ESR imaging on a solid-tumor-bearing mouse using spin-labeled dextran.

Imaging of a tumor with ESR was tried using two different types of spin probes, a low molecular weight spin probe, CPROXYL, and a polymer spin probe, TEMPO-DX. Spin probes were administered to a mouse bearing a solid tumor that was a transplanted Ehrlich's ascites carcinoma in the back, using two methods, conventional intraperitoneal injection and continuous intravenous injection with a micro-feeder. First, the accumulation of the probe was examined by X-band ESR. CPROXYL, which was administered to a mouse intraperitoneally, was exclusively retained in urine, showing that it was rapidly excreted into the bladder, while TEMPO-DX was absorbed from the peritoneal cavity with difficulty to the vessel. Using continuous intravenous injection, CPROXYL was also rapidly excreted, but it was confirmed that TEMPO-DX concentrated in tumor tissue because it has a long half-life in vivo. In addition, measurement of ESR imaging was done to measure the distribution of spin probes with continuous intravenous injection. The strongest spot of CPROXYL was observed on ESR images, showing the accumulation at the bladder, while the spot of TEMPO-DX was observed in the solid tumor of the back of the mouse. These results suggest that TEMPO-DX could stay much longer than a low molecular weight spin probe in vivo and concentrate at the tumor. TEMPO-DX may be useful for developing specific ESR imaging agents for tumor.

Studies of aloe. VI. Cathartic effect of isobarbaloin.

The cathartic effect of isobarbaloin, a stereoisomer of barbaloin (compound principally responsible for the cathartic activity of Aloe), was examined in male rats by oral administration. Individual differences in sensitivity in the laxative activity of isobarbaloin and barbaloin was not found. The cathartic activity (ED50) of isobarbaloin in barbaloin positive rats was 19.2 mg/kg, nearly equal to that of barbaloin (19.5 mg/kg). Also, isobarbaloin administered orally was demonstrated to decompose to aloe-emodin-9-anthrone (active metabolite of barbaloin) as well as to barbaloin. Therefore, it is considered that the mechanism underlying the cathartic effect of isobarbaloin is the same as that of barbaloin.

Spatiotemporal ESR-CT study on the metabolism of spin-labeled polysaccharide in a mouse.

A spatiotemporal ESR-CT study, rapid three dimensional ESR imaging by which distribution and metabolism of radicals in a small region in a living body can be followed, was carried out by intravenously administering spin-labeled polysaccharides to mice. The in vivo lifetime of spin-labeled hydroxyethylstarch (TEMPO-HES) was shorter than that of spin-labeled dextran (TEMPO-DX), suggesting that the clearance of their spins depended on the stability of the polymer chains to the hydrolysis. Spatiotemporal image at the kidney domain of a mouse showed that low molecular weight TEMPO-DX was excreted into the kidney and then was transferred to the bladder. Time dependence of the ESR intensity of TEMPO-HES at certain points in the liver domain had a maximum showing that TEMPO-HES was taken into the liver and then was decomposed there by the metabolism, but the rate of decrease in the intensity was different in each part in the liver because of the difference in metabolic function. The rate of decrease of TEMPO-DX in the blood was slowed by the prior administration of dextran, meaning that the intake of TEMPO-DX into the liver from the blood was suppressed by the dextran.

Dynamic electron spin resonance (ESR) imaging of the distribution of spin labeled dextran in a mouse.

The nitroxide group 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) was covalently bound to a dextran via an N-acetamido linkage to produce a novel spin probe, TEMPO-dextran (TEMPO-DX), which circulates for long time periods in an animal without metabolic degradation. TEMPO-DX was stable in mice, while small TEMPO analogs quickly disappeared after administration. Since dextran was reduced somewhat in size during synthesis, the resultant spin-labeled dextran could be excreted through the kidney. A strong L-band electron spin resonance signal was obtained shortly after intravenous administration of TEMPO-DX into the tail vein of a mouse, from which three-dimensional images of specific organs were calculated. The signal was found to persist for well over 1 h.

Neuronal and vascular pathology produced by verocytotoxin 2 in the rabbit central nervous system.

To study the pathogenesis of the central nervous system (CNS) involvement associated with verocytotoxin-producing Escherichia coli infection, we developed an animal model by administering verocytotoxin 2 to rabbits either intravenously or intrathecally. After an interval of 2-9 days, the rabbits became paralyzed in a dose-dependent manner and in the absence of renal impairment. The minimal intravenous and intrathecal doses that produced these neurological signs were 250 and 0.4 ng/kg, respectively. After intravenous administration, most of the toxin was cleared from the serum within 24 h, with concomitant transition of a small amount into the cerebrospinal fluid. Pathological examination revealed that neurons in various CNS regions showed atrophy, cytoplasmic hyperchromasia and nuclear pyknosis as early as 6 h after administration. The distribution of affected neurons was constant and irrespective of the route of administration. Abnormalities of the blood vessels, such as the thickening of arterioles walls, were noted from 2 days after administration. The vascular lesions became more prominent after the intrathecal injection, which caused thrombosis and multiple infarction. Selective deposition of the toxin on the vessel walls was demonstrated immunohistochemically. Thus, the pathological manifestations of verocytotoxin 2 neurotoxicity consisted essentially of two types of lesions, early neuronal and late vascular, both of which might have developed under the influence of the toxin that had entered the CNS by crossing or circumventing the blood-brain barrier.

[A consideration in regard to 16 patients died at home].

We had given the service to the patients who desired home care. And now we made inquiries about 16 cases died at home. They were satisfied with our service and death at home. But in regard to the question "If you die at home, Are you satisfied?¿, half the number answered "NO". We should recognize the present condition that we can not fulfill the needs of patients and their families.

[Medical economy of home care in private hospital].

In order to analyze the economics of home care, we calculated the revenue and expenditures of both hospitalization and home care in a private hospital. The home care expenses were about 44.7% of the institutional care expenses. It became clear that home care saved medical expenses. The expenses of patients were about 33.7% less with home care. In conclusion, home care may be favorable medical care if some problems are solved. The home care expenses of a cancer patient on the institutional side were about 3.4 times more that those of a patient with other diseases. If we support home care of cancer patients alone, it would be an excessive burden on them, because patients are too few at present to organize and keep a team. As a private hospital, we support home care regardless of disease. However, medical economic expenses as a total turned out to be about 17% short of the necessary expenses.

A novel spin probe with long life in vivo for ESR imaging.

A dextran-bonded nitroxide radical (TEMPO-DX) was synthesized to obtain a radical with long life in vivo for ESR imaging. TEMPO-DX was injected intravenously into a rat tail vein and the decrease in ESR intensity in the collected, circulating blood was followed. The result showed that the half life of TEMPO-DX in vivo was 30 min, the longest value reported so far and more than 30 times longer than the corresponding radicals of the six-membered piperidine ring, which means that the bonding of a radical to the polymer greatly prolonged life. The stabilities of TEMPO-DX against the reduction with L-ascorbic acid and the rat liver homogenate were also examined and compared with those of the 3-carbamoyl-2,2,5,5-tetramethylpyrolidin-1-yloxy (CPROXYL) known as a radical stable in vivo. TEMPO-DX was shown not to be as stable as CPROXYL, thus in vivo stability of TEMPO-DX arises from the fact that it is slowly absorbed into the tissues where the radicals are quenched. An ESR image of the mouse head domain was obtained only by an intravenous injection of TEMPO-DX solution into the tail vein.

Studies of aloe. V. Mechanism of cathartic effect. (4).

Aloe-emodin-9-anthrone(AE-anthrone), produced from barbaloin in the rat large intestine, caused not only an increase in the intestinal water content but also stimulated mucus secretion. This might play an important role in the occurrence of diarrhea. It was demonstrated that the amount of AE-anthrone produced in the rat large intestine(maximal amount: 568 micrograms/rat at 4 h after injection) was enough to cause both of these effects, which were observed following intracecal administration of barbaloin (31.1 mg/kg). These results together with our previous data, which showed a relationship between increase in the intestinal water content and the stimulation of peristalsis, confirm that AE-anthrone is the principal agent responsible for the cathartic effect of barbaloin. We also propose that the increase in water content is a more important factor than stimulation of peristalsis in the induction of diarrhea by barbaloin.

Studies of aloe. IV. Mechanism of cathartic effect. (3).

Charcoal transport, as an indicator of the degree of peristalsis, and water content in the large intestine after the intracaecal administration of barbaloin, were measured simultaneously in the same rat. Charcoal transport was significantly accelerated at both 3.5 and 6.5 h after the administration of barbaloin. At 6.5 h, diarrhea instead of normal faeces was observed. Moreover, at 1 h before the acceleration of charcoal transport, a marked increase in the relative water content of the large intestine was observed. It appears that the increase in water content of the large intestine induced by barbaloin precedes the stimulation of peristalsis, attended by diarrhea. Therefore, it is suggested that the increase in water content is a more important factor than the stimulation of peristalsis in the diarrhea induced by barbaloin.

Rat selection test with respect to laxative activity induced by barbaloin.

As individual differences in sensitivity in the laxative activity of barbaloin were observed in rats, a selection test for barbaloin (31.1 mg/kg, p.o.) was carried out between 1989 and 1991. The annual percentages of rats exhibiting a reaction positive to barbaloin were almost constant (about 57%). We propose that rats positive to barbaloin should be selected for studies on the laxative effect of barbaloin.

Sulfated N-Myristoyl Chitosan as a Surface Modifier of Liposomes.

Sulfated N-myristoyl chitosan (S-M-chitosan), which is strongly electrolytic and water soluble as well as partly hydrophobic due to long alkyl chains, was synthesized to be used as a liposome-surface modifier. The effects of the treatment with an aqueous S-M-chitosan solution on the stability of the liposome suspension prepared from hydrogenated egg yolk lecithin were examined on several points. A suspension of large liposomes prepared by the Bangham method was precipitated by standing for a day, but the precipitation was restrained when the sample was treated with S-M-chitosan solution. The turbidity of a small liposome suspension was changed greatly after the suspension was freeze-thawed, but the change was small in the treated sample. A similar result was obtained when the suspension was freeze-dried following the addition of water. These results come from the facts that the surface of the liposome was coated with S-M-chitosan and negatively charged as ascertained by the measurement of zeta potential and the electron microscopic observation. The repulsive force between charges was considered to be the origin of the stabilization. It was also shown from an ESR experiment that the treatment suppressed the elution rate of the material incorporated into the liposomes.

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VIII. Isotope labeling of ginsenoside Rb2.

To clarify the pharmacokinetics of absorption, distribution and excretion of ginsenoside Rb2 (Rb2), one of the major saponins of the root of Panax ginseng, following oral administration to rats, a tritium (3H) labeling of Rb2 was examined. The C-12 position of Rb2 was labeled with 3H-sodium borohydride (3H-NaBH4) and 12-3H Rb2 and 12-3H-epi Rb2 was synthesized. This method of specific position labeling of Rb2 may be applicable to other ginsenosides. In the near future, the pharmacokinetics of Rb2 in rats may be clarified with 3H labeled Rb2.

Studies on natural antioxidants in citrus species. I. Determination of antioxidative activities of citrus fruits.

The antioxidative activities of twenty types of citrus fruits were investigated with a screening method which is based on rat liver microsomal lipid peroxidation induced by dihydronicotinamide adenine dinucleotide phosphate (NADPH) and adenosine diphosphate (ADP). The activities of the exocarp were greater than those of the sarcocarp and the activities from immature fruits (collected in July-August) were greater than those from mature fruits. The strongest antioxidative activity was found in ponkan (Citrus reticulata Blanco) collected in July.

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VII. Comparison of the decomposition modes of ginsenoside-Rb1 and -Rb2 in the digestive tract of rats.

In order to clarify some similarities and differences of decomposition modes between 20(S)-protopanaxadiol (20(S)-ppd) saponins, represented by ginsenoside Rb1 (Rb1) and ginsenoside Rb2 (Rb2), the decompositions of Rb1 and Rb2 in the rat gastrointestinal tract, 0.1 N HCl and crude hesperidinase were investigated in detail. As in the case of Rb2 reported previously, Rb1 was hydrolyzed to 20(R,S)-ginsenoside Rg3 in 0.1 N HCl. On the other hand, hydroperoxidation of Rb1 occurred in rat stomach; the major hydroperoxide was separated and identified as the 25-hydroperoxy-23-ene derivative of Rb1 (VIII) by 1H- and 13C-nuclear magnetic resonance and fast atom bombardment mass spectrometry. The decomposition modes of 20(S)-ppd saponins (Rb1 and Rb2) differed from that of 20(S)-protopanaxatriol saponin (Rg1) in rat stomach. In rat large intestine, five decomposition products of Rb1 were observed by thin-layer chromatography, and these were identified as gypenoside XVII (G-XVII), ginsenoside Rd (Rd), ginsenoside F2 (F2), compound K (C-K) and VIII. The decomposition modes of Rb1 and Rb2, both 20(S)-ppd saponins, are considered to be different because of the hydrolysis rate in the terminal sugar moiety at the C-20 hydroxyl group in the rat large intestine. Using crude hesperidinase, Rb1 was decomposed to G-XVII, F2 and C-K, and Rb2 was decomposed to 3-O-beta-D-glucopyranosyl-20-O-[alpha-L-arabinopyranosyl(1----6)-b eta-D- glucopyranosyl]-20-(S)-ppd, F2 and C-K. Consequently, it appears that hydrolysis by beta-glucosidase, which is present in the rat large intestine, is distinct from that by crude hesperidinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VI. The decomposition products of ginsenoside Rb2 in the stomach of rats.

The decomposition of ginsenoside Rb2 (Rb2) in rat stomach (in vivo) and in 0.1 N HCl solution (in vitro) was investigated in detail. By treating with 0.1 N HCl, the acidity of which is similar to that of gastric juice, a part of Rb2 was hydrolyzed to 20(R,S)-ginsenoside Rg3. On the other hand, Rb2 was little decomposed in rat stomach and a small quantity of an unidentified metabolite, which was different from the hydrolyzed products in 0.1 N HCl, was observed. The metabolite was separated into four compounds, which were identified by 1H- and 13C-nuclear magnetic resonance and fast atom bombardment mass spectrometry. These compounds were determined to be 25-hydroxy-23-ene (IV), 24-hydroxy-25-ene (V), 25-hydroperoxy-23-ene (VI) and 24-hydroperoxy-25-ene (VII) derivative of Rb2, respectively. In this study, it is suggested that 20(S)-protopanaxatriol saponins undergo hydrolysis of the C-20 glycosyl moiety and hydration of the side chain, on the other hand, 20(S)-protopanaxadiol saponins undergo oxygenation of the side chain.

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. V. The decomposition products of ginsenoside Rb2 in the large intestine of rats.

The decomposition of ginsenoside Rb2 (Rb2) in the rat large intestine after oral administration was investigated in detail. A part of Rb2 was decomposed and six decomposition products (I-VI) were observed on thin-layer chromatogram. Among them, five products (I-V) were isolated, and identification of these compounds was done by carbon-13 nuclear magnetic resonance (13C-NMR). On the basis of 13C-NMR analysis, these compounds were identified as ginsenoside Rd (I), 3-O-beta-D-glucopyranosyl-20-O- [alpha-L-arabinopyranosyl(1----6)-beta-D-glucopyranosyl]-20(S)- proto-panaxadiol (II), ginsenoside F2 (III), 20-O-[alpha-L-arabinopyranosyl(1----6)-beta-D-glucopyranosyl]-20(S )- protopanaxadiol (IV), and compound K (V), respectively.

Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils.

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.

Studies of aloe. III. Mechanism of cathartic effect. (2).

The mechanism of action of aloe-emodin-9-anthrone, a decomposition product of barbaloin, in causing a significant increase in the water content of the rat large intestine, was investigated. Aloe-emodin-9-anthrone inhibited rat colonic Na+, K(+)-adenosine triphosphatase (ATPase) in vitro, and increased the paracellular permeability across the rat colonic mucosa in vivo. Therefore, it seemed that the increase in water content of the rat large intestine produced by aloe-emodin-9-anthrone was due to both inhibition of absorption and stimulation of secretion without stimulation of peristalsis. Furthermore, pretreatment with loperamide, an antidiarrheal agent, completely prevented the increase of paracellular permeability induced by aloe-emodin-9-anthrone but did not completely reduce the concomitant increase in residual fluid volume. These findings suggest that aloe-emodin-9-anthrone has multiple mechanisms of action involved in the increase of water content in the rat large intestine.

Effect of aclacinomycin on lipid peroxide levels in tissues of mice.

We have examined the lipid peroxide levels in aclacinomycin (ACM)-treated mice by using adriamycin (ADR) as a comparative drug. There was no increase in the lipid peroxide level of the heart at either 3h or 4d after ACM administration (15 mg/kg, i.p.), although the level in the heart of ADR-treated mice was elevated to 257% of that in normal mice. The effect of ACM and its glycoside-type metabolites on the increase of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal lipid peroxidation (in vitro) was weaker than that of ADR. Then, we examined the tissue concentrations of ACM. The AUC0-24h of ACM was the lowest in the heart among the tissues examined, being only 29.3% of that obtained with ADR. However, the concentrations of the glycoside-type metabolites of ACM in all tissues determined were higher than the concentration of ACM. In the heart, the T1/2 and AUC0-24h of ACM glycosides were somewhat higher than those of ADR. In conclusion, ACM and its metabolites do not lead to an increase in lipid peroxide level in the heart of mouse, and the difference in lipid peroxide increment in the mouse heart induced by ADR and ACM is independent of the tissue concentration of the drugs.