Association of hepatitis B virus X protein with mitochondria causes mitochondrial aggregation at the nuclear periphery, leading to cell death.

Hepatitis B virus (HBV) X protein activates many viral and cellular genes in trans and functional disruption of the p53 tumor suppressor gene product occurs when X protein is transiently expressed in the cytoplasm of cultured cells. We have carried out investigations to determine the exact location of X protein in X gene transfected cells by using a fluorescent staining technique as well as by biochemical analyses. Aggregation of mitochondrial structures became evident at the periphery of nucleus in the cytoplasm of X transfected cells. X protein was found associated with the aggregated mitochondrial structures. Furthermore, transiently expressed p53 protein co-localized with X protein in X transfected cells. However, the appearance of aggregated mitochondrial structures at the nuclear periphery was independent of the presence of p53 protein in X transfected cells. X protein expression also caused an appearance of TUNEL positive nucleus, cytochrome c release from mitochondrial, the decrease of mitochondrial membrane potential and the membrane blebbing of X transfected cells, which are characteristic of cell death. Our data suggest that X protein causes an abnormal aggregation of mitochondrial structures in the cell, which may be eventually connected with cell death.

Cytoplasmic retention of the p53 tumor suppressor gene product is observed in the hepatitis B virus X gene-transfected cells.

It has been suggested that hepatitis B virus (HBV) X gene activates X gene expression by disrupting the function of p53 tumor suppressor gene (Takada et al., 1996). To find out their connection, effect of X protein expression on the nuclear localization of p53 protein in human hepatoma cells was examined by the immunofluorescent double-staining technique. The location of transiently-expressed p53 protein was examined in X gene-transfected cells, where X protein was detected in the cytoplasm. The nuclear location of transiently-expressed p53 protein was changed to the cytoplasm by X protein co-expression. Endogenous p53 protein was also observed in the cytoplasm by X protein expression. The transcriptional activation domain of X protein and the carboxy-terminal region of p53 protein were found mutually responsible for the cytoplasmic retention of p53 protein in X gene-transfected cells. Therefore, the cytoplasmic retention of p53 protein may be closely correlated to the function of X protein expressed in transfected cells.

Hepatitis B virus X gene expression is activated by X protein but repressed by p53 tumor suppressor gene product in the transient expression system.

Hepatitis B virus (HBV) X gene is known to exhibit a transcriptional activation function and is considered to play a major role in hepatocarcinogenesis. We determined a 20-bp promoter element for the HBV X gene transcription and found a binding protein to this promoter element, designated as an X-PBP. We then examined the effects of HBV X protein and p53 tumor suppressor gene product on X gene transcription from the 20-bp promoter element using the transient expression technique. Activity of the X gene promoter was stimulated by X protein expression, but, in contrast, was repressed by transfected normal p53 gene. On the other hand, mutant p53 gene product exhibited no repression. Moreover, the p53 repression of X gene transcription was canceled by X protein coexpression. Thus, the effects of X protein and normal p53 product appear to be mutually antagonistic in the regulation of X gene expression. However, mutated promoter elements which failed to bind to X-PBP still responded to X protein or p53, indicating that the process of X transactivation or p53 repression may be independent of X-PBP binding to the promoter element. Our data suggest that X protein could disrupt function of normal p53 protein in X gene-transfected cells.

Effect of compression temperature on the consolidation mechanism of chlorpropamide polymorphs.

The effect of environmental temperature on the compression mechanism of chlorpropamide (CPM) polymorph, forms A and C, was investigated with an eccentric type tabletting machine with two load cells and a noncontact displacement transducer. The temperature of the die was controlled at 0 and 45 degrees C by a thermocontroller. Sample powders (200 mg), which were also controlled at 0 and 45 degrees C by a thermocontroller, were compressed at almost 230 MPa. The tabletting dynamic processes of CPM forms A and C at 0 and 45 degrees C were evaluated by Cooper and modified Heckel analyses. The results suggest that particle brittleness or plasticity was affected by compression at different temperatures. The higher tablet hardness of form A at 45 degrees C was thought to be caused by the increased plasticity of primary particles, whereas that of form C at 45 degrees C was ascribed to the decreased size of the secondary particles.

Effect of humidity on solid-state isomerization of various kinds of lactose during grinding.

The effect of humidity on isomerization during grinding of alpha-monohydrate, alpha-anhydrate and beta-anhydrate of lactose was investigated. Samples were ground in an agate centrifugal ball mill at 270 rev min-1 at room temperature (21 degrees C) and at 5 and 60% relative humidity. Crystallinity of the ground lactose was measured by Hermans' method from the powder X-ray diffraction profiles. The alpha- and beta-lactose content of the ground lactose was measured by using angular rotation spectrophotometry. The crystalline lactose samples were transformed into noncrystalline solids by mechanical stress during grinding. After grinding, the adsorbed water content of all ground lactose samples increased, and the isomerized amount increased with increase of the water content during grinding at 5 and 60% relative humidity. The results suggest that the isomerization rate of alpha-monohydrate during grinding may depend on the crystallinity, but those of alpha- and beta-anhydrate depend on the content of adsorbed water.

Effect of geometric factors on hydration kinetics of theophylline anhydrate tablets.

The effect of tablet thickness on the hydration kinetics of types I and II theophylline anhydrate tablets at 95% relative humidity and 35 degrees C was studied with various kinetic equations. Samples of 1- and 2-cm-diameter tablets (1 g) were compressed at 1000 kg/cm2. Types I and II tablets expanded by 11-17% in volume during hydration to the monohydrate. The thickness expansion of all tablets exceeded the diameter expansion. After the hygroscopicity test, the final expansion ratio of type I tablets was more than that of type II tablets. The hydration kinetics analysis suggested that the hydration of theophylline anhydrate tablets proceeds as follows: Hydration of the 1-cm-diameter type I tablet followed the two-dimensional phase boundary equation, and that of the 1-cm-diameter type II tablet followed the three-dimensional phase boundary equation, but those of the 2-cm-diameter types I and II tablets followed the equation for two-dimensional growth of nuclei. The hydration kinetics of tablets does not depend on the nature of bulk powder but on geometrical factors (tablet thickness and porosity).

Effects of tableting pressure on hydration kinetics of theophylline anhydrate tablets.

The effects of tableting pressure on hydration kinetics of types I and II theophylline anhydrate tablets at 95% relative humidity, 35 degrees C, have been studied by using various kinetic equations. Relations between tablet expansion and hydration were studied. Samples of 2 cm diameter tablets (1 g) were compressed at 5, 10 and 20 MPa. The hydration of types I and II tablets decreased with increased tableting pressure. The time required for 50% hydration of 2 cm diameter tablets, compressed at various pressures suggests that the tablet hydration rate was affected by the tableting pressure. Types I and II tablets expanded 11.37-16.75% in volume during hydration to the monohydrate. The thickness expansion of the tablets exceeded the diameter expansion as the tablet structure was not uniform owing to the orientation of particles during the compression. The final expansion ratio of the tablets increased with increased tableting compression pressure. The Hancock Sharp constant (m) and fitting of the kinetic data to a suitable model suggested that the hydration of theophylline anhydrate tablets followed the two-dimensional phase boundary equation (type I tablets) or the three-dimensional phase boundary equation (type II tablets).

Isomerization of lactose in solid-state by mechanical stress during grinding.

Isomerization during grinding of solid-state alpha-monohydrate, alpha-anhydrate and beta-anhydrate of lactose was investigated. Samples were ground in an agate centrifugal ball-mill at 270 rev min-1 at room temperature (20 degrees C). The crystallinity of ground lactose was measured by Hermans' method from the powder X-ray diffraction profiles. The alpha- and beta-lactose content of the ground lactose was obtained from the specific rotation measured by using angular rotation spectrophotometry. The crystalline lactose samples were transformed into non-crystalline solids by mechanical stress during grinding. After grinding, the water content of all ground lactose samples increased, and the samples had about 2 mol water per lactose after 10 h grinding. After 10 h grinding of alpha-monohydrate and alpha-anhydrate, 10 and 15%, respectively, of alpha-lactose was transformed into beta-lactose by the mechanical treatment. After 10 h grinding of beta-lactose, 20% of beta-lactose was transformed into alpha-lactose. The results suggest that crystalline lactose was transformed into a non-crystalline solid, and water was adsorbed on the non-crystalline lactose. The non-crystalline solids of alpha- or beta-lactose were then transformed into their counterparts by the mechanochemical effects of grinding.

Effects of temperature and pressure during compression on polymorphic transformation and crushing strength of chlorpropamide tablets.

The effects of temperature on the polymorphic transformation of chlorpropamide during compression and on the physical properties of the tablet have been investigated. A heater and liquid nitrogen pool were mounted on the die of a single punch eccentric tableting machine, and the die temperature was controlled by a thermocontroller. A tableting machine with two load cells (upper and lower punches) and a non-contact displacement transducer were used to measure compression stress, distance and energy. The X-ray diffraction profiles of the deagglomerated compressed sample powder were measured to calculate the polymorphic content. The amount of form C transformed from form A at 45 degrees C was about twice that at 0 degree C with the same compression energy. The amount of form A transformed from form C by compression at 45 degrees C was almost the same as that at 0 degree C. This suggests that the mechanochemical effect of form A depended on the compression temperature, but that of form C was independent of temperature. The crushing strength of tablets of form A was about twice that of form C, even at the same porosity. The plots of log (crushing strength of tablet) against porosity of form A tablets compressed at 0 and 45 degrees C were linear with the same slope; the slope for form C tablets compressed at 45 degrees C was less than that at 0 degree C.

Effect of surface characteristics of theophylline anhydrate powder on hygroscopic stability.

The hygroscopicity of theophylline anhydrate has been investigated by gas adsorption and hydration kinetic methods. Type 1 theophylline anhydrate was obtained by recystallization from distilled water at 95 degrees C, and type II was obtained by dehydration of theophylline monohydrate. The X-ray diffraction pattern of types I and II agreed with the data of theophylline anhydrate. However, the diffraction peaks of the (200) and (400) planes of type I were much stronger than those of type II. The particles of type I were clear crystalline-like single crystals. However, the particles of type II had many cracks. The gas affinity balance (H/N) of type II, measured by gas adsorption, was about 7 times that of type I. After the hygroscopicity of types I and II had been tested at various levels of relative humidity (RH) at 35 degrees C, type I was stable at less than 82% RH, but transformed into the monohydrate at more than 88% RH. Type II was stable at less than 66% RH and transformed into the monohydrate at less than 75% RH. The hydration data of type I at 88% RH and type II at 75% RH were calculated for hydration kinetics using various solid-state kinetic models, but no particular model could be preferred from these data.

Effects of the mechanical energy of multi-tableting compression on the polymorphic transformations of chlorpropamide.

The effects of the mechanical energy of tableting compression on the polymorphic transformation of chlorpropamide have been examined. A single-punch eccentric tableting machine with a load cell and a non-contact displacement transducer were used to measure compression stress, distance and energy. An amount of 100 mg of the stable form A or the meta-stable form C of the drug was loaded into the press and the sample compressed with a compression stress of 196 MPa at room temperature (20 degrees C). The compression cycle was repeated from 1 to 30 times. The powder X-ray diffraction profiles of the deagglomerated compressed sample powder were measured to calculate the polymorphic content. The results on forms A and C suggested that both forms were transformed into each other in the solid state by mechanical energy during tableting. The contents of forms A and C reached equilibrium at a constant value above 100 J g-1 of compression energy after more than 10 cycles. After 30 tableting cycles of forms A and C, the contents of A, C and the non-crystalline solid were almost constant at about 45, 25 and 30%, respectively. The compression energies were estimated to be about 500-600 J g-1. From the results it seems that the transformation mechanism of forms A and C during tableting were as follows. The crystal form of A or C was converted to a non-crystalline solid by the mechanical energy, and the solid was then transformed into form A or C.

Comparison of disposition parameters of quinidine and quinine in the rat.

The difference in disposition of quinidine (Qd) and its diastereomer quinine (Qn) after intravenous administration was examined in rats at doses ranging from 5 to 20 mg/kg. Dose-dependent kinetics in total clearance and in distribution volume of tissue based on a two-compartment model was observed for Qd; there was no evidence of nonlinearity for Qn. However, there was no significant difference between Qd and Qn for blood clearance at doses of 5 and 10 mg/kg, at which the blood clearances were almost equal to hepatic blood flow for both Qd and Qn since the excretion of Qd and Qn into the urine and bile was minimal. This indicates the elimination of these diastereomers to be non-restrictive in the liver. A concentration dependence in unbound volume of tissue distribution and in plasma protein binding was observed for Qd; there was no concentration dependence for Qn. Although affinity of the drug for components on or within the blood cells was not concentration-dependent for either Qd or Qn, a significantly higher binding capacity for Qn than for Qd was observed attributable to blood cell binding. Based on these results, it is suggested that a larger number of binding sites exist for Qn than for Qd in the body. However, the dissociation binding constant for Qd is much lower than for Qn, resulting in a higher binding of Qd and Qn at low concentrations, with a reversal at high concentrations.

Prediction of hepatic first-pass metabolism and plasma levels following intravenous and oral administration of barbiturates in the rabbit based on quantitative structure-pharmacokinetic relationships.

Based on the concept of physiological pharmacokinetics, the hepatic first-pass metabolism and plasma levels following intravenous and oral administration of barbiturates in the rabbit was predicted based on the relationships between principle kinetic parameters and lipophilicity (chloroform-water partition coefficient). Good log-log linear relationships between kinetic parameters and lipophilicity were obtained for the seven barbiturates examined. The values of correlation coefficient were improved slightly by using the corrected values for partition coefficients of nonionic molecules in the cases of principle parameters such as drug-protein and drug-blood cell affinity, intrinsic hepatic clearance, and unbound volume of distribution. There was also a good linear relationship between absorption rate constant (mean absorption time) and lipophilicity. The mean hepatic transit time was negligible for the most lipophilic drug (hexobarbital) examined; this suggests that the mean absorption time for these barbiturates does reflect the absorption process. The available fraction in relation to hepatic first-pass metabolism was well predicted from the lipophilicity by both well-stirred and parallel-tube models, and the difference in the values predicted by both models was minimal. There were good relationships between predicted and observed values for plasma levels after intravenous and oral administration, except for two (cyclobarbital and phenobarbital) of the seven drugs used. The great difference between predicted and observed values for these two drugs was considered due to substituent effects in liver metabolism.

Pharmacokinetic study of carbamazepine and its epoxide metabolite in humans.

A major metabolite of carbamazepine (CBZ), CBZ-10, 11-epoxide (EPO), has been reported to possess anticonvulsant properties. Therefore, the present study was undertaken in order to develop a pharmacokinetic model to predict the behavior of EPO in the body after administration of CBZ. The serum concentration-time curves after oral administration of solution of CBZ (200 mg) or EPO (150 mg) in six healthy subjects showed the characteristic "nose", suggesting that disposition of CBZ or EPO could be described by the two-compartment model. The kinetic parameters of disposition for CBZ and EPO were calculated by the method of Wagner, assuming the absolute bioavailabilities of CBZ and EPO to be 1.0 and 0.81, respectively. Total body clearance and elimination rate constant of EPO were very much larger than those of the parent drug but there was no statistically significant difference in the distribution volume between CBZ and EPO. The formation rate of EPO was calculated by a deconvolution method, and obeyed Michaelis-Menten kinetics. Based on these findings, a pharmacokinetic model of the fate of CBZ and EPO in humans was developed and the time courses of CBZ and EPO in serum after oral administration of three tablet preparations and a solution containing 200 mg of CBZ were simultaneously fitted to this model by solving the differential equations by the Runge-Kutta-Gill method. There was good agreement between calculated and observed serum values, suggesting that the present model is appropriate to describe the formation and disposition of EPO from CBZ. The formation rate constant of EPO (Vmax/Km/V1) was approximately one-fifteenth of the elimination rate constant of EPO. This suggested a flip-flop model in which the formation of EPO was rate-limiting in humans. The observation that the serum concentrations of EPO after administration of CBZ were one-tenth to one-twentieth of those of the parent drug was well explained by the flip-flop kinetics of EPO, together with the large differences in total body clearance and elimination rate constant between CBZ and EPO.