Intrinsic factor-mediated binding of cyanocobalamin to cholestyramine.

The intrinsic factor-mediated binding of cyanocobalamin to cholestyramine was studied in vitro under varying conditions of pH, added electrolyte, and bile salt. The intrinsic factor-cyanocobalamin complex was adsorbed strongly by the resin at pH 3 in the presence of neutral salt and low concentrations of glycocholic acid. An increase in glycocholic acid concentration as well as neutral pH decreased the observed binding, although significant amounts of cyanocobalamin-intrinsic factor complex remained bound after incubation at pH 6.8. Cyanocobalamin alone was not adsorbed by the resin.     

Ion-exchange resins as potential phosphate-binding agents for renal failure patients: effect of the physicochemical properties of resins on phosphate and bile salt binding

The effect of resin type, degree of cross-linking, bead size, and surface area on the phosphate and bile salt binding characteristics of five strongly basic Dowex anion-exchange resins in the chloride form was studied. The maximum uptake of phosphate (expressed as uptake of phosphorus) from sodium phosphate solutions was 137, 82, 86, 138, and 76 mg of phosphorus per gram of dry Dowex resins XF 43311, XY 40013, XF 43254, XY 40011, and XY 40012, respectively. The presence of simulated gastric or intestinal fluids resulted in small but insignificant alterations in phosphorus uptake by the resins. The resins all bound similar amounts of phosphorus and taurocholate (80-100% of the total phosphorus and taurocholate in solution) at physiological concentrations of phosphate and bile salt. Dowex resins XY 40013 and XF 43254, with identical physicochemical properties, but different bead sizes and surface areas, bound similar amounts of the bile salt sodium taurocholate at all taurocholate concentrations, indicating that binding was not restricted to the surface sites on the resin bead. The 2% cross-linked resins bound 3-4 times more taurocholate than the 8% cross-linked resins (at high taurocholate concentrations); the smaller pore size of the latter resins probably presents a greater mechanical exclusion barrier than the larger pore size of the 2% cross-linked resins.     

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.     

Relative in vitro efficacy of the phosphate binders lanthanum carbonate and sevelamer hydrochloride

The high tablet burden and poor compliance associated with phosphate-binding drugs has led to a search for more potent agents. In vitro-binding studies were performed on the recently introduced binder, lanthanum carbonate (LC; Fosrenol), to compare its phosphate-binding affinity with sevelamer hydrochloride (SH; Renagel). Langmuir equilibrium binding affinities (K(1)) for LC and SH were established using different phosphorus (5-100 mM) and binder (134-670 mg per 50 mL) concentrations at pH 3-7, with or without salts of bile acids present (30 mM). At all pH levels, LC had a higher binding affinity for phosphate than SH. For LC, K(1) was 6.1 +/- 1.0 mM(-1) and was independent of pH. For SH, K(1) was pH dependent, being 1.5 +/- 0.8 mM(-1) at pH 5-7 and 0.025 +/- 0.002 mM(-1) at pH 3, that is, >200 times lower than for LC. In the presence of 30 mM bile salts, SH lost 50% of its phosphate, whereas no displacement of phosphate occurred for LC. These findings indicate that LC binds phosphate more effectively than SH across the pH range encountered in the gastrointestinal tract, and has a lower propensity for bound phosphate to be displaced by competing anions in the intestine.     

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.     

Bile salt-reinforced alginate-chitosan beads

A polymeric delayed release protein delivery system was investigated with albumin as the model drug. The polysaccharide chitosan was reacted with sodium alginate in the presence of calcium chloride to form beads with a polyelectrolyte. In this study, attempts were made to extend albumin release in the phosphate buffer at pH 6.8 from the alginate-chitosan beads by reinforcing the matrix with bile salts. Sodium taurocholate was able to prevent albumin release at pH 1.2, protecting the protein from the acidic environment and extending the total albumin release at pH 6.8. This effect was explained by an interaction between the permanent negatively charged sulfonic acid of sodium taurocholate with the amino groups of chitosan. Mild formulation conditions, high bovine serum albumin (BSA) entrapment efficiency, and resistance to gastrointestinal release seem to be synergic and promising factors toward the development of an oral protein delivery form.     

Factors affecting the bioaccessibility of fluoride from seafood products

Fluoride is considered important for health because of its beneficial effect on the prevention of dental caries and on bone development in the child population. However, excessive intake has negative effects. The main pathway for exposure is oral, through consumption of drinking water, and some food products. Therefore its bioaccessibility (quantity of the element solubilized during the digestive process) is a parameter to be considered when estimating the risk/benefit associated with this element. The aim of the present study was to evaluate the influence of the digestion phase, gastrointestinal digestion factors (pH, pepsin and bile salt concentrations) and the presence of cations on the bioaccessibility of fluoride from seafood products.The results show that the solubilization of fluoride takes place entirely during the gastric phase. Its bioaccessibility is strongly influenced by conditions that favor the formation of insoluble complexes of fluoride with other elements present in the matrix. The factors that are most influential in reducing its bioaccessibility are the increase in pH in the gastric phase, the presence of cations, especially in the intestinal phase, and a low concentration of bile salts.     

Effect of Competing Anions on Binding of Bile Salts by Cholestyramine

The binding of bile salts by cholestyramine may be influenced by other anions, as the Langmuir adsorption coefficients for three bile salts tested were similar to the model anion, citrate. However, the selectivity coefficient indicated preferential binding of cholate anion in comparison to citrate anion. Binding experiments confirmed cholestyramine's preference for bile salts as the presence of other anions reduced but did not prevent the binding of cholate anion. Binding of cholate anion was reduced in direct relationship to the citrate anion concentration. Prior exposure of cholestyramine to citrate anion caused the binding of cholate anion to decrease slightly. Sequential exposure of cholestyramine to simulated gastric fluid and simulated intestinal fluid containing cholate anion resulted in a decrease in cholate binding which was attributed to competition with anions present in the gastrointestinal fluids. Components of tomato juice and orange juice, fluids commonly used to enhance ingestion of cholestyramine, also reduced the binding of cholate anion.     

Wetting properties of bile salt solutions and dissolution media

The objective of this study was to determine the extent to which specific bile salt solutions and compendial dissolution media differ in their ability to wet a model surface. Solutions were examined in the concentration range of bile salts found in the gastrointestinal tract and at pH values approximating those of the stomach and small intestine. Wetting was evaluated from measurement of the surface tension of the solutions and contact angles of sessile drops on poly(methyl methacrylate). Compendial dissolution media had higher surface tensions and contact angles than bile salt solutions at 10 mM. Individual bile salts at 10 mM varied in surface tension lowering and contact angles. The contact angle-concentration profiles achieved plateau values at 2.5 mM. Dewetting was observed at low bile salt concentrations at pH 7.5. Calculated adhesion tension and interfacial tension were consistent with this behavior. The effect was attributed to the influence of the substrate surface charge on the orientation of the adsorbed bile salt molecule. Adhesion tension profiles showed that from low (<0.5 mM) to moderate (2 mM) concentrations preferential bile salt adsorption to the liquid-vapor interface occurred, but at higher values (>2 mM) the preference shifted toward the solid-liquid interface. These results have implications in the design of physiologically based dissolution media.     

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 salt- and lysophosphatidylcholine-induced membrane damage in human erythrocytes.

The interaction of bile salts and lysophosphatidylcholine (LPC) with membranes has implications both in understanding the aetiology of a number of gastrointestinal disorders, including gastritis, gastric ulcers and colonic cancer, and in enhancing drug absorption by various epithelia. The membrane toxicity of nine bile salts (the sodium (S) salts of chenodeoxycholate (CDC), deoxycholate (DC) and cholate (C) and their glycine (G) and taurine (T) conjugates) and LPC was determined using erythrocyte haemolysis as a model parameter. Washed human erythrocytes were incubated for 15-60 min at 20 degrees C with media buffered at pH 8, 7 and 6. Bile salt toxicity was shown to be a function of type, concentration, pH and contact time with the membrane. At pH 7 toxicity decreased in the order LPC greater than unconjugated dihydroxy salts (SDC and SCDC) greater than conjugated deoxycholates (SGDC and STDC) greater than conjugated chenodeoxycholates (SGCDC and STCDC) greater than unconjugated trihydroxy salt (SC) greater than conjugated trihydroxy salts (SGC and STC). Incubation with equimolar combinations of bile salts (SDC+SCDC; STCDC+SGDC; SDC+STDC) indicated that the resultant damage was an additive function of the damage induced by the individual bile salts.     

Quaternized colestipol, an improved bile salt adsorbent: in vitro studies

Colestipol.HCl (col-HCl) was quaternized with methyl iodide to form col-CH3l. The in vitro binding capacities of the quaternized and protonated resins in water and in Tris-HCl buffer (0.0015 and 0.0025 M, pH 7.0) at approximately 22 degrees C for sodium glycocholate (NaGC) was determined by reversed-phase HPLC. The binding capacities were found to depend on the adsorption medium. In water, the binding capacity of col-CH3l was 30% greater than that of its protonated form. In Tris-HCl buffer at pH 7.0, the binding capacities of the resins were similar. When the quaternized colestipol was converted to its chloride form, the binding capacity for NaGC in Tris-HCl increased significantly and was 30% greater than that for its protonated analogue. In Cotazym 65B-water, a medium used to test the binding capacity of the resins in the presence of various agents (to try to simulate intestinal conditions), the binding capacity of the quaternized resin was again greater than that of its protonated form. Quaternization thus increases the in vitro binding capacity of colestipol for the glycocholate anion.     

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.     

Effects of bile salts on gastrointestinal absorption of pravastatin

This study aimed to examine the effects of bile salts and formulations on the absorption through gastrointestinal tract of pravastatin, which has low bioavailability. Pravastatin sodium physical mixtures and solid dispersions were prepared using various bile salts. The physicochemical characteristics and permeation profiles were investigated using pravastatin sodium-bile salt physical mixtures and solid dispersions. Pravastatin in the physical mixture did not achieve amorphous state, whereas that in the solid dispersion was completely converted to amorphous state. The permeation enhancement factors ranged between 1.13 and 11.9 with the addition of bile salts, and the permeation flux of pravastatin sodium greatly increased as the sodium cholate (NaC) concentration increased from 5 to 10 mM. Pravastatin sodium permeation fluxes [μg/(cm(2) h)] from solid dispersions (drug-NaC = 1:49) (20.8 ± 2.7) were much higher than those from physical mixtures (4.7 ± 3.1) and commercial tablets (3.5 ± 1.2) (p < 0.05). The dissolution rates of pravastatin sodium from solid dispersions in pH 1.2 were much lower than those from physical mixtures and commercial products, whereas more than 97% of pravastatin sodium was dissolved at 5 min in pH 6.8. On the basis of the results, it was concluded that pravastatin sodium solid dispersions containing bile salts could enhance drug absorption.     

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.     

Preparation and Characterization of Reconstructed Small Intestinal Brush Border Membranes for Surface Plasmon Resonance Analysis

PURPOSE: To prepare the surface generated by small intestinal brush border membrane vesicles (BBMVs) for the surface plasmon resonance (SPR) analysis, which allows the real-time measurement of binding events occurring on the intestinal membrane. METHODS: BBMVs were isolated from Sprague-Dawley rats, suspended in HEPES-buffered saline, and flowed over the surface of a SPR sensor chip composed of dextran derivatives modified with lipophilic residues. The surface coverage was determined from binding of bovine serum albumin to BBMV-immobilized sensor chip. The performance of BBMVs immobilized was evaluated by their interaction with otilonium bromide and bile salts. RESULTS: The stable BBMV surface was achieved when BBMV suspension was flowed over the sensor chip for 8 h at a rate of 2 microl/min. The flow of otilonium bromide resulted in an increased SPR signal because of its binding to calcium channel, which is known to be distributed over the gastrointestinal tact. When bile salts were flowed over ileal and duodenal BBMV surfaces, respectively, a slightly higher SPR signal was observed in the ileal BBMV surface, indicating the specific interaction of bile salts with bile acid transporters. CONCLUSIONS: BBMV surfaces may be useful for the estimation of binding events on the intestinal membrane by SPR analysis, especially for the drugs that are orally administrated.     

Functional group contribution of bile salt molecules to partitioning of a quaternary ammonium N,N-dimethyl derivative of propranolol

A quaternary ammonium N,N-dimethyl derivative of propranolol was extracted from pH 7.4 phosphate buffer into 1-octanol as ion-pairs with 12 different bile salts. The binding number, n, and the extraction constant, Ke, were determined. To obtain group contribution values of the bile salt molecule from the ion-pair extraction data, multiple linear regression analysis by the Free-Wilson technique was applied. The results showed that the fundamental premise of the functional group's contribution to the ion-pair extraction is valid. The functional groups of counterions contribute to the partitioning of the ammonium compound independently and additively in this system.     

Correction of drug binding defects in uremia in vitro by anion exchange resin treatment

Serum protein binding of weakly acidic drugs is impaired in uremia, but that of basic drugs tends to be normal. Treatment of uremic serum with anion exchange resin (Amberlite CG-400, acetate form) corrected binding defects for three acidic drugs (nafcillin, salicylate and sulfamethoxazole) but did not affect the binding of two basic drugs (trimethoprim and quinidine). Resin treatment of normal human serum did not alter the binding of these five drugs. Extraction of the acetate buffer eluate from resin exposed to uremic serum with n-butyl chloride at acidic pH (3.0) resulted in a fraction that could induce similar binding defects in normal human serum. The factor(s) responsible for binding defects in uremia appears to be lipid soluble, weakly acidic, and dialyzable. It is believed to be tightly bound to albumin at physiologic pH, but dissociates from it at acidic pH. These findings further support the previously proposed hypothesis that drug-binding defects in uremia are due to accumulation of certain endogenous metabolic product(s).     

Effect of colestipol,a new bile acid sequestrant,on the absorption of phenprocoumon in man

Summary The effect of colestipol, a basic anion-exchange resin, which lowers the serum cholesterol level, has been examined on the absorption of phenprocoumon in four human volunteers,in vivo. Plasma concentrations of phenprocoumon were determined after the simultaneous ingestion either of 8 g colestipol or 4 g placebo (microcrystalline cellulose) and 12 mg phenprocoumon according to a randomized crossover repetition design. The plasma levels were not affected by colestipol, suggesting that it had no effect on the absorption of phenprocoumon. —In vitro, the phenprocoumon-binding capacity of colestipol was the same as that of cholestyramine, except in buffers at pH 5 when there was a marked decrease in the colestipol binding.     

Effects of bile salts on propranolol distribution into liposomes studied by capillary electrophoresis

The objective of this study was to study the effect of four different bile salts, cholate (C), deoxycholate (DC), taurocholate (TC), monoketocholate (MKC), on the membrane binding of a cationic model drug, propranolol, using capillary electrophoresis. The apparent distribution coefficient of propranolol in a buffer/liposome system, in the absence and presence of various concentrations of the bile salts, was measured using capillary electrophoresis frontal analysis. At bile salt concentrations which did not disrupt the liposomes, the bile salts increased the apparent distribution coefficient of propranolol in a concentration-dependent manner, to various extents (DC > C > TC > MKC). The mechanisms for these increases were inferred from studies of ion pairing between bile salts and propranolol using mobility shift affinity capillary electrophoresis and from zeta potential measurements. The bile salts ion-paired with propranolol to different extents as indicated by the estimated complexation constants (K range: 30-58 M⁻¹). This was found to have a minor effect on the membrane distribution of propranolol only. The major effect is proposed to be due to the insertion of bile salt into the liposomal membranes leading to a more negatively charged membrane surface thereby providing stronger electrostatic interactions with the positively charged propranolol.