Aggregation of DNA enhanced by the protoberberine alkaloids, coralyne and berberine.

The aggregation of DNA caused by coralyne was studied by spectroscopic, viscosity and electric birefringence measurements. Aggregation was markedly enhanced over a narrow range of coralyne to DNA phosphate ratio and then followed by precipitation. The electric birefringence measurements indicated that the ratio at the maximal aggregation varied depending on the concentration of coralyne, finally reaching 1:1 at higher concentrations. The particles (type I) for such enhanced aggregation were estimated to be prolate ellipsoids 1700-4000 A in length with a diameter of 1400-4000 A. At higher coralyne concentrations, another particle (type II) was formed which was a thick rod-like particle 1700-4000 A in length with a diameter of 120-210 A. These dimensions indicate that type I and II particles consist respectively of several tens of thousands and some hundreds of molecules of DNA. On the other hand, berberine did not produce such a marked aggregation of DNA, and the result was a thick rod-like particle 1700-4000 A in length with a diameter of 300-1000 A. The enhancement by coralyne and berberine is discussed in terms of intermolecular interactions.

Disposition of glycyrrhizin in the perfused liver of rats.

The disposition of glycyrrhizin (GLZ) in the perfused liver of rats after dosing in the range of 0.5-30.0 mg was investigated and a pharmacokinetic model was devised to interpret the results. The uptake rate of GLZ into the liver with respect to the unbound GLZ concentration (Cf) in the perfusate followed a Michaelis-Menten type equation with a Km,up of 1.17 micrograms/ml and Vmax,up of 13.9 micrograms/min/g of liver. The efflux clearance (0.044 ml/min/g of liver) from the liver was independent of the Cf in the liver. The biliary excretion rate at a steady-state Cf level in the liver followed a Michaelis-Menten type equation with a substrate inhibition constant (Ki,B) of 42.3 micrograms/ml, Km,B of 1.68 micrograms/ml, and Vmax,B of 3.11 micrograms/min/g of liver. The proposed model, with the holding time fitted to biliary excretion at each dose, accurately described both the perfusate concentration-time profile and the cumulative biliary excretion profile.

Marchantin A trimethyl ether: its molecular structure and tubocurarine-like skeletal muscle relaxation activity.

Marchantin A is a novel macrocyclic bis(bibenzyl)ether isolated from the liverwort Marchantia species. An X-ray study of its derivative, marchantin A trimethyl ether, revealed that the molecule possesses convex and concave surfaces, with a central hole on the concave surface. The centroid-centroid separations of opposing benzene rings are 8.80 and 4.55 A. A pharmacological study showed that the skeletal muscle relaxation activity is about 3.5 times less potent than that of d-tubocurarine. A comparison of the X-ray structure of marchantin A trimethyl ether and that of O,O',N-trimethyltubocurarine reported by Sobell et al. revealed that the molecules share almost the same macrocyclic bis(bibenzyl)ether skeleton structure, portions of which may therefore be crucial for the skeletal muscle relaxation activity.

Uptake of glycyrrhizin by isolated rat hepatocytes.

The mechanism of uptake of glycyrrhizin (GLZ) by isolated rat liver cells was studied. The uptake rate was dependent on the unbound GLZ concentration. The initial uptake rate with respect to the unbound GLZ concentration reflected the operation of both saturable and nonsaturable processes, which followed Michaelis-Menten type kinetics; the process involves a Km of 11.3 microM, Vmax of 0.112 nmol/min/10(6) cells, and a first-order rate constant (Kd) of 0.195 nmol/min/10(6) cells/mM. GLZ adsorption on the cell membrane occurs at two types of binding sites with a linear adsorption coefficient = 2.81 nmol/10(6) cells/mM and a dissociation constant = 18.3 microM and its adsorption capacity = 0.12 nmol/10(6) cells describing specific adsorption. GLZ uptake did not require the presence of Na+ in the incubation medium and was not significantly inhibited by ouabain. The Arrhenius plot of uptake of 10 microM GLZ presented a single straight line in the range of 4-37 degrees C, with an activation energy of 15.9 kcal/mol. An energy requirement was also demonstrated, as all metabolic inhibitors studied (rotenone, antimycin A, 2,4-dinitrophenol, and KCN) significantly reduced the uptake of 10 microM GLZ (p < 0.01). The uptake was competitively inhibited by glycyrrhetinic acid (GLA), taurocholate (TCA), and probenecid (PBC) with inhibition constants, Ki, of 13.7, 48.5, and 115.9 microM, respectively, and it was noncompetitively inhibited by bromosulfophthalein (Ki 9.2 microM) and indocyanine green (Ki 13.5 microM) only at low GLZ concentrations (5 and 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent pharmacokinetics of glycyrrhizin in rats.

The dose-dependent pharmacokinetics of glycyrrhizin (GLZ) was investigated by measuring drug disappearance from plasma and biliary excretion in rats. The decline in plasma concentration was biexponential after an i.v. dose of 5, 10, 20, or 50 mg/kg. Dosage, however, had a marked effect on the pharmacokinetics, with a greater-than-proportional increase in area under the plasma concentration curve (AUC) at doses of 20 and 50 mg/kg, even though the increase was proportional at doses of 5 and 10 mg/kg. There was also a significant increase of the steady-state distribution volume (Vdss), as well as significant decreases in total body (CLtot) and biliary (CLB) clearances, at 20 and 50 mg/kg from those at 5-10 and 5-20 mg/kg, respectively. The AUC, Vdss, and renal clearance (CLR) at a given dose showed no significant difference between rats with and without bile fistulas. The plasma unbound fraction (fp) (0.006-0.026) increased with increasing plasma GLZ concentration over the observed range (2-900 micrograms/ml). No significant change in Vdss for unbound GLZ was observed between the doses, indicating that the distribution of GLZ into tissues is not changed by an increase in dose. On the other hand, a dose dependency in CLtot for unbound GLZ was observed and confirmed to be attributed to dose dependency in CLB for unbound GLZ since there was no significant difference in CLR or metabolic clearance for unbound GLZ between the doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycyrrhizin binding site on human serum albumin.

The binding site of glycyrrhizin (GLZ) on human serum albumin was detected through competitive displacement experiments with GLZ and ibuprofen (IBU) (diazepam site), warfarin (WAR), salicylate (SAL) (digitoxin site), or deoxycholic acid (DCA) by means of an ultrafiltration technique. The specific binding of GLZ was subject to competitive inhibition by IBU, WAR, SAL, or DCA (1 or 4 mM). The extent of displacement was in the order of: DCA greater than IBU greater than WAR greater than SAL. Conversely, the specific bindings of WAR and DCA and the low-affinity bindings of IBU and SAL were subject to competitive inhibition by GLZ (1 or 4 mM). The extent of inhibition by GLZ was in the order of DCA greater than IBU greater than WAR not equal to SAL. In addition, the low-affinity IBU binding and the specific DCA binding showed mutual competitive inhibition at 4 mM, with almost identical displacements. It was concluded that the specific GLZ binding site on human serum albumin may be located mostly within the low-affinity IBU binding site area (probably the same as the specific DCA binding site area) and partially within the specific WAR binding site area and the low-affinity SAL binding site area.

Prediction of glycyrrhizin disposition in rat and man with liver failure by a physiologically based pharmacokinetic model.

Two physiologically based pharmacokinetic models A and B incorporating enterohepatic recycling, which succeeded previously in predicting the disposition of glycyrrhizin (GLZ) in normal rats and subjects, were applied to predict GLZ disposition in plasma and tissues of chronically CCl4-intoxicated rats, and serum of humans with hepatitis after i.v. dosing. The prediction by model A with the direct excretion of GLZ from liver into gut lumen gave fairly good agreement with the observed time courses of GLZ concentrations in blood and tissues in the intoxicated rats. The human serum disposition was predicted by model B, to which was added a gallbladder for the excretion from liver into gut lumen to model A by assuming continuous delaying transfer from the gallbladder. An attempt to predict the serum dispositions in five human subjects by considering individual differences in serum free fraction, biliary excretion ratio, and intestinal absorption clearance was successful in model B. Thus, scale-up of the disposition kinetics of GLZ from rat to man with liver failure was successful.

Prediction of glycyrrhizin disposition in rat and man by a physiologically based pharmacokinetic model.

Three physiologically based pharmacokinetic models A--C, incorporating enterohepatic recycling, were developed to predict glycyrrhizin (GLZ) disposition in rat plasma and tissues, and human serum. Model A, which included fourteen compartments (artery, vein, tissues except brain, and gut lumen) with the assumption of direct excretion of GLZ from the liver into the gut lumen gave fairly good agreement between the observed and predicted disposition profiles in rat, but was unsuitable in man, where elimination is very rapid. Models B and C for man were obtained by adding a gallbladder compartment (drug storage organ) for the excretion from the liver into the gut lumen and by assuming continuous transfer from the storage compartment or instantaneous emptying from it during meal ingestion as the excretion process from the gallbladder into the gut lumen, respectively. The agreement between the observed and predicted serum concentration time-course profiles was better with model C than model B, especially in the terminal elimination phase, where secondary peaks appeared. However, it was thought that the observed serum disposition can be sufficiently well predicted by model B. In conclusion, prediction in rat was successful in all compartments except the brain, which shows a negligible distribution. Scale-up of the disposition kinetics of GLZ from rat to man was also successful.

Pharmacokinetics of glycyrrhetic acid, a major metabolite of glycyrrhizin, in rats.

The pharmacokinetics of glycyrrhetic acid (GLA) was examined in rats after bolus i.v. injection at a dose of 2, 5, or 12 mg/kg. The decline in plasma concentration was generally biexponential at each dose, but the terminal disposition became much slower with increase of dose. A greater than proportional increase in plasma GLA concentration was observed with increase of dose, suggesting a dose-dependency of GLA disposition. Apparent total body clearance decreased significantly with increase of dose. On the other hand, the apparent steady-state distribution volume after i.v. administration was unaffected by dose. The plasma disposition at each dose fitted well to a two-compartment pharmacokinetic model with Michaelis-Menten elimination. It was concluded that the pharmacokinetics of GLA in the rat is dose-dependent owing to a saturable elimination rate. The plasma level of GLA after glycyrrhizin (GLZ) i.v. dosing (100 mg/kg) in the control rats (without biliary fistulization) sustained the concentration range of 1.5-3 micrograms/ml during 1-48 h, but that in the rats with biliary fistulization declined with time. It was suggested that the sustained plasma level of GLA is accounted for by the intestinal reabsorption of GLA produced from GLZ and GLA-conjugates during the enterohepatic recycling of both.

Binding of glycyrrhizin to human serum and human serum albumin.

The binding of glycyrrhizin (GLZ) to human serum and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in both human serum and HSA. The association constants (K) for the specific bindings were very similar: 1.31 x 10(5) M-1 in human serum and 3.87 x 10(5) M-1 in HSA. The number of binding sites (n) and the linear binding coefficient (phi) in HSA were 1.95 and 3.09 x 10(3) M-1, respectively. When the human serum protein concentration was assumed to be 4.2% (equal to the measured serum albumin concentration), n in human serum was 3.09, which is similar to the n value in HSA, and phi in human serum was 0.71 x 10(3) M-1, which is reasonably close to that for HSA. The binding pattern of GLZ with human serum protein on Sephadex G-200 column chromatography showed that GLZ binds to only the albumin fraction. It was concluded that the GLZ-binding sites in human serum exist mainly on albumin and GLZ binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.

Biliary excretion and enterohepatic cycling of glycyrrhizin in rats.

The enterohepatic cycling of glycyrrhizin was examined using rats with and without biliary fistulization. The plasma decay in the control rats without fistulization following an iv dose of 100 mg/kg of glycyrrhizin, was generally biphasic. However, secondary peaks were observed in all rats in the elimination phase, i.e., 0.5 to 12 h following dosing. The plasma concentrations in the rats with biliary fistulization administered the same dose showed a biexponential decline. The AUC and CLtot were significantly higher and lower in the control rats, respectively. The biliary excretion was 80.6 +/- 9.9% of the administered dose, and intestinal absorption was confirmed by using the bile collected after iv dosing. From these results, we concluded that glycyrrhizin was predominantly secreted from the liver into the bile, and that the secondary peaks in the elimination phase, the higher AUC, and the lower CLtot in the control rats were due to the effects of enterohepatic recycling of glycyrrhizin. Furthermore, the transport of the drug from the liver to the bile appears to be a saturable process.