Supercritical fluid chromatography with tandem mass spectrometry combined with postcolumn compensation and one-step acetone protein precipitation to evaluate the bioavailability of probucol solid dispersion tablet.

Solid dispersion technology was used to improve the bioavailability of probucol due to its low hydrophilicity and high lipophilicity. In this study, a highly rapid and sensitive supercritical fluid chromatography with tandem mass spectrometry method was optimized and validated for the determination of probucol in beagle dog plasma with diazepam as an internal standard. The analyte and internal standard were extracted by acetone and then separated on a polar 2-ethylpyridine phase column (100 mm × 3 mm, 1.7 µm) at a flow rate of 1.0 mL/min using CO2 (≥99.99%) and methanol (95:5, v/v) as the mobile phase. The mass transition ion-pair was m/z 515.6→236.2 and 285.2→193.1 for probucol and internal standard, respectively. Excellent linearity was observed over the concentration range of 5-5000 ng/mL (r2 ≥ 0.9999) with a lower limit of quantification of 5 ng/mL. The intra- and inter-day accuracy and precision for all quality control samples were within ±15%. The proposed method was accurate, rapid and reproducible, which was successfully applied to a bioavailabilty evaluation of probucol solid dispersion tablets.

G5-PEG PAMAM dendrimer incorporating nanostructured lipid carriers enhance oral bioavailability and plasma lipid-lowering effect of probucol.

This work aimed to improve the oral bioavailability and plasma lipid-lowering effect of probucol (PB) by constructing a combined drug delivery system (CDDS) composed of nanostructured lipid carrier (NLC) and PEGylated poly(amidoamine) dendrimer (PEG-PAMAM). PEG-PAMAM with dendrimer generations of 5 (G5-PEG) or 7 (G7-PEG) were incorporated in PB-NLCs to form PB-CDDSs, PB-NLCs/G5-PEG and PB-NLCs/G7-PEG. The resultant two kinds of PB-CDDSs were characterized by particle size, zeta potential, drug encapsulation efficacy, PB release rates, and physical stability. Formulation effects of NLC and CDDS on the cellular uptake of hydrophobic drug were explored in Caco-2 cells by fluorescent Cy5 dye as a hydrophobic drug model. Furthermore, in vivo pharmacokinetics of the PB-CDDS composed of G5-PEG and PB-NLCs were investigated in a low density lipoprotein receptor knockout (LDLr-/-) mouse model, including plateau plasma PB concentrations after oral administration of multiple doses, and bioavailability after oral administration of a single dose of different PB formulations. In addition, lipid-lowering effect of PB-NLCs/G5-PEG was studied. The results indicate that both G5-PEG and G7-PEG significantly improved aqueous solubility of PB. The two PB-CDDSs exhibited similar particle size (around 150nm) as PB-NLCs, but slower PB burst release rate, higher total PB release amount, and better particle morphology and storage stability than PB-NLCs. In comparison with traditional NLC, CDDS dramatically enhanced cellular uptake of Cy5 into Caco-2 cells. In vivo results demonstrate that PB-NLCs/G5-PEG had the highest plateau plasma PB concentration and oral bioavailability, and the greatest cholesterol-lowering effect in comparison with PB suspensions and PB-NLCs. Therefore, G5-PEG incorporating NLC can be exploited as a promising drug delivery system to improve oral bioavailability and lipid-lowering effect of PB.

Application of phospholipid complex technique to improve the dissolution and pharmacokinetic of probucol by solvent-evaporation and co-grinding methods.

To enhance the aqueous solubility and thus oral bioavailability of a poorly water-soluble drug, probucol (PB), probucol-phospholipid complex (PB-PC) was formulated by solvent-evaporation or co-grinding methods. The complexes were characterized by differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), solubility, oil-water partition coefficient and in vitro dissolution. The DSC, IR and PXRD data confirmed the formation of phospholipid complex. Furthermore, the results indicated hydrogen bond formation between PB and PC molecules play an important role in the formation of PB-PC without the formation of a new compound. The water solubility of PB in the complexes was improved from 0.005 to 17.76 or 1.65 µg/mL (by solvent-evaporation or co-grinding methods respectively). As a result of it, the improved dissolution was shown in the prepared complexes. The PB-PC complexes by both solvent-evaporation and co-grinding methods exhibited higher peak plasma concentration (16,625.7 or 5343.3 vs. 2628.4 ng mL(-1)), increased AUC0-48 h (145,863.2 or 77,477.0 vs. 34,435.9 ng mL(-1)h) when compared with the commercial product, suggesting improved bioavailability of the drug. The study therefore suggests that the phospholipid complexes have possibilities in enhancing the therapeutic efficacy of PB which may be due to its improved aqueous solubility, dissolution behavior and thus bioavailability.

Effects of probucol, a typical hERG expression inhibitor, on in vivo QT interval prolongation in conscious dogs.

The cholesterol-lowering drug, probucol, is known to induce QT interval prolongation and torsades de pointes in patients. Recent in vitro studies have indicated that probucol reduces hERG expression in the plasma membrane and does not directly block human ether-a-go-go-related gene (hERG) channels. The present study was performed to investigate the effects of probucol on in vivo QT interval prolongation. Epicardial electrocardiograms were recorded in conscious dogs given oral single or repeated (7 days) doses of probucol (100mg/kg), and in combination with moxifloxacin (20mg/kg). QTc intervals were analyzed by a probabilistic method with individual rate collection formulae. Values of change in QTc (QTc) interval and its integration from 1 to 21 h (AUC1-21h) were calculated to evaluate drug-induced QT prolongation. A single dose of probucol slightly but significantly increased the AUC1-21h QTc interval on days 2 and 3. The QT prolongation was markedly augmented by repeated doses of probucol in a time-dependent manner, despite the lack of increase in plasma concentration. The combination of probucol and moxifloxacin produced additive effects on QT interval prolongation. These results suggest that long-term exposure to the hERG expression inhibitor, probucol, is required to evaluate its maximal effects on in vivo QT interval prolongation. A combination of direct and indirect hERG inhibitors may produce simple additive effects on QT interval prolongation.

A new function of Vitamin E-TPGS in the intestinal lymphatic transport of lipophilic drugs: enhancing the secretion of chylomicrons.

The purpose of this study was to examine whether Vitamin E-TPGS had a function in promoting the secretion of chylomicrons in enterocytes. Therefore, we simulated the human intestinal epithelial cells by Caco-2 cell model to study the effect of Vitamin E-TPGS on the chylomicron secretion in vitro. Meanwhile, chylomicron flow blocking rat model and mesenteric lymph cannulated rat model were used for the studies in vivo to evaluate the effect and probucol was chosen as the model drug. The results of cell experiments indicated that Vitamin E-TPGS at low concentration had a strong enhancement on the secretion of chylomicrons. The results of animal experiments indicated that Vitamin E-TPGS could significantly enhance the lymphatic transport of probucol by the same role consistently with the results obtained from cell experiments. However, the role would reach the plateau and saturate after a concentration dependent increase. These studies first demonstrate the function of Vitamin E-TPGS in the intestinal lymphatic transport of lipophilic drugs, which can significantly promote the secretion of chylomicrons by the intestinal epithelial cells.

[Determination of probucol in human plasma by HPLC-MS/MS].

OBJECTIVE: To develop a specific and sensitive method for the determination of probucol in human plasma using HPLC-MS/MS technique. METHODS: Probucol were extracted from plasma by ethyl ether: dichloromethane (1:1, V/V), with physcion as an internal standard. The analytes went through the column of ultimate CN (50 mm x 4.6 mm, 5 microm) with mobile phase acetonitrile: water: ammonia water = 97:3:0.05 (adjusted pH = 7.2 with formic acid). Probucol was analyzed with a negative mode. The ion pairs being detected were 515.5-->236.1 (probucol) and 283.0-->239.9 physcion, respectively. RESULTS: The established method was able to determine probucol in human plasma over the range of 2.5-6000 ng/mL, with a method recovery ranging from 93.02% to 104.12%. The intra and inter day variances were below 4.67% and 5.72%. CONCLUSION: The HPLC-MS/MS method for analyzing probucol was validated. It is sensitive and suitable for pharmacokinetic studies of probucol.

Enhanced bioavailability of probucol following the administration of solid dispersion systems of probucol-polyvinylpyrrolidone in rabbits.

Disks of probucol and solid dispersion systems of probucol-polyvinylpyrrolidone (PVP) in various weight ratios were prepared. Dissolution of probucol was markedly increased in the solid dispersion systems in J.P. XV disintegration media No. 1 (pH 1.2) and No. 2 (pH 6.8). The concentrations of probucol after the dissolution of the disks of solid dispersion systems showed supersaturation. Following the administration of disks of solid dispersion systems in rabbits, a marked increase in the area under the plasma concentration time curve (AUC) was observed. When the weight ratio of PVP to probucol was larger, a larger AUC was observed. When disks of the 1 : 9 solid dispersion system (weight ratio of probucol : PVP=1 : 9) containing 50 and 100 mg probucol were respectively administered, AUC values were approximately proportional to the dose. AUC values following the administration of disks of the 1 : 9 solid dispersion systems containing 15 mg probucol (total weight: 150 mg) and 500 mg probucol were approximately equal. The mean half life (t(1/2)) was 12 h when disks of the 1 : 9 solid dispersion system were administered, whereas the t(1/2) was 35 h when probucol disks were administered. The markedly increased dissolution of probucol in solid dispersion systems resulted in a marked increase in its bioavailability.

Effects of alpha tocopherol and probucol supplements on allergen-induced airway inflammation and hyperresponsiveness in a mouse model of allergic asthma.

OBJECTIVE: We investigated the role of antioxidants in airway hyperresponsiveness to acetylcholine using young asthma model mice, which were sensitized and stimulated with ovalbumin. METHODS: The mice had been fed either a normal diet, an alpha-tocopherol-supplemented diet or a probucol-supplemented diet 14 days before the first sensitization. They were immunized with antigen at intervals of 12 days and, starting from 10 days after the second immunization, they were exposed to antigen 3 times every 4th day using an ultrasonic nebulizer. Twenty-four hours after the last antigen inhalation, airway responsiveness to acetylcholine was measured and bronchoalveolar lavage fluid (BALF) was collected. A blood and lung tissue study was also carried out. RESULTS: Twenty-four hours after the last antigen challenge, both IL-4 and IL-5 in the BALF of alpha-tocopherol-supplemented mice were significantly decreased. The IL-5 level in probucol-supplemented mice was also decreased, but there was no difference in IL-4 levels. The serum IgE level was decreased in probucol-supplemented mice. Differential cell rates of the fluid revealed a significant decrease in eosinophils due to antioxidant supplementation. Airway hyperresponsiveness to acetylcholine was also repressed in antioxidant-supplemented mice. In histological sections of lung tissue, inflammatory cells and mucus secretion were markedly reduced in antioxidant-supplemented mice. We investigated the antioxidant effect on our model mice by examining 8-isoprostane in BALF and lung tissue, and acrolein in BALF; however, our experiment gave us no evidence of the antioxidant properties of either alpha-tocopherol or probucol contributing to the reduction of airway inflammation. CONCLUSION: These findings indicate that alpha-tocopherol and probucol suppress allergic responses in asthma model mice, although these two drugs cause suppression in different ways that are unrelated to antioxidation.

Evaluation of pharmacokinetic interaction between cyclosporin A and probucol in rats.

PURPOSE: The purpose of this study was to clarify the mechanism of pharmacokinetic interaction between cyclosporin A and probucol in clinical cases. METHODS: The whole blood concentration of cyclosporin A was measured after oral administration of cyclosporin A with or without probucol in rats. Cyclosporin A was administered as three types of solutions: the contents of the conventional formulation (Sandimmun capsule) diluted with corn oil and the contents of the new microemulsion preconcentrate formulation (Neoral capsule) diluted with saline or corn oil. The solubility of cyclosporin A and another lipophilic agent tacrolimus in water with or without probucol was also measured. RESULTS: The area under the blood concentration-time curve (AUC) after the administration of Sandimmun (corn oil) and Neoral (corn oil) was significantly decreased to 26% and 41% of the control by coadministration of probucol. However in the case of Neoral (saline), it was unchanged. The terminal elimination rate constant was not affected by probucol in any type of cyclosporin A solution. The solubility of cyclosporin A or tacrolimus in water dropped to 49% or 16% of the respective control in the presence of probucol. CONCLUSION: The interaction between cyclosporin A and probucol is caused by the decreased absorption of cyclosporin A partly based on the lowered solubility in the presence of probucol.

Effects of probucol on atherosclerosis of apoE-deficient or LDL receptor-deficient mice.

The effect of probucol on atheroma formation was evaluated using mouse models for atherosclerosis with different diet protocols. Dietary administration of probucol (0.5 %, wt/wt) for 12 weeks reduced total plasma cholesterol levels in both apolipoprotein E (apoE)-deficient mice fed a western diet and in low-density lipoprotein receptor (LDLR)-deficient mice fed a Paigen diet by 60 % and 30 % to 60 %, respectively. Probucol treatment also significantly reduced high-density lipoprotein (HDL) levels in apoE-deficient mice, but not in LDLR-deficient mice. Atherosclerotic plaques in the aortic sinus of probucol-treated apoE-deficient mice were two-fold larger than those in untreated apoE-deficient mice, while the lesions in probucol-treated LDLR-deficient mice were similar to those in untreated LDLR-deficient mice. A strong negative correlation between HDL cholesterol levels and lesion sizes at the aortic sinus was observed in apoE-deficient mice, but not in LDLR-deficient mice. Thus, in contrast to LDLR-deficient mice, probucol had a strong proatherogenic effect in the aortic sinus of apoE-deficient mice associated with the reduction of HDL levels in spite of the reduction of total plasma cholesterol levels. The varying effects of probucol on atherogenesis depend upon the portion of aorta and which animal model is evaluated, implicating that complex cellular events are involved in the effect of probucol.

Inhibition of VCAM-1 expression in the arterial wall is shared by structurally different antioxidants that reduce early atherosclerosis in NZW rabbits.

We previously established that probucol decreases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expression that normally accompanies atherogenesis. To determine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expression and whether the degree of VCAM-1 inhibition correlates with the reduction of atherosclerosis or the antioxidant protection of LDL. Atherogenesis and VCAM-1 mRNA and protein were determined in four groups of NZW rabbits (n = 6;-8) fed 0.5% cholesterol alone or supplemented with 0.1% vitamin E, a low dose (0.04;-0.075%) of probucol yielding the same degree of antioxidant protection of plasma LDL as vitamin E, or a high dose (0.5%) of probucol, and in normocholesterolemic rabbits. After 81 days, extensive atherosclerosis and a greater than 4-fold up-regulation of VCAM-1 mRNA was seen in rabbits on high cholesterol diet, mostly in the intima. Treatment with vitamin E, high-dose probucol, and low-dose probucol significantly decreased VCAM-1 mRNA by 49.0, 74.9, and 57. 5%, respectively, and reduced atherosclerosis in adjacent segments of the thoracic aorta to a similar degree as reported by previous studies. Immunocytochemistry confirmed that lesions of antioxidant-treated animals also contained less VCAM-1 protein. Neither the degree of VCAM-1 inhibition nor the extent of atherosclerosis correlated with the degree of antioxidant protection of plasma LDL.In summary, treatment with structurally unrelated antioxidants conveyed different degrees of antioxidant protection to plasma LDL but significantly reduced VCAM-1 expression in vivo and inhibited atherogenesis. This is consistent with the assumption that antiatherogenic effects of antioxidants may in part be mediated by interference with oxidation-dependent intracellular signaling.

Combined effects of probucol and benzafibrate on lipoprotein metabolism and liver cholesteryl ester transfer protein mRNA in cholesterol-fed rabbits.

Probucol decreases and bezafibrate increases plasma high density lipoprotein-cholesterol (HDL-C) levels in humans. This study was performed to determine whether the HDL-C-lowering effects of probucol could be reversed by treatment with bezafibrate in hypercholesterolemic rabbits. Forty-nine normolipidemic Japanese White rabbits were divided into 5 groups [group 1: normal chow; group 2: 0.2% cholesterol (Ch) diet; group 3: 0.2% Ch and 1% probucol diet; group 4: 0.2% Ch and 1% bezafibrate diet; group 5: 0.2% Ch and 1% probucol plus 1% bezafibrate diet] and treated for 8 weeks. Plasma lipids, cholesteryl ester transfer protein (CETP) activity in the lipoprotein-deficient plasma fraction, CETP mRNA in liver tissue and plasma drug concentrations were investigated. Serum total cholesterol (TC) increased after the rabbits in groups 2, 3, 4 and 5 were fed Ch, but overall, no significant differences were observed in serum TC and triglyceride (TG) among these groups. Serum HDL-C levels increased (p<0.01) in the bezafibrate-treated group, but a significant (p<0.05) reduction in HDL-C was observed in both the Ch + probucol (group 3) and Ch + probucol plus bezafibrate (group 5) groups; no significant difference was observed between groups 3 and 5. Significant correlation (p<0.01) was found between serum low density lipoprotein cholesterol (LDL-C) levels and plasma probucol concentrations in groups 3 and 5, but no correlation was found between plasma concentrations of probucol/bezafibrate and serum HDL-C levels. CETP activity in the lipoprotein-deficient plasma fraction increased in the Ch-, Ch + probucol-, and Ch + probucol and bezafibrate-fed groups (groups 2, 3 and 5, respectively), whereas a significant reduction in this activity was observed in the Ch + bezafibrate-fed group (group 4). An analysis of covariance showed that the CETP activity responded more sensitively to drug treatment than did the serum HDL-C level. CETP mRNA in liver tissue was assessed by Northern blotting at 8 weeks, but no changes were observed among the 5 groups. Probucol decreased and bezafibrate increased serum HDL-C levels, through CETP activity without affecting liver CETP mRNA levels, and the decrease in HDL-C levels produced by probucol could not be reversed by bezafibrate.

Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E-deficient mice: possible mechanisms of action.

BACKGROUND: The effects of probucol and a phytosterol mixture (FCP-3PI) on atherosclerotic lesion formation, plasma lipoproteins, hepatic and lipoprotein lipase activities, antioxidant enzyme activities, and plasma fibrinogen were investigated in apolipoprotein E-knockout (apoE-KO) mice. METHODS AND RESULTS: Three groups of 8 mice were fed a diet containing 9% (wt/wt) fat (controls) or the foregoing diet supplemented with either 1% (wt/wt) probucol (the probucol group) or 2% (wt/wt) FCP-3PI (the FCP-3PI group) for 20 weeks. Compared with controls, atherosclerotic lesion size was 3 times greater in the probucol group, whereas it was decreased by half in the FCP-3PI group. Probucol treatment resulted in high plasma probucol concentrations, which correlated (r=0.69) with the lesion area. HDL cholesterol was reduced (>75%) in the probucol group and slightly increased (14%) in the FCP-3PI-treated group. Postheparin lipoprotein lipase (LPL) activity was significantly reduced in both treatment groups, but only FCP-3PI significantly decreased hepatic lipase activity. Plasma fibrinogen was increased 42% by probucol and decreased 19% by FCP-3PI relative to controls. Probucol significantly increased plasma glutathione reductase, glutathione peroxidase, and superoxide dismutase activities (P<0.05). In contrast to findings in apoE-KO mice, there was no probucol-induced atherosclerosis in their wild-type counterparts fed the same dose for the same period of time. CONCLUSIONS: Antiatherogenic activity of FCP-3PI in apoE-KO mice is associated with an increase in HDL cholesterol concentration along with decreases in hepatic lipase activity and plasma fibrinogen concentrations. Proatherogenic effects of probucol may be related to increased plasma fibrinogen, decreased HDL cholesterol concentrations along with decreased LPL activity, or its direct "toxicity" due to very high plasma concentration. Our studies demonstrate that the antioxidant and cholesterol-lowering properties of probucol do not prevent atherogenesis in this particular animal model.

[A comparison of the hypolipidemic action of probucol at doses of 500 and 1000 mg/day in moderate hyperlipoproteinemia]. / Sopostavlenie gipolipidemicheskogo deistviia probukola v doze 500 i 1000 mg/sut pri umerennoi giperlipoproteidemii.

AIM: Evaluation of effectiveness of hypolipidemic action of probucol in doses 500 and 1000 mg/day and comparison of probucol blood concentrations on the treatment month 3 and 6. MATERIALS AND METHODS: Probucol (Akrikhin, Russia) was given to 41 patients with primary hypercholesterolemia in a dose 500 mg/day. 3 months later the patients were divided into two groups. Group 1 patients exhibited a > 10% decrease in cholesterol levels and continued to take probucol in the dose 500 mg/day. Group 2 patients were crossed over to higher cholesterol dose--up to 1000 mg/day. Lipids levels were measured by enzyme tests, apoproteins--by immunoturbidimetry and immunodiffusion, probucol concentrations--by high-performance liquid chromatography. RESULTS: After 3 months of treatment, cholesterol lowered by 14.3 and 9.2% in groups 1 and 2, respectively. After 6 months, by 19.7 and 12.9%, respectively. Probucol concentrations in blood were higher after 6 months of treatment than after 3 months in both groups. No significant differences existed between the groups by probucol concentrations in 3 and 6 months. CONCLUSION: Hypolipidemic effect of probucol depended on the individual features of lipoproteins metabolic disorders rather than the drug blood concentration. Larger probucol doses fail to reduce cholesterol further.

Formation of malonaldehyde in the presence of probucol, an anti-atherosclerosis drug.

Formation and inhibition of malonaldehyde (MA) from blood plasma lipids oxidized by Fenton's reagent in the absence or presence of probucol [4,4'-(isopropylidenedithio)bis(2,6-di-tert-butylphenol)] and L-ascorbic acid were investigated. The amount of MA formed was quantitatively analysed by gas chromatography. L-Ascorbic acid inhibited MA formation by about 30% at the level of 4.0/micromol, but the amount of MA formed was increased by the presence of probucol. When 3.0 micromol oxidized probucol was hydrolysed at pH 1. 3 and 5, 2616.5 nmol, 287.5 nmol and 103.9 nmol MA were recovered, respectively. This is the first report of quantitative analysis of MA formed from probucol on oxidation.

Measurement of reactive oxygen species by chemiluminescence in diet-induced atherosclerosis: protective roles of vitamin E and probucol on different radical species.

We have investigated the effects of a high-cholesterol diet on the production of different reactive oxygen species in rabbit aortic rings and evaluated the protective effects of vitamin E and probucol in preventing peroxidative changes. Twenty-five male albino rabbits were divided into five groups. Control rabbits were fed a vitamin E-poor rabbit chow. Rabbits in the second group were given a vitamin E-poor diet supplemented with 2% cholesterol. Other groups received either 50 mg/kg vitamin E, 1% probucol, or both, in addition to 2% cholesterol for 4 weeks. Reactive oxygen species formation in aortic rings was measured by enhanced chemiluminescence using luminol and lucigenin. (The results were given as cpm/mg wet weight.) Further differentiation of radical species involved in luminol-enhanced chemiluminescence was performed using sodium azide and L-nitroarginine, a selective inhibitor of nitric oxide production. Our results indicated that cholesterol feeding increased lucigenin and luminol chemiluminescence, where the contribution of free radicals inhibited by sodium azide (radicals originating from endothelial cells or from phagocytes) were 53% and peroxynitrite 24%. Both vitamin E and probucol were effective as scavengers of free radicals, but the effect of vitamin E was more pronounced. In conclusion, the present study demonstrated excessive generation of reactive oxygen species within the atherosclerotic vessel. Peroxidative changes could be prevented by vitamin E and probucol treatment, but vitamin E seemed to be more efficient.

Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice.

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.

Probucol treatment attenuates the aortic atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

We examined the effect of probucol on the aortic atherosclerosis already developed in Watanabe heritable hyperlipidemic (WHHL) rabbits at the initiation of treatment. In WHHL rabbits treated with probucol for 5 months from 8 months old, the lesion area in the aorta was significantly (P < 0.05) reduced when compared with that in untreated animals as well as animals at age 8 months. In contrast, plasma cholesterol levels in the probucol-treated group and untreated group during the experiment were not significantly different. LDL prepared from rabbits receiving probucol for 5 months showed resistance to oxidation by copper ions. Plasma CETP activity was significantly (P < 0.05) increased by probucol treatment. An immunohistochemical study showed that macrophages were abundant in the atherosclerotic lesions of untreated rabbits whereas smooth muscle cells were predominant in lesions of probucol-treated rabbits. These results suggest that the atherosclerotic lesion in WHHL rabbits can regress when treated by probucol and that the attenuation of atherosclerosis in this animal involves effects of probucol other than a decrease in plasma cholesterol, for example anti-oxidant activity.

Endothelial dysfunction in streptozotocin-diabetic rats is not reversed by dietary probucol or simvastatin supplementation.

Oxidative stress and dyslipidaemia are key features of diabetes mellitus and may be involved in mediating the vascular endothelial dysfunction associated with this disease. The aim of this study was to examine the effect of dietary lipid-lowering and antioxidant agents on vascular endothelial function and oxidative stress. Diabetic male Sprague-Dawley rats (i.v. streptozotocin, 45 mg/kg) were fed for 4 weeks on a standard diet or on a diet supplemented with either the lipid-lowering antioxidant probucol (1% w/w in diet) or the 3-hydroxy 3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitor simvastatin (0.01% w/w in diet). Responses to noradrenaline, acetylcholine, and sodium nitroprusside were assessed in small mesenteric arteries (mean internal diameter 300+/-5 microm, n = 80) mounted on a small vessel myograph. Plasma concentrations of total cholesterol and triglycerides were significantly raised in standard-fed diabetic rats and significantly reduced in probucol and simvastatin-fed diabetic rats 8-epi-prostaglandin (PG)F2alpha, an indicator of oxidative stress, was raised in liver and aorta from diabetic rats compared to controls. Probucol supplementation reduced 8-epi-PGF2alpha in aorta and liver of diabetic rats but increased 8-epi-PGF2alpha content in plasma and aorta from control animals. The abnormal relaxation to acetylcholine in arteries from the diabetic rats (pEC550 diabetic 6.763+/-0.172 vs control 7.541+/-0.175; p < 0.05) was not improved by probucol or simvastatin. These data, therefore, do not support a role for oxidative stress or dyslipidaemia in mediating the impaired ACh-induced endothelium-dependent relaxation of small mesenteric arteries from the streptozotocin-diabetic rat.

Effect of LDL-apheresis on the pharmacokinetics of the lipophilic antilipidemic agent probucol.

The effect of LDL-apheresis on the pharmacokinetics of antilipidemic agents has not been evaluated thoroughly. In this study, we investigated the effect of LDL-apheresis on the pharmacokinetics of probucol, a lipophilic antilipidemic agent, by studying its distribution and changes in the blood concentration of probucol after LDL-apheresis. The concentrations of lipoproteins were measured before and after LDL-apheresis in eight patients with familial hypercholesterolemia taking probucol. Concentrations of probucol in the various lipoprotein fractions and plasma were measured by HPLC. The serum concentrations of probucol before and after LDL-apheresis were 39.8 +/- 3.3 and 16.5 +/- 1.6 micrograms/ml, and the correlation coefficient between the changes in the serum probucol concentration and those in the serum cholesterol concentration before and after LDL-apheresis was significant (r = 0.73, P < 0.01). Changes in the probucol and cholesterol concentrations after LDL-apheresis were mainly found in the LDL fraction. The calculated reductions in the serum contents of probucol and cholesterol were similar to the contents of probucol and cholesterol in the irrigation fluid of the dextran sulfate column. These data suggest that changes of probucol concentration in plasma by LDL-apheresis are mainly due to reductions in the LDL fraction.