Colonic Absorption and Bioavailability of the Pentapeptide Metkephamid in the Rat

The concept of delivering systemically active peptide drugs to the colon in order to improve their oral absorption requires reasonable peptide permeability of the large intestinal wall and stability against the activity of the colonic microflora. In addition, the role of hepatic extraction needs to be addressed. In this study the absorption of the pentapeptide metkephamid following single pass perfusion of rat ascending colon was investigated by monitoring its disappearance from the large intestine and simultaneous appearance in the portal vein, the hepatic vein and the aorta. In addition its stability against colonic microflora was tested in vitro using pig caecal contents. Metkephamid was absorbed from the large intestine and appeared in the blood circulation; peptide concentrations in the portal vein increased over-proportionally with increasing perfusate concentrations (0.1 – 4.6 mmol/L) from 0.19 µg/mL ± 0.12 (SD, n = 7) to 31.6 µg/mL + 20.65 (SD, n = 4), respectively, and thus suggesting a saturable transport or metabolism. Concentrations in the hepatic vein were significantly lower than in the portal vein, hepatic extraction ratios were 0.35 ± 0.14, 0.61 ± 0.18 and 0.62 ± 0.28 (SD, n = 4) for 0.1, 0.5 and 1.0 mM metkephamid perfusate concentrations, respectively. In the anaerobic colon metabolism model the degradation half-life of the peptide was 14.9 hours, thus, indicating relative stability in the bacterial environment of the colon. The results of the present study encourage further investigations on colonic delivery of peptide drugs.     

Why is it Challenging to Predict Intestinal Drug Absorption and Oral Bioavailability in Human Using Rat Model

Purpose To study the correlation of intestinal absorption for drugs with various absorption routes between human and rat, and to explore the underlying molecular mechanisms for the similarity in drug intestinal absorption and the differences in oral bioavailability between human and rat.Materials and Methods The intestinal permeabilities of 14 drugs and three drug-like compounds with different absorption mechanisms in rat and human jejunum were determined by in situ intestinal perfusion. A total of 48 drugs were selected for oral bioavailability comparison. Expression profiles of transporters and metabolizing enzymes in both rat and human intestines (duodenum and colon) were measured using GeneChip analysis.Results No correlation (r ² = 0.29) was found in oral drug bioavailability between rat and human, while a correlation (r ² = 0.8) was observed for drug intestinal permeability with both carrier-mediated absorption and passive diffusion mechanisms between human and rat small intestine. Moderate correlation (with r ² > 0.56) was also found for the expression levels of transporters in the duodenum of human and rat, which provides the molecular mechanisms for the similarity and correlation of drug absorption between two species. In contrast, no correlation was found for the expressions of metabolizing enzymes between rat and human intestine, which indicates the difference in drug metabolism and oral bioavailability in two species. Detailed analysis indicates that many transporters (such as PepT1, SGLT-1, GLUT5, MRP2, NT2, and high affinity glutamate transporter) share similar expression levels in both human and rat with regional dependent expression patterns, which have high expression in the small intestine and low expression in the colon. However, discrepancy was also observed for several other transporters (such as MDR1, MRP3, GLUT1, and GLUT3) in both the duodenum and colon of human and rat. In addition, the expressions of metabolizing enzymes (CYP3A4/CYP3A9 and UDPG) showed 12 to 193-fold difference between human and rat intestine with distinct regional dependent expression patterns.Conclusions The data indicate that rat and human show similar drug intestinal absorption profiles and similar transporter expression patterns in the small intestine, while the two species exhibit distinct expression levels and patterns for metabolizing enzymes in the intestine. Therefore, a rat model can be used to predict oral drug absorption in the small intestine of human, but not to predict drug metabolism or oral bioavailability in human.     

Mass Balance Approaches for Estimating the Intestinal Absorption and Metabolism of Peptides and Analogues: Theoretical Development and Applications

A theoretical analysis for estimating the extent of intestinal peptide and peptide analogue absorption was developed on the basis of a mass balance approach that incorporates convection, permeability, and reaction. The macroscopic mass balance analysis (MMBA) was extended to include chemical and enzymatic degradation. A microscopic mass balance analysis, a numerical approach, was also developed and the results compared to the MMBA. The mass balance equations for the fraction of a drug absorbed and reacted in the tube were derived from the general steady state mass balance in a tube: dM/dZ = {[(2/R)(P _w + k _r)]CV _L}/v _z, where M is mass, z is the length of the tube, R is the tube radius, P _w is the intestinal wall permeability, k _r is the reaction rate constant, C is the concentration of drug in the volume element over which the mass balance is taken, V _L is the volume of the tube, and v _z is the axial velocity of drug. The theory was first applied to the oral absorption of two tripeptide analogues, cefaclor (CCL) and cefatrizine (CZN), which degrade and dimerize in the intestine. Simulations using the mass balance equations, the experimental absorption parameters, and the literature stability rate constants yielded a mean estimated extent of CCL (250-mg dose) and CZN (1000-mg dose) absorption of 89 and 51%, respectively, which was similar to the mean extent of absorption reported in humans (90 and 50%). It was proposed previously that 15% of the CCL dose spontaneously degraded systemically; however, our simulations suggest that significant CCL degradation occurs (8 to 17%) presystemically in the intestinal lumen. Insulin (M _r = 5700), which is metabolized in the intestine primarily by -chymotrypsin, was chosen for the second application of theory. The simulations show that the intestinal absorption of insulin is approximately 1% of the administered dose. Further, the extent of insulin oral absorption may not exceed 2% even if effective enzyme inhibitors are dosed concurrently since simulations show that insulin absorption is permeability limited. The steady-state macroscopic and microscopic simulation results were comparable and, for the antibiotics, were similar to published clinical results. Therefore, both approaches are useful for estimating the extent of oral peptide absorption and intestinal reaction from in vitro and in situ results.     

Impact of Regional Intestinal pH Modulation on Absorption of Peptide Drugs: Oral Absorption Studies of Salmon Calcitonin in Beagle Dogs

Purpose. To investigate the relationship between the modulation of intestinal pH and the oral absorption properties of a model peptide drug, salmon calcitonin (sCT), in conscious beagle dogs. Methods. Studies were performed to characterize the disintegration of the formulation, intestinal pH changes, and the appearance of the peptide in the blood. Enteric-coated formulations containing sCT and various amounts of citric acid (CA) were tethered to a Heidelberg capsule (HC) and given orally to normal beagle dogs. Blood samples were collected and analyzed by radioimmunoassay (RIA). Intestinal pH was continuously monitored using the Heidelberg pH capsule (HC) system. The integrity of the HC-delivery system tether was verified by fluoroscopy. Results. The intra-individual variation in gastric emptying (GE) of the delivery system was large. There were also large inter-individual differences in the disintegration and absorption properties of the various formulations. However, the peak plasma concentrations of sCT were always observed when the intestinal pH declined. The average baseline intestinal pH was 6.1 ± 0.2 (mean ± SEM, n = 12). The intestinal pH reduction was 2.6 ± 0.4 (mean ± SEM, n = 12, ranged from 0.5 to 4.0 units from baseline). There was a good correlation between the time to reach the trough intestinal pH (t_pH,min) and time to reach the peak plasma concentration (t_conc,max) of sCT (t_conc,max = 0.95 × t_pH,min + 14.1, n = 11, r² = 0.91). Plasma C_max and area under the curve (AUC) increased with increasing amounts of CA in the formulations. Conclusions. The results of these studies demonstrate that the oral absorption properties of a model peptide drug, sCT, can be modulated by changing intestinal pH. sCT is a substrate for the pancreatic serine protease trypsin which has maximal activity at pH 5 to 6. Reducing intestinal pH presumably stabilizes sCT in the GI tract enabling greater absorption of the intact peptide.     

Comparison of the Pharmacokinetics of an Ondansetron Solution (8 mg) When Administered Intravenously,Orally, to the Colon,and to the Rectum

Ondansetron, an antagonist of the serotonin type 3 (5-HT₃) receptor, is indicated for the treatment of chemotherapy-induced emesis. This study compares the pharmacokinetics, especially the bioavailability, of an Ondansetron 8-mg solution when administered intravenously, orally, to the colon via nasogastric intubation, and to the rectum using a retention enema. Six healthy, male volunteers received ondansetron infused into the colon during the first treatment period. These subjects then received the remaining three treatments in random order, with a minimum 1-week washout period between treatments. Serial plasma samples were obtained for up to 24 hr after dosing in each treatment period. Absolute bioavailability after the oral dosing, colonic infusion, and rectal administration averaged 71 ± 14, 74 ± 26, and 58 ± 18%, respectively. These values were not significantly different (P > 0.05). Values of T _max and C _max were also not significantly different among the nonparenteral routes. Mean absorption half-lives were 0.66, 1.1, and 0.75 hr after the oral, colonic, and rectal administrations, respectively. These results indicate that ondansetron is well absorbed in the intestinal segments studied including the upper small intestine, the colon, and the rectum and that sustained-release and suppository formulations of ondansetron are feasible.     

Enhanced Bioavailability of Cefoxitin Using Palmitoylcarnitine. II. Use of Directly Compressed Tablet Formulations in the Rat and Dog

The performance of tablets containing the absorption enhancer palmitoylcarnitine chloride (PCC) and the antibiotic cefoxitin (CEF) was determined by direct placement of tablets in the rat stomach, small intestine, and colon. While the bioavailability (F) of tablets containing 12 mg CEF without PCC ranged from 0.6 to 3.9%, the addition of 24 mg PCC resulted in an enhanced CEF bioavailability in the rat colon (mean ± SD: F = 57 ± 19%) and rat jejunum (F = 71 ± 16%) but not in the rat stomach. Following oral administration to dogs, tablets of 200 mg CEF without or with 600 mg PCC resulted in the same low bioavailabilities (7.0 ± 10.3 and 7.0 ± 3.6%, respectively). However, when these tablets were enteric coated, PCC improved CEF bioavailability from 2.44 ± 1.84 to 29.0 ± 13.4%. Therefore, the use of enteric-coated direct compressed tablets containing PCC and direct compression excipients improved the peroral bioavailability of a poorly absorbed compound.     

The Site of Absorption in the Small Intestine Determines Diltiazem Bioavailability in the Rabbit

Purpose. Since the ability of the small intestine to biotransform a drug may decrease in distal segments of the intestine, this study aimed to assess whether the site of administration in the small intestine could affect the systemic bioavailability of diltiazem and its two active metabolites, N-desmethyldiltiazem (MA) and desacetyldiltiazem (Ml). Methods. Five mg/kg of diltiazem were administered into the lumen of the proximal (0–30 cm, n = 9) or the distal (150–180 cm) small intestine (n = 7) of anesthetized New Zealand rabbits. Blood samples were drawn from the femoral artery for 6 hours, and diltiazem, MA and M1 were assayed by HPLC. Results. The area under the curve (AUC₀ )of diltiazem administered into the distal small intestine was larger than that estimated when diltiazem was given in the proximal segment (14.20 ± 2.82 vs 8.14 ± 0.88 µg.min/ml, p < 0.05), due to a lower diltiazem oral clearance (440 ± 78 vs 660 ± 55 ml/min/kg, p < 0.05). The AUC_{0 360} of MA was not affected by the site of diltiazem administration, but the AUC_{0 360} of M1 was increased when diltiazem was administered in the distal segment of the small intestine. When administered into the distal segment of the intestine, the molar sum of diltiazem and its active metabolites was 48% greater than when delivered into the 0–30 cm segment of the small intestine; as a consequence, absorption of diltiazem in distal segments of the small intestine may enhance its pharmacological response. Conclusions. The site of absorption into the intestine modulates the bioavailability of diltiazem and its two active metabolites.     

Carrier-mediated intestinal absorption of valacyclovir,the L-valyl ester prodrug of acyclovir. 1. Interactions with peptides,organic anions and organic cations in rats

The mechanism of intestinal transport of valacyclovir (VACV), the L-valyl ester prodrug of acyclovir, was investigated in rats using an in situ intestinal perfusion technique. VACV demonstrates an oral bioavailability that is three to five time greater than acyclovir, concentration dependent, and saturable in humans. Homogenate and perfused buffer stability results demonstrated that VACV was increasingly unstable with increasing pH. VACV was converted to ACV in a concentration dependent manner during a single pass through the intestinal segment. Perfusions were performed at 37°C, pH 6.5, and under iso-osmotic conditions (290±10 mOsm L⁻¹). Intestinal outlet concentrations were corrected for VACV that was converted to ACV during the perfusion. The effective dimensionless intestinal permeability (P_e*) of VACV was concentration dependent, saturable (intrinsic K_m = 1.2±0.7 mM), and significantly reduced (p <0.05) in the presence of peptide analogues (amoxicillin, ampicillin, cefadroxil, and cephradine), by the organic anion, p -amino hippuric acid and by the organic cation quinine. VACV transport was not inhibited by classical nucleoside competitive substrates or inhibitors or by valine. These results suggest that H⁺–oligopeptide, H⁺–organic cation, and organic anion transporters are involved in the small-intestinal uptake of VACV. The permeability of VACV in the colon was very low, indicating that VACV is predominantly absorbed from the small intestine. VACV P_e* was not altered in the presence of glucose-induced convective fluid flow, suggesting that carrier-mediated, transcellular uptake is the predominant absorption pathway of VACV in rat small intestine. Based on these results, the oral bioavailability of VACV appears to be significantly influenced by the preabsorptive conversion of VACV to the poorly absorbed ACV, by the involvement of multiple transporters in VACV small-intestinal uptake, and by the low permeability of VACV in the colon. © 1998 John Wiley & Sons, Ltd.     

Biopharmaceutical Approaches for Developing and Assessing Oral Peptide Delivery Strategies and Systems: In Vitro Permeability and In Vivo Oral Absorption of Salmon Calcitonin

Purpose. To evaluate a biopharmaceutical approach for selecting formulation additives and establishing the performance specifications of an oral peptide delivery system using sCT as a model peptide. Methods. The effect of formulation additives on sCT effective permeability and transepithelial electrical resistance (TEER) was evaluated in side-by-side diffusion chambers using rat intestinal segments. Baseline regional oral absorption of sCT was evaluated in an Intestinal and Vascular Access Port (IVAP) dog model by administration directly into the duodenum, ileum, and colon by means of surgically implanted, chronic catheters. The effect of varying the input rate and volume of the administered solution on the extent of sCT absorption was also evaluated. Citric acid (CA) was utilized in all studies to cause a transient reduction in local pH. In vitro samples and plasma samples were analyzed by radioimmunoassay (RIA). Two oral delivery systems were prepared based on the results of the in vitro and IVAP studies, and evaluated in normal dogs. Results. Maximal permeability enhancement of sCT was observed using taurodeoxycholate (TDC) or lauroyl carnitine (LC) in vitro. Ileal absorption of sCT was higher than in other regions of the intestine. Low volume and bolus input of solution formulations was selected as the optimal condition for the IVAP studies since larger volumes or slower input rates resulted in significantly lower sCT bioavailability (BA). Much lower BA of sCT was observed when CA was not used in the formulation. The absolute oral bioavailability (mean ± SD) in dogs for the control (sCT + CA) and two proprietary sCT delivery systems was 0.30% ± 0.05%, 1.10 ± 0.18%, and 1.31 ± 0.56%, respectively. Conclusions. These studies demonstrate the utility of in vitro evaluation and controlled in vivo studies for developing oral peptide delivery strategies. Formulation additives were selected, the optimal intestinal region for delivery identified, and the optimal release kinetics of additives and actives from the delivery system were characterized. These methods were successfully used for devising delivery strategies and fabricating and evaluating oral sCT delivery systems in animals. Based on these studies, sCT delivery systems have been fabricated and tested in humans with favorable results.     

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.     

Prediction of the Oral Absorption of Low-Permeability Drugs Using Small Intestine-Like 2/4/A1 Cell Monolayers

Purpose. To characterize the paracellular route of 2/4/A1 monolayers and to compare the permeabilities of incompletely absorbed oral drugs in 2/4/A1 with those in Caco-2 monolayers. Methods. The cells were cultivated on permeable supports. The 2/4/A1 expression of genes associated with tight junctions was compared with that in the small intestine using RT-PCR. The aqueous pore radii were determined using paracellular marker molecules. The permeabilities of a series of incompletely absorbed drugs (defined as having a fraction absorbed 0 to 80%) after oral administration to humans were studied. Results. Occludin and claudin 1 and 3 were expressed in 2/4/A1. The pore radius of 2/4/A1 was 9.0 ± 0.2 Å, which is similar to that in the human small intestine, although the pore radius was smaller (3.7 ± 0.1 Å) in Caco-2. The relationship between permeability and fraction absorbed of 13 drugs was stronger in 2/4/A1 than in Caco-2. The relationships were used to predict the intestinal absorption of another seven drugs. The prediction was more accurate in 2/4/A1 (RMSE = 15.6%) than in Caco-2 (RMSE = 21.1%). Further, Spearman's rank coefficient between FA and permeability was higher in 2/4/A1. Conclusion. The improved 2/4/A1 cell culture model has a more in vivo-like permeability and predicted the oral absorption of incompletely absorbed drugs better than Caco-2 cells.     

Analysis of Intestinal Perfusion Data for Highly Permeable Drugs Using a Numerical Aqueous Resistance—Nonlinear Regression Method

Purpose. To develop, validate and apply a method for analyzing the intestinal perfusion data of highly permeable compounds using the Numerical Aqueous Resistance (NAR) theory and nonlinear regression (NAR-NLR) and to compare the results with the well-established Modified Boundary Layer (MBL) Analysis. Methods. The NAR-NLR method was validated and the results were compared to the MBL analysis results using previously reported cephradine jejunal perfusion data. Using the Single Pass Intestinal Perfusion (SPIP) method, the concentration dependence of intestinal permeability was investigated for formycin B, proline, and thymidine, three compounds reported to be absorbed by carrier-mediated transport processes. The MBL and NAR-NLR analyses were then applied to the three sets of SPIP data. Results. The results demonstrate that the intrinsic MBL transport parameters were highly variable and, in one case, the analyses failed to give a statistically significant Michaelis constant. The MBL mean dimensionless wall permeabilities (P*_w) were greater than the NAR-NLR P*_w and were also highly variable. In all cases, the NAR-NLR variability was significantly lower than the MBL variability. The extreme variability in the MBL-calculated P*_w is due to the sensitivity of P*_w when the fraction of unabsorbed drug (C_m/C_o) is low or, alternatively, when P*_w approached the aqueous permeability, P*_aq. Conclusions. The NAR-NLR method facilitates the analysis of intestinal perfusion data for highly permeable compounds such as those absorbed by carrier-mediated processes at concentrations below their K_m. The method also allows for the use of a wider range of flow conditions than the MBL analysis resulting in more reliable and less variable estimates of intestinal transport parameters as well as intestinal wall permeabilities.     

Dose-Dependent Intestinal Absorption and Significant Intestinal Excretion (Exsorption) of the β-Blocker Pafenolol in the Rat

The elimination of [³H]pafenolol and metabolites was investigated in fasted and fed rats. Separate groups received intravenous doses (0.3 and 3.0 µmol/kg) and oral doses (1 and 25 µmol/kg). After iv administration of pafenolol, the excretion of unchanged drug into urine and feces was about 50 and 25–30% of the given dose, respectively. The predominating mechanism for the excretion of pafenolol into feces was intestinal excretion (exsorption) directly from blood into gut lumen, since only about 3% of a given iv dose was recovered as pafenolol in the bile. When the oral dose was raised from 1 to 25 µmol/kg, the mean (±SD) bioavailability, calculated from urine data, increased from 14 ± 9 to 30 ± 11% (P < 0.05) in the starved rats and from 14 ± 3 to 16 ± 3% in the fed animals. In parallel, the fraction absorbed from the gut (f _a) increased from 19 ± 9 to 31 ± 10% in the starved rats and from 16 ± 4 to 19 ± 5% in the fed animals, respectively. This indicates that the low bioavailability is due primarily to poor intestinal uptake.     

Age-Dependent Intestinal Absorption of Valproic Acid in the Rat

The absorption of valproic acid (VPA) across isolated perfused segments of jejunum, ileum and colon was examined in situ in 14-day-to 24-month-old Fischer-344 rats. Within each age group, the intrinsic absorptive clearance (C1_a) of VPA at a perfusate concentration of 1 mg/ml was highest in the jejunum, lowest in the colon, and intermediate in the ileum. When intestinal Cl_a was normalized for the dry weight of the segment, within-group variability decreased. In all segments, VPA Cl_a normalized by dry weight decreased during development (20 to 90 days) and remained relatively constant during aging (90 days to 24 months). The mechanism of valproate absorption (active vs. passive) was examined across age in everted intestinal sacs prepared from each of the three segments. Data were consistent with active transport of VPA in the jejunum and ileum of rats of all ages, and in the colon of pre-weanling animals. Colonic absorption of VPA appeared to occur by passive diffusion in adult rats. In contrast, colonic absorption of d-glucose occurred only by passive diffusion in all age groups. These data indicate that, during development, significant alterations in the rate of VPA absorption occur throughout the rat intestine. Furthermore, while active transport of VPA by the small intestine was present throughout the age range investigated, active transport by the colon became negligible by the time of weaning.     

Intestinal Absorption of Octreotide Using Trimethyl Chitosan Chloride: Studies in Pigs

Purpose. To investigate the enhancing effect of trimethyl chitosan chloride (TMC) on the enteral absorption of octreotide and to delineate the required doses of both TMC and peptide in vivo in juvenile pigs. Methods. Six female pigs (body weight, 25 kg) were operated to induce a stoma at the beginning of their jejunum and to insert an in-dwelling fistula for intrajejunal (IJ) administration of the formulations. A silicone cannula was inserted at the jugular vein for blood sampling. One week after surgery the pigs received IJ octreotide solution administrations with or without TMC at pH 7.4 or chitosan HCl at pH 5.5. For determining bioavailability (F) values, the pigs also received an octreotide solution intravenously (IV). Blood samples were taken from the cannulated jugular vein and subsequently analyzed by radioimmunoassay. Results. Intrajejunal administration of 10 mg octreotide without any polymer (control solution) resulted in F values of 1.7 ± 1.1% (mean ± SE). Chitosan HCl 1.5% (w/v) at pH 5.5 led to a 3-fold increase in F compared to the control (non-polymer containing) formulations. Co-administration of octreotide with 5 and 10% (w/v) TMC at pH 7.4 resulted in 7.7- and 14.5-fold increase of octreotide absorption, respectively (F of 13.9 ± 1.3% and 24.8 ± 1.8%). IJ administration of 5 mg octreotide solutions resulted in low F values of 0.5 ± 0.6%, whereas co-administration with 5% (w/v) TMC increased the intestinal octreotide bioavailability to 8.2 ± 1.5%. Conclusions. Cationic polymers of the chitosan type are able to enhance the intestinal absorption of the peptide drug octreotide in pigs. In this respect, TMC at neutral pH values of 7.4 appears to be more potent than chitosan HCl at a weak acidic pH of 5.5.     

Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model.

The purpose of present study was to determine the intestinal absorption and metabolism of genistein and its analogs to better understand the mechanisms responsible for their low oral bioavailability. The Caco-2 cell culture model and a perfused rat intestinal model were used for the study. In both models, permeabilities of aglycones (e.g., genistein) were comparable to well absorbed compounds, such as testosterone and propranolol. In the Caco-2 model, permeabilities of aglycones were at least 5 times higher (p < 0.05) than their corresponding glycosides (e.g., genistin), and the vectorial transport of aglycones was similar (p > 0.05). In contrast, vectorial transport of glucosides favored excretion (p < 0.05). Limited hydrolysis of glycosides was observed in the Caco-2 model, which was completely inhibited (p < 0.05) by 20 mM gluconolactone, a broad specificity glycosidase inhibitor. In the perfused rat intestinal model, genistin was rapidly hydrolyzed (about 40% in 15 min) in the upper intestine but was not hydrolyzed at all in the colon. Aglycones were rapidly absorbed (P*(eff) > 1.5), and absorbed aglycones underwent extensive (40% maximum) phase II metabolism via glucuronidation and sulfation in the upper small intestine. Similar to the hydrolysis, recovery of conjugated genistein was also region-dependent, with jejunum having the highest and colon the lowest (p < 0.05). This difference in conjugate recovery could be due to the difference in the activities of enzymes or efflux transporters, and the results of studies tend to suggest that both of these factors were involved. In conclusion, genistein and its analogs are well absorbed in both intestinal models, and therefore, poor absorption is not the reason for its low bioavailability. On the other hand, extensive phase II metabolism in the intestine significantly contributes to its low bioavailability.     

Stool Water Content and Colonic Drug Absorption: Contrasting Effects of Lactulose and Codeine

Purpose. By varying stool water content using lactulose and codeine, we investigated the influence of luminal water content on the absorption of quinine, a transcellular probe, and ^5lCr-EDTA, a paracellular probe, from the distal gut. Methods. Sixteen volunteers entered a three-way cross-over trial in which absorption of probe markers from a timed-release delivery system was determined following treatment with lactulose 20 mls tds (increasing water content), or codeine 30 gms qds (decreasing water content), and compared with control untreated values. Stool water content was assessed by freeze drying stool samples. Site of release was determined by gamma scintigraphy, and absorption was measured by plasma levels and urinary recovery of the marker probes. Results. Lactulose accelerated ascending colon transit (3.7 ± 0.8 vs 4.5 ± 1.4 hrs, p < 0.05), increased stool water content (75 ± 2 vs 71 ± 2%, p < 0.01), caused greater dispersion of released material (dispersion score 3.4 ± 0.3 vs 1.8 ± 0.2, p < 0.01), and enhanced absorption of the transcellular probe quinine (4.66 ± 0.78 vs 3.02 ± 0.63%, p < 0.05) compared to control. Conversely codeine slowed ascending colon transit (8.9 ± 1.8 hrs), reduced stool water content (61 ± 2 vs 71.2%, p < 0.05), and tended to diminish absorption (2.60 ± 0.77 vs 3.02 ± 0.63%, p = 0.20). Within the ascending colon specifically, there was a significant trend for treatments increasing luminal water content to enhance quinine absorption (medians: codeine = 1.2%, [n = 8] < control = 2.3%, [n = 5] < lactulose = 3.2%, [n = 7], p < 0.01). Delivery site also had an important influence on absorption, with more distal release resulting in less absorption in the control arm (medians: small intestine = 4.4% [n = 5] > ascending colon = 2.3% [n = 5] > transverse colon = 1.5% [n = 6], p < 0.005). Conclusions. Lactulose accelerates transit, increases stool water content, and enhances drug absorption from the distal gut whilst codeine slows transit, decreases stool water content, and tends to diminish absorption, compared to controls. We conclude that water content may be an important determinant in colonic drug absorption.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

First-pass Metabolism of Peptide Drugs in Rat Perfused Liver

To elucidate the extent and mechanisms of the first-pass metabolism of peptide drugs in the liver after oral administration, a liver perfusion study was performed in rats using metkephamid, a stable analogue of methionine enkephalin, and thyrotropin-releasing hormone (TRH), as model peptides. The fraction of intact metkephamid recovered after single-pass constant perfusion through rat liver reached steady-state very quickly, and it was concluded that metkephamid was hydrolysed enzymatically at the surface of hepatocytes or endothelial cells of microvessels, or both, rather than being taken up by hepatocytes. The fraction of metkephamid recovered intact was approximately 40% under protein-free conditions but increased to 70–75% on addition of bovine serum albumin (BSA) to the perfusate. The fraction of metkephamid bound to BSA was approximately 50% under these conditions, implying that only the free fraction of metkephamid in the plasma was metabolized in the liver. Calculations based on the tube model showed that approximately 30–35% of metkephamid absorbed from the intestine undergoes first-pass metabolism before entering the systemic circulation in-vivo. In contrast, the fraction of TRH metabolized in the liver was less than 10%, indicating a remarkably low contribution of first-pass metabolism to the bioavailability of TRH. These results show that hepatic first-pass metabolism of metkephamid contributes to its low systemic bioavailability. After intestinal absorption free metkephamid is rapidly hydrolysed on the surface of hepatocytes or endothelial cells, rather than being taken up by hepatocytes. This information has important implications in the oral delivery of many kinds of peptide.     

A pharmacological comparison of [3H]-granisetron binding sites in brain and peripheral tissues of the mouse

The affinities of a range of structurally diverse 5-HT₃ receptor agonists and antagonists for [³H]-granisetron binding sites have been measured in membrane homogenates prepared from central and peripheral tissues of the mouse. By comparing the affinities of compounds across these tissues, the question of whether intea-species 5-HT₃ receptor subtypes exist in the mouse has been addressed.In entorhinal cortex and brainstem, [³H]-granisetron bound to a single high affinity saturable binding site (K_d 0.47 ± 0.14 and 0.60 ± 0.05 nM; B _max 20 ± 6 and 7 ± 2 fmol (mg protein)^–1 respectively; mean ±SEM; n = 3). In distal and proximal colon, the specific binding of [³H]-granisetron was best fitted to a 2-site model. K_d values obtained for the high affinity site were similar to those obtained in brain tissue (distal colon: 0.47 ± 0.09 nM, n = 4; proximal colon: 0.39 ± 0.09 nM, n = 4). In salivary gland, 2-sites were evident in 2 out of 4 experiments. The K_d value (calculated from the high affinity site in the 2-site model) was approximately 10-fold less than in brain or colon (3.3 ± 1.1 nM, n = 4). B _max values were 7 ± 2, 4 ± 1 and 71 ± 16 fmol (mg protein)^–1 for distal colon, proximal colon and salivary gland respectively. For all tissues the estimated affinity of the low affinity site was variable, and B _max values could not be reliably calculated.Extensive comparative studies performed with 17 different 5-HT₃ receptor agonists and antagonists in the five tissues did not reveal differences in affinity for any compound between the entorhinal cortex and the brainstem nor between the two regions of the colon. However, MDL72222, R-zacopride, d-tubocurarine, and GR80284 apparently had significantly lower affinity for colon than brain binding sites. Also, MDL72222, 2-methyl-5-HT, GR80284, 1-(m-chlorophenyl)-biguanide, metoclopramide, and granisetron had significantly lower affinity for the salivary gland binding sites than the brain binding sites. In an attempt to replicate these observations, we conducted a second study using the compounds which had shown the largest inter-tissue differences in affinity keeping as many variables as possible constant. Simultaneous comparative assays on entorhinal cortex, colon and salivary gland homogenates taken from the same mice showed that the differences that were apparent in the initial comparative study were not maintained. In conclusion, we can find no clear evidence for the existence of tissue-specific subtypes of the 5-HT₃ high affinity binding site for [³H]-granisetron in the mouse in the tissues tested. However, a low affinity binding site for [³H]-granisetron was detected in peripheral tissues.