Quantitative enzyme immunoassay for human granulocyte colony stimulating factor (G-CSF).

Human granulocyte colony stimulating factor (G-CSF) was detected at the pM level by a simple sequential sandwich enzyme immunoassay. The polyclonal antiserum obtained by immunizing rabbits with recombinant G-CSF did not recognize other colony stimulating factors. The EIA developed gave satisfactory reproducibility as judged by an intra-assay precision of 4.3-6.2% and an interassay precision of 6.2-9.0%. Results from this assay procedure correlated well with those of a bioassay. The method could be performed within 6 h.

Pharmacokinetic studies of intravenous glycosylated recombinant human granulocyte colony-stimulating factor in various hematological disorders: inverse correlation between the half-life and bone marrow myeloid cell pool.

The pharmacokinetics of an intravenous bolus dose of glycosylated recombinant human G-CSF (rhG-CSF) was examined in 15 patients with various hematological disorders and 3 normal volunteers. The elimination half-life of rhG-CSF varied with the disorder. The half-life of an initial dose of rhG-CSF (2 micrograms/weight kg) was significantly prolonged in patients with aplastic anemia (2.7 +/- 0.3 h, n = 3) and myelodysplastic syndrome-refractory anemia (2.0 +/- 0.3 h, n = 3) when compared with those in normal controls (0.9 +/- 0.5 h, n = 3). In contrast, in patients with acute myelogenous leukemia which was overt leukemia from myelodysplastic syndrome-refractory anemia with excess of blasts in transformation, the half-life was shortened after chemotherapy (0.2 +/- 0.1 h, n = 3). The half-life of rhG-CSF in 2 patients with acute lymphoblastic leukemia in complete remission was prolonged (2.0 and 2.7 h) at the time of marrow-suppression after chemotherapy and then shortened (0.5, 1.0 h, respectively) in the recovery phase. The half-life of rhG-CSF was very weakly, inversely correlated with absolute neutrophil count in blood (n = 24, r2 = 0.32, P < 0.01), and was inversely correlated with the absolute count of bone-marrow myeloid cells (nucleated cell count in bone-marrow aspirates x the percentage of myeloid cells/100) of patients with aplastic anemia and myelodysplastic syndrome-refractory anemia (n = 12, r2 = 0.63, P = 0.002). These results suggest that the half-life of intravenously administered rhG-CSF (2 micrograms/kg) reflects the size of the myeloid cell compartment in vivo, and support the hypothesis that receptor-mediated consumption mainly accounts for the clearance of exogenous G-CSF.

Radioimmunoassay for erythropoietin using anti-recombinant erythropoietin antibody with high affinity.

A sensitive radioimmunoassay (RIA) for the detection of erythropoietin (EPO) was developed using anti-recombinant EPO antibody with high affinity. The sensitivity was 100 amol/tube (5 mIU/ml) and it was possible to detect a serum EPO level between 5 and 200 mIU/ml. This method enabled us to measure native EPO as well as recombinant EPO. With this method we determined serum EPO levels in healthy individuals and patients with chronic renal disease, rheumatoid arthritis and iron deficiency anemia. Values in patients with chronic renal disease were lower than those in healthy individuals, while values in patients with rheumatoid arthritis, or iron deficiency anemia were significantly higher than those in healthy individuals.

Interrelationship of cardiovascular effects, plasma levels of nicorandil, and vascular cGMP formation in conscious rats.

The relationship between the dual activity of nicorandil (KATP channel-opening activity and nitrate-like action), plasma levels, and changes in vascular cGMP levels and cardiovascular parameters was investigated in conscious rats. Nicorandil (3 mg kg(-1), p.o.) was rapidly absorbed and caused a significant reduction in blood pressure, lasting for at least 1 h, increases in heart rate and femoral blood flow, and decreases in femoral vascular resistance. These were entirely abolished by intravenous glibenclamide (20 mg kg(-1)). The plasma concentration of nicorandil reached a maximum 30 min after dosing. After administration of nicorandil, a correlation was observed between blood pressure and plasma nicorandil level or femoral vascular resistance. A significant increase (P < 0.05) in the cGMP content of the thoracic aorta occurred 15 min after administration of nicorandil, and persisted for at least 2 h. These results imply that nicorandil induces vasodilatation by opening KATP channels in peripheral resistance vessels, leading to overt reduction of blood pressure, but acts on conductance vessels mainly through nitrate-like activity.

Structure of G-CSF: significance of the sugar chain.

1. The carbohydrate chain protects rhG-CSF from polymerization and/or conformational alterations associated with physicochemical changes, elevation of pH or temperature fluctuations. 2. The carbohydrate chain of rhG-CSF prevents loss of its biological activity in normal human serum by inhibiting proteinase activity. 3. These facts indicate that the carbohydrate chain of rhG-CSF has a markedly important role in maintaining the stability of the protein itself as well as in effecting the exertion of its biological activity.

[The significance of serum gamma-seminoprotein in prostatic cancer].

The level of serum gamma-seminoprotein (gamma-Sm) was determined by enzyme immunoassay using an EIA gamma-Sm test kit in 32 patients with prostatic cancer (before treatment for 12 and after treatment was started for 20), 24 patients with benign prostate hypertrophy and 22 patients with other urogenital cancer. A gamma-Sm level of over 4.0 ng/ml was considered to be positive. The positive rate was 43.8% in prostatic cancer patients (83.3% before and 20.0% after treatment), 25.0% in benign prostate hypertrophy and 0% in other urogenital cancer. Since the positive rate of prostatic acid phosphatase (PAP) was 34.3% in prostatic cancer patients (75.0% before and 10.0% after treatment) and 16.7% in benign prostate hypertrophy patients, gamma-Sm may be more sensitive but less specific as an indicator of prostatic cancer in PAP. In 9 patients with prostatic cancer before treatment, the levels of serum gamma-Sm and PAP were serially determined for up to 11 months. The level of gamma-Sm decreased in 7 patients, and PAP in all patients after hormone therapy. One patient showed a consistently positive gamma-Sm level and the level of the others became positive only for gamma-Sm during follow-up. There was a statistical correlation between the levels of serum gamma-Sm and PAP in patients with prostatic cancer (r = 0.595, p less than 0.01), in patients with benign prostate hypertrophy (r = 0.882, p less than 0.01) and also in the patients in both groups together (r = 0.590, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

[A phase II study of Futraful suppository in head and neck cancer].

A phase II study of FT-207 suppository (Futraful Suppository) for head and neck cancer was conducted at the Kagoshima University Hospital Dept. of Otorhinolaryngology and its 6 affiliated hospitals. Forty cases with head and neck cancer were entered in the trial, of which 37 were perfectly evaluable. Partial response (PR) (16.7%) was achieved in one of 6 patients with maxillary cancer, in 2 of 9 cases with oral cancer (22.2%), in one of 7 with laryngeal cancer (14.3%), in 3 of 13 with pharyngeal cancer, while 2 of the same 13 achieved a complete response (CR) (38.5%), for a total response rate of 24.3%. FT-207 suppository was continuously administered as standard, at a dose of 750 mg each twice daily, or 1,500 mg/day. The response rate was dose-dependent; 2 CR cases with a total dose of 105 g for administration over a total period of more than 150 days. Adverse effects appearing in 35.0% of the overall were mainly anorexia, nausea and vomiting; none of them, however, were serious.

Cloning of a cDNA for a 1-aminocyclopropane-1-carboxylate synthase that is expressed during development of female flowers at the apices of Cucumis sativus L.

In cucumber (Cucumis sativus L.) plants, it has been reported that a high correlation existed between the evolution of ethylene from the apices and the development of female flowers. We isolated three distinct cDNA encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase from cucumber. Among of these, only CS-ACS2 mRNA was detected at the apices of gynoecious cucumber in which female flowers were developing. The expression of the CS-ACS2 gene was examined in the apices of three cucumber cultivars (Rensei, Ougonmegami 2-gou and Shimoshirazu) by RNA gel blot analysis. In these cultivars, both the timing and the levels of expression of the CS-ACS2 transcript were correlated with the development of female flowers at the nodes. Furthermore, the timing of the induction of expression of the CS-ACS2 gene at the apex corresponded to that of the action of ethylene in induction of the first female flower at the apex of gynoecious cucumber plants. These results suggest that the development of female flowers might be regulated by the level of CS-ACS2 mRNA at the apex.

Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites.

The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.

Isolation of a MADS-box gene (ERAF17) and correlation of its expression with the induction of formation of female flowers by ethylene in cucumber plants (Cucumis sativus L.).

Ethylene regulates sex expression in cucumber (Cucumis sativus L.) plants. When the apices of monoecious cucumber seedlings (cv. Shimoshirazu-jibai) were treated with the ethylene-releasing compound, ethephon, female flowers were induced at the nodes. To clarify the action of ethylene in the regulation of sex expression, we attempted to isolate genes whose expression changed during induction of the formation of female flowers at the apices of these cucumber plants upon treatment with ethephon. Using the differential-display method, we identified 20 clones (#1 to #20) that reflected differences in the accumulation of transcripts in apices treated or not treated with ethephon. Sequence analysis of cDNA fragments revealed that the cDNA #17 had the sequence of a MADS-box gene. We isolated the full-length cDNA and showed that it included both a MADS box and a K box, and the corresponding gene was designated ERAF17. We examined the expression of ERAF17 in the apices of cv. Shimoshirazu-jibai and in those of a gynoecious cultivar (Rensei). In these cultivars, the timing and levels of expression of the ERAF17 transcript were correlated with the development of female flowers. Induction of the synthesis of the ERAF17 transcript by ethephon occurred within 4 h of the start of treatment and continued for 4 days at least. Expression of ERAF17 at apices was localized in the floral buds of the gynoecious cultivar, and expression was maintained in female flowers thorough their development. Our results suggest that the induction of the formation of female flowers by ethylene might be regulated by the expression of ERAF17 in floral buds at the apices of cucumber plants and that expression of this gene might also be involved in the development of female flowers.

Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay.

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.

Metabolic activation of 1alpha-hydroxyvitamin D3 in human liver microsomes.

1. 1Alpha-hydroxyvitamin D3 (1alpha-OH-D3) is a synthetic prodrug of the active form of vitamin D3, and requires the hydroxylation at the C-25 position before eliciting its biological activity. 2. 25-Hydroxylation activities for 1alpha-OH-D3 were present in both microsomal and mitochondrial fractions of human liver. 3. To determine the P450 enzyme(s) involved in microsomal 25-hydroxylation, 14 P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2D6-Met, 2E1, 3A4, 4A11) were tested for their 25-hydroxylation activity of 1alpha-OH-D3. None catalysed the 25-hydroxylation reaction. 4. 1Alpha-OH-D3 in a high concentration (2.5 ng ml(-1)) showed small but significant inhibition of the catalytic activities of CYP2C8, 2C9-Cys, 2C19, 2D6-Val and 2E1 for their typical substrates. However, 1alpha-OH-D3 in a clinically used low concentration will not significantly affect drug metabolism catalysed by the 14 P450s tested. 5. In summary, the 25-hydroxylation activity of 1alpha-OH-D3 that localizes in the microsomal fraction appears to be attributable to a cytochrome P450 other than the microsomal forms tested in this study.

Radioimmunoassay method for the determination of 22-oxacalcitriol, a novel analog of 1 alpha,25-dihydroxyvitamin D3 having valuable clinical potency, in rat and human plasma.

22-Oxacalcitriol is a synthetic analog of 1 alpha,25-dihydroxyvitamin D3, which is expected to be a novel agent for the treatment of patients with secondary hyperparathyroidism, psoriasis, and/or breast cancer. A sensitive and practical RIA for 22-oxacalcitriol in rat and human plasma has been developed. A rabbit antiserum raised against 22-oxacalcitriol 3-hemiglutarate conjugated with bovine serum albumin was used in combination with a tritium-labeled 22-oxacalcitriol having high specific radioactivity. A plasma sample was extracted with ethyl ether and the extract was successively purified with Bond Elut C18 and a normal-phase HPLC prior to the RIA to remove interfering substances. Accuracy of the RIA was assessed by various studies including serial dilution and recovery tests. Intra- and inter-assay coefficients of variation were lower than 10%, and the quantification limit was low enough for practical use (33 pg/ml plasma). The present RIA will be useful for the pharmacokinetic studies of 22-oxacalcitriol in both preclinical and clinical stages.

Microaggregate of immunostained macrophages in noninflamed gastroduodenal mucosa: a new useful histological marker for differentiating Crohn's colitis from ulcerative colitis.

OBJECTIVE: In 10% of cases it may be difficult to differentiate Crohn's colitis from ulcerative colitis. Distinguishing the two conditions is important because they are distinct entities with different therapeutic implications. Noncaseating granulomas are usually considered diagnostic of Crohn's disease. We previously reported that the presence of a microaggregate of immunostained macrophages within the noninflamed gastroduodenal mucosa was a characteristic finding of Crohn's disease. The aim of this study was to determine whether a microaggregate of immunostained macrophages can be a reliable marker for differentiating Crohn's colitis from ulcerative colitis. METHODS: We investigated the presence of microaggregates of immunostained macrophages and epithelioid cell granulomas in biopsy specimens taken from the noninflamed gastroduodenal mucosa of 22 known Crohn's colitis patients and 23 established ulcerative colitis patients. The incidence of microaggregates and granulomas was compared between these two groups. RESULTS: Microaggregates and granulomas were detected only in the Crohn's colitis patients. In addition, the presence of microaggregates was more frequent than that of granulomas in Crohn's colitis patients (54.5% and 18.2%, respectively, 95% confidence interval for the difference: 10.0-62.7%). CONCLUSION: Detecting a microaggregate of immunostained macrophages in a biopsy specimen taken from noninflamed gastroduodenal mucosa seems to be a useful method for differentiating Crohn's colitis from ulcerative colitis.

Vascular levels and cGMP-increasing effects of nicorandil administered orally to rats.

We examined a relation between cyclic guanosine monophosphate (cGMP) production in thoracic aorta, as an indicator probably reflecting the vascular response, and the vascular as well as plasma levels of nicorandil administered orally to rats. Nicorandil (3 mg/kg) given orally was rapidly absorbed, reaching the maximal plasma (approximately 2,600 ng/ml) and vascular concentrations (approximately 176 ng/g) at 15 min after the dosing and thereafter decreased rapidly. Even 2 h after the dosing, the level of the vascular cGMP formation in vivo remained significantly higher (approximately 1,000 fmol/mg increase from the control level) in the nicorandil-treated group, compared with the vehicle-treated one, and was enough to develop pronounced muscle relaxation in in vitro aortic preparations. However, it seems that the vascular cGMP increase in vivo was not always correlated to the plasma concentration of nicorandil, because the plasma concentration (approximately 750 ng/ml corresponding to 3.5 microM) at 2 h after the dosing, caused only relatively low cGMP production (300-400 fmol/mg increase from the control level), when tested in in vitro aortic preparations. Our study may indicate, therefore, that the vascular cGMP elevation in vivo is due to the content of nicorandil effectively remaining at its vascular targets of action as well as the plasma nicorandil concentration.

Synthesis and pharmacokinetics of 1 alpha-hydroxyvitamin D3 tritiated at 22 and 23 positions showing high specific radioactivity.

A novel synthesis of a radioactive compound of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-(3)H4]1 alpha OHD3) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22,23-(3)H4]1 alpha OHD3 (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studies with rats. In the pharmacokinetics studies, the plasma concentration level of the active form of vitamin D3, 1 alpha,25-dihydroxy-vitamin D3 [1 alpha,25(OH)2D3], after oral or intravenous administration of [22,23-(3)H4]1 alpha OHD3 (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1 alpha,25(OH)2D3 tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1 alpha OHD3 (1) over the treatment of 1 alpha,25(OH)2D3 (2).