Possible mechanism for the anti-atherosclerotic action of the calcium channel blocker AE0047 in cholesterol-fed rabbits.

1. The present study was designed to investigate the anti-atherosclerotic effect of AE0047, a calcium channel blocker, and to compare it with that of nilvadipine in cholesterol-fed rabbits. Furthermore, the effects of AE0047 on low-density lipoprotein (LDL) oxidation were studied in vitro. 2. A 7 week treatment period with AE0047 (3 and 10 mg/kg, p.o.) led to a dose-dependent reduction in the lipid deposition area by Oil Red-O staining (surface index) without affecting serum lipid levels. There was no reduction in the surface index following treatment with the same dose of nilvadipine (10 mg/kg). 3. In a vehicle-administered high-fat diet group of rabbits, levels of total cholesterol (TC) and esterified cholesterol (EC) and calcium content in the aorta were increased approximately two- to three-fold over those of the normal diet group. Increased levels of TC and EC and calcium content were reduced to the same levels as the normal diet group by AE0047 treatment, whereas nilvadipine did not affect TC and EC levels. 4. In an in vitro study, AE0047 (10 micromol/L) inhibited LDL oxidation and the aggregation of apolipoprotein (Apo) B-100 induced by Cu2+. Furthermore, AE0047 inhibited the degradation of oxidized LDL by macrophages. In contrast, the same dose of nilvadipine (10 micromol/L) did not inhibit either LDL oxidation or the aggregation of ApoB-100. 5. In summary, AE0047 inhibited LDL oxidation, resulting in a decrease of its uptake into macrophages and an inhibition of cholesterol esterification. This leads to an anti-atherosclerotic effect of AE0047. Thus, AE0047 may have therapeutic potential in preventing cardiovascular disease in hypertensive patients.

Therapeutic effects of AE0047, a novel calcium antagonist, on progression of brain damage after stroke in stroke-prone spontaneously hypertensive rats.

1. The potential of AE0047, a novel calcium antagonist, to remedy brain damage of stroke-prone spontaneously hypertensive rats (SHRSPs) with signs of stroke was compared with those of nicardipine and hydralazine. 2. AE0047 (1 and 3 mg/kg/day) given daily to diseased SHRSPs prevented mortality and improved neurological symptoms. Histological examination also supported the effectiveness of AE0047 against the progression of the disease. 3. Nicardipine (10 mg/kg/day) and hydralazine (10 mg/kg/day) were less effective than AE0047 in a dose equal to or more than the hypotensive dose, respectively. 4. AE0047 may be beneficial for treating the acute stage of stroke in humans by virtue of its long-lasting hypotensive action and undefined direct actions on the cerebral vasculature.

Effects of long-term treatment with the calcium antagonist AE0047 on cerebrovascular autoregulation and hypertrophy in spontaneously hypertensive rats.

Chronic hypertension is associated with structural and functional changes in the cerebrovascular bed, which influence cerebral circulation and its autoregulation. We examined whether or not long-term treatment of spontaneously hypertensive rats (SHRs) with the new calcium antagonist AE0047 would reverse structural changes in the cerebral vasculature and normalize the elevated lower blood pressure (BP) limit of the cerebral blood flow (CBF) autoregulation. Treating 6-month-old SHRs with a diet containing either 0.013 or 0.04% AE0047 for 8 weeks reduced BP and, at the higher dose, maintained BP at a level similar to that of age-matched Wistar-Kyoto (WKY) rats. At the end of the treatment period, CBF was measured by using the hydrogen-clearance method, and the lower limit of the autoregulation curve was estimated by repeated CBF measurement with stepwise reduction of BP through bleeding. This limit was significantly higher in untreated SHRs than in WKY rats (111 +/- 8 vs. 60 +/- 8 mm Hg). AE0047 caused a significant and dose-dependent shift in the elevated lower BP limit, which decreased to 83 +/- 9 and 75 +/- 6 mm Hg at the low and high dose, respectively. In perfusion-fixed proximal, intermediate, and distal portions of the middle cerebral artery, media thickness/external diameter (M/ED) ratios were significantly greater in untreated SHRs than in WKY rats. In AE0047-treated animals, M/ED ratios in all portions tended to be reduced halfway between those for untreated SHRs and those for WKY rats, but with no statistical significance. These results suggest that long-term treatment of patients with hypertension with AE0047 will normalize the autoregulatory threshold while preserving CBF and thereby improve tolerance to BP reduction, but the potential to ameliorate structural alterations may be small.

Effects of AE0047 on cerebral ischaemia and oedema after middle cerebral artery occlusion in cats.

1. AE0047 is a new dihydropyridine calcium antagonist with protective effects against cerebral ischaemia and the occurrence of stroke in several animal models. 2. In the present study we investigated whether AE0047 would improve the reduced cerebral blood flow (CBF) and oedema formation in cats subjected to middle cerebral artery (MCA) occlusion and compared it for efficacy with other dihydropyridine calcium antagonists with different moieties, such as nilvadipine and nicardipine. 3. Middle cerebral artery occlusion reduced local CBF (ICBF), as measured by the hydrogen clearance method, while dry weight measurement showed that water content in the cortical tissues surrounding each ICBF measurement electrode had increased after 4 h ischaemia. 4. Both AE0047 (10 micrograms/kg) and nilvadipine (30 micrograms/kg), given intravenously 20 min after MCA occlusion, produced an approximate 10% hypotensive response and significantly increased ICBF in severely and moderately ischaemic regions, grouped according to the initial reduced flow values. However, nicardipine (5 micrograms/kg bolus followed by infusion of 3 micrograms/kg per min for 60 min) failed to mitigate the reduction in ICBF despite an increase in the ICBF of the contralateral cortex. In addition, AE0047 tended to prevent an increase in cortical water content in severely ischaemic regions, whereas water content in both nilvadipine- and nicardipine-treated groups tended to increase. 5. These results suggest that dihydropyridine calcium antagonists act differently on cerebral ischaemia and oedema formation in a manner dependent on their side-chain structures and that AE0047 effectively attenuates ischaemic brain damage without aggravating oedema.

Preventative and therapeutic effects of AE0047 on renal injury in stroke-prone spontaneously hypertensive rats.

1. The present study was designed to investigate the preventative and therapeutic effects of AE0047 on renal injury compared with those of nitrendipine in stroke-prone spontaneously hypertensive rats (SHRSP). 2. In the preventative study, drug administration was started before the appearance of renal injury, such as proteinuria. Treatment for 6 weeks with AE0047 (1 and 3 mg/kg, p.o.) led to a dose-related reduction in systolic blood pressure (SBP). Nitrendipine, at doses of 10 and 30 mg/kg, also lowered SBP to a similar degree to that seen with AE0047 at 1 and 3 mg/kg, respectively. 3. In the vehicle-administered SHRSP group, urinary excretion of protein (Uprotein V) increased progressively from 14 weeks of age for another 6 weeks. AE0047 at both doses maintained Uprotein V within normal levels throughout the experimental period. However, the elevation of Uprotein V was only inhibited in the 30 mg/kg nitrendipine-treated group. Urinary N-acetyl-beta-D-glucosaminide (NAG) activity in the vehicle-treated SHRSP group was elevated. Urinary NAG activity remained at a low level only in AE0047-treated groups. 4. Histopathological examination revealed severe lesions (i.e. fibrinoid necrosis, proliferative vasculitis and glomerular lesions) of the kidney in SHRSP. AE0047 treatment at each dose attenuated the development of renal lesions in SHRSP. In contrast, nitrendipine, at 10 mg/kg, was ineffective against the development of renal lesions. Although nitrendipine at 30 mg/kg suppressed the development of renal lesions, this effect was still weaker than that seen with AE0047 at 1 mg/kg. 5. In the therapeutic study, drugs were administered to 17-week-old SHRSP with moderate renal damage for 10 days. Treatment with AE0047 (1 and 3 mg/kg) produced dose-dependent decreases in Uprotein V. In the nitrendipine-treated group, Uprotein V tended to decrease but the changes were not significant. 6. Histopathological studies revealed that 3 mg/kg AE0047 improved renal lesions, such as fibrinoid necrosis, proliferative vasculitis and glomerular lesions, whereas 30 mg/kg nitrendipine did not. 7. Taken together, the results indicate that AE0047 is capable of preventing proteinuria as well as renal lesions, in part via a mechanism independent of its depressor action on SBP. Furthermore, AE0047 improves proteinuria and renal lesions in proteinuria-established SHRSP. Thus, AE0047 may have therapeutic potential in suppressing either the development or the progression of renal disease in hypertensive patients.

Effects of the calcium antagonist AE0047 on the development of neurological deficit and infarction after middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats.

AE0047 [4-(4-benzhydrylpiperazino)phenethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate dihydrochloride] is a new dihydropyridine calcium antagonist with protective effects against cerebral ischaemia and the occurrence of stroke in several animal models. We investigated the effects of AE0047 on focal ischaemia induced by middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. AE0047 at a dose causing 20 or 40% systemic hypotension (1 or 3 mg kg-1) was given orally twice, 15 min and 24 h after occlusion. The neurological status of animals was investigated 2, 24 and 48 h after occlusion. Infarct area of brain was measured 48 h after occlusion. Middle cerebral artery occlusion resulted in the progressive deterioration of neurological status and large infarction in middle cerebral artery territories with 40% mortality. AE0047 dose-dependently attenuated the deterioration of neurological status, and reduced mortality to 0 or 10%. AE0047 significantly reduced infarct size and left/right hemispheric area ratio, an index of brain swelling. These results suggest that AE0047 has the ability to ameliorate ischaemic cerebral stroke in hypertensive patients.

AE0047-mediated calcium channel blocking in vascular smooth muscles.

1. This experiment was designed to pharmacologically characterize a novel calcium channel blocker, AE0047. 2. After 1-hr treatment with each drug (10(-6) M), K(+)-induced contraction in rat aortic strip was clearly depressed by nifedipine and manidipine and slightly depressed by AE0047. After a wash out of the preparation in drug-free medium, the inhibition of K(+)-induced contraction by nifedipine or manidipine was abolished or unchanged, respectively. In contrast, AE0047-produced inhibition was reinforced with time after removal of the drug. 3. A cell membrane depolarization-induced 45Ca uptake into tissue was depressed completely by nifedipine, but, if it was washed out, merely 20% inhibition of control remained. AE0047-produced inhibition became prominent after drug removal. Manidipine did not have the same inhibitory effect after wash out. 4. A receptor-binding study indicated that affinity of AE0047 and manidipine for the dihydropyridine-sensitive Ca channel receptor was lower than that of nifedipine. AE0047, unlike nifedipine and manidipine, inhibited [3H]PN200-110 binding more strongly when a 4-hr preincubation was used than without extended incubation. 5. The drug molecule of AE0047 was highly partitioned into the lipid bilayer of the synaptosome in canine cerebral cortices. In the synaptic membrane and liposomes, both prepared from canine cerebral cortices, the respective partition coefficients of the drug were 6997 +/- 2309 and 422 +/- 28 against 1395 +/- 161 and 24 +/- 2 of nitrendipine. 6. AE0047 showed slower onset of inhibition against K(+)-induced contraction and enhanced Ca influx compared with manidipine and nifedipine. These results may suggest that AE0047 requires a long period of time to occupy the dihydropyridine-sensitive sites within the Ca channel, which was detected by decreased specific [3H]PN200-110 binding, and to inhibit K(+)-induced Ca influx into rat aorta.

Prostaglandin E1 prevents apoptotic cell death in superficial dorsal horn of rat spinal cord.

Neuronal degeneration, distinguished morphologically and biochemically from necrosis, was induced in the dorsal horn of the lumbar spinal cord by chronic constriction of the sciatic nerve in rats. Neuronal apoptosis in the territory of the spinal cord which receives afferent excitatory inputs from the sciatic nerve was confirmed by TUNEL-staining and electrophoresis of genomic DNA. The morphological changes including the appearance of dark neurones, as identified by toluidine-blue staining, were almost completely blocked by 10 microg/kg of the prostaglandin E (EP) receptor agonist lipo-PGE1, incorporating PGE1 in lipid microspheres for chemical stability and targeted delivery, but not by 10 microg/kg of carbacyclin a prostacyclin (IP) receptor agonist. Lipo-PGE1 also blocked the "ladder type" fragmentation of genomic DNA extracted from tissue in the affected area of the spinal cord. Since the regional blood flow in the subfield of the spinal cord was neither influenced by the chronic injury nor by application of the vasodilative prostaglandin, the effect of lipo-PGE1 must have been achieved via another mechanism. These results demonstrate that the neuronal apoptosis in spinal cord induced by sciatic constriction injury is more effectively inhibited by the EP receptor agonist PGE1 than by the IP receptor agonist carbacyclin.

Intravenous immunoglobulin (IVIG) treatment of experimental colitis induced by dextran sulfate sodium in rats.

We investigated the therapeutic effect and immunological action mechanism of IgG in experimental colitis induced by 3% dextran sulfate sodium in rats. Intravenous injection of homologous (rat) IgG (400 mg/kg per day) caused a significant suppression of occult blood discharge and ulcerative lesions in the colon, while no suppressive effect was observed in the case of heterologous (human) IgG. The positive effect of rat IgG on the lesions was also clearly shown by the histological examinations. Generation of proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-alpha) and IL-1alpha, in the lesions was found to be inhibited by administration of rat IgG. Little or no suppressive action was exerted by human IgG. Careful examination of recruited T cells and macrophages, both of which are thought to play important roles in the development of ulcerative colitis, indicated that rat IgG, but not its human counterpart, decreased the number of immunocompetent cells in colonic mucosa. Meanwhile, in an in vitro study, both forms of IgG were shown to suppress production of TNF-alpha and IL-1alpha from lamina propria mononuclear cells isolated from rat colon. These findings suggest that, mainly by suppressing recruitment of immunocompetent cells into the lesions, homologous IgG may reduce the occurrence of colitis.

Proliferative effect of dextran sulfate sodium (DSS)-pulsed macrophages on T cells from mice with DSS-induced colitis and inhibition of effect by IgG.

The authors have previously reported that homologous immunoglobulin (Ig)G reduces the occurrence of dextran sulfate sodium (DSS)-induced colitis, mainly by suppressing recruitment of immunocompetent cells into colitis lesions. However, the mechanisms of cell recruitment and of its suppression by IgG remain unclear. In addressing these questions, this study focused on the activation of T cells in the presence of macrophages. The authors found that [3H]-thymidine uptake of T cells from DSS-induced colitis mice, but not from normal mice, was significantly enhanced when cultured with DSS-pulsed macrophages. From the profile of cytokine production, it was suggested that T helper 1 (Th1)-type cells become predominant during stimulation. Addition of homologous IgG significantly suppressed T cell proliferation in a dose-dependent manner, while no suppressive effect was observed with heterologous IgG. Mouse IgG F(ab')2, but not Fc, fragments partially mimicked the suppressive effect of whole IgG. These findings provide evidence that Th1-type cells may play an important role in the development of DSS-induced colitis and that homologous IgG exerts its protective action at least in part through the F(ab')2 portion.

Secretion of active Fc fragments of immunoglobulin E directed by the yeast invertase signal sequence in mammalian cells.

The human Fc fragments of immunoglobulin E (IgE-Fc) expressed in the mammalian COS cells, those mainly consisting of C epsilon 3-C epsilon 4 chains with or without Cys-328 known to be responsible for interchain disulfide bonding, were secreted into the culture medium directed by the yeast Saccharomyces cerevisiae invertase (SUC2) signal sequence (SUC2/Ig2 or SUC2/Ig, respectively) as well as by the human interleukin 2 receptor alpha chain signal sequence (IL2R/Ig2 or IL2R/Ig, respectively). For the binding activity to the human soluble alpha chain of high affinity receptor for IgE, IgE-Fc with Cys328, SUC2/Ig2 and IL2R/Ig2, were active but had smaller activity than native IgE. By comparison IgE-Fc without Cys328, SUC2/Ig and IL2R/Ig, showed lower activity than SUC2/Ig2 and IL2R/Ig2. Immunoprecipitation analysis revealed both SUC2/Ig and SUC2/Ig2 had molecular weights (M(r)s) and degrees of glycosylation similar to IL2R/Ig and IL2R/Ig2, respectively. These results suggested that in mammalian cells the SUC2 signal sequence was functional in directing the heterologous multimeric proteins to the endoplasmic reticulum, resulting in secretion of the active proteins. This finding might show merits on heterologous protein secretion systems.

Synthesis and pharmacological activity of triazole derivatives inhibiting eosinophilia.

In order to develop novel antiasthmatic agents based on a new mechanism of action, a series of 3-substituted 5-amino-1-[(methylamino)(thiocarbonyl)]-1H-1,2,4-triazole derivatives were synthesized and evaluated in a model in which eosinophilia was induced in the airway through intravenous (iv) injection of Sephadex particles on days 0, 2, and 5. After screening of several hundred derivatives, we finally identified the highly potent eosinophilia inhibitor 5-amino-3-(4-chlorophenyl)-1-[(methylamino)(thiocarbonyl)]-1H-tria zole (23c, GCC-AP0341), which had ID50 values of 0.3 and 0.07 mg/kg when administered orally (os) and intraperitoneally (ip), respectively. This compound showed complete inhibition of the hypersensitivity induced by ascaris inhalation at an ip dose of 1 mg/kg as well as low toxicity, with an LD50 value of > 2.0 g/kg in mice. Extensive study of its mechanism of action revealed that 23c inhibited eosinophil survival induced by interleukin-5 (IL-5), but had little or no effect on leukotriene D4 (LTD4) or platelet-activating factor (PAF)-induced responses. Taken together, these results suggest 23c as a novel candidate for the treatment of chronic asthma. Further studies are now underway.

Antithrombin III prevents blood pressure elevation and proteinuria induced by high salt intake in pregnant stroke-prone spontaneously hypertensive rats.

In pregnant stroke-prone spontaneously hypertensive rats, salt-loading causes symptoms similar to those of human preeclampsia, such as hypertension and proteinuria. To seek evidence of the therapeutic potential in preeclampsia of antithrombin III (AT III), which is a serine protease inhibitor active on various enzymes of the coagulation cascade, we examined the effect of consecutive treatment with AT III on hypertension and proteinuria in this animal model. Salt-loading (2% NaCl diet) caused a significant elevation of systolic blood pressure on day 15-17 and of urinary protein excretion on day 17-19 of gestation, as compared with animals fed a normal diet. AT III, administered i.v. at a dose of 60 or 300 U/kg/d for 10 d from day 9-11 to 18-20, attenuated these pathological changes in a dose-dependent manner. Histological examination of the kidney revealed that AT III prevented the occurrence of arteriosclerosis and thickening of the capillary basement membrane. However, the pathological changes induced by salt-loading were not attributable to activation of the blood coagulation system. These results demonstrate that AT III has preventive action against salt-induced hypertension and proteinuria in pregnancy through a mechanism largely independent of its anticoagulant action. AT III may thus be beneficial for the treatment of clinical symptoms of preeclampsia.

Antithrombin III prevents renal dysfunction and hypertension induced by enhanced intravascular coagulation in pregnant rats: pharmacological confirmation of the benefits of treatment with antithrombin III in preeclampsia.

We tested the hypothesis that enhanced intravascular coagulation in pregnancy could produce clinical symptoms similar to those of preeclampsia, such as hypertension, proteinuria, and edema. Having confirmed this, we then examined whether the pathological changes caused by intravascular coagulation could be suppressed by administration of antithrombin III (AT III), an endogenous inhibitor active to thrombin and factor X a. Intravascular coagulation was induced in Wistar rats on day 16-20 of pregnancy by 1-h arterial infusion of tissue thromboplastin (TP) through the left ventricle of the heart. One hour after the end of the infusion period, organ blood flows were measured by the radioactive ((57)Co-labeled) microsphere method, and fibrin deposition in organs was measured by radiolabeling with [(125)I] fibrinogen injected before TP infusion. Infusion of TP produced fibrin deposition in the kidney, lung, and liver, but not in the myometrium and placenta, as well as an 80% decrease in renal blood flow (RBF), with oliguria and proteinuria. TP also caused an increase in blood pressure (BP) accompanied by an increase in plasma renin activity (PRA), both of which were suppressed by bilateral nephrectomy before TP infusion. The prophylactic administration of AT III concentrates (60 or 300 U/kg intravenously (i.v.), followed by infusion of 30 or 150 U/kg/2 h, respectively) prevented all pathological changes in a dose-dependent manner. AT III increased placental blood flow regardless of the state of coagulation. These findings suggest that intravascular coagulation plays a significant part in the pathophysiology of preeclampsia and that AT III concentrates may have therapeutic potential in the treatment of this condition.

[Immunological effect of immunoglobulin on experimental colitis induced by dextran sulfate].

To clarify the mechanism of action of immunoglobulin (IgG) in intravenous immunoglobulin therapy for ulcerative colitis (UC) patients, we studied the effect of IgG on the dynamics of immunocompetent cells in the colonic mucosa of experimental colitis induced by dextran sulfate sodium (DSS) in rats. The administration of the same species' IgG suppressed the mucosal infiltration of immunocompetent cells (activated T cells, macrophages and neutrophils), although the different species' IgG didn't. We have already shown that the same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha, IL-1 alpha and IL-8) in the colonic mucosa of experimental colitis induced by DSS. In the present report, we demonstrated the different species' IgG, as well as same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha and IL-1 alpha) from lamina propria mononuclear cells of rat large intestine in vitro. Therefore, it was considered that the suppression of cytokine production was a consequence of the decreased immunocompetent cells in colitis mucosa. Furthermore, we demonstrated that the DSS-treated antigen presenting cells (APCs) activated antigen-specific T cells as a possible mechanism underlying the colitis induced by DSS and the same species' IgG inhibited this activation of T cells.

[Protective effect of human macrophage colony-stimulating factor on fungal infection (2). In vitro effect of human macrophage colony-stimulating factor on systemic aspergillosis and in vitro effect on the activities of macrophage].

We studied the protective effect of human macrophage colony-stimulating factor (M-CSF) of fungal infection due to systemic aspergillosis in normal mice. We also examined the effect of M-CSF against the activities of mouse peritoneal macrophage which were relating to the phagocytosis, the killing, the production of superoxide after contacting with phorbol myristate acetate and the production of nitric oxide after contacting with interferon-gamma in vitro. M-CSF improved the median survival time and the survival rate of systemic aspergillosis. Combination therapy with M-CSF and amphotericin-B (AMPH-B) showed the therapy with either M-CSF or AMPH-B alone. M-CSF enhanced the activities of phagocytosis and the killing of ingested Candida albicans H and spores of Aspergillus fumigatus K by macrophage. Furthermore, M-CSF promoted the production of superoxide and nitric oxide in macrophage. These results indicate that M-CSF can enhance the fungicidal activity of macrophages by activation in vivo, thereby preventing the dissemination of fungal infection.

Protective effects of AE0047, a novel calcium antagonist, on incidence of stroke and hemodynamic disturbances in stroke-prone spontaneously hypertensive rats.

To test for evidence of stroke-preventive action, we initially examined the effect on salt-loaded stroke-prone spontaneously hypertensive rats of chronic treatment with 4-(4-benzhy-drylpiperazino)phenethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate dihydrochloride (AE0047), a novel calcium antagonist with slow onset and long-lasting hypotensive effect, and compared its efficacy with that of the calcium antagonist nicardipine and the vasodilator hydralazine. We then used radioactive microspheres to study the effects of these three agents on hemodynamic impairment to clarify the mechanism of the test drug's putative preventive action. In a 12-week repeated-administration study using animals of initial age 9 weeks; all vehicle-treated subjects died within 37 days as a result of severe hypertension and stroke, whereas those treated with AE0047, at doses of 1 or 3 mg/kg/day, remained free of stroke and showed no signs of hemorrhage, brain softening or the cerebrovascular lesions typical in this animal model. Significant suppression of the development of hypertension was not noted at the lower of these doses nor at a nicardipine dose of 10 mg/kg/day, which failed to prevent stroke in most cases. A 10-mg/kg/day dose of hydralazine did suppress the development of hypertension but failed to prevent death in half of all cases. In the hemodynamic study, 4-week treatment with AE0047 averted the marked decreases in cardiac output and blood flow in the brain, heart, kidneys and adrenal glands observed in the control group, as well as the accompanying rise in total peripheral resistance before and after stroke.(ABSTRACT TRUNCATED AT 250 WORDS)

[Protective effect of human macrophage colony-stimulating factor (hM-CSF) on fungal infection (1). In vivo effect of hM-CSF on systemic candidiasis and in vitro effect of hM-CSF on macrophages activities].

We studied the protective effect of human macrophage colony-stimulating factor (hM-CSF) on fungal infection due to systemic candidiasis in vivo and the activities of macrophages in vitro, in order to demonstrate the usefulness of M-CSF on fungal infection. The effect of hM-CSF on systemic candidiasis was examined by using normal and immunosuppressed mice. In addition, the effects of hM-CSF on the activity of reticuloendothelial system (RES) organ and on the phagocytic activity and NBT reduction activity of mouse macrophage were also examined in vitro. HM-CSF improved the survival rate of systemic candidiasis in both normal and immunosuppressed mice. Combination therapy with hM-CSF and fluconazole showed higher survival rate more than in the therapy with either hM-CSF or fluconazole alone. Furthermore, hM-CSF enhanced the activity of RES organ, phagocytosis by macrophages and NBT reduction by macrophages, significantly. These results indicate that hM-CSF enhances the phagocytic cactivity and candicidal activity by macrophages in vivo, thereby preventing dissemination of fungal infection.

Antimicrotubule effects of the novel antitumor benzoylphenylurea derivative HO-221.

The antitumor action of HO-221, a novel benzoylphenylurea derivative, was studied. The in vitro cytotoxic strength of HO-221 was investigated, as measured by IC50 values, compared with those of other drugs with different action mechanisms, using Chinese hamster lung (CHL) cells, mouse leukemia L1210 cells and human promyelocytic leukemia HL-60 cells. Morphological alterations following treatment were observed under a phase contrast microscope, and the mitotic index was determined at regular intervals to check for accumulation of metaphase cells. HO-221 was found to have a very strong toxic effect on all cell types, equal to that of the spindle poisons used as controls. HO-221 also produced the same specific morphological changes as the spindle poisons, with a significant accumulation of metaphase cells. A chromosome analysis of treated cells showed that HO-221 frequently induced polyploid and aneuploid cells, but without accompanying chromosome-breaking activity. An in vivo mouse bone marrow micronucleus assay was also carried out. The assay allowed the in vivo identification of a chromosome breaker or a spindle poison through the measurement of the relative sizes of micronuclei produced and erythrocytes. HO-221 was found frequently to induce relatively large micronuclei, an action regarded as specific to spindle poisons. It was thus demonstrated that HO-221 acts as a spindle poison both in vitro and in vivo. In order to investigate the mechanism of this action, a study of tubulin assembly using purified calf brain tubulin was carried out, which demonstrated clearly that HO-221 inhibits microtubule assembly. A detailed investigation of the action mechanism of HO-221 as a spindle poison is now called for.

Inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation: liquid heat treatment and polyethylene glycol fractionation.

A method for the heat treatment of human IgG solution at 60 degrees C for 10 h was established. Human immunodeficiency, mumps, vaccinia and 4 other viruses were added to the IgG solution in 33% sorbitol and heated at 60 degrees C. Those viruses were inactivated within 1 h. Heat-treated intravenous IgG (IVIG-H) was prepared by heat treatment and polyethylene glycol (PEG) fractionation. Conventional nonheated intravenous IgG (IVIG-C) was prepared from the same source paste by the fractionation method. No physicochemical or biological difference was observed between the heated and control IVIG preparations.