Molecular mechanism for the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat, and lisinopril.

The affinity of three substrates for the intestinal peptide carrier is explained based on their three-dimensional (3D) structural data. The kinetic transport parameters of three ACE-inhibitors, enalapril, enalaprilat, and lisinopril, have been determined in an in vivo system using rat intestine. The observed kinetic transport parameters (+/- asymptotic standard error) of enalapril are: 0.81 (+/- 0.23) mM, 0.58 (+/- 0.37) mumol/h per cm2, and 0.56 (+/- 0.04) cm/h for the half-maximal transport concentration (KT), the maximal transport flux (Jmax) and the passive permeability constant (Pm). Enalaprilat was transported by passive diffusional with a Pm of 0.51 (+/- 0.04) cm/h. For lisinopril the kinetic transport parameters were 0.38 (+/- 0.19) mM, 0.12 (+/- 0.07) mumol/h per cm2, and 0.18 (+/- 0.02) cm/h for KT, Jmax, and Pm, respectively. The affinity of the ACE-inhibitors for the intestinal peptide carrier has been evaluated based on their ability to inhibit the transport rate of cephalexin. The inhibition constants (Ki) of enalapril, enalaprilat and lisinopril were 0.15, 0.28 and 0.39 mM, respectively. 3D structural analysis of lisinopril using molecular modelling techniques reveals that intramolecular hydrogen bond formation is responsible for decreased carrier affinity.

Evaluation of a chronically isolated internal loop in the rat for the study of drug absorption kinetics.

A model is described to study absorption kinetics of drugs in the unanesthetized rat. The surgical technique consists of a long-term isolation of an intestinal segment inside the animal. This isolated loop is used in perfusion experiments. In this model the effects of anesthesia and surgical trauma on absorption kinetics are absent. In addition, this model allows for cross-over experimental schemes. Absorption kinetics are evaluated on the basis of steady-state blood levels (Css) of the perfused drug, since the animal can be used in experiments over a long time period. Steady-state blood levels can be used as a measure of the absorption if the compound under investigation shows linear elimination kinetics. Dantrolene sodium was used as a model compound to evaluate this technique. The elimination of dantrolene sodium followed linear kinetics after different intravenous doses in the same rat. The half-life of elimination (t1/2 beta) of dantrolene sodium was approximately 45 min. Perfusions of two different concentrations of dantrolene sodium in the same rat showed that an increase of the perfusate concentration results in a proportional increase in the Css. A prerequisite for performing cross-over experiments is that the absorption characteristics of the isolated segment are constant during the experimental period. This model showed a constant absorption of dantrolene sodium on consecutive days, over a two-week period, in the same rat.

Molecular determinants of recognition for the intestinal peptide carrier.

Computer-aided conformational analysis was used to characterize the pharmacophore for the intestinal peptide carrier. The active analog approach to pharmacophore building was applied as implemented in the SYBYL software package. Conformational analysis and MOPAC calculations were used to determine the lowest energy conformation of carrier substrates, as well as the conformations of compounds that displayed a common pharmacophoric geometry (i.e., inhibitors and inactive structural analogs). A pharmacophore map was calculated, and based on structural mutualities and functional topology, three substrate groups were suggested: compounds that bind to the transporter and are transferred across the membrane; compounds that show affinity for the peptide carrier (i.e., known to inhibit transport of substrates) but are not transferred across the membrane; and compounds that contain the pharmacophoric geometry but show no affinity for the carrier. Affinity for the peptide transporter can be diminished or abolished in either of three ways: esterification of the free carboxylic acid moiety; introduction of a second negative group; and intramolecular steric hindrance of the free carboxylic acid by either side chains with a positively charged nitrogen function or groups capable of hydrogen bond formation.

Comparison of four experimental techniques for studying drug absorption kinetics in the anesthetized rat in situ.

Theophylline absorption kinetics were determined using in situ perfusion techniques. The objectives of this study were to obtain information on the effective permeability constant (ke) of theophylline and its variance during the course of an experiment, and to study the dependence of ke on the experimental technique used. Four in situ intestinal perfusion techniques were compared in the rat: single-pass perfusion, recirculating perfusion, oscillating perfusion, and the closed loop method. The absorption of theophylline appeared to be strongly dependent on the hydrodynamics in the lumen. Constant values and similar coefficients of variation for ke values were obtained for the single-pass perfusion, the recirculating perfusion, and the oscillating perfusion methods. The closed-loop method suffered both from a dropping value of ke with time and a relatively large coefficient of variation.

Intestinal absorption of drugs. I: The influence of taurocholate on the absorption of dantrolene in the small intestine of the rat.

The influence of sodium taurocholate (1) on the intestinal absorption of the lipophilic drug dantrolene (2) was studied in vivo in a chronically isolated internal loop in the rat. Concentrations of 2 were kept below the saturation concentration in saline. Absorption kinetics of 2 were evaluated on the basis of steady-state blood levels, which develop during single-pass perfusions, and on the basis of the rate of disappearance of the drug from the perfusate during recirculating perfusions. Compound 1 at a concentration of 10 mM in the perfusate induced a twofold reduction of the absorption rate compared with the same concentration of 2 in saline. Pretreatment of the absorptive surface with a 10 mM solution of 1 had no detectable effect on the absorption rate of 2 in saline. After perfusions with 10 mM solutions of 1, the perfusate concentration of proteins, phosphorus, and hexoses in the effluent was increased. The reduction of the absorption rate can be ascribed mainly to a reduction of the thermodynamically active concentration of 2 as calculated from the phase-separation model. In addition, 10 mM 1 seems to temporarily increase the barrier function of the mucous layer.

In vitro, ex vivo, and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil.

Caco-2 monolayers (in vitro), rat intestinal sheets mounted in modified Ussing Chambers (ex vivo), and in situ intestinal perfusion of rat ileum were used as models to determine and compare the absorption characteristics of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir) and its bis(pivaloyloxymethyl)-ester prodrug [bis(POM)-PMEA, adefovir dipivoxil]. Although metabolism of adefovir dipivoxil was more pronounced in the ex vivo and in situ models than in the Caco-2 system, the transport of 'total adefovir' [= adefovir dipivoxil and its metabolites mono(POM)-PMEA and adefovir] was comparable in the three models. Compared with transport of the parent compound (adefovir), use of adefovir dipivoxil resulted in a significant increase in transport of total adefovir in the in vitro ( approximately 100-fold) and the in situ ( approximately 10-fold) models; in contrast, the ex vivo method failed to demonstrate a remarkable transport enhancement when using the ester prodrug. Similar to the results obtained in the Caco-2 model, the inclusion of the P-glycoprotein inhibitor verapamil resulted in transport enhancement during in situ perfusion of rat ileum with adefovir dipivoxil; however, no effect of verapamil could be observed in the ex vivo model. The results of this study confirm the utility of both the in vitro and in situ models to assess intestinal transport and metabolism of adefovir dipivoxil. The ex vivo model appeared to be less appropriate because of its inability to discriminate transport following administration of adefovir or adefovir dipivoxil and because of the absence of an effect of verapamil on transport when using adefovir dipivoxil.

Comparison of bioavailability in man of tamoxifen after oral and rectal administration.

The bioavailability of tamoxifen from 40 mg suppositories was tested in six male volunteers and compared with that of tamoxifen (Nolvadex) tablets. Plasma concentrations of tamoxifen and its major metabolites, 4-hydroxytamoxifen and N-desmethyltamoxifen, were measured by extraction from plasma obtained at different times after administration, separated by HPLC, converted on-line to fluorescent phenanthrene derivatives and quantified with a fluorescence detector. The mean relative bioavailability from the suppositories was 28%; the addition of a surfactive agent diminished the bioavailability to 13%. Simulation of repeated administration of 40 mg suppositories suggests a mean steady state plasma concentration for tamoxifen of approximately 70 ng ml-1, i.e. 30% of the steady state value after simulated oral administration. Rectal administration of tamoxifen leads to a lower bioavailability than that by oral administration and therefore cannot be recommended when used in equivalent doses.

Intestinal absorption of drugs. The influence of mixed micelles on on the disappearance kinetics of drugs from the small intestine of the rat.

The solubilization of the hydrophilic drugs paracetamol and theophylline, and the lipophilic drugs dantrolene, griseofulvin and ketoconazole has been determined in mixed micellar aqueous dispersions composed of 10 mM taurocholate + 5 mM oleic acid. The solubilization of dantrolene and paracetamol has also been determined in aqueous (mixed) micellar dispersions of 1 g L-1 lysophosphatidyl-choline (LPC), or taurocholate/LPC. The influence of these (mixed) micelles on the absorption of the model drugs from solution was studied in the rat chronically isolated internal loop. Absorption kinetics of the drugs were evaluated on the basis of the disappearance rate of the drug dissolved in the perfusion medium in this loop. Absorption experiments with taurocholate/oleic acid in the perfusate resulted in a reduction of the disappearance rate for the lipophilic drugs and the hydrophilic drug theophylline. This could partly be ascribed to the decreased fraction of drug free in solution as a result of its micellar solubilization for dantrolene, griseofulvin and ketoconazole, but the decrease in the disappearance rate of theophylline was unexpected. Taurocholate/oleic acid, LPC and taurocholate/LPC micelles had no effect on the disappearance of paracetamol. The disappearance rate of dantrolene in the presence of LPC alone was not altered, in spite of the decreased fraction of the drug free in solution owing to its micellar solubilization. In contrast, taurocholate/LPC micelles caused a reduction in the rate of disappearance of dantrolene, as expected according to the phase-separation model. In-vitro, taurocholate and taurocholate/LPC reduced the molecular cohesion of porcine intestinal mucus, whereas LPC alone did not exhibit an effect on the gel structure of mucus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the mucoadhesive polymer polycarbophil on the intestinal absorption of a peptide drug in the rat.

The absorption across rat intestinal tissue of the model peptide drug 9-desglycinamide, 8-arginine vasopressin from bioadhesive formulations was studied in-vitro, in a chronically isolated internal loop in-situ and after intraduodenal administration in-vivo. A controlled-release bioadhesive drug delivery system was tested, consisting of microspheres of poly(2-hydroxyethyl methacrylate) with a mucoadhesive Polycarbophil-coating, as well as fast-release formulation consisting of an aqueous solution of the peptide in a suspension of Polycarbophil particles. Using the controlled-release system, a slight improvement of peptide absorption was found in-vitro in comparison with a non-adhesive control system, but not in-situ or in-vivo. In contrast, bioavailability was significantly increased in all three models from the Polycarbophil suspension in comparison with a solution of the drug in saline. The effect appeared to be dose-dependent, indicative of intrinsic penetration-enhancing properties of the mucoadhesive polymer. A prolongation of the absorption phase in-vitro and in the chronically isolated loop in-situ suggested that the polymer was able to protect the peptide from proteolytic degradation. This could be confirmed by degradation studies in-vitro. The duration of the penetration enhancing/enzyme inhibiting effect was diminished with increasing complexity of the test model, in the same way as was previously found for the bioadhesive effect. This interrelationship suggests that the observed improvement in peptide absorption and the mucoadhesive properties of this polymer are associated. The development of a fast-release oral dosage form for peptide drugs on the basis of Polycarbophil appears to be possible.

Oral bioavailability and in vitro stability of pivampicillin, bacampicillin, talampicillin, and ampicillin in horses.

OBJECTIVES: To determine the oral bioavailabilities of 3 ampicillin esters (pivampicillin, bacampicillin, and talampicillin) and ampicillin sodium, and to determine in vitro stability of the ampicillin esters in ileal contents (pH 8.3 to 8.5). DESIGN: A crossover design to administer the 4 drugs orally, and ampicillin i.v. to all horses in the study. ANIMALS: 4 healthy adult horses. PROCEDURE: The drugs were administered intragastrically to the horses at a dosage equimolar to 15 mg of ampicillin/kg of body weight. Also, ampicillin sodium was administered i.v. at the same dosage. Blood samples were taken up to 12 hours after drug administration, and ampicillin concentrations in plasma were determined. For the in vitro study, the ampicillin esters were incubated at 37 C in ileal contents obtained from ponies with cecal fistulas. After incubation, the remaining intact ester and the formed ampicillin were measured. RESULTS: Absolute oral bioavailability was 31, 39, 23, and 2% for pivampicillin, bacampicillin, talampicillin, and ampicillin sodium, respectively. In the in vitro study, 90% decomposition of the ester took place in 30, 60, and 5 minutes, for pivampicillin, bacampicillin, and talampicillin, respectively. CONCLUSIONS: Pivampicillin and bacampicillin are promising candidates for oral antibiotic treatment of horses. The rapid decomposition of ampicillin esters is caused by chemical hydrolysis at the high pH of equine ileal contents.

Bioavailability of pivampicillin and ampicillin trihydrate administered as an oral paste in horses.

Pivampicillin was administered as an oral paste to five healthy adult horses, and an oral paste with ampicillin trihydrate was administered to three horses. Pivampicillin was administered to both starved and fed horses, ampicillin trihydrate was administered to fed horses only. The dose of pivampicillin was 19.9 mg/kg, and the dose of ampicillin trihydrate was 17 mg/kg. Both doses are equivalent on a molecular basis to 15 mg/kg ampicillin. Ampicillin concentrations in plasma were determined up to 24 hours after administration. After administration of pivampicillin to starved and fed horses the mean areas under the plasma concentration-time curve (AUCs) were 23.0 and 19.3 micrograms.h.ml-1, respectively. After administration of ampicillin trihydrate to fed horses the mean AUC was 0.7 microgram.h.ml-1. The peak plasma concentrations were 4.8, 6.7, and 0.1 microgram/ml, after administration of pivampicillin to starved and fed horses and of ampicillin trihydrate to fed horses, respectively. There was no statistically significant difference in peak plasma concentration or AUC between pivampicillin administered to starved or fed horses. It is concluded that pivampicillin administered as an oral paste at a dose of 19.9 mg/kg gives satisfactory plasma concentrations in both starved and fed horses, whereas ampicillin trihydrate produces negligible plasma concentrations. Pivampicillin binds to feedstuffs at the pH found in the horse's stomach and small intestine. After incubation for 6 h at pH 6, approximately 15% remains in solution, and after incubation for 3 h at pH 1.9, approximately 40% remains in solution. Ampicillin, which binds to feedstuffs to a lesser extent, has a lower bioavailability than pivampicillin. Therefore, binding to feedstuffs does not seem to be a critical factor in the absorption of aminopenicillins.

Is a diurnal rhythm in bioavailability caused by a rhythm in intestinal absorption?

This study was undertaken to elucidate the influence of diurnal rhythmicity in rats an the intestinal transport step of two model compounds. One of these compounds is a lipophilic drug (griseofulvine) and the other hydrophilic (theophylline). For this purpose, chronically isolated loops in the rat intestine in the proximal ileum were perfused for approximately 24 hours with a solution of theophylline or griseofulvine in phosphate buffered saline. The water flux was also monitored during perfusion. No significant difference between the disappearance rate at day and at night was shown in the perfusion experiments performed, irrespective of the compound under investigation. Only a normal bias in disappearance rate was shown throughout the perfusion experiments. In the waterflux an influx of 2-5 ml/hr was shown; a diurnal rhythm however was completely absent. We conclude therefore that the rhythmicity in plasma levels found in drug disposition experiments are caused by other influential factors, such as difference in food between morning and evening, difference in transit time or gastric emptying or difference in the amount or composition (pH!) of the gastric or intestinal fluid. Based on our experiments, however, these variations are not caused by diurnal variation in the passive transport step in the mucosa.

The interdigestive colonic motor complex of the dog.

Interdigestive colonic motor patterns of conscious dogs chronically implanted with strain gauge force transducers showed regularly recurring motor complexes. These motor complexes consisted of a sequence of 8-12 tonic contractions (frequency: 1.3/min), on which phasic contractions (frequency: 5-6/min) were superimposed. Complex duration was about 7 min, cycle duration about 32 min. These interdigestive motor complexes of the canine colon were not related to the small intestinal motor complexes and must therefore be considered as a unique and organ-specific motor pattern.

Carrier-mediated transport mechanism of foscarnet (trisodium phosphonoformate hexahydrate) in rat intestinal tissue.

New findings are presented on the specific transport mechanisms of foscarnet (trisodium phosphonoformate hexahydrate) in rat small intestinal tissue and proof for the partial participation of the Na(+)-phosphate co-transport system in foscarnet transport. The transport of the free acid form of foscarnet, phosphonoformic acid (PFA), was studied in rat small intestine by applying Ussing chambers. Transport studies in both mucosal (m)-to-serosal (s) and s-to-m directions revealed polarization of PFA transport. In m-to-s studies, nonlinear concentration-dependent transport was observed and described by the following transport parameters (estimate +/- asymptotic standard error): 0.84 +/- 0.13 mumol/h.cm2, 1.13 +/- 0.29 mM and 0.22 +/- 0.05 cm/h for the maximal transport rate (Jmax), the half-maximal transport concentration (Kt) and the passive membrane permeability constant (Pm), respectively. PFA transport (1.0 mM) was reduced to 72% and to 56% in the presence of the structural analogs phosphate and arsenate (10 mM), respectively. Bidirectional transport studies of PFA at 38 degrees C and 4 degrees C revealed a higher decrease in transport rate for the m-to-s studies than for the s-to-m studies. The combined results of the experiments described in this study demonstrate that PFA transport across rat small intestine is partly passive, using both the paracellular and transcellular pathways, and partly carrier-mediated, involving the phosphate co-transport system.

Prolonged storage of aminophylline suppositories. The impact on physical parameters and bioavailability.

Prolonged storage of aminophylline/cocoa butter suppositories at 30 degrees resulted in a marked decrease in bioavailability, after administration to a panel of human volunteers. Not more than 20% of the drug content was absorbed during the first 8 hours and the maximum plasma level was obtained 5-7 hours after administration. It was concluded that storage conditions should be considered well and storage instructions should be given to the patient.

Intestinal absorption of drugs. III. The influence of taurocholate on the disappearance kinetics of hydrophilic and lipophilic drugs from the small intestine of the rat.

The influence of sodium taurocholate (TC) on the intestinal absorption of drugs was studied in vivo in a chronically isolated internal loop in the rat. The hydrophilic drugs paracetamol (PA) and theophylline (TP) and the lipophilic drugs griseofulvin (GF) and ketoconazole (KE) were used as model drugs. The drug concentrations were kept below the saturation concentration. Absorption kinetics of the drugs were evaluated on the basis of disappearance rates of the drug from luminal solutions in the intestinal loop. Concentrations of TC above the critical micelle concentration (CMC) did not affect the absorption rate of the hydrophilic drugs PA and TP; the barrier function of the intestinal wall for PA and TP was not altered in the presence of taurocholate. The addition of concentrations of TC above the CMC in the perfusion solution resulted in a reduction of the absorption rate of GF and KE. The reduction in the absorption kinetics of GF in the presence of TC correlated well with the reduction of the drug-free fraction in solution due to micellar solubilization. For KE this relation was less clear. It was not possible to determine, on the basis of the phase-separation model, to what extent the fraction of the drug incorporated in TC micelles contributes to the overall diffusion of GF and KE across the preepithelial diffusion barrier. It was concluded that TC exhibits only a minor, if not negligible, effect on the barrier function of the aqueous diffusion barrier adjacent to the intestinal wall.

Kinetics of acetaminophen after single- and multiple-dose oral administration as a gradient matrix system to healthy male subjects.

The in vivo characteristics of two formulations of a recently developed controlled-release system, the Gradient Matrix System (GMS-1 and GMS-2), with acetaminophen as a model drug compound have been determined in healthy volunteers both after separate single- and multiple-dose administration. Values for the mean residence time (MRT) were increased from 5.2 h for an oral solution to 10.2 and 13.3 h for two GMS formulations after single dosing. Peak plasma concentrations were lower for the two GMS formulations after single dosing compared to the oral solution. The bioavailability, relative to the oral solution, was 91 per cent and 84 per cent for the two GMS formulations tested. After multiple dosing of one of the GMS formulations over 5 days, no change in AUC compared to the single dose AUC occurred. Steady state was reached within 2-3 days of twice daily dosing of the GMS formulation. The peak-trough-fluctuation (per cent PTF) was 44 per cent. No signs of dose dumping were observed in fasted subjects. A plateau-like plasma drug concentration profile at steady state was maintained with the GMS formulation.

Comparison of in vitro and in vivo release characteristics of acetaminophen from gradient matrix systems.

An effort was made to correlate the in vivo and in vitro release data of acetaminophen from two formulations of a recently developed controlled-release system, the Gradient Matrix System (GMS-1 and GMS-2). The in vivo release curves, obtained by deconvolution of the plasma concentration time plots, showed a small inter-subject variability. GMS-1 with fastest in vitro release also showed fastest in vivo release. A good relationship was only found after time-scaling of the release data.

Bioavailability of paracetamol after oral administration to healthy volunteers. Influence of caffeine on rate and extent of absorption.

The absorption rate and the bioavailability of two commercially available paracetamol tablets were investigated in a panel of seven volunteers; one of these tablets contained a combination of 50 mg caffeine and paracetamol. Considering the urinary excretion data, it is concluded that the tablets release their contents completely; the absolute bioavailability, however, calculated from plasma concentrations, is lower than 100%, indicating a first-pass effect. A marked interindividual variation in first-pass effect was noticed. No general influence of caffeine on the extent of absorption of paracetamol could be established; there is, however, a slightly positive influence of caffeine on the absorption rate of paracetamol in six out of seven volunteers. It was concluded that this positive influence on absorption rate is not responsible for the established enhancement of paracetamol analgesia by caffeine.

Structure-transport relationship for the intestinal small-peptide carrier: is the carbonyl group of the peptide bond relevant for transport?

PURPOSE: The objective of this study was to determine the influence of the peptide bond with emphasis on the carbonyl group on the interaction with and transport by the intestinal small-peptide carrier. Therefore enalapril, a known substrate for the small-peptide carrier, has been modified to an analogue with a reduced peptide bond, enamipril. The transport characteristics of both compounds have been determined. METHODS: The in vitro transport studies were performed using rat ileum in Ussing chambers. The transport of enalapril and enamipril were measured in a concentration range from 0.5-8 mM in both directions across the ileum. in the presence and absence of inhibitors. The interaction with the small-peptide carrier was studied by evaluating the ability of enalapril and its analogue enamipril to inhibit the transport rate of amoxycillin. RESULTS: Enalapril shows, besides passive diffusion (P(m)3.06+/-0.14 . 10(-6)cm/s), saturable transport kinetics (Jmax = 16+/-5 nmol/h.cm2, Km = 1.86+/-0.64 mM) which can be inhibited with 10 mM cephalexin. The analogue with a reduced peptide bond does not show saturable transport from the mucosal to the serosal side, and cephalexin does not inhibit the flux of enamipril. However, the transport of enamipril from the serosal to mucosal side of the intestinal membrane is saturable and can be inhibited by 100 microM verapamil. Although enamipril is not a substrate for the small-peptide carrier in contrast to enalapril, both enalapril and enamipril are able to inhibit the active transport of amoxycillin with a K(i) of 0.41+/-0.24 mM and 0.24+/-0.12 mM respectively. CONCLUSIONS: The reduction of the peptide bond of enalapril results in a compound, enamipril, which does not show polarized and saturable transport from the mucosal to the serosal side of the intestinal tissue. Also because the transport of enamipril cannot be inhibited by cephalexin, the analogue with the reduced peptide bond is no longer a substrate for the intestinal small-peptide carrier. Therefore, it can be concluded that the carbonyl group is an essential structural requirement for transport by the small-peptide carrier. In contrast, the interaction with the small-peptide carrier is still present, shown by the inhibition of the fluxes of amoxycillin. Reduction of the peptide bond of enalapril resulted in a new substrate for the P-glycoprotein efflux pump.