Effects of salicylate and other enhancers on rectal absorption of erythropoietin in rats.

To develop a new treatment for patients with anaemia in which erythropoietin (EPO) can be given without injection, the effects of promoters of the rectal absorption of EPO were studied. Recombinant human (rHu) EPO (5000 units) in a dosing solution or in a rectal suppository was placed in the rectum of healthy rats and changes in serum EPO levels were monitored by an enzyme-linked immunosorbent assay. Without a promoter, rHuEPO was not absorbed. Sodium glycocholate, sodium caprate, and sodium salicylate in the solution of rHuEPO increased the absorption of rHuEPO. Sodium salicylate or sodium caprate in the suppository with rHuEPO also increased its absorption. The bioavailability of rHuEPO in a suppository containing 5% sodium salicylate compared with that by an intravenous injection was 1.2%. rHuEPO given in rectal suppositories containing sodium salicylate and inserted once a day for 6 consecutive days increased erythropoiesis in peripheral blood.

Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties.

Erythropoietin (Epo) has three N-linked sugar chains. Codons for asparagine at N-glycosylation sites in genomic human Epo DNA were replaced with those for glutamine. The wild-type Epo gene and seven mutants that lacked N-glycosylation sites in every possible combination were introduced into baby hamster-kidney cells. To study the role of the N-linked sugars in Epo biosynthesis, Epo protein expressed transiently was measured by an enzyme-linked immunoassay. The elimination of all three N-glycosylation sites decreased Epo production to 10% of that of the wild-type Epo. Wild-type and mutant Epos produced by stably transfected cells were partially purified to investigate their properties. Removal of N-glycosylation sites changed affinity of Epo to the receptor. The in vitro activity of Epo that lost all N-glycosylation sites was comparable with that of the wild-type Epo, while the in vivo activity severely decreased. These results indicate that N-linked sugars of Epo have two major functions; N-linked sugars are important for 1) proper biosynthesis and/or secretion and 2) expression of the in vivo activity probably by enhancing survival in the circulation. N-Linked sugars of Epo affect binding affinity of the ligand to the receptor but do not play a key role in expression of the in vitro activity.

Factor with erythroid burst-promoting activity in human urine unlike other hematopoietic growth factors.

A factor with burst-promoting activity (BPA) stimulates the formation of erythroid bursts in the presence of erythropoietin, acting on early erythroid progenitor cells (erythroid burst-forming units, or BFU-E). Here we investigated the biological properties of this factor partially purified from the urine of anemic patients. The human urinary factor did not cause the formation of late erythroid progenitor cells (erythroid colony-forming units, or CFU-E) or enhance such colony formation in the presence of erythropoietin. Thus, the urinary factor was a different substance from erythroid potentiating activity and from activin, which act on both BFU-E and CFU-E. The urinary factor promoted the colony formation of BFU-E from both humans and mice, but the human hematopoietic growth factors such as recombinant interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor did not stimulate the formation of BFU-E derived colonies from mice. The results suggested that the factor in the urine of anemic patients was different from the hematopoietic growth factors identified so far.

Renal anemia in polycystic kidney disease mouse.

DBA/2FG-pcy mice developed the chronic renal failure by the progressive polycystic formation at five months old. Their bilaterally enlarged kidneys occupied about 15% of the body weight. It was about 9 times larger than the normal kidney of DBA/2N mice. A large number of various-sized cysts appeared in cortex and medulla of bilateral light-yellow kidneys of sponge-like shape. Blood urea nitrogen and creatinine increased. Red blood cells (746 +/- 39 x 10(4)/mm3), hemoglobin (8.8 +/- 0.4 g/dl) and hematocrits (31.1 +/- 1.5%) were lower than those of normal control mice. Serum erythropoietin level and reticulocytes did not increase. In addition, the treatment with exogenous erythropoietin improved the anemia in DBA/2FG-pcy mice. It was suggested that the anemia in DBA/2FG-pcy mice was due to the disorder of erythropoietin production caused by the progressive polycystic formation in kidneys.

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth.

Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm3 per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed.

Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using a ceramic bed reactor.

Ceramic pieces composed of 99.5% Al2O3, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl2, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (phi = H = 150 mm), increasing dilution rate to 0.5 day-1 could reduce product inhibition at 3-4 x 10(5) cells/ml. Cellular productivity of 560 IU/10(6) cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.

The analysis of the defense mechanism against indigenous bacterial translocation in X-irradiated mice.

The defense mechanism against indigenous bacterial translocation was studied using a model of endogenous infection in X-irradiated mice. All mice irradiated with 9 Gy died from day 8 to day 15 after irradiation. The death of mice was observed in parallel with the appearance of bacteria from day 7 in various organs, and the causative agent was identified to be Escherichia coli, an indigenous bacterium translocating from the intestine. Decrease in the number of blood leukocytes, peritoneal cells and lymphocytes in Peyer's patches or mesenteric lymph nodes was observed as early as 1 day after irradiation with 6 or 9 Gy. The mitogenic response of lymphocytes from various lymphoid tissues was severely affected as well. The impairment of these parameters for host defense reached the peak 3 days after irradiation and there was no recovery. However, in vivo bactericidal activity of Kupffer cells in mice irradiated with 9 Gy was maintained in a normal level for a longer period. It was suggested that Kupffer cells play an important role in the defense against indigenous bacteria translocating from the intestine in mice.

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone.

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.

14 alpha-hydroxyandrost-4-ene-3,6,17-trione as a mechanical-based irreversible inhibitor of estrogen biosynthesis.

Various derivatives of androst-4-ene-3,17-dione derived from microbial transformation were evaluated as inhibitors of human placental aromatase. 14 alpha-Hydroxyandrost-4-ene-3,6,17-trione was the most potent inhibitor showing a time-dependent, pseudo-first-order inactivation of aromatase in the presence of reduced nicotinamide adenine dinucleotide phosphate with apparent Ki of 1.3 microM and Kinact of 0.23 min-1. This compound also inhibited aromatase in rat ovary and suppressed serum estradiol levels in in vivo experiments.

DNA breaks and repair in the mouse leukemia L1210 cells exposed to three different types of interstrand DNA cross-linkers.

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal 'hit' directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5-10 micrograms/ml MMC and with 10-100 micrograms/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 micrograms/ml MMC and 30 micrograms/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 micrograms/ml MMC and slightly inhibited to 70% of control in those treated with 100 micrograms/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 micrograms/ml SN-07.

Effects of a M1 muscarinic receptor agonist on the central cholinergic system, evaluated by brain microdialysis.

The effects of a novel M1-receptor agonist, AF102B (FKS-508; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine), on the central cholinergic system in vivo were evaluated by determination of acetylcholine (ACh) content in the rat brain after microwave irradiation and by measurement of ACh release with microdialysis perfusion in freely moving rats. Intraperitoneal administration of AF102B resulted in a significant decrease of ACh content in the brain, while AF102B produced an increase of in vivo ACh release. The present results suggest that ACh content in the brain after treatment with muscarinic agents may be related to the changes of ACh release, in which both M1 and M2 muscarinic receptors may be involved.

The role of carbohydrate in recombinant human erythropoietin.

1. Recombinant human erythropoietin has N-linked sugar [Tsuda et al., (1988) Biochemistry 27, 5646-5654]. Here we have demonstrated the presence of O-linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Gal beta(1-3)GalNAc. 2. To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoietin from which N-linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N-linked sugar abolished the in vivo biological activity completely, indicating that the presence of N-linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin. 3. Incubation of asialo-erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 degrees C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.

Central muscarinic activities of an M1-selective agonist: preferential effect on reversal of amnesia.

Effects of FKS-508 (AF102B; cis-2-methylspiro (1,3-oxathiolane-5,3')-quinuclidine), a novel M1-selective agonist, on central muscarinic responses in mice were examined in comparison with oxotremorine. FKS-508 was slightly less potent (6 times) in reversal of scopolamine-induced amnesia (passive avoidance failure), but far less potent (260 and 55 times) in producing hypothermia and tremor than oxotremorine. These results show that the selective M1 agonist FKS-508 differentiates highly between the central muscarinic effects.

Beneficial effects of FKS-508 (AF102B), a selective M1 agonist, on the impaired working memory in AF64A-treated rats.

The effects of FKS-508 [AF102B; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine], a selective M1 muscarinic receptor agonist, were examined to predict the possible activity on memory disorders using a T-maze and radial-arm maze task in experimental amnesia models. The amnesia models were produced by bilateral intracerebroventricular injection of ethylcholine aziridinium ion (AF64A), a selective cholinotoxin, in rats. Repeated administrations of FKS-508 (5 mg/kg/day, i.p.) for 5 weeks significantly ameliorated impaired performance of AF64A-treated rats (AF64A-rats) in a delayed alternation task in the T-maze. Repeated administrations of FKS-508 (1 and 5 mg/kg/day, p.o.) for 5 weeks significantly ameliorated acquisition failures of AF64A-rats in a radial-arm maze task. Single administration of FKS-508 (1 and 5 mg/kg, p.o.) significantly reduced the incorrect choices of AF64A-rats in a radial-arm maze task with 6 hr-delay time. No abnormalities in general behaviors, such as loss of appetite and ataxia, were observed in rats treated with FKS-508 repeatedly during 5 weeks. Our present results showed that FKS-508 can ameliorate memory impairments in AF64A-rats with central cholinergic hypofunction without causing any behavioral abnormalities. FKS-508 may be considered as a candidate for the clinical examination of the cholinergic hypothesis of senile dementia of the Alzheimer type.

Nucleotide sequence of SN-07 chromophore binding site.

DNase I footprinting was used to investigate binding sites for SN-07 chromophore on DNA fragments prepared from pBR322. Six sites were protected on about 150 base pair DNA fragments by SN-07 chromophore, but not by related anthracycline antibiotics from DNase I digestion. All the protected sites contained the dinucleotide sequence 5'-GC-3', but no other regularities could be discerned. A drug-induced conformational change of DNA was suggested by enhancement of DNase I sensitivity between the protected sites. These results support covalent interaction of the carbinolamine function of SN-07 chromophore to 2-amino group of guanine residues.

Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants.

We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = approximately 2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.