 1999 and 2008, were retrospectively analyzed according to the age distribution of enrolled patients. The age distribution of the Japanese cancer population was derived from Cancer Statistics in Japan 2003 and Annual Report on Health, Labour and Welfare 2003-2004. RESULTS: In the Japanese cancer population, the estimated median age of cancer patients is 70 years, and 66% of cancer patients are aged 65 years or more. The estimated median age of cancer patients in all registration trials conducted in Japan was 59 years, whereas it was 55 years in the registration trials conducted overseas. The proportion of patients aged 65 years or more enrolled in registration trials conducted in Japan was 35%; this number was 28% in registration trials conducted overseas. CONCLUSION: Elderly patients are underrepresented in oncology registration trials in Japan.

Antiviral effects of amino acid derivatives with the fluorene substituent on murine leukemia viruses.

Two new amino acid derivatives with the fluorene substituent, when administered ip to female inbred ICR-CD1 mice inoculated with Friend murine leukemia virus, significantly inhibited virus-induced splenomegaly, reduced viable virus titers in spleen and plasma, and significantly prolonged survival time. These compounds also inhibited multiplication of the strains of the Friend and Moloney murine leukemia viruses in a cell culture system. The action of these compounds on murine leukemia virus was presumely different from that of tilorone.

Antiviral effect of N-phenylacetoaminomethylene-DL-p-nitrophenylalanine (A-101) on murine leukemia viruses.

N-Phenylacetoaminomethylene-DL-p-nitrophenylalanine (A-101), when administered ip to male DDY mice infected with Friend leukemia virus, significantly inhibited virus-induced splenomegaly, reduced viable virus titers in spleen and plasma, and significantly prolonged survival time. A-101 also inhibited multiplication of the Friend and Moloney viruses in tissue culture systems.

Possible mechanism of inhibition of experimental pulmonary metastasis of mouse colon adenocarcinoma 26 sublines by a sialic acid: nucleoside conjugate.

As described previously (I. Kijima-Suda et al., Cancer Res., 46: 858-862, 1986), a sialyltransferase inhibitor, 5-fluoro-2',3'-isopropylidene-5'-O-(4-N-acetyl-2,4-dideoxy-3,6,7,8-tetra -O- acetyl-1-methoxycarbonyl-D-glycero-alpha-D-galactooctapyranosyl)ur idine (KI-8110), inhibits pulmonary metastasis of murine colon adenocarcinoma 26 sublines of high (NL-17) and low (NL-44) metastatic potential. To investigate the mechanism of this inhibition, the effect of KI-8110 on the metastatic cascade, especially on the interaction between tumor cells and platelets which may play a crucial role in tumor cell metastasis, was examined. NL-17 cells induced irreversible platelet aggregation in heparinized human platelet-rich plasma in vitro. This activity was reduced by pretreatment of the tumor cells with KI-8110. Inhibition of aggregation was also induced by the treatment of tumor cells with neuraminidase or Limax flavus agglutinin, a lectin specific for sialic acid. Sialic acid, fucose, sialyllactose, and bovine submaxillary mucin inhibited this tumor cell-induced platelet aggregation, while galactose, mannose, lactose, alpha 1-acid glycoprotein, fetuin, and asialo-bovine submaxillary mucin did not. KI-8110 also inhibited platelet-derived growth factor-dependent growth of NL-17 cells, but showed no effect on insulin or epidermal growth factor-dependent growth of the tumor cells. Platelet-derived growth factor-induced phosphorylation of membrane protein was reduced by treatment of NL-17 cells with KI-8110. The same result was obtained in the neuraminidase-treated membrane fraction of NL-17 cells. These results suggest that KI-8110 inhibits experimental tumor cell metastasis by inhibiting the interaction between tumor cells and host platelets in at least two pathways, and this may be due to a reduction of sialic acid contents of the membrane surface of tumor cells.

Inhibition of experimental pulmonary metastasis of mouse colon adenocarcinoma 26 sublines by a sialic acid:nucleoside conjugate having sialyltransferase inhibiting activity.

The total and sialidase-releasable sialic acid contents of murine colon adenocarcinoma 26 sublines of high (NL-17) and low (NL-44) metastatic potential were found to be positively correlated with their ability to undergo metastasis. Furthermore, sialyltransferase activity of intact NL-17 cells was higher than that of NL-44 cells. These findings suggest that sialic acid on the cell surface may play a role in the metastasis of these cells. Therefore, the effect of a sialyltransferase inhibitor, 5-fluoro-2',3'-isopropylidene-5'-O-(4-N-acetyl-2,4-dideoxy-3,6,7,8-tetra -O -acetyl-1-methoxycarbonyl-D-glycero-alpha-D-galactooctapyranosyl)u ridine (Kl-8110), on the experimental lung metastasis of NL-17 or NL-44 cells was examined. Kl-8110 inhibited the transfer of sialic acid to its endogenous acceptor in NL-17 and NL-44 cells. NL-17 or NL-44 cells were injected into the tail veins of mice, and the metastasis-inhibiting activity of Kl-8110 was evaluated on the basis of both the lung weight and the number of pulmonary surface nodules about 3 wk after the tumor cell injection and of the survival ratio of mice inoculated with the tumor cells. Pretreatment of tumor cells with Kl-8110 together with i.v. injection of Kl-8110 caused significant inhibition of pulmonary metastasis of both NL-17 and NL-44 cells. Inhibition of metastasis and prolongation of survival were also observed on i.v. injection of Kl-8110 without pretreatment of the tumor cells with Kl-8110, but the degree of inhibition was lower than that in the case of the two treatments together. Kl-8110 itself had neither cytostatic nor cytotoxic effects on NL-17 and NL-44 but reduced the retention of tumor cells in the lungs. This antimetastatic effect of Kl-8110 may be due to modification of the tumor cell surface resulting from inhibition of sialyltransferase by Kl-8110. In addition, a beta-linked sialic acid:nucleoside conjugate (Kl-8111) and an equimolar mixture of Kl-8110 and Kl-8111 (Kl-414) also inhibited the metastatic ability of NL cells to the same extent as Kl-8110 did.

Lectin-like molecules on the murine macrophage cell surface.

Lectin-like molecules on the surface of murine peritoneal exudate macrophages induced by thioglycolate or an anti-tumor streptococcal preparation, OK-432, were investigated and isolated. Furthermore, their sugar-binding specificities and their role in macrophage-mediated tumor cytotoxicity were examined. A neoglycoprotein, D-galactose (Gal)-bovine serum albumin, bound to these murine peritoneal macrophages. This binding of Gal-bovine serum albumin was inhibited by D-galactose, and by complex-type oligosaccharides (unit B) and high mannose-type oligosaccharides (unit A) prepared from porcine thyroglobulin. When thioglycolate-elicited macrophages were activated by lipopolysaccharide and/or the culture supernatant of concanavalin A-activated mouse spleen cells, they became tumoricidal and the number of the lectin-like molecules on the macrophage surface was found to increase. Since the binding and cytotoxic activities of these tumoricidal macrophages toward tumor cells were partially inhibited by D-galactose, the D-galactose-binding lectin-like molecules on the surface of tumoricidal macrophages might play an important role in macrophage-mediated cytotoxicity. These lectin-like molecules were then isolated from solubilized murine peritoneal exudate cells labeled with pyridoxal 5'-phosphate and sodium [3H]borohydride by affinity chromatography on columns of asialo unit B oligosaccharide-Sepharose 4B and/or beta-D-galactose-Bio-Gel P-100. The proteins bound to the asialo unit B oligosaccharide-Sepharose 4B column and eluted specifically were found to have approximate molecular weights of 79 000 and 18 000, and the protein bound to and eluted from the beta-D-galactose-Bio-Gel P-100 column had an approximate molecular weight of 77 000. These isolated proteins bound to the surface of glutaraldehyde-fixed tumor cells, and their binding was inhibited by D-galactose and also by D-mannose. Since most of the 77 kDa protein bound to the asialo unit B oligosaccharide-Sepharose 4B, this protein was assumed to be identical with the 79 kDa protein. These results suggest that the lectin-like molecules on murine macrophages have wide specificity and that one lectin-like molecule can bind both D-galactose and D-mannose.

Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP.

The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.

Deletion of 12 carboxyl-terminal residues from pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase affects steroid metabolism.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase (3alpha/beta,20beta-HSD) is 80-85% identical to human, rat, and mouse carbonyl reductases. However, pig 3alpha/beta,20beta-HSD contains an extra 12 amino acids at its COOH-terminus that these other mammalian carbonyl reductases lack. We constructed a pig 3alpha/beta,20beta-HSD mutant, G278opal, which lacks these amino acids and found that compared to wild-type 3alpha/beta,20beta-HSD, G278opal has a 10-fold lower catalytic efficiency for testosterone and progesterone. G278opal also has lower 3alpha- and 20beta-reductase and increased 3beta-reductase activity compared to wild-type 3alpha/beta,20beta-HSD. Binding of NADPH to G278opal was similar to that of wild-type 3alpha/beta,20beta-HSD. The recently determined three-dimensional structure of 3alpha/beta,20beta-HSD, without a steroid substrate, shows the 12 COOH-terminal amino acids in a random configuration. Our data indicate that the 12 COOH-terminal amino acids have a role in steroid metabolism suggesting that binding of steroid to wild-type 3alpha/beta,20beta-HSD induces a conformational change in which the 12 COOH-terminal amino acids interact with the steroid substrate.

Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats.

Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

TNF-mediated cytotoxicity. Importance of intracellular cGMP level for determining TNF-sensitivity.

Previous work on the cytolytic action of activated macrophages indicated that tumor necrosis factor (TNF) showed synergistic cytolytic activity with NO, which has been shown to act as a cyclic GMP (cGMP) generator [Higuchi et al., J. Immun. 144, 1425-1431, (1990)]. In this study, we investigated the relationship between the accumulation of intracellular cGMP and the cytotoxic action of TNF. It was demonstrated that TNF-mediated cell lysis was closely related to the background level of intracellular cGMP, and that the accumulation of cGMP within TNF resistant cells induced TNF sensitivity. We reached these conclusions on the basis of the following results; (1) agents (sodium nitroprusside and isobutylmethylxantine) that cause the accumulation of cGMP intracellularly increased the TNF-sensitivity of TNF-resistant cells; (2) the addition of dibutyryl cGMP to TNF-resistant cells increased the TNF-sensitivity; and (3) treatment at 40 degrees C or agents such as interferon gamma and actinomycin D, that synergistically kill tumor cells together with TNF, potentially increased the cGMP level. Therefore, intracellular cGMP may be one of the key molecules that lead to cell death caused by TNF.

The relationship between phospholipid methylation and calcium influx in murine lymphocytes stimulated with native and modified Con A.

Native Con A and two chemical derivatives, divalent dimeric Con A and monovalent dimeric Con A. induced a transient increase of phospholipid methylation, Ca2+ influx, and also increased DNA synthesis in murine lymphocytes. For each of the individual mitogens, the dose-response curves for these three activities were very similar. However, there were major differences between the dose-response curves for Con A and each of its two chemical derivatives. On the other hand, the time course of phospholipid methylation for each lectin reached a maximum at about 10 min after the addition of lectin, and then gradually decreased to control levels. In like manner, Ca2+ influx reached its maximum at approximately 5 min. The lectin-stimulated increase in phospholipid methylation occurred in calcium-free medium, while the inhibitor of phospholipid methylation, 3-deaza-SIBA, also suppressed the increased calcium influx. This suggests that the Ca2+ influx might be regulated by early phospholipid methylation. Further, in the absence of calcium, the methylated phospholipids do not undergo Con A-accelerated breakdown by phospholipase A2. This suggests that the increased influx of calcium is necessary for the activation of phospholipase A2, an enzyme that hydrolyses methylated phospholipids to yield arachidonic acid and lysolecithin. Blocking any of these biochemical steps also blocked subsequent DNA synthesis, suggesting that the pathway may be required for the activation of lymphocytes.

Purification and cDNA cloning of a novel factor produced by a human T-cell hybridoma: sequence homology with animal lectins.

We have purified a novel immunoregulatory factor (BMPG: bone-marrow proteoglycan) produced by a T-cell hybridoma, with a monoclonal antibody column. Using an oligonucleotide probe corresponding to the partial amino acid sequence of BMPG, we cloned, sequenced, and expressed a cDNA for BMPG. BMPG has 222 amino acid residues with a 16 N-terminal signal sequence, so the mature form has 206 amino acid residues. BMPG was found to have unique characteristics: it has three types of sugar chains and it shows a marked homology with animal lectins including the human asialoglycoprotein receptor, chicken hepatic lectin and the homing receptor of lymphocytes.

Damage to mitochondrial respiration chain is related to phospholipase A2 activation caused by tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to be cytotoxic to tumor cell lines in vitro, but the mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we used various inhibitors to investigate the sequence of events that lead to cytotoxic effects of TNF on L.P3 cells, a highly sensitive, murine fibroblast cell line. Our results indicate that mitochondrial respiration chains are damaged by a hydroxyl radical at an early stage of the cell lysis after TNF treatment. This event is followed by the activation of phospholipase A2, and finally leads to cell lysis.

Molecular cloning of a novel actin-binding protein, p57, with a WD repeat and a leucine zipper motif.

A 57 kDa protein (p57) was obtained during the study on phosphatidylinositol-specific phospholipase C. Its cDNA was isolated from calf spleen and human leukemia cell line HL60 libraries and cloned. In the primary structures of p57, they have two unique amino acid sequence motifs, a WD repeat and a leucine zipper motif. Furthermore, p57 shared sequence similarity (40%) with coronin, an actin-binding protein responsible for chemotaxis, cell motility, and cytokinesis of Dictyostelium discoideum, which has only the WD repeat. p57 also showed an actin-binding activity and was mainly expressed in immune tissues. From these results, we conclude that p57 is a coronin-like novel actin-binding protein in mammalian cells but may also have a different function from coronin.

Antiinflammatory activity of some 2,3-dihydrobenzofuran-5-acetic acids and related compounds.

A series of 2,3-dihydrobenzofuran-5-acetic acids and related compounds was prepared as potential antiinflammatory agents. As measured by the carrageenan-induced edema method for the preliminary screening test, introduction of a methyl group alpha to the acetic acid function enhanced the antiinflammatory activity, and alpha-(7-chloro-2,2-dimethyl-2,3-dihydrobenzofuran)-alpha-methyl-5-acetic acid (13a) showed the most potent activity in this series.

N-acylphenylalanines and related compounds. A new class of oral hypoglycemic agents.

N-Benzoyl-DL-phenylalanine (1) was found to possess hypoglycemic activity. A series of the analogues of compound 1 were prepared and evaluated for their blood glucose lowering activity. Both the steric effects of the phenylalanine moiety and the effects of variations in the acyl moiety were investigated. This study elucidated some of the structure-activity relationships and led to the development of N-(4-ethylbenzoyl)-D-phenylalanine (34), which was 50 times more potent than the initial compound 1.

N-(cyclohexylcarbonyl)-D-phenylalanines and related compounds. A new class of oral hypoglycemic agents. 2.

A series of analogues of N-(cyclohexylcarbonyl)-D-phenylalanine (5) have been synthesized and evaluated for their hypoglycemic activity. Relationships were studied between the activity and the three-dimensional structure of the acyl moiety, which was characterized by high-resolution 1H NMR spectroscopy and MNDO calculations. The role of the carboxyl group of the phenylalanine moiety was also studied by comparing the activities of the enantiomers, the decarboxyl derivative, the esters, and the amides of the phenylalanine derivatives. Thus, the structural requirements for possessing hypoglycemic activity was elucidated and a highly active compound, N-[(trans-4-isopropylcyclohexyl)carbonyl]-D-phenylalanine (13) was obtained, which showed a 20% blood glucose decrease at an oral dose of 1.6 mg/kg in fasted normal mice.

Loss of heterozygosity and microsatellite instability in ductal carcinoma in situ of the breast.

To investigate the alterations of genetic instabilities in carcinogenesis of the breast, we analyzed the allelotypic profile of 65 ductal carcinomas in situ (DCIS), compared with that of 207 invasive ductal carcinomas (IDC) of the breast. These studies were performed by means of examining microsatellite-length polymorphisms at seven loci (AluVpa, ESR, D11S988, D13S267, D16S398, D17S1159, and D17S855) from microdissected paraffin sections. Allelic loss or imbalance, considered a loss of heterozygosity (LOH), tended to be more frequently seen in IDC than in DCIS. In particular, the frequency of LOH at the 17p locus was significantly higher in IDC than in DCIS (42 vs. 23%, P=0.022). LOH in DCIS was most frequently seen at D16S398 (26%). LOH frequency at D16S398 in low- and intermediate-grade DCIS was higher than that in high-grade DCIS, while LOH frequencies at D11S988 and D17S1159 in low- and intermediate-grade DCIS was lower than those in high-grade DCIS. LOH frequency at D11S988 in non-comedo type DCIS was lower than that in comedo type DCIS. Furthermore, the frequency of microsatellite instability (MSI) at only one locus in DCIS (28%) was statistically higher than that in IDC (6%) (P<0.001), while there was no difference between the frequency of MSI at multiple loci in DCIS (6%) and that in IDC (3%). Together, these observations indicate that chromosomal losses of 16q may occur in low- and intermediate-grade DCIS and those of 11p and 17p may occur high-grade DCIS, and that MSI occurring at only one locus is not yet clear and MSI at multiple loci is uncommon in not only IDC but also DCIS of the breast.

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes.

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification and characterization of membrane-bound and cytosolic phosphatidylinositol-specific phospholipases C from murine thymocytes.

Membrane-bound and cytosolic phosphatidylinositol (PI)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographies on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolic enzyme increased more than 1,800- and 1,400-fold, respectively, compared with those of microsomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70,000 by gel filtration. These purified enzymes also hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2 in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphatidylcholine enhanced the activity of the membrane-bound enzyme, while phosphatidylcholine (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.