A scanning calorimetric study of the effect of clover saponin on liposomal phospholipid membrane

The effect of clover saponin on the phase transition of liposomal lipid bilayers of dimyristoyl phosphatidylcholine was investigated with differential scanning calorimetry. The thermograms of the liposomal bilayers incorporated with the clover saponin were obtained, and the enthalpy changes and the sizes of cooperative unit of the transition were calculated. The results showed that incorporation of the clover saponin into the liposomal bilayers effectively reduced the transition temperature at which the transition from solid state to liquid-crystalline state occurs, and broadened the thermogram peaks. It also reduced the size of cooperative unit of the transition. These results indicate that the clover saponin might have significant effect on the fluidity of biological membranes.     

New conformational polymorph of hydrochlorothiazide with improved solubility

Context: To characterize a new conformation of hydrochlorothiazide (HCT) with better solubility and establishing its relationship with previously reported form I, obtained during attempted crystallization experiments.

Objective: The aim of present investigation is to unveil a new conformational polymorph (form IA) having a higher solubility compared to commercially available form I.

Materials and methods: New form (IA) was obtained from slow evaporation as well as by solvent–antisolvent method and was then characterized by DSC, FTIR, PXRD and SCXRD. Equilibrium solubility profile shows that it is more soluble than form I.

Results: Appearance of phase transition endotherm at 215.87?°C in DSC spectra indicated the existence of new polymorph which was further confirmed by FTIR and PXRD. Single crystal study showed significant difference in various bond angles and torsion angles of the two forms. The solubility exhibited by form IA was (938?µg/mL) compared to form I (791?µg/mL) in water.

Discussion: Complete structural analysis and molecular arrangements in the unit cell along with the DSC and FTIR data confirm the existence of new conformer of HCT.

Conclusion: This study reveals the existence of a new conformational polymorph of HCT molecule having higher solubility could prove to be promising in pre-formulation.     

The Cryopreservation of Liposomes. 1. A Differential Scanning Calorimetry Study of the Thermal Behavior of a Liposome Dispersion Containing Mannitol During Freezing/Thawing

The thermal behavior of water in liposome dispersions and in liposome dispersions containing mannitol at subzero temperatures was investigated with differential scanning calorimetry (DSC). The cooling curves from 20 down to - 60°C for a liposome dispersion (bilayer composition PL100H/DCP), monitored at cooling rates of 5 and 10°C/min, showed several heat flows related to water crystallization. All lipid-containing dispersions showed water crystallization at temperatures below –40°C. The magnitude of this heat flow strongly depended on the experimental variables. Cooling rate, particle size, lipid concentration, and location and nature of the cryoprotectant all influenced the water crystallization behavior as shown in the DSC cooling curve. Different fractions of water–presumably related to their location in the dispersion–could be distinguished. It is concluded that DSC provides a valuable tool for the detection of changes in the physical state of water in liposome dispersions during freezing/thawing. The insights gained from these DSC studies may make it possible to select–on the basis of rational considerations rather than by trial and error–optimum conditions for the cryopreservation of liposomes containing water-soluble drugs.     

Effects of Concanavalin A on the isolated perfused rat liver

Summary In the isolated perfused liver, Concanavalin A provoked a significant decrease of flow rate within 2 to 4 min which was dose-dependent and could be partly inhibited by specific antagonists.Furthermore it was found that the lectin led to a decline of the respiration, an increase of the lactate/pyruvate ratio and a release of the transaminases into the medium. It was suggested that Concanavalin A displaced endothelial cells in the liver capillaries, which occluded the vessels and decreased the flow rate. The decreased respiration was considered to be secondary to this effect.     

Interaction of the chlorinated hydrocarbon insecticide lindane or DDT with lipids--a differential scanning calorimetry study

The interaction of DDT and lindane with glycosphingolipids and phospholipids was investigated by employing differential scanning calorimetry. The degree of perturbation produced by lindane is stronger than that of DDT and depends also on the lipid.     

Use of rat liver slices for the study of oxidative DNA damage in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells.

Tissue slices are a useful biological system for lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat liver slices, in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl(3)), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37 degrees C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2'-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in liver slices and isolated nuclei, Fe/NTA (1 mM/4 mM) induced high levels of TBARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TBARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate. Liver slices incubated with 1 mm BrCCl(3), BrB or 2-NP had elevated TBARS but had little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl(3), BrB or 2-NP, levels of TBARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH showed marked elevation of TBARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants.     

The Reversibility of Absorption Promoter Interaction with Red Blood Cell Membranes Studied with Differential Scanning Calorimetry

Absorption promoters, or adjuvants, are used to enhance the gastrointestinal absorption of poorly absorbed drugs such as macromolecules. In the present work, adjuvant–membrane interactions have been studied by differential scanning calorimetry (DSC) using red blood cell (RBC) membranes as model membrane. These interactions caused temperature shifts, amplitude changes, and broadening of the RBC transitions. Because more than one transition may be simultaneously affected by a given adjuvant, complex overlappings occur. Gaussian modeling and nonlinear regression analysis, therefore, were used to resolve these transitions. A correlation, which may serve as an indicator of adjuvant potency, was found between adjuvant concentration and induced transition temperature shifts. Further, these shifts recovered to baseline after successive washings with buffer (for most adjuvants). Sodium lauryl sulfate induced transition alterations, however, never recovered. Thus the DSC might be useful in monitoring reversible adjuvant–membrane interactions.     

Interaction of aromatic aldehydes with isolated rat liver mitochondria

It has been suggested that aromatic aldehydes may reduce cytochrome c [Wolf et al. Fedn Proc.39 (3), 1013 (1980)]. Therefore, interaction of the aromatic aldehydes, p-anisaldehyde, benzaldehyde, p-tolualdehyde, p-carboxybenzaldehyde, p-chlorobenzaldehyde and p-nitrobenzaldehyde, with rat liver mitochondria was examined in vitro. Although both pyruvate/malate- and succinate-mediated respiration, as well as that mediated by other citric acid cycle intermediates, were inhibited by the aromatic aldehydes (0.5 to 1.0 mM), cytochrome c oxidase was not inhibited by aromatic aldehydes (1.0 to 20 mM). There was a marked inhibition of suecinic dehydrogenase and both ADP- and DNP-stimulated respiration by benzaldehyde (2 to 20 mM). Since both pyruvate/malate- and succinate-mediated respiration were inhibited by the aromatic aldehydes without inhibition of cytochrome c oxidase, several sites of inhibition, possibly both at the site of transport of substrates and the active enzymes, may exist. Benzaldehyde, 300 μM, inhibited pyruvate/malate-mediated state 3 respiration by 50% which suggests that no additional functional group or metabolism to another species is required for these inhibitory effects.     

应用高效液相色谱法中的高速扫描技术测定全血中环孢素A浓度

目的建立一种运用高速扫描技术测定血中环孢素A浓度的HPLC法.方法取全血2.0 ml,加盐酸溶液2 ml后,以3 ml乙醚两次提取;分离乙醚液后用氢氧化钠液洗涤后蒸干;以乙腈/水溶液60 μl重组,再用正己烷洗涤后进样20 μl.色谱条件Elite 220 mm×4.6 mm C18柱,柱温65℃,以甲醇-水(88∶12)为流动相,流速1.0 ml·min-1,扫描波长205～250 nm(间隔5 nm),积分波长210 nm.结果CsA浓度在50～1000 ng·     

阿德福韦酯-PEG6000固体分散体的制备

目的将难溶性药物阿德福韦酯制备成固体分散体,以增加体外溶出度。方法以聚乙二醇6000(polyethylene glycol 6000,PEG6000)为载体,采用熔融法制备阿德福韦酯固体分散体;配合差示扫描量热(differential scanning calorimetry,DSC)与X-射线衍射(X-ray diffraction,XRD)观察药物在载体中的存在状态;考察相对湿度(relative humidity,RH)75%40℃放置3个月固体分散体对溶出度的变化及载体-药物质量比对溶出的影响。结果阿德福韦酯以无定型状态存在于固体分散体中,相对湿度RH75%40℃放置3个月固体分散体对溶出度改善明显,载体-药物质量比不同,药物的溶出度不同。结论将阿德福韦酯制成固体分散体能显著增加阿德福韦酯的体外溶出度。     

A Mechanistic Investigation of an Amorphous Pharmaceutical and Its Solid Dispersions,Part I: A Comparative Analysis by Thermally Stimulated Depolarization Current and Differential Scanning Calorimetry

PURPOSE: To explore using thermally stimulated depolarization current (TSDC), in comparison to differential scanning calorimetry (DSC), for the characterization of molecular mobility of an amorphous pharmaceutical new chemical entity (LAB687), an amorphous polymer (PVPK-30), and their combination as solid dispersions at different % drug loadings. METHODS: Amorphous drug was prepared by quenching from the melt. Solid dispersions containing 10-60% of drug in polymer were prepared by solvent evaporation method. Glass transition temperatures (Tg) were determined by DSC and TSDC. RESULTS: In comparison to a single T. obtained from DSC for the drug substance, TSDC shows two overlapping relaxations. Both peaks correspond to a-relaxations that are associated with the glass transition, with the second peak corresponding to the rigid fraction that is difficult to be detected by DSC because it is associated with only small changes in heat capacity. Two overlapping relaxations were also observed for the polymer vs. one Tg by DSC. The lower temperature relaxation is believed to be a beta-relaxation, whereas the higher temperature transition corresponds to an alpha-relaxation. For the solid dispersions, one single peak was obtained for each of the 20% and 30% dispersions in excellent agreement with the DSC results. However, at the 40% drug load, a small shoulder was observed by TSDC at the low temperature of the main peak. This shoulder becomes more pronounced and overlaps with the main peak as the drug load increases to 50% and 60%. Agreement between the Tg values calculated by the Gordon-Taylor equation and the DSC and TSDC experimental data, especially for the 20% and 30% drug loading, indicate ideal miscibility. At higher drug loads, only by TSDC was it possible to detect the saturation level of the drug in the polymer. CONCLUSIONS: TSDC proved to be very sensitive in detecting small reorientational motions in solids and in separating overlapping events with only slight differences in molecular motion exhibited as broad events in DSC. This allowed for detection of the rigid fraction of the amorphous drug, the sub-glass transition beta- relaxation in the polymer, and the limit of miscibility between the drug and the polymer in the solid dispersions.     

Predictions of bisphenol A hepatic clearance in the isolated perfused rat liver (IPRL): impact of albumin binding and of co-administration with naproxen

1.?This study aimed (i) to characterise hepatic clearance (CL) of bisphenol A (BPA) and naproxen (NAP) administered alone or in binary mixtures to highlight the influence of a binding to albumin (ALB) using an isolated perfused rat liver (IPRL) system; and (ii) to compare results of prediction algorithms with measured clearance rates.

2.?The IPRL system and liver microsomes were used to determine the metabolic constants of BPA and NAP either in the presence or absence of ALB. In this study, the IPRL was used as proxy for the in vivo situation. Accordingly, diverse in vitro-to-in vivo and in vivo-to-in vivo extrapolations (IVIVEs) were made to predict CL of BPA determined in situ/in vivo with ALB from metabolic data determined without ALB by using different binding correction methods (i.e., direct and conventional scaling as well as a novel scaling considering an ALB-facilitated uptake mechanism).

3.?The addition of ALB significantly influenced the liver kinetics of BPA and NAP either administered alone or in binary mixtures, which was reflected in the Michaelis-Menten constants. Analysis of concomitant exposures of BPA and NAP gave a fully competitive inhibition. Furthermore, the IVIVE method based on the ALB-facilitated uptake mechanism provided the most accurate predictions of CL_{in vivo} as compared with the other IVIVE methods when the impact of ALB is considered.

4.?Our findings support the notion that high binding to ALB reduces the biotransformation of BPA and NAP when administered alone or in mixtures in the IPRL system. However, the free drug concentration in liver in vivo is probably higher than expected since the IVIVE method based on a potential ALB-facilitated uptake mechanism is the most robust prediction method. Overall, this study should improve the physiologically-based pharmacokinetic (PBPK) modelling of chemical–drug interactions.     

Preparation and phase behaviour of surface-active pharmaceuticals: self-assembly of DNA and surfactants with membranes. Differential adiabatic scanning microcalorimetric study

Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipid-DNA self-assemblies, as alternative models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg(2+) and N-alkyl-N,N,N-trimetylammonium ions (C(n)TMA, n=12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexes with Mg(2+) and C(12)TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodisperce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast, divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on the structure and physicochemical properties of DNA-Mg(2+)-liposomes or DNA-cationic surfactant-liposome systems is emphasized by proposing the structure of their molecular self-organizations with further implications in gene transfer research.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies

Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresorufin indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a significant NADPH oxidation catalyzed by CYP2E1 supersomes, strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present findings may contribute to understand better the physiological function of CYP450 in relation with inflammation and other physiological processes in which HA may have a relevant role.     

Cyclosporin A and erythromycin: A study of a drug interaction in the in situ perfused rat liver model

Using the in situ perfused rat liver model, the effect of erythromycin (Ery) on the disposition of cyclosporin A (CyA) and the major human metabolite, AMl, was investigated. Prior to perfusion experiments, oral dosing was carried out for three days on three groups of male Sprague—Dawley rats (300–350 g), involving pretreatment with water (control and H₂O/Ery groups) or erythromycin (Ery oral group). On the fourth day, perfusion experiments took place using standard techniques, with the addition of 20 mg Ery to the H₂O/Ery and Ery oral groups, and 2.5 mg CyA to all groups. Perfusate and bile samples were collected and assayed for CyA and AMl by HPLC. Results indicated that inhibition of CyA metabolism had occurred as the CyA concentration in perfusate was significantly higher in both Ery groups at all times compared to the control group, and the levels of AMl in both perfusate and bile were significantly lower than in the control group. There was also a marked reduction in the apparent metabolic clearance of CyA in the Ery groups. It was concluded that AMl production had been inhibited by Ery, the most likely mechanism being inhibition of the isoenzyme CYP3A with which Ery forms a stable complex.     

HPLC法测定大鼠UGT1A6的酶活性及体外动力学分析

目的:建立灵敏、稳定的高效液相色谱法,以对硝基酚为探针底物,评价体外大鼠肝微粒体中UGT1A6酶活性。方法:色谱柱为Agilent Zorbax SB-C18柱(250 mm×4.6 mm,5μm),流动相为20 mmol.L-1磷酸二氢钾缓冲液-甲醇(50∶50,V/V),流速0.8 mL.min-1,柱温30℃,检测波长280 nm。对硝基酚与大鼠肝微粒体在37℃共同孵育一段时间后,加入冰乙腈终止反应,于4℃下12000 r.min-1离心15 min后,吸取上清进行HPLC分析,以双倒数作图法计算酶动力学参数。结果:对硝基酚保留时间为8.95 min,线性范围为0.5～100μmol.L-1,回归方程为Y=5973.12X+571.58(r=0.9999),最低检测限(LOD)为0.1μmol.L-1(S/N≥3),最低定量限(LLOQ)为0.5μmol.L-1,回收率为104.5%～105.5%,日内、日间RSD均小于10%,温孵体系中其他内源性物质不干扰测定;计算酶动力学参数Km为24.63μmol.L-1,Vmax为18.87 nmol.min-1.mg.protein-1。结论:该方法灵敏度高、稳定性好,适合体外对硝基酚的测定,可用于大鼠体外UGT1A6酶活性的评价及酶动力学研究。     

Interaction of the diuretics torasemide and U-37883A with the KATP channel in rat isolated aorta

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N–1-adamantyl-N’-cyclohexylhydrochloride) with the ATP-sensitive K⁺ channel (K_ATP channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K_ATP channels; this group is lacking in furosemide. U-37883A blocks several types of K_ATP channels. The interaction with the vascular K_ATP channel was probed in binding studies, ⁸⁶Rb⁺ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K_ATP channel inhibitor [³H]glibenclamide and of the opener [³H]P1075 with IC₅₀ values of 19 and 45 μM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In ⁸⁶Rb⁺ efflux experiments, the loop diuretics, at μM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was inhibited by U-37883A and torasemide with IC₅₀ values of 0.06 and 130 μM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC₅₀ values between 6 and 10 μM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 μM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 μM. The data show that torasemide interferes specifically with the binding of the K_ATP channel modulators [³H]glibenclamide and [³H]P1075 and with the K_ATP channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K_ATP channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced ⁸⁶Rb⁺ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs. Received: 28 January 1998 / Accepted: 20 April 1998