A phase I dose-escalation study of eribulin and S-1 for metastatic breast cancer.

BACKGROUND: We evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, recommended dose for phase II (P2RD), and preliminary anticancer activity of a combination eribulin and S-1 therapeutic in metastatic breast cancer patients pretreated with anthracycline and taxane. METHOD: Patients aged 20-74 years were recruited. In level 1, patients received S-1 (65 mg m(-2)) from day 1 to 14, and eribulin (1.1 mg m(-2)) on day 1 and 8 in a 21-day cycle. In level 2, eribulin was increased to 1.4 mg m(-2). In level 3, S-1 was increased to 80 mg m(-2). RESULTS: Twelve patients were enrolled into three cohorts. Planned dose escalation was completed, with one case exhibiting dose-limiting toxicity (grade 3 hypokalaemia) at level 3, without reaching the MTD. The P2RD was determined to be level 2 (eribulin 1.4 mg m(-2) and S-1 65 mg m(-2)). The most common grade 3 or 4 toxicity was neutropenia (83.3%), followed by febrile neutropenia (25.0%). Five of eleven patients (41.7%) with measurable disease had a partial response. Pharmacokinetics were characterised by dose-dependent elimination and nonlinear exposure. CONCLUSION: Dose level 3 was not tolerated owing to febrile neutropenia development. Thus, intermediate dose level 2 was recommended for further evaluation. Preliminary antitumour activity warrants further investigation in this setting.

Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.

A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels.

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

Expression of Sia alpha 2-->6Gal beta 1-->4GlcNAc residues on sugar chains of glycoproteins including carcinoembryonic antigens in human colon adenocarcinoma: applications of Trichosanthes japonica agglutinin I for early diagnosis.

The N-linked sugar chain structures of the carcinoembryonic antigen (CEA) produced by liver metastases of colon cancers and the normal counterpart of CEA purified from human adult feces (NFA-2) were previously determined comparatively (K. Fukushima, T. Ohkura, M. Kanai, M. Kuroki, Y. Matsuoka, A. Kobata, and K. Yamashita. Glycobiology, 5:105-115, 1995). Seventy-five % of NFA-2 contained complex type sugar chains with Gal beta 1-->3GlcNAc residues, in contrast to the sugar chains of CEA, in which over 90% of the oligosaccharides contained Gal beta 1-->4GlcNAc residues, and Sia alpha 2-->6Gal beta 1-->4GlcNAc residues were detected in 18 to 65% of the oligosaccharides. The expression of Sia alpha 2-->6Gal beta 1-->4GlcNAc residues on CEA molecules in sera and tissues was investigated using Trichosanthes japonica agglutinin I (TJA-I), which interacts with Sia alpha 2-->6Gal beta 1-->4 GlcNAc residues. Ten purified CEA samples bound to a TJA-I column while seven NFA-2 samples passed through the column. Various concentrations of serum CEA samples from patients with metastatic colon cancers exclusively bound to the TJA-I column, reflecting that CEA molecules exfoliated into the blood circulation comprise sugar chains with Sia alpha 2-->6Gal beta 1-->4GlcNAc residues. In histochemical studies involving biotinylated TJA-I, normal mucosa (n = 20) and benign adenomas (n = 20) were not stained, and 83% of well and moderately differentiated colon adenocarcinomas (n = 53) reacted with TJA-I, although poorly differentiated ones (n = 9) and mucinous specimens (n = 10) were negative. Because over 90% of colon adenocarcinomas can be differentiated, TJA-I staining might be applicable to the early diagnosis of colon cancers.

Species-specific beta-N-acetylgalactosaminylation of serum IgG proteins.

Lectin blot analysis of bovine, goat, human, rabbit and mouse serum immunoglobulin G (IgG) samples revealed that Wisteria floribunda agglutinin (WFA) binds to the heavy chains of bovine, goat and human serum IgG proteins but not those of the rabbit and mouse proteins. WFA-positive light chain bands were also detected in bovine, goat and human serum IgG samples only after the filters were treated with Arthrobacter ureafaciens sialidase. The WFA-binding to these IgG proteins was abolished by treatment of the filter with sialidase and then beta-N-acetylhexosaminidase or N-glycanase prior to incubation with the lectin. WFA-agarose column chromatography of the oligosaccharides released by hydrazinolysis from the IgG samples followed by reduction with NaB3H4 revealed that 0.15, 0.09 and 0.07% of the total oligosaccharides from bovine, goat and human serum IgG samples bind to the column, respectively. Partial characterization of WFA-positive bovine IgG oligosaccharides by Bio-Gel P-4 column chromatography suggested that the major oligosaccharide is of non-fucosylated biantennary complex-type. These results indicate that beta-N-acetylgalactosaminylation occurs to N-linked sugar chains of heavy and light chains of IgG proteins in a species-specific manner.

Distribution of glycoproteins with beta-N-acetylgalactosaminylated N-linked sugar chains among bovine tissues.

Only a small number of glycoproteins has been reported to contain N-linked sugar chains with GalNAcbeta1-->4GlcNAc structure. Our previous studies showed that most glycoproteins from bovine milk fat globule membranes contain beta-N-acetylgalactosaminylated N-linked sugar chains [Sato et al., J. Biochem. 114 (1993) 890-900]. In order to study how widely this glycosylation occurs, lectin blot analysis of membrane glycoproteins from 12 bovine tissues was performed using Wistaria floribunda agglutinin (WFA), which interacts with oligosaccharides terminating with N-acetylgalactosamine. The WFA-positive bands were detected in samples from most tissues except for intestine although the number and reactivity of bands to lectin varied among the tissues. Upon pretreatment of blotted filters with Bacillus beta-N-acetylgalactosaminidase or N-glycanase, no lectin binding was observed. WFA-agarose column chromatography of oligosaccharides released by hydrazinolysis from membrane glycoproteins of bovine tissues except for intestine revealed that a few to 18% of the released oligosaccharides bind and are eluted from the column with 100 mM N-acetylgalactosamine. These results indicate that many glycoproteins from a variety of bovine tissues contain N-linked sugar chains with GalNAcbeta1-->4GlcNAc structure, suggesting a wider occurrence of this glycosylation in bovine tissues.

Overall distribution of substance P nerves in the rat cornea and their three-dimensional profiles.

The overall distribution of substance P-like immunoreactive (SPI) fibers, examined by using whole-mounts of the rat cornea, was investigated by means of indirect immunofluorescence. SPI fibers entered the cornea from two levels; one from the middle layer of the sclera and the other from the episclera. From the sclera, a thick SPI fiber trunk, extending to the central part, subdivided into smaller SPI fiber bundles and approached the epithelium. The SPI fiber bundles from the episclera were smaller than those from the sclera. However, both fiber bundles formed a dense fiber network in the uppermost part of the stroma. This fiber plexus dissociated into SPI fibers extending to the superficial part of the epithelium where they formed an abundant arborization of fine SPI fibers. The results suggest that these fibers originate from SPI neurons in the trigeminal ganglion.

Leucine-enkephalin-like immunoreactivity in the chicken retina with a special reference to its fine structures.

Leucine-enkephalin-like immunoreactivity (LEI) in the chicken retina was investigated using light and electron microscopic immunohistochemistry. A flat-mount technique for light microscopy clearly demonstrated that LEI cells were distributed widely throughout the entire retina, without any differences between the central and peripheral retinal regions. Frozen sections for light microscopy confirmed the previous findings in pigeons that LEI is localized in amacrine cells. The present electron microscopic observations confirmed the findings of light microscopy and revealed that LEI terminals make synaptic contract mainly with vesicle-containing processes that seemed to belong to the amacrine cells. The present study further suggests that LEI terminals are both pre- and postsynaptic elements onto vesicle-containing processes mentioned above.

Retrospective survey of urea cycle disorders: Part 2. Neurological outcome in forty-nine Japanese patients with urea cycle enzymopathies.

We analyzed neurological data, including DQ or IQ, EEG, and CT scan, in 49 patients with urea cycle enzymopathies, all of whom were included in a retrospective survey from 1978-1988 in Japan. We classified 3 groups depending on age-at-onset: group 1 (0-28 days, N = 11), group 2 (29 days-5 years, N = 31), and group 3 (greater than 5 years, N = 7). The least DQ or IQ score and the highest CT score, representing the most severe brain damage was found in group 1, and the highest DQ or IQ and the least CT score was found in group 3. Intermediate scores of both parameters were found in group 2. There was a negative correlation between these 2 parameters (r = -0.82, P less than 0.01). Abnormal EEG during the attack-free period was predominantly observed in patients with CT abnormalities compared to those with a normal CT scan (P less than 0.01). Approximately 40% of the patients, mostly in groups 2 and 3 (92.8%) had normal findings in all 3 parameters. Thus, the magnitude of developmental abnormalities is clearly related to the degree of brain damage and to the age-at-onset of these diseases.

Retrospective survey of urea cycle disorders: Part 1. Clinical and laboratory observations of thirty-two Japanese male patients with ornithine transcarbamylase deficiency.

In a retrospective survey done from 1978-1988 in Japan, 32 male patients with ornithine transcarbamylase (OTC) deficiency were identified. We classified a neonatal and 2 late-onset groups, depending on clinical manifestations and the age at onset; group 1 (0-28 days; N = 10), group 2 (29 days-5 years; N = 13), and group 3 (greater than 5 years; N = 9). Compared to findings in the group 2 patients, there was a higher rate of mortality and a higher incidence of mental retardation in association with a great decrease in enzyme activity in group 1. In group 3, the mortality rate and enzyme activities were similar to those in group 1. However, patients in this group were asymptomatic prior to the first episode. Enzyme activities were measured mostly in autopsy samples. The serum citrulline levels (enzyme product) were highest in this group. Thus, the mutant enzymes were apparently labile with greater activities in vivo than in vitro. Treatments, including a protein-restricted diet, arginine supplementation, and sodium benzoate administration, resulted in a favorable prognosis for survivors with partial enzyme deficiency. We wish to emphasize that the incidence of late onset of this disease is higher than heretofore considered.

Heterogeneity of liver disorder in type B Niemann-Pick disease.

Patients with type B Niemann-Pick disease (NPD) are known to be complicated with varying degrees of prognosis-determining liver dysfunction. To see heterogeneity of the dysfunction histologically, we performed liver biopsies on three NPD patients from three different families, who were diagnosed by enzyme assay of acid sphingomyelinase (ASM) and analysis of the ASM gene. In a severe case, of a female patient in her childhood, the liver showed definite fibrosis despite her age. In contrast, in a very mild case, of an adult male patient, the liver showed little fibrosis, though the ballooning of hepatocytes and infiltration of foamy histiocytes were observed in the tissue. Three homo-allelic mutations (S436R, A599T, and S231P) were identified in the patients. Thus, various hepatic phenotypes in type B NPD were shown to be caused by the heterogeneity of liver lesions originating from different ASM gene mutations.

Ontogeny of substance P-containing structures in the ocular tissue of the rat: an immunohistochemical analysis.

The ontogeny of the substance P-like immunoreactivity (SPI) containing system in the ocular tissue of the rat was examined by means of the indirect immunofluorescence method. SPI-containing amacrine cells first appeared at postnatal day 4 and displaced SPI-containing amacrine cells at postnatal day 5. After this time, they developed markedly reaching their maximum content and distribution at postnatal day 14. On the other hand, SPI-containing cells in the trigeminal ganglion were first seen at gestational day 17 and reached their maximum content at birth. SPI-containing fibers in the cornea and uvea were first observed at gestational days 17-19. The SPI-containing fibers in the iris reached their maximum content at birth, while those in the cornea, choroid and ciliary body were fully developed at postnatal day 3. In the adult rats, numerous SPI structures were still seen in the ocular tissue, retina, cornea and uvea. These findings suggest that SPI might play some role in the developing ocular tissue in addition to its neurotransmitter or neuromodulator role in the adult, because SPI structures appear in the retina before establishment of synaptogenesis and in the cornea and uvea during the fetal stage.

Hemodynamics in the prone jackknife position during surgery.

We examined the hemodynamic changes occurring with prone jackknife positioning during colorectal surgery. The operative procedure was restorative proctocolectomy with ileal J-pouch anal anastomosis in five patients with adenomatosis coli and six patients with ulcerative colitis and anoabdominal resection of the rectum with colonic J-pouch anal anastomosis in eight patients with rectal cancer. Nineteen patients (10 men and 9 women aged 41 +/- 19 years) were monitored with arterial and Swan-Ganz catheters during positioning. Measurements were obtained in the supine and prone positions (1 minute, 3 minutes), and the jackknife position (1, 3, 5, and 10 minutes), as well as before and after adoption of the Lloyd-Davies position (1, 3, 5, and 10 minutes). Turning the patient from the supine position to the prone position resulted in a significant decrease in the cardiac index (CI). However, following head-down rotation, the CI increased and returned to the value seen in the supine position (p less than 0.05). Heart rate (HR) slowed and mean arterial pressure (MAP) increased in the prone jackknife position. We concluded that the extent of the changes in cardiac function presented no serious problems.

Evaluation of hydration states of protein in freeze-dried amorphous sugar matrix.

A model to analyze the hydration state of protein in freeze-dried amorphous sugar matrix was proposed, based on the assumptions that there is a limit to the amount of protein that a given amount of amorphous sugar could embed and that the freeze-dried sugar-protein mixture is composed of the four components, i.e., sugars with and without hydrogen bonding to proteins and proteins with and without hydrogen bonding to sugar. Bovine serum albumin and three kinds of disaccharides, i.e., sucrose, maltose, and trehalose, were used as samples. Using the analytical equations derived from the model and experimental sugar content dependencies of the water sorption at various relative humidities, the amount of hydration water for bovine serum albumin, and the minimum amount of sugar to embed the protein were determined. On the basis of these results, the degree of interaction between the three sugars and protein was discussed, with respect to their stabilizing effect on the protein.

On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon.

Adsorption of proteins on solid surfaces and their interaction are major concerns in a number of fields such as biology, medicine, biotechnology and food processing, and play an important role from various points of view. Based on practical viewpoints, information on the conformation of the adsorbed protein as well as adsorption characteristics is essential for a system's performance. Although there are still many problems to be solved, extensive studies in recent years, owing to the development in instrumentation and instrumental techniques, reveal the adsorption behavior of proteins in detail. Here, we stress the importance and interesting aspect of protein adsorption on solid surfaces by reviewing findings that have been obtained in recent years.

Kinetic analysis for synthesis of a dipeptide precursor using an immobilized enzyme in water-immiscible organic solvents.

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetener aspartame, was synthesized, using thermolysin immobilized onto Amberlite XAD-7, both in ethyl acetate and in tert-amyl alcohol. The initial rates for synthesis of Z-AspPheOMe in the organic solvents were predicted on the basis of a model proposed for an aqueous/organic biphasic reaction and compared with the experimentally observed substrate concentration dependencies. The experimental synthetic rates using the enzyme immobilized at a high enzyme concentration were lower than the calculated ones over a wide range of the substrate concentration. It was suggested as a reason for this discrepancy that the enzyme molecules form compact aggregates and those existing inside the aggregates cannot be utilized for reaction. The experimental results with the enzyme immobilized at a low concentration in ethyl acetate coincided well with the calculated ones. On the other hand, when tert-amyl alcohol was used, the experimental results were different in tendency irrespective of the amount of enzyme loaded, probably due to the fact that a distinct water phase does not exist around the enzyme aggregates inside the support.

Kinetics and equilibrium for thermolysin-catalyzed syntheses of dipeptide precursors in aqueous/organic biphasic systems.

The initial kinetics for the syntheses of N-(benzyloxycarbonyl)-L-alanyl-L-phenylalanine methyl ester (ZAPM) and N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZDPM) in an aqueous/organic biphasic system, using free thermolysin were elucidated, both experimentally and theoretically. As model organic solvents, ethyl acetate and tert-amyl alcohol were used. The substrate concentration dependencies of the initial rate of syntheses for ZAPM and ZDPM observed in the biphasic system were well simulated using the overall partition coefficients of the substrates and product taking into consideration the effect of the formation of ion-pair complexes between the acid and amine components of the substrate, the initial rate equations determined in an aqueous buffer saturated with the organic solvent, and the pH dependence of the rate constant. The equilibrium yield for the synthesis of ZDPM was also in good agreement with the calculated result using the overall partition coefficients and equilibrium constant measured in the aqueous buffer.

Stability of immobilized thermolysin in organic solvents.

Various factors affecting the stability of thermolysin immobilized by cross-linking with glutaraldehyde were elucidated, particularly in the water-immiscible organic solvents such as ethyl acetate and tert-amyl alcohol. The main reason for enzyme inactivation in water-immiscible organic solvents was found to be autolysis in the water phase, which may surround the enzyme immobilized inside the support. By contrast, in water-miscible organic solvents thermal denaturation was the predominant cause of enzyme inactivation. Courses of inactivation were expressed by second-order kinetics in the initial stage, after which inactivation proceeded at a slower rate. The extent of autolysis was found to strongly depend on the kind of organic solvent, the water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support. Thermolysin was immobilized onto Amberlite XAD-7 as a compact aggregate inside the support which may increase the stability of the enzyme. Finally, it was shown that the stability of the immobilized enzyme could be correlated with the logP value for water-miscible organic solvents and with the solubility of water for water-immiscible organic solvents.

Analysis for partition equilibrium of amino acid derivatives in aqueous/organic biphasic systems.

The overall partition coefficients of the acid and amine components of amino acid derivatives, the substrates for proteinase-catalyzed synthesis of oligopeptide precursors, in an aqueous/organic biphasic system were studied both experimentally and theoretically. In a single-component system containing either an acid or amine component, the overall partition coefficient was well expressed by a model using the acid dissociation constant and partition coefficients for the non-ionized and ionized forms of the component. The overall partition coefficient in a binary-component system containing both acid and amine components was well simulated by the model when the formation of ion-pair complexes in the organic solvent phase was taken into consideration in addition to the partitioning of the non-ionized and ionized forms of the components. The formation of ion-pair complexes in the organic solvent phase was indicated by an analysis using Fourier transform infrared ray spectroscopy (FT-IR). In addition to the partition equilibrium, the aqueous-phase pH change after partitioning could be predicted by the model.

Analysis of monoglyceride synthetic reaction in a solvent-free two-phase system catalyzed by a monoacylglycerol lipase from Pseudomonas sp. LP7315.

Kinetics of monoglyceride synthesis catalyzed by a monoacylglycerol lipase (MGL) isolated from Pseudomonas sp. LP7315 was studied at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in a glycerol phase containing a small amount of water. The initial rate of the synthetic reaction depended on several factors: the amounts of fatty acid and glycerol, and the concentration of MGL in the glycerol phase. To analyze the effects of these factors, a kinetic model was developed based on the assumption that the adsorption equilibrium of MGL molecules at the interface between the two phases is the crucial factor for the synthetic reaction. The model was found to yield good approximations of the initial synthetic rate under various reaction conditions. The analysis suggests that the adsorption behavior of MGL onto the interface had a large effect on the initial rate of the monoglyceride synthesis.