Prediction of dissolution-absorption relationships from a dissolution/Caco-2 system

While the analysis of in vitro dissolution-in vivo absorption relationships from oral solid dosage forms provides biopharmaceutical insight and regulatory benefit, no well developed method exists to predict dissolution-absorption relationships a priori to human studies. The objective was to develop an integrated dissolution/Caco-2 system to predict dissolution-absorption relationships, and hence the contributions of dissolution and intestinal permeation to overall drug absorption for fast and slow formulations of piroxicam, metoprolol, and ranitidine. Dissolution studies were conducted on fast and slow dissolving immediate-release formulations of piroxicam, metoprolol tartrate, and ranitidine HCl. Dissolution samples were treated with concentrated buffers to render them suitable (i.e., isotonic and neutral pH) for Caco-2 monolayer permeation studies. The dissolution/Caco-2 system yielded a predicted dissolution-absorption relationship for each formulation which matched the observed relationship from clinical studies. The dissolution/Caco-2 system's prediction of dissolution or permeation rate-limited absorption also agreed with the clinical results. For example, the dissolution/Caco-2 system successfully predicted the slow piroxicam formulation to be dissolution rate-limited, and the fast piroxicam formulation to be permeation rate-limited. Moreover, the system predicted this change from dissolution rate-limited absorption for slow piroxicam to permeation rate-limited absorption for fast piroxicam, in spite of piroxicam's high permeability and low solubility. The dissolution/Caco-2 system may prove to be a valuable tool in formulation development. Broader evaluation of such a system is warranted.     

Prediction of dissolution-absorption relationships from a continuous dissolution/Caco-2 system

The objectives were 1) to design a continuous dissolution Caco-2 system to predict the dissolution-absorption relationships for fast and slow dissolving formulations of piroxicam, metoprolol tartrate, and ranitidine HCl, and compare the predicted relationships with observed relationships from clinical studies; 2) to estimate the effect of croscarmellose sodium on ranitidine dissolution-absorption relationships; and 3) to estimate the effect of solubilizing agents on piroxicam dissolution-absorption relationships. A continuous dissolution/Caco-2 system was constructed from a dissolution apparatus and a diffusion cell, such that drug dissolution and permeation across a Caco-2 monolayer would occur sequentially and simultaneously. The continuous system generally matched observed dissolution-absorption relationships from clinical studies. For example, the system successfully predicted the slow metoprolol and slow ranitidiine formulations to be permeation-rate-limited. The system predicted the slow piroxicam formulation to be dissolution-rate-limited, and the fast piroxicam formulation to be permeation-rate-limited, in spite of piroxicam’s high permeability and low solubility. Additionally, the system indicated croscarmellose sodium enhanced ranitidine permeability and predicted solubilizing agents to not modulate permeability. These results suggest a dissolution/Caco-2 system to be an experimentally based tool that may predict dissolution-absorption relationships from oral solid dosage forms, and hence the relative contributions of dissolution and permeation to oral drug absorption kinetics.     

In Vitro and In Situ Permeability of a 'Second Generation' Hydroxypyridinone Oral Iron Chelator: Correlation with Physico-Chemical Properties and Oral Activity

Purpose. The in vitro and in situ transport of CGP 65015 ((+)-3-hydroxy-1-(2-hydroxyethyl) -2-hydroxyphenyl-methyl-1 H-pyridin-4-one), a novel oral iron chelator, is described. The predictive power of these data in assessing intestinal absorption in man is described. Methods. Caco-2 epithelial monolayer and in situ rat jejunum perfusion intestinal permeability models were utilized. In vivo iron excretion and preliminary animal pharmacokinetic experiments were described, lonization constants and octanol/aqueous partition coefficients were measured potentiometrically. Solubilities and intrinsic dissolution rates were determined using standard procedures. Results. Caco-2 cell (P_app 0.25 X 10^–6 cm.s^–1) and rat jejunum (P_w 0.4) permeabilities of CGP 65015 were determined. The log D(pH 7.4) of CGP 65015 was 0.58 and its aqueous solubility was > 0.5 mg.ml^–1 (pH 3–9). The intrinsic dissolution rate of CGP 65015 in USP simulated intestinal fluid was 0.012 mg.min^–1.cm^–2. CGP 65015 promotes iron excretion effectively and dose dependently in animals. Conclusions. Caco-2 and rat intestinal permeabilities predict incomplete oral absorption of CGP 65015 in man. Preliminary rat pharmacokinetics support this. Physico-chemical data are, also, in line and suggest that CGP 65015 may, in addition, be solubility/dissolution rate limited in vivo. Nevertheless, early animal pharmacological data demonstrate that CGP 65015 is a viable oral iron chelator candidate.     

Analysis of Drug Permeation Across Caco-2 Monolayer: Implication for Predicting In Vivo Drug Absorption

Purpose. The aim of the present work is to characterize in vitro drug permeation processes across Caco-2 monolayer and to identify the advantages of this cultured cell system in predicting in vivo drug absorption after oral administration. Methods. The passive permeability of various drugs through Caco-2 monolayer was measured using Ussing-type chambers and compared with that of the isolated rat jejunum and colon. The in vivo drug permeability to the intestinal membrane was estimated by means of an intestinal perfusion study using the rat jejunum. Results. In Caco-2 monolayer, drug permeability increased with increasing drug lipophilicity and showed a good linear relationship with the in vivo permeability. In contrast, in the isolated jejunum and colon, the permeability of high lipophilic drugs was almost constant and, propranolol, a drug with the highest lipophilicity, hardly passed through the jejunal membrane in vitro. As a result, there was no significant relationship between in vitro and in vivo drug permeability in rat jejunum. However, the amount of drugs accumulated in the jejunal mucosa increased with increasing drug lipophilicity even under the in vitro condition. Conclusions. The permeation and the accumulation studies suggested that the rate-limiting process of in vitro permeation of lipophilic drugs through the intestinal membrane differs from that of in vivo drug absorption. On the other hand, drug permeation through Caco-2 monolayer, which consists of an epithelial cell layer and a supporting filter, is essentially the same process as that of in vivo drug absorption. We concluded that the simple monolayer structure of a cultured cell system provides a distinct advantage in predicting in vivo drug absorption.     

Regional Drug Delivery I: Permeability Characteristics of the Rat 6-Day-Old Air Pouch Model of Inflammation

Purpose. To determine the permeability characteristics of the rat air pouch model of inflammation using permeability extremes within which the NSAIDs S[ + ] ibuprofen, piroxicam and diclofenac could be evaluated. Methods. Permeability was calculated using concentration data obtained following intrapouch and intravenous administration of [³H]-water, [¹⁴C]-urea, [¹⁴C]-inulin and [¹²⁵I]-albumin and compared to similar data obtained for the three NSAIDs. Results. Similar permeability values (5–6.5 ml hr^–1) were obtained for the three NSAIDS which fell between the permeability extremes of the molecular weight markers [³H]-water (9.7 ml hr^–1), [¹⁴C]-urea (6.8 ml hr^–1), [¹⁴C]-inulin (1.0 ml hr^–1) and [¹²⁵I]-albumin (0.6 ml hr^–1). Coadministration of equipotent anti-inflammatory doses of the NSAIDs did not affect local blood flow to the air pouch (as assessed by urea kinetics) but did reduced vascular permeability (as assessed by albumin flux into the pouch). Conclusions. Comparison of the NSAIDs with the permeabilities of the molecular weight markers indicates that a perfusion rate limitation probably exists. Systemic absorption is complete over the first two hours following intrapouch administration of the NSAIDs, therefore albumin flux into the pouch is insufficient to materially affect the permeability of the NSAIDs. However, subsequently (post 5hr) albumin concentration in the pouch rises sufficiently to lower the effective flux of the NSAIDs.     

Effects of Structural Modifications on the Intestinal Permeability of Angiotensin II Receptor Antagonists and the Correlation of In Vitro,In Situ,and In Vivo Absorption

Purpose. The effects of structural modifications on the membrane permeability of angiotensin II (Ang II) receptor antagonists and the usefulness of in vitro and in situ intestinal absorption models in predicting in vivo absorption or bioavailability were investigated. Methods. Intestinal permeability was determined in vitro using Caco-2 cell monolayers and in situ using a perfused rat intestine method. Several physicochemical parameters were either measured or computed, and correlated with intestinal permeation. Results. Permeation coefficients (Pa) across Caco-2 cell monolayers correlated well with both in situ absorption rate constants (ka) and in vivo bioavailability or % absorption. For these Ang II antagonists, Pa values larger than 3 × 10^–6 cm sec^–1 and in situ ka values of 2 × 10^–4 min^–1 cm^–1 or above were associated with good in vivo absorption. Structural modifications at the R₅ position, where a COOH group was substituted with either a CHO or CH₂OH group, enhanced the permeability of the Ang II receptor antagonists up to 100-fold. There were good correlations between permeability and log P(octanol/buffer), log P_HPLCcharge, solvation/desolvation energy and assigned hydrogen bonding potential. Conclusions. The correlations obtained in this study indicate that both the Caco-2 cell model and the in situ perfused rat intestine could be used to predict intestinal absorption in vivo. Structural modifications of the Ang II antagonists had a significant impact on the intestinal permeability. Charge, solvation energy, and hydrogen bonding are predominant determinants of intestinal permeability and oral bioavailability of these compounds.     

Mechanism of Intestinal Absorption of Ranitidine and Ondansetron: Transport Across Caco-2 Cell Monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability coefficients (P _app) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. P _app values for ranitidine and ondansetron (bioavailability of 50 and 100% in humans, respectively) were 1.03 ± 0.17 × 10^–7 and 1.83 ± 0.055 × 10^–5 cm/sec, respectively. The P _app value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.     

Effect of Size and Charge on the Passive Diffusion of Peptides Across Caco-2 Cell Monolayers via the Paracellular Pathway

Purpose. To evaluate the effect of size and charge on the permeation characteristics of peptides across the intestinal mucosa. Methods. The lipophilicities of neutral, positively and negatively charged capped amino acids (Asn, Lys, Asp), tripeptides (Ac-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) and hexapeptides (Ac-Tip-Ala-Gly-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) were estimated using an immobilized artificial membrane. The diffusion coefficients used to calculate the molecular radii were measured by NMR. The transport characteristics of the model peptides were determined across Caco-2 cell monolayers. Results. When model compounds having the same charge were compared, permeation was highly size-dependent (capped amino acids > tripeptides > hexapeptides), suggesting transport predominantly via the paracellular route. For example, the flux of the negatively charged Asp amino acid (P_app = 10.04 ± 0.43 × 10^–8 cm/s) was 3 times greater than that observed for the Asp-containing hexapeptide (P_app = 3.19 ± 0.27 × 10^–8 cm/s). When model compounds of the same size were compared, permeation across the cell monolayer was charge-dependent (negative < positive neutral). For example, the neutral, Asn-containing tripeptide (P_app = 25.79 ± 4.86 × 10^–8 cm/s) was substantially more able to permeate the Caco-2 cell monolayer than the negatively charged Asp-containing tripeptide (P_app = 7.95 ± 1.03 × 10^–8 cm/s) and the positively charged Lys-containing tripeptide (P_app = 9.86 ± 0.18 × 10^–8 cm/s). The permeability of the cell monolayer to peptides became less sensitive to net charge as the size of the peptides increased. Conclusions. A positive net charge of hydrophilic peptides enhances their permeation across the intestinal mucosa via the paracellular pathway. With increasing molecular size, molecular sieving of the epithelial barrier dominates the transport of peptides, and the effect of the net charge becomes less significant.     

Comparison of HT29-18-C1 and Caco-2 Cell Lines as Models for Studying Intestinal Paracellular Drug Absorption

Purpose. To compare the permeability characteristics of HT29-18-C₁ colonic epithelial cell line with Caco-2, an established model of intestinal drug transport. Methods. Cell lines were grown as epithelial monolayers. Permeability was measured over a range of transepithelial electrical resistance (R_t) using a group of drug compounds. Results. HT29-18-C₁ develop R_t slowly when grown in culture, allowing permeability to be measured over a wide range (80–600 ·cm²). In contrast, Caco-2 monolayers rapidly develop R_t of 300 ·cm² and require Ca²⁺-chelation to generate R_t equivalent to human intestine (60–120 ·cm²). Permeability of atenolol, ranitidine, cimetidine, hydrochlorothiazide and mannitol across HT29-18-C₁ decreased 4–5 fold as R_t developed from 100–300 ·cm² indicating they permeate via the paracellular route. In contrast, ondansetron showed no difference in permeability with changing R_t consistent with transcellular permeation. Permeability profiles across low R_t HT29-18C₁ and pulse EGTA-treated Caco-2 monolayers were the same for all 5 paracellular drugs suggesting that transient Ca²⁺ removal does not alter selectivity of the tight junctions. Permeabilities of cimetidine, hydrochlorothiazide and atenolol across 100 ·cm² HT29-18-C₁ monolayers reflect more closely those reported for the human ileum in vivo than did mature Caco-2 monolayers. Conclusions. HT29-18-C₁ monolayers can be used to study drug permeability at R_t values similar to human intestine without the need for Ca²⁺ chelation. As such, they offer a useful alternative to Caco-2 for modelling intestinal drug absorption.     

In Vivo Assessment of Intestinal,Hepatic, and Pulmonary First Pass Metabolism of Propofol in the Rat

Purpose. The relative contribution of the intestinal mucosa, liver and lung to the in vivo disposition of propofol in the rat was investigated. Methods. Propofol (4.9–5.1 mg · kg^–l) was administered to groups of rats (n = 4) via the intra-arterial, intravenous, hepatic portal venous and oral routes. The AUC's of propofol were estimated and the fractions of the administered dose escaping first pass metabolism by the gut wall (f_G), liver (f_H) and lung (f_L) were calculated. In addition, transport experiments were carried out using Caco-2 cell monolayers to rule out the possibility that intestinal permeability is limiting the oral absorption of propofol. Results. Values for f_G, f_H and f_L were the following: 0.21 ± 0.07, 0.61 ± 0.13, and 0.82 ± 0.09, respectively. The apparent permeability coefficient of propofol across Caco-2 cell monolayers was 24.2 ± 0.3 × 10^–6 cm · sec^–1, which is similar to the apparent permeability coefficient obtained for propranolol (30.7 ± 1.7 × 10^–6 cm · sec^–1), a compound known to easily cross the intestinal epithelial membranes. The formation of propofol glucuronide, a major metabolite of propofol, could not be demonstrated during the flux experiments across the Caco-2 cell monolayers. Conclusions. The intestinal mucosa is the main site of first pass metabolism following oral administration of propofol in the rat. Intestinal metabolism could therefore also contribute to the systemic clearance of propofol.     

Intestinal Absorption Screening of Mixtures from Combinatorial Libraries in the Caco-2 Model

Purpose. Understanding how chemical structures influence transport across the intestinal mucosa will greatly enhance the discovery of orally available drugs. In an attempt to accelerate defining such relationships between structure and transport, six arbitrary mixtures of N-substituted glycine (NSG) peptoids containing 24 physicochemically diverse compounds were evaluated in the Caco-2 model of intestinal absorption. Methods. Samples were analyzed by HPLC and the areas of the peaks representing the components of each mixture were summed to measure 'aggregate' apparent permeability coefficients (P _app) a score of the influence of the common structural element within each mixture towards absorption. Mass spectrometry was used to identify the chemical structure of Caco-2 permeable compounds. Results. Three linear trimeric mixtures were examined and, for each mixture, none of the components was detected in receiver chambers. It was concluded that the components of these mixtures each had a P _app value less than 0.8 × 10^–6 cm/sec, a permeability less than mannitol. Three dimeric mixtures were examined and they exhibited aggregate P _app values of 9.2 × 10^–6 , 14 × 10^–6 and 6.9 × 10^–6 cm/sec. These transport rates reflected the transport of most of the components of each mixture. Furthermore, the components of the dimeric mixtures which were transported at a rate greater than mannitol were apparently transported by passive mechanisms. Conclusions. This study demonstrated that mixtures can be used to study structure-transport relationships in the Caco-2 model. The information obtained from this type of study will be integrated into the design of future chemical libraries. Other potential uses of chemical mixtures with the Caco-2 model are also discussed.     

Mechanisms of Transport and Structure-Permeability Relationship of Sulfasalazine and Its Analogs in Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. Methods. Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. Results. Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BLAP) than from apical to basolateral direction (APBL) at 100, 200, and 500 M, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4×10^–5 cm/s, 1.9×10^–8 cm/s, and 369 M, respectively. The efflux of sulfasalazine was completely blocked at 4°C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. Conclusions. The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.     

Drug Absorption Limited by P-Glycoprotein-Mediated Secretory Drug Transport in Human Intestinal Epithelial Caco-2 Cell Layers

The hypothesis was tested that the operation of an ATP-dependent export pump localized at the apical (brush border) surface of the intestinal epithelium may limit substrate absorption kinetics. Human intestinal Caco-2 cell-layers display saturable secretion of vinblastine from basal to apical surfaces (K _m, 18.99 ± 5.55 µM; V _max, 1285.9 ± 281.2 pmol cm^–2 hr^–1) that is inhibited by verapamil, consistent with the expression of the ATP-dependent P-glycoprotein drug efflux pump at the apical brush border membrane. Inhibition of P-glycoprotein by a variety of modulators (verapamil, 1,9-dideoxyforskolin, nifedipine, and taxotere) is associated with an increased vinblastine absorptive permeability. Vinblastine absorption displayed a nonlinear dependence upon luminal (apical) vinblastine concentration, and vinblastine absorption increased markedly at concentrations where vinblastine secretory flux was saturated (>20 µM). Upon inhibition of P-glycoprotein by verapamil and 1,9-dideoxyforskolin, vinblastine absorption increased and was linearly dependent on vinblastine concentration. The limitation of P-glycoprotein substrate absorption by active ATP-dependent export via P-glycoprotein is discussed, together with the possibility that other classes of substrate may be substrates for different ATP-dependent export pumps.     

In Vitro Percutaneous Absorption of Arildone,a Highly Lipophilic Drug,and the Apparent No-Effect of the Penetration Enhancer Azone in Excised Human Skin

Purpose. Arildone, a novel lipophilic antiviral drug when evaluated in Clinical Trials showed limited skin absorption and antiviral efficacy. These studies were conducted to explain the apparent poor absorption characteristics and attempt to promote skin absorption by using Azone, a penetration enhancer. Methods. Standard in vitro skin permeation methods using excised human skin were employed to characterise the absorption of Arildone. ¹⁴C-Arildone was used to estimate the distribution in skin layers by scintigraphic and autoradiographic procedures. Results. The aqueous solubility and distribution constant values for Arildone were 2 µg ml^–1 and 5 × 10⁵ (isopropyl myristate/water), respectively. Absorption through full thickness skin or stratum corneum-viable epidermal membranes (diffusional resistant dermis removed), from a propylene glycol vehicle, was slow and the addition of Azone had no effect on the permeation rate. Distribution studies showed accumulation of Arildone in the stratum corneum. The concentration of Arildone in the viable epidermis was estimated from sectioning the skin and was found to be in sufficient amounts (400 µg cm^–3) to have potential antiviral activity. Conclusions. The apparent accumulation of Arildone in the stratum corneum suggested that the hydrophilic skin region presented the main barrier to permeation. Azone which affected the permeability of the stratum corneum was therefore not effective at enhancing Arildone absorption. Vehicles which readily permeate and enhance the transfer of lipophilic drugs from the stratum corneum into the viable epidermis were recommended.     

An in Vitro Study of Diamorphine Permeation Through Premature Human Neonatal Skin

The permeation kinetics of diamorphine through human premature neonatal cadaver skin over a range of gestational ages between 24 and 36 weeks was investigated using small diffusion cells. A strong inverse correlation was noted between the apparent permeability coefficient and the gestational age of the skin (P < 0.01; n = 26). The calculated apparent permeability coefficients decreased with gestational age from 6.0 × 10 ^–2 cm · hr^–1 at 24 weeks' gestation to 5.2 × 10^–6 cm · hr^–1 at 36 weeks' gestation. The amount of diamorphine remaining bound within the skin at the end of the in vitro experiments did not change significantly with gestational age of the skin. Diamorphine was subject to degradation over the course of the in vitro experiments to produce significant amounts of 6-mono-acetylmorphine and evidence is presented to suggest that this was due to residual skin esterase activity. It is calculated that the steady-state flux rate of diamorphine through neonatal skin observed in these experiments would be sufficient to obtain a therapeutic plasma concentration of morphine assuming a 2-cm² area for application and a delivery rate of 15 µg hr ^–1 kg^–1. However, the prolonged half-life of morphine in the premature neonate would result in a delay of some hours before the attainment of this level.     

In Vitro Permeability Across Caco-2 Cells (Colonic) Can Predict In Vivo (Small Intestinal) Absorption in Man—Fact or Myth

Purpose. To evaluate and optimize the use of Caco-2 cell monolayers to predict thein vivo absorption of a broad range of compounds in man. Methods. Caco-2 cells are derived from human adenocarcinoma colon cells and spontaneously differentiate when grown on porous polyethylene terephthalate membranes (PETP) in a 12 well format to form monolayers of polarized cells possessing function similar to intestinal enterocytes. Transport experiments were conducted using 21 day cultured cells in a shaking water bath at 37°C. Radiolabeled mannitol was used to determine monolayer integrity. Apparent permeability coefficient (P_app) was calculated from the appearance of drug in the receiver side. Results. A strong correlation was observed between in vivo human absorption and in vitro P_app for a variety of compounds (R = 0.95, N = 35). For compounds that are substrates of p-glycoprotein (Pgp), use of a Pgp inhibitor resulted in a better estimate of absorption in humans. The results of this study suggest that the overall ranking of compounds with P_app < 1 × 10^–6 cm/sec, between 1–10 × 10^–6 cm/ sec, and > 10 × 10^–6 cm/sec can be classified as poorly (0–20%), moderately (20–70%) and well (70–100%) absorbed compounds, respectively. Conclusions. These data suggest that Caco-2 cells developed under the culturing and transport conditions defined herein can be used to predict in vivo human absorption of compounds regardless of transport mechanism, viz., transcellular, paracellular and carrier-mediated.     

In Vitro and In Situ Absorption of SDZ-RAD Using a Human Intestinal Cell Line (Caco-2) and a Single Pass Perfusion Model in Rats: Comparison with Rapamycin

Purpose. To compare the intestinal absorption and active efflux protein susceptibility of a new immunosuppressive agent (SDZ-RAD) with that of its analog rapamycin. Methods. Caco-2 cell monolayers were used to examine bidirectional transport of the two compounds at low micromolar concentrations. Single pass rat intestinal perfusion was also used to examine steady state permeability. Results. Rapamycin and SDZ-RAD showed a distinct preference for transport in the basolateral to apical direction of Caco-2 monolayers as efflux was >20 times greater than apical to basolateral transport. Efflux of SDZ-RAD was completely inhibited by verapamil while efflux of rapamycin was mostly inhibited by verapamil and partially inhibited by probenecid. Passive permeability was shown to be 20 × 10^–6 cm/sec for SDZ-RAD and 10 × 10^–6 cm/sec for rapamycin. In situ rat studies also showed the permeability of rapamycin to be half that of SDZ-RAD with permeabilities of 12.6 × 10^–6 for rapamycin and 24.8 × 10^–6 cm/sec for SDZ-RAD. Conclusions. SDZ-RAD and rapamycin are strong substrates for P-gp-like mediated efflux. Rapamycin is also partially removed from cells by a second efflux system that is not responsive to SDZ-RAD. When these efflux pumps are inhibited SDZ-RAD is likely to be absorbed across the intestine at a faster rate than rapamycin.     

Proliposomes of exemestane for improved oral delivery: Formulation and in vitro evaluation using PAMPA, Caco-2 and rat intestine

The aim of the present study was to develop proliposomal formulations to enhance the oral bioavailability of exemestane by improving solubility, dissolution and/or intestinal permeability. Proliposomal powder formulations were prepared using different ratios of drug (exemestane), distearoyl–phosphatidylcholine (DSPC), cholesterol and dimyristoyl–phosphatidylglycerol (DMPG) by solvent evaporation method. The effect of phospholipid composition and drug:lipid ratio on in vitro performance of proliposomes was studied. Proliposomes were characterized for their particle size distribution, thermal characteristics by differential scanning calorimetry (DSC) and dissolution behavior. Further, the formulated proliposomes were subjected to in vitro permeation or transport studies using different models such as rat intestine, parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell line. Proliposomes provided enhanced exemestane dissolution due to incorporation into the phospholipid bilayers and change in the physical state from crystalline to amorphous. The in vitro transport studies in rat intestine, PAMPA and Caco-2 models revealed that the proliposomes were successful in enhancing the permeation of exemestane. These proliposomal formulations of exemestane could provide improved oral bioavailability due to enhanced solubility, permeability and hence absorption.     

Evaluation of the Caco-2 Monolayer as a Model Epithelium for Iontophoretic Transport

Purpose. To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium. Methods. The apparent permeability co-efficient (Papp) of mannitol, thyrotrophin releasing hormone (TRH), dexamethasone and a range of sizes of fluorescein isothiocyanate (FITC) dextrans across Caco-2 monolayers was measured under passive and electrically stimulated conditions. Trans-epithelial electrical resistance (TEER) was determined throughout. Transmission electron micrographs (TEM) of the monolayers were taken. Confocal laser scanning microscopy (CLSM) was used to visualize the iontophoretic transport route of FITC-Dextran (MW = 20 kDa) across a Caco-2 monolayer. Results. Application of 14.3 -Eq.cm^–2 across the monolayer evoked a transient drop in TEER. The drop in TEER was accompanied by statistically significant increases in fluxes of all the agents in the mucosal to serosal direction except for FD-70. TEM of test samples exhibited tight junction dilatation, in addition to intracellular vacuolisation. The iontophoresis of FD-20 was visualised with confocal laser scanning microscopy and was localised in paracellular spaces of the monolayer. Conclusions. The fluxes of mannitol, TRH, dexamethasone, FD-4, FD-10 and FD-20 across the Caco-2 monolayer were significantly enhanced when electric field was applied. The iontophoretic effect appeared to be directly upon tight junctions     

Use of the InteliSite® Capsule to Study Ranitidine Absorption from Various Sites Within the Human Intestinal Tract

Purpose. The purpose of this study was to evaluate the extent of ranitidine absorption from an externally activated drug-delivery system in two distinct regions of the intestine (jejunum and ileum) in healthy human volunteers. This investigation also was designed to evaluate the utility of the InteliSite ^® capsule for studying regional intestinal drug absorption in humans. Methods. The intestinal absorption of ranitidine from the jejunum and ileum was compared in eight, healthy volunteers in this open-label, two-way crossover study. In two of the eight volunteers, absorption from the colon also was studied. Subjects swallowed the capsule containing ranitidine solution (121 mg) and 100 Ci of ^99mTc-DTPA. The endcap of the capsule contained 20 Ci of ¹¹¹In-DTPA. At the desired intestinal site, the capsule was activated by the application of an external RF magnetic signal (6.78 MHz operating frequency) and the ranitidine solution was released. Blood samples were collected from a forearm vein for 12 hours after capsule activation. Results. The capsule released the ranitidine solution when activated in the jejunum, ileum and colon (visualized by the gamma camera). There was no difference in the extent of ranitidine absorption or ranitidine pharmacokinetics when the capsule was activated in the jejunum or ileum. Conclusions. This study demonstrates the utility of a novel, externally activated drug-delivery system to assess site-specific intestinal drug absorption in humans. Results indicate that use of the InteliSite ^® capsule method to evaluate site-specific intestinal ranitidine absorption in humans yields data similar to that obtained previously by means of oral intubation studies.