Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first-line treatment for patients with metastatic colorectal cancer (WJOG4407G).

BACKGROUND: FOLFIRI and FOLFOX have shown equivalent efficacy for metastatic colorectal cancer (mCRC), but their comparative effectiveness is unknown when combined with bevacizumab. PATIENTS AND METHODS: WJOG4407G was a randomized, open-label, phase III trial conducted in Japan. Patients with previously untreated mCRC were randomized 1:1 to receive either FOLFIRI plus bevacizumab (FOLFIRI + Bev) or mFOLFOX6 plus bevacizumab (mFOLFOX6 + Bev), stratified by institution, adjuvant chemotherapy, and liver-limited disease. The primary end point was non-inferiority of FOLFIRI + Bev to mFOLFOX6 + Bev in progression-free survival (PFS), with an expected hazard ratio (HR) of 0.9 and non-inferiority margin of 1.25 (power 0.85, one-sided α-error 0.025). The secondary end points were response rate (RR), overall survival (OS), safety, and quality of life (QoL) during 18 months. This trial is registered to the University Hospital Medical Information Network, number UMIN000001396. RESULTS: Among 402 patients enrolled from September 2008 to January 2012, 395 patients were eligible for efficacy analysis. The median PFS for FOLFIRI + Bev (n = 197) and mFOLFOX6 + Bev (n = 198) were 12.1 and 10.7 months, respectively [HR, 0.905; 95% confidence interval (CI) 0.723-1.133; P = 0.003 for non-inferiority]. The median OS for FOLFIRI + Bev and mFOLFOX6 + Bev were 31.4 and 30.1 months, respectively (HR, 0.990; 95% CI 0.785-1.249). The best overall RRs were 64% for FOLFIRI + Bev and 62% for mFOLFOX6 + Bev. The common grade 3 or higher adverse events were leukopenia (11% in FOLFIRI + Bev/5% in mFOLFOX6 + Bev), neutropenia (46%/35%), diarrhea (9%/5%), febrile neutropenia (5%/2%), peripheral neuropathy (0%/22%), and venous thromboembolism (6%/2%). The QoL assessed by FACT-C (TOI-PFC) and FACT/GOG-Ntx was favorable for FOLFIRI + Bev during 18 months. CONCLUSION: FOLFIRI plus bevacizumab was non-inferior for PFS, compared with mFOLFOX6 plus bevacizumab, as the first-line systemic treatment for mCRC. CLINICAL TRIALS NUMBER: UMIN000001396.

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker.

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

Effects of rapessed oil on activity of methylmalonyl-CoA carboxyltransferase in culture of Streptomyces fradiae.

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.

'Green mice' as a source of ubiquitous green cells.

The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an 'enhanced' GFP (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.

Ultrastructural changes during myocardial hypertrophy and its regression: long-term effects of nifedipine in adult spontaneously hypertensive rats.

Nifedipine (20 mg/kg/day) was given to 15-week-old spontaneously hypertensive rats for 20 weeks (SHR-N, n = 8). Comparison was done with sex-matched 15-week-old SHR (SHR-15, n = 7), untreated 35-week-old SHR (SHR-C, n = 10), 15-week-old normotensive Wistar-Kyoto rats (WKY-15, n = 15), and 35-week-old WKY (WKY-15, n = 5). Light and electron microscopic data on the subepicardial, middle and subendocardial layers and papillary muscles of the left ventricle were compared among the five rat groups. In SHR-N, blood pressure was significantly reduced by nifedipine, but was higher than in WKY-35 (199 +/- 11 mmHg vs 121 +/- 13 mmHg). The left ventricular weight/body weight ratio was much lower in SHR-N than in SHR-C, and was even below the baseline value in SHR-15. In addition, cardiac myocyte diameter was much smaller in each myocardial layer of SHR-N than in SHR-C, and was similar to the findings in SHR-15, but still larger than in WKY-35. The interstitial area ratio was markedly reduced in SHR-N and did not differ from that in SHR-15 or even WKY-15, while capillary density was significantly greater than in SHR-C and comparable to that in WKY-35. In SHR-C, large fibrotic foci were common, and many hypertrophic cardiac myocytes showed various degenerative changes including those of mitochondria and widening of the intermyofibrillar spaces. These changes were rarely seen in SHR-N. The intracellular volume ratio of myofibrils did not differ between SHR-N and WKY-35, but was significantly decreased in SHR-C, whereas that of mitochondria did not differ between SHR-N and SHR-C or WKY-35. These findings indicate that despite only a moderate suppression of hypertension, long-term nifedipine treatment caused regression of left ventricular hypertrophy, with cardiocyte hypertrophy, interstitial fibrosis, degenerative changes, and subcellular remodeling being reversed to the baseline levels in SHR-15. In addition, the capillary density was increased to that seen in WKY-35.

Cloning and characterization of the human Calmegin gene encoding putative testis-specific chaperone.

The putative chaperone Calmegin is required for sperm fertility in mouse and the relevance of the gene to certain cases of human male infertility has been suggested. In the present paper, we have isolated and characterized the human homolog cDNA of the mouse germ cell-specific Calmegin. The entire coding region of the human cDNA showed 80% identity with the previously reported mouse Calmegin. The predicted amino acid sequence showed strong conservation of the two sets of internal repetitive sequences (Ca2+ binding motif), and the hydrophilic COOH terminus, which corresponds to the putative endoplasmic reticulum (ER) retention motif. Our finding will support diagnosis of male infertility. Northern blotting analysis of various human tissues showed that the transcript was 3 kb in length and was expressed exclusively in the testis. Using the fluorescence in situ hybridization (FISH) technique, human Calmegin gene was mapped to chromosome 4q28.3-q31.1.

Enhancement of cephamycin C production using soybean oil as the sole carbon source.

Vegetable oils were investigated to evaluate their potential to act as the sole carbon source for production of cephamycin C in shake and jar-fermentor cultures. Soybean oil was the best carbon source for cephamycin C production. Bioautography and HPLC analyses showed that cephamycin C was exclusively produced even when soybean oil was used as the sole carbon source. The optimal pH and initial concentration of soybean oil was 7.5 and 7 g/l, respectively. Both pH and the pH-control agent affected cephamycin C production, and among phosphoric acid, acetic acid and sulfuric acid, phosphoric acid was associated with the best production. Soybean oil was slowly consumed after the soluble nitrogen source was consumed. When the initial soybean oil concentration was 7 g/l, cephamycin C production was maximal, 2.0 g/l, which was twice as high as that from starch. The product yield from soybean oil was 4.7 times higher than that from starch. These results show that vegetable oils, which are cheaper than other carbon sources, could be used as the sole carbon source in the production of antibiotics.

Left ventricular function of the heart regressed by nifedipine in spontaneously hypertensive rats.

Left ventricular (LV) performance of the pharmacologically regressed heart in hypertension is still unclear. We compared LV function of the heart regressed by nifedipine with that of the hypertrophied heart in spontaneously hypertensive rats (SHR). Nifedipine (30 mg/kg/day in food) was given to 15-week-old male SHR for 20 weeks (n = 12). Age- and sex-matched SHR served as controls (n = 12). LV catheterization was performed using a micromanometer and cardiac output was determined by the thermodilution method. Hemodynamic studies were performed after washout of nifedipine (24 h), when blood pressure had returned to the untreated level. Peak pumping ability was assessed during acute volume loading with saline. Nifedipine significantly decreased blood pressure in conscious animals (222 +/- 11 to 201 +/- 12 mmHg, p < 0.01) and reduced LV weight (1.20 +/- 0.07 to 1.07 +/- 0.05g, p < 0.01). After washout of nifedipine, LV systolic and end-diastolic pressures, dp/dtmax and cardiac output determined under pentobarbital anesthesia were similar in the treated and untreated groups. Peak pumping ability during acute preload elevation was also similar in the 2 groups. Plasma norepinephrine was unaltered, and plasma renin activity was significantly lower in the treated rats (p < 0.05). These results indicate that nifedipine regressed LVH with a minimal reduction of blood pressure and without evidence of neurohumoral activation or volume retention. In conclusion, LV function of the heart regressed by nifedipine was preserved after a spontaneous rise in blood pressure and during acute preload elevation.

BCR/ABL oncoprotein-targeted antitumor activity of antisense oligodeoxynucleotides complementary to bcr/abl mRNA and herbimycin A, an antagonist of protein tyrosine kinase: inhibitory effects on in vitro growth of Ph1-positive leukemia cells and BCR/ABL oncoprotein-associated transformed cells.

We investigated whether antisense oligodeoxynucleotides complementary to bcr/abl mRNA or protein kinase antagonists display antitumor activity on Ph1-positive leukemia cell lines. bcr/abl antisense oligomers showed inhibitory effects on the in vitro growth of Ph1-positive leukemia cell lines in liquid culture, and further displayed an inhibitory effect on transformed murine hematopoietic cells using transfection with a retroviral vector expressing P210bcr/abl oncoprotein. However, in vitro treatment with a bcr/abl antisense oligomer did not completely abolish the expression of bcr/abl mRNA and did not display the desired "killing effect" on Ph1-positive leukemia cells. On the other hand, investigation of the effect on Ph1-positive leukemia cells by various types of protein kinase antagonists revealed that herbimycin A, a protein tyrosine kinase antagonist, displays preferential and remarkable suppression of the growth of Ph1-positive leukemia cells and P210bcr/abl associated transformed cells by virtue of suppressing bcr/abl protein tyrosine kinase activity. These results may provide important future insights in developing a new category of antitumor therapy by targeting oncogene products.

Potent platelet antiaggregant action of an antiarrhythmic peptide (AAP).

A potent platelet antiaggregant action of an antiarrhythmic peptide (AAP) was demonstrated to be a cause of the antithrombotic effect. AAP (10, 20 or 40 mg/kg, i.v.) inhibited ex vivo platelet aggregation induced by collagen in a dose-dependent manner. AAP also inhibited the platelet aggregation of platelet-rich plasma (PRP) induced by collagen, Ca-ionophore A-23187, adenosine diphosphate (ADP), thrombin or arachidonic acid in vitro. The IC50 was 2.5 mM for collagen, 1.7 mM for A-23187, 5 mM for ADP, 0.4 mM for thrombin and 0.15 mM for arachidonic acid. The aggregation inhibitory activity of the peptide on washed platelet (WP), in a Ca2+-free medium, was stronger than on PRP. The IC50 was 1 mM for collagen and 20 microM for A-23187. No significant difference was found between antiaggregant activities of platelet-free plasma (PFP) from AAP-treated rats and PFP from normal rats supplemented with AAP. The direct action of AAP on platelets was also supported by the incorporation of AAP into platelet cytoplasma which caused a decrease of Ca2+-dependent 3':5'-cyclic nucleotide phosphodiesterase (Ca-PDE) activity. It was considered that AAP showed its platelet aggregation inhibitory activity by decreasing intracellular Ca2+ concentration through the inhibition of Ca-PDE activity.

Immunotherapy of cancer with cell wall skeleton of Myocabacterium bovis-Bacillus Calmette-Guérin: experimental and clinical results.

Adjuvant and antitumor activities of CWS prepared from cells of mycobacteria, nocardia, and corynebacteria were examined. Oil-attached CWS of M. bovis BCG (BCG-CWS) stimulated the generation of cell-mediated cytotoxic effector cells in mice. Tumor growth was suppressed in mice inoculated intradermally with a mixture of oil-attached CWS and living tumor cells. Systemic and specific tumor immunity was demonstrated in mice in which tumor growth was suppressed. Tumor growth was also suppressed by oil-attached CWS of BCG or N. rubra in autochthonous autografts of spontaneous mammary adenocarcinoma and methylcholanthrene-induced fibrosarcoma in mice. The intravenous injection of oil-attached BCG-CWS prevents the appearance of lung cancer in rabbits by the instillation of chemical carcinogens. It was also shown that treatment with oil-attached BCG-CWS was able to elevate the immunologically depressed state of tumor-bearing mice to a normal level, as determined by a cell-mediated cytotoxicity assay that empolyed chromium release as the standard. Preliminary results suggest that oil-attached BCG-CWS is useful as an immunotherapeutic agent for both lung cancer and for malignant melanoma, leukemia, Hodgkin's disease, and other neoplastic diseases and that this agent operates without any significant complications.