Efficacy and immunomodulatory actions of ONO-4641, a novel selective agonist for sphingosine 1-phosphate receptors 1 and 5, in preclinical models of multiple sclerosis.

ONO-4641 is a next-generation sphingosine 1-phosphate (S1P) receptor agonist selective for S1P receptors 1 and 5. The objective of the study was to characterize the immunomodulatory effects of ONO-4641 using preclinical data. ONO-4641 was tested in both in-vitro pharmacological studies as well as in-vivo models of transient or relapsing-remitting experimental autoimmune encephalomyelitis (EAE). In vitro, ONO-4641 showed highly potent agonistic activities versus S1P receptors 1 and 5 [half maximal effective concentration (EC(50) ) values of 0·0273 and 0·334 nM, respectively], and had profound S1P receptor 1 down-regulating effects on the cell membrane. ONO-4641 decreased peripheral blood lymphocyte counts in rats by inhibiting lymphocyte egress from secondary lymphoid tissues. In a rat experimental autoimmune encephalomyelitis (EAE) model, ONO-4641 suppressed the onset of disease and inhibited lymphocyte infiltration into the spinal cord in a dose-dependent manner at doses of 0·03 and 0·1 mg/kg. Furthermore, ONO-4641 prevented relapse of disease in a non-obese diabetic mouse model of relapsing-remitting EAE. These observations suggest that ONO-4641 may provide therapeutic benefits in the treatment of multiple sclerosis.

Stabilization of egg phosphatidylcholine liposomes by the insertion of sulfatide.

The characteristic effects of sulfatide and its derivatives on the stability of small unilamellar vesicles of egg phosphatidylcholine liposomes in phosphate-buffered saline were investigated by measuring the leakage of carboxyfluorescein that had been entrapped in these vesicles. We found that both the sulfate group and a long acyl chain, such as lignoceric acid, of the sulfatide are essential for the stabilization. The sulfatide derivatives that contain a somewhat shorter acyl chain such as stearic acid had no effect to suppress the leakage of carboxyfluorescein. The galactose residue of sulfatide is not essential to suppress the leakage. 1H-NMR study using a paramagnetic shift reagent demonstrated that the distribution of phosphatidylcholine in the vesicles containing sulfatide is homogeneous, which seems to contribute to the stability of the membrane.

Five jumps per day increase bone mass and breaking force in rats.

The effects of jump training on bone morphological and mechanical properties were investigated in immature bones of female Fischer 344 rats. Five-week-old rats were divided into control or five jump-trained groups comprised of 5-, 10-, 20-, 40-, and 100-jump groups, representing the number of jumps per day. The rats were jump-trained 5 days/week for 8 weeks, and the height of jump was increased to 40 cm progressively. The femur and tibia in the 5-jump group had significantly greater fat-free dry weights per body weight and maximum loads at the fracture tests than those in the control group. The tibia in the 5-jump group also had significantly larger cortical area at the cross-sectional analysis. Although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 10-, 20-, and 40-jump groups. The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.

Modification of acyl-plasmin-streptokinase complex with polyethylene glycol. Reduction of sensitivity to neutralizing antibody.

Acyl-plasmin-streptokinase complex has advantages as a 'site' directed fibrinolytic agent with the active site protected from the plasma protease inhibitors. But, in clinical use, the fibrinolytic potential of this acyl-enzyme complex is modified or abolished by the presence of streptokinase antibodies in the patients. Therefore, better therapeutic agents are required. In this work, chemical modification of the acyl-plasmin-streptokinase complex with polyethylene glycol was found to result in marked resistance to neutralization with streptokinase antibodies.

Sequence of an intestinal cDNA encoding human motilin precursor.

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.

Effects of a new aldose reductase inhibitor, (2S, 4S)-6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-ca rboxamid e (SNK-860), on the slowing of motor nerve conduction velocity and metabolic abnormalities in the peripheral nerve in acute streptozotocin-induced diabetic rats.

The effects of a new aldose reductase inhibitor (ARI), (2S,4S)-6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-ca rboxamide (SNK-860), on the slowing of motor nerve conduction velocity (MNCV) and metabolic abnormalities in sciatic nerve were investigated in acute streptozotocin (STZ)-induced diabetic rats. MNCV in the diabetic rats was significantly slower 2 weeks after STZ injection. In the following 2 weeks, treatment with SNK-860 improved MNCV in a dose-dependent manner. The efficacy of 1 mg/kg SNK-860 was equipotent to that of 20 mg/kg sorbinil. Four weeks after STZ injection, increases in sorbitol levels, decreases in myo-inositol levels, and reductions in Na+, K(+)-adenosine triphosphatase (ATPase) activity were observed in sciatic nerves of diabetic rats. Administration of SNK-860 for 14 days beginning 2 weeks after the induction of diabetes inhibited these metabolic abnormalities in a dose-dependent manner. SNK-860 restored all of these parameters to normal levels at a dose of 2 mg/kg. In addition, close correlations were observed between MNCV and sorbitol levels (r = -.95) and between MNCV and myo-inositol levels (r = .93) in the sciatic nerve; a close correlation was also observed between sorbitol and myo-inositol levels in the sciatic nerve (r = -.86). Therefore, it is suggested that the effect of SNK-860 on the slowing of MNCV results from normalizing the above-mentioned metabolic abnormalities in the sciatic nerve of diabetics. Thus, SNK-860 may be useful in the treatment of diabetic neuropathy.

Effects of four stereoisomers of beta-hydroxyglutamic acid on identifiable giant neurons of an African giant snail (Achatina fulica Férussac).

The effects of four stereoisomers of beta-hydroxyglutamic acid (erythro-L-BHGA, threo-L-BHGA, erythro-D-BHGA and threo-D-BHGA) and glutamic acid (L-Glu and D-Glu) were compared on six identifiable giant neurons of Achatina fulica Férussac, namely, PON (periodically oscillating neuron), VIN (visceral intermittently firing neuron), d-RPLN (dorsal-right parietal large neuron), RAPN (right anterior pallial neuron), FAN (frequently autoactive neuron) and v-RCDN (ventral-right cerebral distinct neuron). On PON and VIN, erythro-L-BHGA was the most inhibitory (effective potency quotient (EPQ) of the substance: 1.0). Its minimum effective concentration (MEC) for both neurons was the same at 3 X 10(-5) M. The effect of threo-L-BHGA was weaker (EPQ in relation to the most effective substance: 0.3-0.1). Erythro- and threo-D-BHGAs were almost ineffective on PON (EPQ: less than 0.03) though they had some effects on VIN (EPQ: 0.1-0.03 for erythro-D-BHGA and 0.3-0.1 for threo-D-BHGA). On d-RPLN, erythro-L-BHGA was the most excitatory (MEC: 10(-4)M). Threo-L-BHGA and threo-D-BHGA also had some effects (EPQ: 0.1 and 0.3 respectively), while erythro-D-BHGA was ineffective. On RAPN, erythro- and threo-L-BHGAs were the most excitatory (MEC: 10(-4) M). Threo-D-BHGA was also excitatory (EPQ: 0.3), while erythro-D-BHGA was ineffective. Threo-L-BHGA was the most inhibitory on FAN and v-RCDN (MEC: 10(-4) M in FAN and 10(-5) M in v-RCDN).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of compounds related to beta-hydroxyglutamic acid (BHGA) on identifiable giant neurones of an African giant snail (Achatina fulica Férussac).

The present study aimed to further elucidate the pharmacological features, with respect to sensitivity to L-BHGA agonists, of the receptors sensitive to beta-hydroxy-L-glutamic acid (L-BHGA) in five Achatina giant neurones: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these neurones, d-RPLN and RAPN were depolarized by L-BHGA, while PON, VIN and v-RCDN were inhibited. Threo-beta-hydroxy-DL-aspartic acid markedly depolarized d-RPLN and RAPN (effective potency quotient (EPQ) in relation to the more effective L-BHGA isomer: 1 for d-RPLN and 0.3 for RAPN). This compound produced only slight inhibitory effects on PON, VIN and v-RCDN with EPQs calculated to be less than 0.03, less than 0.03 and 0.03, respectively. On the other hand, erythro-beta-hydroxy-DL-aspartic acid at 10(-3) M was almost ineffective, except on v-RCDN where it elicited some slight inhibitory effects (EPQ: 0.01). L-Aspartic and D-aspartic acid at 10(-3) M, also had almost no effect except for slight effects of D-aspartic acid on d-RPLN (EPQ: 0.1). N-Methyl-L- and N-methyl-D-aspartic acid were slightly effective only on v-RCDN (EPQ: less than 0.01 and 0.01, respectively). The other compounds, including beta-hydroxypyrroglutamic acid (cyclic BHGA) and proline derivatives, were almost ineffective at 10(-3) M; very weak effects were occasionally observed on some neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of long-term treatment with a new aldose reductase inhibitor, (2S,4S)-6-fluoro-2',5'-dioxospiro-[chroman-4,4'-imidazolidine]-2-carbox amide (SNK-860), on peripheral neuropathy in streptozotocin-induced diabetic rats.

We studied the long-term effects of a new aldose reductase inhibitor, (2S,4S)-6-fluoro-2',5'-dioxospiro-[chroman-4,4'-imidazolidine]-2- carboxamide (SNK-860), on functional, biochemical, and structural changes in peripheral nerve of streptozotocin (STZ)-induced diabetic rats. During the experimental period of 26 weeks, the delayed motor-nerve conduction in diabetic rats was significantly prevented by SNK-860 treatment, and elevated sorbitol levels and reduced myo-inositol levels were normalized to 100% and 71% of control levels, respectively. Teased nerve fiber studies demonstrated that the frequency of abnormal fibers was significantly reduced in treated diabetic rats. Morphometric analysis of myelinated fibers also disclosed prevention of axonal atrophy, distorted axonal circularity and preservation of large-sized fibers following SNK-860 treatment. These results suggest that long-term treatment with SNK-860 has a beneficial preventive effect on the development of experimental diabetic neuropathy.

Pharmacokinetic studies of human urinary kininogenase in healthy volunteers and animals.

Plasma concentrations of human urinary kininogenase (HUK) were determined in healthy volunteers during and after intravenous (iv) infusion by enzyme immunoassay (EIA). Plasma kinin concentrations were also determined by radioimmunoassay (RIA), and related to HUK concentrations. When HUK was infused [at 0.04, 0.075, 0.15, and 0.3 p-nitroaniline unit (PNAU)/body] over 30 min, plasma HUK concentration rapidly increased and reached a maximum at the end of dosing. Then, the concentration of HUK in plasma decreased biexponentially, and the elimination half-life of the terminal phase was found to be approximately 170 min. The area under the curve of concentration versus time from 0 to 180 min (AUC0-180min) and the maximum concentration (Cmax) increased in proportion to the dose, whereas the pharmacokinetic parameters [mean residense time (MRTinf) = 200-270 min, plasma clearance (CLp) = 2.5-3.3 mL/min/kg, volume of distribution at steady state (Vdss) = 470-730 mL/kg] did not very significantly within the dose range of the present study. On the other hand, when HUK was infused (at 0.15 PNAU/body), plasma kinin concentrations reached approximately 2 ng of bradykinin eq/mL 15 min after the onset of administration. This concentration was maintained during the dosing period, after which kinin was rapidly eliminated, and its concentration returned to baseline at 10 min after dose withdrawal. Plasma kinin concentrations at 15 to 30 min after the onset of dosing (at 0.075, 0.15, and 0.3 PNAU/body) increased in proportion to the dose. The pharmacokinetic parameters of HUK (MRTinf, CLp, Vdss) were compared with those of rats, rabbits, and dogs (log-log plots of body weight versus MRTinf, CLp, and Vdss). The Vdss value showed a good correlation (r = 0.996 for n = 4) with the body weight of respective animal species, the correlation with CLp was weak (r = 0.911), and MRTinf did not exhibit any correlation.

Effect of Clostridium perfringens-derived wound healing substance as compared with epidermal growth factor on the growth and morphological transformation of BALB/3T3 A31-1-1 cells.

Clostridium perfringens-derived wound-healing substance (WHS), having growth-stimulating activity, was examined to determine its effect on the growth and morphological transformation of BALB/3T3 A31-1-1 cells. WHS accelerated the cell growth at the exponential growth phase, shortening the doubling time by 8-18%. The maximum cell density of the treated cultures was slightly higher than that of the control culture, and the cell number decreased in the same way as the control cells did. On the other hand, the cells treated with epidermal growth factor (EGF) or insulin showed growth rates similar to that of the control cells during the exponential growth phase, and after the control cells attained the maximum cell number, the number of the treated cells continued to increase gradually for more than 4 days and then decreased. Under the experimental conditions of the two-stage transformation assay, application of WHS at the tumor-initiation or promotion stage did not accelerate the formation of transformed foci. Although treatment with EGF at the initiation stage induced no enhancement, marked enhancement of morphological transformation was observed in the treatment at the promotion stage. These results indicate that the mode of action between WHS and EGF or insulin is different on the growth-stimulating activity and morphological transformation of BALB/3T3 A31-1-1 cells.

Synthesis and cytokinin activity of fluorescent 7-Phenylethynylimidazo[4,5-b]pyridine and its riboside.

7-Phenylethynylimidazo[4,5-b]pyridine and its riboside have been newly developed as fluorescent carbon-substituted cytokinin analogues. Palladium-catalyzed coupling of 7-iodo-3-(tri-O-acetyl-beta-D-ribofuranosyl)imidazo[4,5-b]pyridine with phenylacetylene followed by ammonolysis afforded the 7-phenylethynyl riboside via its tri-O-acetate. Acid hydrolysis of the riboside provided its free base, which showed a marked enhancement in fluorescence intensity in an aqueous alkaline solution. The free base and its riboside were more active than the corresponding 6-phenylethynylpurine and its riboside, respectively, in Amaranthus betacyanin and tobacco callus bioassays. Surprisingly, the imidazo[4,5-b]pyridine base exhibited strong cytokinin activity comparable to that of N(6)-benzyladenine in the tobacco callus bioassay. This compound would be useful for studying localization and transport of cytokinins in cells or tissues of plants.

Effects of an aldose reductase inhibitor, SNK-860, on the histopathological changes of retinal tissues in a streptozotocin-induced diabetic rat model.

In order to clarify the mechanism of retinal tissue damage in diabetes mellitus, the effects of the inhibition of aldose reductase on the pathologic changes in the retina of streptozotocin-induced diabetic (STZ-diabetic) rats were examined histologically and histochemically. The STZ-diabetic animals were maintained with and without peroral administration of an aldose reductase inhibitor, SNK-860, and their retinas were examined microscopically after 12 months. Several abnormal changes observed; folding and edema in the retina, loss of pericytes in the retinal capillary walls, and thickening of basement membranes in the retinal capillaries, were significantly inhibited by SNK-860. Some of these changes were similar to those that had been previously noted in diabetic and galactosemic rats. These data suggest that the enhanced polyol metabolism may be involved in the diabetic changes of the retina.

Influence of propagermanium (SK-818) on chemically induced renal lesions in rats.

A histopathological study was performed to examine the influence of propagermanium and germanium dioxide (GeO2) on chemically induced renal lesions in rats. Animals were treated with adriamycin or mercuric chloride to induce glomerular or proximal tubular damage, and then given drinking water containing propagermanium (480 or 2,400 ppm solution) or GeO2 (300 or 1,500 ppm solution: equivalent to propagermanium in terms of germanium contents). The distal tubular epithelium after 8 weeks dosage with the 1,500 ppm solution of GeO2 was characterized by vacuolization and deposits of PAS-positive material not only in adriamycin-treated rats, but also in normal rats. In contrast, propagermanium administration was not associated with any alternation in the changes induced by adriamycin or mercuric chloride. We previously clarified that propagermanium had no biochemical influence on the renal function of these renal injured rats. The histological demonstration that this compound does not exert renal toxicity, even when given at a high dosage to renal injured rats, further indicates that it would not exacerbate renal dysfunction already present. This confirms that propagermanium may be a safe compound for use in individuals with compromised kidneys.

[Measurement of small bowel transit time in dogs with acetaminophen and indocyanine green].

We developed an experimental method to measure the gastric and the small bowel transit times in dogs without using X-ray nor radioisotopes. The dog with the ileostomy was given food containing acetaminophen (APAP) and indocyanine green (ICG), then we sampled blood and ileal contents of the dog. We could measure the gastric transit time by detecting plasma APAP and the small bowel transit time by detecting ICG in the intestinal contents. Using this experimental system, we examined the times after feeding milk or solid meal. The gastric transit time after taking solid meal was longer than that after taking milk, but this difference was not significant, and the small bowel transit time after taking solid meal was significantly shorter than that after taking milk.