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Objective To evaluate the safety, efficacy and pharmacokinetics of alemtuzumab in Japanese patients, we conducted a phase I study in patients with relapsed or refractory B-cell chronic lymphocytic leukemia. Methods Six patients received alemtuzumab by intravenous infusion every other day three times a week for 12 weeks. The dose was gradually escalated on daily basis (3, 10 and then 30 mg) until the patient tolerated. The primary objective was to evaluate the safety of alemtuzumab in Japanese patients and the secondary objectives were to evaluate the overall response rate and the pharmacokinetics. Results The major treatment-emergent adverse events were anemia, neutropenia (6/6 patients each) and thrombocytopenia (5/6 patients) in hematologic adverse events, and nausea, vomiting, decreased appetite, cytomegalovirus test positive and pyrexia (4/6 patients) in non-hematologic adverse events. As serious adverse events, cytomegalovirus infection, pulmonary tuberculosis and diffuse large B-cell lymphoma were reported in 1/6 patient each. The overall response rate was 33% (95% confidence interval: 4-78) (1/6 patient each achieved complete response and partial response, respectively) and 3/6 patients had stable disease and 1/6 patient had progressive disease. The median time to response was 2.9 months. After last intravenous dosing (Week 12) of alemtuzumab 30 mg every other day three times a week, Cmax, tmax, AUC0-τ and t1/2 were higher and CL and Vss were lower than the values observed after the first dose. Conclusions The efficacy, safety and pharmacokinetics results observed with alemtuzumab in Japanese patients were generally similar to those reported in overseas clinical studies. Alemtuzumab at 30 mg by intravenous infusion every other day three times a week for 12 weeks should be safe and effective similarly in Japanese B-cell chronic lymphocytic leukemia patients. Clinical trial registration no NCT00923182.

Light is the strongest synchronizer controlling circadian rhythms. The intensity and duration of light change throughout the year, thereby influencing body weight, food preferences, and melatonin secretion in humans and animals. Although the expression of clock genes has been examined using human samples, it currently remains unknown whether bright light during the daytime affects the expression of these genes in humans. Therefore, we herein investigated the effects of bright light exposure during the daytime on clock gene expression in the hair follicular and root cells of the human scalp. Seven healthy men (20.4 ± 2.2 years old; 172.3 ± 5.8 cm; 64.3 ± 8.5 kg; BMI 21.7 ± 3.1 kg/m2, mean ± SD) participated in this study. Subjects completed 3-day experimental sessions twice in 1 month during which they were exposed to bright and dim light conditions. The mRNA expression of Per1–3, Cry1–2, Rev-erb-α (Nr1d1), Rev-erb-β (Nr1d2), and Dec1 was analyzed using branched DNA probes. No significant changes were observed in the expression of Per1, Per2, Per3, Cry1, Cry2, Rev-erb-α (Nr1d1), or Dec1 following exposure to bright light conditions. However, the expression of Rev-erb-β (Nr1d2) tended to be stronger under bright light than dim light conditions. These results suggest that the bright light stimulus did not influence the expression of clock genes in humans. Long-lasting bright light exposure during the daytime may be required to change the expression of clock genes in humans.

Secreted Frizzled-related protein 2 (Sfrp2) or Stromal Cell Derived Factor-5 (SDF-5) is highly expressed in the developing limbs. Here we showed the single Sfrp2 inactivation in mice resulted in syndactyly and preaxial synpolydactyly, predominantly in the hindlimbs. Tails were often kinked. A penetrance of the syndactyly was highest in 129/SvJ or CBA/N × 129/SvJ background and the phenotype was haploinsufficient. Preaxial synpolydactyly was seen in homozygous mutants in C57BL/6 × 129/SvJ. Of note, syndactyly showed retarded apoptosis of the second and the third interdigital spaces; concomitantly, mesodermal Msx2 expression was down-regulated. Impaired digital anlagen maturation was also noticeable in the same position. Preaxial synpolydactyly of the Sfrp2 mutants was a non-mirror image type and Shh independent. Although joint formation was not disrupted, chondrocyte maturation was preaxially disturbed. Our results suggest that the Sfrp2 deleted mice can be a useful animal model to study human syndactyly/preaxial synpolydactyly defects. Developmental Dynamics 237:2506–2517, 2008. © 2008 Wiley-Liss, Inc.

The circadian clock is driven by transcriptional oscillation of clock genes in almost all body cells. To investigate the effect of cell type-specific intracellular environment on the circadian machinery, we examined gene expression profiles in five peripheral tissues. As expected, the phase relationship between expression rhythms of nine clock genes was similar in all tissues examined. We also compared relative expression levels of clock genes among tissues and unexpectedly found that quantitative variation remained within an approximately three-fold range, which was substantially smaller than that of metabolic housekeeping genes. Interestingly, circadian gene expression was little affected even when fibroblasts were cultured with different concentrations of serum. Together, these findings support a hypothesis that expression levels of clock genes are quantitatively compensated for the intracellular environment, such as redox potential and metabolite composition. However, more comprehensive studies are required to reach definitive conclusions.

頸椎症性脊髄症に対して宮崎式脊柱管拡大術を施行し, 術後6か月以上経過を観察した35例 (平均観察期間;1年10か月) の手術成績を検討した。JOAスコアは3～15点, 平均8.7点から11～17点, 平均12.5点に改善した。平均改善率は49%であった。術前のJOAスコアと術後JOAスコアの問には強い相関がみられた (r=0.60, p

A series of A-ring pyrrole compounds of duocarmycin bearing 5-membered heteroarylacryloyl groups (thienylacryloyl and pyrrolylacryloyl) and heteroarylcarbonyl groups were synthesized and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. Most of the thienylacrylates displayed in vitro anticellular activity equivalent to 4'-methoxycinnamates. Among the 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of methoxy-thienylacrylates, compound 11b, having 4'-methoxy-2'-thienylacryloyl as segment-B (Seg-B), showed remarkably potent antitumor activity and low peripheral blood toxicity in vivo, which were equal to those of 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4'-methoxycinnamates, compared with the A-ring pyrrole derivatives having the trimethoxyindole skeleton in Seg-B. On the other hand, the 2'-pyrrolylacrylates having a double bond as spacer showed 10(2)- to 10(3)-fold stronger anticellular activity than 2'-pyrrolecarboxylates (IC50 < 0.3 nM, 72 h-exposure). The 8-O-acetate and 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 2'-pyrrolylacrylates exhibited an antitumor effect at a lower dose compared with the 8-O-[(N-methylpiperazinyl] derivatives of 4'-methoxycinnamate (1j). Moreover, it was expected that the antitumor activity would be increased by the strength of the extra hydrogen bond formed between the nitrogen of the pyrrole amido group and DNA, owing to the increase of the number of N-methyl-2'-pyrrolecarboxamide units. However, 2'-pyrrolylacrylates having three N-methyl-2'-pyrrolecarboxamide units showed nearly equal antitumor activity to 2'-pyrrolylacrylates having only one N-methyl-2'-pyrrolecarboxamide unit.

Pulse field gel electrophoresis using a contour-clamped homogeneous electric field was applied for the analysis of DNA-fragmenting activity of antitumor agents towards human uterine cervix carcinoma HeLa S3 cells. Duocarmycins (DUMs), novel antitumor antibiotics with ultrapotent cell growth-inhibitory activities, caused DNA fragmentation at 10 times their IC50 values at 2 h exposure. At 100 times their IC50 values, the size of the smallest fragments was about 245 kilobase pairs (kbp). DUMA, DUMB1 and DUMB2 exhibited similar DNA fragmentation patterns, suggesting similar action mechanisms. DNA fragmentation was also detected in cells treated with radical producers, intercalators and topoisomerase inhibitors. Two bands of about 1800 and 1500 kbp were commonly detected in the cells treated with DUMs and these agents. In addition, fragments of about 900 kbp were detected in the cells treated with a topoisomerase inhibitor, 4'-(9-acridinylamino)methane-sulfon-m-anisidine, and fragments in the broad size range between 700 and 245 kbp in the cells treated with radical producers, bleomycin and neocarzinostatin. DUMs showed a characteristic DNA fragmentation pattern, since both types of fragments induced by the topoisomerase inhibitor and the radical producers were simultaneously detected, suggesting a novel mode of interaction with DNA. DNA-crosslinking agents and mitotic inhibitors did not induce DNA fragmentation under these conditions. The pulse field gel electrophoresis is potentially useful for characterizing DNA-cleaving activity of various antitumor agents at the cellular level.

The effects of magnesium aluminum silicate (MAS) addition on the sorption of bromhexine HCl to polyethylene film in tablets were studied. The addition of MAS prevented the sorption of bromhexine HCl to polyethylene film. In order to investigate the mechanism, the interaction between bromhexine HCl and MAS was studied by the powder X-ray diffraction method. It was observed that bromhexine HCl was preferentially adsorbed to the surface of MAS rather than to polyethylene film. The adsorption was accelerated at high temperature and reduced pressure conditions. The sorption of bromhexine base and bromhexine HCl to packaging material were compared using tablet dosage forms. The sorption of bromhexine base to polyethylene film was greater than that of bromhexine HCl.

The anticellular and antitumor activities of novel antitumor antibiotics, duocarmycins (DUMs), were examined against human and murine tumor cells. DUMs consist of live compounds, A, B1, B2, C1, and C2, which possess a pharmacophore similar to that of CC‐1065, a previously isolated antibiotic. Among them, DUMA exhibited ultrapotent growth‐inhibitory activity with an IC50 value of 6 pM against human uterine cervix carcinoma HeLa S3 cells. DUMA and DUMB1 also inhibited the growth of adriamycin (ADM)‐resistant lines of human nasopharynx carcinoma KB cells and breast carcinoma MCF‐7 cells as well as their sensitive lines. DUMs inhibited the growth of s.c.‐inoculated murine tumors such as B16 melanoma, sarcoma 180, M5076 sarcoma and colon 26. DUMs were also significantly effective in increasing the lifespan of i.p.‐inoculated B16 melanoma‐bearing mice, although their effect was marginal against other i.p.‐inoculated tumors. As a whole, DUMB1 exhibited superior activity to the other four compounds. DUMB1 rapidly inhibited the incorporation of [3H]‐TdR into macromolecules of HeLa S3 cells as compared with that of [3H]UR or [3H]leucine. DNA strand breaks were detected in DUMB1 ‐treated HeLa S3 cells by agarose gel electrophoresis with a contour‐clamped homogeneous electric field apparatus. These results indicate that DUMs possess interesting biological activities as DNA‐targeting antitumor antibiotics.

The interaction between polyethylene containers and bromhexine·HCl solid dosage forms prepared by different methods was studied. It was found that bromhexine·HCl tablets prepared by the wet method (kneading) had more drug remaining than those prepared by the dry method. The sorption of drug to polyethylene was influenced by the kneading solvents used, and the most effective solvent in preventing sorption was methanol, which has high solubility for bromhexine·HCl. A group mixture of bromhexine·HCl with crystalline cellulose was effective inhibiting the sorption of bromhexine to polyethylene. This was explained in terms of the monomolecular dispersion of bromhexine molecules within the network of cellulose molecules in the ground mixture.

The interaction between bromhexine HCl and polyethylene containers was studied in solutions and in tablet packaging systems. Various bromhexine HCl aqueous solutions of different pHs were stored in polyethylene containers at 25 degrees C. The remaining amount of bromhexine HCl was determined by high performance liquid chromatography. It was observed that the transition of bromhexine HCl to polyethylene was inhibited by the addition of some surfactants into the solution, and by lowering the solution pH. In the solid dosage forms, influences of the additives were also studied. It was observed that the decrease of the content of bromhexine HCl in the solid dosage form was inhibited by the addition of acids (citric acid, tartaric acid) and surfactant (sodium dodecyl sulfate) and accelerated by the addition of sodium bicarbonate, similarly to those in solutions. The results indicated that the transition to polyethylene films was influenced by the molecular state of bromhexine HCl.

A series of 3-substituted A-ring pyrrole compounds of duocarmycin were synthesized and evaluated for in vitro anticellular activity against HeLa S 3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. These compounds were evaluated on the peripheral blood toxicity and delayed lethal toxicity. Further, to expand our investigation of their peripheral blood toxicity, the toxicity to bone marrow cells (CFU-GM, CFU-Meg) was investigated. Among 3-substituted A-ring pyrrole compounds of duocarmycin bearing a 5',6',7'-trimethoxy-2'-indolecarboxyl group as segment-B (Seg-B), several analogues showed remarkably potent antitumor activity with low peripheral blood toxicity. The 3-formyl compound 12h, one of such analogues, showed stronger antitumor activity with lower toxicity to bone marrow cells compared to DU-86 (2a), an active metabolite of KW-2189 (2b). However, compound 12h caused delayed death. On the other hand, the 3-bromo compound 15f, one of the 3-substituted A-ring pyrrole derivatives bearing a 4'-methoxycinnamoyl group as Seg-B, showed the most potent antitumor activity among the 4'-methoxycinnamate analogues with low toxicity to bone marrow cells. Furthermore, compound 15f did not cause delayed death similarly to 2d. These results would indicate the importance of the C-3 substituents of A-ring pyrrole duocarmycin derivatives for exhibiting antitumor activity and decreasing toxicity.

Background: Although out-of-lab investigation of the human circadian clock at the clock gene expression level remains difficult, a recent method using hair follicle cells might be useful. While exercise may function as an entrainment cue for circadian rhythms, it remains unclear whether exercise affects human circadian clock gene expression. Methods: Efforts to observe apparent effects of exercise on clock gene expression require that several specific conditions be met: intense exercise should be habitually performed at a relatively uncommon time of day over an extended period; and any relative phase shift thereby observed should be validated by comparison of exercise and no-exercise periods. Wake-up and meal times should be kept almost constant over the experimental period. The present study was conducted using a professional fighter who met these strict criteria as subject. Facial hair samples were collected at 4-h intervals around the clock to ascertain rhythms of clock gene expression. Results: During a period in which nighttime training (from 20:00 to 22:00) was habitually performed, circadian clock gene expression was phase-delayed by 2 to 4 h compared with that during a no-exercise period. Maximum level and circadian amplitude of clock gene expression were not affected by the nighttime training. Conclusion: Our trial observations illustrate the possibility that heavy physical exercise might strongly affect the circadian phase of clock gene expression. Exercise might be therefore effective for the clinical care of circadian disorders. The results also suggest that athletes may require careful scheduling of heavy physical exercise to maintain normal circadian phase and ensure optimal athletic performance.