The pharmacokinetics and safety profiles of belimumab after single subcutaneous and intravenous doses in healthy Japanese volunteers.

WHAT IS KNOWN AND OBJECTIVES: Belimumab is a recombinant human monoclonal antibody that binds and antagonizes the biological activity of soluble B-lymphocyte stimulator (BLyS) protein. BLyS appears to play a role in the pathogenesis of systemic lupus erythematosus, and the biological profile of belimumab suggests that it may have a therapeutic benefit in the treatment for the disease. In this healthy Japanese subjects study, we investigated the pharmacokinetics and safety of a single subcutaneous and intravenous injection of belimumab administered as a 200 mg/mL liquid formulation. METHODS: This was an open-label, randomized, parallel-group, single-dose study in healthy Japanese subjects. Each subject received a single intravenous infusion or a subcutaneous injection of 200 mg belimumab. The pharmacokinetic parameters and safety parameters including local tolerance (injection site), biomarkers, immunogenicity and adverse events were evaluated up to 70 days post-dosing. RESULTS: After a single intravenous or a subcutaneous administration of 200 mg belimumab, all 16 subjects completed the study. There were no serious adverse events or adverse events related to injection site reactions. All seven adverse events were considered mild or moderate in intensity and deemed unrelated to belimumab except for cellulitis following intravenous administration. The bioavailability of the single subcutaneous dose of 200 mg belimumab in the subjects was estimated to be 77·5%. Time to the maximum serum concentration after subcutaneous injection was 6·5 days (median). The geometric mean terminal half-life was comparable between the two administration routes (17·7 days intravenous and 15·9 days subcutaneous). Serum immunoglobulin G level decreased slightly after each treatment. No subjects were found to produce antibelimumab antibodies. WHAT IS NEW AND CONCLUSIONS: A favourable absolute bioavailability in healthy Japanese subjects was seen following a subcutaneous injection of 200 mg belimumab. Considering the intersubject variability, exposures were consistent with those previously observed in healthy non-Japanese subjects. Safety and biomarker data were also consistent with previous non-Japanese clinical studies.

The pharmacokinetic and safety profiles of zanamivir after single and repeat intravenous administration in healthy Japanese males.

WHAT IS KNOWN AND OBJECTIVE: Neuraminidase inhibitors are important options for the treatment of infection by the influenza virus. For the treatment of severe influenza, parenteral administration of a neuraminidase inhibitor may be desirable. This study was conducted to evaluate the pharmacokinetic and safety profiles of intravenous zanamivir, an influenza viral neuraminidase inhibitor, in Japanese subjects to further characterize these profiles particularly following relatively high-doses when compared with inhalation doses and to provide reassurance that there are no marked differences with profiles reported for other ethnically different populations. METHODS: Single doses of 100, 300, 600 mg zanamivir were administered to healthy Japanese men in a randomized, double-blind, ascending dose, placebo-controlled, incomplete three-period cross-over study. In period 3, subjects were given 600 mg of zanamivir on day 1, followed by a 60 h washout period and then a 5-day course of 600 mg zanamivir twice daily. Each subjects received two of three active dosages of zanamivir from 100, 300 and 600 mg, and placebo. RESULTS: Adverse events reported in the study were all mild in intensity and resolved without any treatment. The mean AUC0-∞ values after single intravenous administration of 100, 300 and 600 mg were 16768, 53462 and 100400 ng·h/mL, respectively, demonstrating dose proportionality. No accumulation or time variance was observed after 5 days of twice-daily administration of 600 mg zanamivir. Urinary concentrations of zanamivir after single doses ranging from 100 to 600 mg indicated that over 94% of the zanamivir administered was excreted in urine within 24 h. WHAT IS NEW AND CONCLUSION: Single and 5-day BID repeat dosing of 600 mg were safely administered in Japanese healthy subjects. The pharmacokinetic profile of zanamivir after intravenous administration was consistent with previously reported findings in non-Japanese subjects.

High levels of Ca(2+)-independent endonuclease activity capable of producing nucleosomal-size DNA fragmentation in non-adherent marrow mononuclear cells from patients with myelodysplastic syndromes and acute myelogenous leukemia.

The endogenous endonucleases capable of producing nucleosomal-size DNA fragmentation are considered candidates of the key enzyme of apoptosis. We examined these activities in the nuclear fraction of non-adherent marrow mononuclear cells (NonAd-MNCs) from patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) using a nuclear autodigestion method. We detected Ca2+/Mg(2+)-dependent endonuclease activity in all samples examined. In contrast, Ca(2+)-independent activity with the ability to produce nucleosomal-size DNA fragmentation was found only in samples from a proportion of patients with MDS (12 of 26 consecutive cases) and all the patients with AML (n = 6), but not in the samples from control group patients (n = 10). This activity was correlated with the percentage of bone marrow (BM) blast cells to some extent. Although the levels of these endogenous endonuclease activities seem not to be correlated directly with the susceptibility of the cells to apoptosis, we postulate that the Ca(2+)-independent endonuclease activity may be associated with apoptosis and/or cell proliferation. Further follow-up study of these patients may be meaningful to clarify the prognostic significance of the Ca(2+)-independent endonuclease activity in patients with MDS.

Marked apoptosis of human myelomonocytic leukemia cell line P39: significance of cellular differentiation.

Myelodysplastic syndrome (MDS)-derived leukemia cell line P39/Tsugane could be induced to apoptosis by a variety of agents including metabolic inhibitors, a calcium ionophore and differentiation-inducing agents. As evaluated by characteristic morphological changes and oligonucleosomal lengths DNA ladder, the levels of apoptosis in P39 cells induced by actinomycin D, or A23187, were far greater than in other myeloid lines examined in this study. When 22-oxa-1 alpha, 25(OH)2D3 (D3), dimethyl sulfoxide (DMSO) and all-trans retinoic acid (RA) were used as differentiation-inducers, varying degrees of apoptosis were seen. D3 induced monocytoid differentiation, but not apoptosis above the control level. On the other hand, RA induced profound apoptosis concomitant with the progressive expression of differentiation markers. Studies on morphology, functions and phenotypes of P39 cells exposed to differentiation inducers suggest that the incidence of apoptosis was not affected by the process of differentiation, but cells in the process of varying degrees of differentiation may die via apoptosis. Moreover, RA-treated P39 cells are unique in the simultaneous occurrence of profound apoptosis and differentiation. We propose that RA-treated P39 differentiation model is ideally suited for the study of MDS.

Translocon-associated protein alpha transcripts are induced by granulocyte-macrophage colony-stimulating factor and exhibit complex alternative polyadenylation.

The cloning of full length cDNA for the translocon-associated protein alpha subunit, previously called signal sequence receptor alpha, is reported as a result of differential display experiments in search of genes induced by granulocyte-macrophage colony-stimulating factor. Its messenger RNA was more abundant in growing cells than in either factor-deprived cells or quiescent cells and comprised four species, each having microheterogeneity, as a result of complex alternative polyadenylation apparently dependent on arrays of non-canonical polyadenylation signals. Radiation hybrid mapping of the gene showed that the gene is on the short arm of chromosome 6.

A kinetics approach to the characterization of an IgM specific for the glycolipid asialo-GM1.

The unique features of protein recognition of membrane-anchored glycolipids were investigated by surface plasmon resonance (SPR) monitoring of antibody interactions with glycolipids contained in liposomes. Several positive hybridomas belonging to the IgM and IgG classes were identified when tested for binding to the glycosphingolipid asialo-GM1 (Gal beta1-3GalNAcl beta1-4Gal beta1-4Glc beta1-1-Ceramide). Preliminary screening by enzyme immunoassay and thin layer chromatography (TLC) followed by immunostaining indicated that only those of the IgM type showed specificity for this glycosphingolipid. One of the IgMs, H2G10, was purified and further characterized using a SPR technique that involved antibody binding to liposomal asialo-GM1. This method generated kinetic and affinity constants for the interaction and confirmed the specificity of H2G10 for the terminal galactose of asialo-GM1. Interestingly, inhibition of antibody binding to asialo-GM1 liposomes by the asialo-GM1 tetrasaccharide reduced the total amount of bound antibody but increased the affinity of the antigen-antibody interaction due to an inverse relationship between tetrasaccharide concentration and the H2G10 dissociation rate constant. We believe that this effect is due to the selective inhibition of lower valency binding by the tetrasaccharide which, in turn, promotes higher avidity antibody-carbohydrate interactions. The observation that slower dissociation rate constants were also observed at high antigen to antibody ratios supports this interpretation. These results highlight the insight that kinetic data can provide in efforts to promote and inhibit high avidity interactions such as those involving proteins and carbohydrates.

Quantitative analyses of binding affinity and specificity for glycolipid receptors by surface plasmon resonance.

In recent years, rapid expansion has been seen of the application of SPR to a wide range of biomolecular interactions. For protein-carbohydrate interactions, SPR techniques offer the means of presenting glycolipids, and potentially glycoproteins, in an environment that closely resembles their in vivo situation. It is already clear that the technology can provide much additional insight into these interactions because it monitors the interactions under conditions that approach physiologic ones and can allow for the investigation of parameters that are not accessible by conventional approaches. Strategies for the immobilization of glycolipids on BIACORE sensor chips are not limited to the two approaches described here. For example, liposomes containing biotinylated phospholipid could be captured by avidin or streptavidin surfaces; this strategy has been used for the capture of natural membrane vesicles. Also, Biacore AB has announced plans to introduce a version of the CM5 chip that has been derivatized with an undisclosed group that mediates the capture of large amounts of liposomes.

Structure of extrachromosomal circular DNAs generated by immunoglobulin light chain gene rearrangements.

Recombination at the immunoglobulin kappa or lambda light chain locus generates extrachromosomal circular DNAs. We have isolated circular DNAs from adult mouse spleen cells and prepared a circular DNA clone library. We characterized four J kappa-positive and one J lambda 1-positive clones. The J kappa-clones contained both coding and signal joints of V kappa-J kappa joining, and the J lambda 1-clone contained a signal joint of V lambda 1-J lambda 1 joining. Genomic organization of the V kappa gene families used in these joints suggested the excision of circular DNA preceded by inversion. A specific dinucleotide (P) insertion in the coding joint was observed in two clones. Three coding joints were out of frame and one clone had an in-frame coding joint, although possibly combined with a pseudo-V kappa gene. These kappa-positive circular DNAs are possibly excised from the chromosome by secondary recombinations which replace non-productive primary rearrangements.

Excision products of immunoglobulin gene rearrangements.

We have isolated circular DNAs from splenocytes of euthymic and athymic mice, and prepared the DNA libraries of 1.5 X 10(6) clones. Hundreds of clones homologous to immunoglobulin (Ig) heavy chain segments (DSP2 and DQ52-JH) or light chain segments (J kappa and J lambda) have been identified. Southern hybridization predicted that three of 12 euthymic mouse clones homologous to DQ52-JH and four of 10 athymic mouse clones homologous to DSP2 contained reciprocal recombination products of D-J joining. Some of these clones were characterized by sequencing: two clones contained the precise excision product of the recombination of a DSP2 segment with either JH2 or JH3 segment; two clones showed imprecise ligation of the DSP2-JH2 coding joint and precise ligation of the DFL16.1-JH3 reciprocal joint in the same molecule. They seem to represent a replacement of the pre-existing DSP2-JH2 rearrangement by joining an upstream DFL16 segment to a downstream JH3 segment. The presence in extrachromosomal DNA of a reciprocal recombination product of DH-JH joining is consistent with the view that immunoglobulin genes, like T cell receptor (TCR) genes, can be rearranged in B cell lineage by the looping-out and excision of chromosomal DNA.

DNase I hypersensitivity analysis of the human CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene.

Human C/EBPepsilon is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue-specific manner; it is expressed almost exclusively in myeloid cells. To understand the mechanism by which the expression of C/EBPepsilon gene is controlled, we cloned a large genomic region surrounding the C/EBPepsilon gene and performed a DNase I hypersensitivity analysis of this locus. These sites probably represent areas of binding of proteins modulating gene transcription. Hypersensitive (HS) regions in 30 kb of DNA surrounding the C/EBPepsilon gene were examined in C/EBPepsilon high-expressing (NB4, HL-60), low-expressing (Jurkat), very-low-expressing (KG-1), and non-expressing (K562) hematopoietic cells as well as in non-hematopoietic-non-expressing cells (MCF-7, DU 145, PC-3). Three HS sites were detected near the first exon of C/EBPepsilon gene. They were found only in hematopoietic cells and were especially prominent in C/EBPepsilon expressing cells, suggesting that these sites play an important role in transcribing the gene. These hypersensitive bands did not change when the cells were cultured with retinoids. Gel-shift assays using 200 bp of nucleotide sequences that encompassed the hypersensitive sites and nuclear extracts from NB4 and Jurkat cells (C/EBPepsilon expressing) as well as K562 and MCF-7 cells (non-expressing) showed different retarded bands on gel electrophoresis. A fourth HS site, located about 11 kb upstream of exon 1, was found only in cells highly expressing C/EBPepsilon. Two sites, one about 4.5 kb upstream of exon 1 and another about 8.5 kb downstream of exon 2, were positive only in non-expressing cell lines, suggesting that repressors may bind in these areas. Taken together, we have found six specific DNase I hypersensitive sites in the region of C/EBPepsilon that may be involved in regulating transcription of this gene.

The relationship between homovanillic/vanillylmandelic acid ratios and prognosis in neuroblastoma.

A total of 44 cases with neuroblastoma cases (excluding true positive cases detected in mass screenings) who were born from 1979 to 1991, and had data concerning the clinical stage and values of vanillylmandelic acid (VMA) and homovanillic acid (HVA) at diagnosis (microg/mg creatinine) were followed up until the end of 1994. Deaths were confirmed using the record of vital statistics of the Hokkaido Government. The 60-month survival rate of those who had an HVA/VMA ratio of 1-2 was 80.0%. Conversely, those with ratios <1 or >2 had respective survival rates of 24.1% and 5.3%. Most of those with a ratio >2 died within 24 months of diagnosis. Many of the cases with a ratio <1 lived over 12 months but died within about 36 months. Many tumors of those cases with a ratio of 1-2 originated in extra-adrenal glands, and had negative n-myc amplification. Most of the patients with a ratio >2 were diagnosed at 1 year of age or older. The HVA/VMA ratio at diagnosis is useful in estimating both the survival period and the prognosis.

Initial medical management of patients severely irradiated in the Tokai-mura criticality accident.

A nuclear criticality accident occurred in Japan on September 30, 1999, which resulted in severe exposure of three victims to mixed flux of neutrons and gamma-rays. Estimated average doses for the three victims were 5.4 Gy of neutrons and 8.5 Gy of gamma-rays for Patient A, 2.9 Gy of neutrons and 4.5 Gy of gamma-rays for Patient B, and 0.81 Gy of neutrons and 1.3 Gy of gamma-rays for Patient C. They then suffered the consequences of the effects of ionizing radiation resulting in acute radiation syndrome. In Patients A and B, bone marrow failure was so severe that they received haematopoietic stem cell transplantation. The graft initially took successfully in both patients, although in Patient B it was later taken over by his own haematopoietic cells. They also suffered from severe skin lesions, later exhibited gastrointestinal bleeding and eventually died of multiple organ failure 82 and 210 days after the accident, respectively. The survival of these patients beyond the period of agranulocytosis means that bone marrow failure per se caused by exposure to ionizing radiation may now be overcome. Patient C also developed bone marrow failure and was treated with granulocyte colony-stimulating factor as well as supportive care. He recovered without major complications and is now under periodical follow-up. Remarkably, during the prodromal phase, all the patients exhibited hypoxaemia, two of whom also showed interstitial oedema of the lungs. In Patient C these manifestations improved within a week. The circumstances of the accident and the initial medical treatment of the victims are described.

[Relationship between the cell cycle and production of hCG in choriocarcinoma cells].

Human choriocarcinoma cells in vitro (ENAMI-1) were exposed to various concentrations of MTX (10(-8)M, 10(-7)M, 10(-6)M, 10(-4)M) for 48 hr, and after removal of MTX, cells were harvested and measured by FCM every 24 hr for 96 hr. Although the growth of cells were inhibited in MTX more than 10(-7)M in concentration, hCG-beta levels were elevated markedly. It was showed that hCG positive cells were accumulated in G1-phase by flow cytometric two color analysis with monoclonal antibody. In the presence of MTX more than 10(-7)M in concentration, the cells were accumulated in S-phase. After removal of 10(-7)M MTX, S-phase cells migrated toward G2 + M phase on the other hand in the presence of MTX more than 10(-6) M in concentration cells stopped moving to G2 + M phase. Immunoperoxidase with monoclonal antibodies against BrdU and hCG-beta has got the same phenomena resulted by FCM. It was confirmed that hCG was an useful marker for choriocarcinoma.

Determination of radionuclides produced by neutrons in heavily exposed workers of the JCO criticality accident in Tokai-mura for estimating an individual's neutron fluence.

In the Tokai-mura criticality accident, three workers were heavily exposed. Biological materials, such as blood, urine, vomit and hair, were collected from the workers and analyzed for radioactivities, produced by the neutron irradiation. Activation products. such as 24Na, -K and 82Br, were found in these materials by gamma-ray spectrometry. The radionuclide of the highest activity observed in biological materials was 24Na, e.g. the concentrations of this nuclide in the blood samples from the three patients at the accident time were 169, 92 and 23 Bq/ml, respectively. The concentrations of stable sodium in the same samples were determined by ICP-AES to obtain specific activities of 24Na (concentration ratio between the produced 24Na and stable 23Na), which are essential for estimating the neutron fluences and radiation doses. The specific activities of 24Na obtained for the three patients through the blood analysis were 8.2 x 10(4),4.3 x 10(4) and 1.2 x 10(4) Bq24Na/g23Na. Based on these values, individual's neutron fluences were estimated to be 5.7 x 10(11), 3.0 x 10(-1) and 0.85 x 10(11) cm(-2), respectively.

Bioassay for neutron-dose estimations of three patients in the JCO criticality accident in Tokai-mura by measuring beta-ray emitters.

The measurement of beta-emitters in biological samples (hair, urine and bone) from three patients in the JCO criticality accident was performed to assess the neutron dose to individuals. The result of the measurements of 32P in hair and urine collected immediately after the accident showed that sufficient 32P activities had been induced in the hair by fast neutrons and in the urine by thermal neutrons to know the severity of the exposure to the individuals and to the position. From the measurement of 32P and 45Ca in bone from various anatomical parts of two patients who died 82 and 210 days after the accident, it was suggested that the distribution of the induced beta-emitters activities could prove the position and posture of the patients at the moment of exposure.

Initial symptoms of acute radiation syndrome in the JCO criticality accident in Tokai-mura.

A criticality accident occurred on September 30, 1999, at the uranium conversion plant in Tokai-mura (Tokai-village), Ibaraki Prefecture, Japan. When the criticality occurred, three workers saw a "blue-white glow," and a radiation monitor alarm was sounded. They were severely exposed to neutron and gamma-ray irradiation, and subsequently developed acute radiation syndrome (ARS). One worker reported vomiting within minutes and loss of consciousness for 10-20 seconds. This worker also had diarrhea an hour after the exposure. The other worker started to vomit almost an hour after the exposure. The three workers, including their supervisor, who had no symptoms at the time, were brought to the National Mito Hospital by ambulance. Because of the detection of gamma-rays from their body surface by preliminary surveys and decreased numbers of lymphocytes in peripheral blood, they were transferred to the National Institute of Radiological Sciences (NIRS), which has been designated as a hospital responsible for radiation emergencies. Dose estimations for the three workers were performed by prodromal symptoms, serial changes of lymphocyte numbers, chromosomal analysis, and 24Na activity. The results obtained from these methods were fairly consistent. Most of the data, such as the dose rate of radiation, its distribution, and the quality needed to evaluate the average dose, were not available when the decision for hematopoitic stem cell transplantation had to be made. Therefore, prodromal symptoms may be important in making decisions for therapeutic strategies, such as stem-cell transplantation in heavily exposed victims.

[A case of rhabdomyolysis demonstrated by Tc-99m methylene diphosphate scintigraphy].

Technetium-99m phosphate compounds are useful for bone scintigraphy. Furthermore they occasionally demonstrated acute myocardial infarction and myocarditis as positive lesions. Also accumulations in the extraskeletal muscle have been reported using these compounds. This case was a 47-year-old male and had localized rhabdomyolysis, caused by compartment syndrome. We report the usefulness of technetium-99m methylene diphosphonate to diagnose the location of rhabdomyolysis.

[Multiparameter analysis of uterine cancer cells using flow-cytometry-computer].

In recent progress, Laser-based flow cytometric technique makes it possible to do quantitative determination of nuclear DNA-RNA content and proteins on monodisperse cell population simultaneously. Gynecologic cytology specimens that derived from the cervix and the endometrium were stained with a combination of propidium iodide for nuclear DNA-RNA and fluorescein isothiocyanate for protein in cells, then analyzed using a flow cytometry system with computer. These results included assessment of the presence or absence of malignant cells. They also demonstrated the dispersion contour of the normal squamous cell population, and inflammatory responding cell population. Measurements of clinical specimens were resulted with a 9.6% false negative rate and a 27.0% false positive rate (80.6% in accuracy rate) by comparison with routine visual cytologic evaluation of the same samples.

[Effects of cis-DDP in children with refractory malignant tumors].

Eighteen children with refractory malignant tumors were treated with cis-DDP. They included 10 neuroblastomas, 2 rhabdomyosarcomas, and one each of hepatoblastoma, yolk sac tumor, osteosarcoma and pinealoma. Of 7 cases treated only with cis-DDP, 2 were NC, 6 PD and none were CR or PR. Of 11 cases treated with cis-DDP combined with other agents, 7 were PR, 2 NC, 2 PD and none were CR. The major side effect of cis-DDP was renal toxicity, but this was not serious and only transient. Three cases were treated with the continuous intra-arterial infusion method. These included 2 neuroblastomas and one yolk sac tumor. This treatment was therefore highly effective for children with malignant tumors, and few side effects were observed.

[Cell kinetic effects of cis-platinum on cancer cells in a culture system and in cases of cervical cancer using monoclonal antibody of BrdU].

Flow cytometric analyses were done in cervical squamous cell carcinoma cell line (SKG-IIIa) and in cancer cells of cervical cancer cases treated with CDDP using monoclonal antibody of BrdU. 1) In fundamental studies using culture system, accumulation of late S phase cells and of mid S phase cells was observed after 48 hours at the dose of 0.2 micrograms/ml and 0.5 micrograms/ml of CDDP, respectively. 2) Accumulation of early S phase cells was observed at the condition of 1 microgram/ml and 3 micrograms/ml of CDDP. All S phase cells decreased gradually thereafter 3) In clinical studies, accumulation of early S phase cells was observed in cancer cells of cervical cancer cases after intra-arterial infusion of CDDP. However, cell cycle recycling was observed in tumor cells which retained their low sensitivity to CDDP after 21-day treatment with intra-arterial infusion of CDDP.