Effect of Anions on Adsorption of Bile Salts by Colestipol Hydrochloride

The Langmuir affinity constant and adsorptive capacity for the adsorption of citrate anion or cholate anion by colestipol hydrochloride at pH 7.5, 37°C, were similar. Prior exposure of colestipol hydrochloride to citrate anion caused the adsorption of cholate anion to decrease slightly in comparison to a control utilizing only cholate anion. The concentration of citrate anion was found to be directly related to the decrease in cholate anion adsorption. Simultaneous exposure of colestipol hydrochloride to citrate and cholate anions at pH 7.5, 37°C, resulted in the same adsorption of cholate anion as sequential exposure to citrate anion followed by cholate anion. Sequential exposure of colestipol hydrochloride to simulated gastric fluid and simulated intestinal fluid containing cholate anion resulted in a small decrease in cholate adsorption which was attributed to competition with phosphate anion in simulated intestinal fluid. Pepsin in the simulated gastric fluid did not affect adsorption of cholate anion from simulated intestinal fluid. Preexposure to components of tomato juice and orange juice also slightly reduced the adsorption of cholate anion by colestipol hydrochloride.     

Equilibrium and Kinetic Factors Influencing Bile Sequestrant Efficacy

In vitro bile salt binding equilibria and kinetic studies were performed with cholestyramine to determine how these factors influence bile sequestrant efficacy in vivo. Chloride ion at physiologic concentrations caused more than a twofold reduction in glycocholate (GCH) binding, compared to binding in the absence of salt, over a range of GCH concentrations and was also observed to displace bound GCH. In addition, chloride ion displaced from cholestyramine as a result of bile salt binding was measured using a chloride selective electrode, and the results show that bile salt binding is due to ion exchange. Comparison of the results of the equilibrium binding experiments to human data shows that the effect of anion binding competition alone cannot account for the lack of efficacy of cholestyramine. Consideration of other effects, such as additional binding competition or poor availability for binding, based on data from the literature, shows that adequate bile salt binding potential exists and that these interferences are not major factors influencing resin efficacy. In kinetic studies, both binding uptake of GCH and displacement of GCH from cholestyramine by chloride ion were relatively rapid, indicating that cholestyramine should equilibrate rapidly with bile salts in the GI tract. Based on these findings, it is suggested that the low efficacy of cholestyramine is a result mainly of its relatively poor ability to prevent bile salt reabsorption in the ileum.     

Bile acid salt binding with colesevelam HCl is not affected by suspension in common beverages

It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages.     

Binding of bile acids to cholestyramine at gastric pH conditions.

The binding of bile salts to cholestyramine was studied under varying conditions of pH and added electrolyte. The taurine-conjugated bile salts were strongly absorbed by the anion-exchange resin at low pH and in the presence of chloride anions. Glycocholic acid binding was very weak at low pH but increased strongly with increasing pH. The presence of chloride ions strongly decreased the amount of glycocholate bound by the anion-exchange resin.     

High and low affinity binding of [3H]cholate to rat liver plasma membranes

The transport of bile acids across sinusoidal and canalicular membranes of hepatocytes is characterized as carrier mediated. Such a carrier should specifically bind bile acids at physiological concentrations. We examined the binding of [3H]cholate to rat liver plasma membranes using a microcentrifugation technique and detected high (KD = 1.23 +/- 0.44 microM, Bmax = 21.8 +/- 3.3 pmol/mg protein) and low (KD = 1.97 +/- 1.33 mM, Bmax = 41.5 +/- 25.3 nmol/mg protein) affinity binding sites. Maximal binding was achieved within 15-45 sec and was stable for 2 min at 37 degrees. Binding to the high affinity site was reversible, was not Na+ dependent or attributable to vesicular uptake, and exhibited a broad pH optimum. Binding to this site was negligible or not detected in liver mitochondrial and microsomal fractions, was saturable, and was inhibited by other bile acids. The IC50 values for bile acids as inhibitors of [3H]cholate binding at the high affinity site were: taurocholate, 1.9 nM; glycodeoxycholate, 3.1 nM; chenodeoxycholate, 5.6 nM; taurochenodeoxycholate, 7.3 nM; glycochenodeoxycholate, 11 nM; lithocholate, 13 nM; taurodeoxycholate, 20 nM; glycocholate, 3.6 microM; and deoxycholate, 5.6 microM. [3H]Cholate specific binding was inhibited by 10(-5) M bromosulfophthalein, bilirubin and indocyanin green. These data support the hypothesis that the high affinity binding site represents a carrier which is shared by bile acids and nonbile acid organic anions.     

Cholestyramine feeding lowers number of colonic apoptotic cells in rat

Secondary bile acids that are formed in the colon by bacterial action have the potential property of eliciting pathological conditions. Apoptosis of mucosal epithelial cells is recognized as an adaptation that may counteract such pathologies. Cholestyramine, an anion exchange resin that sequesters bile salts in the gut, could decrease levels of secondary bile salt stress and thus conserve the potency of the protective action. Two groups of rats were studied: those fed 4% cholestyramine and those fed regular rat food. Rats were fed cholestyramine for 7, 14, 21, or 28 d. All animals were evaluated for cell death (apoptosis) using in situ TUNEL staining, and confirmed with single-stranded DNA (ssDNA). The effect of cholestyramine on the proliferating cell nuclear antigen (PCNA) in colonic crypt cells was also examined. Our data shows that animals fed cholestyramine for 28 d show evidence of a significant decrease in the levels of apoptotic cells in their large intestines, particularly goblet cells, when compared with the control animals and no change in cell proliferation. Thus, cholestyramine may serve as an alternative in attenuating apoptosis associated with inflammatory disorders that can result in significant enterocyte and goblet-cell death.     

Effects of novel bile salts on cholesterol metabolism in rats and guinea-pigs

Novel bile salts (quaternary ammonium conjugates) inhibited cholic acid binding and transport in everted ileal sacs in vitro. The cationic piperazine conjugate of lithocholic acid (di-iodide salt, compound 8, BRL 39924A) appeared most active, inhibiting binding by 29% and transport by 59% in guinea-pig ileum (200 microM). BRL 39924A also inhibited taurocholate uptake into guinea-pig ileal sacs and cholate uptake into rat ileal sacs and was selected for further study in vivo. In hyperlipidaemic rats, BRL 39924A significantly raised cholesterol 7 alpha-hydroxylase activity and decreased hepatic accumulation of exogenous cholic acid. HDL cholesterol concentration in the serum increased and the level of VLDL plus LDL cholesterol decreased. In hyperlipidaemic guinea-pigs. BRL 39924A lowered serum total cholesterol and triglyceride levels. Although metabolic changes were less than those achieved with the bile acid sequestrant, cholestyramine, the doses of BRL 39924A used were much lower (100-500 mg/kg body wt). Selective inhibition of receptor mediated bile acid uptake may be associated with local side-effects but these novel bile salts are useful pharmacological tools to examine the effects of receptor blockade on lipoprotein metabolism.     

In Vitro Adsorption of Bile Salts by Colestipol Hydrochloride

The acid–base titration of colestipol hydrochloride exhibits no sharp inflection points, indicating a weakly basic anion-exchange copolymer. The swelling of colestipol hydrochloride in water and the adsorption of cholate anion are inversely related to pH and are, therefore, related to the ionization state of the copolymer. The Langmuir adsorption parameters at pH 7.5 and 37°C are similar for cholate, glycocholate, and taurocholate anions. Adsorption capacity was not related to particle size and exceeded the adsorptive capacity of the external surface by three orders of magnitude. Therefore, it is believed that the swelling of colestipol hydrochloride makes extensive internal surface area available for adsorption of bile salts. The rate of adsorption depends on the concentration of sodium cholate to which the colestipol hydrochloride is exposed. Adsorption was complete within 5 min when the concentration was below the adsorptive capacity. In contrast, adsorption at levels of sodium cholate at or above the adsorptive capacity was not complete within a 3-hr test period.     

Bile acid binding and hypocholesterolemic activity of a new anion exchange resin from 2-methylimidazol and epichlorohydrin

A new anion exchange resin with an imidazolium salt on a epoxide polymer skeleton was synthesized. This white powder material was odorless and tasteless. The in vitro sodium cholate binding of this resin was much more potent than that of cholestyramine. The hypocholesterolemic activity of this resin in cholesterol-fed rabbits proved to be 4.3 times more potent than that of cholestyramine. These results suggest that effective reduction of plasma cholesterol may be achieved with lower doses of this resin.     

In vitro methods can forecast the effects of intragastric residence on dosage form performance

Intragastric conditions can affect the performance of solid dosage forms. For two cases, the ability of in vitro methods to forecast these effects was investigated: first, the ability of cholestyramine to sequester bile salts in the fed small intestine and, second, disintegration times of hard gelatin capsules. After incubating cholestyramine for 90 min in milk gradually digested with pepsin, the binding of taurocholates from fed state simulating intestinal fluid onto the resin became non-specific and the affinity constant was reduced from 220 l/mole (without prior incubation) to 60 l/mole. These data are consistent with the comparatively poor performance of cholestyramine products when administered in the fed state. Scintigraphic studies showed that intragastric disintegration times of hard gelatin capsules are delayed in both the fasted and fed states according to the degree of cross-linking. These results were satisfactorily predicted by the in vitro disintegration times in fasted state simulating gastric fluid and in milk gradually digested with pepsin, whereas results were poorly predicted in compendial media. This work illustrates that recently proposed methods for simulating intragastric environment may be useful in predicting the performance of solid dosage forms.     

Uranium(VI) solubility and speciation in simulated elemental human biological fluids

The complete understanding of the human body response to uranium contamination exposure is vital to the development of exposure analysis and subsequent treatments for overexposure. Thermodynamic modeling has traditionally been used to study environmental metal contaminant migration (especially uranium and other radionuclides), allowing examination of chemical processes difficult to study experimentally. However, such techniques are rarely used in the study of metal toxicology. Chemical thermodynamics has a unique and valuable role in developing models to explain metal metabolism and toxicology. Previous computational models of beryllium in simulated biological fluids have been shown to be useful in predicting metal behavior in the human body. However, previous studies utilizing chemical thermodynamics in understanding uranium chemistry in body fluids are limited. Here, a chemical thermodynamic speciation code has been used to model and understand the chemistry of uranium in simulated human biological fluids such as intracellular, interstitial, and plasma fluids, saliva, sweat, urine, bile, gastric juice, pancreatic fluid, and a number of airway surface fluids from patients with acute lung conditions. The results show predicted uranium solubility, and speciation varies markedly between each biological fluid due to differences in fluid composition, ionic strength, and pH. The formation of uranium hydroxide, phosphate (sodium/potassium autunite), and calcium uranate was observed in most of the fluids. The results of this work, supported by experimental validation, can aid in understanding the metabolism and toxic effects of uranium with potential applications to biological monitoring as well as chelation treatment of uranium body burden.     

Polymeric sorbents for bile acids. I: Comparison between cholestyramine and colestipol.

Isotherms for the sorption of bile acids at 20 degrees C, in tris(hydroxymethyl)aminomethane.HCl(tris) and KH2PO4-NaOH (phosphate) buffers (pH 7.4), indicate that the binding by cholestyramine and colestipol is mainly through ionic linkages, although hydrophobic interactions are also of importance. Cholestyramine has a higher sorption capacity for bile acids, in both buffers, than colestipol. The chloride form of cholestyramine has a higher capacity for cholate in tris buffer than the iodide form. Increased ionic strength of the medium leads to decreased amounts of sorption.     

Increasing the in vitro bile acid binding capacity of diethylaminoethylcellulose by quaternization.

Diethylaminoethylcellulose (DEAE-cellulose) was quaternized with methyl iodide (DEAE-cellulose-CH3I), and its in vitro binding capacity for sodium glycocholate, at room temperature, in water, Tris-HCl buffer (0.0015-0.0050 M, pH 7.0), and aqueous NaCl (0.0025 M) was determined by reversed-phase HPLC. Quaternization increased the in vitro bile salt binding capacity of DEAE-cellulose. On a molar basis, the binding capacity was greater than that of cholestyramine, a cholesterol-lowering agent. Increasing the ionic strength of the medium decreased the binding capacities, as expected if ionic interactions are important. However, conversion of DEAE-cellulose-CH3I to its chloride form did not change the binding capacity. The bile salt binding capacity of DEAE-cellulose-CH3I was similar for both sodium cholate and sodium glycocholate.     

Bile acid sequestrants based on cationic dextran hydrogel microspheres. 2. Influence of the length of alkyl substituents at the amino groups of the sorbents on the sorption of bile salts

Cationic dextran hydrogel microspheres with pendant quaternary ammonium groups having alkyl substituents (C(2)-C(12)) at quaternary nitrogen were synthesized. The in vitro sorption of sodium salts of four bile acids (glycocholic, cholic, taurocholic, and deoxycholic acids) with these hydrogels was studied as a function of substituent alkyl chain length and bile acid hydrophobicity. Sorption experiments were performed in phosphate buffer solutions (pH 7.4) containing one bile salt (individual sorption) or mixtures of several bile salts (competitive sorption). Parameters for individual sorption were calculated taking into consideration the stoichiometric and cooperative binding of bile salts to oppositely charged polymer hydrogels. The results show that the increase in the length of the alkyl chain of the substituent leads to an increase in both ionization constant K(0) and overall stability constant of binding K, but decreases the cooperativity parameter u. The competitive sorption studies indicate that the hydrogels display a good affinity for both dihydroxylic and trihydroxylic bile salts. The molar ratio of maximum amounts bound for the two types of bile acid is 2 to 1, which is much lower than those reported for other cationic polymers recommended as bile acid sequestrants. The binding constants for the sorption of bile salts by some dextran hydrogels are 20-30 times higher than those obtained for cholestyramine under similar sorption conditions.     

Interaction of tricyclic antidepressants with cholestyramine in vitro.

The adsorption of amitriptyline, desipramine, doxepin, imipramine, and nortriptyline onto cholestyramine was demonstrated in vitro with use of 1.2 mol/L HCl at 37 degrees C to simulate gastric fluid. Binding to cholestyramine was approximately 80% for each of the tricyclic antidepressants, and this was about the same degree of binding noted with a nonpharmaceutical, non-ionic resin widely used in the diagnostic toxicology laboratory (Amberlite XAD-2). In contrast, five other non-antidepressants (acetaminophen, chlordiazepoxide, procainamide, quinidine, and theophylline) showed only minimal binding to cholestyramine under these conditions. Activated charcoal completely bound all drugs studied. These findings suggest that cholestyramine should be used with caution in patients receiving tricyclic antidepressants. They also suggest that cholestyramine may be a potentially useful adjunctive therapy in treatment of overdose with the tricyclic antidepressants.     

Certain GABA mimetics reduce the affinity of anions required for benzodiazepine binding in a GABA-reversible way

Binding of ³H-flunitrazepam to specific sites on rat-brain membranes is anion-dependent. In the absence of added ions, binding is reduced to less than 10% of maximum, and eight anions (Tris salts) were found to increase binding in a concentration-dependent and saturable manner with affinity constants (Kd) ranging from 0.75 mM (phosphate) to 4.5 mM (chloride). The GABA mimetic THIP (200 μM) increases these Kd values from three- to 10-fold, depending on the anion. The GABA mimetics isoguvacine and piperidine-4-sulfonic acid (P4S) increase the Kd values for chloride ion eight- to 10-fold, while amino-propanesulfonate and imidazole acetate produce smaller (2- to 4-fold) increases. These effects of THIP on anion affinities are reversed in a competitive manner by GABA and muscimol. Although there is no correlation between the ability of the anions to enhance ³H-flunitrazepam binding and their ability to substitute for chloride ion electrophysiologically, it seems possible that the ions bind to sites similar to the chloride channels associated with GABA receptors. All benzodiazepine receptors are probably coupled indirectly to GABA receptors through anion binding sites.     

Choleresis and hepatic transport mechanisms

Summary To investigate, whether binding to micelles has a function in hepatic transport, biliary excretion of three organic anions, phenolphthalein-\-D-glucuronide (PG), dibromosulphthalein (DBSP) and indocyanine green (ICG) was studied in rats during saline, taurocholate or dehydrocholate administration. Taurocholate causes a weak choleresis with formation of biliary micelles, dehydrocholate a strong choleresis with little micelle formation. The two bile salts did not uniformly influence biliary excretion of the organic anions: biliary excretion of ICG (12.9 moles/kg) and DBSP (75.0 moles/kg) was stimulated by both bile salts: ICG excretion most pronounced by taurocholate and DBSP excretion most strongly by dehydrocholate. Biliary output of PG (25.8 and 200 moles/kg) was not stimulated by bile salt administration. Binding of PG, DBSP and ICG to biliary micelles was studied in sedimentation experiments by ultracentrifugation. PG, DBSP and ICG in bile showed a similar sedimentation pattern as ³H-taurocholate in bile, which indicates an association of all three anions with biliary micelles.Thus, the influence of bile salts on biliary transport of organic anions varies with the compound studied and the bile salt used, effects which cannot be explained by differences in binding to biliary micelles.The previously published paper of Vonk et al. (1974) is regarded as paper I in this series.     

Choleresis and hepatic transport mechanisms

Summary To investigate binding of drugs to biliary micelles as a possible factor in the hepatic transport process, interaction of two uncharged compounds, ³H-ouabain and ³H-K-strophanthoside with biliary micelles was studied by ultracentrifugation of bile. The various bile acids normally present in rat bile were predominantly associated with cholesterol containing micelles, but not to the same extent. The tendency of the bile salts to be associated with mixed micelles was the greatest for conjugated chenodeoxycholate, somewhat lower for conjugated deoxycholate and the least for conjugated cholate. The sedimentation patterns of the water-soluble cardiac glycosides, added in vitro, indicated binding to mixed biliary micelles as well as non-cholesterol containing micelles. Also mannitol, a drug used to estimate canalicular bile flow, was found to be associated with both categories of biliary micelles.In spite of the binding of cardiac glycosides to the micelles, administration of taurocholate, which promotes formation of biliary micelles, did not stimulate biliary output of both glycosides. Also administration of the choleretics dehydrocholate and ethacrynic acid failed to enhance biliary output of the glycosides.These results indicate, that binding of drugs to biliary micelles diminishes the free concentration of drugs in bile and confirms earlier studies with organic anions that binding to biliary micelles is not a pertinent factor in the rate of biliary excretion.     

Inhibition of hepatic uptake of bile acids by rifamycins

Summary The effect of rifamycin SV and rifampicin on hepatic bile acid uptake was studied using isolated rat hepatocytes in presence and in absence of albumin. The drugs inhibited cholate uptake more than taurocholate uptake and the inhibition was of non-competitive type. In presence of 3% albumin the inhibitory effect of the drugs was more for cholate and less for taurocholate uptake than in absence of albumin. Neither the binding of bile acids nor that of the drugs to albumin was altered by one another. Thus the effect in presence of albumin cannot be explained by the binding of the drugs and bile acids to albumin alone. It is suggested that albumin interacts with hepatic bile acid uptake process and this interaction with cholate uptake is different from that with taurocholate uptake. This additional and different effect of albumin may explain the effect of the drugs in presence of albumin. The results may be of clinical significance in rifamycins treatments.Part of this publication was presented in the 18th Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (1977)     

In vitro dissolution medium with supramicellar surfactant concentration and its relevance for in vivo absorption

Use of a dissolution medium with supramicellar surfactant concentration is proposed for the in vitro testing of practically insoluble drugs (less than 0.01%). Based on the fact that the model drug tested—palmitoylcatechin—is absorbed in the intestine, a pH 6.8 phosphate buffer (Pharm. Eur.) with the addition of 50 mmol/1 sodium laurylsulfate was used in our work. At that concentration, this surfactive agent had similar solubilizing properties toward the drug as the natural bile salts sodium cholate and sodium taurocholate. Five experimental and commercial palmitoylcatechin preparations, namely four tablet and one capsule formulations, were tested. A preliminary disintegration stage in simulated gastric fluid (a 30-min period was adopted here) was sometimes necessary in order to better correlate dissolution and in vivo absorption data.