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.

Stereospecific interaction of a novel spirosuccinimide type aldose reductase inhibitor, AS-3201, with aldose reductase.

Aldose reductase (AR) is an NADPH-dependent enzyme implicated in diabetic complications. AS-3201 [(R)-(-)-2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3'-pyrrolidine-1,2',3,5'-tetrone] is a structurally novel and potent ARI with an inhibitor constant (K(i) = 10(-)(10) M) 2000-fold lower than that of its optical antipode (S-isomer). To elucidate the inhibition modes and the stereochemical differences in their inhibitory potencies, we examined the interaction of these R- and S-isomers with AR under physiological conditions. Enzyme kinetic analysis, which was performed by using physiological substrates at 37 degrees C, showed that both isomers selectively act on the E-NADP(+) complex in both the forward and reverse reactions of AR. However, fluorometric titration analysis demonstrated that the affinities of the isomers for the E-NADP(+) complex are about the same as those for the E-NADPH complex and the apoenzyme. These results suggested that the selective binding to the E-NADP(+) complex arises from the predominance of this enzyme form during steady-state turnover rather than from binding specificity. Both the competition with a known active site-directed ARI and the protective effect on AR inactivation by N-bromosuccinimide showed that the isomers bind to the active site of the enzyme, but the thermodynamic parameters for the binding to AR indicated that additional hydrogen bonds and/or van der Waals interactions contribute to the energetic stabilization in the E-R-isomer complex. Molecular modeling, together with the deductions from spectroscopic studies, suggested that the succinimide ring and the 4-bromo-2-fluorobenzyl group of the R-isomer are optimally located for formation of a hydrogen-bonding network with AR, and that the latter benzyl group is also effective for the differentiation between AR and aldehyde reductase (a closely related enzyme).

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.

Involvement of muscarinic cholinergic and alpha2-adrenergic mechanisms in growth hormone secretion during exercise in humans.

The purpose of this study was to examine the role of muscarinic cholinergic and alpha2-adrenergic mechanisms in growth hormone (GH) secretion during exercise in humans. The GH responses induced during moderate-intensity exercise (using a cycle ergometer at 60% maximal oxygen uptake, VO2max, for 30 min) without treatment (control) and after the administration of a muscarinic cholinergic antagonist (atropine 1 mg) or after an alpha2-adrenergic antagonist (yohimbine 15 mg) were compared in seven healthy men. Although, serum GH concentration had increased significantly after exercise in the control experiment [mean peak GH concentration 52.64 (SEM 18.60) ng x ml(-1)], the increase was suppressed by the administration of either atropine [mean peak GH concentration 8.64 (SEM 7.47) ng x ml(-1)] or yohimbine [mean peak GH concentration 17.50 (SEM 7.89) ng x ml(-1)]. The area under the curve of serum GH concentration against time was significantly lower in the experiment using these drugs [with atropine, mean area 458 (SEM 409) ng x ml(-1) min], with yohimbine mean area 946 (SEM 435) ng x ml(-1) min] than in the control experiment [mean area 3135 (SEM 1098) ng x ml(-1) x min]. These results suggest that muscarinic cholinergic and alpha2-adrenergic mechanisms are involved in GH secretion during exercise in humans.

Effects of monatepil maleate, a new Ca2+ channel antagonist with alpha1-adrenoceptor antagonistic activity, on cholesterol absorption and catabolism in high cholesterol diet-fed rabbits.

The mechanism of the prophylactic effect against hyperlipidemia by monatepil maleate was investigated in animal models. Monatepil maleate is an antihypertensive agent with Ca2+-channel antagonistic, alpha1-adrenergic receptor-blocking, and lipid peroxidation inhibitory activity. In high cholesterol diet-fed rabbits, monatepil maleate (30 mg/kg, p.o., once daily for 9 weeks) showed a prophylactic effect against increases in total cholesterol and beta-lipoprotein. Monatepil maleate significantly accelerated the clearance of radioactivity from the blood after intravenous injection of low-density lipoprotein (LDL) labeled with [1alpha,2alpha (n)-3H]cholesterol, increasing biliary excretion of [3H]-bile acids without modifying bile acid composition. Furthermore, monatepil maleate tended to inhibit the absorption of orally administered [1alpha,2alpha (n)-3H]cholesterol from the gastrointestinal tract in these rabbits. In Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of hepatic LDL receptor deficiency, monatepil maleate (30 mg/kg, p.o., once daily for 6 months) did not suppress the increase in plasma lipids. These results suggest that the plasma lipid lowering effect of monatepil maleate requires the presence of hepatic LDL receptors. It is also suggested that monatepil maleate improves plasma lipid metabolism through two mechanisms: enhancement of clearance of plasma LDL, which may be mediated by up-regulation of hepatic LDL receptors, and acceleration of conversion of free cholesterol to bile acids in the liver.

Effect of exercise-induced acidosis on aldosterone secretion in men.

The present study was carried out to elucidate whether an exercise-induced increase in plasma hydrogen ion concentration influences aldosterone secretion. Six healthy men (aged 22-25 years) performed two intermittent exercise tests with and without drug administration. The intensities of these exercise tests were 40% maximal oxygen uptake (VO2max) and 90% VO2max, respectively. Administration of 2-mg Dexamethasone and 50-mg Captopril caused an almost complete suppression of adrenocorticotropic hormone (ACTH) and an enhancement of the elevation in renin concentration during exercise, indicating successful inhibition of ACTH release and angiotensin II production during exercise. While the magnitude of the increase in aldosterone in the drug experiment was depressed compared with the control experiment, a significant increase in aldosterone concentration was observed at the end of the 90% VO2max exercise. Whilst the change in aldosterone concentration did not correlate with the change in plasma potassium concentration, there was a significant correlation between aldosterone and plasma hydrogen ion concentrations in the drug experiment. Since the correlation coefficient was low (r = 0.455), the biological meaning of this correlation should be further investigated. These results would suggest that an elevation of plasma hydrogen ion concentration induced by exercise per se appears to be related, at least in part, with increased aldosterone secretion, independent of the pituitary-adrenal axis, and the renin-angiotensin system.

Ouabain-sensitive K(+)-dependent outward current caused by threo-beta-hydroxy-L-glutamic acid on a snail neuron.

1. An analog of L-glutamic acid, threo-beta-hydroxy-L-glutamic acid (threo-L-BHGA), was applied locally to the giant neuron of an Achatina snail by pneumatic brief pressure ejection and induced an outward current (Iout) on the ventral-left cerebral distinct neurone (v-LCDN). The present study aimed to elucidate the ionic mechanisms of the Iout caused by threo-L-BHGA (ItL-BHGA) of v-LCDN and the effects of ouabain on this current under voltage clamp. 2. The reversal potentials of ItL-BHGA (EtL-BHGA) of v-LCDN in varied K+o were fitted to the Nernst equation as ItL-BHGA = IK (K+ current) and were almost unchanged in Cl-o-free and Na+o-reduced (20% of normal) states. The ItL-BHGA is due to the increase in permeability of the neuromembrane to K+(K(+)-dependent) and is neither Na(+)- nor Cl-(-)-dependent. K(+)-channel blockers, a mixture of tetraethyl-ammonium (TEA) and 4-amino-pyridine (4-AP), blocked ItL-BHGA mainly in a noncompetitive and partly in an uncompetitive manner. 3. Unexpectedly, ItL-BHGA of v-LCDN was almost abolished in the Na+o-free state and significantly reduced in the Cl-o-free state. However, an Na(+)-channel blocker, tetrodotoxin, showed a tendency to enhance ItL-BHGA. On the other hand, ItL-BHGA was enhanced in K+o-free state. 4. Ouabain markedly inhibited ItL-BHGA in both noncompetitive and uncompetitive manners. Benzamil, an inhibitor of the Na(+)-Ca2+ exchange applied simultaneously with ouabain could not prevent ouabain inhibition on ItL-BHGA. The currents induced by other putative neurotransmitters, including a K(+)-dependent Iout caused by dopamine on v-LCDN, were not affected by ouabain. 5. According to our previous study, the threo-L-BHGA receptors are not linked with protein kinases or calmodulin. Then, ItL-BHGA could be produced by the receptor K+ channel complex or the receptor-G-protein-K+ channel combination. The present results indicate that the ATPase activity inhibited by ouabain and the presence of extracellular Na+ and Cl- are needed for threo-L-BHGA to activate the K(+)-dependent structure. Furthermore, the K+o-free state, which inactivates the Na(+)-K+ pump, and tetrodotoxin, which suppresses the Na+ channel at least partly, did not affect the structure to be activated.

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.

Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection.

A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The method was applied to the determination of AJ and the metabolites in rat plasma samples.

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.

Effects of L-glutamic acid and its agonists on snail neurones.

The sensitivities of 22 giant neurone types of an African giant snail (Achatina fulica Férussac) to threo-beta-hydroxy-L-glutamic acid (threo-L-BHGA), a derivative of L-glutamic acid (L-Glu), applied by brief pneumatic pressure ejection, were examined under current clamp. The 5 neurone types were depolarized by this compound, whereas 2 were hyperpolarized. The 4 neurone types, PON (periodically oscillating neurone), RAPN (right anterior pallial nerve neurone), d-RPLN (dorsal-right parietal large neurone) and RPeNLN (right pedal nerve large neurone) that are excited by threo-L-BHGA and one type, v-LCDN (ventral-left cerebral distinct neurone), inhibited by this compound, were selected to study their pharmacological features in detail. Effects of the stereoisomers of L-Glu and threo-L-BHGA, and mammalian L-Glu receptor agonists, ejected by brief pressure, on the 5 Achatina neurone types were examined under voltage clamp. d-RPLN produced an inward current (Iin) by L-Glu and threo-L-BHGA, whereas this neurone type was insensitive to D-Glu and erythro-L-, threo-D- and erythro-D-BHGA. This was also excited by AMPA, indicating that the pharmacological features of the L-Glu receptors in this neurone type were similar to those of the mammalian ionotropic AMPA type L-Glu receptors. RAPN produced Iin by L-Glu and threo-L-BHGA. This neurone type was also excited by quisqualic acid and ibotenic acid, indicating that the features of the L-Glu receptors were similar to those of the mammalian metabotropic L-Glu receptors. PON and RPeNLN produced Iin by L-Glu and threo-L-BHGA. These neurone types were also excited by quisqualic acid, AMPA and ibotenic acid, indicating that their L-Glu receptors seemed to be in the mixed type, of the two types mentioned. On the other hand, v-LCDN produced an outward current (Iout) by threo-L- and erythro-L-BHGA, but was insensitive to L-Glu, indicating that the receptors activated by L-BHGA were not L-Glu receptors. This neurone type was also inhibited by quisqualic acid.

Pharmacological characteristics of an outward current produced by beta-hydroxy-L-glutamic acid on a snail neurone.

An outward current (Iout) was produced by stereoisomers of beta-hydroxy-L-glutamic acid (L-BHGA), an L-glutamic acid (L-Glu) derivative, applied by brief pneumatic pressure ejection on an identifiable neurone type, v-LCDN (ventral-left cerebral distinct neurone), of Achatina fulica Férussac. However, L- and D-Glu were almost ineffective on this neurone type. The pharmacological features of this Iout caused by L-BHGA were elucidated in the present study. According to the dose (pressure duration)-response studies on the L-BHGA stereoisomers that produced the Iout, the effective potency of threo-L-BHGA was approximately similar to that of erythro-L-BHGA. The dose (pressure duration)-response curve of quisqualic acid was shifted towards the left direction from those of threo-and erythro-L-BHGA, suggesting that the binding activity of quisqualic acid to the receptors would be stronger than those of the L-BHGA stereoisomers. GABA, glycine and L-homocysteic acid showed an inward current (Iin) on this neurone type, in contrast to the Iout caused by L-BHGA. beta-Alanine and taurine had absolutely no effect. Therefore, no amino acid inhibitory neurotransmitter candidate was found for this neurone type except for L-BHGA. It was assumed that L-BHGA, in either threo-or erythro-configuration, would be an inhibitory neurotransmitter for this neurone type. Mammalian L-Glu receptor antagonists. D(-)-AP-5, (+/-)-CPP, CNQX and L(+)-AP-3, applied by perfusion, showed no effect on the Iout of v-LCDN caused by threo-L-BHGA, indicating that the features of the inhibitory receptor activated by L-BHGA were much different from those of any type of the mammalian L-Glu receptors. Among the inhibitors of ATP-sensitive K+ channel, glipizide significantly inhibited the Iout caused by threo-L-BHGA, whereas tolbutamide did not. Inhibitors of intracellular signal transduction systems, H-7, H-8, H-9, staurosporine, calphostin C, KT5823 and W-7, had no effect on the Iout caused by threo-L-BHGA, suggesting that the receptors activated by threo-L-BHGA would be ionotropic.

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.

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.

Pharmacodynamics and tumoricidal effect of adriamycin entrapped in ceramide sulfate-containing liposomes.

Ceramide sulfate (N-acylsphingosine-1-O-sulfate), which lacks the galactose residue of sulfatide, was examined as a possibly preferable constituent of liposomes for drug delivery. Multilamellar vesicles prepared from phosphatidylcholine, cholesterol, and N-lignoceroylsphingosine-1-O-sulfate in a molar ratio of 5:4:1 efficiently entrapped adriamycin, and the retention of the drug in the liposomes in saline at 4 degrees C for 8 d was nearly 100%. In terms of entrapment efficiency and retention of the drug in liposomes, N-lignoceroylsphingosine-1-O-sulfate was superior to N-stearoylsphingosine-1-O-sulfate. A pharmacodynamic study revealed that the blood level of adriamycin was far higher with the drug encapsulated in N-lignoceroylsphingosine-1-O-sulfate-containing liposomes than with the free drug. The drug level in the heart was remarkably reduced with the liposome-entrapped drug, which is advantageous in reducing the cardiotoxicity of this drug. The effect of N-lignoceroylsphingosine-1-O-sulfate-containing liposomes on the blood level of adriamycin was superior to that of sulfatide-containing liposomes, though the effect of the former on the heart level was comparable to that of the latter. The tumoricidal effect on ascitic P388 leukemia and Lewis lung carcinoma was higher with adriamycin entrapped in N-lignoceroylsphingosine-1-O-sulfate-containing liposomes than with the free drug.

Synthesis and aldose reductase inhibitory activity of 2-substituted 6-fluoro-2,3-dihydrospiro[4H-1-benzopyran-4,4'-imidazolidine]-2',5'-dio nes.

Optically active and racemic 2-substituted 6-fluoro-2,3-dihydrospiro[4H-1- benzopyran-4,4'-imidazolidine]-2',5'-diones were synthesized stereoselectively from (+)-, (-)-, and (+/-)-6-fluoro-3, 4-dihydro-4-oxo-2H-1-benzopyran-2-carboxylic acid [(+)-1, (-)-1, and (+/-)-1], respectively, for evaluation as new aldose reductase inhibitors. Among these compounds, the 2S,4S compounds were found to be more potent inhibitors of aldose reductase in vitro and in vivo than the corresponding 2R,4R enantiomers. The chloromethyl compound [(+)-5] showed highly potent activities in inhibiting cataract formation in the lenses and polyol accumulation in the sciatic nerve of rats.

Synthesis and aldose reductase inhibitory activity of 2-substituted-6-fluoro-2,3-dihydrospiro [4H-1-benzopyran-4, 4'-imidazolidine]-2',5'-diones.

Optically active and racemic 2-substituted-6-fluoro-2,3-dihydrospiro[4H-1-benzopyran-4, 4'-imidazolidine]-2',5'-diones were synthesized from (+)-, (-)-, and (+-)-6-fluoro-3,4-dihydro-4-oxo-2H-1-benzopyran-2-carboxylic acid. These compounds were then evaluated for in vitro and in vivo aldose reductase inhibitory activity. The 2S,4S isomers were found to be more potent aldose reductase inhibitors than the other corresponding stereoisomers. Among these compounds, (2S,4S)-6-fluoro-2,3-dihydro-2',5'-dioxospiro[4H-1-benzopyran-4, 4'-imidazolidine]-2-carboxamide ((+)-1b, SNK-860, CAS 105300-43-4) showed the most potent in vitro and in vivo activity.

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.

Efficient gene transfer with less cytotoxicity by means of cationic multilamellar liposomes.

A simple procedure for the preparation of cationic multilamellar vesicles (MLV) consisting of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine in a molar ratio of 1:2:2 was devised. When bacteriophage lambda DNA was encapsulated into these liposomes, entrapment efficiency was found to be nearly 100%, and digestibility of the DNA was less than 10%. Upon encapsulation of the plasmid pCH110 into cationic MLV, efficient expression was comparable to that obtained with cationic vesicles prepared by reverse-phase evaporation method (REV). Cytotoxicity of the present liposomes was less than that of cationic REV and far less than that of Lipofectin.

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.